APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/00/$04.0010

Sept. 2000, p. 4022–4028 Vol. 66, No. 9

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Virulence Genes in Environmental Strains of Vibrio choleraeSOUMEN CHAKRABORTY,1 ASISH K. MUKHOPADHYAY,1 RUPAK KUMAR BHADRA,2

AMAR NATH GHOSH,1 RUPAK MITRA,1 TOSHIO SHIMADA,3 SHINJI YAMASAKI,1,4

SHAH M. FARUQUE,5 YOSHIFUMI TAKEDA,3 RITA R. COLWELL,6,7*AND G. BALAKRISH NAIR1

National Institute of Cholera and Enteric Diseases, Beliaghata, Calcutta 700 010,1 and Indian Institute of ChemicalBiology, Calcutta 700 032,2 India; National Institute of Infectious Diseases, 1-23-1 Toyama,3 and Research Institute,International Medical Center of Japan,4 Shinjuku-ku, Tokyo 162, Japan; International Centre for Diarrhoeal Disease

Research, Bangladesh, Dhaka-1212, Bangladesh5; and Center of Marine Biotechnology, University of MarylandBiotechnology Institute, Baltimore, Maryland 21202,6 and Department of Cell and Molecular Biology, University of

Maryland, College Park, Maryland 207427

Received 9 March 2000/Accepted 21 June 2000

The virulence of a pathogen is dependent on a discrete set of genetic determinants and their well-regulatedexpression. The ctxAB and tcpA genes are known to play a cardinal role in maintaining virulence in Vibriocholerae, and these genes are believed to be exclusively associated with clinical strains of O1 and O139serogroups. In this study, we examined the presence of five virulence genes, including ctxAB and tcpA, as wellas toxR and toxT, which are involved in the regulation of virulence, in environmental strains of V. choleraecultured from three different freshwater lakes and ponds in the eastern part of Calcutta, India. PCR analysisrevealed the presence of these virulence genes or their homologues among diverse serotypes and ribotypes ofenvironmental V. cholerae strains. Sequencing of a part of the tcpA gene carried by an environmental strainshowed 97.7% homology to the tcpA gene of the classical biotype of V. cholerae O1. Strains carrying the tcpA geneexpressed the toxin-coregulated pilus (TCP), demonstrated by both autoagglutination analysis and electronmicroscopy of the TCP pili. Strains carrying ctxAB genes also produced cholera toxin, determined by mono-sialoganglioside enzyme-linked immunosorbent assay and by passage in the ileal loops of rabbits. Thus, thisstudy demonstrates the presence and expression of critical virulence genes or their homologues in diverseenvironmental strains of V. cholerae, which appear to constitute an environmental reservoir of virulence genes,thereby providing new insights into the ecology of V. cholerae.

Vibrio cholerae is known to be an autochthonous inhabitantof brackish waters and estuarine systems (4, 13). Among the193 currently recognized O serogroups of V. cholerae (43), onlyserogroups O1 and O139 have caused epidemics of cholera.The other serogroups of V. cholerae, collectively referred to asnon-O1 non-O139 serogroups, have not been associated withepidemics but can cause sporadic diarrhea (30) and are ubiq-uitously distributed in the aquatic environment (22, 26). Thissharp distinction between serogroups which can cause choleraand those which are not associated with cholera is related tothe observation that more than 95% of the strains belonging toserogroups O1 and O139 produce cholera toxin (CT), which iscentral to the disease process. In contrast, more than 95% ofthe strains belonging to non-O1 non-O139 serogroups do notproduce CT (15). Another important virulence factor of V.cholerae is the toxin-coregulated pilus (TCP), which is an ad-hesin that is coordinately regulated with CT production (39).TCP is the only V. cholerae pilus that has been demonstrated todate to have a role in colonization of the gut mucosa of humans(9) and of infant mice (39), the latter being an experimentalcholera model.

It has been presumed that CT and TCP are exclusivelyassociated with clinical strains of V. cholerae, notably thosebelonging to serogroups O1 and O139, whereas reports on theincidence of CT among environmental strains of V. cholerae

are rare (24). Similarly, TCP has rarely been reported amongenvironmental strains of V. cholerae, suggesting that TCP isassociated only with virulent V. cholerae O1 or O139. Recently,the presence of tcpA in some non-O1 toxigenic strains (8, 32)and in two nontoxigenic, non-O1 non-O139 strains has beenpublished (27).

The genes encoding CT form part of the genome of a lyso-genic filamentous bacteriophage, designated CTXf. The piluscolonization factor TCP is also known to act as a receptor forCTXf, which can infect nontoxigenic V. cholerae, leading tothe emergence of new toxigenic strains (42). The tcpA gene ispart of a pathogenicity island of about 39.5 kb known as the V.cholerae pathogenicity island (VPI) (16). The structural fea-tures of VPI are suggestive of a bacteriophage origin, and thereis at least one report describing the production of a bacterio-phage designated VPIf (17). This supports the current hypoth-esis that some pathogenic bacteria have evolved from non-pathogenic strains of the same species via horizontal transferof virulence genes (5).

To understand the ecology of the V. cholerae serogroupsassociated with cholera, it is important to determine the originand distribution of virulence genes among environmentalstrains. In the study reported here, isolation and analysis ofunique strains of V. cholerae of environmental origin whichpossess virulence gene(s) homologues are described. Thesehomologues appear to be variants, or intermediates, in theevolution of virulence genes.

(Part of this paper was presented at the 34th Joint Confer-ence on Cholera and Other Bacterial Enteric Infections Panel,U.S.-Japan Cooperative Medical Sciences Program, held at

* Corresponding author. Mailing address: Center of Marine Bio-technology, University of Maryland Biotechnology Institute, 701 EastPratt Street, Baltimore, MD 21202. Phone: (703) 306-1000. Fax: (703)306-0109. E-mail: colwell@umbi.umd.edu.

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Shonan Village, Japan, between 30 November and 3 December1998.)

MATERIALS AND METHODS

Collection and processing of environmental samples. Water, sediment, andplankton samples collected from three different freshwater lakes and pondslocated in the eastern part of Calcutta (longitude 88°209E, latitude 22°329N),India, were examined from July 1997 to June 1998, as described previously (3,25). Water samples were filtered using Whatman no. 1 filter paper and subse-quently filtered through a 0.45-mm-pore-size membrane (Millipore Corp., Bed-ford, Mass.) using vacuum pressure of 15 to 20 lb/in2. The membrane was cut intoeight pieces and vortexed in 2 ml of 10 mM phosphate-buffered saline (PBS, pH7.4) for 3 min. One milliliter of the suspension was added to 10 ml of alkalinepeptone water (APW) containing peptone (1%, wt/vol) and NaCl (1%, wt/vol)(pH 8.5) contained in 20-ml screw-cap glass tubes, for enrichment at 37°C withshaking (100 rpm) for 16 to 18 h.

Sediment samples were added to 100 ml of distilled water until the finalvolume reached 200 ml, mixed well, and allowed to settle. A 10-ml amount of theslurry was centrifuged at 2,000 rpm for 8 min at room temperature to removeparticulate matter, and 1 ml of slurry was added to 10 ml of APW (pH 8.5) forenrichment, as described above. Plankton samples were collected using a 20-mmplankton net. The samples were further concentrated using Whatman no. 1 filterpaper, with the paper containing the plankton then being washed with 3 ml ofPBS. The suspension was homogenized using a glass homogenizer. One milliliterof the homogenized sample was added to 10 ml of APW (pH 8.5) for enrichment.The enriched samples from each of the components, i.e., sediment, water, andplankton, were screened for virulence genes by PCR, as described below.

Isolation of single-cell clones containing virulence genes. A search for strainspossessing the virulence gene(s) was performed when APW (pH 8.5)-enrichedsamples yielded a positive PCR amplicon for any of the virulence genes of V.cholerae sought in this study. Each sample (20 ml) was streaked on thiosulfate-citrate-bile-sucrose agar (TCBS) (Eiken, Tokyo, Japan) and tellurite tauro-cholate gelatin agar (TTGA) (trypticase agar base, 10 g; NaCl, 10 g; sodiumtaurocholate, 5 g; sodium carbonate, 1 g; gelatin, 30 g; agar, 15 g per liter;potassium tellurite, 1% [wt/vol]; pH 8.5) plates. In addition, the enriched sampleswere also serially diluted and plated on Luria agar (LA; Difco, Detroit, Mich.)supplemented with 1% (wt/vol) NaCl. The rationale for using TCBS, TTGA, andLA concurrently was to search for strains of V. cholerae as well as other hetero-trophic bacterial flora which might carry the virulence genes that were beingsought. The plates were incubated overnight at 37°C. LA plates which contained30 to 300 colonies were selected, and each colony was assigned a number.One-third of the colonies were randomly selected for further analysis. A part ofeach selected colony was inoculated into 2 ml of Luria broth (LB) and grown at37°C to prepare DNA for confirmation of the presence of the virulence gene byPCR. To prepare template DNA, 1 ml of the culture was centrifuged, resus-pended in sterile distilled water, and boiled for 10 min. Similarly, each colonyselected from the TCBS and TTGA plates was inoculated into LB, incubated at37°C with shaking, and processed to obtain template DNA for PCR as describedabove.

Serology. The identity of V. cholerae was confirmed as described previously(30). The 24 V. cholerae strains which were found to possess one or another of thevirulence genes sought were examined for agglutination by the somatic O antigenserogrouping scheme for V. cholerae developed at the National Institute ofInfectious Diseases, Tokyo, Japan (43).

PCR and sequencing. Three pairs of primers (ctxA, tcpA [classical variant;henceforth designated tcpA-C], and tcpA [El Tor variant; henceforth designatedtcpA-E]) were used in the first set of multiplex PCR (18), and two pairs ofprimers (ctxB and sto [encodes heat-stable enterotoxin]) were used in the secondset of multiplex PCR, as described elsewhere (28, 29). The cycling conditions forthe PCR assay included an initial denaturation at 94°C for 5 min, followed by 30cycles of 1.5 min of denaturation at 94°C, 1.5 min of primer annealing at 60°C(for the first set) and 1 min at 55°C (for the second set), and 1.5 min of primerextension at 72°C. PCR assays were also performed to detect the V. choleraeregulatory genes toxR and toxT. The primers used for amplification of toxR andtoxT were those described elsewhere (2, 21). Cycling conditions for PCR includedan initial denaturation at 94°C for 5 min, 30 cycles of 0.5 min at 94°C (denatur-ation), 0.5 min at 64°C (primer annealing), and 0.5 min at 72°C (primer exten-sion) for toxR; and 25 cycles of 1 min at 94°C (denaturation), 1 min at 50°C(primer annealing), and 1 min at 72°C (primer extension) for toxT. All PCRassays were performed using an automated thermal cycler (Biometra, Gottingen,Germany).

Sequencing of double-stranded DNA from purified PCR products was carriedout using the Taq dye terminator sequencing kit (Perkin-Elmer) and an auto-mated DNA sequencer (ABI Prism 377), following the manufacturer’s instruc-tions. Both strands were sequenced using the same forward and reverse primers,which were used for amplifying the classical biotype-specific tcpA. The sequenceswere aligned using the DNAsis software program (Hitachi), and searches fornearly identical sequences were performed using the Basic Local AlignmentSearch Tool (BLAST) program available on the National Center for Biotech-nology Information network server.

DNA extraction. A modification of the method of Murray and Thompson (23)was used for DNA extraction. In brief, cells from an 18-h LB culture werecollected and resuspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA [pH8.0]), treated with 10% (wt/vol) sodium dodecyl sulfate and freshly preparedproteinase K (Sigma Chemical Co., St. Louis, Mo.), and incubated at 37°C for1 h. After incubation, 10% cetyl trimethyl ammonium bromide in 0.7 M NaClwas added and incubated at 65°C for 10 min. The aqueous phase was treated withphenol-chloroform, and the DNA pellet was washed with 70% ethanol. Theextracted nucleic acid was suspended in TE and treated with RNase at 37°C for30 min.

Probes and hybridization. The presence of virulence-associated genes wasconfirmed by using specific DNA probes. The ctxA probe consisted of a 540-bpXbaI-ClaI fragment of ctxA cloned in pKTN901 using EcoRI linkers (14). TheDNA fragment used as the probe for tcpA in the Southern blot hybridization wasgenerated by PCR using primers described elsewhere (18). The rRNA geneprobe consisted of a 7.5-kb BamHI fragment of the Escherichia coli rRNA clonepKK3535 (7).

Genomic DNA from representative environmental strains of V. cholerae wasdigested with the appropriate restriction endonuclease, and the fragments wereelectrophoretically separated in a 0.8% agarose gel using TAE buffer (40 mMTris-acetate, 1 mM EDTA). DNA was transferred to a Hybond N1 membrane(Amersham International, PLC, Buckinghamshire, England) using 103 SSC (13SSC is 0.15 M NaCl plus 0.015 M sodium citrate) by vacuum transfer (Amer-sham). The membrane was washed with 103 SSC and dried at room tempera-ture. DNA was covalently immobilized to the membrane using alkali fixation.Southern blotting with probes conjugated to horseradish peroxidase to allowhybridization to be detected with a chemiluminescent substrate (Amersham) wasperformed as described elsewhere (41). The membrane was washed and exposedto X-ray film (Fuji Film, Fuji, Japan) and developed following the manufacturer’sinstructions.

The rRNA probe was labeled by random priming with a random primer DNAlabeling kit (BRL) and [a-32P]dCTP (3,000 Ci/mmol) (Amersham). Southernblots were hybridized with the labeled probe, and autoradiographs were devel-oped as described elsewhere (7).

Autoagglutination. The hydrophobicity of V. cholerae is greatly increased inbroth culture due to the expression of pili, which cause visible clumping ofbacteria, leaving a pellet at the bottom of the tube and a clear supernatant. Thisphenomenon is known as autoagglutination and has previously been shown to becorrelated with the expression of TCP (39). To determine whether environmen-tal V. cholerae strains possessing tcpA-C expressed pili, the strains were grown inLB (pH 6.8) supplemented with 1% (wt/vol) NaCl and incubated at 37°C for18 h.

Electron microscopy. Agar media were used to examine the expression of thepilus by the environmental strains of V. cholerae possessing tcpA. The media usedincluded colonization factor antigen (CFA) agar (34) and LB supplemented with20 g of Bacto agar (Difco) per liter. The strains were incubated at 25°C for 24 or36 h. Samples were taken from the different agar media at the designated timesand processed. Bacterial suspension (5 ml in 10 mM phosphate-buffered saline[pH 7.4]) was deposited on a 300-mesh copper grid coated with a film of pyelovarand stabilized with a thin layer of carbon. After about 1 to 2 min, the excess fluidwas blotted and stained with 2% (wt/vol) uranyl acetate for 1 min. Grids wereexamined using a Philips 420T transmission electron microscope.

Detection of CT by GM1 ELISA. To detect expression of CT by the environ-mental strains, the cells were grown either in AKI (containing [per liter] Bac-topeptone, 15 g; NaCl, 5 g; yeast extract, 5 g; sodium bicarbonate, 3 g; pH 7.5[11]) or in YEP (containing [per liter] yeast extract, 4 g; Bactopeptone, 15 g;NaCl, 5 g; pH 7.5 [10]) medium at 37°C, with shaking, for 16 h. After centrifu-gation, the supernatant was examined for the presence of CT by a monosialo-ganglioside (GM1) enzyme-linked immunosorbent assay (ELISA) as describedby Svennerholm and Holmgren (37). Pure CT (lot no. 19H4022), obtained fromSigma Chemical Co., St. Louis, Mo., was used as the positive control.

Animal passage. The rabbit ileal loop model was used for animal passage of V.cholerae strain SCE188 (ctxAB1 tcpA), as described previously (19). The cellsgrown in YEP were introduced into the rabbit ileum and incubated for 18 h.Fluid from the rabbit ileal loop was plated on TCBS, typical cholera organism-like colonies were picked, and their identity was reconfirmed both by biochemicaltests and by the multiplex PCR described above. The rabbit-passaged strainswere grown in AKI and YEP, and CT from the culture supernatant was mea-sured by GM1 ELISA (37).

Nucleotide sequence accession number. The nucleotide sequence of the tcpAgene from environmental strain SCE5 of V. cholerae has been deposited with theDNA Data Bank of Japan (DJBB) with accession number AB012946.

RESULTS AND DISCUSSION

Presence of ctxA, tcpA, toxR, and toxT in environmentalstrains of V. cholerae. A total of 122 samples (44 water, 34sediment, and 44 plankton samples) collected from three sitesin Calcutta between July 1997 and June 1998 were analyzed byconventional bacteriology and by multiplex PCR assays after

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enrichment of the samples in APW (pH 8.5). Two multiplexPCR assays were designed to detect five known virulence genesof V. cholerae, including tcpA-E, tcpA-C, ctxA, ctxB, and sto(gene encoding the heat-stable enterotoxin of V. cholerae). Ofthe 122 enriched APW (pH 8.5) samples analyzed using thetwo sets of multiplex PCR, five water and four plankton sam-ples examined at different time intervals were positive for ei-ther tcpA or ctxAB; none were positive for sto. None of thesediment samples were positive for any of the virulence genessought. A total of 19 strains of V. cholerae positive for tcpA andanother 5 strains positive for ctxA and ctxB were isolated (Ta-ble 1). These 24 virulence gene-positive strains of V. choleraewere isolated after examining approximately 4,800 colonies (anaverage of 200 colonies per search) from either TCBS, TTGA,or LA. Of the 19 environmental V. cholerae strains examined,the size of the tcpA amplicon in 17 strains matched the size ofthe tcpA amplicon (617 bp) of the reference classical strain (V.cholerae O395), while in 2 strains the size of the tcpA amplicon(471 bp) matched that of the reference El Tor strain (V. chol-erae VC20) (Fig. 1). All 19 strains were, however, negative forthe 301-bp ctxA and 460-bp ctxB amplicons, indicating thatthese strains did not have the genetic potential to produce CT.Furthermore, five environmental strains of V. cholerae whichwere positive for ctxA and ctxB were negative for tcpA with theset of primers used in this study (18). This is contrary to thecurrent assumption that most CT-positive strains are also pos-itive for TCP, since TCP is known to be the receptor for CTXfinfection of V. cholerae. All 24 strains were positive for toxR, atranscriptional activator of many virulence genes in V. cholerae(20, 21). In contrast, toxT was found in only three strains(SCE4, SCE5, and SCE6) positive for tcpA and all five strainspositive for ctxAB (Table 1).

The results of PCR assays were confirmed by Southern hy-bridization when representative strains positive for tcpA orctxA were hybridized with the respective probes. Strains posi-

tive for tcpA-C (SCE4, SCE5, and SCE6) revealed two frag-ments after digestion with PstI and probing with tcpA (Fig. 2).Southern blot hybridization using the ctxA probe after diges-tion of DNA of the representative strains SCE188 and SCE223with PstI revealed two different restriction patterns, as shownin Fig. 2.

Analysis of ribotypes. Analysis of BglI restriction patterns ofconserved rRNA genes (ribotype) in the environmental strainsrevealed clonal diversity, and 10 different ribotypes (A throughJ) were detected (Fig. 3). The distribution of ribotypes amongthe strains belonging to different serogroups is shown in Table1. Two strains, SCE4 and SCE5, which belonged to differentserogroups (O8 and O11, respectively) belonged to a singleribotype (A). Another O8 strain belonged to a different ri-

TABLE 1. Details of environmental strains of V. cholerae possessing virulence genes isolated in this study

Strain Date of isolation(mo/day/yr) Source Serogroup Ribotypea

Presence of virulence gene:

ctxAB tcpAb toxR toxT

SCE4 7/7/97 Plankton, freshwater lake O8 A 2 1 1 1SCE5 7/7/97 Plankton, freshwater lake O11 A 2 1 1 1SCE6 7/7/97 Plankton, freshwater lake O8 A 2 1 1 1SCE188 12/2/97 Water, fish farm O44 F 1 2 1 1SCE200 12/2/97 Water, fish farm O44 ND 1 2 1 1SCE201 12/2/97 Water, fish farm O44 ND 1 2 1 1SCE223 1/8/98 Plankton, freshwater pond O27 G 1 2 1 1SCE225 1/8/98 Plankton, freshwater pond O35 B 2 1 1 2SCE226 1/8/98 Plankton, freshwater pond O35 G 2 1 1 2SCE227 1/8/98 Water, fish farm O35 B 2 1 1 2SCE228 1/8/98 Plankton, fish farm O35 B 2 1 1 2SCE256 2/10/98 Water, fish farm O42 C 2 1 1 2SCE257 2/10/98 Water, fish farm O42 D 2 1 1 2SCE258 2/10/98 Water, fish farm O42 D 2 1 1 2SCE259 2/10/98 Water, fish farm O42 D 2 1 1 2SCE260 2/10/98 Water, fish farm O42 ND 2 1 1 2SCE261 2/10/98 Water, fish farm O42 ND 2 1 1 2SCE263 2/10/98 Water, fish farm O10 E 2 1 1 2SCE264 2/10/98 Water, fish farm O42 C 2 1 1 2SCE265 2/10/98 Water, fish farm O42 C 2 1 1 2SCE340 5/5/98 Plankton, freshwater pond O69 H 2 1 1 2SCE341 5/5/98 Plankton, freshwater pond O69 H 2 1 1 2SCE354 5/19/98 Water, freshwater pond O27 I 1 2 1 1SCE359 5/19/98 Water, fish farm O8 J 2 1 1 2

a The various ribotype patterns designated A to J are shown in Fig. 3. ND, not determined.b The tcpA from strains SCE340 and SCE341 belonged to the El Tor variant, while tcpA from the other strains belonged to the classical variant.

FIG. 1. PCR analysis of tcpA and ctxA of genomic DNA from representativeenvironmental strains of V. cholerae isolated from enriched APW (pH 8.5)samples. Lane 1, environmental strain of V. cholerae (SCE5) possessing tcpA-C;lane 2, environmental strain of V. cholerae (SCE340) possessing tcpA-E; lane 3,environmental strain of V. cholerae (SCE188) possessing ctxA; lane 4, V. choleraeO1 Ogawa, El Tor (positive control for ctxA and tcpA-E); lane 5, V. cholerae O1,Ogawa, classical (positive control for ctxA and tcpA-C).

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botype (J). Four strains belonging to the O35 serogroup sharedtwo different ribotypes (B and G). Strains belonging to sero-group O42 shared ribotypes C and D. A toxigenic strain,SCE223, shared the same ribotype with a nontoxigenic strain,SCE228. Ribotyping was performed to determine whetherstrains of V. cholerae isolated from a given APW (pH 8.5)enrichment broth of a particular sample were siblings. Forexample, strains of V. cholerae with different serogroups (O27and O35) as well as different ribotypes (B and G) were isolatedfrom an APW (pH 8.5) enrichment of plankton samples col-lected from a freshwater pond in Calcutta on 8 January 1998.Similarly, APW enrichment of plankton samples collectedfrom a freshwater lake on 7 July 1997 yielded three strains withtwo different serogroups (O8 and O11) but a single ribotype(A). We also isolated two strains of V. cholerae from an en-richment culture of a sample of water from a fish farm taken on10 February 1998 which had the same serogroup (O42) andribotype (C).

Nucleotide sequence of tcpA of environmental strains of V.cholerae resembling classical tcpA. Among several putative col-onization factors of V. cholerae, TCP has been shown to beessential for colonization in the infant mouse model as well asin human volunteers (1, 9, 38–40). Thus, the function of TCPin colonization of the human intestinal epithelium is well es-tablished, as is its partial homology to type 4 or N-methylphe-nylalanine pili, the long surface filaments found in a variety ofpathogenic bacteria, notably Neisseria gonorrhoeae, Moraxellabovis, Pseudomonas aeruginosa, and Dichelobacter nodosus (6,10, 35). To determine whether the 617-bp tcpA amplicon was

similar to the tcpA of classical V. cholerae epidemic strains, wesequenced this amplified fragment of DNA and determinedthe extent of similarity between the nucleotide sequences ofthe amplified DNA and the reported sequence of classical tcpA(6). The nucleotide sequence data of the tcpA-like amplicon ofstrain SCE5 (V. cholerae serogroup O11), obtained from twosequencing reactions of two independent amplicons, yieldedreadable sequences of 597 bases, with 97.7% identity to thetcpA gene sequence of the classical V. cholerae O1 (6). Notably,only 14 bases differed. Furthermore, it was found that thederived amino acid sequence of TcpA in environmental strainshad an identity of 98.5% when compared with the reportedamino acid sequence of TcpA that is found in classical strainsof V. cholerae O1, with differences discernible only at positions104, 144, and 154 (Fig. 4). Homology between the environmen-tal V. cholerae TcpA amino acid sequence and V. cholerae ElTor TcpA sequence (31) was 80.4%, with 39 of the deducedTcpA residues of the environmental strain (SCE5) differingfrom those of TcpA of the V. cholerae El Tor biotype (Fig. 4).

The close similarity of most of the tcpA genes found inenvironmental strains of V. cholerae to the classical V. choleraetcpA is interesting, despite the fact that the current cholerapandemic is caused by the El Tor biotype. Recent epidemio-logical data from Bangladesh, where classical V. cholerae ex-isted until 1991 (36), show the absence of this biotype (A. K.Siddique, personal communication). However, the data ob-tained in this study indicate that a tcpA gene similar to theclassical type is present in environmental non-O1, non-O139 V.cholerae strains. An environmental reservoir of tcpA genes ofthe classical type strongly suggests the possibility of a reemer-gence of the classical biotype via gene transfer events in theenvironment. The classical biotype transiently reemerged in

FIG. 2. Southern blot hybridization of PstI-digested genomic DNA fromenvironmental strains of V. cholerae using tcpA (A) and ctxA (B) probes.

FIG. 3. BglI restriction patterns of rRNA genes in environmental strains of V.cholerae isolated in Calcutta. Ribotype patterns A through J produced by differ-ent strains are shown (see Table 1 for details). Numbers indicating the molecularsizes of bands correspond to a 1-kb DNA ladder (Bethesda Research Labora-tories).

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Bangladesh in 1983 as the predominant epidemic strain, about10 years after its apparent replacement by the El Tor biotype(33).

Expression of tcpA. To determine whether the tcpA geneswere expressed, the 19 strains of V. cholerae that were positivefor the tcpA amplicon were examined further. It was found thatthree strains (SCE4, SCE5, and SCE6) exhibited the autoag-glutination phenotype when incubated in LB containing 1%NaCl at pH 6.5 with aeration at 37°C. The conditions weredifferent from those reported for expression of classical tcpA,which included growth at 30°C at pH 6.5 in LB (39). Autoag-glutination was not observed in any other broth medium orcultural conditions in the other 16 tcpA-positive strains exam-ined. The 16 tcpA-positive but autoagglutination-negativestrains were negative for the virulence regulator toxT.

Transmission electron microscopy of negatively stainedspecimens of SCE5 performed to visualize the pilus revealedthat growth on CFA agar for 24 h at 25°C resulted in produc-tion of pili attached to the surface of the bacteria (Fig. 5A). Piliin bundles were observed after incubation for 36 h (Fig. 5B).Thus, we were able to demonstrate expression of Tcp by threestrains by testing the autoagglutination phenotype and also tovisualize pili of SCE5 by electron microscopy. Interestingly,strains positive for both tcpA and toxT showed the autoagglu-tination phenotype, whereas strains positive for tcpA but neg-ative for toxT did not autoagglutinate. In epidemic strains of V.cholerae, the tcpA gene is located in a 39.5-kb DNA segmentalong with other physically linked genes involved in Tcp bio-genesis (16). It was concluded that the complete VPI is notpresent in the remaining 16 tcpA-positive environmentalstrains of V. cholerae.

Expression of CT. Strains SCE188, -200, and -201 expressedCT in both AKI and YEP media, used for optimal productionof CT from El Tor and classical V. cholerae, respectively (11,12). However, the amount of CT antigen produced by SCE188and SCE201 was higher in YEP than in AKI, while the yield ofCT from SCE200 was the same whether grown in YEP or AKI.Despite possessing DNA fragments with sequences very simi-

lar to that of CT genes, two of the environmental isolates,SCE223 and SCE354, did not produce detectable amounts ofCT when grown in either YEP or AKI. Passage of strainSCE188 in a rabbit ileum resulted in positive fluid accumula-tion and isolation of strains that produced twofold more CTthan the wild type. This result (Table 2) suggests that selectionfor strains producing larger amounts of CT in the rabbit ileumoccurs in both environmental and epidemic strains (19).

Conclusion. In this study, the occurrence and distribution ofselected virulence-associated genes in environmental strains ofV. cholerae that had been isolated in Calcutta, India, weredemonstrated. These environmental V. cholerae strains wereneither O1 nor O139, nor did they carry together the genes forthe major virulence factors CT and TCP. Nevertheless, thesestrains constitute a potential reservoir of virulence genes in theenvironment. Diverse serogroups of V. cholerae are shown, forthe first time, to harbor these genes. What is most exciting isthat molecular characterization of microbial ecosystems pro-vides useful information about the ecology of V. cholerae, abacterium autochthonous to riverine, coastal, and estuarineecosystems but, at the same time, pathogenic for humans.Environmental studies of V. cholerae have been done with theexpectation that V. cholerae strains possessing the entire com-plement of virulence genes would be isolated. Now it is con-cluded that virulence genes are dispersed among environmen-tal strains of V. cholerae and may be ferried about, given thefact that most of the virulence genes that were studied arelocated on mobile elements. Indeed, the potential for “mixingand matching” of genes in the environment or in the humanintestine, leading to new pathogenic variants, must now beaddressed. Ribotypes of the strains isolated in this study wereshared by strains belonging to more than one serogroup, andconversely, a particular serogroup comprised more than oneribotype. Toxigenic strains and nontoxigenic strains belongingto an identical ribotype were also detected, further supportingthe hypothesis of gene transfer among vibrios in the environ-ment. Further studies on the ecology and evolution of V. chol-

FIG. 4. Multiple alignment of pilin amino acid sequence of TcpA-C, TcpA of SCE5, and TcpA-E. The alignment was created by the DNAsis (Hitachi) program.The shaded areas indicate identical residues, while unshaded areas indicate dissimilar residues. The GenBank accession numbers for tcpA-C and tcpA-E are M33514and U89807, respectively.

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erae will surely provide new insights into the epidemiology ofcholera.

ACKNOWLEDGMENTS

This work was supported, in part, by the Japan International Coop-eration Agency (JICA/NICED Project 054-1061-E-O) and by the Na-tional Institutes of Health (grant 1RO1A1392901).

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Strain orcomponent tested

OD492

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SCE188 0.76 6 0.27 0.35 6 0.03SCE188a 1.78 6 0.56 0.77 6 0.20SCE200 0.69 6 0.24 0.69 6 0.13SCE201 0.89 6 0.09 0.64 6 0.11SCE223 0.17 6 0.07 0.18 6 0.10SCE354 0.06 6 0.01 0.06 6 0.01VC20 2.43 6 0.12 1.81 6 0.42SCE18 0.11 6 0.04 0.03 6 0.02Bufferb 0.13 6 0.04 0.01 6 0.12Medium 0.17 6 0.02 0.22 6 0.03CT (1 ng) 0.64 6 0.03 0.59 6 0.08CT (0.1 ng) 0.33 6 0.01 0.25 6 0.02

a VC20 was a positive control, SCE18 was a negative control, and SCE188awas animal passaged.

b Components (buffer, medium, CT) were tested without strains.

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