detection and characterisation of - diva portal130396/fulltext01.pdfmethods this report comprises...
TRANSCRIPT
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