INTRODUCTION

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Vibrio cholerae, a noninvasive gram-negative bacterium, is the causative agent of the diarrheal disease cholera. The specificity of the somatic O antigen of V. cholerae resides in the polysaccharide moiety of the lipopolysaccharide in the outer membrane, which forms the basis of the serological classification of this organism (36). The V. cholerae strains causing cholera epidemics have, until recently, been confined to serogroup O1, which consists of two biotypes, classical and El Tor. The classical biotype was responsible for cholera epidemics until 1961, when the El Tor biotype displaced it. V. cholerae strains other than O1, which are collectively called non-O1 vibrios, cause only sporadic infections and are believed to lack the potential to cause epidemics (26). In late 1992, for the first time in the history of cholera, a non-O1 strain, V. cholerae O139 Bengal (henceforth designated O139B), caused an epidemic in the Indian subcontinent (1, 32) which persisted for about 1 year (28). Strains isolated from different parts of India and Bangladesh during the epidemic were found to be of clonal origin, and several lines of evidence have suggested that strain O139B arose from the El Tor biotype (5-7, 9, 10) by the acquisition of a 35-kb novel DNA segment which replaced most of the O1-antigen-encoding gene cluster, the rfb locus, of the recipient strain (8, 11). Thus, serogroup O139 combines the virulent properties of epidemic strains (V. cholerae O1) with the outer appearance of nonepidemic strains (V. cholerae non-O1). In subsequent outbreaks O139B was again replaced by El Tor O1 strains (27, 28). Surprisingly, the genomes of El Tor strains isolated immediately before and after the O139B outbreak showed extensive restriction fragment length polymorphism (RFLP) among themselves and with the genome of O139B (30, 42). Alterations in a well-characterized clonal strain within such a short period are unexpected.

Recently, a resurgence in Calcutta, India (25), of V. cholerae O139 (henceforth designated O139R) having altered antibiotic sensitivity compared to O139B was recorded. The genomes of O139R strains were examined in the present study to investigate whether the observed phenotypic changes relative to O139B are associated with alterations in the genome. Gross plasticity in the organization of the genomes of V. cholerae strains belonging to different serovars and biovars has recently been found by using the intron-encoded enzyme I-CeuI, which provides an excellent tool for the rapid examination of the organization of genomes of related species of bacteria. V. cholerae strains belonging to serovars O1 and O139 have 9 I-CeuI sites in their genomes, and those belonging to serovars non-O1 and non-O139 have 10 I-CeuI sites in their genomes (30). The results presented here show that the clonality of O139 is not retained and that an amplification of rrn operon has occurred in the genomes of O139R strains relative to those of O139B strains. Moreover, there are three copies of the cholera toxin (CTX) genetic element in the genomes of O139R strains, and the organization of these elements and the direct-repeat sequence (RS) connecting the core regions are considerably different from those of O139B strains.

	MATERIALS AND METHODS

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Bacterial strains and growth condition. The V. cholerae strains used in this study are described in Table 1. All strains were obtained from the National Institute of Cholera and Enteric Diseases, Calcutta, India. The cells were grown in a gyratory shaker at 37°C in Luria-Bertani broth and maintained as described previously (33). Bacterial strains were tested for their susceptibility to streptomycin (10 µg) and sulfamethoxazole (23.75 µg)-trimethoprim (1.25 µg) (SXT) by the disc diffusion method (28).

Preparation of high-molecular-weight genomic DNA and enzyme digestion. Intact bacterial chromosomal DNA was prepared as described previously (22, 34). Briefly, V. cholerae cells in the logarithmic phase of growth were suspended in a 10 mM Tris-HCl (pH 7.6) buffer containing 1 M NaCl. Agarose plugs were prepared by mixing equal volumes of bacterial cells and molten 1% low-melting-point agarose (Pharmacia, Uppsala, Sweden). Bacterial cells embedded in agarose were lysed in the presence of RNase, treated with proteinase K, and stored in 0.5 M EDTA (pH 9.0) at 4°C. The agarose plugs containing intact genomic DNA were treated with phenylmethylsulfonyl fluoride (final concentration, 1 mM) to inactivate proteinase K and washed with 10 volumes of TE (10 mM Tris-HCl, 1 mM EDTA [pH 8.0]) to remove the phenylmethylsulfonyl fluoride. The agarose slice containing intact genomic DNA was digested with NotI, SfiI, or I-CeuI (New England Biolabs, Beverly, Mass.) as directed by the manufacturer and subjected to pulsed-field gel electrophoresis (PFGE).

PFGE. Electrophoresis was carried out in a Pulsaphor Plus system with a hexagonal electrode array (Pharmacia). The enzyme-digested DNA was separated on a 1% FastLane agarose gel (FMC, Rockland, Maine) with 20 mM Tris-acetate (pH 8.3)-0.5 mM EDTA as the running buffer at 5 to 10 V/cm (depending upon the size of the fragment that needed to be well resolved) and various pulse times (depending upon experimental conditions). Phage  multimeric DNA and yeast chromosomal DNA were used as molecular mass markers. The gels were stained with ethidium bromide.

Molecular genetic methods. About 1 µg of a 780-bp NdeI fragment from the ctxA gene or 2.3-kb BglII-PstI DNA segment containing the RS, cep, and part of orfU in plasmid pCVD15 (12) or cosmid pSXT1 (40) containing SXT resistance genes was nick translated with [-³²P]dCTP (Amersham, Little Chalfont, United Kingdom) and used as probes in Southern blot hybridization. The restriction fragment of DNA excised from the gel was directly labeled with [-³²P]dCTP by the random-priming method with a kit from New England Biolabs. End labeling of DNA fragments following I-CeuI or NotI digestion was done by incubating the agarose blocks in a buffer containing Klenow enzyme and [-³²P]dCTP (for I-CeuI) or [-³²P]dCTP plus dGTP (for NotI) and subjected to PFGE followed by autoradiography. For Southern blot hybridization, genomic DNA was digested with restriction endonucleases, separated by electrophoresis, transferred to Nytran membranes, and hybridized with labeled DNA probe at 60°C. The filters were washed under high-stringency conditions (0.1× SSC [1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate] at 60°C), air dried, and exposed to Kodak X-OMAT AR5 films as described previously (7).

	RESULTS

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Antibiotic sensitivity of O139R. One of the features that differentiates O139B from strains belonging to the El Tor biotype of O1 vibrios, from which it is believed to have originated, is its sensitivity to SXT (29). A self-transmissible, site-specific 62-kb conjugative transposon encodes the functions that confer SXT and streptomycin resistance to O139B strains (40). From an examination of the SfiI-digested, PFGE-separated profiles of the genomes of Escherichia coli and V. cholerae El Tor strains, it was concluded that SXT-sensitive strains have two closely migrating genomic DNA fragments of about 400 kb and that SXT-resistant strains have only one of these fragments (40). The SfiI digestion profile of the genomes of SXT-resistant (CO457) and -sensitive (VC44) El Tor O1 strains (Fig. 1A) used in the present study confirmed this conclusion. However, the SfiI digestion profiles of the genomes of O139R strains have a single DNA fragment in the 400-kb region (Fig. 1A), which, according to the above criterion, should indicate that like O139B, O139R is resistant to SXT. While the resurgent O139 strains retained their streptomycin-resistant phenotype, they were sensitive to SXT (Table 1). Since the SfiI digestion profiles of the genomes of the O139R strains and of the O139B strains are similar, and since the streptomycin-resistant phenotype is retained in O139R isolates, the possibility of deletion of the 62-kb genetic element from the genomes of the resurgent strains is unlikely. This was confirmed by Southern blot hybridization of PstI-digested genomic DNA of O139 strains with the gene encoding SXT resistance as a probe. The SXT probe hybridized with seven fragments of the PstI-digested plasmid pSXT1 (Fig. 1C, lane d) and with six fragments (>23, 15, 12, 5, 2, and 1 kb) of the genomic DNA of SXT-resistant O139B (lane c), as reported previously (40). However, the PstI-digested genome of representative O139R strains hybridized with the first four fragments only (lanes a and b). Thus, a 3-kb (2 kb plus 1 kb) deletion from the 62-kb element in the resurgent strains makes them sensitive to SXT while retaining the streptomycin resistance. The SXT probe did not hybridize with PstI-digested genomic DNA of the SXT-sensitive biotype El Tor strain VC44, which does not possess the 62-kb element (data not shown).

View larger version (71K): [in this window] [in a new window]   FIG. 1.   (A) PFGE of NotI- and SfiI-digested genomic DNA of V. cholerae strains. The enzyme-digested DNA was electrophoresed on 1% FastLane agarose with a Pulsaphor Plus System with pulse time interpolated between 5 and 25 s for 22 h at 10 V/cm at 3°C. The gels were stained with ethidium bromide. Lanes: a and g, AS192 (O139R); b and h, AS197 (O139R); c and i, AS199 (O139R); d and j, SG24 (O139B); e, VC44 (El Tor, O1); f, CO457 (El Tor, O1). Numbers indicate the molecular size markers in kilobases. Arrowheads indicate RFLP between AS192 and O139R and O139B strains. The arrowhead in lane e indicates two SfiI fragments in the 400-kb region (see the text for details). (B) Autoradiogram of NotI-digested, end-labeled, PFGE-separated V. cholerae O139 genomic DNA. The conditions of electrophoresis were as described for panel A. Lanes: a, SG24 (O139B); b, AS192 (O139R); c, AS197 (O139R); d, AS199 (O139R). The open arrowheads indicate RFLP between O139B and O139R genomes, and the solid arrowheads indicate the CTX genetic element locus (see the text). The ctxAB genes are located in 84- and 7-kb NotI fragments in the genomes of strains SG24 (O139B) and AS192 (O139R) (lanes a and b, solid arrowheads) and in a 97-kb NotI fragment of O139R genomes (lanes c and d, solid arrowheads). Numbers in the margin indicate the molecular size markers in kilobases. (C) Southern blot analysis of the hybridization of SXT-encoding genes (pSXT1) with SXT-resistant or -sensitive O139 strains. The cosmid pSXT1 (as control) and the chromosomal DNA of O139 strains were digested with PstI, electrophoresed, and analyzed by Southern blot hybridization with pSXT1 as a probe. Lanes: a, AS197 (O139R); b, AS210 (O139R); c, SG24 (O139B); d, pSXT1. M denotes molecular size markers in kilobases. The open arrowheads indicate the deletion of 2- and 1-kb PstI fragments of the SXT element from the genomes of O139R strains. The PstI fingerprint of O139B strain SG24 is identical to that of different O139B strains, as reported previously (40).

NotI digestion profiles of the genomes of O139R strains. When the intact genomic DNAs of O139R and O139B strains were digested with NotI, end labeled, and analyzed by PFGE, RFLP was recorded between the resurgent strains and O139B strains (Fig. 1A). It has been reported (7) that the ctxA gene hybridized with two NotI fragments of 84 and 7 kb (Fig. 1B, lane a) of the O139B genome. However, only one 97-kb NotI fragment hybridized with the O139R genome (Fig. 1B, lane d, and Fig. 2). The 7-kb fragment is generated due to the presence of a NotI site in the RS flanking the core region of the CTX genetic element (Fig. 2B) of O139B (6, 7). The absence of this fragment suggests that the NotI site in the RS is lost in O139R. Although the genome of resurgent strain AS192 showed distinct RFLP with that of other O139R and O139B strains (Fig. 1A and B), the ctxA gene hybridized with the 84- and 7-kb NotI fragments like O139B (Fig. 2). Thus, unlike other resurgent clones, the organization and location of CTX genetic element in the genome of AS192 have not changed from that of O139B (see Fig. 4).

View larger version (34K): [in this window] [in a new window]   FIG. 2.   (A) Southern blot hybridization of different restriction enzyme-digested genomes of V. cholerae O139 strains with ctxA or RS as a probe. With the ctxA probe, NotI, lanes: a, AS192; b, AS197; c, AS199; HindIII, lanes: a, SG24; b, AS192; c, AS197; d, AS199; AvaI, lanes: a, AS192; b, AS197; c, AS199; PstI, lanes: d, AS192; e, AS197; f, AS199; BglII or EcoRV or NotI-HindIII, lanes: a, SG24; b, AS192; c, AS197; d, AS199. RS probe: BglII, lanes: a, AS192; b, AS197; c, AS199, d, SG24. Hybridization was carried out at 60°C, and the filter was washed under high-stringency conditions (0.1× SSC at 60°C). (B) Schematic representation of the origin of the 97-kb NotI and 23-kb BglII fragments in O139R and the 23-kb HindIII fragment of O139B and AS192 genomes in Southern hybridization with the ctxA gene probe (panel A). The open boxes represent core regions of the CTX genetic element. Thick and thin arrows indicate RS of O139B and O139R origin, respectively. Thin lines represent chromosomal DNA. A, AvaI; B, BglII; E, EcoRV; H, HindIII; N, NotI.

rrn operons in O139R. In all the bacterial genomes examined so far (19-21, 23), including the classical and El Tor biotypes of V. cholerae (17, 30), the I-CeuI sites are located only in the rrn operons (19-21, 23, 30). The number of I-CeuI sites in the genome is taken as the measure of the number of rrn operons present in the genome. There are nine I-CeuI sites and hence nine rrn operons in the genome of V. cholerae O1 and O139B strains (30). When genomic DNA of O139R strains was digested with I-CeuI and size fractionated by PFGE, eight restriction fragments of about 1,400, 1,000, 460, 245, 170, 103, 87, and 80 kb, with no detectable RFLP with O139B or themselves, could be resolved in ethidium bromide-stained gels (Fig. 3a). Two additional fragments of 6 and 5.5 kb in O139R and one of 6 kb in O139B were resolved in the autoradiogram of end-labeled DNA (Fig. 3b). This result indicates that in contrast to 9 rrn operons in O139B (30), the genome of O139R has 10 rrn operons. Since the number and location of rrn operons in any bacterium appear to be highly conserved, the presence of an additional copy of the rrn operon in the genome of O139R is unexpected.

View larger version (44K): [in this window] [in a new window]   FIG. 3.   PFGE of I-CeuI-digested genomes of V. cholerae O139R and O139B strains. (a) The genomes of representative O139R and O139B strains were digested with I-CeuI and electrophoresed with a pulse time interpolated between 5 and 100 s for 24 h at 10 V/cm at 3°C, and the gels were stained with ethidium bromide. (b) Autoradiogram of I-CeuI-digested, end-labeled, PFGE-separated genomic DNA of V. cholerae O139R and O139B. The pulse time described in panel a was followed by ramping between 10 and 150 s for another 12 h at 5 V/cm. Two more I-CeuI fragments in the O139R genome and one more in the O139B genome could be resolved under these pulse conditions by autoradiography. The numbers represent the I-CeuI fragment sizes in kilobases.

Organization of the CTX genetic element in O139R. The genes encoding the A and B subunits of CTX (ctxAB), along with other virulence genes encoding core pilin (cep), accessory cholera enterotoxin (ace), and zonula occludence toxin (zot), as well as an unknown open reading frame (orfU), are located on a 4.5-kb central core region of the CTX genetic element (Fig. 4) (4, 31, 37). The core region is flanked by an RS of about 2.4 to 2.8 kb, having one BglII site and one EcoRV site that are conserved in all O1 and O139B strains examined (7, 31). The RS of O139B strains has, in addition to the conserved BglII and EcoRV sites, a NotI site (6, 7). The hybridization of ctxA with only one 97-kb NotI fragment of O139R (Fig. 2A) indicated either that its genome has only one copy of the CTX genetic element or that multiple copies from which the NotI site in the RS is lost are present in tandem. There is no HindIII or EcoRI site in the CTX genetic element of the V. cholerae genome (6, 15, 16, 24, 31, 38), and so these enzymes were used to ascertain whether multiple copies of the element are separated or arranged in tandem (38). In a Southern blot hybridization with the ctxA gene as probe and the HindIII- or EcoRI-digested genome of O139B, a 23-kb HindIII fragment (Fig. 2) and a 23-kb EcoRI fragment (data not shown) lit up in the autoradiogram as expected. On the other hand, the ctxA gene hybridized with three HindIII fragments of 13.5, 8.6, and 7.3 kb (Fig. 2B) and with three EcoRI fragments of 23, 17, and 7.3 kb (data not shown) of the O139R genome. These observations confirm that there are three copies of the CTX genetic element in the genomes of O139R strains, and the presence of the 7.3-kb common HindIII and EcoRI fragment, comprising one core region (4.5 kb) and one RS segment (2.8 kb), shows that these elements are present in tandem. Southern blot hybridization with the ctxA gene probe of AvaI- or PstI-digested genomic DNA (Fig. 2A) also confirmed this possibility.

View larger version (19K): [in this window] [in a new window]   FIG. 4.   Schematic representation of the organization of the CTX genetic element in V. cholerae O139R and O139B strains (not drawn to scale). Open boxes are coding regions of the corresponding genes comprising the core region of the element; thick arrows indicate RS of O139B origin; hollow arrows indicate RS of O139R origin; and thin lines represent chromosomal DNA. The 780-bp NdeI fragment of the ctxAB gene was used as a probe.

	DISCUSSION

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Resurgence of V. cholerae O139 with altered sensitivity to a number of antibiotics compared to O139B within a short period is unexpected in well-characterized clonal strains. The present study shows that in O139R strains clonality has not been retained and that the organization of the CTX genetic elements in the resurgent strains is different from that in O139B. Furthermore, an amplification of rrn operons has taken place in the genomes of O139R strains, including strain AS192, which showed extensive RFLP with other O139 isolates. This strain is sensitive to SXT and resistant to streptomycin and, unlike other resurgent strains, has not undergone any alteration in the organization of the CTX genetic element compared to that in O139B strains. Strain AS192 perhaps represents a different clone from those belonging to the O139B and O139R variants.

Resistance to SXT is used as a presumptive diagnosis of O139B strains (41), and all cells resistant to SXT possess a 62-kb self-transmissible, site-specific, and chromosomally integrating genetic element (40). The results presented in this report showed that the SXT-sensitive resurgent O139 strains, including AS192, still possess the 62-kb element, from which a 3-kb segment has been deleted. From the fact that all O139B strains are resistant and the resurgent isolates are sensitive to SXT, it is tempting to speculate that the sensitive phenotype might be of selective advantage to the organism for survival in the environment, maintaining its epidemic potential.

The gene encoding CT (ctxAB) is located in a 7.0- to 9.7-kb CTX genetic element in the genome of toxigenic V. cholerae strains, frequently in multiple copies arranged in tandem, but is absent in nontoxinogenic vibrios (31). Attachment sites that flank this element promote its integration into the genome by a site-specific mechanism. Several studies have indicated that even in strains of clonal origin, the CTX genetic elements can undergo rapid rearrangement in the form of amplification or deletion (7, 39). The O139B genome has two copies of CTX genetic elements in tandem, with the core regions being connected by two RS segments. In agreement with a recent report (35), the present study demonstrates that the genome of O139R strains has three copies of the element present in tandem. The present study also demonstrates that the RS connecting core regions of the elements is different from that in O139B strains (Fig. 4); it has gained HindIII and EcoRI and lost NotI and BglII sites. In all strains where the elements are present in tandem, only one HindIII or EcoRI fragment hybridizes, since there is no HindIII or EcoRI site either in the core or in the RS element of all O1 and O139B strains examined (6, 7, 24). Since the RS in the CTX genetic elements of O139R strains contains HindIII and EcoRI sites (Fig. 4), these enzymes can no longer be considered diagnostic for differentiating biotypes. Besides, the BglII site, which is conserved in the RS of all O1 and O139B strains, is lost in the RS of O139R strains. Surprisingly, RS of O139B origin is retained upstream of the three CTX genetic elements of O139R strains and two copies of it are present in tandem compared to one in O139B strains.

The number and location of rrn operons in enteric bacteria appear to be highly conserved. In contrast to seven rrn operons in the genomes of different enteric gram-negative organisms (20), the genomes of V. cholerae O1 and O139B have nine (17, 30). V. cholerae strains belonging to non-O1 and non-O139 serogroups, which are autochthonous organisms in the aquatic environment, have 10 rrn operons in their genome (30), as in the O139R genome investigated in the present study. Intraspecies variation in the number of rrn operons has recently been found in V. cholerae (30). The present study shows variation in the number of rrn operons even within the same serovar. While Sharma et al. (35) could not detect any difference in the ribotype between O139R and O139B strains, Faruque et al. (13) have shown that a 3-kb BglI fragment containing the rRNA gene is unique to reemerging V. cholerae O139 strains isolated in Bangladesh during 1995 and 1996. The 3-kb BglI fragment cannot originate from the generation of a BglI site in one of the rrn-containing fragments in the genomes of O139 strains isolated during 1992 and 1993, because the rRNA gene restriction patterns of two clones, apart from the 3-kb BglI fragment, are identical (13). It was also reported that the strains belonging to a new ribotype also belonged to a new ctx genotype (13). It appears that the reemergent O139 strains described by Faruque et al. (13) are probably of the same genotype as the O139R isolates of Indian origin. Thus, an amplification of rrn operons in the genomes of O139R strains has occurred. However, Basu et al. (3) have shown that very recent Bangladesh O139 isolates have similar ribotype and ctx genotype to O139B but that all these strains are sensitive to streptomycin. Thus, the Bangladesh O139 strains studied by Basu et al. are different from O139B and O139R strains and the strains described by Faruque et al. (13).

Spontaneous duplication of rrn operons by homologous recombination has been found in Salmonella typhimurium during laboratory maintenance (2, 14). This has not happened in O139R strains since the cells were minimally subcultured. Besides, the genomes of all the O139R isolates produced identical I-CeuI cleavage profiles (Fig. 3). No change in the number of rrn operons was detected in the genomes of O139B strains, which were repeatedly subcultured in the laboratory since 1992. The number of rrn operons is directly proportional to the selective advantage of the survival of the organisms in a continually fluctuating environment (18). Many different factors in the aquatic environment affect the growth and survival of V. cholerae cells. Furthermore, in the course of the transition from a typical environmental source such as water to the human intestine, these bacteria are exposed to a series of environmental changes. The observed modulation in the number of rrn operons might be necessary for the survival of the organism in the human intestine as well as for maintaining the genomic plasticity through rrn-mediated rearrangements which have been shown to be characteristic of V. cholerae (30). It has been predicted that the possibility of genome rearrangements is greater when there are more repeats of the rrn operons (18).