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MICR 420
Emerging and Re-EmergingInfectious Diseases
Lecture 3:Challenge of Antibiotic Resistance
S. aureus (MRSA), E. faecalis (VRE), A. baumannii
Dr. Nancy McQueen & Dr. Edith Porter
Overview
Challenge of antibiotic resistance S. aureus
Methicillin resistance
E. faecalis Vancomycin resistance
A. baumannii MDR
Morphology Virulence factors Diseases Diagnosis
Culture, biochemicals PCR: toxin production Antibiogram
Penicillin binding protein 2a latex agglutination assay
Challenge of Antibiotic Resistance
Antibiotic resistance Resistance genes Transfer of resistance genes
Antibiotic Resistance Enzymes that degrade or modify the antibiotic
-lactamases modifying enzymes like acetyltransferases
Alteration of the antibiotic target Penicillin binding proteins
Inhibition of antibiotic entry Porin mutations LPS modifications
Efflux pumps Tetracycline efflux Multidrug resistance efflux
Resistant bacteria are NOT more virulent but they are harder to destroy!
Antibiotic
Central cavity with promiscuous binding site
Pore
Funnel connection to an outer membrane channel
Cell membrane
Resistance Genes
Plasmid and chromosomal encoded E.g., Plasmid: beta-lactams, aminoglycosides E.g.,Chromosomal: fluoroquinolones, tetracyclines
Spontaneous mutations At low doses of antibiotics
Acquisition of resistance genes
Acquisition of Resistance Genes
Inherited Horizontally
Conjugation Cell to cell contact
Transduction Virus-mediated
Transformation Uptake of free DNA
Lessefficient
Occurs also between distantly related genera such as between
gram-positive and gram-negative bacteria!
Major Causes for Increased Antibiotic Resistance
Improper use in medicine Incomplete therapy Inappropriate use for viral
infections Unnecessary use of broad-
spectrum antibiotics Low dose use in animal
husbandry to promote animal growth
Use in agriculture leads to unwanted distribution of aerosols
After Stuart B. Levy in “The Challenge of Antibiotic Resistance”
Distribution of Antibiotics Manufactured in the U.S.(Currently about 50 Million Pounds per Year)
HumanAnimalAgriculture
Detection of Antibiotic Resistance
Conventional antibiotic disk diffusion (Kirby Bauer) and determination of minimal inhibitory concentrations
DNA microarrays for detection of plasmid-mediated antimicrobial resistance (and virulence factor) genes
Active Learning Exercise
What role do transposons play in antibiotic resistance?
Staphylococcus aureus
Staphylococcus aureus Gram positive cocci In clusters Facultative anaerobic Catalase positive Mannitol positive Coagulase positive Often beta-hemolytic Normal flora (low numbers)
Nostrils Pharynx Perineal
MSA Agar Plates
S. aureus
S. epidermidis
S. aureus: Virulence Factors
Coagulase Various enzymes Protein A
Binds antibodies via Fc receptor Various toxins
Enterotoxins Exfoliative toxin (epidermiolysin) Toxic shock syndrome toxin
Wound InfectionsPurulent infections
Toxin specific diseases
S. aureus Food Intoxication
Uptake of preformed enterotoxin Nausea, vomiting, diarrhea Self limited Short duration
Staphylococcal Exfoliative Toxin: SSSS
Staphylococcal scalded skin syndrome Cleaves cell adhesion molecules of
keratinocytes (desmoglein)
(http://dermatlas.med.jhmi.edu/derm/)
Toxic Shock Syndrome
First described in menstruating women using certain types of tampons
High fever, rash, skin peeling in palms, shock, multiple organ failure
Staphylococcus TSST production triggered in these tampons
TSST resorption through vaginal mucosa
Toxic Shock Syndrome Pathogenesis
Superantigen mediated Uncontrolled immune response to
staphylococcal and streptococcal toxins Early signs
Fever Dizziness Confusion Flat red rash over large areas of the body
Shock and multi-organ failure
Superantigens
Activate numerous T Helper cells simultaneously
T Helper cells release numerous proinflammatory cytokines
Bacterial Superantigens
Staphylococcal superantigens S. aureus Over 20 described TSST, exfoliatins, enterotoxins
Streptococcal S. pyogenes (Group A beta-hemolysing
streptococci) Exotoxin A and C, and others
New Threats by S. aureus Nosocomial infections (healthcare-associated
infections, HAIs) 15% of all isolates; 8% of all HAIs
Spread of MRSA in the community Vancomycin resistant MRSA
Methicillin Resistance High level resistance encoded by MecA Encodes alternative penicillin binding protein PBP2a
Structural changes in transpeptidase penicillin-binding proteins Plays role in peptidoglycan synthesis Low affinity of binding to beta-lactams
Carried by staphylococcal cassette chromosome mec (SCCmec) Unique mobile genetic element Integrated into the S. aureus chromosome Composed of the mec gene complex encoding methicillin
resistance and the ccr gene complex that encodes recombinases responsible for its mobility
These elements also carry various resistance genes for non-beta-lactam antibiotics
Spread of MRSA S. aureus in the Community Adam H et al.
Fatal case of post-influenza, community-associated MRSA pneumonia in an Ontario teenager with subsequent familial transmission. Can Commun Dis Rep. 2007 Feb 15;33(4):45-8.
van der Flier M et al. Fatal pneumonia in an adolescent due to community-acquired
methicillin-resistant Staphylococcus aureus positive for Panton-Valentine-leukocidin. Ned Tijdschr Geneeskd. 2003 May 31;147(22):1076-9.
Francis JS et al. Severe community-onset pneumonia in healthy adults caused by
methicillin-resistant Staphylococcus aureus carrying the Panton-Valentine leukocidin genes. Clin Infect Dis. 2005 Jan 1;40(1):100-7.
First Reports of Vancomycin Intermediate and Resistant MRSA
Hososaka Y et al. Nosocomial infection of beta-lactam antibiotic-induced vancomycin-resistant Staphylococcus aureus (BIVR). J Infect Chemother. 2006 Aug;12(4):181-4.
Resistance mechanism described for VISA Sequential point mutations in key global regulatory
genes associated predominately with cell wall thickening and restricted vancomycin access to its site of activity in the division septum (Howden et al., 2010)
Enterococcus faecalis
Enterococcus faecalis Gram positive cocci In pairs and chains Catalase negative Non-hemolytic Facultative anaerobic Bile esculin positive Normal flora in intestine Often highly resistant to
antibiotics Nosocomial infections
Urinary tract infection Wound infections Endocarditis
Vancomycin resistance observed
E. faecalis Virulence factors
Surface protein (ESP)
Adherence Antiphagocytic Variations
observed Escapes immune
response
Vancomycin Resistant E. faecalis (VRE)
Vancomycin binds to an essential substrate at a late stage of the biosynthetic pathway of peptidoglycan (Reynolds 1989) D-Ala-D-Ala in peptidoglycan
precursor Vancomycin resistance is
caused by the production of depsipeptide D-Ala-D-Lac, which replaces D-Ala-D-Ala (Gin and Zhanel, 1996)
Results in a more-than-1,000-fold lowering of the affinity of vancomycin for its target (Reynolds and Courvalin, 2005).
New Threats by E. faecalis Spread of VRE in the hospital setting Zubaidah et al. Hospital-acquired vancomycin-resistant enterococci: now
appearing in Kuala Lumpur Hospital. Med J Malaysia. 2006 Oct;61(4):487-9. Comert et al. First isolation of vancomycin-resistant enteroccoci and spread of a
single clone in a university hospital in northwestern Turkey. Eur J Clin Microbiol Infect Dis. 2007 Jan;26(1):57-61.
Vonberg et al. [Prevention and control of the spread of vancomycin-resistant enterococci : Results of a workshop held by the German Society for Hygiene and Microbiology.] Anaesthesist. 2007 Feb;56(2):151-7.
Huang et al. Risk of acquiring antibiotic-resistant bacteria from prior room occupants. Arch Intern Med. 2006 Oct 9;166(18):1945-51.
Bar et al. Systemic inflammatory response syndrome in adult patients with nosocomial bloodstream infections due to enterococci. BMC Infect Dis. 2006 Sep 26;6:145.
Furtado et al. Risk factors for vancomycin-resistant Enterococcus faecalis bacteremia in hospitalized patients: an analysis of two case-control studies. Am J Infect Control. 2006 Sep;34(7):447-51.
Acinetobacter baumannii
Acinetobacter baumannii Pleomorphic gram-
negative or gram-variable coccoid rods Short, fat rods, resemble
cocci and diplococci Can be confused with
Neisseria spec.
Non-motile Non-fermenter Biofilm production
contributes to antibiotic resistance (Dallo SF, Weitao T 2010)
http://www.cyberspaceorbit.com/AB_picenh2.jpg
Threats by Acinetobacter baumannii
Increasingly isolated in hospital settings (war and natural disaster victims) Wound infections Septicemia
Increasingly resistant production of
carbapenemases (metallobetalactamases, CRAB)
Worldwide spreading of resistant strains
http://www.bioquell.com/Pictures/Acinetobacter%20map.jpg
Outbreaks of infections withcarbapenem resistant A. baumannii
World Wide Spread of Acinetobacter Infection. 2010 Apr 1. Successful treatment of three children with post-
neurosurgical multidrug-resistant Acinetobacter baumannii meningitis. Ozdemir H et al (Turkey)
J Infect Dev Ctries. 2010 Mar 29;4(3):164-7. In vitro antimicrobials activity against endemic Acinetobacter baumannii multiresistant clones. Rodriguez CH et al. (Buonos Aires)
Scand J Infect Dis. 2010 Mar 26. The microbiological characteristics of patients with crush syndrome after the Wenchuan earthquake. Wang T et al. (China)
J Hosp Infect. 2010 Mar 18. Emergence of an extreme-drug-resistant (XDR) Acinetobacter baumannii carrying bla(OXA-23) in a patient with acute necrohaemorrhagic pancreatitis. Grosso F et al. (Portugal)
J Med Assoc Thai. 2009 Dec;92 Suppl 7:S173-80. Clonal spread of carbapenem resistant Acinetobacter baumannii in the patients and their environment at BMA Medical College and Vajira Hospital. Phumisantiphong U et al. (Thailand)
Increasing Nosocomial Infections with Drug Resistant A. baumannii
Search Terms* 1988 1998 2008 2009
AB & infection 1 27 181 172
AB & nosocomial 0 16 72 78
AB & resistant 1 23 186 226
Take Home Message
Increasing antibiotic resistance is a major threat to public health.
Of particular concern are MRSA, VISA and VRSA, VRE, and A. baumannii.
Antibiotic resistance is mediated by expression of enzymes inactivating the antibiotic, alteration of the drug target, prevention of drug entry into the cell, and drug efflux pumps.
Inappropriate drug use is the major cause.
Resources Microbiology: An Introduction, by Tortora, Funke and Case; Pearson
Prentice Hall; 9th ed, 2007, Chapter 11; pp 436t, 591, 593, 606. 633b
Current Issues: pp 62 -71 Alekshun MN and Levy SB (2007) Molecular mechanisms of
antibacterial multidrug resistance. Cell 128(6):1037-50 Emerging Infectious Diseases: pp28 – 30 http://www.microbelibrary.org CDC fact sheets http://dermatlas.med.jhmi.edu/derm/ Hiramatsu K, Cui L, Kuroda M, Ito T. The emergence and evolution
of methicillin-resistant Staphylococcus aureus. Trends Microbiol. 2001 Oct;9(10):486-93.
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