APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 2010, p. 4890–4895 0099-2240/10/$12.00 doi:10.1128/AEM.00636-10 Copyright © 2010, American Society for Microbiology. All Rights Reserved.
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Genetic Characterization of Vibrio vulnificus Strains from Tilapia Aquaculture in Bangladesh䌤§ Zahid H. Mahmud,1† Anita C. Wright,2† Shankar C. Mandal,1 Jianli Dai,3 Melissa K. Jones,2 Mahmud Hasan,4 Mohammad H. Rashid,4 Mohammad S. Islam,1 Judith A. Johnson,3,5 Paul A. Gulig,6 J. Glenn Morris, Jr.,3 and Afsar Ali3* Environmental Microbiology Laboratory, Laboratory Sciences Division, International Centre for Diarrhoeal Disease Research, Dhaka 1212, Bangladesh1; Food Science and Human Nutrition Department, University of Florida, Gainesville, Florida 326112; Emerging Pathogens Institute3 and Department of Fisheries,4 University of Dhaka, Dhaka 1000, Bangladesh; and Department of Pathology5 and Department of Molecular Genetics and Microbiology,6 University of Florida, Gainesville, Florida 32610 Received 12 March 2010/Accepted 11 May 2010
Outbreaks of Vibrio vulnificus wound infections in Israel were previously attributed to tilapia aquaculture. In this study, V. vulnificus was frequently isolated from coastal but not freshwater aquaculture in Bangladesh. Phylogenetic analyses showed that strains from Bangladesh differed remarkably from isolates commonly recovered elsewhere from fish or oysters and were more closely related to strains of clinical origin. virulence-correlated gene (vcg) locus (31, 41, 42), and repetitive sequence in the CPS operon (12). DiversiLab repetitive extrageneic palindromic (rep-PCR) analysis also confirmed these genetic distinctions and showed greater diversity among clinical strains (12). Wound infections associated with tilapia in Israel implicated aquaculture as a potential source of V. vulnificus in human disease (6, 40). Tilapia aquaculture is increasing rapidly, as shown by a 2.8-fold increase in tons produced from 1998 to 2007 (Food and Agriculture Organization; http://www.fao.org /fishery/statistics/en). Therefore, presence of V. vulnificus in tilapia aquaculture was examined in Bangladesh, a region that supports both coastal and freshwater sources of industrial-scale aquaculture. V. vulnificus strains were recovered from market fish, netted fish, and water samples, and the phylogenetic relationship among strains was examined relative to clinical and environmental reference strains collected elsewhere. V. vulnificus in Bangladeshi aquaculture. Tilapia in Bangladesh is cultured in conjunction with shrimp and other local fish species. Freshwater ponds are closed to outside water sources except for occasional flooding, while coastal ponds connect to the Bay of Bengal (salt water source). Phosphate and urea fertilizers are used monthly to promote algal growth to feed the cultured fish. Market tilapia fish (Oreochromis niloticus) were obtained in Dhaka. Netted fish and water samples from tilapia aquaculture were obtained in May to July 2008 from freshwater (Matlab) and coastal (Satkhira) ponds (n ⫽ 6) and were collected in sterile bags according to the procedure of the American Public Health Association (2). Subsurface water was collected at 0.5-m depth about 50 m from shore, and water temperature, salinity, total dissolved solids, dissolved oxygen, conductivity, and water pH were recorded. Samples were transported directly to the International Centre for Diarrhoeal Disease Research, Bangladesh in Dhaka and processed within 24 h of collection. Gill, muscle, and intestine tissues were dissected aseptically, washed extensively with sterile normal saline (0.9% [wt/vol] NaCl, pH 7.5), homogenized
Vibrio vulnificus causes severe wound infections and lifethreatening septicemia (mortality, ⬎50%), primarily in patients with underlying chronic diseases (10, 19, 23) and primarily from raw oyster consumption (21). This Gram-negative halophile is readily recovered from oysters (27, 35, 43) and fish (14) and was initially classified into two biotypes (BTs) based on growth characteristics and serology (5, 18, 39). Most human isolates are BT1, while BT2 is usually associated with diseased eels (1, 39). An outbreak of wound infections from aquacultured tilapia in Israel (6) revealed a new biotype (BT3). Phenotypic assays do not consistently distinguish biotypes (33), but genetic analyses have helped resolve relationships (20). A 10locus multilocus sequence typing (MLST) scheme (8, 9) and a similar analysis of 6 loci (13) segregated V. vulnificus strains into two clusters. BT1 strains were in both clusters, while BT2 segregated into a single cluster and BT3 was a genetic mosaic of the two lineages. Significant associations were observed between MLST clusters and strain origin: most clinical strains (BT1) were in one cluster, and the other cluster was comprised mostly of environmental strains (some BT1 and all BT2). Clinical isolates were also associated with a unique genomic island (13). The relationship between genetic lineages and virulence has not been determined, and confirmed virulence genes are universally present in V. vulnificus strains from both clinical and environmental origins (19, 23). However, segregation of several polymorphic alleles agreed with the MLST analysis and correlated genotype with either clinical or environmental strain origin. Alleles include 16S rRNA loci (15, 26, 42), a
* Corresponding author. Mailing address: 2055 Mowry Road, Emerging Pathogens Institute (EPI), University of Florida at Gainesville, Gainesville, FL 32610. Phone: (352) 273-7984. Fax: (352) 2736890. E-mail:
[email protected]. † These authors contributed equally to the manuscript. § Supplemental material for this article may be found at http://aem .asm.org/. 䌤 Published ahead of print on 21 May 2010. 4890
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TABLE 1. Distribution of V. vulnificus in tilapia and water samples collected in Bangladesh aquaculture Coastal water parametersa
V. vulnificus recovery Sample (total no.)
Market fish (18) Netted fish (12) Water (12)
No. of positive samples
No. of isolates recovered
Temp (oC)
Salinity (ppt)
pH
Conductivity (S/cm)
Total dissolved solids
Dissolved oxygen
8 8 8
18 9 6
NAb NA 33.2 ⫾ 0.8
NA NA 5.5 ⫾ 0.4
NA NA 8.0 ⫾ 0.24
NA NA 9,900 ⫾ 170
NA NA 5,300 ⫾ 370
NA NA 7.9 ⫾ 0.7
a Measurement of parameters is described in the Materials and Methods, and water conditions represent the average of results for six ponds for samples collected between May and July 2008. b NA, not applicable.
by a grinder for 1 min, and suspended in sterile normal saline. V. vulnificus was isolated from alkaline peptone water enrichment by streaking onto thiosulfate-citrate-bile-salt-sucrose (TCBS) agar (BD) and CHROMagar Vibrio (CV; CHROMagar, Paris, France) with overnight growth at 37°C. Speciesspecific identification was confirmed by vvhA PCR, as previously described (38). This is the first report to examine the distribution of V. vulnificus in Bangladesh aquaculture. All positive samples were from coastal aquaculture (Table 1), including market fish (44%) and netted fish and water samples from aquaculture ponds (66%), whereas no positive samples were from freshwater (not shown). Isolation frequency was distributed equally among the different fish tissues (see Table S1 in the supplemental material). Statistical analysis of recovery rates in Bangladesh aquaculture is precluded by the small sample sizes, but V. vulnificus concentrations in positive samples were generally lower for netted fish (3 to 15 most probable number [MPN]/ g⫺1) than for market samples (16 to 100 MPN/g⫺1) and somewhat higher on CV than on TCBS agar (data not shown). V. vulnificus is an obligate halophile, and its prevalence in coastal aquaculture is likely due to increased salinity at these sites. Coastal water was also higher for temperature, pH, total dissolved solids, and dissolved oxygen than freshwater, which may also contribute to the survival of V. vulnificus. Recovery of 33 isolates in Bangladesh contrasted with the even more limited ability to obtain strains from aquaculture in Israel (6). Phenotypic characterization. All strains from Bangladeshi aquaculture were BT1 (Table 2; see Table S1 in the supplemental material) based on tests for oxidase, lysine decarboxylase, arginine dihydrolase, ornithine decarboxylase, lactose fermentation, sucrose fermentation, salicin fermentation, D-sorbitol fermentation, cellulose fermentation, o-nitrophenyl-D-galactopyranoside, and D-mannitol fermentation (5, 18, 39). These results are similar to prior observations that describe V. vulnificus isolates from water, fish, and shellfish sources as predominantly biotype 1 (15, 25, 28, 44). Susceptibility to five antibiotics commonly prescribed in Bangladesh was tested by the Kirby-Bauer method using reference strain ATCC 27562 and included gentamicin (CN10), furazolidone (FR100), trimethoprim sulfamethoprim (SXT 25), cephalothin (CF 30), and ciprofloxacin (CIP 5). Of 33 V. vulnificus isolates tested, 28 were resistant to cephalothin and 2 were resistant to gentamicin. No multidrug-resistant strains were identified, contrasting with a recent finding that most V. vulnificus strains (n ⫽ 151) from the United States were resistant to multiple antibiotics (3). However, that study assayed a greater number
and broader spectrum of antibiotics (n ⫽ 26), which may account for the differential findings. Quinolone and fluoroquinolone resistance has also been reported in zoonotic serovars of BT2 strains from eels (30).
TABLE 2. Summary of V. vulnificus strain distribution relative to source, biotype, and genotype Typing methoda
Strain distribution by source and type 关% (no.)兴b Clinical
Environment
Bangladesh
Biotypes (n ⫽ 124) 1 2 3 Total
93.3 (42) 2.2 (1) 4.4 (2) 100 (45)
88.9 (40) 8.9 (4) 2.2 (1) 100 (45)
100 (33) 0 0 100 (33)
vcg genotypes (n ⫽ 105)c C E Total
66.7 (22) 33.3 (11) 100 (33)
2.6 (1) 97.4 (38) 100 (39)
100 (33) 0 100 (33)
MLST (n ⫽ 96)d I II III Total
17.1 (7) 78.0 (32) 4.9 (2) 100 (41)
86.3 (19) 9.2(2) 4.5 (1) 100 (22)
0 100 (33) 0 100 (33)
rep-PCR (n ⫽ 105)e 1 2 3 4 5 6 7 8 NC Total
6.1 (2) 6.1 (2) 12.1 (4) 0 15.2 (5) 9.1 (3) 24.2 (8) 24.2 (8) 3.0 (1) 100 (33)
0 0 2.6 (1) 0 17.9 (7) 0 76.9 (30) 0 2.6 (1) 100 (39)
0 3.0 (1) 39.3 (13) 6.1 (2) 27.3 (9) 6.1 (2) 0 0 18.2 (6) 100 (33)
c
a Typing methods are described in the text. Not all strains were subjected to every analysis, and results for individual strains are described in detail in Table S1 in the supplemental material. b The distribution of strains is shown as the percentage of total reference clinical strains, total reference environmental strains, or total for each assay, with number of strains in parentheses. c Biotypes and vcg allele types are based on typing of Bangladeshi strains from this study and from previously described reference strains in Table S1 in the supplemental material. d MLST data are from the phylogram in Fig. 1, using sequences from Bangladeshi strains in the present study in conjunction with the online MLST database for glp (glucose-6-phosphate isomerase), pyrC (dihydroorotase), and dtdS (threonine dehyrogenase) genes as described in the text. e The rep-PCR data are from the dendrogram in Fig. 2. NC, strains that did not form clusters of more than one strain.
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FIG. 2. Dendrogram of rep-PCR analysis. Strains are grouped by DiversiLab rep-PCR software for ⬎85% similarity but ⬍95% similarity of two or more strains based on the Pearson correlation coefficient and unweighted pair group method with arithmetic mean to automatically compare the rep-PCR-based DNA fingerprints of unknown isolates. Strains include V. vulnificus isolates from Bangladeshi aquaculture in this study (boxed), and from previously described (12) clinical strains isolated from blood (boxed and shaded) or wound infections (shaded) and environmental strains from other sources (not boxed or shaded). Stains from eels (E) or related to the outbreak of wound infections from Israel (I) are labeled in the left portion of the figure, and all strains are described in detail in Tables S1 and S2 in the supplemental material.
Genetic characterization. Molecular typing systems included a PCR-based assay (41) that previously correlated vcg alleles with either clinical (C-type) or environmental (E-type) strain origin (12, 31). A commercial DiversiLab rep-PCR typing system (bioMe´rieux) developed for typing Salmonella enterica was also used with an existing database of 85 strains of V. vulnificus
in accordance with the manufacturer’s instructions (12). This rep-PCR system detects amplified fragments using a microfluidics chip (Agilent 2100 bioanalyzer) and web-based software (DiversiLab version 2.1.66) using the Pearson correlation coefficient and the unweighted pair group method with arithmetic mean to automatically compare the DNA fingerprints of
FIG. 1. Multilocus sequencing typing (MLST) of V. vulnificus strains isolated from tilapia and water samples from Bangladesh. Three target genes, glp, dtdS, and pyrC, were used in this study. MLST data obtained from Bangladeshi fish and water isolates were compared with MLST of clinical and environmental strains derived from the online MLST library described at http://pubmlst.org/vvulnificus/ (22) and listed in Table S1 in the supplemental material. The major clusters of MLST are numbered as I, II, and III.
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unknown isolates. An abbreviated MLST scheme derived from the method of Bisharat et al. (8) used glp (glucose-6-phosphate isomerase) from chromosome I and pyrC (dihydroorotase) and dtdS (threonine dehyrogenase) from chromosome II. These loci provide as much discrimination of clusters as was achieved with the full set of 10 loci. In particular, pyrC has several mutations that clearly differentiate between the two major clusters (9). Purified PCR products (Qiagen MiniElute 96 UF PCR purification kit) were sequenced from both ends using the Big Dye Terminator v3.1 cycle sequencing kit (Applied Biosystems), and sequences were assembled using phred and phrap (16, 17). Concatenated sequences were compared to published sequences (22) using MEGA 4.1 (36) and neighbor joining (32) with the maximum composite likelihood method (37) in a bootstrap test of 1,000 replicas. V. parahaemolyticus rooted the phylogenetic tree, and bootstrap values greater than 50% are shown at the nodes. Genetic analyses of Bangladeshi V. vulnificus isolates showed that they differed remarkably from fish or environmental isolates collected elsewhere (see Tables S1 and S2 in the supplemental material). As summarized in Table 2, all Bangladeshi isolates were vcg C-type and segregated into MLST cluster II (Fig. 1), containing most (78%) of the clinical strains. In contrast, environmental isolates that were not from Bangladesh in the present study, including all other fish isolates, were mostly vcg E-type (97%) and were in either MLST cluster I or III (70%). Bangladeshi strains were represented in multiple rep-PCR clusters (2, 3, 5, and 6) that mostly (76%) contained clinical strains (Fig. 2). A large group of Bangladeshi strains (27%) grouped with other fish or fish-related isolates in rep-PCR cluster 5. None of the Bangladeshi strains was present in rep-PCR cluster 7, consisting predominantly of oyster strains (70%), or in cluster 8, which was comprised solely of strains from clinical origin. The remaining Bangladeshi strains either did not cluster or clustered with one other Bangladeshi strain. Discussion. These results showed that V. vulnificus strains recovered from Bangladesh aquaculture are genetically different from strains that are commonly associated with oysters or fish aquaculture elsewhere. All Bangladeshi strains exhibited a genetic profile (vcg C-type, MLST cluster II, multiple rep-PCR clusters) that was more typical for strains from clinical origin. In contrast, only 33% of isolates from tilapia aquaculture in Israel (n ⫽ 360) were positive for the vcg C-type allele, while 98% of clinical BT3 strains from wound infections (n ⫽ 44) were positive for this allele (11). However, the association of a particular genotype with increased virulence is still speculative without more comprehensive animal studies. DePaola et al. (15) noted significant differences between clinical and environmental strains in the log CFU/g of V. vulnificus recovered from liver samples following subcutaneous inoculation of irontreated mice, as did a similar study with a more limited number of strains (34). Studies to investigate these relationships are ongoing in our laboratories. Bangladeshi strains also resembled clinical strains in their greater overall diversity compared to isolates from eels or from tilapia in Israel (4, 7, 9). Bisharat et al. (9) speculated that the V. vulnificus genome is extremely plastic, sustaining pervasive changes probably through horizontal gene transfer from related or even from more distant bacterial species. They sug-
APPL. ENVIRON. MICROBIOL.
gested that interspecific hybrids could be the ancestor of most clinical strains, and that “clonal expansion of such hybrids would also increase the availability of divergent sequence for typical recombination events.” Genetic hybrids similar to BT3 were not observed in Bangladeshi strains, based on our MLST analysis of three genes; however, rep-PCR did group some Bangladeshi strains with other fish isolates. The underlying cause of the association of specific genotypes with aquaculture is unknown. Human disease is unlikely to contribute to the evolution of V. vulnificus, whereas fish can be colonized with V. vulnificus at densities that greatly exceed those seen in seawater or shellfish (14). The high nutrient conditions and increased host density that accompany tilapia aquaculture may further contribute to V. vulnificus populations and provide advantageous conditions for recombination events and/or clonal expansion of the particular lineages. Climate change may exacerbate these events, as evidenced by observations of seasonal shifts in genetic profiles of V. vulnificus populations that correspond to increased water temperatures (24) and by the coincidence of unusually high water temperature with the outbreak of wound infections caused by BT3 clones in Israeli aquaculture (29). We hypothesize that distinct environmental factors or specific industry practices associated with coastal aquaculture in Bangladesh may contribute to the emergence of genetically more diverse and potentially more virulent strains of V. vulnificus. Genomic sequence comparison of V. vulnificus strains from Bangladesh will be needed to unveil the basis for evolution of virulence in this species in relationship to the ecological niche presented by coastal aquaculture in Bangladesh. Technical support was provided by Michael Hubbard. This publication made use of the Vibrio vulnificus Multi Locus Sequence Typing website (http://pubmlst.org/vvulnificus/) developed by Keith Jolley and sited at the University of Oxford (22). The development of this site has been funded by the Wellcome Trust. REFERENCES 1. Amaro, C., and E. G. Biosca. 1996. Vibrio vulnificus biotype 2, pathogenic for eels, is also an opportunistic pathogen for humans. Appl. Environ. Microbiol. 62:1454–1457. 2. APHA. 1998. American Public Health Association (1998) recommended procedure for the examination of seawater and shellfish, 4th ed. American Public Health Association, Washington, DC. 3. Baker-Austin, C., J. V. McArthur, A. H. Lindell, M. S. Wright, R. C. Tuckfield, J. Gooch, L. Warner, J. Oliver, and R. Stepanauskas. 2009. Multi-site analysis reveals widespread antibiotic resistance in the marine pathogen Vibrio vulnificus. Microb. Ecol. 57:151–159. 4. Biosca, E. G., C. Amaro, J. L. Larsen, and K. Pedersen. 1997. Phenotypic and genotypic characterization of Vibrio vulnificus: proposal for the substitution of the subspecific taxon biotype for serovar. Appl. Environ. Microbiol. 63:1460–1466. 5. Biosca, E. G., J. D. Oliver, and C. Amaro. 1996. Phenotypic characterization of Vibrio vulnificus biotype 2, a lipopolysaccharide-based homogeneous O serogroup within Vibrio vulnificus. Appl. Environ. Microbiol. 62:918–927. 6. Bisharat, N., V. Agmon, R. Finkelstein, R. Raz, G. Ben-Dror, L. Lerner, S. Soboh, R. Colodner, D. N. Cameron, D. L. Wykstra, D. L. Swerdlow, and J. J. Farmer III. 1999. Clinical, epidemiological, and microbiological features of Vibrio vulnificus biogroup 3 causing outbreaks of wound infection and bacteraemia in Israel. Israel Vibrio Study Group. Lancet 354:1421–1424. 7. Bisharat, N., C. Amaro, B. Fouz, A. Llorens, and D. I. Cohen. 2007. Serological and molecular characteristics of Vibrio vulnificus biotype 3: evidence for high clonality. Microbiology 153:847–856. 8. Bisharat, N., D. I. Cohen, R. M. Harding, D. Falush, D. W. Crook, T. Peto, and M. C. Maiden. 2005. Hybrid Vibrio vulnificus. Emerg. Infect. Dis. 11: 30–35. 9. Bisharat, N., D. I. Cohen, M. C. Maiden, D. W. Crook, T. Peto, and R. M. Harding. 2007. The evolution of genetic structure in the marine pathogen, Vibrio vulnificus. Infect. Genet. Evol. 7:685–693. 10. Blake, P. A., M. H. Merson, R. E. Weaver, D. G. Hollis, and P. C. Heublein.
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