Hybrid Vibrio vulnificus

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Hybrid Vibrio vulnificus

Naiel Bisharat,* I. Cohen,? lind M. Harding,* Falush,*

Derrick W. Crook,* Tim Peto,* and C. Maiden*

*University of Oxford, Oxford, United Kingdom; and ?Tel Aviv University,

Ramat Aviv, Israel

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The recent emergence of the human-pathogenic Vibrio vulnificus in Israel was

investigated by using multilocus genotype data and modern molecular

evolutionary analysis tools. We show that this pathogen is a hybrid organism

that evolved by the hybridization of the genomes from 2 distinct and

independent populations. These findings provide clear evidence of how

hybridization between 2 existing and nonpathogenic forms has apparently led

to the emergence of an epidemic infectious disease caused by this pathogenic

variant. This novel observation shows yet another way in which epidemic

organisms arise.

Vibrio vulnificus, a ubiquitous inhabitant of marine and estuarine

environments, is considered one of the most dangerous waterborne pathogens.

The case-fatality rate for V. vulnificus septicemia may reach 50% (1). Human

infection is generally acquired through eating contaminated raw or

undercooked seafood or through contamination of wounds by seawater or marine

animals (2). Infected persons with preexisting liver disease,

hemochromatosis, or compromised immune systems are at particularly high risk

for fatal septicemia (3-8).

Human infections are sporadic and almost entirely caused by strains of

biotype 1, while biotype 2 strains have been reported to cause disease

mainly among eels and rarely infect humans (9). During the summer of 1996, a

major outbreak of systemic V. vulnificus infections started among Israeli

fish market workers and fish consumers (10,11). Molecular studies showed

that the disease outbreak was caused by a previously undescribed biotype

that exhibited a distinct phenotypic and molecular pattern, designated

biotype 3 (11).

The origins of this emergent infectious disease have not been fully

understood, although it was originally thought to arise mostly from human

behavior and work practices (10). On the basis of these assumptions, new

fish-handling procedures were introduced (11,12). However, disease

continued, although at a lower incidence. Therefore, studies were undertaken

to determine whether this novel outbreak of disease was caused by a specific

lineage or clone. The emergence of this new biotype could not be resolved by

conventional microbiologic and molecular typing approaches. We investigated

this outbreak by combining a multilocus sequence typing approach (13) with

molecular evolutionary analyses.

Materials and Methods

Bacterial Isolates

To study the emergence of this new biotype, we examined a collection of 159

V. vulnificus isolates that represented all 3 biotypes from human disease

and environmental sources that originated in Israel (n = 64), the United

States (n = 54), Denmark (n = 7), Germany (n = 6), Spain (n = 5), Sweden (n

= 5), Japan (n = 8), South Korea (n = 2), Singapore (n = 2), Thailand (n =

1), Indonesia (n = 1), and Taiwan (n = 1). In addition, 3 well-characterized

reference strains that represented the 3 biotypes were included, the ATCC

27562 strain (biotype 1, isolated from human blood in the USA), the E-39

strain (biotype 2, isolated from diseased eel in Spain), and ATCC BAA-86

(biotype 3, isolated from human blood in Israel). Biotype 1 strains (n = 82)

consisted of 39 isolates from human disease and 43 environmental isolates;

biotype 2 strains (n = 15) consisted of 13 isolates from diseased eels, 1

from an infected person, and 1 from diseased shrimp. Biotype 3 strains were

isolated from samples from persons with invasive disease in Israel (n = 61)

and from fish-pond water (n = 3). Isolates were grown on blood agar plates

and incubated overnight at 35°C in aerobic conditions. The lists of the

isolates used in this study and their sources can be accessed at

http://pubmlst.org/vvulnificus.

DNA Extraction

The DNeasy kit (QIAGEN GmbH, Hilden, Germany) was used to extract DNA with

the gram-negative bacterial protocol as recommended by the manufacturer.

Briefly, several colonies from a bacterial culture were picked off into

phosphate-buffered saline solution and centrifuged at 7,500 rpm (5,000 x g)

for 10 min. The cell pellet was resuspended in 180 ?L of tissue lysis

buffer, then 20 ?L of proteinase K (10 mg/mL) was added, and the sample was

incubated at 55°C until the tissue was completely lysed. Then 200 ?L of

lysis buffer was added and incubated at 70°C for 10 min. The DNA in the

clear viscous lysates was precipitated with ethanol 95% (vol/vol) and added

to DNeasy mini columns. Ethanol 70% (vol/vol)-based buffers (AW1 and AW2)

were added sequentially to the columns and centrifuged at 8,000 rpm (6,000 x

g). The supernatants were discarded, and the DNA was resuspended in AE

buffer and used for amplification.

Multilocus Sequence Typing (MLST)

This bacterium has 2 chromosomes. Fourteen housekeeping genes (7 from each

chromosome) that encoded enzymes responsible for intermediary metabolism

were identified by searching the genome database

(http://www.ncbi.nlm.nih.gov/genomes/MICROBES/Complete.html) of V.

vulnificus strain CMCP6, with gene sequences from other bacteria. Genetic

loci were chosen for further investigation on the basis of the following

criteria: chromosomal location, suitability for primer design, and sequence

diversity in pilot studies. Ten loci were chosen for the MLST scheme, 5 from

each chromosome. The following were chosen from the large chromosome: glp,

the encoding glucose-6-phosphate isomerase; gyrB, the encoding DNA

gyrase-subunit B; mdh, the encoding malate-lactate dehydrogenase; metG, the

encoding methionyl-tRNA synthetase; and purM, the encoding

phosphoribosylaminoimidazole synthetase. The following were chosen from the

small chromosome: dtdS, the encoding threonine dehydrogenase; lysA, the

encoding diaminopimelate decarboxylase; pntA, the encoding transhydrogenase

alpha subunit; pyrC, the encoding dihydroorotase; and tnaA, the encoding

tryptophanase. Their chromosomal location suggested that it was unlikely for

any of the loci to be coinherited in the same recombination event, as the

minimum distance between loci was 300 kb (Table).

Amplification and Nucleotide Sequence Determination

Polymerase chain reaction (PCR) products were amplified with oligonucleotide

primer pairs designed from the V. vulnificus genome sequence. These primers

provided reliable amplification from a diverse range of samples (available

from http://pubmlst.org/vvulnificus). Each 50-?L amplification reaction

mixture was made up of 10 ng of V. vulnificus chromosomal DNA, 100 pmol of

each PCR primer (MWG Biotech, Ebersberg, Germany), 10 x PCR buffer with 1.5

mmol/L MgCl2 (QIAGEN GmbH), 0.5 U of Taq DNA polymerase (QIAGEN GmbH), and

1.6 mmol/L deoxynucleoside triphosphates (ABgene, Epsom, UK). The reaction

conditions were denaturation at 94°C for 1 min, primer annealing at 50°C for

45 s and extension at 72°C for 1 min for 30 cycles. The amplification

products were purified by precipitation with 20% polyethylene glycol and 2.5

mol/L NaCl (14), and their nucleotide sequences were determined at least

once on each DNA strand by using internal nested primers (available from

http://pubmlst.org/vvulnificus) and ABI PRISM BigDye Terminators v 3.0

Reaction Mix (Applied Biosystems, City, CA) in accordance with the

manufacturer's instructions. Unincorporated dye terminators were removed by

precipitation of the termination products with sodium acetate (3 mol/L, pH

5.2) and 95% ethanol, and the reaction products were separated and detected

with an ABI PRISM 3730 DNA Analyzer (Applied Biosystems). Sequences were

assembled from the resultant chromatograms with the STADEN suite of computer

programs and edited to resolve any ambiguities (15). For each locus, every

different sequence was assigned a distinct allele number in order of

identification; these sequences were internal fragments of the gene, which

contained an exact number of codons. Each isolate was therefore designated

by a 10-integer number (the allelic profile), which corresponds to the

allele numbers at the 10 loci in the following order: glp, gyrB, mdh, metG,

purM, dtdS, lysA, pntA, pyrC, and tnaA. Isolates with the same allelic

profile are assigned to the same sequence type (ST), which were numbered in

the order of their identification (ST-1, ST-2, and so on). The data have

been deposited (http://pubmlst.org/vvulnificus).

Inferring the Population Structure and Ancestral Sources

The program STRUCTURE was used to define the population structure and

identify the ancestral sources of the 10 gene fragments from all the

strains. STRUCTURE is a recently developed program that implements a

Bayesian model approach for inferring population structure and ancestral

sources from multilocus genotype data (16). Of the 4,326 nucleotides

sequenced for each isolate from the 10 genes, 447 nt were polymorphic. For

the purposes of the analysis, these nucleotides were used by STRUCTURE as

individual loci. STRUCTURE can infer the population structure by using a

variety of models, including the linkage model (17), which incorporates

linkage disequilibrium due to correlations in ancestry between loci that

reflects admixture between populations. This approach has recently been used

to elucidate the structure and evolution of populations of the human

pathogen Helicobacter pylori (18), and we have used the same method here.

For the purposes of the analysis, the nucleotide sequence of the 10

housekeeping gene fragments of 2 clinical strains of V. vulnificus, CMCP6

and YJ016, whose complete genome sequence has been recently completed

(http://www.ncbi.nlm.nih.gov/genomes/MICROBES/Complete.html), were added to

all the data sets.

Results

Genotypes Identified

The 159 isolates were resolved into 70 STs, 56 of which were present only

once in the entire collection. Eighty-two isolates (51.6%) were represented

by 1 of 4 STs: ST-8 was the most common and occurred 62 times (39%); ST-6

occurred in 11 isolates (6.9%); ST-32 occurred in 5 isolates (3.1%); ST-16

occurred in 4 isolates (2.5%). The remaining 21 isolates resolved into 10

sequence types. Strains of biotype 1 (n = 82) resolved into 66 STs. Biotype

2 (15 isolates) resolved into 4 STs; ST-6, ST-9, ST-10, and ST-48. ST-6 was

the most common, occurring 11 times and consisting of all the

indole-negative isolates. All biotype 3 strains (n = 62) were genetically

identical and belonged to ST-8.

Population Structure and Ancestral Sources

Figure 1

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Figure 1. Triangle plots of STRUCTURE results. Only 2 populations were

identified (A and ...

Figure 2

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Figure 2. Results of a Bayesian cluster analysis by STRUCTURE...

Figure 3

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Figure 3. A neighbor-joining tree of representative isolates from the 2

populations is plotted with the inferred ancestral sources...

The observed sequence variation between the 2 chromosomes was comparable.

Initial analysis of sequence data from the 10 gene fragments showed that

extensive recombination had occurred within all the genetic loci under study

(data not shown). Constructing phylogenetic trees in the presence of

recombination is problematic because different parts of the sequence may

have different phylogenetic histories. Therefore, we analyzed the data with

the program STRUCTURE. First, we tested the assumption that the 3 V.

vulnificus biotypes represent 3 distinct predetermined populations of this

pathogen (K = 3). The results of multiple analyses with STRUCTURE were

incompatible with this assumption; in all cases, only 2 populations were

identified, populations A and B (Figure 1 and data not shown). Further,

while biotype 1 was present in both populations, biotype 2 was present only

in population A (Figure 1A). Biotype 3 occupied an intermediate position

between the 2 populations (Figure 1A). Figure 1B shows that an

overrepresentation of human disease isolates occurred in population B and an

overrepresentation of environmental isolates occurred in population A. And

Figure 1C shows that both populations were globally distributed.

To identify the evolutionary processes underlying the emergence of the

genotype responsible for the Israeli outbreak, we repeated the STRUCTURE

analysis assuming only 2 populations (K = 2) (based on the findings from the

first STRUCTURE analysis). This analysis identified the ancestral sources of

the individual strains (Figure 2). Each strain is represented by a thin

vertical line partitioned into 2 (K = 2) most likely predetermined

populations or genetic ancestries. Each line shows the proportion of

polymorphic sites inherited from each of the 2 populations (shown in green

and red). It shows that most biotype 1 and 2 strains have predominant

contribution from 1 of the 2 genetic ancestries. However, strains of biotype

3 have almost equal contributions from both genetic ancestries. This

analysis was further detailed to identify the ancestral sources of each of

the polymorphic sites in each of the 10 gene fragments (Figure 3).These

analyses confirmed that, notwithstanding the subdivision of V. vulnificus

populations into 2 populations, recombination had occurred between these

populations and that the Israeli outbreak genotype is a hybrid, with some

genes originating from 1 population and some from another, while some genes

have representation from both.

Discussion

We have shown that a hybrid virulent organism that acquired genes from 2

distinct and independent populations has caused the disease outbreak in

Israel. To achieve this analysis, we studied large, carefully assembled,

collections of V. vulnificus isolates. The human strains were collected from

infected patients in Israel, the United States, Europe, and Southeast Asia.

The environmental strains were collected from environmental sources in the

United States, the Pacific Ocean, the Baltic Sea, inland fish farms in

Israel, and eel farms in Europe.

The division of V. vulnificus populations into 2 major groups is consistent

with results of multilocus enzyme electrophoresis studies (19). However,

those studies placed the Israeli electrophoretic type within 1 of the 2

groups, in contrast to our findings, which placed the Israeli genotype in an

intermediate position between the 2 populations (Figure 1A and Figure 3).

Hybridization within bacterial populations, i.e., the process whereby a

hybrid results from the hybridization of the genomes of 2 or more

populations of a species or an organism, has been the focus of much

attention by scientists in the last decade (20-25); these events, which may

be intra- or interspecies, could alter the genetic distances and the

phylogenetic relationships within bacterial populations. The magnitude by

which these events occur is crucially dependent on ecologic factors;

different populations of a species must be present within the same niche for

genetic exchange to have an impact on genetic variation (26). Multiple

sampling of fish-farm water and fish documented the abundance of biotype 1

strains (11 and data not shown). These biotype 1 strains, representing both

populations of V. vulnificus, were never implicated in disease among

fish-farm fish, according to the Central Fish Health Laboratory, Kibbutz Nir

, Israel (www.moag.gov.il/english), or among humans in Israel (11).

These observations are consistent with finding that these populations are

not pathogenic to either humans or fish. The finding that this hybrid

variant (biotype 3) was the only implicated organism in all disease cases

from 1995 to 2003 is indicative of its pathogenicity. Furthermore, the

finding that all 62 biotype 3 strains were genetically identical could

suggest that this hybrid clone may have evolved by a relatively recent

genome hybridization event.

Hybrid variants have been recently described among populations of

Staphylococcus aureus (27) and Chlamydia trachomatis (28). However, our

findings show the first bacterial variant that is clearly more pathogenic

than the existing forms of the organism, i.e., the Israeli hybrid clone, is

more pathogenic than the existing biotype 1 strains within the Israeli

aquaculture system. These findings are consistent with observations among

influenza viruses (29). This phenomenon has also recently been described

also among populations of mosquitoes (30), in which hybridization between

existing forms of a relatively nonpathogenic organism has apparently led to

the emergence of a novel pathogenic variant that poses a particular threat

to human health.

The Israeli genotype spread extensively after its emergence in 1995, and by

2003, most of the fish farms in Israel were the sources of V. vulnificus

cases. This finding is consistent with the idea that this pathogen is

circulating freely within the underground brackish water reservoirs that

supply these fish farms. Despite the widespread use of inland fish farming

around the world, no similar outbreaks have been reported. During the 1970s

and 1980s, the introduction into Israel of stocks of Tilapia spp. from

Africa, the Far East, and South America (31-33) (for experimental and

commercial purposes) may have contributed to the evolution of this hybrid

clone. In view of the widespread fish-trading industry, this hybrid clone

may eventually emerge through exports of Israeli tilapia stocks, in remote

geographic locations. In conclusion, these observations demonstrate the

power of molecular and population genetic approaches in investigating the

emergence of a novel pathogen and defining its nature. Our results show

another way by which epidemic infectious diseases arise.

Acknowledgments <cut>

References <cur>

http://www.cdc.gov/ncidod/EID/vol11no01/04-0440.htm