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FLORIDA INTERNATIONAL UNIVERISTY
Miami, Florida
AN ETHNOPHARMACOLOGICAL APPROACH TO MULTIDRUG-RESISTANT
STAPHYLOCOCCUS AUREUS: EVALUATION OF ITALIAN PLANTS USED IN
THE TRADITIONAL HEALING OF SKIN DISEASE
A dissertation submitted in partial fulfillment of the
requirements for the degree of
DOCTOR OF PHILOSOPHY
in
BIOLOGY
by
CASSANDRA LEAH QUAVE
2008
To: Dean Kenneth Furton College of Arts and Sciences This dissertation, written by Cassandra Leah Quave, and entitled An Ethnopharmacological Approach to Multidrug-Resistant Staphylococcus aureus: Evaluation of Italian Plants used in the Traditional Healing of Skin Disease, having been approved in respect to style and intellectual content, is referred to you for judgment. We have read this dissertation and recommend that it be approved.
_________________________________________ Lisa W. Plano
_________________________________________
Lidia Kos
_________________________________________ Kelsey Downum
_________________________________________
B.M. Golam Kibria
_________________________________________ Bradley C. Bennett, Major Professor
Date of Defense: March 12, 2008
The dissertation of Cassandra Leah Quave is approved.
_________________________________________
Dean Kenneth Furton College of Arts and Sciences
_________________________________________
Dean George Walker University Graduate School
Florida International University, 2008
ii
© Copyright 2008 by Cassandra Leah Quave
All rights reserved.
iii
DEDICATION
I dedicate this dissertation to my family. To my husband, Marco, for his
boundless love, personal sacrifice, and constant support and encouragement as I pursued
this endeavor. To my children, Donato and Isabella, for the infinite joy that they have
brought to my life. To my granny, Eva Grayce, for always believing that I could reach
my goals. To my sister, Marnie, for bringing me closer to God. To my nephew Trevor,
for showing me the beauty of resilience. To my best friend, Mandy, for being my faithful
confidant and enduring source of laughter and comfort. To my mother-in-law, Milagros,
for teaching me the value of patience and the true meaning of family. Lastly, to my
parents, Cecilia and Raymond, for encouraging my curiosity and teaching me that the key
to success is perseverance and hard work.
iv
ACKNOWLEDEGMENTS
I wish to sincerely thank all of the members of my dissertation committee for
their support and guidance. I would especially like to thank my major professor, Dr.
Bennett, for his patience and understanding as I juggled motherhood and graduate school.
His family friendly outlook relieved my apprehension and helped me to complete this
project while still enjoying those precious first years of my children’s lives. His advice
and support in scientific writing and presentation was indispensable, as was the training
that he provided in botanical taxonomy and field methods.
I would like to extend my heartfelt gratitude to Dr. Lisa Plano for her assistance
in procuring bacterial isolates and interpreting the microbiological data. Her advice and
support in the development of this project and in grant writing was incredibly valuable. I
also thank Dr. Golam Kibria for his valuable assistance in the statistical analysis of my
data.
This research project was a rather immense undertaking, and the assistance of
dedicated volunteers was vital to its success. Volunteers were helpful in different aspects
of plant collection, preparation, and analysis. Donato Caputo, Donato Carbone, Asunta
Di Leo, Renato Rivarosa, and Donato Millito assisted in plant collections. Rosanna
Caputo, Milagros Prieto, Fabrizio Caputo, Gemmina Caputo, Antonello Fiore, and
Ettorino Caputo assisted in the recruitment of interviewees. Marco Caputo built the plant
drier used to dry bulk samples and voucher specimens in the field. Carlo and Cristina
Allamprese provided the containers for shipping plant materials. Dr. Carmine Colacino,
Dr. Bradley C. Bennett, and Dr. Andrea Pieroni assisted in the identification of plant
specimens. Traci Pantuso, Marco Caputo, Jana Rose, and Susan Mendez assisted in the
v
laboratory preparation and analysis of plant extracts. Myron Georgiadis assisted in
analyzing δ-toxin with mass spectrometry. Dr. Kalai Mathee allowed use of the sonicator
in her lab during biofilm experiments.
Several people offered their valuable time and expertise to train me in different
research techniques: Dr. Bradley C. Bennett (plant collection and identification), Dr.
Andrea Pieroni (ethnobotanical field methods), Dr. James Graham (plant extraction), Wei
Lin (cell culture), Dr. Horacio Priestap (HPLC), Dr. Michael Otto (δ-toxin assay), Dr.
Robert Perez and Prof. Steve Davis (biofilm assays).
The project described was supported in part by grant number F31AT004288 from
the National Center of Complementary and Alternative Medicine. The content is solely
the responsibility of the author and does not necessarily represent the official views of the
National Center for Complementary and Alternative Medicine or the National Institutes
of Health. This research was also supported by grants from MBRS RISE - NIH/NIGMS
R25GM061347, RISE Biomedical Research Initiative summer research award, Botany in
Action, Anne Chatham Fellowship in Medicinal Botany, USDA CSREES
20053842215940 and NIH/NCAAM 1T32AT01060-01 (PI: B.C. Bennett).
vi
ABSTRACT OF THE DISSERTATION
AN ETHNOPHARMACOLOGICAL APPROACH TO MULTIDRUG-RESISTANT
STAPHYLOCOCCUS AUREUS: EVALUATION OF ITALIAN PLANTS USED IN
THE TRADITIONAL HEALING OF SKIN DISEASE
by
Cassandra Leah Quave
Florida International University, 2008
Miami, Florida
Professsor Bradley C. Bennett, Major Professor
One-third of botanical remedies from southern Italy are used to treat skin and soft
tissue infections (SSTIs). Methicillin-resistant Staphylococcus aureus (MRSA), a
common cause of SSTIs, is responsible for increased morbidity and mortality from
infections. Therapeutic options are limited by antibiotic resistance. Many plants possess
potent antimicrobial compounds for these disorders. Validation of traditional medical
practices is important for the people who rely on medicinal plants. Moreover,
identification of novel antibiotics and anti-pathogenic agents for MRSA is important to
global healthcare.
I took an ethnopharmacological approach to understand how Italian medicinal
plants used for the treatment of SSTIs affect MRSA growth and virulence. My hypothesis
was that plants used in folk remedies for SSTI would exhibit lower cytotoxicity and
greater inhibition of bacterial growth, biofilm formation and toxin production in MRSA
than plants used for remedies unrelated to the skin or for plants with no ethnomedical
application. The field portion of my research was conducted in the Vulture-Alto Bradano
vii
area of southern Italy. I collected 104 plant species and created 168 crude extracts. In the
lab, I screened samples for activity against MRSA in a battery of bioassays. Growth
inhibition was analyzed using broth microtiter assays for determination of the minimum
inhibitory concentration. Interference with quorum-sensing (QS) processes, which
mediate pathogenicity, was quantified through RP-HPLC of δ-toxin production.
Interference with biofilm formation and adherence was assessed using staining methods.
The mammalian cytotoxicity of natural products was analyzed using MTT cell
proliferation assay techniques.
Although bacteriostatic activity was limited, extracts from six plants used in
Italian folk medicine (Arundo donax, Ballota nigra, Juglans regia, Leopoldia comosa,
Marrubium vulgare, and Rubus ulmifolius) significantly inhibited biofilm formation and
adherence. Moreover, plants used to treat SSTI demonstrated significantly greater anti-
biofilm activity when compared to plants with no ethnomedical application. QSI activity
was evident in 90% of the extracts tested and extracts from four plants (Ballota nigra,
Castanea sativa, Rosmarinus officinalis, and Sambucus ebulus) exhibited a significant
dose-dependent response. Some of the plant remedies for SSTI identified in this study
can be validated due to anti-MRSA activity.
viii
TABLE OF CONTENTS
CHAPTER PAGE I. INTRODUCTION...................................................................................................1
The target pathogen: Staphylococcus aureus..........................................................2 A historical perspective on the emergence of drug-resistance in S. aureus............5 Anti-pathogenic properties of natural products: interference with quorum- sensing processes.....................................................................................................6 An ethnopharmacological approach........................................................................9 The study site: Vulture-Alto Bradano area of southern Italy................................10 Summary................................................................................................................11 References..............................................................................................................15
II. DERMATOLOGICAL REMEDIES IN THE TRADITIONAL PHARMACO URE-ALTO BRADANO, INLAND POEIA OF VULTSOUTHERN ITALY.............................................................................................20 Abstract..................................................................................................................20 Background............................................................................................................21 Methods .................................................................................................................24 Results and Discussion..........................................................................................25
The folk medical construct........................................................................25 Botanical remedies.....................................................................................26 Malva sylvestris (Malvaceae) – malva..........................................27
Marrubium vulgare (Lamiaceae) – maruggē................................27 Matricaria recutita (Asteraceae) – camomilla..............................28
Zootherapy and remedies of mineral or industrial origin..........................29 Aged pig fat...................................................................................30 Garden slug....................................................................................30 Human breast milk.........................................................................31
Ethnoveterinary remedies..........................................................................31 Copper sulphate.............................................................................32 Hypericum perforatum (Hypericaceae) – iperico..........................32
Traditional knowledge: current trends and future projections...................33 Conclusion .............................................................................................................34 Acknowledgements ...............................................................................................35 References..............................................................................................................73
III. EFFECTS OF EXTRACTS FROM ITALIAN MEDICINAL PLANTS ON PLANKTONIC GROWTH, BIOFILM FORMATION AND ADHERENCE IN METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS.....................78 Abstract..................................................................................................................78 Introduction...........................................................................................................79 Materials and Methods..........................................................................................82 Field research.............................................................................................82
ix
Extraction of natural products...................................................................84 Bacteria and culture conditions.................................................................85 Growth inhibition......................................................................................86 Biofilm formation and adherence..............................................................87 Mammalian cytotoxicity............................................................................88 Statistical analyses.....................................................................................89 Results...................................................................................................................90 Effects on planktonic growth of MRSA....................................................90
Mammalian cytotoxicity............................................................................90 Biofilm inhibition......................................................................................91
Discussion..............................................................................................................92 Cytotoxicity and creation of a therapeutic index.......................................92 Bacteriostatic activity for MRSA: Erodium malacoides...........................93 Effects on MRSA biofilm formation and adherence.................................94
Arundo donax (Poaceae) – canna..................................................94 Ballota nigra (Lamiaceae) – erba cane.........................................95 Juglans regia (Juglandaceae) - noce..............................................96 Leopoldia comosa (Hyacinthaceae) – cipuljdin............................96 Marrubium vulgare (Lamiaceae) – maruggē................................97 Rubus ulmifolius (Rosaceae) – more.............................................98
Conclusion.............................................................................................................99 Acknowledgements .............................................................................................100 References............................................................................................................130
IV. QUORUM SENSING INHIBITORS FOR METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS FROM ITALIAN MEDICINAL PLANTS..............................................................................................................138 Abstract................................................................................................................138 Introduction .........................................................................................................139 Materials and Methods........................................................................................141 Plant material and extraction...................................................................141 Bacteria and culture conditions...............................................................142 Determination of minimum inhibitory concentrations (MICs)...............142 Quantification of δ-toxin production.......................................................143 Statistical analysis....................................................................................144 Results .................................................................................................................145 Discussion............................................................................................................146 Acknowledgements .............................................................................................147 References............................................................................................................164
V. SUMMARY.........................................................................................................166 VITA................................................................................................................................168
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LIST OF TABLES
TABLE PAGE
CHAPTER 2 Table 1. Table 2. Table 3. CHAPTER 3 Table 1. Table 2. Table 3. Table 4. CHAPTER 4 Table 1. Table 2. Table 3. Table 4.
Data on the sampled communities...........................................................44Popular uses of botanical materials for dermatological conditions and topical (external) applications...........................................................45 Popular uses of animal, mi eral, or industrial derived materials for ndermatological conditions and topical (external) applications...............67
Percent inhibition of growth of 6 S. aureus isolates at a test concentration of 512 μg/ml with unfractionated plant extracts.............112Results from One-way ANOVA tests indicate that there was no significant difference in the inhibition of planktonic growth (p > 0.05) between ethnobotanical usage groups. There was, however, a significant difference in the inhibition of biofilm formation and mammalian cytotoxicity (p< 0.005) between groups.....120Effect of plant extracts on growth and biofilm formation in MRSA (ATCC 33593) and on mammalian cytotoxicity (human T lymphoblastoid cell line). The MIC50 for growth was tested in the range of 8-512 μg/ml, whereas the IC50 for biofilm formation and mammalian cytotoxicity was tested from 4-128 μg/ml due to issues with the percent excipient (DMSO) in the test solution. A dash (-) repres nts that no ICerange tested............................................................................................121
50 was identified within the concentration
Results from a multiple comparison analysis performed using the Bonferroni method. A significant difference in activity of Groups 1 and 3 is indicated for the anti-biofilm data. A significant difference in activity of Groups 2 and 3 is indicated for the mammalian cytoxicity data........................................................................................129 Antibiogram for MRSA PFT USA500/NRS385...................................155Inhibition of δ-toxin and minimal inhibitory concentrations of plant extracts against MRSA (strain I.D. NRS385; PFT USA500)...............156Summary statistics on δ-toxin inhibition for each ethnobotanical usage group............................................................................................163Results from One-way ANOVA tests indicate that there was no significant difference in the inhibition of quorum sensing (p>0.05) between ethnobotanical usage groups....................................................163
xi
LIST OF FIGURES
FIGURE PAGE
CHAPTER 1 Figure 1. Figure 1a. Figure 1b. Figure 2 CHAPTER 2 Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. CHAPTER 3 Figure 1. Figure 2. Figure 3.
Scanning electron micrographs of a MRSA isolate.................................13Magnification x 20,000............................................................................13Magnification x 4,300..............................................................................13Diagram of the accessory gene regulator system in Staphylococcus aureus and its interaction with the regulatory transcript sarA based on data presented in publications by various authors...............................14
Map of the study region: the Vulture-Alto Bradano region of Basilicata, southern Italy..........................................................................37Number of plant-, animal-, and mineral-derived remedies for the topical treatment of medical conditions in humans..................................38Mecca Maria Leonarda (63 yrs. old) with dried chamomile (Matricaria recutita) stems that she uses in a decoction to treat eye infections...................................................................................39Vito Sabato (74 yrs. old) with a garden slug (Arion hortensis) that he uses to treat warts, inflammations, and calluses on the skin...............40A young shepherd (23 years old) from Sant’ Andrea uses a mixture of red wine vinegar and copper sulphate to rinse the cracked hooves of his ass. He also applies the crushed mature fruits of Sambucus ebulus (Iervolē) to open wounds on his livestock as an insect repellent.................................................................41Verderammē (copper sulphate) is used in ethnoveterinary remedies for injured or infected hooves of livestock...............................42Mean and standard deviation of the number of remedies cited by gender and age categories.......................................................................43
Dried figs (Ficus carica L. [Moraceae]) are stuffed with almonds (Prunus dulcis (Mill.) D.A. Webb [Rosaceae]) and placed onto a slice of giant reed stem (Arundo donax L.[Poaceae]), then stored for the winter to be used in a decoction against colds and respiratory ailments. Zia Giovannina (95 years-old) of Ginestra has used this remedy since childhood..........................................................................102The white hemicellulose membrane found at the node of the giant reed (Arundo donax L. [Poaceae]) is used as a haemostatic agent for minor lacerations..............................................................................103Test for biofilm formation and adherence. The graph represents planktonic growth as measured by OD600nm readings for MRSA strain ATCC 33593................................................................................104
xii
Figure 3a. Figure 3b. Figure 4. Figure 5. Figure 6. Figure 7. Figure 8. Figure 9. Figure 9a. Figure 9b. Figure 9c. Figure 9d. Figure 9e. Figure 9f. CHAPTER 4 Figure 1. Figure 1a. Figure 1b.
Extracts were tested for the ability to prevent biofilm formation by adding the test extract to the wells at 0 hours post inoculation and performing the staining process at 40 hours post-inoculation.........104 Extracts were tested for the ability to disrupt established biofilms (promote desorption) by aspirating the liquid content of wells at 20 hours post-inoculation, and then adding the extract and fresh tryptic soy broth. The staining process was performed at 40 hours.......................................................................................................104Ethnobotanical use categories for plants whose extracts demonstrated ≥50% growth inhibiting activity for MRSA at concentrations of 128- 512 μg/ml..........................................................105Comparison of mean bacteriostatic activity of plant extracts from different ethnobotanical groups. Group 1: plants used for SSTI; Group 2: plants used for medicinal purposes not involving the skin; Group 3: plants with no medicinal application......................................106Comparison of mean mammalian cell growth inhibition by plant extracts from different ethnobotanical groups. Group 1: plants used for SSTI; Group 2: plants used for medicinal purposes not involving the skin; Group 3: plants with no medicinal application........................107Ethnobotanical use categories for plants whose extracts demonstrated ≥50% inhibition for MRSA biofilm formation at concentrations of 8- 128 μg/ml..............................................................108Comparison of mean inhibition of biofilm formation by plant extracts from different ethnobotanical groups. Group 1: plants used for SSTI; Group 2: plants used for medicinal purposes not involving the skin; Group 3: plants with no medicinal application.......................109The percent inhibition of extracts for planktonic growth, biofilm formation and adherence in MRSA........................................................110Arundo donax, aqueous extract of nodes...............................................110Ballota nigra, aqueous extract of aerial parts........................................110Juglans regia, ethanolic extract of immature fruits...............................110Leopoldia comosa, ethanolic extract of bulbs........................................110Marrubium vulgare, ethanolic extract of roots......................................111Rubus ulmifolius, ethanolic extract of roots...........................................111
Mass spectroscopic analysis of HPLC fractions containing derformylated and formylated δ-toxin. Peaks matching the spectrogram presented in the study by Somerville et al. (2003) are highlighted..............................................................................................148Absorbance at 280nm of NRS385 (PFT USA500) supernatant fractionated by HPLC............................................................................148Mass spectrogram of peak 1, deformylated δ-toxin (molecular mass 2979.2 Da).....................................................................................148
xiii
Figure 1c. Figure 2. Figure 3. Figure 3a. Figure 3b. Figure 3c. Figure 3d. Figure 4. Figure 4a. Figure 4b. Figure 4c. Figure 4d.
Mass spectrogram of peak 2, formylated δ-toxin (molecular mass of 3007.4 Da).........................................................................................149Comparison of mean quorum sensing inhibition of plant extracts from different ethnobotanical groups. Group 1: plants used for SSTI; Group 2: plants used for medicinal purposes not involving the skin; Group 3: plants with no medicinal application.......150HPLC chroma gram of δ-toxin after treatment with different toconcentrations of plant extract...............................................................151EtOH extract of Ballota nigra stems......................................................151EtOH extract of Castanea sativa leaves.................................................151EtOH extract of Rosmarinus officinalis leaves, stems, and flowers......152EtOH extract of Sambucus ebulus leaves...............................................152Percent inhibition of δ-toxin peak area after treatment with different concentrations of plant extract...............................................................153EtOH extract of Ballota nigra stems......................................................153EtOH extract of Castanea sativa leaves.................................................153EtOH extract of Rosmarinus officinalis leaves, stems, and flowers......154EtOH extract of Sambucus ebulus leaves...............................................154
xiv
LIST OF ACRONYMS
TERM ACRONYM
3-(4,5-dimethylthiazol-2-yl)-2,5,-diphenyl-2H-tetrazolium bromide MTT
Accessory gene regulator agr
American Type Culture Collection ATCC
Autoinducing peptide AIP
Centers for Disease Control and Prevention CDC
Community-associated methicillin-resistant Staphylococcus aureus CA-MRSA
Degrees Celsius °C
Distilled Water dH2O
Dimethyl Sulfoxide DMSO
Ethyl Alchol EtOH
Fairchild Tropical Botanic Gardens FTBG
Glycopeptide Intermediate Staphylococcus aureus GISA
Gylcopeptide Resistant Staphylococcus aureus GRSA
Healthcare-associated methicillin-resistant Staphylococcus aureus HA-MRSA
Hours h
Inhibitory Concentration IC
Optical Density OD
Minutes m
Methicillin Resistant Staphylococcus aureus MRSA
Minimum Inhibitory Concentration MIC
Multi-Locus Sequence Typing MLST
National Institute of Health NIH
xv
National Center for Complementary and Alternative Medicine NCCAM
Panton-Valentine Leucocidin PVL
Penicillin binding protein PBP
Pulsed-Field Gel Electrophoresis PFGE
Pulsed-Field Type PFT
Quorum sensing QS
Quorum sensing inhibitor QSI
Ribonucleic acid RNA
Reverse phase high performance liquid chromatography RP-HPLC
Ribonucleic acid III activating protein RAP
Skin and soft tissue infection SSTI
Staphylococcal accessory regulator sar
Staphylococcal chromosomal cassette SCC
Stratified random sampling SRS
Traditional knowledge TK
Toxic shock syndrome TSS
Toxic shock syndrome toxin 1 TSST-1
Tryptic Soy Broth TSB
United States US
United States Department of Agriculture USDA
World Health Organization WHO
xvi
CHAPTER I
INTRODUCTION
The increasing frequency of antibiotic-resistant bacterial infections poses an
alarming dilemma to healthcare today. Antibiotic resistant “superbugs” threaten public
health worldwide and are associated with rising numbers of patients suffering from
serious infection and even death. Likewise, healthcare costs associated with this
pandemic are steadily growing (Coast et al., 1996).
The predominant force augmenting the rate of resistance is the misuse of
antimicrobial drugs. Examples of misuse include the overzealous application of
antibiotics in agriculture, over-prescription of antibiotics by physicians, over-the-counter
acquisition and self-prescription of antibiotics, lengthy prophylactic antibiotic therapy,
early termination of antibiotic therapy, patient “stockpiling” antibiotics for later use and
recent trends in the household use of antibacterial soaps and cleaning products (Levy,
2002). Overuse of antibiotics leads to the evolution of resistance (Coast et al., 1996).
Emergence of highly resistant bacterial strains has compromised the use of newer
generations of antibiotics including penicillins, cephalosporins, aminoglycolsides,
fluoroquinolones, trimethroprim and trimethroprim-sulfamethoxazole combinations
(Levy, 2002).
In this study, I use techniques from ethnopharmacology to explore the possible
activity of natural products from medicinal plants against a common drug resistant
pathogen. The logic behind this approach is based on the assumption that medicinal
plants used in the folk medical treatment of skin and soft tissue infection (SSTI) should
demonstrate some valid activity against a pathogen responsible for many skin infections:
1
methicillin-resistant Staphylococcus aureus (MRSA). This assumption is reinforced by
the fact that many traditional remedies for the skin have been proven effective due to
antibacterial, antifungal, or anti-inflammatory activity (Cuéllar et al., 1998; Erdemoglu et
al., 2003; Matu and van Staden, 2003). Traditional medicines used for a particular
condition, such as SSTI, are more likely to show greater in vitro and clinical efficacy than
others.
The target pathogen: Staphylococcus aureus
Each year in the US, there are an estimated 292,000 hospitalizations with a
diagnosis of S. aureus infection. Of these approximately 126,000 hospitalizations are
related to MRSA (Kuehnert et al., 2006a). The prevalence of MRSA infections in the US,
combined with the limited therapeutic options offered by existing classes of antibiotics,
makes this pathogen an important target for drug screening. Moreover, the elucidation of
activity against MRSA is important to the validation and conservation of traditional
medical knowledge.
Staphylococcus aureus, a gram-positive bacterium (Figure 1), is a ubiquitous
colonizer of the skin and mucous membranes of humans and animals. Approximately
32% (89.4 million persons) of the US population are colonized with S. aureus, and
approximately 0.8% (2.3 million persons) are colonized with MRSA (Kuehnert et al.,
2006b).
Staphylococcus aureus is a pathogen that causes two types of syndromes:
intoxications and infections. Staphylococcal intoxication involves the release of toxins
into the host, causing symptoms such as fever, rash and multi-organ dysfunction in
2
staphylococcal toxic shock syndrome (TSS), acute exfoliation of the skin in
staphylococcal scalded skin syndrome, and nausea, vomiting, abdominal cramping and
diarrhea in staphylococcal food poisoning (Genigeorgis, 1989).
Staphylococcal infection causes invasive disease through a breach in the host’s
barriers – most commonly the skin. In fact, S. aureus is the most prominent etiological
agent for skin and soft tissue infections (SSTIs), and can cause such conditions as
folliculitis, furuncle, carbuncle, paronychia, bullous impetigo, nonbullous impetigo and
cellulites (Fauci et al., 1998). There were an estimated 12 million outpatient healthcare
visits for suspected S. aureus SSTIs in the United States from 2001-2003 (McCaig et al.,
2006). In 2004, approximately 76% of purulent SSTIs seen in adults treated in emergency
departments from 11 US cities were caused by S. aureus. Of these infections 78% were
caused by MRSA and overall MRSA caused 59% of all SSTIs, including non-purulent
infections (Moran et al., 2006).
In addition to these SSTIs, S. aureus infection is the most common cause of post-
operative brain abscess, subdural empyema, spinal epidural abscess, endophthalmitis
(Callegan et al., 2002), septic intracranial thrombophlebitis, acute bacterial endocarditis,
acute osteomyelitis and chronic osteomyelitis (Fauci et al., 1998). It is also associated
with otitis media (Nawasreh and Fraihat, 2001).
S. aureus infections are categorized into two basic groups: community-associated
and healthcare-associated. Community-associated methicillin-resistant S. aureus (CA-
MRSA) are both genotypically and phenotypically different from healthcare-associated
MRSA (HA-MRSA). CA-MRSA have unique methicillin-resistance chromosomal
cassettes (SCCmec IV and V) and carry the Panton-Valentine leukocidin (PVL) virulence
3
factor (Johnson et al., 2007; Vandenesch et al., 2003). CA-MRSA, unlike HA-MRSA, are
broadly susceptible to antibiotic classes other than the ß-lactams. In the US, two major
clones of CA-MRSA have been identified through pulsed-field gel electrophoresis
(PFGE), and have been named USA300 (Johnson et al., 2007) and USA400 (Stemper et
al., 2006) by the Centers for Disease Control and Prevention (CDC). The virulent
USA300 clone is the predominant cause of SSTIs in the US (Johnson et al., 2007).
CA-MRSA are mainly responsible for SSTIs in children and young adults.
Outbreaks are also common in certain distinctive populations, such as school children,
athletes, prison inmates, and Native Americans (Levison and Fung, 2006). Risk factors
for the acquisition of CA-MRSA include crowding, close contact, lack of cleanliness,
compromised skin, and contaminated fomites (Stemper et al., 2006).
HA-MRSA, on the other hand, are usually multidrug-resistant and carry the
SCCmec types I, II, and III (Johnson et al., 2007). HA-MRSA infections usually affect
patients during hospitalization, after surgery, and during stays in long-term care facilities.
HA-MRSA infections are also common in patients who have indwelling catheters for
dialysis or other implanted medical devices. Two predominant HA-MRSA clones have
been identified by PFGE and named USA100 and USA200 by the CDC. The proportion
of HA-MRSA infection in the US is on the rise: 2% of S. aureus infections in intensive-
care units were due to MRSA in 1974, 22% in 1995, and 64% in 2004 (Klevens et al.,
2006). Invasive MRSA infections affect an estimated 94,360 persons each year and are
associated with approximately 18,650 deaths in the US. Of these invasive infections,
about 86% are healthcare-associated and 14% are community-associated (Klevens et al.,
2007).
4
A historical perspective on the emergence of drug-resistance in S. aureus
Within only four years of the 1941 introduction of Penicillin, β-lactamase-mediated
resistance was reported in Staphylococci. β-lactamase inhibits the bacterial penicillin-
binding proteins (PBPs) that make the bacteria susceptible to these classes of antibiotics.
The transcription of staphylococcal β-lactamase (most of which are penicillinases) is
induced by the presence of β-lactam in growth medium. Today, less than 2% of S. aureus
strains remain sensitive to penicillin due to the widespread production of β-lactamase
(Fauci et al., 1998).
In 1960, methicillin, the β-lactamase-stable semisynthetic penicillin, was
introduced. Within one year the first MRSA strains were reported. From the 1980s to
1998, the proportion of MRSA isolates rose from close to zero to approximately 70% in
Korea and Japan, 40% in Belgium, 30% in the U.K. and 28% in the U.S.A. (Smith and
Coast, 2002). Today, MRSA is the most frequent cause of nosocomial infection (Coimbra
et al., 2003; Mangeney et al., 2002; Otto et al., 1999).
The introduction of vancomycin in the fight against MRSA provided a short respite,
but glycopeptide intermediate Staphylococcus auerus (GISA) soon emerged (Cetinkaya
et al., 2000; Srinivasan et al., 2002). Although multidrug-resistant strains of S. aureus
have been primarily associated with nosocomial transmission, recent studies have also
demonstrated alarming new trends in community-associated transmission (Aiello et al.,
2003; Almer et al., 2002).
More disconcerting, perhaps, than the rapid emergence of multidrug-resistant
strains of S. aureus and other pathogens, is the slow rate of new drug development. It
5
usually requires ten or more costly years of laboratory, animal and human trials for which
only 4-5% of new drugs make it out of the lab and into clinical use (Levy, 2002). It takes
an estimated $900 million to bring a drug to market and pharmaceutical companies are
experiencing difficulty in recooping their investment (Nelson, 2003). In addition, the
drug discovery boom that followed the discovery of penicillin only 50 years ago is
waning, and new drugs often lack structural uniqueness – offering similar routes of
resistance to bacteria that carry resistance genes for other existing antibiotics.
Anti-pathogenic properties of natural products: interference with quorum-sensing
processes
A major barrier to antibiotic development is the rapid acquisition of bacterial
resistance. It has been argued by many that drugs interfering with cell-to-cell
communication, such as quorum-sensing, would have the advantage of suppressing
virulence factors without actually killing the bacteria, or select for drug resistance
(Mayville et al., 1999; Otto et al., 1999; Podbielski and Kreikemeyer, 2004; Winzer and
Williams, 2001). Quorum sensing (QS) in S. aureus is controlled by the accessory gene
regulator (agr) system. This four gene operon is composed of agrA, a cytoplasmic
regulator, agrB, which encodes a membrane-associated protease, agrC, a membrane-
associated signal kinase, and agrD, a signal peptide (Garvis et al., 2002; Podbielski and
Kreikemeyer, 2004).
The basic pathway leading to increased transcription of the P2 and P3 promoters is
illustrated in Figure 2. The agr operon undergoes constant transcription from a low
activity promoter. During transcription, the signal pre-pro-peptide AgrD is exported out
6
of the cell membrane through the membrane associated protease AgrB. This transport
through AgrB is complemented by the N- and C-terminal cleavage of AgrD, producing a
five amino acid intramolecular thiolactone ring. In a cell-density phenomenon (quorum
sensing), as the population of S. aureus approaches the end of its exponential growth
phase, the AgrD peptide pheromone, or autoinducing peptide (AIP), reaches a
concentration above threshold. After this, AgrD pheromone interacts with the AgrC
signal kinase, activating the ATP→ADP phosphorylation of the cytoplasmic regulator
AgrA. AgrA~P then induces an increase in the transcription rate of the promoters P2 and
P3. The activation of P2 results in a positive auto-regulatory loop, amplifying the
transcription of the agr operon through transcription of RNAII. The activation of P3,
instead, results in the amplification of RNAIII transcription (Dufour et al., 2002; Garvis
et al., 2002; Otto, 2004; Podbielski and Kreikemeyer, 2004; Yarwood and Schlievert,
2003).
RNAIII is responsible for the initiation of transcription of certain target genes either
directly or indirectly through the regulation of the staphylococcal accessory regulatory
(sar) transcription regulators (Bronner et al., 2004). During the post-exponential phase,
RNAIII acts to repress the transcription of several cell-wall associated proteins, including
protein A, coagulase, and fibronectin binding protein. In addition, RNAIII activates the
production of several exoproteins, including alpha-toxin, β-hemolysin, δ-toxin, toxic
shock syndrome toxin-1 (TSST-1), and leucotoxins. In essence, RNAIII modulates the
expression of virulence factors at both the transcriptional and translational levels
(Bronner et al., 2004).
7
The sar regulatory locus is responsible for the expression of 3 overlapping
transcripts: sarA, sarC, and sarB. SarA encodes the transcriptional regulator sarA, which
binds to the agr locus promoters P2 and P3, enhancing RNAII and RNAIII transcription,
thus altering the synthesis of virulence factors (Bronner et al., 2004; Garvis et al., 2002).
The expression of sarA is growth-phase dependent; the multi-δ-promoters of the sarA
operons permit expression during all growth phases, but a peak in the expression of sarA
normally occurs during the late exponential phase (Bronner et al., 2004; Manna and
Cheung, 2003).
This QS-activated phenomenon results in an increase in the transcription rate of
the P2 and P3 promoters via both agr and sar mediated pathways. In turn, these promoters
act to promote RNAII and RNAIII. RNAIII, which is activated via a hypothetical RNA
III activating protein (RAP) pathway, down-regulates the transcription of cell-wall
constituents, and up-regulates the transcription of extracellular toxins, such as the protein
which causes food poisoning.
Many of the virulence factors produced by S. aureus, such as α-toxin, β-toxin, δ-
toxin, serine protease, DNase, fibrinolysin, enterotoxin B and toxic shock syndrome
toxin-1 (TSST-1), are controlled by the agr system (Novick et al., 1993; Otto et al.,
1999). While measurement of agr activity has traditionally been undertaken via Northern
Blot analyses of RNA III – the intracellular regulatory molecule of the agr system – new
rapid techniques for measurement of RNA III activity at the translational level have been
developed (Otto and Götz 2000). RP-HPLC can be used for the quantitative measurement
of δ-toxin, a virulence factor which is encoded in the RNA III molecule.
8
An ethnopharmacological approach
There is a clear need for a faster route to the discovery and development of
structurally unique antimicrobial compounds. Synthesis of more compounds similar to
those in existing antibiotic classes are unlikely to keep multidrug-resistant pathogens in
check for even a short period of time. One possible solution may lie in taking an
interdisciplinary approach to this problem. An aim of the field of ethnopharmacology is,
in fact, to approach such problems through research on natural products, incorporating
techniques from such diverse fields as anthropology, genetics, ecology, botany,
microbiology and natural product chemistry (Cox, 1990; Cox and Heinrich, 2001; Ellen,
1995).
The traditional use of plants against SSTIs is a common practice in the popular
medicine of many cultures, although the precise causation of disease and mechanism of
cure is not always understood (Grierson and Afolayan, 1999; Srinivasan et al., 2001). In a
previous study conducted in the ethnic Albanian communities of the Vulture-Alto
Bradano region of southern Italy, I found that about one-third of traditional plant
remedies were used in topical applications to the skin (Pieroni et al., 2002c). Likewise,
Bennett et al. (2002) found that nearly one-third of the 211 Shuar medicinal species
(from Amazonian Ecuador) were employed to treat skin ailments. In fact, approximately
one third of all traditional medicines are applied for the treatment of wounds and skin
disorders, compared to only 1-3% of modern drugs (Mantle and Gok, 2001). The topical
application of many traditional botanical preparations for the treatment of various skin
conditions has proven effective due to the presumed antimicrobial and anti-inflammatory
properties of the plants.
9
The study site: Vulture-Alto Bradano area of southern Italy
Southern Italy has a rich cultural history that has been augmented by both historic
immigration from Greece and Albania and recent immigration flux from Eastern Europe
and northern Africa. In this unique cultural landscape, wild and locally cultivated plants
play important roles in many aspects of daily life. Still today, plants are commonly
gathered in the wild as sources of food and medicine. This tradition of preparing
botanical remedies in the household have been studied at length in ethnic Albanian
communities situated in southern Italy by myself and research colleagues (Pieroni, 2003;
Pieroni et al., 2004a; Pieroni et al., 2002a; Pieroni et al., 2002b; Pieroni et al., 2002c;
Pieroni and Quave, In Press). In addition, I have also studied the cosmetic application of
plants by migrant communities in central Italy (Pieroni et al., 2004b). Traditional
cosmetics represent an important aspect of traditional medicine since the intent of their
application is not necessarily limited to beautification purposes, but dermato-therapeutic
uses as well (Pieroni et al., 2004b; Saikia et al., 2006).
The Vulture-Alto Bradano area, located in the center of southern Italy’s Basilicata
province, offers a unique study location due to several important factors. The area is
characterized by a strong dependence on small-scale agriculture and small communities
maintain strong connections with the land. The majority of households maintain family
vineyards and home gardens, where medicinal as well as food plants are regularly
cultivated. In addition, the tradition of collecting wild botanicals for household use is
prevalent.
The folk-medical construct of the region ranges from spiritualistic (personalistic) to
naturalistic, and has been detailed in two publications (Quave and Pieroni, 2002; Quave
10
and Pieroni, 2005). The treatment of many folk-illnesses involves the integration of
magico-spiritual prayer rituals and the topical application of plants or their derivatives.
This is especially true for those folk-illnesses involving the skin. Since most of this
previous work involved ethnic Albanian communities, little is known about the folk-
medical practices of the autochthonous Italian communities in the region. This study
focuses on the folk-medical practices of autochthonous, or native, Italians regarding the
collection and use of botanicals for home remedies, especially those pertaining to SSTIs.
Summary
I conducted an ethnopharmacological study focused on the identification and
evaluation of botanicals used in the traditional treatment of SSTI in ten communities in
southern Italy. Three groups of plants (those used to treat skin disease, those used for
general medicinal purposes, and those without any reported use in these communities)
were collected and analyzed using in vitro models for potential anti-staphylococcal
properties. The studies presented here are the product of a multidisciplinary project
designed to investigate the impact of extracts from plants used in Italian folk medicine for
SSTI on MRSA isolates. My hypothesis was that plants used in folk remedies for SSTI
would exhibit lower mammalian cytotoxicity and greater inhibition of bacterial growth,
biofilm formation and toxin production in MRSA than plants from the other two test
groups. These findings are presented in three chapters:
Chapter II is has been published in the Journal of Ethnobiology and
Ethnomedicine. This chapter specifically addresses the results from the field component
of this study. I conducted field research in southern Italy during the spring and summer of
11
2006. I documented the traditional use of 38 plants in the treatment of various skin
ailments. This study is the first systematic documentation of dermatological remedies in
the Vulture-Alto Bradano area of southern Italy.
In Chapter III, I address the in vitro activity of the plant extracts on
staphylococcal growth and biofilm formation. I also discuss the in vitro cytotoxicity of
plant extracts against a human T lymphoblastoid cell line. This chapter has been
formatted for submission to the Journal of Ethnopharmacology. I report anti-biofilm
activity in MRSA for extracts from six plants for the first time.
Chapter IV examines the quorum sensing inhibiting (QSI) activity on MRSA by
these extracts. This chapter has been formatted for submission to Phytotherapy Research.
I describe the first use of a RP-HPLC assay for δ-toxin quantification in a natural product
screening study for QSIs. I also report the dose-dependent QSI activity for extracts from
4 plants for the first time.
Chapter V summarizes the results of this study, offering an analysis of the
difference in activity between the three aforementioned groups of plants collected. I
discuss the relevance of certain plants to the traditional folk medical construct of the
study area and address the efficacy of certain botanic remedies.
This research is aimed at examining the validity of traditional Italian remedies for
SSTIs as related to their efficacy against MRSA growth and pathogenicity. Furthermore,
based on the results obtained in this study, I propose the further investigation of specific
plant species in future drug discovery studies for MRSA.
12
(a)
(b)
Figure 1. Scanning electron micrographs of a MRSA isolate. (a) Magnification x
20,000. (b) Magnification x4,300. [Images taken by CLQ in 2005.]
13
Cell envelope
Extracellular toxins
Cell-wall
+
-
AgrB AgrC
AgrD AgrA
AgrA - P
ATP ADP
P
+
+
AgrD cleavage
agr operon RNAII
Extracellular environment
SarA
+ sarA
RNAIII P3 P2 agrB agrD agrC agrA
Figure 2. Diagram of the accessory gene regulator system in Staphylococcus aureus and
its interaction with the regulatory transcript sarA based on data presented in publications
by various authors (Lyon and Novick, 2004; Podbielski and Kreikemeyer, 2004; Winzer
and Williams, 2001; Yarwood and Schlievert, 2003).
14
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19
CHAPTER II
DERMATOLOGICAL REMEDIES IN THE TRADITIONAL
PHARMACOPOEIA OF VULTURE-ALTO BRADANO, INLAND SOUTHERN
ITALY
Abstract
Dermatological remedies make up at least one-third of the traditional
pharmacopoeia in southern Italy. The identification of folk remedies for the skin is
important for both the preservation of traditional medical knowledge and in the search for
novel antimicrobial agents in the treatment of skin and soft tissue infection (SSTI). My
goal is to document traditional remedies from botanical, animal, mineral and industrial
sources for the topical treatment of skin ailments. In addition to SSTI remedies for
humans, I also discuss certain ethnoveterinary applications.
Field research was conducted in ten communities in the Vulture-Alto Bradano
area of the Basilicata province, southern Italy. I sampled 112 interviewees, stratified by
age and gender. After obtaining prior informed consent, I collected data through semi-
structured interviews, participant-observtaion, and small focus group techniques.
Voucher specimens of all cited botanic species were deposited at FTG and HLUC
herbaria located in the US and Italy.
I report the preparation and topical application of 116 remedies derived from 38
plant species. Remedies are sued to treat laceration, burn wound, wart, inflammation,
rash, dental abscess, furuncle, dermatitis, and other conditions. The pharmacopoeia also
includes 49 animal remedies derived from sources such as pigs, slugs, and humans.
Ethnoveterinary medicine, which incorporates both animal and plant derived remedies, is
20
addressed. I also examine the recent decline in knowledge regarding the dermatological
pharmacopoeia.
The traditional dermatological pharmacopoeia of Vulture-Alto Bradano is based
on a dynamic folk medical construct of natural and spiritual illness and healing.
Remedies are used to treat more than 45 skin and soft tissue conditions of both humans
and animals. Of the total 165 remedies reported, 110 have never before been published in
the mainland southern Italian ethnomedical literature.
Background
The folk-medical treatment of dermatological conditions is prevalent in southern
Italy and elsewhere. Dermatological conditions are particularly common in rural agro-
pastoral communities, where skin abrasions and small cuts are regularly exposed to
bacteria found in the soil and animal feces. At least one-third of all traditional remedies
used in the south Italian folk pharmacopoeia are directly relevant to the skin.
Skin and soft tissue infection (SSTI) caused by multidrug-resistant bacteria, such
as methicillin-resistant Staphylococcus aureus (MRSA), are an ever-increasing source of
death and high healthcare costs worldwide. In the US alone, over 126,000
hospitalizations each year are due MRSA infections [1]. Moreover, invasive MRSA
infections affect an estimated 94,360 individuals each year in the US and are associated
with approximately 18,650 deaths, exceeding mortality estimates for AIDs [2]. Natural
products from botanical sources used in traditional folk remedies for the skin may offer a
new route for combating multidrug-resistant bacterial infections through the elucidation
of biological compounds with novel mechanisms of action. Natural products can work in
21
a complementary synergistic manner to impact multiple biological targets [3, 4]
Furthermore, validation of traditional remedies could rekindle community interest in
preserving local traditional medical knowledge.
Ten communities in the Vulture-Alto Bradano area of the Basilicata (historically
known as Lucania) province were the focus of this study. Basilicata, a region of southern
Italy bordering the Tyrrhenian and Ionian Seas, is divided into two Provinces: Potenza
and Matera. It is bounded by the regions of Puglia (north and east), Calabria (south), and
Campania (west). The territory is roughly divided into a mountainous western section,
which is dominated by the Appennino Lucano, an eastern section of wide valleys and low
hills, and flat plains to the south along the Ionian Sea.
Basilicata region covers 9,992 km2, and based on a 2001 census of The Italian
National Statistical Institute (ISTAT), Basilicata has a population of about 600,000.
ISTAT also reports that the Basilicata region has the lowest percentage of urban
population (17%, calculated in the period of 1997-1999), the highest life expectancy
(75.7 years, calculated in the period 1991-1995), and presents the lowest utilization of
allopathic medical services (23.9% among men, 32.5% among women, calculated in
1997) in all of Italy [5].
Basilicata has been influenced by historical immigration flux from both Greece
and Albania. Today, immigrants come mainly from Eastern Europe or from Northern
Africa. The majority of recent immigrants into the smaller communities, such as those
included in this study, come from Eastern Europe seeking work as housekeepers and
caretakers for the elderly, whereas North Africans typically seek employment in the
larger cities.
22
The Vulture-Alto Bradano area of northern Basilicata is characterized by its
proximity to the dormant volcano, Monte Vulture (1,326 m.a.s.l.). The soil of this region
is particularly rich due to the presence of tuffaceous-clayey-volcanic soil (produced by
the explosive volcano ca. 830,000 years ago) and the local economy is founded in large
part on agriculture. Durum wheat, olives, and the aglianico variety of grapes are the
predominant crops. Small-scale pastoral activity is also important, and goat and sheep
cheeses are produced locally. Regional industry, other than agriculture, is scarce, but
some local artisans produce ceramics and a substantial portion of the population between
20-45 years old are currently employed in the Fiat automobile factory or other associated
factories.
Based on data from historic Albanian communities in the Vulture-Alto Bradano,
traditional knowledge related to household remedies corresponds with age and gender.
The likelihood of traditional knowledge transmission is affected by work patterns, which
influences the amount of time an individual spends in the natural environment performing
agricultural work. Women are the primary carriers of knowledge for folk remedies, as
well as the primary providers of household medicine. Older generations are more
knowledgeable of plant sources of both wild foods and medicines [6-10]. Furthermore,
because of the strong link between traditional knowledge and age, the level of traditional
knowledge in small communities is on a steep decline as the oldest generation dies. This
reflects a sense of urgency to record folk remedies, which are normally passed down
through oral tradition, before they are lost forever.
In the past 50 years, only a few ethnobotanical studies focused on Basilicata [6-
16]. The aim of this study is to collect ethnomedical data regarding the application of
23
traditional remedies from botanical, animal, and mineral sources for the topical treatment
of the skin. In addition to remedies for humans, I also discuss certain ethnoveterinary
remedies that are particularly important for the care of livestock in the study area.
Socioeconomic dynamics are discussed in relation to the transmission of knowledge for
folk remedies across generations. Botanical species identified as components to
traditional remedies for SSTIs in this study will be evaluated for their potential in
modifying MRSA growth and virulence in subsequent studies.
Methods
Field research was conducted in ten communities situated in the Vulture-Alto
Bradano area of Basilicata, Italy from April-July of 2006 (Figure 1). One hundred and
twelve interviewees were recruited for the study. Interviewees were equally stratified by
four age groups (21-35, 36-50, 51-70, 71+ years) and gender in order to compare
traditional knowledge levels across different generations. Prior informed consent, as
approved by the Florida International University Institutional Review Board (#120505-
01), was obtained before conducting interviews. I followed the ethical guidelines adopted
by the ICE/International Society of Ethnobiology [17] and Italian Association of Ethno-
Anthropologists (AISEA) [18]. I conducted interviews in Italian, took notes, and made
audio or video recordings of the interviewees when possible. Data was collected using
semi-structured interviews, participant observation, and focus groups. Interviewees were
questioned about the medicinal uses of the local flora and fauna, particularly those related
to the skin. Demographic data regarding each community sampled in the study is reported
in Table 1.
24
All plants mentioned by interviewees were collected and identified following the
standard work of the Italian flora [19]. Familial nomenclature follows the current
Angiosperm Phylogeny Group [20]. Voucher specimens were deposited at the Università
degli Studi della Basilicata (HLUC) in Potenza, Italy and Fairchild Tropical Botanic
Gardens (FTG) in Miami, FL, USA. Bulk samples (100-300 g per species) were collected
for bioassays. Plant materials were shipped to the USA under USDA permit #DP63438.
The transcription of vernacular names of traditional remedies described in the local
pharmacopoeia follows the rules of the Italian language. The neutral centralized vowel
‘schwa’ of the southern Italian (Calabro-Neapolitanean) dialect spoken in the studied
area, often marked by some linguists as (’) has been denoted in this study with the
symbol “ē”. The collected data have been compared with all of the ethnobotanical studies
conducted thus far in mainland southern Italy, including Abruzzo, Molise, Campania,
Puglia, Calabria and Basilicata.
Statistical analyses of data, including calculation of mean values and standard
deviations, were calculated using Microsoft Excel. Differences between means were
analyzed with a one way ANOVA, followed by a series of 2-sample t-tests on SPLUS
software. Differences were considered significant with p-values < 0.05.
Results and Discussion
The folk medical construct
Skin and soft tissue infections (SSTIs) are treated in different manners, depending
on the perceived causation of the illness. Illnesses with a naturalistic (biological)
causation are treated in a non-ritual context and involve the topical application of either a
25
plant, animal, mineral, or industrial product as the therapeutic agent. This is usually
carried out by the individual being treated or by the female head of household (mother or
grandmother).
Illnesses of perceived spiritualistic causation are treated much differently. Some
manifestations of inflammation on the skin, for example, are believed to be caused by
malevolent spirits, such as mal vjntē (wind illness) or fuoco morto (dead fire illness).
These cases are treated ritualistically, and a plant, animal, mineral, or industrial product is
employed strictly as a ritual object in the ceremony, rather than as the therapeutic agent.
Furthermore, the ritual treatment can be performed only by specific healers (most of
whom are women) who are recognized in the community for their healing powers. The
treatments for south Italian spiritual illnesses in Basilicata have been discussed in
previous publications [21-23].
Botanical remedies
I documented the topical application of 38 plant species from 27 families,
comprising 116 distinct remedies (Table 2). Plants are listed alphabetically by species.
The vernacular and English names are also listed, as well as the author citation of each
species. Detailed information regarding the cultivation status, part(s) used, preparation,
application and popular use is provided. A consensus index is included and is specific to
each individual remedy cited. References to similar remedies in the ethnomedical
literature on mainland southern Italy are noted.
Some of the more common uses of these remedies include analgesic, vulnerary
(especially for burn wounds), toothache, anti-inflammatory, anti-sting/anti-itch (for insect
26
bites), antiseptic (for lacerations), anti-furuncle, haemostatic, suppurative, emollient, and
anti-abscess. Roughly 14% of plant-based remedies are applied for the treatment of
abscess and furuncle (Figure 2). Here I discuss three of the most frequently cited plant
remedies: common mallow, white horehound and German chamomile.
Malva sylvestris (Malvaceae) - malva
Common mallow is an herbaceous species common throughout most of Europe,
except in the most northern regions. It is one of the most important medicinal species in
the southern Italian folk pharmacopoeia. Its use as a panacea is reinforced by a local
saying, La malva, da ogni mal' ti salva, (the common mallow saves you from every
disease). Common mallow is most often cited for its restorative properties for cold, flu,
and stomach-ache and as a post-partum depurative [8, 13]. In these cases, a decoction of
the aerial parts is drunk.
It is also important in topical remedies for the treatment of toothache due to dental
abscess or decay, heat- and diaper- rash, bruise, furuncle, abscess, and mastitis. The
application of remedies from this plant to a number of SSTIs, most of which are typically
associated with bacterial infection, suggests that this plant may exhibit antibacterial
properties and should be subjected to further study.
Marrubium vulgare (Lamiaceae) - maruggē
White horehound is a perennial herb native to Europe, northern Africa, and
temperate Asia. Much like common mallow, white horehound is also an extremely
important species in the folk pharmacopoeia of southern Italy. It, too, is considered a
27
panacea and is associated with the following saying, A maruggē, ognē malē struggē (the
white horehound destroys every disease). In previous studies on south Italian
ethnopharmacology, the use of white horehound decoctions as an expectorant,
hepatoprotective agent, and cure-all has been described [8, 9, 13]. The Commission E
monographs approve of white horehound use for treating loss of appetite and dyspepsia
[24].
A decoction of the aerial parts can be used as a rinse to treat several important
SSTIs, including general dermatitis, athlete’s foot, furuncle, abscess, cyst, and wart in
both humans and animals. Investigation of the anti-MRSA potential of this species and its
phytochemical components could be promising.
Matricaria recutita (Asteraceae) - camomilla
German chamomile is best known for its anxiolytic and sedative properties. In
the Vulture-Alto Bradano, it is also commonly used as a wash for skin conditions such as
rash, acne and dermatitis and also as an anti-conjunctivital eye-wash (Figure 3).
Application of German chamomile decoctions for the treatment of eye inflammation and
infection has been broadly reported in Italy. German chamomile has demonstrated in
vitro anti-staphylococcal activity [25] and has been found to promote wound healing in
vivo [26]. The German Commission E has approved the use of German chamomile in
external applications for the treatment of skin and mucous membrane inflammation,
bacterial skin disease, and ano-genital inflammation [24].
28
Zootherapy and remedies of mineral or industrial origin
The treatment of ailments with remedies made from animals and their products is
known as zootherapy [27]. Since ancient times, zootherapy has been integral to the
traditional pharmacopoeias of many cultures [28-30]. Despite its prevalence in traditional
medical practices worldwide, the phenomenon of zootherapy has often been neglected in
comparison to the literature regarding medicinal plant therapies. Animal-based remedies
represent an important portion of traditional medicine practiced today in southern Italy.
The sources of these remedies include human, pig, bee, chicken, and slug mucous.
Remedies of mineral and industrial origin are also important, and include sources
such as copper sulphate, salt, soot, and old gas, among others. They are employed in the
treatment of burn wound, furuncle, facial wrinkle, abscess, broken bone, bruise, chapped
skin, conjunctivitis, bronchitis, wart, acne, stomach ulcer, callus, cradle-cap, and
dermatitis.
Forty-nine remedies from 30 animal, mineral, or industrial sources are used in
topical applications (Table 3). Remedy sources are listed alphabetically by English name.
Vernacular names are also listed, along with detailed information regarding the
preparation, application and popular use of the remedy. A consensus index is included
and is specific to each remedy cited. References to similar remedies in the ethnomedical
literature on mainland southern Italy are noted. Here, I discuss some of the most unusual
of these remedies, including aged pig fat, garden slug and human breast milk.
29
Aged pig fat
Aged pig fat, locally known as sugna fracidē, is prepared by aging the dorsal back
fat of a pig in a cool dark place, such as a cantina. It is an important component of
several remedies for skin conditions. In addition to its emollient properties, it is also
reported to be a useful vulnerary agent in the treatment of both animals and humans. It is
also applied together with the leaves of Rubus ulmifolius to treat furuncle, and to draw the
pus out of purulent abscess. A similar therapy with R. ulmifolius was reported in
Campania, another area of southern Italy [31].
Garden slug
Therapies involving the slug Arion hortensis, locally known as u’ marruculē or
lummachē senza guscio (snail without a shell), has been briefly mentioned in previous
work [13]. The most popular use of this gastropod is to treat gastritis or stomach ulcer by
swallowing it whole and alive. A clear mucous produced by the slug is rubbed onto the
skin to treat dermatitis, inflammations, calluses, and acne (Figure 4). The mucous is
thought to promote wound healing. In addition, a special ritual is incorporated in the
treatment of wart. Mucous from a live slug is first rubbed onto the wart, and then the slug
is hung out in the sunshine to dry out and die. It is believed that once the slug has dried
up, the wart should as well. A similar remedy for wart involving mucous from an
unidentified snail is mentioned in Guarrera’s work in Latium, Central Italy [32]. This
may, however, be a reference to the same species of garden slug.
30
Human breast milk
Human breast milk is a popular zootherapeutic remedy, and is applied in the
treatment of conjunctivitis, chapped nipples, and cradle-cap. The topical application of
breast milk to infected eyes was cited by nearly one-third of the interviewees. This
remedy was also cited in a previous study on the Lucanian Dolomite region of Basilicata
[13]. This use of breast milk may be well-founded due to the antibacterial properties of
xanthine oxidase and IgA antibodies found in the colostrum and milk [33, 34].
Ethnoveterinary remedies
Ethnoveterinary practices have been reported in the Italian regions of Basilicata
[15], Calabria [35], Tuscany [36], Romagna [37], Marche, Abruzzo, and Latium [38,
39]. Much of this data on Italian ethnoveterinary medicine has been summarized in a
useful database [40].
In Vulture-Alto Bradano, the topical application of traditional remedies to treat
wounds and inflammation in livestock was particularly important in the smallest
communities surveyed, primarily in the localities of Montemarcone Alto, Sant’Andrea,
and Pierno. Many families in these communities are economically dependent on
agropastoral activities, and the health of their animals is a central concern (Figure 5).
Plant, animal, and mineral (or industrial) derived remedies for ethnoveterinary
applications are indicated with the symbol ‘♣’ in Tables 2 and 3. Here, I briefly discuss
two important sources of ethnoveterinary treatments: copper sulphate and St. John’s
Wort.
31
Copper sulphate
Copper (II) sulphate (verderammē) is a bright blue-green mineral sold locally as a
chemical fungicide for plants (Figure 6). However, it is also one of the most frequently
cited traditional ethnoveterinary remedies. While it is also used for the treatment of
swollen feet in humans on occasion, the burning sensation it produces is a significant
detractor. It is prepared either as a ground powder, a mixture with vinegar, or a mixture
with water and salt and is then applied to cracked hooves or to the chapped skin
surrounding the hooves of livestock. This ethnoveterinary remedy has also been reported
in the western Basilicata Dolomite mountain range [12].
Hypericum perforatum (Hypericaceae) - iperico
An oleolite made from an olive oil infusion of the aerial parts of St. John’s Wort
(known locally as erva pē rē cuttē or iperico) is massaged into the legs of horses and
asses to treat tendonitis. This remedy has also been described in Tuscan ethnoveterinary
practices [36]. In other studies, oleolite preparations of the aerial parts or flowering tops
have been reported for wound healing and to restore skin damaged by burns [38, 41, 42]
or for treating mastitis in milk-producing goats [31]. Oleolite preparations of a similar
species, H. perfoliatum, have been reported in Sicily for use as a disinfectant and to
promote healing of burn wounds [43]. The anti-staphylococcal properties of Hypericum
species are well known [44-46] and are likely relevant to these traditional therapies.
32
Traditional knowledge: current trends and future projections
The mean number of remedies quoted by interviewees is reported by age group
and gender (Figure 7). A generation gap in knowledge regarding folk remedies for SSTI’s
was evident. There is a statistically significant difference (p<0.05) in knowledge of folk
remedies for the skin between the youngest subset of women sampled (21-35 years old)
and women 36 years and older. This may be explained by recent shifts in the
socioeconomic patterns of the region. Prior to the opening of the Fiat automobile factory
and all of the associated parts factories in the region, women seldom participated in the
workforce. When the factories opened nearly 15 years ago, a large shift in women’s
socioeconomic placement occurred. Young women entered the workforce and no longer
regularly participated in outdoor activities, such as upkeep of the family gardens and
vineyards, thus creating a significant gap between them and nature. It also fractured the
daily interaction with the older women, who are the primary keepers of traditional
knowledge and responsible for the oral passage to younger generations. This
socioeconomic shift, undoubtedly, played a significant role in the decline of passing
traditional knowledge on to this youngest generation of women.
There was no significant difference between men in different age groups and men
and women were roughly equivalent in oldest subsets sampled (51-70 and 71+ years old).
This lack of difference in traditional knowledge for men may be explained by historic
emigration patterns. A large portion of men from the oldest subsets sampled emigrated to
northern Italy and Switzerland in their youth for work. Later in life, these men returned to
the community of their birth, but the substantial number of years away from their native
environment and traditions was detrimental to their level of traditional knowledge. In
33
addition, younger men, like the young women, did not acquire much traditional
knowledge due to these socioeconomic dynamics.
Another pattern in traditional knowledge levels related to the size of communities
was notable. Younger men from the smaller communities (primarily the localities), who
were actively involved in agriculture – either in farming crops or pastoral activities, had
much higher levels of traditional knowledge for skin remedies than young men from
larger, less isolated communities. For example, one young shepherd from the locality of
Sant’Andrea was able to describe more than 40 remedies related to the skin. Young men
who either worked in factory jobs or were unemployed from a larger community, like
Ripacandida, could typically only cite 4 to 7 remedies. This reflects the importance of
daily interaction with the environment and its role in the acquisition and retention of
traditional knowledge of plant derived remedies.
The likelihood of a continued decline in traditional knowledge transmission
across generations is great and will be most notable amongst women. Socioeconomic
factors and shifts in the dynamics of women’s roles in the home have perhaps the greatest
influence on this paradigm. Stronger dependence on allopathic, rather than traditional
medical care is expected in the coming years.
Conclusion
I identified 165 remedies derived from plant, animal, mineral and industrial
sources with particular relevance to skin and soft tissue infection. Of the remedies
reported, 110 have never before been published in the mainland southern Italian
ethnomedical literature. Moreover, some of the botanical sources for SSTI remedies have
34
never been investigated for their antimicrobial properties and further investigation of the
phytochemical constituents of these species is recommended.
Transfer of knowledge regarding household medicine is declining, especially
amongst women. These findings are important because women have traditionally been
the keepers of knowledge regarding folk remedies and the providers of household
medicine. This loss of knowledge is intrinsically linked with shifting socioeconomic
dynamics in the area and is not expected to be reversed. This reinforces the importance of
recording ethnomedical and ethnobiological data now before it is lost with the passing of
the oldest generations.
Acknowledgements
Funding for this study was provided by: NIH/NCCAM 1F31AT004288-01A1 (PI:
C.L. Quave), Botany in Action, Anne Chatham Fellowship in Medicinal Botany, USDA
CSREES 20053842215940 and NIH/NCAAM 1T32AT01060-01 (PI: B.C. Bennett). I
thank the many volunteers who assisted in the undertaking of this study. Donato Caputo,
Donato Carbone, Asunta Di Leo, Renato Rivarosa, and Donato Millito assisted in plant
collections. Rosanna Caputo, Milagros Prieto, Gemmina Caputo, Antonello Fiore, and
Ettorino Caputo assisted in the recruitment of interviewees. Fabrizio Caputo assisted in
the collection of demographic data from the communities. Marco Caputo built the plant
drier used to dry bulk samples and voucher specimens in the field. Carlo and Cristina
Allamprese provided the containers for shipping plant materials. Dr. Carmine Colacino
assisted in the taxonomic identification of plant specimens. We especially thank all of the
35
study participants who graciously shared a wealth of knowledge regarding the traditional
medical practices of their communities.
36
Figures
Figure 1. Map of the study region: the Vulture-Alto Bradano region of Basilicata,
southern Italy.
37
0
5
10
15
20
25
Abs
cess
& fu
runc
le
Arth
ritis
Bre
ast
Bro
ken
bone
s
Bru
ise
Der
mat
itis
Dry
ski
n
Eye
con
ditio
ns
Faci
al w
rinkl
es
Foot
& le
g
Hae
mor
rhoi
d
Hai
r & s
calp
Hea
dach
e
Infla
mm
atio
n
Inse
ct s
tings
Lace
ratio
n &
wou
nds
Pai
n
Rhe
umat
ism
Shi
ngle
s
Sor
e th
roat
Spi
ritua
l Illn
ess
Toot
hach
e &
mou
th
War
t
Condition Treated
Num
ber o
f Rem
edie
s
Plant-derived remedies Animal- or mineral-derived remedies
Figure 2. Number of plant-, animal-, and mineral-derived remedies for the topical
treatment of medical conditions in humans.
38
39
Figure 3. Mecca Maria Leonarda (63 yrs. old) with dried chamomile (Matricaria
recutita) stems that she uses in a decoction to treat eye infections.
Figure 4. Vito Sabato (74 yrs. old) with a garden slug (Arion hortensis) that he uses to treat warts, inflammations, and calluses on
the skin.
40
40
Figure 5. A young shepherd (23 years old) from Sant’ Andrea uses a mixture of red wine
vinegar and copper sulphate to rinse the cracked hooves of his ass. He also applies the
crushed mature fruits of Sambucus ebulus (Iervolē) to open wounds on his livestock as an
insect repellent.
41
Figure 6. Verderammē (copper sulphate) is used in ethnoveterinary remedies for injured
or infected hooves of livestock.
42
43
0
5
10
15
21-35 36-50 51-70 71Age (Years)
Mea
n #
of R
eme
20
25
30
+
dies
Kno
wn
MaleFemale
Figure 7. Mean and standard deviation of the number of remedies cited by gender and
age categories.
Tables Table 1. Data on the sampled communities [47-49].
Community Elevation (m.a.s.l.)
Number of Families
Number of Inhabitants
Number of Interviewees
% of Population Sampled
Forenza 836 1,051 2,546 15 0.6% Frusci (locality of Avigliano) 925 70 192 8 4.2% Ginestra 675 280 726 10 1.4% Montemarcone Alto (locality of Avigliano) 900 15 46 5 10.8% Pierno (locality of San Fele) 960 24 52 5 9.6% Rapone 838 473 1,203 9 0.7% Rionero in Vulture 647 4,738 13,423 4 0.03% Ripacandida 622 744 1,767 14 0.8% Sant’Andrea (locality of Atella) 716 55 176 27 15.3% Scalera (locality of Filiano) 670 185 488 15 3.07% Total - 7,635 20619 112 0.54%
44
44
Table 2. Popular uses of botanical materials for dermatological conditions and topical (external) applications.
Botanical Taxon
(voucher specimen
code)
Botanical Family
Vernacular name in Vulture-
Alto Bradano
English Name Status Part(s)
Used Preparation
and Application Popular
Use
Consensus Index ♦<10%
♦♦ 10-20% ♦♦♦ 21-30% ♦♦♦♦ 31-40% ♦♦♦♦♦ >40%
Records of similar use in other Italian
ethno-botanical studies
conducted in mainland
southern Italy Aesculus hippocastanum L.
Sapinda-ceae
castagna bastarda
horse chestnut
W
fruit
Fresh fruits are split in half and seeped in alcohol for 40 days and then used to massage swollen or sore legs.
Analgesic; anti-oedemic
♦
None
bark and leaves
A decoction is made of the bark and leaves then mixed with olive oil or pig fat to make an ointment. It is applied topically.
Anti-haemor-rhoid
♦ None
Allium cepa L. (CQ-206)
Alliaceae cipudda onion C bulb Slice of bulb is warmed, and then a drop of olive oil and sugar is added. This is then topically applied.
Anti-furuncle; anti-abscess
♦ [9, 13]
45
45
bulb Slice of fresh
bulb is rubbed on burned skin.
Vulnerary (for burn wounds)
♦ None
bulb membrane
Applied topically (like a Band-Aid) to small lacerations.
Haemo-static
♦♦ [8]
leaves Fresh leaves are used to tie strips of bark from Ulmus minor L. around the affected limb.
Splint for broken or severely bruised bones
♦ None
Allium sativum L.
Alliaceae agljē garlic C bulb Piece of bulb is crushed and rubbed onto nipple of breast-feeding mother.
Wean baby from breast milk
♦ None
Bulb is boiled, and then while still hot, it is rubbed onto a callus.
Anti-callus; analgesic (for callus)
♦ [31, 50]
Pieces of fresh bulb (small cloves) are kept in mouth to suck on.
Against sore throat
♦ None
Fresh pieces of bulb are chewed.
Against toothache;
♦ [50, 51]
46
46
anti-gingivitis
Fresh bulb is rubbed onto the skin where stung by an insect.
Anti-sting/anti-itch (for insect bite)
♦♦ [9, 13, 31, 38]
Arum italicum Mill. (CQ-175)
Araceae frugnē n cuttē
Italian lords and ladies
W leaves Fresh leaf applied topically. It will “stick” to the skin.
Analgesic (for insect stings and burns); vulnerary (for burn wounds)
♦♦ None
Arundo donax L. (CQ-146)
Poaceae cannē giant reed W white hemi-cellulose membrane at node
Topically applied to lacerations.
Haemo-static
♦♦♦♦ [8, 9, 13, 50]
Topically applied to mouth sores on lips (but not inside the mouth or on tongue).
Anti-herpes (of the mouth)
♦ None
stem Stem is cut so that it will fit onto the fingers. These canned are worn to protect the fingers during the grain
Physical protection of fingers
♦♦ None
47
47
harvest.
Green stem is cut into 9 tooth-size pieces which are used in the ritual healing of toothache.
Against toothache
♦ None
Ballota nigra L. (CQ-160)
Lamia-ceae
erba cane black horehound
W aerial parts
Decoction is used as a skin wash.
Anti-inflamma-tory
♦ None
Haemo-static
♦ [13]
Brassica oleracea L.
Brassica-ceae
cavolo cabbage C leaf Heated and topically applied for pain relief.
Anti-rheumatic; anti-arthritis; analgesic
♦ [38, 50]
Fresh leaf is wrapped around injured area of limb, and then held in place with a bandage. Frequently used on horse legs.
♣ Analgesic and anti-inflamma-tory for tendonitis
♦ None
Fresh leaf applied to engorged breast of lactating woman.
anti-lactic; anti-mastitis
♦ [13]
Cannabis Cannaba- canapē hemp W fibers Hemp fibers Cast for ♦ None
48
48
sativa L. ceae (stoppē) are coated in well-beaten egg white and used to make a hard cast (stoppata) for setting bones.
broken or severely bruised bones
Capsicum annuum L.
Solana-ceae
pepperoni picantē
sweet pepper
C fruit Dried and rubbed onto nipple of breast-feeding mother.
Wean baby from breast milk
♦ None
Cyclamen hederifolium L. (CQ-186)
Myrsina-ceae
ciclamino hardy cyclamen
W tuber Black skin of fresh tuber is removed, and then the rest of tuber is grated to make a poultice for topical applications.
Anti-wart ♦ None
Ecballium elaterium (L.) A. Richard (CQ-169)
Cucurbita-ceae
u cuppē ti ridzē
squirting cucumber
W leaf Heated and topically applied.
Anti-abscess
♦ None
Fresh leaf is wrapped around painful limb.
Anti-rheumatic; anti-inflamma-tory; analgesic
♦ None
Ficus carica L. (CQ-173)
Moraceae fēchē fig C latex from immature fruit
Topically applied.
Anti-callus; anti-corn (feet)
♦ [38]
49
49
Anti-wart
♦♦ [9, 13, 15, 38,
50, 51]
Anti-itch for itchy dermatitis; anti-inflamma-tory
♦ None
Anti-sting/anti-itch (for insect bites)
♦ [13, 31, 38]
Topically applied to mouth sores on lips (but not inside the mouth or on tongue).
Anti-herpes (of the mouth)
♦ None
leaf
Fresh leaf is placed on breast as compress.
Anti-mastitis
♦ None
Foeniculum vulgare ssp. vulgare Mill. (CQ-196)
Apiaceae fēnocchē fennel W seeds Seeds are chewed by another person and the resulting aromatic breath is blown into the inflamed or red eyes of the patient.
Anti-conjuncti-vitis
♦ None
50
50
Hordeum vulgare L.
Poaceae orzo barley C fruits (grains)
Boiled and placed in cloth to be used as warm compress that is applied to the throat.
Against sore throat
♦ None
Hypericum perforatum L. (CQ-183)
Hyperica-ceae
erva pē rē cuttē; iperico
St. John’s Wort
W aerial parts
Fresh aerial parts are used to make an oleolite (heated in olive oil). When the oil has cooled, it is used to massage injured or swollen horse legs.
♣ Anti-inflamma-tory; emollient; vulnerary
♦ [31]
Juglans regia L. (CQ-181)
Juglanda-ceae
noce walnut SC bark Decoction used as mouth wash or gargle.
Against toothache
♦ None
flowers Topically applied as ritual object in spiritual healing of mal d’arco (rainbow illness).
Against “Rainbow illness”
♦ None
leaves Fresh leaves topically applied to areas of swelling from varicose veins. This is done at night and the
Heals varicose veins; reduces swelling of legs
♦
[51]
51
51
leaves are held in place with a bandage.
Malva sylvestris L. (CQ-156)
Malva-ceae
malvē common mallow
W aerial parts (or just stems)
Decoction – once cooled, it is used as a wash for infants. Some also make the decoction together with the dried aerial parts of Matricaria recutita.
Anti-dermatitis (babies); Against heat- and diaper-rash in babies
♦ None
Decoction is used as a mouth wash.
Against toothache (dental abscess)
♦
[31, 38, 52]
leaves Fresh leaf applied topically; changed 2-3 times a day.
Anti-bruise
♦ None
Fresh leaf is heated over a fire and applied topically as compress; compress is changed 2-3 times a day.
Anti-furuncle; anti-abscess
♦♦ [15, 50]
Fresh leaves are boiled and placed into cloth
Anti-mastitis
♦♦ [50]
52
52
sack to use as a warm compress; compress is changed 2-3 times a day.
Leaves are boiled and placed into a cloth sack to use as a compress that is held on the jaw or cheek.
Against toothache (dental abscess)
♦ None
Leaves are boiled and packed into the space where rotten teeth have fallen out or areas of acute pain from toothache.
Against toothache (dental abscess)
♦ None
Marrubium vulgare L. (CQ-170)
Lamia-ceae
marruggē white horehound
W aerial parts
Decoction is made and used as a wash for the affected area. The decoction is left on the skin to air dry. This is repeated 2-3 times a day until healed.
♣ Anti-furuncle; anti-abscess; anti-cyst (used on humans and livestock)
♦♦♦ [13, 52]
Anti- ♦ [50]
53
53
dermatitis
Anti-fungal (for foot fungus)
♦
None
Anti-haemor-rhoid
♦
[52]
Anti-wart
♦
None
Panacea
♦♦♦♦♦ [8, 9, 50, 52]
Matricaria recutita L. (CQ-118)
Astera-ceae
camomilla German chamo-mile
W stems Dried, then made into decoction that is used as an eye wash. Eyes are washed 3 times a day until healed. A cloth compress soaked in this decoction water can also be applied to the eyes.
Anti-conjuncti-vitis; anti-inflamma-tory (of eye)
♦♦♦ [9, 13, 15]
aerial parts (or just stems)
Dried, then made into decoction that is used as a facial wash.
Face cleansing; anti-acne
♦ [42]
Dried, then made into decoction that is used to wash or bathe babies with heat-
Anti-dermatitis; anti-rash; anti-inflamma-
♦ None
54
54
or diaper-rash. Some also make the decoction together with the dried aerial parts of Malva sylvestris L.
tory
Dried, then made into decoction for washing hair.
Wash for oily hair
♦ None
Hair lightener
♦
None
Olea europaea L. (CQ-197)
Oleaceae aulivē olive C leaves Decoction with rosemary leaves (Rosmarinus officinalis L.) is used as a foot bath.
Relieve foot swelling
♦ None
oil
Topically applied to burn wound. This must be done in secret immediately after burn injury occurs.
Vulnerary and emoillient (for burn wounds)
♦♦ [38, 50, 51]
Rubbed onto the skin several times a day to treat the folk illness Fuoco di Sant’Antonio (St. Anthony’s Fire),
Anti-shingles; anti-inflamma-tory
♦ None
55
55
or shingles.
Rubbed onto the skin of babies with diaper- or heat -rash.
Anti-dermatitis; anti-inflammatory
♦ None
Rubbed onto the crown of baby’s head, then hair is brushed with soft brush or comb.
Against cradle-cap (in infants)
♦ None
Warm oil is applied to animal hooves.
♣ Vulnerary for cracked hooves or chapped skin near hooves
♦ None
Topical application to diaper area on infants.
Against diaper rash
♦ None
Topical applications to dry skin.
Emollient
♦♦ [51]
Rubbed into the hair and scalp in ritual treatment for scabies and lice.
Anti-scabies; anti-lice
♦ [15]
56
56
Origanum heracleoticum L. (CQ-207)
Lamia-ceae
rigano oregano W leaves Dried leaves are mixed with olive oil and made into a compress that is applied to jaw.
Against toothache
♦ [9]
Oryza sativa L.
Poaceae riso rice C grains Rice grains (store bought) are boiled until over-cooked, then applied topically.
Suppura-tive; anti-furuncle; anti-abscess
♦ None
Parietaria diffusa Mert. & Koch (CQ-212)
Urticaceae erba vjntē pellitory of the wall
W aerial parts
A decoction of aerial parts is used as a mouth wash.
Against toothache; anti-gingivitis
♦ None
Plantago major L. (CQ-225)
Plantagina-ceae
cintu nirvi common plantain
W leaves Fresh leaves are crushed with a stone then applied topically.
Anti-rheumatic; anti-arthritic; analgesic; anti-inflamma-tory
♦♦ None
anti-abscess; anti-boil; suppura-tive
♦♦♦♦ [9, 15, 31, 52]
Vulnerary (for burns)
♦♦♦ None
57
57
Rosmarinus officinalis L. (CQ-113)
Lamia-ceae
rosamarēnē rosemary C, W leaves Decoction with olive leaves (Olea europaea L.) is used as a foot bath.
Relieve foot swelling
♦ None
Rubus ulmifolius Schott (CQ-164)
Rosaceae mora elmleaf blackberry
W leaves A layer of aged pig fat (la sugnē fracidē) is placed on affected area of skin, and then fresh leaves are placed on top of the fat. This is wrapped with a bandage to hold the leaf and fat in place. (Some also cite the application of the leaf directly to furuncle and abscess without using pig fat.)
Anti-furuncle; vulnerary (for laceration); anti-abscess; anti-carbuncle; suppura-tive
♦♦ [31, 38]
Fresh leaves are applied topically to skin to treat dog bite. The leaves are held in place with a bandage.
Vulnerary and anti-infective (for dog-bite)
58
♦ None
root A decoction of the fresh root is made for use as a hair wash.
Prevents/stops hair loss
♦ None
58
Rumex crispus L. (CQ-171)
Polygona-ceae
patsa curled dock
W leaves Leaves are warmed over a fire; the cortex is removed and placed in olive oil. This is topically applied to burn wounds.
Vulnerary (for burn wounds)
♦ None
Ruta graveolens L.
Rutaceae ruta rue W aerial parts
Dried then made into a decoction that is used as an external wash for humans and animals.
♣ anti-furuncle; anti-abscess; anti-acne
♦ None
Decoction of aerial parts is drunk or used as an external wash.
Panacea
59
♦ None
Sambucus ebulus L. (CQ-180)
Adoxa-ceae
Iervolē dwarf elder
W flowers When flowers turn black, they are placed in shoes and walked in (without socks) for 3 days.
Prevents formation of blisters and foot sores
♦ None
fruit Juice from mature fruits is applied to open wounds in animals to detract flies and other insects
♣ Fly and insect repellent for open wounds
♦ [39]
59
from the wound.
leaves Fresh leaf is wrapped around foot before putting on shoes.
Prevents formation of blisters; sweat absorbent; toughens foot skin
♦ [13]
Sambucus nigra L. (CQ-151)
Adoxa-ceae
savēuchē elder W flowers Dried then made into infusion for use as a foot bath.
Vulnerary for foot sores; emollient for dry, cracked feet
♦ [15, 42]
leaves Fresh leaves topically applied.
Anti-rheumatic; analgesic; Anti-inflamma-tory
♦
60
[8, 51]
Anti-sting/anti-itch (for insect bites)
♦ [9, 50]
Haemo-static for laceration
♦ None
Anti-boil; anti-
♦ [38]
60
abscess; suppura-tive
Solanum lycopersicum L.
Solana-ceae
pomodoro tomato C fruit Fruit pulp is rubbed onto the skin.
Heals insect stings (from wasps and bees)
♦
None
Anti-callus
♦ None
Solanum tuberosum L.
Solana-ceae
potata potato C tuber Fresh slice of tuber is topically applied.
Vulnerary (for burn wounds)
♦♦
[8, 38, 42, 50]
Fresh slice of tuber is topically applied.
Anti-bruise
61 ♦ None
Fresh slice of tuber is grated and applied to eyes.
Anti-inflamma-tory (for eyes); eye moistur-izer
♦ [8, 50, 53]
Fresh skin of tuber is peeled off and applied as a compress on the jaw or cheek over area of toothache pain.
Analgesic (against tooth-ache)
♦ None
61
Fresh slice of tuber is placed on the forehead until headache is relieved.
Anti-headache
♦
[50]
Tussilago farfara L. (CQ-202)
Astera-ceae
stampē kavalēl
coltsfoot W leaves Fresh leaf topically applied to affected area.
Anti-furuncle; suppura-tive
♦
[8, 38]
Vulnerary (for burn wounds)
♦ None
Fresh leaves applied as ritual objects in healing ceremony for pesta cane (dog paw print illness) in which the leaves are laid in a cross over the lower back of infants that won’t grow.
Against pesta cane (dog paw print illness)
62
♦ None
Ulmus minor L. (CQ-195)
Ulmaceae olmo elm W bark Strips of bark are used to make a splint and the bark is tied around the affected limb with the leaves
Splint for broken or severely bruised bones
♦ None
62
of Allium cepa L.
Umbilicus rupestris (Salisb.) Dandy (CQ-157)
Crassula-ceae
favē alla mersē; grassossē; cuppētieddē; grassognē
navelwort W leaves Fresh leaf is topically applied to affected area. Most cite the extraction, or “peeling off” of the leaf “skin” (epithelium) before using.
Anti-corn (feet); anti-callus (feet)
♦♦ [38]
Heal ingrown toenails
♦♦
None
Anti-furuncle; anti-abscess; suppura-tive
♦
[8, 9, 13, 54]
Relieves foot swelling; anti-inflamma-tory
♦
63
[13]
Vulnerary (also for laceration)
♦
None
Fresh leaf is heated and applied to jaw or cheek.
Against toothache
♦ None
63
Urtica dioica L. (CQ-179)
Urticaceae ardiga common nettle
W leaves Fresh leaves are topically applied.
Anti-rheumatic; anti-arthritis
♦
[31, 42, 52]
Decoction used as hair wash.
Wash for oily hair; strengthen weak hair
♦ [15, 42]
Verbascum sinuatum L. (CQ-218)
Scrophu-lariaceae
varvaschē wavyleaf mullein
W leaves Decoction in red wine (Vitis vinifera var. aglianico) is used as a mouth wash and gargle. It isn’t swallowed because it is considered poisonous.
Analgesic (for toothache and mouth sores)
♦
None
Vitis vinifera var. aglianico (CQ-209)
Vitaceae vētē grape vine C vinegar Cloth is bathed in vinegar and placed on forehead.
Febrifuge
64
♦ None
Hair is washed first with soap and water, then with vinegar.
Wash for oily hair
♦ [8]
Mixed with salt and rubbed on skin.
Against skin nodules; anti-cyst
♦ None
64
Mixed with salt
and used as mouth wash.
Against toothache; anti-gingivitis
♦ None
Mixed with verderame (oxidized copper, a fungicide used on cultivated plants) and used to treat canero del fatone, or cracked/ wounded horse hooves.
♣ Heals cracked or wounded horse hooves
♦ None
wine Used to wash wounds and lacerations (for humans and animals).
♣ Anti-septic
♦♦♦♦
65
[12]
Decoction with the leaves of Verbascum sinuatum L. is used as a mouth wash and gargle. It isn’t swallowed because it is considered poisonous.
Analgesic (for toothache and mouth sores)
♦ None
Mixed with Anti- ♦ None
65
bread crumbs, and then rubbed onto skin to treat contact dermatitis caused by a stinging caterpillar crawling on skin.
dermatitis; anti-inflamma-tory
Applied topically in ritual healing of mal vjntē (wind illness).
Against mal vjntē (wind illness)
♦ None
wine or vinegar
Mixed with durum wheat flour, used to coat a rough canvas material (stoppē) to make a hard cast
Cast for broken or severely bruised bones
♦ None
66
Status: C: cultivated; SC: semi-cultivated (including plants managed in the wild); W: wild. Consensus Index/ Spontaneous Quotation
Frequency for remedies: ♦: quoted by < 10% of informants; ♦♦ quoted by 10- 20% of informants; ♦♦♦: quoted by 21-30% of informants;
♦♦♦♦: quoted by 31-40% of informants; ♦♦♦♦♦: quoted by > 41% of informants. ♣: Ethnoveterinary application.
66
Table 3. Popular uses of animal, mineral, or industrial derived materials for dermatological conditions and topical (external)
applications.
Ingredient
Vernacular name in Vulture-Alto
Bradano Preparation and Application Popular Use
Consensus Index ♦<10%
♦♦ 10-20% ♦♦♦ 21-30% ♦♦♦♦ 31-40% ♦♦♦♦♦ >40%
Records of similar use in other Italian
ethnobotanical studies
conducted in mainland
southern Italy Ash cinerē Ashes from fireplace are added to a pail of
water and mixed. This is used for washing hair, dishes, and clothing.
Hair wash
♦ None
Breastfeeding lattē alla reversē Nursing mothers with mastitis must breast-feed their infants using the “reverse” or “football” position.
Anti-mastitis
67
♦♦ None
Brick or Tile mattone caldo A brick or clay tile is heated, and then applied to the skin over the affected area.
Heals bruises; anti-mastitis
♦ None
mattone fredo A cold tile is held on the abdomen for gastritis.
Anti-gastritis
♦ None
Cigarette filter cica della sigaretta Filter of cigarette (tobacco is removed) is placed inside mouth and applied directly to the area of toothache pain.
Analgesic (for toothache)
♦ None
Cooked cream (from cow’s milk)
panna cottē
Cooked cream is applied topically. Anti-furuncle ♦ [13]
Copper sulphate (chemical fungicide for cultivated plants)
verderammē Mixed with vinegar from Vitis vinifera var. aglianico and used to treat canero del fatone, or cracked/ wounded horse hooves.
♣ Heals cracked or wounded animal hooves and the chapped skin
♦ None
67
around the hooves
Mixed with water and sometimes salt. It is used to soak injured or swollen feet. This is rarely used on humans because it produces a “burning” sensation of the skin.
Heals chapped skin and swelling of feet
♦♦
None
♣ Heals cracked or wounded animal hooves and the chapped skin around the hooves
♦♦ [12]
Ground into a powder form that is sprinkled onto animal hooves.
♣ Heals cracked or wounded animal hooves and the chapped skin around the hooves
♦ [13]
Cow’s milk u latte freschē
Fresh milk is used to wash facial skin or dry skin.
Anti-wrinkle; emollient
68 ♦ None
Dog hair pelo di cane Take a hair from the dog that bit you and apply topically to bite wound.
Heal dog bite ♦ None
Egg white bianco di uova Egg white is beaten well, and then used to coat hemp (Cannabis sativa L.) fiber (stoppē) to make a hard cast (stoppata) for setting bones.
Cast for broken or severely bruised bones
♦ None
Egg Yolk rosso di uovo Egg yolk is mixed with the yeast from fresh pasta dough and is then applied topically.
Anti-furuncle; anti-abscess
♦ None
Hedgehog bile
bile dē riccē Bile of a freshly killed hedgehog is fed to babies that won’t stop crying
Calms crying babies
♦ None
Honey miele Topically applied to the nipple between breast-feeding sessions.
Emollient for chapped nipples (of
♦ None
68
breast-feeding mothers)
Human breast milk
lattē dē mama Fresh milk is expressed into the infected eyes of babies (also sometimes for adults). Some state the milk must be from a mother who nurses a son. Others say the milk must be from a mother nursing her first born child. Less commonly, fresh milk from an ass or goat is used.
Anti-conjunctivitis ♦♦♦ [13]
Breast milk is expressed and rubbed onto the nipple.
Heals chapped skin of nipple (for breast-feeding women)
♦ None
Breast milk is expressed and rubbed onto an infant’s scalp. This is followed by brushing the hair of the infant to remove the excess skin.
Against cradle-cap
69
♦ None
Human urine piscē Used to wash and clean lacerations. Haemostatic; antiseptic
♦♦ [13]
First urine of the day is used to wash eyes Anti-conjunctivitis
♦ None
Knife (must have a black handle)
coltello con u manicho nero
The knife is used to make the mark of a cross (indentations – not cuts) onto the skin over the area of an insect sting. The metal of this knife will “absorb the poison” and take away the sting.
Heals insect stings; analgesic
♦ None
Matches (sulfur based)
fiammifirē dē zolfē Match is lit and held close to a laceration. Haemostatic
♦ None
Match is lit and used to burn warts. Anti-wart
♦ None
Pasta dough (that pasta rubattē Pasta dough is stolen in secret, and then Anti-wart ♦ None
69
is stolen) rubbed on the skin. The pasta dough must then be hidden or thrown away in secret. When the dough “dries up”, so will the wart.
Pasta dough (homemade)
pasta fattē in casē Pasta dough is rolled out into a thin strip that is applied topically to areas of muscle pain.
Analgesic (for muscle pains or injured joints)
♦ None
Pork fat (from dorsal back fat – it is aged and kept in cantina hanging on a hook)
sugna fracidē Applied topically to sore chapped nipples (for breast-feeding women) or to chapped skin in general.
Emollient ♦ None
A thin layer of fat is placed on affected area of skin, and then fresh leaves of Rubus ulmifolius Schott are placed on top of the fat.
Anti-furuncle; vulnerary (for lacerations); anti-abscess; suppurative
♦♦ [31]
Layer of fat is applied to the chest. This is then covered with a warm towel.
Anti-bronchitis
70
♦ None
Fat is rubbed onto animal wounds and lacerations to protect the injured area and promote healing.
♣ Vulnerary ♦ [12]
Layer of fat is applied over the burn wound. Or, the fat may first be boiled, and then placed on burn wound.
Vulnerary (for burn wounds)
♦ None
Applied topically to the udders of milk-producing animals, especially cows.
♣ Emollient for chapped udders; protects against flies
♦ None
Pork lard lardo di maiale Lard is boiled, and then when cooled it is applied topically to burn wounds.
Vulnerary (for burn wounds)
♦ None
70
Pumice stone powder
pietra pomice Powder from pumice stone is topically applied to wounds.
Vulnerary
♦ None
Rock (unidentified)
pietra Rock is rubbed onto burn wounds.
Vulnerary (for burn wounds)
♦ None
Salt (that is stolen) sale rubbattē A handful of salt is stolen in secret, and then rubbed on the skin. The salt must then be hidden or thrown away in secret.
Anti-wart ♦ None
Salt and water bagnē a sale e acqua A warm bath of salt and water is used to soak feet.
Heals swollen, sore feet
♦♦ None
Slug (Arion hortensis Férussac)
marruculē; lummachē senza guscio
Live slug is swallowed whole. Anti-ulcer (stomach ulcer); anti-gastritis
♦♦♦ None
Mucous from live slug is rubbed onto skin, then animal is hung out in sunshine – when it dries up, the wart will also “dry up”.
Anti-wart
71 ♦♦♦ [13]
Mucous from live slug is rubbed onto skin.
Vulnerary; against dermatitis; anti-inflammatory
♦♦
None
Anti-callus
♦ None
Mucous from live slug is rubbed onto skin for facial cleansing.
Anti-acne; emollient
♦ None
Soot from fireplace
fuliggine Black soot is topically applied to wounds. Vulnerary
♦ None
Tractor oil (old or burnt oil)
olio bruggiatē Old tractor oil is mixed with ashes from burnt hay to make a poultice which is applied topically to lacerations and wounds in animals
♣ Cicatrizing ♦ None
71
72
(especially for horses and mules).
Old tractor oil is brushed onto animal’s (usually for horses, mules, and donkeys) coat to detach blood-sucking insects known as tafani from the animal’s skin.
♣ Insecticide ♦ None
Water acqua bollente For fingertip-pricks (such as with a needle), the affected fingertip is quickly dipped into boiling water three times.
Against pains from fingertip-prick
♦ None
Water foam schiumma d’acqua Foam from water coming from natural spring fountains is topically applied to warts.
Anti-wart ♦ None
Wood affected by woodworms (Anobium punctatum)
leuna imbracētatē Powder from this wood is rubbed onto the top of an infant’s head.
Against cradle-cap ♦ None
Powder from this wood is topically applied to diaper area in babies.
Against diaper- or heat-rash
♦ [13]
Consensus Index/ Spontaneous Quotation Frequency for remedies: ♦: quoted by < 10% of informants; ♦♦ quoted by 10-20% of
informants; ♦♦♦: quoted by 21-30% of informants; ♦♦♦♦: quoted by 31-40% of informants; ♦♦♦♦♦: quoted by > 40% of
informants. ♣: Ethnoveterinary application.
72
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41. Pieroni A, Quave CL, Villanelli ML, Mangino P, Sabbatini G, Santini L, Boccetti T, Profili M, Ciccioli T, Rampa LG, et al: Ethnopharmacognistic survey on the natural ingredients used in folk cosmetics, cosmeceuticals and remedies for healing skin diseases in the inland Marches, Central-Eastern Italy. Journal of Ethnopharmacology 2004, 91:331-344.
42. De Feo V, Senatore F: Medicinal plants and phytotherapy in the Amalfitan Coast, Salerno Province, Campania, southern Italy. Journal of Ethnopharmacology 1993, 39:39-51.
43. Lentini F: The role of ethnobotanics in scientific research. State of ethnobotanical knowledge in Sicily. Fitoterapia 2000, 71:S83-S88.
44. Gibbons S, Moser E, Hausmann S, Stavri M, Smith E, Clennett C: An anti-staphylococcal acylphloroglucinol from Hypericum foliosum. Phytochemistry 2005, 66:1472-1475.
45. Gibbons S, Ohlendorf B, Johnsen I: The genus Hypericum--a valuable resource of anti-Staphylococcal leads. Fitoterapia 2002, 73:300-304.
46. Po Shiu WK, Gibbons S: Anti-staphylococcal acylphloroglucinols from Hypericum beanii. Phytochemistry 2006, 67:2568-2572.
47. Comune di Avigliano. 2007:http://www.comune.avigliano.pz.it.
48. Conoscere La Basilicata. I Comuni Lucani. Consiglio Regionale Della Basilicata 2007:http://www.consiglio.basilicata.it/conoscerebasilicata/cultura/percorsi/schede/comuni.asp.
49. Comuni-Italiani.it. 2007:http://en.comuni-italiani.it.
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50. Passalacqua NG, Guarrera PM, De Fine G: Contribution to the knowledge of the folk plant medicine in Calabria region (Southern Italy). Fitoterapia 2007, 78:52-68.
51. De Feo V, Aquino R, Menghini A, Ramundo E, Senatore F: Traditional phytotherapy in the Peninsula Sorrentina, Campania, southern Italy. Journal of Ethnopharmacology 1992, 36:113-125.
52. Guarrera P, Leporatti L: Ethnobotanical remarks on Central and Southern Italy. Journal of Ethnobiology and Ethnomedicine 2007, 3:23.
53. Guarrera PM: Food medicine and minor nourishment in the folk traditions of Central Italy (Marche, Abruzzo and Latium). Fitoterapia 2003, 74:515-544.
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CHAPTER III
EFFECTS OF EXTRACTS FROM ITALIAN MEDICINAL PLANTS ON
PLANKTONIC GROWTH, BIOFILM FORMATION AND ADHERENCE OF
METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS
Abstract
One-third of botanical remedies from southern Italy are used to treat skin disorders
and wounds. Many plants possess potent antimicrobial agents and provide effective
remedies for these disorders. Staphylococcus aureus, a common cause of skin infections,
has generated increasing concern among health care professionals due to the prevalence
of drug resistant strains. Identification of novel antibiotics and anti-biofilm agents for
methicillin-resistant S. aureus (MRSA) is important to healthcare on a global scale. The
aim of this study was to investigate plants from different ethnobotanical usage groups for
inhibition of growth, biofilm formation and adherence in MRSA and mammalian
cytotoxicity.
I categorized plants into 3 ethnobotanical groups: (1) folk remedies for skin and soft
tissue infection, (2) folk remedies not involving the skin, and (3) plants with no
ethnomedical application. I screened 168 plant extracts, representing 104 botanical
species, for activity against MRSA (ATCC 33593). I employed broth microtiter dilution
methods to determine the MIC after 18 hours growth using an optical density (OD600nm)
reading. The impact of extracts on biofilm formation and adherence was tested by
growing biofilms for 40 hours, then fixing and staining with crystal violet. After washing,
10% Tween 80 was added and OD570nm readings were taken. Cytotoxicity of extracts was
determined using a MTT assay with a human T lymphoblastoid cell line.
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Several plants used in Italian folk medicine demonstrated strong interference with
biofilm formation and adherence, though very few demonstrated strong inhibition of
growth (MIC≤128 μg/ml). Moreover, many extracts responsible for potent anti-biofilm
activity (≤32 μg/ml) actually promoted growth of planktonic cells. Despite the limited
bacteriostatic activity of unfractionated plant extracts, six plant extracts with potent anti-
biofilm effects were identified and merit further investigation.
1. Introduction
The increase of microbial resistance to antibiotics threatens public health on a
global scale as it reduces the effectiveness of treatments and increases morbidity,
mortality, and health care costs (Coast et al., 1996). Evolution of highly resistant bacterial
strains has compromised the use of newer generations of antibiotics including penicillins,
cephalosporins, aminoglycolsides, fluoroquinolones, trimethroprim and trimethroprim-
sulfamethoxazole combinations (Levy, 2002).
Although the active constituents may occur in lower concentrations, plant extracts
may be a better source of antimicrobial compounds than synthetic drugs (Cox and Balick,
1994). The phenomenon of additive or synergistic effects is often crucial to bioactivity
(Aqil et al., 2006a; b; Kamatou et al., 2006) in plant extracts and in some cases, the
activity is lost in purified fractions (Cos et al., 2006). Development of bacterial resistance
to synergistic drug combinations, such as those found in plants, may be slower than for
single drug therapies.
Only a small percent of plants have been investigated for their bioactivity.
Furthermore, most of these studies have concentrated on the growth-inhibitory potential
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of plant products. New assays investigating other potential roles, such as the mediation of
pathogenicity via quorum sensing inhibition, have recently emerged (Adonizio et al.,
2006; Rasmussen and Givskov, 2006). Investigations into the anti-pathogenic potential of
natural products may open new avenues for drug development in the control of antibiotic
resistant pathogens.
The use of plants against skin disease is a common practice in the popular medicine
of most cultures, although the precise causation of disease and mechanism of cure is not
always understood (Grierson and Afolayan, 1999; Srinivasan et al., 2001). While 1-3% of
pharmaceutical drugs are used to treat skin disorders and wounds, 33% of botanical
therapies in non-Western societies are used to this end (Bennett et al., 2002; Mantle and
Gok, 2001). The topical application of many botanical remedies for the treatment of
various skin diseases has proven effective due to the antimicrobial and anti-inflammatory
properties of the plants.
Southern Italy has a rich cultural history that has been augmented by recent
immigration flux from Eastern Europe and North Africa. The Basilicata Province, also
known as Lucania, is characterized by small scale agriculture and its inhabitants maintain
strong ties with the land. The demographic and socioeconomic aspects of this region are
described in Chapter 2. Wild plants are regularly gathered as sources of food and
medicine (Figures 1-2). Medicinal plants make up an integral facet of home healthcare in
Basilicata. At least one-third of all medicinal plants utilized in the folk-pharmacopoeia of
this region are applied towards the treatment of skin and soft tissue infection (SSTI).
Staphylococcus aureus, a gram-positive species, is a ubiquitous colonizer of the
skin and mucous membranes of humans and animals. It is frequently associated with skin
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disease and is a common target for natural product drug screenings. Many strains of S.
aureus carry resistance genes for penicillin antibiotics, tetracyclines, methicillin and now,
vancomycin. Methicillin resistant S. aureus (MRSA) presents a significant threat to
public health in the USA. A surveillance study on MRSA found that an estimated 94,360
patients in the USA had invasive MRSA infections in 2005, resulting in an estimated
18,650 deaths (Klevens et al., 2007), surpassing the mortality estimates for AIDS in the
USA.
Staphylococcus aureus causes two types of syndromes in humans and animals:
intoxications and infections. Staphylococcal infection causes invasive disease through a
breach in the host’s barriers – most commonly the skin. In fact, S. aureus is the most
prominent etiological agent for skin and soft tissue infections. It can cause such
conditions as folliculitis, furuncle, carbuncle, paronychia, bullous impetigo, nonbullous
impetigo and cellulitis (Fauci et al., 1998).
Biofilm-related infections caused by staphylococci are among the leading causes of
nosocomial infection in the USA (Kong et al., 2006). Biofilms comprise sessile microbial
communities that embed themselves within a self-made extracellular polymeric matrix.
This phenotype confers some protection to bacteria against host defenses and impedes the
delivery of certain large molecule antimicrobials (Lewis, 2001). Moreover, bacteria
associated with biofilms grow slowly, and this restricts the efficacy of many antibiotic
classes, including the β-lactams (Lewis, 2005). The first stages of staphylococcal biofilm
formation involve adherence of planktonic bacterial cells to cellular or prosthetic
surfaces, such as surgically implanted medical devices, including catheters, plates,
screws, artificial joints and cardiac valves. Biofilms present significant therapeutic
81
barriers for many antibiotics and the discovery of agents which could prevent their
formation or adherence would be of great use. A few studies on the antibiotic potential of
natural products from Mediterranean plants have already shown promising results in
bacteriostatic activity for drug resistant staphylococci (Gibbons, 2004; Gibbons et al.,
2002; Gibbons et al., 2003; Oluwatuyi et al., 2004). The objectives of this study were to
evaluate extracts of south Italian plants for effects on growth, biofilm formation and
adherence in MRSA and mammalian cytotoxicity.
My hypothesis was that plants used for the specific treatment of SSTI would
demonstrate higher mean activity for growth and biofilm inhibition, and lower mean
mammalian cytotoxicity than plants used in folk remedies not involving the skin or plants
with no ethnomedical application.
2. Materials and Methods
2.1 Field research
I conducted field research in ten communities situated in the Vulture-Alto Bradano
area of Basilicata, Italy from April-July of 2006 (see Chapter 2, Figure 1 for a map of the
study location). Random sampling techniques were employed to recruit 112 interview
subjects. Interviewees were equally stratified by four age groups (21-35, 36-50, 51-70,
71+ years) and gender in order to analyze levels of traditional knowledge (TK) across 4
generations. Prior informed consent, as approved by the Florida International University
Institutional Review Board (#120505-01), was obtained prior to conducting interviews. I
followed the ethical guidelines adopted by the ICE/International Society of Ethnobiology
(ISE, 2007) and Italian Association of Ethno-Anthropologists (AISEA) (DEA, 2007). I
82
conducted interviews in Italian, took notes, and made audio or video recordings of the
interviewees when possible. I collected data using semi-structured interviews, participant
observation, and focus groups. I questioned interviewees about the medicinal uses of the
local flora, particularly those related to the skin. The ethnobotanical applications of the
species included in this study are summarized in Table 1. For the purposes of statistical
analysis, all plants were categorized into one of three groups: Group 1= plants used for
SSTI (n=25), Group 2= plants used medicinally, but not for the skin or topical
applications (n=281), and Group 3= plants with no reported ethnomedical application
(n=51).
All plants mentioned by interviewees were collected and identified by the first
author following the standard work of the Italian flora (Pignatti, 2002). Familial
nomenclature follows the current Angiosperm Phylogeny Group (Stevens, 2001
onwards). I followed procedures from the 2003 WHO Guidelines on Good Agricultural
and Collection Practices for Medicinal Plants (WHO, 2003) for the collection of bulk and
voucher specimens. I collected plants from wild populations on public land in the village
of Ginestra in the Vulture-Alto Bradano area of southern Italy. Permission for collection
and export of materials was obtained from local government officials in the village of
Ginestra. I collected plants when in flower and during the season associated with
traditional collection practices. I avoided collection near areas associated with high levels
of pesticide exposure, such as drainage canals, grain fields, roadsides and active pastures.
Voucher specimens (Bridson and Forman, 1992) of quoted botanical species were
deposited at the Herbarium Lucanum, Univeristà degli Studi della Basilicata (HLUC) in
Potenza, Italy and Fairchild Tropical Botanic Gardens (FTG) in Miami, FL, USA. Bulk
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specimens were composed only of the targeted plant species; parts from other plants and
soil were removed prior to drying and vacuum storage. Bulk materials were separated by
part (e.g., leaves, fruits, stems), cut into small fragments and dried in a plant drier using a
heat (35° C) and air flow source. A total of 100-300 g of dry sample was prepared for
each plant part selected for investigation. After drying, plant materials were packed into
plastic bags with several silica packets [Süd-Chemie SORB-IT®] and vacuum sealed to
prevent mold growth. Plant materials were then shipped to the USDA PPQ APHIS
inspection center in Miami, FL, USA under permit #DP63438.
2.2 Extraction of natural products
Dry plant materials were ground into a fine powder using a W-17-J Fitzpatrick
Homoloid Machine (homogenizer). I cleaned the machine between samples using a
compressed air pump. Ground materials were stored in Ziploc freezer bags at -80ºC until
extraction. I made ethanolic extracts of all plant samples by soaking in 95% EtOH using a
ratio of 1g (plant material):10 ml (EtOH) for 72 h. Flasks were agitated daily. After 72 h,
I vacuum filtered extracts and the volumes were measured.
I transferred extracts to round-bottom flasks for rotary evaporation using the Büchi
Rotavapor R-205 system. The heating bath temperature was set at 40ºC and the cooling
water condenser coils were set to 2.3 ºC. The rotation speed was 45 rotations per minute.
After all of the solvent had evaporated, I stored the flasks in a -80ºC freezer.
Extracts were lyophilized using the Labconco Freeze-Dry System/ Freezone 4.5
machine then weighed to calculate the percent yield of product. The product was scraped
out of the flask and placed in a scintillation vial, then stored in a -20ºC freezer. For
84
bioassay screening, I reconstituted 50 mg of product with 5 ml of dimethyl sulfoxide
(DMSO), bringing it to a stock concentration of 10 mg/ml. Extracts were stored in the
dark at -20ºC.
While all plant materials were extracted in EtOH using the above described method,
some plants were also selected for aqueous extraction based on their traditional
preparation. The traditional preparations were made as decoctions using a ratio of 1g
(plant material): 50 ml (dH2O). I boiled materials on a hot plate with a magnetic stir rod
for 30 minutes. The decoction was vacuum filtered and the extract volume was measured,
rotary evaporated, frozen, then lyophilized and reconstituted with sterile dH2O.
Once reconstituted in DMSO (or dH2O for aqueous extracts), I filtered extracts in a
biosafety cabinet using 5ml syringes and sterile 0.2 μm nylon filters. I placed sterile
filtered extracts in sterile scintillation vials and stored in the dark at 4ºC. The excipient
(DMSO or dH2O) made up less than 5.1% of the final test solution for MIC assays and
less than 1.2% for MTT and biofilm assays.
2.3 Bacteria and culture conditions
Methicillin-resistant Staphylococcus aureus (ATCC 33593) was grown in Tryptic
Soy Broth (BD Diagnostic Systems, Franklin Lakes, NJ, USA) or on agar plates for 18 h
at 37°C. A 0.5 McFarland Standard was used to create inoculum densities of 1.5 x 108
cfu/ml in phosphate buffered saline using the direct suspension method (Isenberg, 2004)
for MIC and biofilm assays. To test the mean percent inhibition of growth against a
collection of six S. aureus strains, I also used a methicillin sensitive isolate (ATCC
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25923), a glycopeptide intermediate isolate (NRS 37), and three additional MRSA
isolates with different drug resistance profiles (NRS194, NRS382 and NRS 385).
2.4 Growth inhibition
I determined the effect of plant extracts on staphylococcal growth by using a
microtiter broth method (Amsterdam, 1996) in sterile flat-bottom 96-well polystyrene
plates. I used serial dilution techniques to determine the MIC50 of extracts at
concentrations of 8-512 μg/ml after 18 h growth. In addition, I tested negative (growth)
controls (cells + TSB), positive controls (cells + TSB + antibiotics – vancomycin,
ampicillin, and trimethroprim-sulfamethoxazole), vehicle controls (cells + TSB +
DMSO), and media controls (TSB). I performed all tests in triplicate. Optical density
readings were taken using a KC4 microplate reader at 600 nm at 0 and 18 hours post-
inoculation. Results are reported as the MIC50 for growth at 18 hours post-inoculation. To
account for the effect of extract color on the OD600nm reading, a formula for calculating
percent inhibition was used. The mean % inhibition of replicate tests was used to
determine the final MIC50 value.
% inhibition = 100018
0181 xODgcODgcODtODt
⎟⎟⎠
⎞⎜⎜⎝
⎛⎟⎟⎠
⎞⎜⎜⎝
⎛−−
−
ODt18 = optical density (600 nm) of the test well at 18 hours post-inoculation
ODt0 = optical density (600 nm) of the test well at 0 hours post-inoculation
ODgc18 = optical density (600 nm) of the growth control well at 18 hours post-
inoculation
ODgc0 = optical density (600 nm) of the growth control well at 0 hours post-inoculation
86
2.5 Biofilm formation and adherence
I employed a modified microtiter dish system (Christensen et al., 1985; Schadow et
al., 1988; Yarwood et al., 2004) to test the effect of plant extracts on biofilm formation
and adherence. I performed assays in sterile flat-bottom 96-well polystyrene plates (BD
Falcon Tissue Culture Plates No. 351172). I used serial dilutions to determine the IC50 of
extracts at sub-inhibitory (for growth) concentrations of 4-128 μg/ml after 40 h growth. I
included negative controls (cells + TSB), positive controls (cells + TSB + antibiotics –
vancomycin, ampicillin, and trimethroprim-sulfamethoxazole), vehicle controls (cells +
TSB + DMSO), and media controls (TSB) and (TSB + extract). All tests were performed
in triplicate.
To test for the effects of extracts on biofilm formation, the appropriate
concentration of extract was added to the test wells prior to inoculation. Plates were
placed in a 37°C incubator and bacteria were shaken for 40 h. The contents of the wells
were then aspirated, rinsed 3 times with PBS, and fixed by drying for 1 h in the 37°C
incubator. Once the wells were fully dry, I added 200 μl of 0.1% crystal violet stain to
wells to stain for 15 m. The excess stain was rinsed off with tap water and 200 μl of 10%
Tween 80 was added to wells. I pulled off the stain adhering to the biofilm biomass with
the Tween 80 and transferred to new 96-well plates for spectrophotometric analysis (KC4
microplate reader at OD570nm).
To test for the effects of extracts on biofilm adherence, biofilms were established in
the 96-well plates by growing shaking for 20 h at 37°C. At 20 h post-inoculation,
planktonic cells and TSB were aspirated and fresh TSB was added with the appropriate
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concentration of test extract. Plates were then placed back into the 37°C incubator on a
shaker for 20 h. The staining methods were the same as those described above (Figure 3).
Replicate absorbance readings for each concentration tested were averaged and the
average of the media control (TSB + extract) was subtracted. This value was then divided
by the mean absorbance of the vehicle control and multiplied by 100. The concentration
at which the extract depleted the biofilm biomass by at least 50% was labeled as the IC50.
2.6 Mammalian cytotoxicity
I evaluated plant extracts for their toxic potential in an in vitro test with the CCRF-
CEM human T lymphoblastoid cell line (ATCC CCL-119) using a 3-(4,5-
dimethylthiazol-2-yl)-2,5,-diphenyl-2H-tetrazolium bromide (MTT) cell proliferation
assay. This assay was used to evaluate the population-level response of human cells to
the toxic potential of plant products in regards to cell proliferation and cell viability.
Growth medium consisting of RPMI 1640 growth medium (ATCC 30-2001), fetal
calf serum (ATCC 30-2020), and penicillin-streptomycin-glutamine solution (10,000
units/ml-10,000 μg/ml-29.23 mg; ATCC 30-2220) was used for establishing and
maintaining the cell culture. Cultures were maintained in log phase growth at a cell
concentration between 2 x 105 – 2 x 106 cells/ml by regular centrifugation of cells and
reconstitution in fresh media. Cultures were incubated at 37°C with 5% CO2 in air
atmosphere and 95% relative humidity. After the cell culture remained uncontaminated
and viable for a period of 14 days, I performed the MTT cell proliferation assay. I
conducted cell counts and viability checks using a hemocytometer before and after each
experiment.
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I used serial dilution techniques to screen extracts in triplicate at concentrations of
4-128 μg/ml using the MTT cell proliferation assay kit (supplied by ATCC). I also tested
negative (growth) controls (cells + growth medium), positive controls (cells +
Campothecin), vehicle controls (growth medium + cells + DMSO), and media controls
(growth medium) and (growth medium + extract). A cell suspension at 1 x 106 cells/ml
was incubated with the appropriate concentration of extract in sterile flat-bottom 96-well
polystyrene plates (BD Falcon Tissue Culture Plates No. 351172) resulting in a total
volume of 100 µl per well. After 24 hours, I added 10 µl of MTT solution to each well
and incubated for an additional 4 hours. Then, I added 100 µl of detergent reagent and
incubated the plates overnight at room temperature in the dark. The absorbance of each
well was read at 570 nm and 655 nm using a KC4 microtiter plate reader.
The replicate absorbance readings for each concentration tested were averaged and
the difference between the readings was taken (ΔOD570nm-655nm). This value was then
divided by the mean absorbance of the negative vehicle control and multiplied by 100.
Values were expressed as a percent of the control mean absorbance. The concentration at
which the extract inhibited the growth of the cells by at least 50% was labeled as the
cytotoxic IC50. This value was used together with the IC50 for growth inhibition to
determine the therapeutic index, in which the following formula was used: Therapeutic
Index = antimicrobial MIC50 /cytotoxic IC50.
2.7 Statistical analyses
The mean values and standard deviations of all replicates described in the above
tests were calculated using Excel software. Differences between means were analyzed
89
with a One-Way ANOVA, followed by multiple comparison analysis using the
Bonferroni method on SPLUS software. Differences were considered significant with p-
values <0.05.
3. Results
3.1 Effects on planktonic growth of MRSA
Plant extracts demonstrated limited bacteriostatic activity (Table 1). Roughly 18%
of the extracts tested demonstrated an IC50 of 256 or 512 μg/ml. Of those demonstrating
activity, the majority were not associated with any particular ethnobotanical application
(Figure 4). The mean percent inhibition of growth for each ethnobotanical usage group
when screened at 512 μg/ml ranged from 15.93-19.46% (Group 1= 19.46%, Group 2 =
15.93%, and Group 3= 18.43%). Based on results from a One-Way ANOVA (p>0.05), I
found that there was no significant difference in the mean bacteriostatic activity of
extracts from plants in each ethnobotanical usage group (Table2, Figure 5).
3.2 Mammalian cytotoxicity
Five extracts from three plants demonstrated strong (≤ 32 μg/ml) cytotoxic
effects: Achillea millefolium, Rosmarinus officinalis, and Cyclamen hederifolium (Table
3). The mean percent inhibition of mammalian cell growth for each ethnobotanical usage
group when screened at 64 μg/ml ranged from 4.9-15.17%. (Group 1= 7.2%, Group
2=15.17%, and Group 3=4.9%).
Based on results from a One-Way ANOVA (p<0.005), I found that there was a
significant difference in the mean cytotoxic activity of extracts from plants in each
90
ethnobotanical usage group (Table2, Figure 6). Multiple comparison analysis using the
Bonferroni method revealed a significant difference between groups 2 and 3. The
cytotoxicity of plants categorized for medicinal applications unrelated to the skin was
significantly greater than activity from plants without any ethnomedical applications
(Table 4).
3.3 Biofilm inhibition
Extracts from 10 plants exhibited an IC50 ≤32 μg/ml for inhibition of biofilm
formation: Lonicera alpigena, Castanea sativa, Juglans regia, Ballota nigra, Rosmarinus
officinalis, Leopoldia comosa, Malva sylvestris, Cyclamen hederifolium, Rosa canina var.
canina, and Rubus ulmifolius (Table 3). The mean percent inhibition of biofilm formation
for each ethnobotanical usage group when screened at 64 μg/ml ranged from 0.58-8.73%
(Group 1=8.73%, Group 2=6.6%, and Group 3=0.58%). Plants used in folk remedies for
SSTI made up 38% of plants exhibiting an IC50 of 8-128 μg/ml for MRSA biofilm
formation (Figure 7).
Based on the results of a One-Way ANOVA test (p<0.005), the mean anti-biofilm
activity of extracts from plants in each ethnobotanical usage group was not the same
(Table 2). Multiple comparison analysis using the Bonferroni method revealed a
significant difference between groups 1 and 3. The anti-biofilm activity of plants
categorized for medicinal applications to the skin was significantly greater than activity
from plants without any ethnomedical applications (Table 4, Figure 8).
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4. Discussion
Here, I discuss the creation of a cytotoxicity index and its usefulness in selecting
crude plant extracts for further phytochemical analysis. I also offer a detailed discussion
of the bacteriostatic, anti-biofilm, and cytotoxic properties of 8 plants: Erodium
malacoides, Rosmarinus officinalis, Arundo donax, Ballota nigra, Juglans regia,
Leopoldia comosa, Marrubium vulgare, and Rubus ulmifolius. This is the first report of
anti-biofilm activity for these plants.
4.1 Cytotoxicity and creation of a therapeutic index
Antibacterial activity should be discriminated from non-specific toxicity via
incorporation of a parallel evaluation for cytotoxicity on mammalian cell lines (Cos et al.,
2006). One of the most popular assays for determining the cytotoxic potential of plant
products is the MTT cytotoxicity assay (Ali et al., 2001). This assay is based on the
reduction of MTT by active cells (Denizot and Lang, 1986; Mosmann, 1983).
The intention of cytotoxicity screening is not to predict the nature of all possible
toxic effects of an active plant extract, but to estimate the risk of failure that the extract
poses in future in vivo toxicity studies (Gray and Edwards, 2005). Furthermore,
cytotoxicity screening is useful in the identification of efficacious compounds during
initial, high throughput antimicrobial screening studies.
A therapeutic index was created to reflect the ratio of antimicrobial activity to
mammalian cytotoxicity. The most promising extracts are those with low therapeutic
index values (i.e., with antimicrobial IC50 at very low concentrations and cytotoxic IC50 at
very high concentrations). Approximately 90% of the extracts tested were non-toxic to
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the human T lymphoblastoid cell line. Thus, the therapeutic index for most extracts with
any level of growth inhibitory effects on MRSA were relatively good, and ranged from 1-
4 (Table 3).
Rosmarinus officinalis (EtOH extract of rosemary leaves, stems, and flowers)
demonstrated both growth inhibition (MIC50=512 μg/ml) and inhibition of biofilm
formation (IC50=16 μg/ml). Rosemary is well known for its use as a food (spice) and
cosmetic applications (perfume and emollient for the skin). The in vitro effects of
rosemary extracts on cytotoxicity (Ahernea et al., 2007) of human cell lines and on
growth for a broad-spectrum of bacteria have already been reported in other studies
(Bozin et al., 2007; Dorman and Deans, 2000; Fu et al., 2007; Luqman et al., 2007;
Packer and Luz, 2007; Tsai et al., 2007). The effect of rosemary extract on biofilm
formation and adherence in MRSA has not been previously reported.
4.2 Bacteriostatic activity for MRSA: Erodium malacoides
An ethanolic extract of the aerial parts of Erodium malacoides (Mediterranean
Stork’s Bill) demonstrated an IC50 of 128 μg/ml for growth inhibition and a cytotoxic
IC50 of 64 μg/ml (therapeutic index value = 2). There is no ethnobotanical use associated
with this species in the field study region. Bacteriostatic effects for S. aureus have been
identified in other members of this genus, including E. glaucophyllum (L.) Aiton (Gohar
et al., 2003), and E. stephanianum Willd.(Yeung, 1985), E. chrysanthum L’Hér ex DC.,
E. circutarium L’Hér ex Aiton, and E. hymenodes L’Hér (Karel and Roach, 1951). This
activity may be due to the polyphenol geraniin (Gohar et al., 2003).
93
4.3 Effects on MRSA biofilm formation and adherence
A significant dose-dependent response in biofilm inhibition was evident in 6 of
the 168 extracts tested: Arundo donax, Ballota nigra, Juglans regia, Leopoldia comosa,
Marrubium vulgare, and Rubus ulmifolius (Figure 9). Here, I offer a discussion of the
ethnomedicinal applications of these plants in southern Italy and review the literature for
other reports of anti-staphylococcal activity.
4.3.1 Arundo donax (Poaceae) - canna
The giant reed is commonly used in the construction of musical instruments and
the production of industrial cellulose. It is also used for several other purposes in
southern Italy, especially to support grape vines. This use was so important to local
agriculture in the past that land was often divided along the border lines of reed colonies.
Thin slivers of the reed are used as skewers for drying figs (Figure 1). The white
hemicellulose membrane located at the nodes of the reed is inserted into fresh lacerations
as a haemostatic agent (Passalacqua et al., 2007; Pieroni et al., 2002c) (Figure 2). Small
tooth-sized pieces of the reed are also cut up and used in the ritual treatment of toothache
(Quave and Pieroni, 2005). Antiproliferative activity against several human cancer cell
lines has been reported for a lectin isolated from the rhizomes of this plant (Kaur et al.,
2005). A. donax is rich in alkaloids, including bufotenidine and gramine. Small amounts
of DMT have also been isolated from the flowers (Duke, 2008).
I found that aqueous extracts of the reed nodes (which contain the white
hemicellulose membrane) demonstrated a marked dose-dependent response for anti-
biofilm activity, both in preventing MRSA biofilm formation and disrupting established
94
biofilms (Figure 9a). These results may suggest that the traditional application of the reed
membrane to fresh lacerations may be useful as a prophylactic for biofilm-related
infection. The effect of giant reed extract on biofilm formation and adherence in MRSA
has not been previously reported.
4.3.2 Ballota nigra (Lamiaceae) – erba cane
Infusions of the aerial parts of black horehound are used in the south Italian
pharmacopoeia as a rinse for skin rashes and are also drunk to promote circulation.
Extensive phytochemical studies (Bertrand et al., 2000; Bruno et al., 1986; Seidel et al.,
1997; Seidel et al., 2000) have been performed on black horehound, and several
phenylpropanoid glycosides have been identified and linked to moderate growth
inhibition in Staphylococcus aureus (Didry et al., 1999). Black horehound also contains
diterpenes, such as marubiin, as well as caffeic and ferulic acid derivatives (Gruenwald et
al., 1998). Potent antioxidant activity has reported in infusions of the aerial parts (Citoglu
et al., 2004; Vrchovska et al., 2007). I prepared several types of extractions of the
different parts of the plant, and found that an aqueous extraction of the aerial parts,
similar to that prepared in the folk medical tradition, was the most effective at inhibiting
both biofilm growth and adherence. Although this extract actually promoted planktonic
growth of this particular MRSA strain by almost twofold, significant dose-dependent
responses for the inhibition of both biofilm formation and adherence were evident (Figure
9b). The effect of black horehound extract on biofilm formation and adherence in MRSA
has not been previously reported.
95
4.3.3 Juglans regia (Juglandaceae) - noce
The common walnut has been reported in the ethnobotanical literature for its use
as an aromatic for cheese and to protect it from parasites (Guarrera et al., 2005b) in Italy,
and as a medicine for rheumatism, fever, fungal infection, hemorrhoid, cough and eczema
in Turkey (Erdemoglu et al., 2003; Kultur, 2007). In this field study, I found that the bark
is prepared in a decoction and gargled for toothache and fresh leaves are applied topically
to reduce the swelling associated with varicose veins. The immature fruits are used for
cosmetic (hair dye) applications (Pieroni et al., 2004b) and the flowers are employed in
ritual healing ceremonies for mal d’arco (rainbow illness) (Quave and Pieroni, 2005).
I found that an ethanolic extract of the immature fruits promoted biofilm
desorption and prevented biofilm formation. Limited growth inhibition was noted at
higher doses 128-512 μg/ml (Figure 9c). Similar activity in staphylococcal growth
inhibition by aqueous extracts of walnut leaves has also been reported (Pereira et al.,
2007). The walnut tree is rich in phenolic compounds (Pereira et al., 2007; Stampar et al.,
2006), including naphtoquinones and flavonoids that are likely responsible for the anti-
bacterial activity of extracts derived from this plant. The use of walnut leaves for mild
skin inflammations has been approved by the German Commission E (Blumenthal et al.,
2000). This is the first report of anti-biofilm activity for MRSA by walnut extract.
4.3.4 Leopoldia comosa (Hyacinthaceae) - cipuldjin
In southern Italy, the tassel grape hyacinth is used in folk remedies for toothache
(bulb is grated and applied topically) (Guarrera et al., 2005c) and for food (Casoria et al.,
1999; Pieroni et al., 2002b). Extracts from the bulbs have demonstrated potent in vitro
96
anti-oxidant activity (Pieroni et al., 2002a). I was unable to find any reports regarding the
phytochemical makeup of this species. Both the aqueous and ethanolic extracts of the
bulb demonstrated potent anti-biofilm activity for this strain of MRSA. The IC50 for
preventing biofilm formation and adherence was 16 and 8 μg/ml, respectively. The IC90
for biofilm adherence was 128 μg/ml (Figure 9d). This is the first report of anti-biofilm
activity for this plant.
4.3.5 Marrubium vulgare (Lamiaceae) - maruggē
White horehound is one of the most frequently quoted medicinal species applied in
south Italian traditional medicine. A local rhyme is associated with this plant: “U’
marrugxhə, ogni mal’ destruggə” (the white horehound destroys every disease). It is used
in the treatment of furuncle, boil, Athlete’s foot, skin infection, dermatitis, foot and
mouth disease (in animals), gastritis, and malaria. It is thought to be particularly good at
cleansing the liver and is used as a hepatoprotectant and general panacea (Pieroni et al.,
2002c; Pieroni and Quave, 2005). Aqueous decoctions or infusions of the aerial parts are
either applied topically as a skin rinse or drunk, depending upon the illness being treated.
I found that an ethanolic extract of the roots was effective in promoting desorption of
established biofilms at very low concentrations (IC50 = 8 μg/ml and IC90=128 μg/ml).
This extract was not, however, very effective at preventing the formation of biofilms on
plastic surfaces. The maximum percent inhibition for biofilm formation was 31% at a
concentration of 128 μg/ml (Figure 9e).
Previous studies have demonstrated that white horehound extracts exhibit
antispasmodic (Schlemper et al., 1996), antioxidant (Berrougui et al., 2006; Matkowski
97
and Piotrowska, 2006; Weel et al., 1999), hypotensive (El Bardai et al., 2001),
insecticidal (Pavela, 2004), and analgesic (Meyre-Silva et al., 2005) properties.
Marrubium species are rich in diterpenes, caffeic acid derivatives, sterols, and flavonoids
(Khanavi et al., 2005; Lazari et al., 1999). One of the principle medicinal components of
horehound extract is marubiin, a furanic labdane diterpene (Blumenthal et al., 2000). It
has been found to exhibit non-selective anti-inflammatory properties in mouse models,
which may support its use as a folk remedy for dermatitis (Stulzer et al., 2006). The
German Commission E approved the use of this herb for the treatment of loss of appetite
and dyspepsia (Blumenthal et al., 2000). All of the horehound extracts that I tested were
non-toxic in the in vitro cytotoxicity assays. The effect of white horehound extract on
biofilm formation and adherence in MRSA has not been previously reported.
4.3.6 Rubus ulmifolius (Rosaceae) - more
The elmleaf blackberry is used in south Italian skin remedies. The leaves are
applied topically for anti-furuncle, vulnerary, anti-abscess, anti-carbuncle, suppurative,
and anti-infective properties (Pieroni and Quave, 2005; Pieroni et al., 2004a). A root
decoction is used as a hair wash to prevent and treat hair loss. Similar ethnobotanical
applications have been reported in other areas of Italy (Guarrera, 2005; Guarrera et al.,
2005a; Guarrera et al., 2005c). Previous phytochemical studies on elmleaf blackberry
have demonstrated strong antioxidant activity (Dall'Acqua et al., 2008). The
antimicrobial properties of this plant have been reported for a broad spectrum of gram +
and gram – bacteria (Panizzi et al., 2002) and fungi (Sisti et al.). Moderate growth
inhibition against a drug sensitive strain of S. aureus (ATCC 25923) has also been
98
reported, and has been linked to the active constituents ferulic acid, gallic acid (Panizzi et
al., 2002), and rubanthrone A (Flamini et al., 2002). In this study, however, I found only
a very low level of growth inhibition when extracts were tested against MRSA. At best,
the MIC50 for EtOH extracts of the roots and woody stems was 512 μg/ml.
An ethanolic extract of roots demonstrated a dose-dependent response for
inhibiting biofilm formation. The response for inhibiting biofilm adherence, however,
was not dose dependent – the best response of 95% inhibition was found at 16 μg/ml, and
steadily decreased to 54% inhibition at a test concentration of 128 μg/ml (Figure 9f). The
standard deviation for replicates of this data was <5% for each concentration tested, thus
this negative trend is not due to testing errors. This is just one example of how complex
the relationship between natural products and biofilm processes are. The effect of elmleaf
blackberry extract on biofilm formation and adherence in MRSA has not been previously
reported.
5. Conclusion
Plant extracts demonstrated limited bacteriostatic activity, and in fact, many
actually promoted planktonic growth. More significantly, extracts from six plants
exhibited strong dose-dependent inhibition of biofilm formation and adherence. I
identified a significant correlation in anti-biofilm activity with medicinal plants used for
the skin when compared with plants with no apparent ethnomedical use. In other words,
medicinal plants for the skin are more effective biofilm inhibitors than plants with no
cited medicinal use. Moreover, 90% of plant extracts assessed in this study were nontoxic
to human cells when tested in vitro. These results validate the efficacy of the topical
99
application of Arundo donax, Ballota nigra, Juglans regia, Leopoldia comosa,
Marrubium vulgare, and Rubus ulmifolius in Italian folk remedies for SSTI.
While these results are based on the analysis of crude, unfractionated extracts, they
also represent the first of many steps towards the development of new anti-biofilm drugs.
Inhibition of biofilms provides a method of controlling the effects of pathogenic bacteria
without strong selection for drug resistance. The emergence of infections due to highly
resistant MRSA capable of forming and many times involving biofilms presents a
significant dilemma to medicine today. The healthcare community is ill-prepared to
effectively treat such infections as the old classes of antibiotics currently in use rapidly
lose their efficacy. Effective treatments for the disruption of established biofilms could
save many lives and decrease healthcare costs related to the treatment and potential
replacement of infected implanted prosthetic devices. Prophylactic agents for biofilms
embedded in surgically implanted devices and catheters could significantly diminish
morbidity and subsequently reduce healthcare costs associated with nosocomial infection.
I recommend further investigation of natural products from the following plants for
anti-biofilm activity in MRSA: Arundo donax, Ballota nigra, Juglans regia, Leopoldia
comosa, Marrubium vulgare, and Rubus ulmifolius.
Acknowledgements
This work was funded by NIH/NCCAM 1F31AT004288-01A1 (PI: C.L. Quave),
MBRS RISE - NIH/NIGMS R25GM061347, RISE Biomedical Research Initiative
summer research award, Botany in Action, Anne Chatham Fellowship in Medicinal
100
Botany, USDA CSREES 20053842215940 and NIH/NCAAM 1T32AT01060-01 (PI:
B.C. Bennett).
I thank Dr. Carmine Colacino of the Herbarium Lucanum in Potenza, Italy for
assisting in the taxonomic identification of plants collected and to the following people
for logistical support during field research: Donato Caputo, Milagros Prieto, Marco
Caputo, Rosanna Caputo, Antonello Fiore, and Donato Carbone. Thanks to Dr. Roberto
Perez and Prof. Steve Davis of the University of Miami Miller School of Medicine in
Miami, FL for providing bacterial isolates and training in biofilm assays and to Wei Lin
of the Florida International University Tissue Culture Facility in Miami, FL for providing
training in cell culture techniques. Special thanks to volunteers who assisted in laboratory
work: Jana Rose, Marco Caputo and Susan Mendez. Lastly, I extend my gratitude to all
of the study participants who graciously shared a wealth of knowledge regarding the
traditional medical practices of their communities.
101
Figure 1. Dried figs (Ficus carica L. [Moraceae]) are stuffed with almonds (Prunus
dulcis (Mill.) D.A. Webb [Rosaceae]) and placed onto a slice of giant reed stem (Arundo
donax L.[Poaceae]), then stored for the winter to be used in a decoction against colds and
respiratory ailments. Zia Giovannina (95 years-old) of Ginestra has used this remedy
since childhood.
102
103
Figure 2. The white hemicellulose membrane found at the node of the giant reed (Arundo
donax L. [Poaceae]) is used as a haemostatic agent for minor lacerations.
well contents aspirated, fresh TSB & extract added
well contents aspirated, rinsed & stained cells & TSB
added
0 5 10 15 20 25 30 35 40
104
Figure 3. Test for biofilm formation and adherence. The graph represents planktonic growth as measured by OD600nm readings for
MRSA strain ATCC 33593. (a) Extracts were tested for the ability to prevent biofilm formation by adding the test extract to the
wells at 0 hours post inoculation and performing the staining process at 40 hours post-inoculation. (b) Extracts were tested for the
ability to disrupt established biofilms (promote desorption) by aspirating the liquid content of wells at 20 hours post-inoculation,
and then adding the extract and fresh tryptic soy broth. The staining process was performed at 40 hours.
Time (hours) (b)
OD600nm
extract, cells, & TSB added
0 5 10 15 20 25 30 35 40
well contents aspirated, rinsed & stained
(a)
OD600nm
104
No reported use49%
Medicinal application to the skin21%
Medicinal application unrelated to the skin
15%
Food6%
Ornamental6%
Agricultural tool3%
Figure 4. Ethnobotanical use categories for plants whose extracts demonstrated ≥50%
growth inhibiting activity for MRSA at concentrations of 128-512 μg/ml.
105
0
10
20
30
40
50
60
70
80
90
100
Perc
ent I
nhib
ition
Group 3Group 2Group 1
Figure 5. Comparison of mean bacteriostatic activity of plant extracts from different
ethnobotanical groups. Group 1: plants used for SSTI; Group 2: plants used for medicinal
purposes not involving the skin; Group 3: plants with no medicinal application.
106
0
10
20
30
40
50
60
70
80
90
100
Perc
ent I
nhib
ition
Group 3Group 2Group 1 Figure 6. Comparison of mean mammalian cell growth inhibition by plant extracts from
different ethnobotanical groups. Group 1: plants used for SSTI; Group 2: plants used for
medicinal purposes not involving the skin; Group 3: plants with no medicinal application.
107
No reported use14%
Medicinal application to the skin38%Medicinal application
unrelated to the skin24%
Food19%
Cosmetic application5%
Figure 7. Ethnobotanical use categories for plants whose extracts demonstrated ≥50%
inhibition for MRSA biofilm formation at concentrations of 8-128 μg/ml.
108
0
10
20
30
40
50
60
70
80
90
100
Perc
ent I
nhib
ition
Group 3Group 2Group 1 Figure 8. Comparison of mean inhibition of biofilm formation by plant extracts from
different ethnobotanical groups. Group 1: plants used for SSTI; Group 2: plants used for
medicinal purposes not involving the skin; Group 3: plants with no medicinal application.
109
0102030405060708090
100
8 16 32 64 128Concentration (μg/ml)
% In
hibi
tion
0102030405060708090
100
8 16 32 64 128Concentration (μg/ml)
% In
hibi
tion
(a) (b)
0102030405060708090
100
8 16 32 64 128Concentration (μg/ml)
% In
hibi
tion
0102030405060708090
100
8 16 32 64 128Concentration (μg/ml)
% In
hibi
tion
(c) (d)
110
111
0102030405060708090
100
8 16 32 64 128Concentration (μg/ml)
% In
hibi
tion
0102030405060708090
100
8 16 32 64 128Concentration (μg/ml)
% In
hibi
tion
(e) (f)
Planktonic Growth Biofilm Formation Biofilm Adherence
Legend:
Figure 9. The percent inhibition of extracts for planktonic growth, biofilm formation and
adherence in MRSA. (a) Arundo donax, aqueous extract of nodes; (b) Ballota nigra,
aqueous extract of aerial parts; (c) Juglans regia, ethanolic extract of immature fruits; (d)
Leopoldia comosa, ethanolic extract of bulbs; (e) Marrubium vulgare, ethanolic extract
of roots; (f) Rubus ulmifolius, ethanolic extract of roots.
Table 1. Percent inhibition of growth of 6 S. aureus isolates at a test concentration of 512 μg/ml with unfractionated plant extracts.
Mean percent inhibition of growth at test concentration of 512 μg/mL for different S. aureus
isolates**
Family Botanic Name (Voucher ID)
Plant Part Ethno-botanical Use*
Extract Solvent
ATCC 25923
ATCC 33593
NRS 37
NRS 194
NRS 382
NRS 385
inflorescence N EtOH 12 45 - - - - leaves S EtOH - - 15 - 11 -
Sambucus ebulus L. (CQ-168)
stems N EtOH 4 19 19 - - - woody parts R EtOH - 14 8 - - -
EtOH - 63 3 - 2 - leaves S dH2O - - - - 3 - EtOH - - - - - - inflorescence S; R dH2O - - - - 7 2
Adoxaceae
Sambucus nigra L. (CQ-151)
infructescence F EtOH - 44 26 - 7 - Alliaceae Allium cepa L.
(CQ-206) leaves; bulbs; roots S; M; F EtOH - 26 - - - -
leaves; stems N EtOH 18 33 36 - 16 - Daucus carota L. (CQ-215) inflorescence;
infructescence N EtOH 5 20 25 - 2 -
Foeniculum vulgare ssp. piperitum (Ucria) Coutinho (CQ-192)
leaves; stems M; F EtOH - 35 4 - 11 -
Foeniculum vulgare ssp. vulgare Mill. (CQ-196)
leaves; stems M EtOH - 29 25 - 9 -
Apiaceae
Tordylium apulum L. (CQ-101)
flowers; leaves; roots; stems
N EtOH 3 12 42 - 18 -
Apocynaceae Vinca major L. (CQ-117)
flowers; leaves; roots; stems
M EtOH 55 29 18 - - -
stems N EtOH - - 35 - 28 - fruits N EtOH - 13 26 - 18 - stalks N EtOH 16 31 51 - 8 -
Aracaeae Arum italicum Mill. (CQ-175)
leaves S EtOH 7 6 43 - 13 - Asphodelaceae Asphodelus microcarpus inflorescence N EtOH - 5 47 - 5 9
112
112
Salzm. & Viv. (CQ-109)
leaves N EtOH 60 98 45 - - 13
Achillea ageratum L. (CQ-219)
leaves; stems; flowers
M EtOH 88 71 42 43 28 42
inflorescence M EtOH - 17 32 - 18 - leaves; stems M EtOH 12 40 23 3 6 -
Achillea millefolium L. (CQ-176)
leaves; stems; flowers
M EtOH - 17 42 3 - -
Anacyclus tomentosus DC. (CQ-167)
leaves; stems; flowers
N EtOH - 6 62 - 18 -
EtOH 56 100 56 11 36 14 basal leaves; roots F dH2O - - 4 - - -
Cichorium intybus L. (CQ-106)
leaves; stems; flowers
F EtOH - 23 20 - 1 -
EtOH 64 84 82 40 56 44 Matricaria recutita L. (CQ-118)
flowers; leaves; roots; stems
S; M dH2O - - - - - -
Scolymus hispanicus L. (CQ-199)
leaves; stems; flowers
N EtOH 14 - 10 - - -
Tussilago farfara L. (CQ-202)
leaves; stems; roots S EtOH 8 20 47 2 10 13
Asteraceae
Urospermum dalechampii (L.) Scop. (CQ-134)
flowers; leaves; roots; stems
N EtOH - 26 33 - - -
Anchusa officinalis L. (CQ-128)
leaves; stems; flowers
N EtOH 34 30 25 6 - -
EtOH 9 31 33 - - 16 Borago officinalis L. (CQ-100)
flowers; leaves; roots; stems
M dH2O 24 - - 1 17 6
Cerinthe major L. (CQ-110)
flowers; leaves; roots; stems
N EtOH - 18 31 - 19 10
Boraginaceae
Echium italicum L. (CQ-162)
leaves; stems; flowers
N EtOH 8 20 47 2 10 13
Brassica rapa subsp. rapa (CQ-104)
flowers; leaves; roots; stems
F EtOH - 21 59 17 24 -
Cardaria draba (L.) Desv. (CQ-140)
flowers; leaves; roots; stems
N EtOH 64 99 60 - 47 34
Brassicaceae
Eruca sativa Mill. (CQ-102)
flowers; leaves; roots; stems
N EtOH - 11 25 - - -
113
113
Sisymbrium officinale (L.) Scop. (CQ-131)
flowers; leaves; roots; stems
N EtOH - 26 50 16 32 12
woody parts N EtOH 47 2 45 - - - Caprifoliaceae Lonicera alpigena L. (CQ-213) leaves N EtOH - 37 52 6 - - Saponaria officinalis L. (CQ-210)
leaves; stems; flowers
N EtOH - 28 1 - 6 -
Silene alba (Mill.) E.H.L. Krause (CQ-123)
leaves; stems; flowers
N EtOH - 16 49 - 20
Caryophylla-ceae
Silene nutans L. (CQ-125)
leaves; stems; flowers
N EtOH 72 36 39 - -
23
-
Cucurbitaceae Ecballium elaterium (L.) A. Richard (CQ-169)
leaves; stems; flowers
S EtOH 9 72 65 28 49 33
114
leaves N EtOH - 10 11 - 15 - Dennstaedtia-ceae
Pteridium aquilinium (L.) Kuhn (CQ-211)
stems N EtOH - 24 4 - - -
leaves; stems N EtOH - 19 35 1 - Dipsacus fullonum L. (CQ-201) flowers N EtOH - 23 21 - -
- -
Knautia arvensis Coult. (CQ-190)
leaves; stems; flowers
N EtOH 3 5 - - -
Dipsacaceae
Knautia lucana Lacaita & Szabo (CQ-166)
leaves; stems; flowers
N EtOH 6 - 16 - -
-
-
Equisetaceae Equisetum arvense L. (CQ-226)
stems; leaves N EtOH 14 20 18 - 25 15
inflorescence O EtOH 18 23 22 - - - stems O EtOH 11 2 30 - 1
Acacia dealbata Link (CQ-115)
leaves; stems O EtOH 35 7 90 - - - 7
Anthyllis vulneraria L. (CQ-147)
leaves; stems; flowers
N EtOH 25 22 44 - 12 35
Astragalus monspessulanus L. (CQ-112)
leaves; stems; flowers; roots
N EtOH 44 17 50 3 24 9
leaves; flowers N EtOH 61 28 37 2 17 -
Fabaceae
Coronilla emerus L. (CQ-137) woody stems N EtOH - 10 31 - 3 1
114
Melilotus alba Medik. (CQ-193)
leaves; stems; flowers
N EtOH - 38 35 3 - -
woody parts N EtOH - 13 - - - - leaves N EtOH 61 72 100 - 3 25
Robinia pseudoacacia L. (CQ-155)
inflorescence N EtOH 18 - 37 - 7 - Spartium junceum L. (CQ-144)
leaves; stems; flowers
A EtOH - 12 12 - - 5
Trifolium repens L. (CQ-138)
leaves; stems; flowers; roots
N EtOH 15 9 100 5 13 23
Vicia craca L. (CQ-149)
leaves; stems; flowers; roots
N EtOH 60 96 63 - 16 -
Vicia faba L. (CQ-103)
leaves; stems; flowers; roots
F EtOH - 19 40 - 11 -
Vicia sativa subsp. angustifolio (CQ-124)
leaves; stems; flowers
N EtOH 78 88 100 29 44 100
Vicia sativa subsp. sativa (CQ-119)
leaves; stems; flowers
N EtOH - 5 35 -
115
- 25
inflorescence O EtOH - 43 59 - 14 18 stems O EtOH - 10 14 - - -
Wisteria sinensis (Sims) Sweet (CQ-126) leaves O EtOH 35 45 50 - 44 32
inflorescence N EtOH 58 100 96 18 49 - leaves N EtOH 85 90 87 59 85 72
Castanea sativa Mill. (CQ-191)
woody parts A EtOH 100 49 97 17 87 58 leaves N EtOH 30 8 95 - 20 5
Fagaceae
Quercus cerris L. (CQ-228) stems; fruits N EtOH 100 26 76 - - -
Gentianaceae Centaurium pulchellum (Sw.) Druce (CQ-217)
leaves; stems; flowers; roots
N EtOH 16 17 20 - 14 -
Erodium ciconium (L.) L’Hér. (CQ-142)
leaves; stems; flowers; roots
N EtOH 91 32 46 - 16 -
Erodium malacoides (L.) L’Hér. ex Aiton (CQ-121)
leaves; stems; flowers
N EtOH 91 32 46 - 16 -
Geraniaceae
Geranium columbinum L. (CQ-129)
leaves; stems; flowers
N EtOH 100 100 100 - 24 -
115
EtOH - 5 - - - - bulbs M; F dH2O 4 4 - - - 6
Hyacinthaceae Leopoldia comosa (L.) Parl. (CQ-105)
leaves; inflorescence N EtOH 5 43 56 - 7 - Hypericaceae Hypericum perforatum L.
(CQ-183) leaves; stems; flowers
S EtOH 100 100 99 9 29 9
immature fruits S; C EtOH - 17 - - 23 - leaves R EtOH - 19 27 - 39 -
Juglandaceae Juglans regia L. (CQ-181)
woody parts N EtOH 65 88 86 - 13 - Juncaceae Juncus articulatus L.
(CQ-216) leaves; fruits N EtOH - 16 33 - 5 -
stems S; M EtOH - 14 - - - - roots N EtOH - 13 88 - 22 23 leaves S; M EtOH 66 23 31 19 19 -
EtOH 11 3 33 6 25 -
Ballota nigra L. (CQ-160)
leaves; stems; flowers
S; M dH2O - - - - 3 -
Clinopodium vulgare L. (CQ-182)
leaves; stems; flowers
N EtOH 15 6 32 14 16 7
EtOH - 21 43 - 8 - leaves; stems; flowers
S; M dH2O - - - - - -
Marrubium vulgare L. (CQ-170)
roots N EtOH - 6 18 - - - Mentha pulegium L. (CQ-200)
leaves; stems; flowers; roots
F EtOH - - 12 - - 1
Mentha spicata L. (CQ-224)
leaves; stems; flowers
F EtOH - 13 34 - - -
Origanum heracleoticum L. (CQ-207)
leaves; stems; flowers
F EtOH 22 26 - - 7 -
Phlomis herba-venti L. (CQ-168)
leaves; stems; flowers
N EtOH - 21 20 - - -
Rosmarinus officinalis L. (CQ-113)
leaves; stems; flowers
F; S EtOH 56 74 99 - - 66
leaves; stems N EtOH 62 44 82 - - - Salvia pratensis L. (CQ-165) inflorescence N EtOH 32 27 48 - - - Salvia virgata Jacq. (CQ-127)
leaves; stems; flowers
N EtOH 82 33 97 77 43 100
Lamiaceae
Stachys tymphaea Hausskn. (CQ-189)
leaves; stems; flowers
N EtOH - 8 36 20
116
46 13
Liliaceae Lilium candidum L. leaves; stems N EtOH - - 27 - - -
116
(CQ-174) inflorescence N EtOH - 25 - - 7 - Alcea rosea L. (CQ-205)
leaves; stems; flowers; roots
O EtOH 66 12 65 - 32 3
EtOH 16 - 2 - - - stems S; M dH2O - 6 14 14 2 -
flowers S; M EtOH - - 65 - 28 27 EtOH 79 97 40 77 74 41
Malvaceae
Malva sylvestris L. (CQ-156)
leaves S; M dH2O 16 - - - - -
leaves N EtOH - 8 38 - 18 - woody parts N EtOH - 11 23 - - -
Moraceae Ficus carica L. (CQ-173)
immature fruits S; F EtOH 31 - 24 - 12 - Myrsinaceae Cyclamen hederifolium
Aiton (CQ-186)
tubers S EtOH - 19 2 - 8 -
Nyctaginaceae Mirabilis jalapa L. (CQ-222)
leaves; flowers; fruits N EtOH - 8 - - - -
leaves M EtOH 48 6 27 19
117
16 - Oleaceae Olea europaea L. (CQ-197) woody parts A EtOH - 35 8 - 11 - Aceras anthropophora R. Br. (CQ-153)
leaves; stems; flowers; roots
N EtOH 27 - 6 - - -
Orchis italica Poir. (CQ-133)
inflorescence; leaves; stems
N EtOH 15 - 19 - 7 -
Orchidaceae
Orchis purpurea Huds. (CQ-132)
inflorescence; leaves; stems
N EtOH 40 - 33 - 17 8
Fumaria officinalis L. (CQ-107)
leaves; stems; flowers; roots
N EtOH - 23 36 - 24 22
Papaver rhoeas subsp. rhoeas (CQ-145)
leaves; stems; flowers; roots
F EtOH - - 34 - 17 -
Papaveraceae
Papaver somniferum L. (CQ-178)
leaves; stems; flowers; roots
M; R EtOH - 14 35 7 22 -
Digitalis ferruginea L. (CQ-227)
leaves; stems; flowers
N EtOH - 7 - - - -
Linaria vulgaris Hill (CQ-223)
leaves; stems; flowers; roots
N EtOH - - 19 - - -
Plantaginaceae
Plantago major L. leaves; stems; S; M EtOH 7 - - - - -
117
(CQ-225) flowers; roots Agropyron repens (L.) P. Beauv. (CQ-208)
leaves; stems; roots M EtOH 54 23 40 - 29 21
stem internodes A; R EtOH - - - - - - EtOH - - 7 - - - stem nodes S dH2O 9 5 - - - 16
Poaceae
Arundo donax L. (CQ-146)
leaves; stems A; R EtOH - - 14 - 4 - Polygonaceae Rumex crispus L.
(CQ-171) leaves; stems; fruits S EtOH 43 87 28 - 8 -
Pottiaceae Syntrichia ruralis (Hedw.) Web. & Mohr (CQ-229)
whole plant N EtOH 22 3 33 - 18 -
Delphinium fissum Waldst. & Kit. (CQ-187)
leaves; stems; flowers; fruits
N EtOH - 15 48 31 29 39 Ranunculaceae
Ranunculus acris L. (CQ-135)
leaves; stems; flowers
N EtOH 7 - 25 -
118
- 15
Crataegus monogyna Jacq. (CQ-116)
leaves; stems; flowers
M EtOH - 17 - - - -
woody parts; leaves M EtOH - 15 - - - - Prunus spinosa L. (CQ-163) fruits N EtOH 11 46 59 36 36 47
fruits N EtOH - 3 - - - - woody parts N EtOH 18 24 30 - 5 -
Rosa canina var. canina (CQ-152)
leaves; stems N EtOH 73 100 100 - 67 69 leaves; stems; flowers
S EtOH 21 - 100 - 40 -
leaves S EtOH 10 10 96 - 52 - roots M EtOH 67 49 69 11 56 33
Rosaceae
Rubus ulmifolius Schott (CQ-164)
woody stems N EtOH - 77 74 11 36 53 Rubiaceae Galium verum L.
(CQ-177) leaves; stems; flowers
N EtOH 6 - 25 - - -
Verbascum sinuatum L. (CQ-218)
leaves; stems; flowers
N EtOH - 19 - - - -
stems M EtOH - - 2 - - - leaves M EtOH - 11 - - - -
Scrophularia-ceae
Verbascum thapsus L. (CQ-172)
inflorescence M EtOH 56 4 5 - - -
118
119
leaves N EtOH - - - - - - Ulmaceae Ulmus minor L. (CQ-195) woody parts M EtOH 25 13 24 1 18 - Parietaria diffusa Mert. & Koch (CQ-212)
leaves; stems; fruits; roots
M EtOH 10 30 8 - 37 22 Urticaceae
Urtica dioica L. (CQ-179)
leaves; stems; flowers
S; M; F EtOH 11 13 13 - 11 -
Valerianaceae Centranthus rubber (L.) DC. (CQ-143)
leaves; stems; inflorescence
M EtOH - 100 34 - 20 -
wine S; F 30 2 13 9 6 4 stems N EtOH - 7 63 - - - fruits F EtOH - 23 10 - - -
Vitaceae Vitis vinifera var. aglianico (CQ-209)
leaves N EtOH 11 8 17 - 18 -
**Strain ID: ATCC 25923 is a MSSA strain from the American Type Culture Collection (ATCC). ATCC 33593 is a MRSA strain
associated with high biofilm formation. The NRS isolates are all clinical MRSA strains from the Network on Antibiotic Resistant
Staphylococcus aureus (NARSA) repository. NRS 37 is also glycopeptide intermediate (GISA).
* Ethnobotanical use of specific plant part(s) in the study region: S = medicinal application to skin; M = medicinal application not
involving the skin; C = cosmetic applications; A = agricultural tool; O = ornamental; R = ritual or spiritual use; F = food; N = no
reported use.
“-“ signifies no inhibitory activity.
119
120
Table 2. Results from One-way ANOVA tests indicate that there was no significant
difference in the inhibition of planktonic growth (p > 0.05) between ethnobotanical usage
groups. There was, however, a significant difference in the inhibition of biofilm
formation and mammalian cytotoxicity (p< 0.005) between groups.
Df Sum of Sq Mean Sq F Value P Value
Planktonic Growth 2 960.1 480.0347 0.7320253 0.48121 Biofilm formation 2 2073.34 1036.669 7.159144 0.00104
Mammalian Cytotoxicity 2 2021.75 1010.875 5.863055 0.003464
Table 3. Effect of plant extracts on growth and biofilm formation in MRSA (ATCC 33593) and on mammalian cytotoxicity
(human T lymphoblastoid cell line). The MIC50 for growth was tested in the range of 8-512 μg/ml, whereas the IC50 for biofilm
formation and mammalian cytotoxicity was tested from 4-128 μg/ml due to issues with the percent excipient (DMSO) in the test
solution. A dash (-) represents that no IC50 was identified within the concentration range tested.
Family Botanic Name (Voucher ID)
Plant Part Ethno-botanical
Use*
Extract Solvent
**MIC50 (Growth)
**IC50 (Biofilm Formation)
**IC50 (Mammalian Cytotoxicity)
Therapeutic Index***
inflorescence N EtOH - - - - leaves S EtOH - - - -
Sambucus ebulus L. (CQ-168)
stems N EtOH - - - - woody parts R EtOH - - - -
EtOH 512 - - 1 leaves S dH2O - - - - EtOH - - - - inflorescence S; R dH2O - - - -
Adoxaceae
Sambucus nigra L. (CQ-151)
infructescence F EtOH - -
121
- - Alliaceae Allium cepa L.
(CQ-206) leaves; bulbs; roots
S; M; F EtOH - - - -
leaves; stems N EtOH - - - - Daucus carota L. (CQ-215) inflorescence;
infructescence N EtOH - - 128 -
Foeniculum vulgare ssp. piperitum (Ucria) Coutinho (CQ-192)
leaves; stems M; F EtOH - - - -
Foeniculum vulgare ssp. vulgare Mill. (CQ-196)
leaves; stems M EtOH - - - -
Apiaceae
Tordylium apulum L. (CQ-101)
flowers; leaves; roots; stems
N EtOH - - - -
Apocynaceae Vinca major L. (CQ-117)
flowers; leaves; roots; stems
M EtOH - - - -
121
stems N EtOH - - - - fruits N EtOH - - - - stalks N EtOH - - - -
Aracaeae Arum italicum Mill. (CQ-175)
leaves S EtOH - - - - inflorescence N EtOH - - - - Asphodelaceae Asphodelus microcarpus
Salzm. & Viv. (CQ-109)
leaves N EtOH 512 - - 1
Achillea ageratum L. (CQ-219)
leaves; stems; flowers
M EtOH 256 - - 1
inflorescence M EtOH - - 16 - leaves; stems M EtOH 512 - 32 16
Achillea millefolium L. (CQ-176)
leaves; stems; flowers
M EtOH - - 32 -
Anacyclus tomentosus DC. (CQ-167)
leaves; stems; flowers
N EtOH - - - -
EtOH 512 - - 1 basal leaves; roots
F dH2O - - - -
Cichorium intybus L. (CQ-106)
leaves; stems; flowers
F EtOH - - - -
EtOH 512 - - 1 Matricaria recutita L. (CQ-118)
flowers; leaves; roots; stems
S; M dH2O - - - -
Scolymus hispanicus L. (CQ-199)
leaves; stems; flowers
N EtOH - - - -
Tussilago farfara L. (CQ-202)
leaves; stems; roots
S EtOH - - - -
Asteraceae
Urospermum dalechampii (L.) Scop. (CQ-134)
flowers; leaves; roots; stems
N EtOH - -
122
- -
Anchusa officinalis L. (CQ-128)
leaves; stems; flowers
N EtOH - - - -
EtOH - - - - Borago officinalis L. (CQ-100)
flowers; leaves; roots; stems
M dH2O - - - -
Cerinthe major L. (CQ-110)
flowers; leaves; roots; stems
N EtOH - - - -
Boraginaceae
Echium italicum L. (CQ-162)
leaves; stems; flowers
N EtOH - - - -
Brassicaceae Brassica rapa subsp. rapa flowers; leaves; F EtOH - - - -
122
(CQ-104) roots; stems Cardaria draba (L.) Desv. (CQ-140)
flowers; leaves; roots; stems
N EtOH 512 - - 1
Eruca sativa Mill. (CQ-102)
flowers; leaves; roots; stems
N EtOH - - - -
Sisymbrium officinale (L.) Scop. (CQ-131)
flowers; leaves; roots; stems
N EtOH - - - -
woody parts N EtOH - 32 - - Caprifoliaceae Lonicera alpigena L. (CQ-213) leaves N EtOH - - - - Saponaria officinalis L. (CQ-210)
leaves; stems; flowers
N EtOH - - - -
Silene alba (Mill.) E.H.L. Krause (CQ-123)
leaves; stems; flowers
N EtOH - - - -
Caryophylla-ceae
Silene nutans L. (CQ-125)
leaves; stems; flowers
N EtOH - -
123
128 -
Cucurbitaceae Ecballium elaterium (L.) A. Richard (CQ-169)
leaves; stems; flowers
S EtOH 512 - 128 4
leaves N EtOH - - - - Dennstaedtia-ceae
Pteridium aquilinium (L.) Kuhn (CQ-211)
stems N EtOH - - - -
leaves; stems N EtOH - - - - Dipsacus fullonum L. (CQ-201) flowers N EtOH - - - - Knautia arvensis Coult. (CQ-190)
leaves; stems; flowers
N EtOH - - - -
Dipsacaceae
Knautia lucana Lacaita & Szabo (CQ-166)
leaves; stems; flowers
N EtOH - - - -
Equisetaceae Equisetum arvense L. (CQ-226)
stems; leaves N EtOH - - - -
inflorescence O EtOH - - - - stems O EtOH - - - -
Acacia dealbata Link (CQ-115)
leaves; stems O EtOH - - - -
Fabaceae
Anthyllis vulneraria L. (CQ-147)
leaves; stems; flowers
N EtOH - - - -
123
Astragalus monspessulanus L. (CQ-112)
leaves; stems; flowers; roots
N EtOH - - 128 -
leaves; flowers N EtOH - - - - Coronilla emerus L. (CQ-137) woody stems N EtOH - - - - Melilotus alba Medik. (CQ-193)
leaves; stems; flowers
N EtOH 512 - - 1
woody parts N EtOH - - - - leaves N EtOH 512 - - 1
Robinia pseudoacacia L. (CQ-155)
inflorescence N EtOH - - - - Spartium junceum L. (CQ-144)
leaves; stems; flowers
A EtOH - - - -
Trifolium repens L. (CQ-138)
leaves; stems; flowers; roots
N EtOH - - - -
Vicia craca L. (CQ-149)
leaves; stems; flowers; roots
N EtOH 512 - - 1
Vicia faba L. (CQ-103)
leaves; stems; flowers; roots
F EtOH -
124
- - -
Vicia sativa subsp. angustifolio (CQ-124)
leaves; stems; flowers
N EtOH 512 - - 1
Vicia sativa subsp. sativa (CQ-119)
leaves; stems; flowers
N EtOH - - - -
inflorescence O EtOH 512 - - 1 stems O EtOH - - - -
Wisteria sinensis (Sims) Sweet (CQ-126) leaves O EtOH 512 - - 1
inflorescence N EtOH 256 16 - 1 leaves N EtOH 256 - - 1
Castanea sativa Mill. (CQ-191)
woody parts A EtOH 512 - - 1 leaves N EtOH - - - -
Fagaceae
Quercus cerris L. (CQ-228) stems; fruits N EtOH - - - -
Gentianaceae Centaurium pulchellum (Sw.) Druce (CQ-217)
leaves; stems; flowers; roots
N EtOH - - - -
Geraniaceae Erodium ciconium (L.) L’Hér. (CQ-142)
leaves; stems; flowers; roots
N EtOH - - - -
124
Erodium malacoides (L.) L’Hér. ex Aiton (CQ-121)
leaves; stems; flowers
N EtOH 128 - 64 2
Geranium columbinum L. (CQ-129)
leaves; stems; flowers
N EtOH 512 - - 1
EtOH - 16 - - bulbs M; F dH2O - 8 - -
Hyacinthaceae Leopoldia comosa (L.) Parl. (CQ-105)
leaves; inflorescence
N EtOH - - - -
Hypericaceae Hypericum perforatum L. (CQ-183)
leaves; stems; flowers
S EtOH 256 128 - 1
immature fruits S; C EtOH - 16 - - leaves R EtOH - - - -
Juglandaceae Juglans regia L. (CQ-181)
woody parts N EtOH 512 - - 1 Juncaceae Juncus articulatus L.
(CQ-216) leaves; fruits N EtOH - - - -
stems S; M EtOH
125
- - - - roots N EtOH - - - - leaves S; M EtOH - - 128 -
EtOH - - - -
Ballota nigra L. (CQ-160)
leaves; stems; flowers
S; M dH2O - 8 - -
Clinopodium vulgare L. (CQ-182)
leaves; stems; flowers
N EtOH - - - -
EtOH - - - - leaves; stems; flowers
S; M dH2O - - - -
Marrubium vulgare L. (CQ-170)
roots N EtOH - - - - Mentha pulegium L. (CQ-200)
leaves; stems; flowers; roots
F EtOH - - - -
Mentha spicata L. (CQ-224)
leaves; stems; flowers
F EtOH - - - -
Origanum heracleoticum L. (CQ-207)
leaves; stems; flowers
F EtOH - - - -
Phlomis herba-venti L. (CQ-168)
leaves; stems; flowers
N EtOH - - - -
Rosmarinus officinalis L. (CQ-113)
leaves; stems; flowers
F; S EtOH 512 16 32 16
Lamiaceae
Salvia pratensis L. leaves; stems N EtOH 512 - - 1
125
(CQ-165) Salvia virgata Jacq. (CQ-127)
leaves; stems; flowers
N EtOH - - - -
Stachys tymphaea Hausskn. (CQ-189)
leaves; stems; flowers
N EtOH - - - -
leaves; stems N EtOH - - - - Liliaceae Lilium candidum L. (CQ-174) inflorescence N EtOH - - - - Alcea rosea L. (CQ-205)
leaves; stems; flowers; roots
O EtOH - - - -
EtOH - - - - stems S; M dH2O - 32 - -
flowers S; M EtOH - - - - EtOH 256 - 128 2
Malvaceae
Malva sylvestris L. (CQ-156)
leaves S; M dH2O - 32 - -
leaves N EtOH - - - - woody parts N EtOH - - - -
Moraceae Ficus carica L. (CQ-173)
immature fruits S; F EtOH
126
- - - - Myrsinaceae Cyclamen hederifolium Aiton
(CQ-186) tubers M EtOH - 8 32 -
Nyctaginaceae Mirabilis jalapa L. (CQ-222)
leaves; flowers; fruits
N EtOH - - - -
leaves N EtOH - - - - Oleaceae Olea europaea L. (CQ-197) woody parts A EtOH - - - - Aceras anthropophora R. Br. (CQ-153)
leaves; stems; flowers; roots
N EtOH - - - -
Orchis italica Poir. (CQ-133)
inflorescence; leaves; stems
N EtOH - - - -
Orchidaceae
Orchis purpurea Huds. (CQ-132)
inflorescence; leaves; stems
N EtOH - - - -
Fumaria officinalis L. (CQ-107)
leaves; stems; flowers; roots
N EtOH - - - -
Papaver rhoeas subsp. rhoeas (CQ-145)
leaves; stems; flowers; roots
F EtOH - - - -
Papaveraceae
Papaver somniferum L. (CQ-178)
leaves; stems; flowers; roots
M; R EtOH - - - -
126
Digitalis ferruginea L. (CQ-227)
leaves; stems; flowers
N EtOH - - - -
Linaria vulgaris Hill (CQ-223)
leaves; stems; flowers; roots
N EtOH - - - -
Plantaginaceae
Plantago major L. (CQ-225)
leaves; stems; flowers; roots
S; M EtOH - - - -
Agropyron repens (L.) P. Beauv. (CQ-208)
leaves; stems; roots
M EtOH - - - -
stem internodes A; R EtOH - - - - EtOH - - - - stem nodes S dH2O - 128 - -
Poaceae
Arundo donax L. (CQ-146)
leaves; stems A; R EtOH - - - - Polygonaceae Rumex crispus L.
(CQ-171) leaves; stems; fruits
S EtOH 512 - - 1
Pottiaceae Syntrichia ruralis (Hedw.) Web. & Mohr (CQ-229)
whole plant N EtOH
127
- - 128 -
Delphinium fissum Waldst. & Kit. (CQ-187)
leaves; stems; flowers; fruits
N EtOH - - 128 - Ranunculaceae
Ranunculus acris L. (CQ-135)
leaves; stems; flowers
N EtOH - - - -
Crataegus monogyna Jacq. (CQ-116)
leaves; stems; flowers
M EtOH - - - -
woody parts; leaves
M EtOH - - - - Prunus spinosa L. (CQ-163)
fruits N EtOH 512 - 128 4 fruits N EtOH - 32 - - woody parts N EtOH - - - -
Rosa canina var. canina (CQ-152)
leaves; stems N EtOH 512 - - 1 leaves; stems; flowers
S EtOH - - - -
leaves S EtOH - - - - roots M EtOH 512 8 - 1
Rosaceae
Rubus ulmifolius Schott (CQ-164)
woody stems N EtOH 512 - - 1 Rubiaceae Galium verum L. leaves; stems; N EtOH - - - -
127
128
(CQ-177) flowers Verbascum sinuatum L. (CQ-218)
leaves; stems; flowers
N EtOH - - - -
stems M EtOH - - - - leaves M EtOH - - - -
Scrophularia-ceae
Verbascum thapsus L. (CQ-172)
inflorescence M EtOH - - - - leaves N EtOH - - - - Ulmaceae Ulmus minor L.
(CQ-195) woody parts M EtOH - - - - Parietaria diffusa Mert. & Koch (CQ-212)
leaves; stems; fruits; roots
M EtOH - - - - Urticaceae
Urtica dioica L. (CQ-179)
leaves; stems; flowers
S; M; F EtOH - - - -
Valerianaceae Centranthus ruber (L.) DC. (CQ-143)
leaves; stems; inflorescence
M EtOH 256 - 64 4
wine S; F - 64 - - stems N EtOH - - - - fruits F EtOH - - - -
Vitaceae Vitis vinifera var. aglianico (CQ-209)
leaves N EtOH - - - - *Ethnobotanical use of specific plant part(s) in the study region: S = medicinal application to skin; M = medicinal application not
involving the skin; C = cosmetic applications; A = agricultural tool; O = ornamental; R = ritual or spiritual use; F = food; N = no
reported use.
** MIC50 and IC50 values expressed as μg/ml.
***Therapeutic index = Growth MIC50/Cytotoxicity IC50. Extracts that were not cytotoxic at a concentration of 128 μg/ml, but
which demonstrated MIC50 for growth in the range of 8-512 μg/ml were assigned a therapeutic index value of 1.
128
Table 4. Results from a multiple comparison analysis performed using the Bonferroni
method. A significant difference in activity of Groups 1 and 3 is indicated for the anti-
biofilm data. A significant difference in activity of Groups 2 and 3 is indicated for the
mammalian cytoxicity data.
Data Set Groups Compared Estimate Standard
Error Lower Bound
Upper Bound
1-2 2.13 3.25 -5.730 9.98 1-3 **** 8.15 2.36 2.460 13.90 Anti-Biofilm
Activity 2-3 6.03 2.75 -0.634 12.70 1-2 -7.97 3.55 -16.50 0.606 1-3 2.30 2.57 -3.91 8.520 Mammalian
Cytotoxicity 2-3**** 10.30 3.01 3.00 17.500
129
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137
CHAPTER IV
QUORUM SENSING INHIBITORS FOR METHICILLIN-RESISTANT
STAPHYLOCOCCUS AUREUS FROM ITALIAN MEDICINAL PLANTS
Abstract
Morbidity and mortality estimates due to methicillin-resistant Staphylococcus
aureus (MRSA) infections continue to rise. Therapeutic options are limited by antibiotic
resistance. In this study, I investigate the potential of plant extracts to inhibit the
pathways responsible for the production of staphylococcal virulence factors. This allows
for the mediation of staphylococcal pathogenesis without affecting growth, thus avoiding
selective forces for drug-resistance.
A cell-density dependent mechanism, known as quorum sensing (QS), is
responsible for controlling protein expression in Staphylococcus aureus. Staphylococcal
QS is encoded by the agr locus and is responsible for the production of virulence factors
such as the α-, ß-, and δ-hemolysins. δ-Hemolysin is a translational protein product of
RNAIII. Quantification of δ-hemolysin produced by S. aureus and found in the culture
supernatants allows for the analysis of agr activity at the translational, rather than
transcriptional, level. RP-HPLC techniques can be applied for the analysis of
staphylococcal culture filtrates.
I investigated the anti-QS activity of 168 crude ethanolic and aqueous extracts
from 104 Italian plants. Plants were categorized into 3 groups: medicinal plants used in
Italian folk remedies for skin and soft tissue infection (SSTI), medicinal plants with
applications unrelated to the skin, and plants with no ethnomedicinal application. Extracts
from four plants (Ballota nigra, Castanea sativa, Rosmarinus officinalis, and Sambucus
138
ebulus) exhibited a dose-dependent response (8-256 μg/ml) in the production of δ-
hemolysin, indicating strong anti-QS activity in MRSA. I found no significant difference
in mean QSI activity among the 3 ethnobotanical usage groups investigated.
Introduction
Infections due to methicillin resistant Staphylococcus aureus (MRSA) pose a
significant threat to hospital patients as the rates of nosocomial infection steadily rise
(Klevens et al., 2006). Moreover, healthcare-associated MRSA (HA-MRSA) are often
multidrug-resistant and therapeutic options are rapidly becoming more limited as new
resistant phenotypes surface (Stemper et al., 2006). One approach to drug discovery for
the treatment of MRSA is through natural products research. Most research on natural
botanic products activity for MRSA is focused on growth inhibition, while some have
focused on inhibition of the multidrug-resistant mechanisms, such as efflux pumps
(Gibbons et al., 2003a; Gibbons et al., 2003b; Gibbons and Udo, 2000; Pereda-Miranda et
al., 2006). No studies on the agr-inhibiting or quorum sensing inhibiting (QSI) activity of
natural botanic products on MRSA have been conducted thus far. Inhibition of
staphylococcal QS pathways could potentially limit the degree of pathogenicity posed by
some MRSA strains by blocking the production of certain virulence factors. Moreover,
the inhibition of staphylococcal pathogenesis could be accomplished without growth
inhibition, thus avoiding selection for drug-resistance.
The staphylococcal QS system is a cell-density-dependent mechanism for
controlling protein expression, including the production of staphylococcal virulence
factors such as the α-, ß-, and δ-hemolysins. It is encoded by the agr locus, which is a
139
quorum-sensing gene cluster of five genes (hld, agrA, agrB, agrC and agrD) (Sakoulas et
al., 2002).
Staphylococcal δ-hemolysin, or δ-toxin, is a translational protein product of
RNAIII. It is a 26-amino-acid polypeptide with surfactant-like properties (Otto and Götz
2000). Translation of hld, the gene for delta-hemolysin, occurs about one hour after
transcription of RNAIII. There are two forms of δ-toxin that can be found in the culture
supernatant: formylated (with an N-terminal methionine) and deformylated. These forms
are represented by two distinct peaks in the RP-HPLC chromatogram (Figure 1). δ-Toxin
accumulates in the culture medium in both forms, and is approximately 90% formylated
and 10% deformylated. This ratio is due to the arrest of deformylated δ-toxin production
during the post-exponential growth phase, whereas formylated δ-toxin continues to
accumulate. Sommerville et al. (2003) suggest that this change may be linked to iron
availability in the culture medium.
Quantification of δ-toxin produced by S. aureus and found in the culture
supernatants allows for the analysis of agr activity at the translational, rather than
transcriptional, level. The identification of agr-inhibiting drugs, or staphylococcal QS-
inhibitors, has been proposed by several research groups as a potential anti-
staphylococcal therapy (Balaban et al., 1998; Dell'Acqua et al., 2004; Mayville et al.,
1999; Otto, 2004; Otto et al., 1999). In 2000, Otto and Götz provided a fast method for δ-
toxin quantification using RP-HPLC techniques for the analysis of staphylococcal culture
filtrates. I apply this method for the first time as a screening tool for identifying plant
extracts with QSI activity for a strain of HA-MRSA known as pulsed-field type (PFT)
USA500.
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USA500 isolates are SCCmecIV and MLST ST8. They are highly multidrug-
resistant (Table 1) and tend to be associated with nosocomial transmission (McDougal et
al., 2003; NARSA, 2007). USA500 is associated with the production of many virulence
factors, including enterotoxins A and B, as well as delta-hemolysin, among others. There
is a critical need for novel therapeutic options in the treatment of highly virulent, MDR
staphylococcal infection, such as those caused by USA500.
In this study, I quantify the amount of δ-hemolysin found in the supernatant of
MRSA cultures treated with plant extracts as a means of measuring the impact of plant
products on the staphylococcal quorum sensing (QS) system. I examine 168 crude
extracts made from 104 Italian plants, representing 44 plant families. Plants were selected
and assigned to one of three groups based on ethnobotanical usage status: group 1
included medicinal plants used in folk remedies for skin and soft tissue infection (SSTI);
group 2 included medicinal plants used in treatments unrelated to the skin; and group 3
included plants with no cited ethnomedicinal application. My hypothesis was that plants
in group 1 would demonstrate a higher mean percent inhibition of δ-hemolysin
production than the other two groups.
Materials and Methods
Plant material and extraction. An ethnobotanical survey of medicinal plants used in the
traditional treatment of skin and soft tissue infections (SSTIs) in the Vulture-Alto
Bradano region of Basilicata, southern Italy was conducted and results are reported in
Chapter 2. Voucher specimens of plants were deposited at the Herbarium Lucanum,
141
Univeristà degli Studi della Basilicata (HLUC) in Potenza, Italy and Fairchild Tropical
Botanic Gardens (FTG) in Miami, FL, USA.
I ground dry plant materials into a fine powder using a homogenizer. I made
ethanolic extracts of all plant samples by soaking in 95% EtOH using a ratio of 1g (plant
material):10 ml (EtOH) for 72 h. Flasks were agitated daily. I made water extracts by
boiling 1g (plant material): 50 ml (dH2O) for 30 minutes. Extracts were vacuum filtered
and rotary-evaporated, then frozen and lyophilized. Stock concentrations of 10 mg/ml of
dry extract in the carrier solvent (DMSO or dH2O) were prepared, sterile filtered (0.2 μm)
and stored in the dark at 4ºC. The excipient (DMSO or dH2O) made up less than 5.1% of
the final test solution for MIC assays and less than 2.5% for δ-toxin assays.
Bacteria and culture conditions. HA-MRSA PFT USA500 (NRS385) was obtained
from the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA)
repository. Bacteria were grown on Tryptic Soy agar plates for 18 h at 37°C. A 0.5
McFarland Standard was used to create inoculum densities of 1.5 x 108 cfu/ml in PBS
using the direct suspension method (Isenberg, 2004) for MIC and δ-toxin assays.
Determination of minimum inhibitory concentrations (MICs). MICs were determined
by the microtiter broth method (Amsterdam, 1996) in sterile flat-bottom 96-well
polystyrene plates. I used serial dilution techniques to determine the MIC50 and MIC90 of
extracts at concentrations of 8-512 μg/ml after 18 h growth. I included negative (growth)
controls (cells + TSB), positive controls (cells + TSB + antibiotics – vancomycin,
ampicillin, and trimethroprim-sulfamethoxazole), vehicle controls (cells + TSB +
142
DMSO), and media controls (TSB). All tests were performed in triplicate. I took optical
density readings using a KC4 microplate reader at 600 nm at 0 and 18 hours post-
inoculation. Results are reported as the MIC for growth at 18 hours post-inoculation. To
account for the effect of extract color on the OD600nm reading, a formula for calculating
percent inhibition was used. The mean % inhibition of replicate tests was used to
determine the final MIC values.
% inhibition = 100018
0181 xODgcODgcODtODt
⎟⎟⎠
⎞⎜⎜⎝
⎛⎟⎟⎠
⎞⎜⎜⎝
⎛−−
−
ODt18 = optical density (600 nm) of the test well at 18 hours post-inoculation
ODt0 = optical density (600 nm) of the test well at 0 hours post-inoculation
ODgc18 = optical density (600 nm) of the growth control well at 18 hours post-
inoculation
ODgc0 = optical density (600 nm) of the growth control well at 0 hours post-inoculation
Quantification of δ-toxin production. I prepared polystyrene 24-well culture plates with
a total volume of 1 ml per well of TSB, an initial sub-MIC test concentration of 64 μg of
extract suspended in DMSO (<1% DMSO in total well volume) and a 0.5 McFarland
standard of the overnight culture of S. aureus prepared in phosphate buffered saline (1X).
Extracts demonstrating significant activity, as exhibited by lower δ-toxin levels, were
also investigated at a range of test concentrations of 8-256 μg/ml. Controls for media,
growth, and growth in the carrier solvent (DMSO) were also performed. Liquid test
cultures were grown for 15 hours at 37°C and aerated by shaking at 150 rpm. All tests
were performed in triplicate.
143
Alliquots of bacteria (2 ml) were centrifuged for 5 min at 14,000 x g with a
microcentrifuge. Supernatants were removed and stored at -20 °C until HPLC analysis.
The concentration of δ-toxin was measured by RP-HPLC with a 1-ml Resource PHE
column (GE Healthcare, Uppsala, Sweden) as previously described (Otto and Götz 2000),
except that 200 μl of supernatant (instead of 500 μl) was injected onto the column using a
Thermo Spectra-System HPLC apparatus, equipped with a Diode Array Detector and
autosampler (Thermo Electron Corporation, San Jose, CA) and ChromQuest 4.1
software.
δ-Hemolysin elutes at a retention time of about 6.4 minutes (deformylated) and
6.8 minutes (formylated) after sample injection as two distinct peaks. Integration of the δ-
toxin peak area was performed at 280 nm. I confirmed the identity of δ-toxin peaks by
peak fractionation and LC-mass spectrometry (Figure 1) using a Thermo Finnigan Deca
XP max ion trap mass spectrometer and surveyor LC with autosampler and diode array
detector (Thermo Electron Corporation, San Jose, CA) using conditions previously
described (Somerville et al., 2003). The peak areas were calculated using ChromQuest
software and the mean % inhibition of δ-toxin production for the replicate tests was
calculated in relation to the mean peak area of the carrier (DMSO) growth controls.
Statistical analysis. All experiments were carried out in triplicate. I analyzed data using
Microsoft Excel and SPLUS software. Differences between the means of the
experimental and control groups were evaluated by a One-Way ANOVA using SPLUS
software.
144
Results
There was a broad low level response of δ-toxin inhibition to the screening test
concentration of 64 μg/ml. QSI activity was apparent in 90% of the extracts tested (Table
2). The mean inhibition of δ-toxin production ranged from 24.32-27.29% for the three
test groups (Table 3). No QSI activity was apparent in aqueous extracts. This suggests
that the active QSI components are predominantly nonpolar in nature. Extracts were not
effective at inhibiting growth of this multidrug-resistant strain of HA-MRSA
(USA500/NRS385). Only 6% of extracts demonstrated a MIC50 at concentrations of 256-
512 μg/ml. None demonstrated a MIC90 at concentrations ≤ 512 μg/ml.
I conducted a One-Way ANOVA to test the null hypothesis that the percent
inhibition of δ-toxin production would be the same for the three plant test groups. The p-
value was > 0.05, thus, I failed to reject the null hypothesis (Table 4). There was no
significant difference in mean agr-activity between different ethnobotanical usage groups
(Figure 2).
Four ethanolic extracts demonstrated significant δ-toxin inhibition, and come
from the following plant species: Ballota nigra, Castanea sativa, Rosmarinus officinalis,
and Sambucus ebulus. Interestingly, each of these species is applied in south Italian folk
remedies for SSTI (see Chapter 2). The HPLC chromatograms and graphs of the percent
inhibition of δ-toxin (by measure of peak area) for these species demonstrate a strong
dose-dependent response (Figures 3 and 4).
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Discussion
Quantification of δ-hemolysin in the supernatant of staphylococcal cultures can be
used as a measure of agr system, or QS, activity (Otto, 2004; Otto and Götz 2000;
Somerville et al., 2003). The agr system controls approximately 150 genes and is critical
to S. aureus virulence (Dunman et al., 2001). While the staphylococcal QS system is a
useful target for the discovery and development of new anti-pathogenic drugs, the
dynamic nature of the agr system must not be ignored. A better understanding of the
effect that agr manipulation can have on the development of infection in vivo is
necessary. For example, inhibiting agr activity during certain times in the infection
process can lead to deleterious effects, such as increased biofilm formation (Harraghy et
al., 2007). In this study, I found that several extracts with high inhibition of δ-toxin also
exhibited a two- to eleven-fold increase in biofilm production.
Based on analyses of δ-hemolysin production, I have offered the first reports of
plant extracts interfering with QS pathways in MRSA. These results indicate that some
degree of QSI activity is evident in 90% of the 168 Italian plant extracts screened,
including those extracts with no growth inhibitory activity.
Plant-based therapies that do not exhibit activity in the standard in vitro
bacteriostatic or bacteriocidal tests are oftentimes dismissed as nonvalid treatments.
However, these data demonstrate that other mechanisms of action may be in play, which
do not necessarily impact bacterial growth, but virulence mechanisms (and thus
pathogenicity), instead. These data give validity to the use of south Italian folk remedies
incorporating Ballota nigra, Castanea sativa, Rosmarinus officinalis, and Sambucus
146
ebulus for the treatment of SSTI. Further investigation, including the fractionation and
isolation of active components from these four species is recommended.
Acknowledgements
This work was funded by NIH/NCCAM 1F31AT004288-01A1 (PI: C.L. Quave),
MBRS RISE - NIH/NIGMS R25GM061347, Botany in Action, Anne Chatham
Fellowship in Medicinal Botany, USDA CSREES 20053842215940 and NIH/NCAAM
1T32AT01060-01 (PI: B.C. Bennett). I thank Dr. Carmine Colacino of the Herbarium
Lucanum in Potenza, Italy for assisting in the taxonomic identification of plants collected.
I also thank Dr. Horacio Preistap and Myron Georgiadis for technical support in the use
of HPLC and MS equipment, respectively.
147
(a)
(b)
148
(c) Figure 1. Mass spectroscopic analysis of HPLC fractions containing derformylated and
formylated δ-toxin. Peaks matching the spectrogram presented in the study by Somerville
et al. (2003) are highlighted. (a) Absorbance at 280nm of NRS385 (PFT USA500)
supernatant fractionated by HPLC. (b) Mass spectrogram of peak 1, deformylated δ-toxin
(molecular mass 2979.2 Da). (c) Mass spectrogram of peak 2, formylated δ-toxin
(molecular mass of 3007.4 Da).
149
0
10
20
30
40
50
60
70
80
90
100
Perc
ent I
nhib
ition
Group 3Group 2Group 1 Figure 2. Comparison of mean quorum sensing inhibition of plant extracts from different
ethnobotanical groups. Group 1: plants used for SSTI; Group 2: plants used for medicinal
purposes not involving the skin; Group 3: plants with no medicinal application.
150
(a)
(b)
151
(c)
(d) Figure 3. HPLC chromatogram of δ-toxin after treatment with different concentrations of
plant extract. (a) EtOH extract of Ballota nigra stems. (b) EtOH extract of Castanea
sativa leaves. (c) EtOH extract of Rosmarinus officinalis leaves, stems, and flowers. (d)
EtOH extract of Sambucus ebulus leaves.
152
0102030405060708090
100
8 16 32 64 128 256Concentration (μg/ml)
% In
hibi
tion
(a)
0102030405060708090
100
8 16 32 64 128Concentration (μg/ml)
% In
hibi
tion
(b)
153
0102030405060708090
100
8 16 32 64 128 256Concentration (μg/ml)
% In
hibi
tion
(c)
0102030405060708090
100
8 16 32 64 128 256Concentration (μg/ml)
% In
hibi
tion
(d)
Figure 4. Percent inhibition of δ-toxin peak area after treatment with different
concentrations of plant extract. (a) EtOH extract of Ballota nigra stems. (b) EtOH extract
of Castanea sativa leaves. (c) EtOH extract of Rosmarinus officinalis leaves, stems, and
flowers. (d) EtOH extract of Sambucus ebulus leaves.
154
155
Table 1. Antibiogram for MRSA PFT USA500/NRS385 (NARSA, 2007).
Antibiotic Drug Sensitivity
Ampicillin R Ampicillin/Sublactam R Cefazolin R Ciprofloxacin R Clindamycin R Erythromycin R Gentamicin R Linezolid S Levofloxacin R Nitrofurantoin S Oxacillin R Penicillin-G R Quinupristin/dalfopristin R Rifampin S Teicoplanin S Tetracycline R Trimethoprim/Sulfamethoxazole R Vancomycin S
Table 2. Inhibition of δ-toxin and minimal inhibitory concentrations of plant extracts against MRSA (strain I.D. NRS385/PFT
USA500).
Family
Botanic Name (Voucher ID) Plant Part
Ethno-botanical
Use*
Extract Solvent
% Inhibition of δ-toxin
Production**MIC50
***
inflorescence N EtOH 45 - leaves S EtOH 48 -
Sambucus ebulus L. (CQ-168)
stems N EtOH 28 - woody parts R EtOH 29 -
EtOH 36 - leaves S dH2O - - EtOH 38 - inflorescence S; R dH2O - -
Adoxaceae
Sambucus nigra L. (CQ-151)
infructescence F EtOH 34 - Alliaceae Allium cepa L.
(CQ-206) leaves; bulbs; roots S; M; F EtOH 22 -
leaves; stems N EtOH 2 - Daucus carota L. (CQ-215) inflorescence;
infructescence N EtOH 39 -
Foeniculum vulgare ssp. piperitum (Ucria) Coutinho (CQ-192)
leaves; stems M; F EtOH - -
Foeniculum vulgare ssp. vulgare Mill. (CQ-196)
leaves; stems M EtOH 8 -
Apiaceae
Tordylium apulum L. (CQ-101)
flowers; leaves; roots; stems
N EtOH 25 -
Apocynaceae Vinca major L. (CQ-117)
flowers; leaves; roots; stems
M EtOH 26 -
stems N EtOH 28 - fruits N EtOH 15 - stalks N EtOH 2 -
Aracaeae Arum italicum Mill. (CQ-175)
leaves S EtOH 22 - Asphodelaceae Asphodelus microcarpus Salzm. & Viv.
(CQ-109) inflorescence N EtOH 19 -
156
140
leaves N EtOH 17 - Achillea ageratum L. (CQ-219)
leaves; stems; flowers M EtOH 66 512
inflorescence M EtOH 41 - leaves; stems M EtOH 23 -
Achillea millefolium L. (CQ-176)
leaves; stems; flowers M EtOH 38 - Anacyclus tomentosus DC. (CQ-167)
leaves; stems; flowers N EtOH 36 -
EtOH 23 - basal leaves; roots F dH2O - -
Cichorium intybus L. (CQ-106)
leaves; stems; flowers F EtOH 8 - EtOH 29 512 Matricaria recutita L.
(CQ-118) flowers; leaves; roots; stems
S; M dH2O - -
Scolymus hispanicus L. (CQ-199)
leaves; stems; flowers N EtOH 23 -
Tussilago farfara L. (CQ-202)
leaves; stems; roots S EtOH 16 -
Asteraceae
Urospermum dalechampii (L.) Scop. (CQ-134)
flowers; leaves; roots; stems
N EtOH 14 -
Anchusa officinalis L. (CQ-128)
leaves; stems; flowers N EtOH 34 -
EtOH 54 - Borago officinalis L. (CQ-100)
flowers; leaves; roots; stems
M dH2O - -
Cerinthe major L. (CQ-110)
flowers; leaves; roots; stems
N EtOH 48 -
Boraginaceae
Echium italicum L. (CQ-162)
leaves; stems; flowers N EtOH 32 -
Brassica rapa subsp. rapa (CQ-104)
flowers; leaves; roots; stems
F EtOH 27 -
Cardaria draba (L.) Desv. (CQ-140)
flowers; leaves; roots; stems
N EtOH 12 -
Eruca sativa Mill. (CQ-102)
flowers; leaves; roots; stems
N EtOH 13 -
Brassicaceae
Sisymbrium officinale (L.) Scop. (CQ-131)
flowers; leaves; roots; stems
N EtOH 20 -
woody parts N EtOH 28 - Caprifoliaceae Lonicera alpigena L. (CQ-213) leaves N EtOH 25 -
157
141
Saponaria officinalis L. (CQ-210)
leaves; stems; flowers N EtOH 4 -
Silene alba (Mill.) E.H.L. Krause (CQ-123)
leaves; stems; flowers N EtOH 43 -
Caryophyllaceae
Silene nutans L. (CQ-125)
leaves; stems; flowers N EtOH 43 -
Cucurbitaceae Ecballium elaterium (L.) A. Richard (CQ-169)
leaves; stems; flowers S EtOH 21 -
leaves N EtOH - - Dennstaedtiaceae Pteridium aquilinium (L.) Kuhn (CQ-211) stems N EtOH 24 -
leaves; stems N EtOH 28 - Dipsacus fullonum L. (CQ-201) flowers N EtOH 28 - Knautia arvensis Coult. (CQ-190)
leaves; stems; flowers N EtOH 48 -
Dipsacaceae
Knautia lucana Lacaita & Szabo (CQ-166)
leaves; stems; flowers N EtOH 6 -
Equisetaceae Equisetum arvense L. (CQ-226)
stems; leaves N EtOH 22 -
inflorescence O EtOH 56 - stems O EtOH 38 -
Acacia dealbata Link (CQ-115)
leaves; stems O EtOH 21 - Anthyllis vulneraria L. (CQ-147)
leaves; stems; flowers N EtOH 28 -
Astragalus monspessulanus L. (CQ-112)
leaves; stems; flowers; roots
N EtOH 36 -
leaves; flowers N EtOH 33 - Coronilla emerus L. (CQ-137) woody stems N EtOH 14 - Melilotus alba Medik. (CQ-193)
leaves; stems; flowers N EtOH 43 -
woody parts N EtOH 32 - leaves N EtOH - -
Robinia pseudoacacia L. (CQ-155)
inflorescence N EtOH 21 - Spartium junceum L. (CQ-144)
leaves; stems; flowers A EtOH 22 -
Trifolium repens L. (CQ-138)
leaves; stems; flowers; roots
N EtOH 4 -
Fabaceae
Vicia craca L. leaves; stems; flowers; N EtOH 19 -
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142
(CQ-149) roots Vicia faba L. (CQ-103)
leaves; stems; flowers; roots
F EtOH 14 -
Vicia sativa subsp. angustifolio (CQ-124)
leaves; stems; flowers N EtOH 22 512
Vicia sativa subsp. sativa (CQ-119)
leaves; stems; flowers N EtOH 29 -
inflorescence O EtOH 36 - stems O EtOH 39 -
Wisteria sinensis (Sims) Sweet (CQ-126)
leaves O EtOH 41 - inflorescence N EtOH 20 - leaves N EtOH 70 512
Castanea sativa Mill. (CQ-191)
woody parts A EtOH 32 512 leaves N EtOH 27 -
Fagaceae
Quercus cerris L. (CQ-228) stems; fruits N EtOH 37 -
Gentianaceae Centaurium pulchellum (Sw.) Druce (CQ-217)
leaves; stems; flowers; roots
N EtOH 21 -
Erodium ciconium (L.) L’Hér. (CQ-142)
leaves; stems; flowers; roots
N EtOH 34 -
Erodium malacoides (L.) L’Hér. ex Aiton (CQ-121)
leaves; stems; flowers N EtOH 7 512
Geraniaceae
Geranium columbinum L. (CQ-129)
leaves; stems; flowers N EtOH - -
EtOH 21 - bulbs M; F dH2O - -
Hyacinthaceae Leopoldia comosa (L.) Parl. (CQ-105)
leaves; inflorescence N EtOH 31 - Hypericaceae Hypericum perforatum L.
(CQ-183) leaves; stems; flowers S EtOH 36 -
immature fruits S; C EtOH - - leaves R EtOH 39 -
Juglandaceae Juglans regia L. (CQ-181)
woody parts N EtOH 17 - Juncaceae Juncus articulatus L.
(CQ-216) leaves; fruits N EtOH 32 -
stems S; M EtOH 76 - roots N EtOH 37 -
Lamiaceae Ballota nigra L. (CQ-160)
leaves S; M EtOH 47 -
159
143
EtOH 47 - leaves; stems; flowers S; M dH2O - -
Clinopodium vulgare L. (CQ-182)
leaves; stems; flowers N EtOH 40 -
EtOH 40 - leaves; stems; flowers S; M dH2O 6 -
Marrubium vulgare L. (CQ-170)
roots N EtOH 40 - Mentha pulegium L. (CQ-200)
leaves; stems; flowers; roots
F EtOH 36 -
Mentha spicata L. (CQ-224) leaves; stems; flowers F EtOH 28 - Origanum heracleoticum L. (CQ-207)
leaves; stems; flowers F EtOH 30 -
Phlomis herba-venti L. (CQ-168)
leaves; stems; flowers N EtOH 16 -
Rosmarinus officinalis L. (CQ-113)
leaves; stems; flowers F; S EtOH 58 256
leaves; stems N EtOH 23 - Salvia pratensis L. (CQ-165) inflorescence N EtOH 58 - Salvia virgata Jacq. (CQ-127)
leaves; stems; flowers N EtOH 42 256
Stachys tymphaea Hausskn. (CQ-189)
leaves; stems; flowers N EtOH 41 -
leaves; stems N EtOH 37 - Liliaceae Lilium candidum L. (CQ-174) inflorescence N EtOH 30 - Alcea rosea L. (CQ-205)
leaves; stems; flowers; roots
O EtOH 18 -
EtOH 22 - stems S; M dH2O - -
flowers S; M EtOH 53 - EtOH 34 -
Malvaceae
Malva sylvestris L. (CQ-156)
leaves S; M dH2O - -
leaves N EtOH 24 - woody parts N EtOH 21 -
Moraceae Ficus carica L. (CQ-173)
immature fruits S; F EtOH 41 - Myrsinaceae Cyclamen hederifolium Aiton
(CQ-186) tubers M EtOH 10 -
160
144
Nyctaginaceae Mirabilis jalapa L. (CQ-222)
leaves; flowers; fruits N EtOH 16 -
leaves N EtOH 40 - Oleaceae Olea europaea L. (CQ-197) woody parts A EtOH 24 - Aceras anthropophora R. Br. (CQ-153)
leaves; stems; flowers; roots
N EtOH 51 -
Orchis italica Poir. (CQ-133)
inflorescence; leaves; stems
N EtOH 49 -
Orchidaceae
Orchis purpurea Huds. (CQ-132)
inflorescence; leaves; stems
N EtOH 57 -
Fumaria officinalis L. (CQ-107)
leaves; stems; flowers; roots
N EtOH 44 -
Papaver rhoeas subsp. rhoeas (CQ-145)
leaves; stems; flowers; roots
F EtOH 25 -
Papaveraceae
Papaver somniferum L. (CQ-178)
leaves; stems; flowers; roots
M; R EtOH 36 -
Digitalis ferruginea L. (CQ-227)
leaves; stems; flowers N EtOH 32 -
Linaria vulgaris Hill (CQ-223)
leaves; stems; flowers; roots
N EtOH 25 -
Plantaginaceae
Plantago major L. (CQ-225)
leaves; stems; flowers; roots
S; M EtOH 12 -
Agropyron repens (L.) P. Beauv. (CQ-208)
leaves; stems; roots M EtOH 19 -
stem internodes A; R EtOH 22 - EtOH 35 - stem nodes S dH2O - -
Poaceae
Arundo donax L. (CQ-146)
leaves; stems A; R EtOH 10 - Polygonaceae Rumex crispus L.
(CQ-171) leaves; stems; fruits S EtOH 25 -
Pottiaceae Syntrichia ruralis (Hedw.) Web. & Mohr (CQ-229)
whole plant N EtOH 45 -
Delphinium fissum Waldst. & Kit. (CQ-187)
leaves; stems; flowers; fruits
N EtOH 29 - Ranunculaceae
Ranunculus acris L. (CQ-135)
leaves; stems; flowers N EtOH 34 -
161
145
Crataegus monogyna Jacq. (CQ-116)
leaves; stems; flowers M EtOH 57 -
woody parts; leaves M EtOH 33 - Prunus spinosa L. (CQ-163) fruits N EtOH 29 512
fruits N EtOH 16 - woody parts N EtOH 44 -
Rosa canina var. canina (CQ-152)
leaves; stems N EtOH 14 512 leaves; stems; flowers S EtOH 10 - leaves S EtOH 17 - roots M EtOH 21 -
Rosaceae
Rubus ulmifolius Schott (CQ-164)
woody stems N EtOH 16 512 Rubiaceae Galium verum L.
(CQ-177) leaves; stems; flowers N EtOH 27 -
Verbascum sinuatum L. (CQ-218)
leaves; stems; flowers N EtOH 34 -
stems M EtOH 31 - leaves M EtOH 40 -
Scrophulariaceae
Verbascum thapsus L. (CQ-172)
inflorescence M EtOH 38 - leaves N EtOH 23 - Ulmaceae Ulmus minor L.
(CQ-195) woody parts M EtOH - - Parietaria diffusa Mert. & Koch (CQ-212)
leaves; stems; fruits; roots
M EtOH 19 - Urticaceae
Urtica dioica L. (CQ-179)
leaves; stems; flowers S; M; F EtOH 36 -
Valerianaceae Centranthus ruber (L.) DC. (CQ-143)
leaves; stems; inflorescence
M EtOH 31 -
wine S; F - - stems N EtOH - - fruits F EtOH 21 -
Vitaceae Vitis vinifera var. aglianico (CQ-209)
leaves N EtOH 22 - * Ethnobotanical use of specific plant part(s) in the study region: S = medicinal application to skin; M = medicinal application not involving the skin; C = cosmetic applications; A = agricultural tool; O = ornamental; R = ritual or spiritual use; F = food; N = no reported use. ** percent inhibition for δ-toxin based on an initial screening concentration of 64 μg/ml for MRSA PFT USA500.
146
*** MIC values are reported as μg/ml for MRSA PFT USA500. “-“ signifies no inhibitory activity.
162
Table 3. Summary statistics on δ-toxin inhibition for each ethnobotanical usage group.
Inhibition of δ-
Toxin Production *Group 1 2 3 Minimum 0 0 0 1st Quarter 1.5 14.5 19 Mean 24.32 26.60 27.29Median 22 26 28 3rd Quarter 37 38 36.25Maximum 76 66 70 Total N 34 23 112 Standard Deviation 20.40 18.69 13.86
*Group 1: plants used in the traditional treatment of SSTIs; Group 2: plants used in the
traditional treatment of disease other than those pertaining to the skin; Group 3: plants
without any quoted medicinal use.
Table 4. Results from One-way ANOVA tests indicate that there was no significant
difference in the inhibition of quorum sensing (p > 0.05) between ethnobotanical usage
groups.
Df Sum of Sq Mean Sq F Value P Value 2 230.24 115.1208 0.4470377 0.6402874
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References Amsterdam, D., 1996. Susceptibility testing of antimicrobials in liquid media. In: V.
Loman (Ed.), Antibiotics in laboratory medicine, Williams and Wilkins, Baltimore, MD, pp. 52-111.
Balaban, N., Goldkorn, T., Nhan, R.T., Dang, L.B., Scott, S., Ridgley, R.M., Rasooly, A., Wright, S.C. and Carlson, J.R., 1998. Autoinducer of virulence as a target for vaccine and therapy against Staphylococcus aureus. Science 280, 438-440.
Dell'Acqua, G., Giacometti, A., Cirioni, O., Ghiselli, R., Saba, V., Scalise, G., Gov, Y. and Balaban, N., 2004. Suppression of drug-resistant staphylococcal infections by the quorum-sensing inhibitor RNAIII-inhibiting peptide. The Journal of Infectious Diseases 190, 318-320.
Dunman, P.M., Murphy, E., Haney, S., Palacios, D., Tucker-Kellogg, G., Wu, S., Brown, E.L., Zagursky, R.J., Shlaes, D. and Projan, S.J., 2001. Transcription profiling-based identification of Staphylococcus aureus genes regulated by the agr and/or sarA loci. Journal of Bacteriology 183, 7341-7353.
Gibbons, S., Oluwatuyi, M. and Kaatz, G.W., 2003a. A novel inhibitor of multidrug efflux pumps in Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 51, 13-17.
Gibbons, S., Oluwatuyi, M., Veitch, N.C. and Gray, A.I., 2003b. Bacterial resistance modifying agents from Lycopus europaeus. Phytochemistry 62, 83-87.
Gibbons, S. and Udo, E.E., 2000. The effect of reserpine, a modulator of multidrug efflux pumps, on the in vitro activity of tetracycline against clinical isolates of methicillin resistant Staphylococcus aureus (MRSA) possessing the tet(K) determinant. Phytotherapy Research 14, 139-140.
Harraghy, N., Kerdudou, S. and Herrmann, M., 2007. Quorum-sensing systems in staphylococci as therapeutic targets. Analytical and Bioanalytical Cehmistry 387, 437-444.
Isenberg, H.D., 2004. Clinical Microbiology Procedures Handbook. ASM Press, Washington, D.C.
Klevens, R.M., Edwards, J.R., Tenover, F.C., McDonald, L.C., Horan, T. and Gaynes, R., 2006. Changes in the epidemiology of methicillin-resistant Staphylococcus aureus in intensive care units in US hospitals, 1992-2003. Clinical Infectious Diseases 43, 387-388.
Mayville, P., Ji, G., Beavis, R., Yang, H., Goger, M., Novick, R.P. and Muir, T.W., 1999. Structure-activity analysis of synthetic autoinducing thiolactone peptides from
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Staphylococcus aureus responsible for virulence. Proceedings of the National Academy of Sciences of the United States of America 96, 1218-1223.
McDougal, L.K., Steward, C.D., Killgore, G.E., Chaitram, J., McAllister, S.K. and Tenover, F.C., 2003. Pulsed-field gel electrophoresis typing of oxacillin-resistant Staphylococcus aureus isolates from the United States: Establishing a national database. Journal of Clinical Microbiology 41, 5113-5120.
NARSA, 2007. http://www.narsa.net. The Network on Atnibiotic Resistance in Staphylococcus aureus.
Otto, M., 2004. Quorum-sensing control in Staphylococci - a target for antimicrobial drug therapy? FEMS Microbiology Letters 241, 135-141.
Otto, M. and Götz , F., 2000. Analysis of quorum sensing activity in staphylococci by RP-HPLC of staphylococcal delta-toxin. BioTechniques 28, 1088-1096.
Otto, M., Submuth, R., Vuong, C., Jung, G. and Gotz, F., 1999. Inhibition of virulence factor expression in Staphylococcus aureus by the Staphylococcus epidermidis agr pheromone and derivatives. FEBS Letters 450, 257-262.
Pereda-Miranda, R., Kaatz, G.W. and Gibbons, S., 2006. Polyacylated oligosaccharides from medicinal Mexican morning glory species as antibacterials and inhibitors of multidrug resistamce in Staphylococcus aureus. Journal of Natural Products 69, 406-409.
Sakoulas, G., Eliopoulos, G.M., Moellering, R.C., Wennersten, C., Venkataraman, L., Novick, R.P. and Gold, H.S., 2002. Accessory gene regulator (agr) locus in geographically diverse Staphylococcus aureus isolates with reduced susceptibility to Vancomycin. Antimicrobial Agents and Chemotherapy 46, 1492-1502.
Somerville, G.A., Cockayne, A., Durr, M., Peschel, A., Otto, M. and Musser, J.M., 2003. Synthesis and deformylation of Staphylococcus aureus δ-Toxin are linked to Tricarboxylic Acid Cycle Activity Journal of Bacteriology 185, 6686-6694.
Stemper, M.E., Brady, J.M., Qutaishat, S.S., Borlaug, G., Reed, J., Reed, K.D. and Shukla, S.K., 2006. Shift in Staphylococcus aureus clone linked to an infected tattoo. Emerging Infectious Diseases 12, http://www.cdc.gov/ncidod/EID/vol12no09/05-1634.htm.
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CHAPTER V
SUMMARY
The specific research aims of this study were to: (1) identify botanicals used in the
traditional folk healing of skin and soft tissue infection (SSTI) in the Vulture-Alto
Bradano area of southern Italy; (2) analyze plant extracts for potential bacteriostatic and
anti-pathogenic properties against multidrug-resistant isolates of Staphylococcus aureus;
and (3) evaluate the mammalian cytotoxicity of these plant extracts. Aim 1 was addressed
in Chapter 2, where a detailed account of ethnobotanical remedies for SSTI was
provided. Aim 2 was addressed in Chapters 3 and 4 in discussions regarding
bacteriostatic, biofilm inhibiting, and quorum sensing inhibiting activities of plant
extracts. Aim 3 was addressed in the discussion of mammalian cytotoxicity featured in
Chapter 3.
The logic behind the design of this study was based on the hypothesis that
medicinal plants used in the traditional treatment of SSTIs would demonstrate more
activity against Staphylococcus aureus, a common cause of SSTI, than plants used for
other ethnomedicinal purposes unrelated to the skin or for plants with no ethnomedicinal
application. This hypothesis was tested with a series of tests, which are discussed in
Chapter 2, 3 and 4.
I have found that while there was no difference in the bacteriostatic or QSI
activity between ethnobotanical usage groups, there was a marked difference in anti-
biofilm activity of plants used for the skin when compared to those with no ethnomedical
application. Some level of QSI activity was common in most of the species tested. There
166
was a significant difference in cytoxicity between plants used for remedies unrelated to
the skin and plants with no ethnomedical application.
The outcomes of this study include the detailed recording of disappearing
traditional medical knowledge in southern Italy and the identification of plant extracts
with anti-staphylococcal properties. I have offered the first report of four plants from
Italian folk remedies for SSTI (Ballota nigra, Castanea sativa, Rosmarinus officinalis,
and Sambucus ebulus) with significant dose-dependent inhibitory activity on the
staphylococcal agr-mediated quorum sensing system. Likewise, I also offer the first
reports of six medicinal plants used in folk remedies for SSTI (Arundo donax, Ballota
nigra, Juglans regia, Leopoldia comosa, Marrubium vulgare, and Rubus ulmifolius) with
potent anti-biofilm activity for MRSA.
This study is important for those people who rely on medicinal plants for
household medicine. The validation of traditional remedies is vital to the reinforcement
and promotion of the conservation of disappearing ethnobotanical knowledge.
Furthermore, the identification of new lead sources for antibacterial drug discovery is
beneficial to public health in general.
167
VITA
CASSANDRA LEAH QUAVE
June 2nd, 1978 Born, Arcadia, Florida
1996-2000 B.S. Biology; B.S. Anthropology & Human Biology Emory University Atlanta, Georgia
2003-2008 The Edmund H. Fulling Award for best contributed oral paper Society for Economic Botany Chicago, Illinois Graduate Student Poster Award Society of Pharmacognosy Oxford, Mississippi
Doctoral Candidate in Biology Florida International University Miami, Florida
PUBLICATIONS AND PRESENTATIONS Quave, C.L. (1999). Paradise lost? Ethnobotany and its relevance to tropical forest conservation in Amazonia. The Emory Anthropologist: A Journal of Undergraduate Research 4: 24-30. Quave, C. and A. Pieroni. (September 2001). Healing with sacred and medicinal plants of three Arbëreshë communities of southern Italy (oral presentation), 3rd International Congress of Ethnobotany. Naples, Italy. Pieroni, A., S. Nebel, C. Quave and M. Heinrich. (2002). Ethnopharmacology of liakra: traditional weedy vegetables of the Arbëreshë of the Vulture area in southern Italy. Journal of Ethnopharmacology 81: 165-85. Pieroni, A., C. Quave, S. Nebel and M. Heinrich. (2002). Ethnopharmacy of the ethnic Albanians (Arbëreshë) in northern Basilicata, Italy. Fitoterapia 73: 217-41. Quave, C. and A. Pieroni. (2002). Traditional healing in the Vulture area of southern Italy. In: Handbuch der Ethnotherapien/ Handbook of Ethnotherapies. C.E. Gottschalk-Batschkus and J.C. Green (Eds.). Pp. 97-118. Institut für Ethnomedizin: Germany.
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Pieroni, A., C.L. Quave and R.F. Santoro. (2004). Folk pharmaceutical knowledge in the territory of the Dolomiti Lucane, inland southern Italy. Journal of Ethnopharmacology 95(2-3): 373-384. Pieroni, A., C.L. Quave, M.L.Villanelli, P. Mangino, G. Sabbatini, L. Santini, T. Boccetti, M. Profili, T. Ciccioli, L.G. Rampa, G. Antonimi, C. Girolamini, M. Cecchi, and M.Tomasi. (2004). Ethnopharmacognostic survey on the natural ingredients used in folk cosmetics, cosmeceuticals and remedies for healing skin diseases in the inland Marches, Central-Eastern Italy. Journal of Ethnopharmacology 91: 331-344. Quave, C.L. and A. Pieroni. (June 2004). Crossing ethnic borders: Ethnopharmacy of the Arbëreshë in Southern Italy (oral presentation), 9th Congress of the International Society for Ethnobiology. Canterbury, England. Pieroni, A. and C.L. Quave. (2005). Traditional pharmacopoeias and medicines among Albanians and Italians in southern Italy: A comparison. Journal of Ethnopharmacology 101(1-3): 258-270. Quave, C.L. and A. Pieroni. (2005). Folk illness and healing in Arbëreshë Albanian and Italian communities of Lucania, southern Italy. Journal of Folklore Research 42(1): 57-97. Pieroni, A. and C.L. Quave. (2006). Functional foods or food-medicines? On the consumption of wild plants among Albanians and south-Italians in Lucania. In: Eating and Healing. Traditional Food as Medicine. ISBN 1-56022-982-9. A. Pieroni and L. Price (Eds.). Pp. 101-129. Haworth Press: Binghamton, USA. Quave, C.L. & B.C. Bennett. (June 2007). The search for new anti-staphylococcal leads: comparing the antibacterial efficiency of Italian plants based on ethnobotanical use categories. (oral presentation). The 48th Annual Meeting of the Society for Economic Botany. Chicago, IL. Quave, C.L. (October 2007). Natural products from Italian medicinal plants: Potential anti-biofilm agents for MRSA. (poster) NIH Graduate Student Research Festival. Bethesda, MD. Quave, C.L. and A. Pieroni. (2007). Traditional health care and food and medicinal plant use among historic Albanian migrants and Italians in Northern Lucania, southern Italy. In: Traveling cultures, plants and medicines. The ethnobiology and ethnopharmacy of migrations. A.Pieroni and I. Vandebroek (Eds.) Pp. 204-226. Berghahn Press, Oxford, U.K. Quave, C.L., A. Pieroni, and B.C. Bennett (2008) Dermatological remedies in the traditional pharmacopoeia of Vulture-Alto Bradano, inland southern Italy. Journal of Ethnobiology and Ethnomedicine 4(5).
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