1

Detection and characterisation of

Vibrio harveyi isolates

Katarina S. Themptander

BSc Biomedical Sciences

May 2005

School of Biological Sciences

Dublin Institute of Technology, Kevin Street, Dublin 8

2

ABSTRACT

Aim Because of the major problems that certain Vibrio specie, especially Vibrio harveyi,

can cause the aquaculture industries a rapid method to identify Vibrio isolates is required.

Early diagnosis of a V. harveyi infection could facilitate disease surveillance, treatment and

prevention in cultured marine animals. Therefore, the use of PCR to aid in the

identification of Vibrio is increasing and a way of extracting DNA in a cheap, fast and easy

way is also of an important requirement to facilitate rapid diagnosis.

Methods This report comprises biochemical profiling and PCR methods in the

characterisation of four isolates of V. harveyi and single isolates of V. tubiashii, V.

alginolyticus, V. anguillarum, V. splendidus, V. tapetis and V. parahaemolyticus.

Strains were examined for adherence to a Hep-2 cell line. Four different DNA extraction

methods were evaluated and compared. The detection limits and the analytical limits of two

PCR methods for Vibrio were determined.

Results The overall findings were that the use of a greater range of biochemical substrates

than are in the API 20E is necessary to identify Vibrio strains, and that none of the strains

tested adhered to Hep-2 cells. All extraction methods successfully produced DNA with the

kit method giving the purest samples. RNA was a contaminant of the other techniques but

this could be overcome by treating extracts with RNase. The rapid microwave extraction

method gave appropriate PCR amplicons when tested.

Conclusion PCR determination of the VH-sequence in combination with VHA and a

distinguishable colonial morphology may be a good choice for the identifying of Vibrio

harveyi.

3

TABLE OF CONTENTS

1.0 INTRODUCTION PAGE No.

1.1: Vibrionaceae 1

1.2. Reservoir and transmission 2

1.3: Vibrio species as fish pathogens 2

1.4: Clinical features of Vibrio infection in aquatic animals 4

1.5: Virulence factors of Vibrio species 4

1.6: Treatment 6

1.7: Isolation and identification of Vibrio species 6

2.0 MATERIAL AND METHODS

2.1 Material

2.1.1: Bacterial isolates 9

2.1.2: Growth media 10

2.1.3: Apparatus 11

2.1.4: Equipment 11

2.1.5: Biochemical identification of bacterial isolates 12

2.1.6: DNA extraction 12

2.1.6.i: Phenol: chloroform extraction 12

2.1.6.ii: High-Pure PCR Template Preparation Kit 13

2.1.7: Agarose Gel Electrophoresis 13

2.1.8: PCR (polymerase chain reaction) 13

2.2 Methods

2.2.1: Biochemical characterisation 15

4

2.2.1.i: General biochemical characterisation 15

2.2.1.ii: Comparison of Vibrio growth on four different media 15

2.2.1.iii: API 20E, biochemical characterisation 15

2.2.2: Tissue adherence test 17

2.2.3: DNA extraction 18

2.2.3.i: Phenol: chloroform extraction according to Montes et al., 2003 18

2.2.3.ii: High-Pure PCR Template Preparation Kit 18

2.2.3.iii: Boiling 19

2.2.3.iv: Microwaving 20

2.2.4: DNA quantification 20

2.2.5: Agarose Gel Electrophoresis 20

2.2.5.i: Visualisation of extracted DNA 20

2.2.5.ii: Visualisation of PCR-products 21

2.2.6: PCR (polymerase chain reaction) 21

2.2.6.i: [MgCl2] optimization 22

2.2.6.ii: 16S rDNA sequence determination 22

2.2.6.iii: VH Amplification 23

2.2.6.iv: Diagnostic Detection Limit 23

2.2.6.v: Analytical Sensitivity 23

3.0 RESULTS

3.1: Phenotypic identification 25

3.1.1: Morphology 25

3.1.3: Tissue adherence test 28

3.2: Biochemical characteristics 29

5

3.2.1: General characteristics 29

3.2.2: API E20, biochemical characterisation 29

3.3: DNA extraction 31

3.3.1: Visualisation of genomic DNA from the four different extracting 32

methods using gel electrophoresis

3.4: 16S rDNA gene determination 34

3.4.1: [MgCl2] optimization 34

3.4.2: Amplicon determination using the 16S rDNA-primers 35

3.5: PCR using V. harveyi VH-1 and VH-2 primers 36

3.5.1: [MgCl2] optimization 36

3.5.2: Amplicon determination using VH-primers 38

3.6 Diagnostic Detection Limit 40

3.7 Analytical Sensitivity 41

4.0 DISCUSSION 43

5.0 CONCLUSION 53

6.0 REFERENCES 54

7.0 APPENDIX 57

6

ACKNOWLEDGEMENTS

I would like to extend my appreciation to the following people, who all played their part in

the realisation of this project.

I would first like to thank my supervisor Mr Patrick McHale for all his support and

patience and for guiding me through this project. Thank you for proofreading and being so

much help.

I would also like to thank you Mr Ted Doody for ordering all the reagents I needed and for

being so helpful. And thank you Fergus Ryan for the help with the molecular work.

A special thanks to Leanne Harris; thank you for all the help and encouragements. Thank

you for lending me reagents and for good advices.

And also big thank you to the rest of the people in the lab for being so helpful and making

me feel welcome.

Last but not least I would like to thank my parents, Jan and Maggie, and my brother

Christofer, for their continued support, patience and endless encouragements throughout

my education.

7

1.0 INTRODUCTION

1.1 Vibrionaceae

Vibrionaceae are a family of Gram-negative rod-shaped bacteria. They are facultative

anaerobes and have a fermentative metabolism. They are separated from the family

Enterobacteriaceae because of a positive oxidase reaction and the presence of polar

flagella.

The taxonomy of the group is in the process of revision due to increasing data obtained

with modern molecular biology techniques, where different genes are examined or where

the whole genome is inspected. Special emphasis has been paid to the 16S rRNA gene,

although other genes, such as the 23S rRNA and gyrB genes, have also been employed

(Dorsch et al., 1992; Gomez-Gil et al., 2004). The sequencing of the 16S rRNA gene is

considered the most reliable tool for the allocation of genera, species, and strains into the

family Vibrionaceae. Following this approach the former three genera (Vibrio, Aeromonas

and Plesiomonas) in the family (Murray et al., 2002) have been replaced by eight genera,

including Vibrio, in the outline of Bergey’s Manual of Systematic Bacteriology (Garrity and

Holt, 2000). To facilitate further studies of vibrios Thompson proposed spliting the

Vibrionaceae into three new families (Thompson et al., 2004). The newly proposed family

Vibrionaceae comprises only the genus Vibrio, with 63 species. The number of Vibrio

species is increasing with new species being described every year (Thompson et al., 2004).

The family is heterogeneous and may require further splitting.

8

1.2 Reservoir and transmission

Vibrios are ubiquitous in aquatic environments and in association with eukaryotes. They

appear at particularly high densities in and/or on marine organisms including corals, fish,

molluscs, seagrass, sponges, shrimp and zooplankton (Thompson et al., 2004).

The exact route by which Vibrio infect fish is unknown but oral transmission is suspected.

The organisms may, in certain circumstances, be able to cross the intestinal wall and cause

systemic disease. During outbreaks the numbers of infectious particles in the environment

will increase dramatically increasing the likelihood that fish will become contaminated.

1.3 Vibrio species as fish pathogens

The first known Vibrio species, V. cholerae, was discovered in 1854 by the Italian

physician Filippo Pacini while he was studying outbreaks of cholera in Florence. V.

cholerae, Vibrio parahaemolyticus and Vibrio vulnificus are the most serious human

pathogens and, in all, twelve Vibrio species known today are involved in human diseases

(Murray et al., 2002).

Many Vibrio species have also been implicated in aquaculture infections. They are the most

important pathogens for reared aquatic organisms such as penaeid shrimps and for several

fish species and molluscs, as well as for corals (Gomez-Gil et al., 2004). The species

associated with disease in fish and shellfish include Vibrio harveyi, Vibrio alginolyticus,

Vibrio splendidus 1 and 2, Vibrio tapetis, Vibrio tubiashii, and Vibrio anguillarum.

Different species infect different types of host animals. In marine fish diseases the main

pathogens are V. anguillarum, Vibrio salmonicida and V. vulnificus (Thompson et al.,

2004). The closely related species V. harveyi and V. campbellii have caused disease in

9

shrimp larvae while Vibrio penaeicida and V. parahaemolyticus have infected juveniles

and adults.

V. harveyi is one of the most commonly isolated marine Vibrio species, and can easily be

found both as free-living organisms or associated with the normal intestinal microflora of

marine animals (Hernandez et al., 2004). Moreover, it is the dominant heterotrophic

species in western Mediterranean seawater and marine bivalves during the warm season. V.

harveyi has been recognised as pathogenic for several crustacean larvae, particularly,

Penaeus species, and is considered responsible for mass mortalities of the bivalve Pinctada

maxima. It has been related to several opportunistic infections of ornamental or edible

cultured fish in the last decade, and recent reports confirm the virulence of some strains for

gilthead sea bream, silver mullet, salmon and seahorse (Pujalte et al., 2003). Although

some strains of V. harveyi are highly pathogenic other strains may be considered as

opportunistic pathogens. Those that do cause disease cause pose a severe threat to the

multi-billion dollar aquaculture industry (Oakey et al., 2003).

V. carchariae was first reported as a pathogen in aquaculture in 1984 after it had been

isolated from a brown shark found dead in captivity. Both V. harveyi and V. carchariae

were shown to be members of the normal microflora in that type of shark. Recently strains

from these species have been shown to be indistinguishable by biochemical or molecular

tests where they showed almost identical 16S rRNA base sequence. It was concluded that

V. carchariae is the junior synonym of V. harveyi (Pedersen et al., 1998).

10

1.4 Clinical features of Vibrio infection in aquatic animals

The signs of vibriosis are similar to many other bacterial diseases of fish. They usually start

with lethargy and a loss of appetite. As the disease progresses, the skin may become

discoloured, red and necrotic. Boil-like sores may appear on the body, occasionally

breaking through the skin surface resulting in large, open sores common around the fins

and mouth. The disease may spread and become systemic. Internal symptoms include

intestinal necrosis, anaemia, ascetic fluid, petechial haemorrhages in the muscle wall, liquid

in the air bladder, haemorrhaging and/or bloody exudates in the peritoneum, swollen

intestine, haemorrhaging in or on internal organs, and pale mottled liver. On the outside,

the external symptoms can be seen in many ways. Some of them are extensive skin lesions,

darkened pigment, eye damage (“pop-eye”), haemorrhaging on the fins and white or dark

nodules on the gills and skin. The infected animal can also show a sluggish behaviour and

spiral or erratic movement (Thompson et al., 2004).

1.5 Virulence factors of Vibrio species

The mode of infection of fish consists of three basic steps: (i) the Vibrio penetrates the host

tissues by means of chemotactic motility; (ii) within the host tissues the bacterium deploys

iron-sequestering systems, and (iii) the bacterium eventually damages the fish by means of

extracellular products. The Vibrio infection starts in the intestinal epithelium and then

invades the blood stream and spreads to other organs eventually leading to death of the fish

(Thompson et al., 2004). The virulence factors implicated in vibriosis are many. They

include the production of flagella (and associated motility), haemolysins, proteases, a

capsule and iron-sequestering siderophores, the presence of a hydrophobic surface antigen

and the ability to adhere to and invade host epithelial cells (Wang and Leung, 2000).

11

Bacterial adherence is an essential step in many infections and the capacity of bacterial

pathogens to adhere to eukaryotic cell surfaces is mediated by macromolecules known as

adhesins. Bacterial fish pathogens like some Vibrio strains have been found to attach to

collagen, fibronectin, fish mucus and fish epithelial cells. The adherence mechanisms of

Vibrio species to fish cells have not been extensively investigated. Internalization and

cytotoxicity are important virulence factors in Vibrio-fish cell interactions and expression

of different adhesins may be dependent on the different environmental conditions as well as

on the host-cell receptors (Wang and Leung, 2000).

Gram-negative bacilli have an intracellular communication system which was first

discovered in Vibrio fischeri (Pesci, 2003). As a free-living organism this marine symbiont

has a dilute cell density and is not luminescent but as an endosymbiont in a light organ of a

fish it can achieve a very high cell density. When that happens populations of V. fisheri

become luminescent and produces a visible light. This type of “Quorum Sensing” allows

the bacteria to act as a group rather than as separated individuals. Recently a new type of

cell-cell signal has been discovered in V. harveyi. This system functions through an acyl-

homoserine lactone signal and a second signal that is different from all of the other signals

known. This second signal is a furanosyl borate diester and is one of the components in a

row of proteins that controls light production in the organism. The gene for this second

signal has even been found in Escherichia coli and in Salmonella enterica. Cell-cell signals

may be crucial in determining expression of virulence traits.

The virulence of salmon-pathogenic V. harveyi strains has been related to duplicate

haemolysin genes, whereas the pathogenicity of strains infecting tiger prawns has been

related to cysteine protease production or the presence of a bacteriophage (Pujalte et al.,

12

2003). Zorilla et al. (2003) found that only V. harveyi strains that developed swarming

produced mortalities in experimentally inoculated sole. Possibly the ability to develop

swarming may be a marker to differentiate between virulent and non-virulent strains.

Expression of genes involved in swarming may be linked to expression of virulence genes.

1.6 Treatment

To avoid the massive loss that a Vibrio infection can cause in reared aquatic animals they

are usually treated with antibiotics in different forms. Antibiotics are used as a prophylactic

treatment in larval tanks in shrimp hatcheries (Karunasagar et al., 1996). The spread of

antibiotic resistance from aquatic cultivations to the natural environment has recently been

reported and it has also been suggested that the massive use of antibiotics together with

high levels of water exchange may favour the proliferation of Vibrio species and enhances

their virulence and disease prevalence (Thompson et al., 2004).

1.7 Isolation and identification of Vibrio species

In order to understand the pathogenic processes involved in a disease and to successfully

treat or prevent it, one must be able to isolate and identify the aetiological agent. Vibrio

species are relatively easy to isolate from marine animals and associated environmental

material although some species require specific growth factors or vitamins. In most cases

salt (NaCl) must be added to the growth media to a concentration equivalent to that of

seawater (the natural environment of the Vibrio species). There are several commercial

media which may be used for the isolation of vibrios, but tryptone soy agar (Oxoid or

Difco) supplemented with 1% or 2% NaCl and marine agar (Difco) generally allow growth

of very healthy colonies after 1-2 days of incubation at 280C. Vibrios grow well at

temperatures between 15 and 300C, depending on the strain under analysis. Thiosulphate-

13

citrate-bile salts sucrose (TCBS) agar is a medium traditionally used to select for V.

cholerae but it will allow growth of many other Vibrio species. Strains which are able to

ferment glucose will form yellow colonies on this medium while other strains will produce

green colonies (Thompson et al., 2004). However, some isolates of Staphylococcus,

Flavobacterium, Pseudoalteromonas and Shewanella may also grow on this medium. A

selective and differential medium for V. harveyi, termed Vibrio harveyi agar (VHA), has

been developed. According to Harris et al. (1996) this agar can differentiate V. harveyi

from 15 other Vibrio species. The selective factors in the medium that separates the Vibrio

species from other families are its high pH and the absence of magnesium ions which are

required by many other marine bacteria. A high concentration of NaCl and an incubation

temperature of 28ºC also seem to favour the marine Vibrio species. Cellobiose and

ornithine are added to the medium to differentiate V. harveyi from other Vibrio species.

V. harveyi ferments cellobiose and decarboxylates ornithine producing colonies with a

distinctive morphology.

Vibrio strains are identified as Gram-negative, oxidase positive, generally catalase positive

motile rod-shaped bacteria. Individual species may be characterised using biochemical

profiles. However, phenotypic identification of the Vibrio species is problematical due to

the great variety of profiles within some species including V. harveyi (Thompson et al.,

2004). The identification of vibrios isolated from the aquacultural environment has been

imprecise and is labour-intensive, requiring many biochemical and/or physiological tests

(Vandenberghe et al., 2003). Even the colonial morphology varies within the genera. Some

species are also very heterogeneous and can, therefore, be hard to identify. V. harveyi is

one of the species that can vary between different isolates. Therefore, rapid methods are

necessary to improve the detection and diagnosis of V. harveyi infections.

14

Serological methods have been used successfully for the identification of Vibrio species in

food and the aquatic environment. Therefore, Robertson et al. (1998) developed plate and

dipstick enzyme-linked immunosorbent assays for the rapid detection of V. harveyi from

penaeid shrimp and water. This ELISA incorporated a polyclonal antiserum which

recognised a wide outer membrane located epitope common between isolates of V. harveyi.

Molecular methods for the identification of Vibrio species have increased lately, especially

the use of Polymerase Chain Reaction (PCR)-based techniques to amplify specific DNA

sequences, as well as digestion of these fragments with restriction enzymes. The most

frequently used molecular methods to identify Vibrio species have been Amplified

Fragment Length Polymorphism (AFLP), Fluorescence In Situ Hybridization, Microarrays,

Multilocus Enzyme Electrophoresis (MLEE), Multilocus Sequence Typing (MLST), Real-

Time PCR, Restriction Fragment Length Polymorphism (RFLP) and Ribotyping. Oakey et

al. (2003) developed specific primers to detect V. harvey usimg PCR. These were relatively

species-specific but some isolates of V. alginolyticus produced positive results with them.

15

2.0 MATERIAL AND METHODS

2.1 MATERIAL

2.1.1 Bacterial isolates

Strains Reference number

V. harveyi 01/021

V. harveyi 01/022

V. harveyi 02/001

V. harveyi 02/109

V. tubiashii NCIMB 2164

V. alginolyticus NCIMB 1903

V. anguillarum NCIMB 329

V. splendidus NCIMB 2231

V. tapetis NCIMB 4600

V. parahaemolyticus NCIMB 10441

E. coli Clinical isolate

All isolates except the E.coli (which was isolated from a human urinary tract infection)

were collected from the Marine Institute, Blanchardstown, Ireland and were clinical

isolates from infected aquatic animals. The Vibrio strains were cultured using the

conditions outlined in Table 2.1.1.A.

16

Table 2.1.1.A Recommended growing conditions for Vibrio species

Vibrio spp. Reference number

Agar Incubation length

Incubation temperature

V. harveyi 01/021 01/022 02/001 02/109

Blood agar with 3.5% NaCl

Overnight 30ºC

V. tubiashii NCIMB 2164 Blood agar with 3.5% NaCl

Overnight 30ºC

V. alginolyticus NCIMB 1903 Blood agar with 3.5% NaCl

Overnight 30ºC

V. anguillarum NCIMB 329 Blood agar with 3.5% NaCl

Overnight 30ºC

V. splendidus NCIMB 2231 Blood agar with 3.5% NaCl

Overnight/48h 15ºC

V. tapetis NCIMB 4600 Blood agar with 3.5% NaCl

Overnight/48h 15ºC

V. parahaemolyticus NCIMB 10441 Blood agar with 0.5% NaCl

Overnight 37ºC

2.1.2 Growth media

1. Blood agar, 0.5% NaCl (Colombia blood agar base) (Oxoid Ltd., CM0331)

2. Blood agar, 3.5% NaCl (Colombia blood agar base) (Oxoid Ltd., CM0331)

3. Tryptic Soy broth, 3.5% NaCl (Difco Laboratories, 0370-17-3)

4. V. harveyi agar (VHA) (Harris et al., 1996)

5. Marine agar (Difco Laboratories, 2216)

6. Thiosulfate-citrate-bile salts-sucrose (T.C.B.S) agar (Oxoid Ltd., CM333)

Since these Vibrio strains were mainly aquaculture pathogens originally isolated from the

marine environment NaCl was added to the blood agar, the tryptic soy agar and the tryptic

soy broth to give them a salinity like that of seawater. VHA is a selective and differential

medium for V. harveyi with a high pH which many Vibrio species but not many terrestrial

bacteria can tolerate. Many marine bacteria except Vibrio species require magnesium

17

which is not present in this medium. V. harveyi can be differentiated from other marine

vibrios by its colonial morphology. The ability of this species to ferment cellobiose and

decarboxylate ornithine will result in colonies that appear greenish, sometimes with the

formation of yellow halos around them. TCBS agar is a medium that is used for isolating

Vibrio species but it is also inhibitory for some of the Vibrio species that are common in the

marine environment.

2.1.3 Apparatus

Autoclave

Electrophoresis apparatus (Consort)

Incubators, 15ºC, 30ºC, 37ºC

Freezers, -20ºC, -80ºC

Microcentrifuge (B Hermle)

Microwave (Sharp)

Refrigerator, 4ºC (Bosch)

Thermocycler, PCR (Hybaid Omn-E)

UV-Visible recording spectrophotometer

Vortex

Waterbath

Weighing balance

2.1.4 Equipment

Bunsen burner

Loop

Microcentrifuge tubes, 1.5ml, 0.5ml (Brand)

18

Microscope slides

Microscope

Sterile swabs (Copan diagnostics)

Sterile pasteur pipettes

Pipettes, automatic

2.1.5 Biochemical identification of bacterial isolates

Catalase reagent, hydrogen peroxide

Oxidase reagent (Difco)

KOH, 3%

Gram stain reagents

Saline

Sterile water

API 20E strips (bioMérieux® sa)

Oil

Kovac’s reagent

API VP 1 and 2 reagents

API TDA reagent

2.1.6 DNA extraction

2.1.6.i Phenol: chloroform extraction according to Montes et al., 2003

Tris:ethylenediaminetetracetate (TE), 10:1 pH8

Potassium acetate, 7.5M

Sodium Dodecyl Sulfate, 10%

Proteinase K

19

Phenol: chloroform: isoamylalcohol, (25:24:1)

Chloroform: isoamylalcohol, (24:1)

Isopropanol

Ethanol, 70%

2.1.6.ii High-Pure PCR Template Preparation Kit (Roche, 1 796 828)

Binding Buffer

Elution Buffer

Inhibitor Removal Buffer

Isopropanol

Lysozyme

PBS, 0.1M pH 7.3

Proteinase K

Wash Buffer

2.1.7 Agarose Gel Electrophoresis

Agarose D-1 (Bio Science)

Ethidium Bromide, 10mg/µl

Loading dye

TBE-buffer, 1X

UV-lamp +screen

2.1.8 PCR (polymerase chain reaction)

Taq DNA polymerase, 5 units/µl (Invitrogen™)

Primer, VH1 and VH2 (Sigma Genosys)

Primer, 63f and 1387r (Sigma Genosys)

20

PCR Reaction Buffer, 10X (-MgCl2) (Invitrogen™)

MgCl2, 50 mM (Invitrogen™)

dNTPs, 2 mM

MilliQ-water

DNA

Table 2.1.8.A 63f/1387r -and VH1/VH2-primer specification

Primer name Conc. per reaction

%GC content

PCR T-annealing

63f forward primer

100 ng/µl ~52 54.8ºC

1387r reverse primer

100 ng/µl ~73 52.5ºC

VH1 forward primer

100 ng/µl 45 64.5ºC

VH2 reverse primer

100 ng/µl ~43 66.6ºC

Table 2.1.8.B 63f/1387r -and VH1/VH2-primer sequences

Primer Sequence 5’ > 3’

63f forward primer CAG GCC TAA CAC ATG CAA GTC

1387r reverse primer GGG CGG WGT GT

VH1 forward primer ACC GAG TTA TCT GAA CCT TC

VH2 reverse primer GCA GCT ATT AAC TAC ACT ACC

21

2.2 METHODS

2.2.1 Biochemical characterisation of Vibrio isolates

2.2.1.i General biochemical characterisation

Gram stains and 3%KOH-tests were performed on all isolates. The KOH-test is used to

separate Gram-positive from Gram-negative bacteria based on differences in their cell wall

structure. Gram-negatives will lyse in the presence of KOH and release their internal

constituents including their deoxyribonucleic acid. This produces a sticky solution.

The presence of cythocrome oxidase activity in the bacteria was confirmed by the oxidase

test. Most Vibrio species are oxidase positive. A catalase test was performed to see whether

the bacteria produced catalase, an enzyme that reacts with hydrogen peroxide to produce

water and oxygen bubbles.

2.2.1.ii Comparison of Vibrio growth on four different media

The Vibrio species were streaked out on blood agar, marine agar, VHA and TCBS-agar

plates using a standard inoculation loop. Some non-Vibrio species, (E. coli, Aeromonas

hydrophila, Stapphylococcus epidermidis, Enterobacter aerogenes and Klebsiella

pneumoniae) were also inoculated onto these plates. A ten-fold dilution series of a 48 hour

culture of V. harveyi 02/001 was prepared and colony counts were performed on aliquots of

dilutions 10-7 to 10-11.

2.2.1.iii API 20E, biochemical characterisation

The API 20E kit characterises bacteria due to their reaction with a series of 20 different

substrates. An identification profile number is generated by the results and a software

program to identify the test bacterium from this. The kit was developed to identify

members of the Enterobacteriaceae in particular, i.e. Gram-negative, oxidase-negative

rods. It has been used, with a modified inoculation protocol, to characterise Vibrio species

22

(Jensen et al., 2003). The principle of the system is that microtubules containing 20

different dehydrated substrates are inoculated with a bacterial suspension that will

reconstitute the media. The strip with the 20 different substrates is incubated and the

interaction of bacterial metabolism with a substrate will spontaneously or, with the help of

added reagents, produces a characteristic colour change in the microtubules.

A bacterial suspension of the test organism was prepared in saline. An incubation box (tray

and lid) was prepared by distributing sterile water into the wells in the tray to create a water

chamber. This provides a humid atmosphere to decrease the possibility of the strip drying

out during incubation. The substrate strip was placed in the tray. The test bacterial

suspension was added to the different microtubules using a pasteur pipette. For the tests

CIT (sodium citrate), VP (creatine sodium pyruvate) and GEL (Kohn’s gelatine) both the

tube and cupule were filled with the bacterial suspension. Only the tube was filled with the

suspension for the other tests. In some of the tests, ADH (arginine), LDC (lysine), ODC

(ornithine), H2S (sodium thiosulfate) and URE (urea), an anaerobic atmosphere is needed

for the metabolic reaction and colour change and this was created by adding mineral oil to

cupule to overlay the bacterial suspension. The incubation box was closed and incubated

between 15-37ºC for 24-48 hours depending on the strain being tested. The reactions were

read. TDA reagent (one drop) was added to the TDA microtubule, Kovac’s-reagent was

added to the IND-test and VP1 and VP2 reagents were added to the VP-test. The TDA-and

IND-tests were read immediately on addition of reagents while the VP-test was read ten

minutes later.

23

2.2.2 Tissue adherence test

The ability of the Vibrio to adhere to tissue was tested by assessing their ability to attach to

a monolayer of Hep-2 culture cells. Hep-2 is an epithelial cell line derived from human

laryngeal tissue. Trac tubes were prepared by treating Hep-2 cells with 0.25% trypsin and

then suspending the cells in growth medium. The cells were counted and a suspension of

105 was prepared. An aliqoute (1 ml) was transferred to a Trac tube containing coverslips

and the preparation was incubated for 18h at 37ºC in a 5% CO2 atmosphere.

For the actual adherence tests bacterial suspensions (1x108 cfu/ml - matching MacFarland

opacity tube number 1) were prepared from 24h cultures. The medium in the Trac tubes

was poured off and the coverslips were washed with PBS (pH 7.2) by pouring some into

the Trac tubes and discarding a few times. For each test, bacterial suspension (1 ml) was

added to the Trac tube from which the growth medium/PBS had been removed and

incubated for one hour. The bacterial suspension was then poured off and the coverslip

washed five times with PBS (pH 7.2) to remove non-adherent bacteria. All PBS was

poured off and replaced with methanol which was left for 90 seconds to fix the cells. The

methanol was then poured off and replaced with 10% Giemsa for 30 minutes. The

coverslips were washed in tap water after staining, washed for approximately 10 seconds in

acid tap water and then twice in tap water. The coverslips were removed and the

monolayers were dehydrated in acetone and cleared in xylol by dipping twelve times into

each solution. One drop of DPX was added to a slide and the coverslip was placed on this

with the monolayer uppermost and another drop of DPX added. A number 1 coveslip was

then placed on this sandwich. The preparations were then examined.

24

2.2.3 DNA extraction

The principle behind the ability to extract DNA from cells is to lyse them and separate the

DNA from other material in the cells like proteins, membranes and organelles. In this study

were four different DNA extraction methods compared.

2.2.3.i Phenol: chloroform extraction according to Montes et al., 2003

Cells were inoculated into 3.5% tryptic soy broth and grown for 48h at the incubation

temperature recommended for the different strains (Table 2.1.1.A). An aliquot (3 ml) of

each sample were centrifuged for 1 minute in 16000xg and the pellet was then resuspended

in 500 µl of TE-buffer (10:1, pH8). To lyse the cells 20 µl 10% SDS and 20 µl proteinase

K (20mg/ml in TE-buffer) were added. The samples were then incubated at 56˚C in a

waterbath for 30 minutes. Then 7.5M potassium acetate (250 µl) was added to each sample

followed by incubation on ice for 15 minutes. To purify the DNA 700 μl of phenol:

chloroform: isoamylalcohol (25:24:1) were added, mixed well and the mixture was

centrifuged for 1 minute. The upper phase was transferred to a new microcentrifuge tube

and 500 μl chloroform: isoamylalcohol (24:1) were added. The samples were mixed with

the help of a vortex and centrifuged for 30 seconds. The upper phase was again transferred

to a new microcentrifuge tube and isopropanol (550 μl) was added to precipitate the DNA.

The samples were mixed carefully by gentle inversion and centrifuged for 1 minute. The

supernatant was removed and the pellet was washed twice with 70% ethanol. The pellet

was redissolved in 30 μl TE-buffer. The DNA samples were stored in a 4˚C refrigerator.

2.2.3.ii High-Pure PCR Template Preparation Kit

The bacterial isolates were inoculated into 3.5% tryptic soy broth and incubated for 48h at

the temperature recommended for the different strains (Table 2.1.1.A). An aliquot (200 µl)

of this culture was transferred to a microcentrifuge tube and centrifuged for 5 minutes at

3000xg. The pellet was resuspended in PBS (200 μl). Lysozyme (5 μl) was added to lyse

25

the cells and the mixtures were incubated for 15 minutes at 37˚C. Binding buffer (200 μl)

and proteinase K (40 μl) were added to each sample and the mixtures were incubated for 10

minutes at 70˚C. Isopropanol (100 μl) was added, the samples were mixed, transferred to

the upper reservoir of the combined High Pure Filter collection tube and centrifuged for 1

minute at 8000xg. The flowthrough and the collection tube were discarded and the upper

reservoir filter was combined with a new collection tube. Inhibitor Removal Buffer (500 μl)

was added to the upper reservoir and centrifuged for 1 minute at 8000xg. The flowthrough

and collection tube were again discarded and a new collection tube was combined with the

upper filter. The sample was washed twice with Wash Buffer (500 μl) that was added to the

upper reservoir and centrifuged for 1 minute at 8000xg. The flowthrough was discarded

and the combined filter and collection tube centrifuged for 10 seconds at maximum speed,

1600xg, to ensure that all of the Wash Buffer had been removed. The collection tube was

discarded and the filter tube was combined with a new 1.5ml microcentrifuge tube.

Prewarmed (70ºC) Elution Buffer (200 μl) was added to the upper reservoir of the filter

tube and centrifuged for 1 minute at 8000xg to elute the DNA. The DNA samples were

stored in a 4ºC refrigerator.

2.2.3.iii Boiling

The test bacteria were inoculated into 3.5% tryptic soy broth and were incubated for 48h at

the temperature recommended for the particular strain (Table 2.1.1.A). An aliquot (1.5 ml)

of this culture was centrifuged for 3 minutes at 14000xg and the pellet was resuspended in

TE-buffer (100 µl). The samples were boiled for 15 minutes and centrifuged again for 3

minutes at 14000xg to spin down the pellet. The supernatant, which contained the extracted

DNA, was removed to a new microcentrifuge tube and stored in a 4ºC refrigerator.

26

2.2.3.iv Microwaving

The test bacteria were inoculated into 3.5% tryptic soy broth and were incubated for 48h at

the temperature recommended for the particular strain (Table 2.1.1.A). An aliquot (1.5 ml)

of this culture was centrifuged for 3 minutes at 14000xg and the pellet was

resuspended in TE-buffer (100 µl). The samples were microwaved at full power for 2

minutes and frozen in -80ºC for 10 minutes. The samples were left to thaw and were then

centrirfuged again for 3 minutes at 14000xg. The supernatant containing the DNA, was

transferred to a new microcentrifuge tube and stored in the4ºC refrigerator.

2.2.4 DNA quantification

The DNA samples were diluted 1:100 (5 µl of the DNA samples +495 µl of distilled milliQ

water) and the absorbances of the solutions were read at wavelength λ260 nm to determine

the DNA concentration and λ280 to determine the protein concentration. All the absorbances

at λ260 were multiplied by 100 to correct for the dilution factor and then by a factor of 50 to

give the DNA concentration in µl/ml. This value was divided by 1000 to give the DNA

concentration in µg/µl.

2.2.5 Agarose Gel Electrophoresis

Agarose gel electrophoresis was used to check whether the extraction methods produced

any DNA and to visualise specific target fragment were amplified with the PCR method.

2.2.5.i Visualisation of extracted DNA

A 0.8% agarose gel was prepared by dissolving 0.8 g agarose in 100 ml of TBE-buffer. The

mixture was heated in a microwave at full power for 2 minutes and swirled at intervals to

help the agarose dissolve. It was cooled down to below 60ºC, ethidium bromide (5 µl) was

added, the solution was poured into a gel tray, and the comb was inserted. The gel was

27

allowed to solidify for about 30 minutes before the comb was removed. The DNA

preparations were adjusted with sterile milliq water to a concentration of 10 µg of DNA per

10 µl. The gel was placed in the geltank and covered with TBE-buffer. Aliquots (12 µl)

containing the diluted DNA (10 µl) 10 µl and loading dye (2 µl ) were added to each well.

An aliquot of a 100 base pair ladder was included in each gel as a size standard. The tank

was connected to the electrophoresis powerpack and the DNA was separated by running

the gel for 1.5h at 110 V and 150 mA.

2.2.5.ii Visualisation of PCR-products

A 2% agarose gel was prepared by dissolving 2 g agarose in 100 ml of TBE -buffer. The

mixture was heated in a microwave at full power for 2 minutes and swirled at intervals to

help the agarose dissolve. It was cooled down to below 60ºC, ethidium bromide (10 µl) was

added, the solution was poured into a gel tray and a comb was inserted to create wells. The

gel was left to solidify for about 30 minutes before the comb was removed. The gel was

placed in the geltank and covered with TBE-buffer. PCR-product (10 µl) and loading dye

(2 µl) were mixed and added to the wells. The tank was connected to the electrophoresis

powerpack and the fragments were separated by running the gel for 1.2h at 110 V and 150

mA.

2.2.6 PCR (polymerase chain reaction)

The Polymerase Chain Reaction is used to amplify a certain sequence of the DNA using a

pair of oligonucleotide primers, each complementary to one end of the DNA target

sequence. The PCR contains three stages with different temperatures and durations, an

initial denaturation temperature, an amplification stage temperature and a final extension

temperature. Three different steps are included in the amplification stage, a denaturation

step that will break the bonds that hold the two strands of the DNA chain together, an

28

annealing step that allows the primers to bind to the complementary sequences on the

strand and a polymerization step.

In this study two different primer pairs were used. The primer pair 63f and 1387r amplify

the 16S rDNA sequence, a sequence that is highly conserved in eubacteria. The other

primer pair, VH-1 and VH-2, is more specific and target a particular sequence only present

within the 16S rDNA gene of V. harveyi.

2.2.6.i [MgCl2] optimization

Magnesium concentration is an important factor that affects the performance of the Taq

DNA polymerase. The different reaction components can affect the amount of free

magnesium and in the absence of Mg2+ the Taq DNA polymerase is inactive. On the other

hand, a too high concentration of free magnesium inhibits and reduces the enzyme fidelity

and may increase the level of non-specific amplifications. Therefore, it is very important to

determine the optimal MgCl2 concentration for each PCR reaction. In this project Mg2+

concentrations in the range 2 mM-3·5 mM and 2 mM-4 mM were examined for the 16S

rDNA primers and VH primers respectively by adding 1, 1.25, 1.5, 1.75 or 2 µl of a 50 mM

MgCl2 stock to 25 µl PCR reactions.

2.2.6.ii 16S rDNA Amplification

The 16S rDNA sequence was amplified using a total PCR-reaction volume of 25 µl. 5 µl

10X PCR Reaction Buffer (-MgCl2), 2.5 mM magnesium chloride, 100 ng/µl primers, 2

mM each dNTP, 1 Unit Taq polymerase and template DNA and MilliQ-water to 25µl. The

PCR-reaction tubes were centrifuged for seconds and then the DNA was added to the PCR

reaction volume. The mixture was centrifuged for a couple of seconds and placed in the

thermal cycler. One PCR cycle consisted of an initial denaturation of 94ºC for 2 min; 35

cycles of 94ºC for 1 min, 55ºC for 45 s and 72ºC for 105 s; and a final extension of 72ºC

29

for 4 minutes (Table 2.2.6). The PCR-products were stored at 4ºC until visualised on a 2%

agarose gel.

2.2.6.iii VH Amplification

A PCR reaction mix was prepared containing 5 µl 10X PCR Reaction Buffer (-MgCl2), 3

mM magnesium chloride, 100 ng/µl primers, 2M each dNTP, 1 Unit Taq polymerase and

template DNA. MilliQ-water was added to a total volume of 25 µl. The PCR reaction tube

was centrifuged for a couple of seconds and placed in the thermal cycler. PCR cycle

parameters were initial denaturation at 94ºC for 2 min, 40 cycles of 94ºC for 1 min and

65ºC for 1 min, and a final extension of 72ºC for 2 minutes (Table 2.2.6). The PCR-

products were stored at 4ºC until visualised on a 2% agarose gel.

2.2.6.iv Diagnostic Detection Limit

Bacterial cell cultures from V. harveyi 02/001 and from V. splendidus NCIMB 2231 were

grown for 24-48 hours in Tryptic Soy broth with 3.5% NaCl at 30ºC and 17ºC respectively.

Dilutions from the original culture were prepared and the DNA were extracted using the

High-Pure PCR template preparation kit from the undiluted cultures and dilutions dilutions

10-1, 10-3, 10-5, 10-7 and 10-8. The DNA samples from these five extractions were subjected

to PCR using both the VH-primers and 16S rDNA-primers. The bacterial dilutions were

also cultured on growth media to determine the number of organisms in each before DNA

extraction.

2.2.6.v Analytical Sensitivity

DNA samples from undiluted cultures extracted with the High Pure kit were diluted down

to 10-5. For each dilution 1 µl template DNA was added to 9 µl of MilliQ-water. From the

10-1 dilution tube 1 µl was added to 9 µl of MilliQ-water to get the dilution 10-2 and then so

on down to a 10-5 dilution. PCR reactions with the two sets of primers were done on the

diluted extracts.

30

Table 2.2.6 Thermocycler programmes, depending on primer, stage design and cycle

number

Initial denaturation Amplification Final extension

PCR program Stage 1

Stage 2 Stage 3

63f /1387r (primer sequences)

94ºC 2 min

94ºC 1 min 55ºC 45 s 72ºC 105 s Cycles: 35

72ºC 4 min

VH1 /2

(primer sequences)

94ºC 2 min

94ºC 1 min 65ºC 1 min Cycles: 40

65ºC 1 min

31

3.0 RESULTS

3.1 Phenotypic identification

3.1.1 Morphology

The four V. harveyi isolates grew well on the VHA producing colonies that were distinctive

from those produced by the other Vibrio species (Table 3.1.1A). Two of the species, V.

tapetis and V. splendidus, failed to grow on this medium. The other species grew on VHA

although poorly in some cases. V. harveyi was easily differentiated from V. alginolyticus.

V. harveyi is phenotypically diverse and differences were observed between colonies of the

four isolates. Some fermented cellobiose more strongly than others. This produced an acid

which resulted in the colonies appearing green with yellow halos. Even the acid-base

indicator bromothymol blue will change the colour of the agar. In an acid environment the

agar will turn from an azure blue colour to yellow. V. alginolyticus does not ferment

cellobiose and are not able to decarboxylate ornithine. This property will result in a pH

change as well but this time the colonies will appear blue or colorless as can bee seen in the

figure 3.1.1.D.

Some non-Vibrio species were also streaked out on the VHA agar. E. coli, A. hydrophila, S.

epidermidis and E. aerogenes did not grow on this medium. K. pneumoniae did grow on

VHA, appearing as sticky green or slightly yellow colonies. On TCBS E. coli, A.

hydrophila and S. epidermidis failed to grow. However, E. aerogenes grew as yellow

colonies with a green halo while K. pneumoniae grew as green colonies with a whiter halo.

Both species grew well on this media producing large colonies.

32

Table 3.1.1.A Colonial morphologies of test strains on various media. Strain Blood agar Marine agar VHA TCBS

V.harveyi 02/001

Spreads haemolytic

White/yellow, sticky, good growth

Small, dark green/yellow

Light green colonies

V. harveyi 02/109

Spreads Haemolytic

White/yellow, sticky, good growth

Yellow colonies, agar yellow

Green, lighter halo, good growth

V. harveyi 01/021

White/grey colonies, spreads haemolytic

White/yellow, sticky, good growth

Brown/yellow, lighter halo

Yellow, ferments sucrose, good growth

V. harveyi 01/022

White/grey colonies, spreads, haemolytic

White/yellow, sticky, good growth

Round, small, yellow colonies agar yellow

Yellow, ferments sucrose, good growth

V. tubiashii NCIMB 2164

Grey colonies, haemolytic

White/yellow, sticky, good growth

No color Yellow colonies, good growth, ferments sucrose

V. alginolyticus NCIMB 1903

Spreads, grey White/yellow, sticky, good growth

Small dots with big halo, no color

Green, big colonies, good growth

V. anguillarum NCIMB 329

White, dry, small colonies

White, dry, small colonies

Small, white colonies

Light yellow, weak growth

V. tapetis NCIMB 4600

White/colorless, haemolytic

White, small, good growth

No growth No growth

V. splendidus NCIMB 2231

White/colorless, haemolytic

No growth No growth No color/weak yellow

V. parahaemolyticus NCIMB 10441

- White/yellow, filamentous, good growth

Small, greenish, weak growth

Green, filamentous

33

Fig. 3.1.1 Vibrio harveyi agar. Three different isolates of V. harveyi and one V. alginolyticus. A V. harveyi 02/109 B V. harveyi 01/021

C V. harveyi 01/022 D V. alginolyticus NCIMB 1903

Recovery of V. harveyi on blood agar 3.5% NaCl, marine agar, VHA and on TCBS was

compared by inoculating aliquots (100 µl) from dilutions of 10-7 to 10-11, incubating under

appropriate conditions and counting the colonies recovered. The salted blood agar gave the

best growth of the bacteria followed by marine agar. TCBS gave a slightly better recovery

rate than the VHA (Table 3.1.1.B). Certain the cultures spread on the marine agar could no

free colonies be distinguished.

Table 3.1.1.B Comparison between different media using V. harveyi 02/001 in a dilution of

10-6.

34

Media cfu/100 µl Estimated

bacterial cfu/ml

Recovery compared

to BA

Blood agar, 3.5% NaCl 37 3.7 x 108 100%

TCBS 26 2.6 x 108 70%

VHA 23 2.3 x 108 62%

3.1.2 Tissue adherence test

Ten Vibrio strains were tested for tissue culture adherence to Hep-2 epithelial cells from

laryngeal tissue. No adhesion to the epithelial cells could be seen in any of the preparations.

A picture over the preparation where V. harveyi were used can be seen in Figure 3.1.2.A.

A known tissue adherent strain of Moraxella catarrhalis were used as a positive control

and is shown in Figure 3.1.2.B.

Figure 3.1.2.A Cell adherence of V. harveyi 02/001 to epithelial cells from human

laryngeal tissue.

Figure 3.1.2.B Positive control: Cell adherence of M. catarrhalis to epithelial cells from

human laryngeal tissue.

35

3.2 Biochemical characteristics

3.2.1 General characteristics

All strains were Gram-negative rods, slightly catalase positive, oxidase and KOH positive.

3.2.2 API 20E, biochemical characterisation

In this biochemical identification test ten Vibrio strains were used. Each isolate was tested

twice to check for reproducibility of the results. Some of the strains gave different profiles

on retesting (Table 3.2.2). All the Vibrio strains were negative in the ONPG test except V.

tapetis. Also V. splendidus gave a positive result the first time but not the second.

36

Table 3.2.2 API 20E, biochemical profiles of ten Vibrio isolates tested twice.

V.harveyi 02/001 1st 2nd

V.harveyi 02/109 1st 2nd

V.harveyi 01/021 1st 2nd

V.harveyi 01/022 1st 2nd

V.tubiashii NCIMB 2164

1st 2nd

V.alginolyticus NCIMB 1903

1st 2nd

V.anguillarum NCIMB 329

1st 2nd

V.tapetis NCIMB 4600

1st 2nd

V.splendidus NCIMB 2231

1st 2nd

V.parahaemolyticus NCIMB 10441

1st 2nd

ONPG - - - - - - - - - - - - - - + + + - - - ADH - - - - - - - - - - - - - - - - - - - - LDC + + + + + + + + - - + + - - - - - - - - ODC + - + + + + + + - - - - - - - - - - - - CIT + - + - + + + + + - + - + - - - - - - - H2S - - - - - - - - - - - - - - - - - - - - URE - - - - - - - - - - - - + + - - - - - - TDA + + + + + + + + + + + + + - - - + + + + IND + + + + + + + + + + + + - - - - + + + + VP - - - - - - - - - - + + + - - - - - + - GEL + + + + + + + + + + + + - - - - + + + + GLU + + + + + + + + + + + + - - - - + + - - MAN + + + + + + + + - - + + - - - - + + + - INO - - - - - - - - - - - - - - - - - - - - SOR - - - - + + + + - - - - - - - - - - - - RHA - - - - - - - - - - - - - - - - - - - - SAC - - - - + + + + + + + + - - - - - - - - MEL - - - - - - - - - - - - - - - - - - - - AMY + + + + + + + + - - + - - - - - + - - - ARA - - - - - - - - - - - - - - - - - - - - OX + + + + + + + + + + + + - - + + + + + +

xxxvii

3.3 DNA extraction

Since the aim of this project was to detect Vibrio harveyi molecularly with the aid of PCR

and specific primers were it necessary to extract the DNA from the isolates used.

Therefore was DNA extracted using 11 different strains. For some reason no DNA was

detected in any of the V. anguillarum extracts. In every extraction, except with the

microwaving where this sample evaporated, V. alginolyticus appeared to give the highest

amount of DNA. All the four methods gave varying amounts of DNA, and the purity

varied with the extractions. The absorbance was read at two wavelengths, λ260 and λ280.

DNA has a maximum of absorbance at the wavelength λ260 and protein at the wavelength

λ280. The ratio between these is a measure of how pure the DNA sample is. The mean

ratio for all of the extraction methods was higher than the optimum ratio between DNA

and protein which suggests there that there was protein contamination in the samples. It

could also be seen that in the samples extracted using the phenol: chloroform method

according to Montes et al., 2003 a much higher DNA concentration was obtained in

comparison with the other extraction methods. The mean concentration in these samples

was 2.4µg/µl. In the others the mean concentration of DNA was 0.96µg/µl for the kit,

0.90µg/µl for the microwaved preparations and 0.77µg/µl for the boiled ones.

xxxviii xxxviii

3.3.1 Visualisation of genomic DNA from the four different extracting methods using gel

electrophoresis

3.3.1 Visualisation of genomic DNA from the four different extracting methods using gel

electrophoresis

Fig. 3.3.1.A Kit (Roche) extraction Fig. 3.3.1.B Phenol: chloroform extraction

1110986 754321 1110986 7543211 2 3 4 5 86 97 101 2 3 4 5 86 97 10

1110986 754321 1110986 754321 1110987654321 1110987654321

1110986 754321 1110986 7543211 2 3 4 5 86 97 101 2 3 4 5 86 97 10

1110986 754321

1110987654321 11109876543211110986 754321

Fig. 3.3.1.C Boiling extraction Fig. 3.3.1.D Microwaving extraction Fig 3.3.1.A-C 1. V. harveyi 02/001 2. V. harveyi 02/109 3. V. harveyi 01/021 4. V. harveyi 01/022 5. V. tubiashii 6. V. alginolyticus 7. V. anguillarum 8. V. tapetis 9. V. splendidus 10. V. parahaemolyticus 11. E. coli Fig 3.3.1.D 1. V. harveyi 02/001 2. V. harveyi 02/109 3. V. harveyi 01/021 4. V. harveyi 01/022 5. V. tubiashii 6. V. anguillarum 7. V. tapetis 8. V. splendidus 9. V. parahaemolyticus 10. E. coli

xxxix

In every extraction method RNA was also observed, in a quite high concentration in some

cases. For three methods: phenol: chloroform, boiling and microwaving it was decided to

treat the samples with an equal volume of RNase to decrease the 1 2 431 2 43

1. V. tapetis 2. V. splendidus 3. V. tapetis, R

possibility of the RNA interfering with the PCR primers. This is not

surprising since the targets for both sets of PCR primers were in the

16S rRNA gene. Also 23S rRNA shows in a quite high

concentration.

The picture (left) shows DNA visualization of V. tapetis and

Nase V. splendidus from the phenol: chloroform extraction before and treated 4. V. splendidRNase treated

us, after RNase treatment.

The fact that the two bands in the middle of the gel disappeared when the extracts were

treated with RNase proved they were RNA. Figure 3.3.1.E illustrates two of the V.

harveyi isolates amplified with VH-primers before and after RNase treatment.

Fig. 3.3.1.E DNA extracted from V. harveyi 02/001 and 02/109 using the phenol:

chloroform method and amplified with VH-primers before and after RNase treatment at

[MgCl2] 3mM

M1 2 3 4

100

400

500

M1 2 3 4 M1 2 3 4

100

400

500

The fact that the expected amplicons of the

VH-sequence at approximately 400 bp are

showing after RNase treatment illustrates

that the 16S rRNA and 23Sr RNA actually

interfered with the primers.

1 V. harveyi 02/001 – no amplified product visible.2 V. harveyi 02/001, RNase treated – PCR product present.3 V. harveyi 02/109 – no amplified product visible. 4 V. harveyi 02/109, RNase treated - PCR product present.M 100 bp DNA ladder

xl

3.4 16S rDNA gene determination

The 16S rDNA gene is present in all eubacteria and contains very highly conserved

nucleotide sequences as well as some variable regions.

3.4.1 [MgCl2] optimization

The optimal magnesium concentration for the primers 63f and 1387r that were used to

amplify the 16S rDNA gene were determined by preparing a magnesium titration in the

range 2 mM-3·5 mM using DNA from one V. harveyi isolate along with one non-harveyi

Vibrio, V. tubiashii. Both DNA template preparations were extracted using the High-Pure

DNA Template Preparation Kit. Amplicons of the expected product size of approximately

1300 bp were detected at the magnesium concentration at 2·5 mM (Figure 3.4.1).

Fig. 3.4.1 [MgCl2] determination for the 16S rDNA primers using DNA templates

extracted with the kit from V. harveyi 02/001 and V. tubiashii NCIMB 2164.

M 100 bp DNA ladder

1 2 63 74 85M 9

100

500

1300

400

1 2 63 74 85M 9

100

500

1300

400

1 2 mM MgCl2 V. harveyi 02/001 2 2·5 mM MgCl2 V. harveyi 02/001 3 3 mM MgCl2 V. harveyi 02/001 4 3·5 mM MgCl2 V. harveyi 02/001 5 Blank 6 2 mM MgCl2 V. tubiashii NCIMB 2164 7 2·5 mM MgCl2 V. tubiashii NCIMB 2164 8 3 mM MgCl2 V. tubiashii NCIMB 2164 9 3·5 mM MgCl2 V. tubiashii NCIMB 2164

xli

3.4.2 Amplicon determination using the 16S rDNA-primers

For the 16S rDNA PCR reactions the DNA extracted with the kit was used. The

magnesium concentration titration showed that the 16S primers bound to the DNA’s

complementary base sequence at a concentration at 2.5mM. Although the PCR reaction

was done using this magnesium concentration only very weak bands were obtained. The

expected 1300 base pair band can be seen in the lanes 1-4, and in lanes 6-10 in Figure

3.4.2. Other smaller fragments in the size of around 100-400 bp were also amplified using

this primer in all of the samples. A band at approximately 100 bp can be seen consistently

in every sample.

Fig. 3.4.2 16S rDNA-primer with a [MgCl2] at 2.5 mM using DNA extracted from 10

Vibrio strains and one E. coli with the High-Pure DNA Template Preparation Kit

M 1 52 63 74 8 9 10 11M 1 52 63 74 8 9

100

1300

400

10 11M 1 52 63 74 8 9 10 11M 1 52 63 74 8 9

100

1300

400

10 11

M 100 bp DNA ladder 1 V. harveyi 02/001 2 V. harveyi 02/109 3 V. harveyi 01/021 4 V. harveyi 01/022 5 V. tubiashii NCIMB 2164 6 V. alginolyticus NCIMB 1903 7 V. anguillarum NCIMB 326 8 V. tapetis NCIMB 4600 9 V. splendidus NCIMB 2231 10 V. parahaemolyticus NCIMB 10441 11 E. coli

xlii

When this experiment, to detect the 16S rDNA gene, would not work out that well it was

decided to concentrate and focus at the more specific VH-primers to be able to detect V.

harveyi.

3.5 PCR using V. harveyi VH-1 and VH-2 primers

3.5.1 [MgCl2] optimization

The optimal concentration of magnesium chloride for the specific VH-primers, VH-1 and

VH-2, was determined by preparing a magnesium chloride titration in the range 2 mM-4

mM. The DNA used came from the kit, boiled and microwaved extractions of two

different isolates from V. harveyi. Amplicons of the expected size in the order of 400 bp

were shown as strongest in the magnesium concentration of 3 mM (Figures 3.5.1.A and

3.5.1.B).

Fig. 3.5.1.A [MgCl2] optimum determination using DNA extracted using the kit method

and the VH-primers.

1 2 63 74 85 9M

100

400

500

1 2 63 74 85 9M

100

400

500

M 100 bp DNA ladder 1 2·5 mM MgCl2 V. harveyi 02/001 2 3 mM MgCl2 V. harveyi 02/001 3 3·5 mM MgCl2 V. harveyi 02/001 4 4 mM MgCl2 V. harveyi 02/001 5 blank 6 2·5 mM MgCl2 V. harveyi 02/109 7 3 mM MgCl2 V. harveyi 02/109 8 3·5 mM MgCl2 V. harveyi 02/109 9 4 mM MgCl2 V. harveyi 02/109

xliii

Fig. 3.5.1.B [MgCl2] optimum determination using DNA from V. harveyi 02/001 and V.

harveyi 02/109 extracted with boiling and microwaving methods, RNase treated, VH

primers.

2 3M 54 6 7 98 10 11 121 M1716151413

100

400

500

2 3M 54 6 7 98 10 11 121 M17161514132 3M 54 6 7 98 10 11 121 M1716151413

100

400

500

100

400

500

2 3M 54 6 7 98 10 11 121 M17161514132 3M 54 6 7 98 10 11 121 M1716151413

100

400

500

2 3M 54 6 7 98 10 11 121 M17161514132 3M 54 6 7 98 10 11 121 M1716151413

100

400

500

100

400

500

M 100 bp DNA ladder 1 2 mM MgCl2 V. harveyi 02/001 Boiled 2 2·5 mM MgCl2 V. harveyi 02/001 Boiled 3 3 mM MgCl2 V. harveyi 02/001 Boiled 4 3·5 mM MgCl2 V. harveyi 02/001 Boiled 5 2 mM MgCl2 V. harveyi 02/109 Boiled 6 2·5 mM MgCl2 V. harveyi 02/109 Boiled 7 3 mM MgCl2 V. harveyi 02/109 Boiled 8 3.5 mM MgCl2 V. harveyi 02/109 Boiled 9 Blank 10 2 mM MgCl2 V. harveyi 02/001 Microwaved 11 2·5 mM MgCl2 V. harveyi 02/001 Microwaved 12 3 mM MgCl2 V. harveyi 02/001 Microwaved 13 3·5 mM MgCl2 V. harveyi 02/001 Microwaved 14 2 mM MgCl2 V. harveyi 02/109 Microwaved 15 2·5 mM MgCl2 V. harveyi 02/109 Microwaved 16 3 mM MgCl2 V. harveyi 02/109 Microwaved 17 3·5 mM MgCl2 V. harveyi 02/109 Microwaved

Stronger bands came up when the DNA from the kit extraction was used even though a

lower concentration of pure DNA was used in these PCR reactions. The DNA extracted

by the boiled and microwaved methods was treated with an equal volume of RNase to

prevent RNA interference with the primers. In an initial attempt to determine the optimum

xliv

PCR magnesium concentration using DNA samples not treated with RNase no

amplification could be seen at all.

3.5.2 Amplicon determination using VH-primers

PCR using the VH primers produced amplicons of the expected approximately 400 bp

size for all four V. harveyi isolates (Figure 3.5.2A). However, a similar product was also

obtained with the V. alginolyticus DNA. The different isolates gave bands that varied in

strength, where isolates 02/001 and 02/109, along with V. alginolyticus, seemed to give

the strongest bands. No product was obtained with DNA from the other four Vibrio

species or the E. coli.

Fig. 3.5.2.A PCR using VH-primer with a [MgCl2] at 3 mM using DNA from 10 Vibrio

strains and one E. coli extracted with the High-Pure DNA Template Preparation Kit.

M 1

100

2

400

3

500

4 5 6 7 8 9 10 11 12M 1

100

2

400

3

500

4 5 6 7 8 9 10 11 12

M 100 bp DNA ladder 1 V. harveyi 02/001 7 V. alginolyticus NCIMB 1903 2 V. harveyi 02109 8 V. tapetis NCIMB 4600 3 V. harveyi 01/021 9 V. splendidus NCIMB 2231 4 V. harveyi 01/022 10 V. parahaemolyticus NCIMB 10441 5 Blank 11 E. coli 6 V. tubiashii NCIMB 2164 12 Blank

xlv

DNA from a subset of the strains extracted using the microwaving method was tested

with the VH-primers (Figure 3.5.2.B). Only the V. harveyi samples produced an

amplicon. The DNA from three non-harveyi Vibrio, V. tubiashii, V. tapetis and V.

splendidus gave no product. Unfortunately, the DNA from the V. alginolyticus evaporated

during extraction and could not be examined. DNA extracted by the microwave, boiling

and phenol: chloroform methods had to be RNase treated before being subjected to PCR.

Fig. 3.5.2.B VH-primers with a [MgCl2] at 3 mM using DNA extracted according to the

microwaving method from four strains of V. harveyi and three strains of non-harveyi

Vibrio species

M 1 52 43 6B 7M 1 52 43 6B 7

100

400

M 1 52 43 6B 7M 1 52 43 6B 7

100

400

B Blank M 100 bp DNA ladder 1 V. harveyi 02/001 2 V. harveyi 02/109 3 V. harveyi 01/021 4 V. harveyi 01/022 5 V. tubiashii NCIMB 2164 6 V. tapetis NCIMB 4600 7 V. splendidus NCIMB 2231

xlvi

3.6 Diagnostic Detection Limit

The sensitivity of the VH-primers was examined using DNA that was extracted from a

series of diluted bacterial cultures, 10-1, 10-3, 10-5, 10-7 and 10-8. Since the diluted bacterial

suspensions were cultured on agar plates as well a calculation could be made to be able to

see how many cells, approximately, could give an amount of DNA that the primers would

be able to amplify the template sequence in. The undiluted salted tryptic soy broth

cultures contained approximately 4x108 colony forming units/ml (Table 3.1.1.B). For

each extraction 1.5 ml of culture was used. This means that in the final volume of 200µl

of elution buffer (kit extraction method) there were 1.5x4x108 (6x108) cells for the

undiluted sample, 6x107 cells in the 10-1 dilution and so on down to the 10-8 dilution that

theoretically only contained 6 bacterial cells. Since only 5 µl of the DNA sample was

used in the PCR reaction it theoretically means that the undiluted PCR sample contained

5/200x6x108 (1.5x107) cells. The PCR reactions and amplicons of the specific sequence

for V. harveyi with the VH-primers can be seen in Figure 3.6.A. There was no PCR

product with the DNA extracted from the undiluted bacterial culture but DNA from all

other dilutions of the culture down to the 10-8 dilution showed the expected amplicon.

Theoretically this last dilution contained DNA extracted from a dilution that had no

bacteria in it. Since the negative control (blank) was negative as expected there could be

conclusively no contamination.

For the 16S rDNA primer pair there was a lot of non-specific bands in the gel. An

amplicon of the expected approximately 1300 bp size can be seen in lanes 1, 2 and 3,

although they are very weak in lanes 1 and 2 (Figure 3.6.B). The strongest PCR

amplification of the 16S rDNA sequence was obtained with the dilution of 10-1. Some

xlvii

non-specific bands were also strongest in the 10-1 dilution. Thus the diagnostic detection

limit would be 1.5x106 cells in this case.

Fig 3.6.A-B Diagnostic Detection Limit determination using the VH-primers (A) and the

16S rDNA-primers (B) together with DNA from diluted bacterial cultures in the range

10-1 to 10-8.

A B

M 1 42 53 6

400

300

M 1 42 53 6

400

300

M 1 4 2 53 6 7

100

500

1300

M 1 4 2 53 6 7

100

500

1300

M 100 bp DNA ladder 1 V. harveyi 02/001, undiluted 1 V. harveyi 02/001, undiluted (1.5x107 cells) 2 V. harveyi 01/001, diluted 10-1 2 V. harveyi 02/001, diluted 10-1 (1.5x106 cells) 3 V. harveyi 01/001, diluted 10-3 3 V. harveyi 02/001, diluted 10-3 (1.5x104 cells)

4 V. harveyi 01/001, diluted 10-5 4 V. harveyi 02/001, diluted 10-5 (1.5x102 cells) 5 V. harveyi 01/001, diluted 10-7 5 V. harveyi 02/001, diluted 10-7(1.5x100 cells) 6 V. harveyi 01/001, diluted 10-8 6 V. harveyi 02/001, diluted 10-8 (0.15 cells) 7 Blank

3.7 Analytical Sensitivity

The analytical sensitivity was determined using a dilution series of the target. A dilution

of the actual DNA was therefore made in the range 10-1 to 10-5 using DNA from V.

harveyi (02/001 and 02/109) extracted using the kit method. The undiluted DNA would

theoretically contain DNA from 1.5x107 cells since only 5 µl of DNA was added to the

actual PCR tube (5/200x1.5x4x108). An endpoint was reached with one of the isolates, V.

harveyi 02/001 (Figure 3.7). The concentration of DNA was enough to be amplified down

xlviii

to the dilution 10-4. At a 10-5 dilution not even a weak band could bee seen. Thus

theoretically 1500 cells could be detected by the PCR whereas 150 cells could not. With

the DNA from V. harveyi 02/109 the PCR product was amplified even using DNA from a

theoretical 150 bacterial cells.

Fig. 3.7 Analytical sensitivity of VH-PCR using DNA dilutions in the range undiluted to

10-5 of V. harveyi 02/001 and 02/109.

M 1 82 73 64 5 9 10 11 12

400

300

M 1 82 73 64 5 9 10 11 12

400

300

M 100 bp DNA ladder 1 V. harveyi 02/001, DNA undiluted 2 V. harveyi 02/001, DNA diluted 10-1

3 V. harveyi 02/001, DNA diluted 10-2

4 V. harveyi 02/001, DNA diluted 10-3

5 V. harveyi 02/001, DNA diluted 10-4

6 V. harveyi 02/001, DNA diluted 10-5

7 V. harveyi 02/109, DNA undiluted 8 V. harveyi 02/109, DNA diluted 10-1

9 V. harveyi 02/109, DNA diluted 10-2

10 V. harveyi 02/109, DNA diluted 10-3

11 V. harveyi 02/109, DNA diluted 10-4

12 V. harveyi 02/109, DNA diluted 10-5

xlix

4.0 DISCUSSION

The aim of this project was to detect Vibrio harveyi and biochemically characterise

different isolates. The detection was made by using PCR together with specific primers

for a sequence within the 16S rDNA gene. This sequence is specific for V. harveyi.

Beside the molecularly detection of V. harveyi were four extraction methods evaluated.

The biochemically characterisation was made by using API 20E. Different growth media

were used to The Vibrio strains were also tested for tissue adherence.

Morphology using different media

The four isolates of V. harveyi showed variations in their colonial morphologies on the

salted blood agar, VHA and TCBS agar. This may reflect a phenotypically diverse nature

of V. harveyi although Harris et al. (1996) stated that the twenty strains of V. harveyi that

they tested displayed very similar morphologies on VHA. The colonies of two isolates,

02/001 and 02/109, swarmed slightly on the salted blood agar but individual colonies

could still be seen. V. harveyi, 01/021 and 01/022 swarmed much more strongly and no

individual colonies could be seen. The other Vibrio species that swarmed on the salted

blood agar was V. alginolyticus. These species seem to be similar at a molecular level and

may share the genes necessary for swarming. This ability may also be an invasive factor

that helps the bacteria to form biofilms or colonise and/or invade fish epithelial cells. The

four V. harveyi isolates were all haemolytic and this is a recognised virulence factor

(Pujalte et al., 2003). Austin et al. (2003) have reported an association between the

presence of a bacteriophage and level of haemolytic activity in V. harveyi strains. It would

l

be interesting to examine the four isolates for the presence of this bacteriophage. All the

Vibrio species grown on salted blood agar were haemolytic except V. anguillarum.

On the marine agar almost every strain looked the same so although this medium may be

good for isolation of Vibrio species it is of little use for differentiating and identifying

particular species.

On the VHA the V. harveyi strains appeared to grow better than the other Vibrio species

that were used in this project although obviously only a relatively small number of strains

were tested. Their ability to ferment cellobiose and decarboxylate ornithine separate them

from the other species and enable them to produce distinctive yellowish green colonies

instead of blue or colourless colonies like the other strains of Vibrio. V. harveyi could be

differentiated from V. alginolyticus using this medium. This could be important since

these two species are difficult to separate even with molecular methods such as the VH-

primer PCR. It would again be desireable to test a greater number of isolates of the two

species on the VHA. This agar was quite selective for the Vibrio species since every

Vibrio grew on it while only one of the non-Vibrio species, K. pneumoniae, did. The

incubation of the cultures at temperatures as low as 15ºC is probably one important factor

that selects the aquatic Vibrio. Moreover, the absence of magnesium chloride that many of

the marine pathogens require for their growth gives it its species selective properties

(Harris et al., 1996). So this medium appears suitable for separating Vibrio from non-

Vibrio strains.

li

On TCBS agar all the Vibrio species grew with similar colonial morphologies. Like the

VHA this agar might be useful for separating Vibrio species from non-Vibrio strains

although two of the non-Vibrio tested did grow on this medium.

Adherence

No adherence could be seen with any of the Vibrio isolates examined in the cell adherence

test although the control M. catarrhalis showed good adherence. In this test epithelial

cells from human laryngeal tissue were used. The Vibrio species that were tested for

adhesion are marine pathogens and isolated from diseased aquatic animal. It would be

more informative and interesting to assess their tissue adhesiveness using fish cell lines.

Unfortunately these were not available for this project.

Biochemical characterisation of the Vibrio species

The biochemical characteristics differ between the different species of Vibrio. One

general biochemical test that was positive for all the Vibrio species is the oxidase test.

According to Thompson et al,. (2004) are the Vibrio species mostly oxidase positive. The

API 20E gave a series of biochemical reactions in different substrates. The results for six

of the tests were similar for all of the 10 different strains tested. These were L-arginine

(ADH), sodium thiosulfate (H2S), inositol (INO), L-rhamnose (RHA), D-melibiose

(MEL) and L-arabinose (ARA). The L-lysine (LDC) test was only positive for the V.

harveyi isolates and for V. alginolyticus. The production of lysine decarboxylase in these

species may be a virulence factor that the bacteria produces to lyse the fish epithelial

cells. The L-ornithine test (ODC) test was only positive for the four V. harveyi isolates.

This supports the use of the substrate in the differential medium for V. harveyi (VHA). D-

sorbitol gave a positive reaction with two of the V. harveyi isolates, 01/021 and 01/022,

lii

while the other two strains reacted negatively on this substrate. The V. harveyi isolates

also reacted differently in the D-sucrose (SAC) test. The isolates 02/001 and 02/109 were

negative in this test while 01/021 and 01/022 were positive. This illustrates one more time

the differences within the V. harveyi species. The citrate test was the most variable test

and gave different reactions in many strains. This lack of reproducibility in the reaction

may be the result of differences in the density of the inoculum as well as on the incubation

time. It could also be the result of how the reaction was read and interpreted. The four V.

harveyi isolates reacted in the same way in all of the tests except in the D-sorbitol and D-

sucrose tests.

Overall, the use of the API 20E system to identify Vibrio is very limited. This is not

surprising since the kit is designed for Enterobacteriaceae not Vibrionaceae. A different

range of substrates would be more suitable for vibrios, perhaps based on the work of

Ottaviani et al. (2003). With the increasing importance of aquaculture and its associated

pathogens it may be timely for a Vibrionaceae biochemical identification kit to be

commercially marketed.

Extraction methods

All four extraction methods assessed produced DNA but in different concentrations. The

method that gave the highest amount DNA was the phenol: chloroform method. The kit

gave a lower concentration of DNA but its purity was the highest. Both the microwaved

and the boiled preparations gave DNA concentrations apparently not much lower than

that extracted by the kit, but these preparations had a higher level of contamination which

would have affected the accuracy of the DNA determination calculation. One could

introduce some purification step into both these extraction techniques to improve the

liii

quality of the DNA produced but this would prolong the extraction processes and increase

their cost. .

Phenol: chloroform extraction

The method chosen was that of Montes et al. (2003). Initial results were poor until the

method was modified by the addition of a Proteinase K digestion step not mentioned in

the published paper. This method gave a high rate of contamination in spite of phenol:

chloroform purification. It is possible that the contamination was, in part as least, due to

mistakes being made when removing the upper-phase during separation steps. This was

found to be technically dificult to do without disturbing the proteinaceous material that

formed at the interface between the upper and lower phases. More practice with the

method may have improved the results. Overall, the method was a relatively laborious one

with a number of transfers between eppendorf tubes and the use of hazardous materials

such as phenol, chloroform and isoamylalcohol. The method gave a high amount of DNA

but also quite a high concentration of RNA with 16S and 23S RNA bands being very

prominent on initial gels (picture, page 37). This was found to interfere with subsequent

PCR amplification.

High-Pure PCR Template Preparation Kit (Roche)

This extraction technique produced the DNA with the highest purity. The samples did not

have to be treated with RNase before PCR amplification with the primers. A higher

concentration of DNA than RNA were obtained in these extractions compared to the other

methods (see fig. 3.3.1A-D). Since a commercial kit was being used the method was most

expensive of the four extraction methods assessed. However, it did give consistently good

DNA for VH-PCR amplification.

liv

Boiling and microwaving methods

These two methods gave similar amount of DNA. Probably since they are based on the

same principle; boiling the cells by in water or by microvaving to extract the DNA. The

DNA from both of the methods had to be RNase treated to get rid of the RNA that

otherwise interfered with the primers.

For the magnesium titration the DNA extracted by the kit method was used where 2 µl

neat DNA was added to a premix of 23 µl. For the boiled and microwaved template

preparations a volume of 2.5 µl neat DNA was used together with an equal volume of

RNase. This mixture was then added to 20 µl of premix. Even though a higher

concentration of DNA was used for the boiled and microwaved extractions weaker PCR

amplicon product bands were obtained. On the other hand a higher concentration of

premix was added to the kit extracted DNA.

RNase treatment

Two bands present on initial gels were shown conclusively to be RNA since they

disappeared on RNase treatment. They were probably representing the bacterial 16S

rRNA and the 23S rRNA. This presence of the RNA interfered with the PCR reaction

(Figure 3.3.1.E). This is not surprising since the targets for both sets of PCR primers were

in the 16S rRNA gene. Obviously complementary sequences for some of the primers

would be present on the 16S rRNA and could bind and inactivate the primers. The two

bands of RNA were even visible in the sample extracted with the kit but the sequences in

these samples could be amplified without RNase treatment.

lv

16S rDNA gene

This gene is present in all eubacteria and contains highly conserved nucleotide sequences

as well as some variable regions. Primers were designed by Oakey et al. (2003) to amplify

this sequence to produce an amplicon product of approximately 1300 bp In this project

these amplicons did not appear as well as they should. In the magnesium concentration

they appeared to amplify correctly and specifically. However, when all the strains were

then tested with the optimised magnesium concentration multiple non-specifics banding

appeared. Very weak band at approximately 1300 bp did show up with some of the

species but not with others. Some of the non-specific amplicons appeared to be amplified

to a much higher concentration. Sometimes these amplicons appeared and sometimes they

did not. Various reagents were changed to try to overcome this problem but it persisted.

The concentration of pure DNA used for the screen gels was higher (2.5x) in the PCR-

reaction, where the very weak amplicons at 1300 bp appeared, than in the magnesium

titration. Perhaps the DNA sample load was too high and meant that salts and other

material, contaminating the DNA extracts were in high enough concentration to interfere

with the amplification. Too high a salt concentration in the DNA sample often tends to

require a lower magnesium concentration. If there was too high a salt concentration in

these samples it may mean that the magnesium concentration used was too high. This

would increase the level of non-specific amplifications due to a reduced fidelity of the

Taq enzyme. In Figure 3.6.B where the diagnostic detection limit was determined using

16S rDNA primers the 1300 bp amplicon band is much stronger in the 10-1dilution than in

undiluted DNA sample. That could mean that the pure DNA contains too much inhibitory

substances, such as too high a salt concentration, so that the sample has to be diluted 10-1

before the 1300 bp sequence will appear. Perhaps a lower concentration of pure DNA in

the actual PCR tube would have produced better results. The extract of V. anguillarum

lvi

did not show at all in the DNA visualisation with gel electrophoresis (Figure 3.3.1.A-D)

and in the λ260 readings these samples gave a very low concentration of DNA (absorbance

readings, appendix). However, the sample did give a strong 1300 bp product in the 16S

rDNA amplification (Figure 3.4.2). Further time would have been required to resolve the

problems with the 16S PCR.

VH primers

The VH primer is supposed, according to Oakey et al., 2003, be specific for V. harveyi.

The primer is designed for one of the variable regions within the 16S rDNA gene that

occur in V. harveyi but not in other bacteria. The primers did not amplify the non-harveyi

Vibrio strains used except from V. alginolyticus. In their study from 2003 it was reported

that there might be a high level of homogeneity between the 16S rRNA genes of V.

alginolyticus and V. harveyi and it was noted that V. alginolyticus (and V. campbellii) is

the most closely related species to V. harveyi. However, it could also be seen that the VH-

1 primer annealing site of the two species was identical. The annealing sequences for VH-

2 are different but still very similar between the two species. Variability in the VH-2

priming site in V. alginolyticus means that some strains will give amplicons with the VH

primers whereas others will not. The specificity seems to depend on the annealing

temperature. Oakey et al. (2003) also reported that the non-specificity increases with a

falling annealing temperature. A temperature at 64ºC gave non-specific bindings and

amplicons from V. alginolyticus and V. campbellii. In contrast an annealing temperature

of 66ºC did not give reproducible amplicons for V. harveyi.

It would be interesting to compare the VH primers with the toxR targeted PCR primers of

Conejero and Hedreyda (2003) using a more extensive collection of isolates.

lvii

Diagnostic Detection Limit

The dilutions were extracted using the High-Pure DNA template kit down to the dilution

10-8 and an endpoint was still not reached when the VH primers were used. This sample,

10-8, would theoretically only contain, since 5 µl of the DNA was used,

5/200x1.5x4x108x10-8 = 0.15 bacterial cells. This appears rather too sensitive on first

consideration. It may be explained by the natural variability inherent in any bacterial plate

count. The term “colony forming unit” is used instead of “colony” because some of the

colonies visible on the agar surface may have grown from a pair or clump of cells rather

than a single cell. Thus the estimated bacterial population would be lower than the actual

one. It is also possible that the bacterial cultures contained a proportion of dead or

dormant cells that would fail to grow on the medium but whose DNA would still be

extracted and provide PCR targets.

For the 16S rDNA primers the endpoint was reached when no amplification was visible at

the dilution 10-3giving an estimated detection level of 6x105 cells. Obviously, this limit

may be an underestimation due to the presence of the non-specific banding. It is generally

more difficult to amplify larger amplicons so one might expect that the figure would be

higher.

Analytical Sensitivity

In this test the DNA extract was diluted down before PCR amplification was done on each

dilution. An endpoint was reached for one of the V. harveyi isolates (02/001) but not for

the other one (02/109) (Figure 3.7). Theoretically this would mean that the latter sample

had a higher concentration of DNA from the beginning since the bands are stronger in

these dilutions, especially the 10-1 and in 10-2 dilutions. This isolate may have grown

lviii

better in the tryptic soy broth or perhaps the cell wall of one of the strains was weaker and

therefore lysed more easily to release its DNA.

5.0 CONCLUSIONS

The aim of this project was to compare DNA extraction methods and subsequent PCR

with biochemical and phenotypical tests in the identification of V. harveyi.

The use of the API 20E kit to identify Vibrio species is limited and would require

supplementary tests.

One can conclude that a combination of the molecular PCR using VH-primers together

with the phenotypic growth on VHA is a good choice for separating V. harveyi from V.

alginolyticus when the latter species gives a positive reaction with these primers. Since

the VHA plates have to be incubated for 24-48h before any growth can be distinguished

this would not be the most rapid way to identify this species. Only pure bacterial cultures

were assessed in this project so one could recommend isolation of colonies on VHA

followed by VH -PCR of colonies giving the characteristic appearance of V. harveyi.

The easiest, cheapest and most rapid way to extract the DNA seemed, in this project, to be

the microwaving method. These samples must be treated with an equal volume of RNase

before PCR testing.

lix

6.0 REFERENCES

Austin B, Pride AC and Rhodie GA. Association of a bacteriophage with virulence in

Vibrio harveyi. Journal of Fish Diseases, 2003; 26: 55-58.

Conejero MJU and Hedreyda CT. Isolation of partial toxR gene of Vibrio harveyi and

design of toxR-targeted PCR primers for species detection. Journal of Applied

Microbiology, 2003; 95: 602-611.

Garrity GM and Holt JN. “A road map to the manual, p. 119-166. In DR Boone et al (ed)

Bergey’s manual of systematic bacteriology, 2nd edition, volume 1, Springer-Verlag KG,

Berlin, Germany, cited in Thompson et al. (2004).

Gomez-Gil B, Soto-Rodríguez S, García-Gasca A, Roque A, Vazquez-Juarez R, L

Thompson F, Swings J. Molecular identification of Vibrio harveyi-related isolates

associated with diseased aquatic organisms. Microbiology, 2004; 150: 1769-1777.

Harris L, Owens L, Smith S. A Selective and Differential Medium for Vibrio harveyi.

Applied and Environmental Microbiology, 1996; 62: 3548-3550.

Hernández G, Olmos J. Molecular identification of pathogenic and non-pathogenic strains

of Vibrio harveyi using PCR and RAPD. Applied Microbiological Biotechnology, 2004;

63: 722-727.

lx

Jensen S, Samuelsen OB, Andersen K, Torkildsen L, Lambert C, Choquet G, Paillard C

and Bergh O. Characterization of strains of Vibrio splendidus and V. tapetis isolated from

corkwing wrasse Symphodus melops suffering vibriosis. Diseases of Aquatic Organisms,

2003; 53: 25-31.

Karunasagar I, Otta SK. Biofilm formation by Vibrio harveyi on surfaces. Aquaculture,

1996; 140: 241-245.

Montes M, Farto R, Pérez MJ, Nieto TP, Larsen JL, Christensen H. Characterization of

Vibrio strains isolated from turbot (Scophthalmus maximus) culture by phenotypic

analysis, ribotyping and 16S rRNA gene sequence camparison. Journal of applied

Microbiology, 2003; 95: 693-703.

Oakey HJ, Levy N, Bourne DG, Cullen B, Thomas A. The use of PCR to aid in the rapid

identification of Vibrio harveyi isolates. Journal of Applied Microbiology, 2003; 95:

1293-1303.

Ottaviani D, Masini L and Bacchiocchi S. A biochemical protocol for the isolation and

identification of current species of Vibrio in seafood. Journal of Applied Microbiology,

2003; 95: 1277-1284.

Pedersen K, Verdonck L, Austin B et al. Taxonomic evidence that Vibrio carchariae

Grimes et al. 1985 is a junior synonym of Vibrio harveyi (Johnson and Shunk 1936)

Bauman et al. 1981. International Journal of Systematic Bacteriology, 1998; 48: 749-758.

lxi

Pesci EC. Quorum Sensing. In Bacterial Protein Toxins. DL Burns, JT Barbieri, BH

Iglewski, R Rappnoli, EC Pesci. American Society of Microbiology, Washington DC.

2003; 4: 55-58

Pujalte MJ, Sitja-Bobadilla A, Macian MC, Belloch C, Alvarez-Pellitero P, Perez-

Sanchez J, Uruburu F and Garay E. Virulence and molecular typing of Vibrio harveyi

strains isolated from cultured Dentex, Gilthead Sea Bream and European Sea Bass.

Systematic and Applied Microbiology, 2003; 26: 284-292.

Robertson PAW, HU H-S and Austin B. An enzyme-linked immunosorbent assay

(ELISA) for the detection of Vibrio harveyi in penaeid shrimp and water. Journal of

Microbiological Methods, 1998; 34: 31-39.

Thompson LF, Iida T, Swings J. Biodiversity of Vibrios. Microbiology and Molecular

Biology Reviews, 2004; 68: 403-431.

Wang HX, Y Leung K. Biochemical characterization of different types of adherence of

Vibrio species to fish epithelial cells. Microbiology, 2000; 146: 989-998.

Zorilla I, Arijo S, Chabrillon M, Diaz P, Martinez-Manzanares E, Balebona MC and

Morinigo MA. Vibrio species isolated from diseased farmed sole, Solea senegalensis

(Kaup), and evaluation of the potential virulence role of their extracellular products.

Journal of Fish Diseases, 2003; 26: 103-108.

lxii

7.0 APPENDIX

Growth media

Blood agar with 3.5% NaCl: 39 g of Columbia base agar (Oxoid) were dissolved in 1000

ml of distilled water. Since the Columbia base agar already consists of 0.5% NaCl only an

extra 3% NaCl was added (30 g). This solution was autoclaved, cooled down to

approximately 60ºC and a bottle of horse blood (100 ml) added. The solution was shaken

a couple of times and then poured into sterile petri dishes.

Tryptic soy broth: 15 g of Tryptic soy broth powder (Oxoid) that already contained 0.5%

NaCl were dissolved in 500 ml of distilled water. 15 g of NaCl were then added to reach a

salt concentration of 3.5%. The solution were autoclaved and poured into sterile petri

dishes.

Marine agar: 55 g of Marine Agar were dissolved in 1000 ml of distilled water. The

solution was autoclaved and then poured into sterile petri dishes.

T.C.B.S cholera medium: 88 g of T.C.B.S powder were dissolved in 1000 ml of distilled

water. The solution was boiled until all ingredients were completely dissolved.

VHA (Harris et al., 1996): All components were mixed in a boiling flask and dissolved in

1000 ml of destilled water. The solution were adjusted to pH9 by the addition of 1M

NaOH and boiled until all of the powder was completely dissolved. The solution was then

left to cool down and dispensed into sterile petri dishes.

Ingredients: D-cellobiose, 2 g (molekula, M53340398); L-ornithine, 2 g (molekula,

M80879264); NaCl, 30 g; Tris[hydroxymethyl]aminomethane, 1.21 g; agar, 20 g;

K2HPO4, 0.075 g; thymol blue, 0.04 g; bromothymol blue, 0.04 g; Bacto Peptone, 0.1 g

(Oxoid LTD., L37); yeast extract, 0.1 g (Oxoid LTD., CM19).

lxiii

Absorbance readings of DNA extracts. High-Pure PCR Template Preparation Kit Phenol: chloroform

Strain λ260 λ280 λ260/ λ280

V. harveyi 02/001

Strain λ260 λ280 λ260/ λ280

0.189 0.181 1.0425

V. harveyi 02/109

V. harveyi 02/001 0.728 0.474 1.5379

0.196 0.185 1.0588

V. harveyi 01/021

V. harveyi 02/109 0.597 0.402 1.4832

0.197 0.185 1.0632

V. harveyi 02/022

V. harveyi 01/021 0.540 0.370 1.4588

0.199 0.187 1.0606

V. tubiashii

V. harveyi 02/022 0.640 0.417 1.5332

0.186 0.179 1.0354

V. alginolyticus

V. tubiashii 0.500 0.353 1.4169

0.217 0.195 1.1124

V. anguillarum

V. alginolyticus 0.682 0.445 1.5320

0.175 0.174 1.0070

V. tapetis

V. anguillarum 0.182 0.179 1.0178

0.183 0.177 1.0316

V. splendidus

V. tapetis 0.421 0.309 1.3614

0.188 0.180 1.0461

V. parahaemolyticus

V. splendidus 0.338 0.261 1.2951

0.189

0.182 1.0403

E. coli

V. parahaemolyticus 0.399 0.294 1.3559

0.181 0.177 1.0234 E. coli 0.184 0.179 1.0273

Boiled Microwaved

Strain λ260 λ280 λ260/ λ280 Strain λ λ λ

V. harveyi 02/001 0.178 0.106 1.6724

V. harveyi 02/109 0.181 0.100 1.8015

V. harveyi 01/021 0.148 0.089 1.6544

V. harveyi 02/022 0.154 0.092 1.6768

V. tubiashii 0.131 0.081 1.6114

V. alginolyticus 0.286 0.163 1.7541

V. anguillarum 0.078 0.058 1.3580

V. tapetis 0.114 0.072 1.5904

V. splendidus 0.116 0.070 1.6637

V. parahaemolyticus 0.124 0.076 1.6314

E. coli 0.185 0.108 1.7057

260 280 260/ λ280

0.221 0.127 1.7344 V. harveyi 02/001

0.245 0.143 1.7055 V. harveyi 02/109

0.143 0.090 1.5943 V. harveyi 01/021

0.161 0.102 1.5740 V. harveyi 02/022

0.188 0.115 1.6337 V. tubiashii

- - - V. alginolyticus

0.070 0.050 1.4093 V. anguillarum

0.124 0.079 1.5585 V. tapetis

0.218 0.136 1.6025 V. splendidus

0.139 0.089 1.5752 V. parahaemolyticus

0.286 0.175 1.6320 E. coli