INTRODUCTION

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Cholera caused by toxigenic Vibrio cholerae is a major public health problem in developing countries. Epidemiological surveillance of cholera and comparative molecular analysis of strains collected during outbreaks have demonstrated clonal diversity among epidemic strains and a continual emergence of new clones of toxigenic V. cholerae (5-7, 21). The mechanisms involved in the emergence of new toxigenic clones have not been adequately explained, although it is assumed that a combination of genetic changes and natural selection caused by unidentified environmental factors as well as the immune status of the host populations is likely to influence the process.

The profuse secretory diarrhea characteristic of cholera is caused by an enterotoxin, cholera toxin (CT), produced by toxigenic V. cholerae when it colonizes the small intestine (20). The genes encoding CT (ctxAB) are part of a larger genetic element (CTX genetic element) consisting of at least six genes (ctxAB, zot, ace, cep, and orfU) comprising the "core" region that is flanked by two or more copies of a repeated sequence (19, 23). It has been demonstrated recently (24) that in the V. cholerae O1 strain P27459 (genetically modified by marker exchange and renamed SM44), the entire CTX element constituted the genome of a filamentous bacteriophage (CTX). The phage could be propagated in recipient V. cholerae strains in which the CTX genome either integrated chromosomally at a specific site, forming stable lysogens, or was maintained extrachromosomally as a replicative form (RF) of the phage DNA (24). Cultures of V. cholerae harboring the RF of CTX produced high titers of the phage in their supernatants. This study indicated that the propagation of CTX may be associated with horizontal gene transfer leading to the origination of novel toxigenic strains of V. cholerae. More recently, it has been reported that during passage of CTX lysogens through the infant-mouse intestine, phage excision and replication occur in vivo (16). The present study was undertaken to analyze the induction of lysogenic CTX in clinical and environmental isolates of toxigenic V. cholerae and to investigate whether cholera pathogenesis in humans is associated with the excretion of CTX particles in the stools. Furthermore, this study investigated the ability of CTX particles derived from naturally occurring V. cholerae strains to infect a CT-negative recipient strain of V. cholerae O1 inside the gastrointestinal tracts of infant mice and under laboratory conditions.

	MATERIALS AND METHODS

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V. cholerae strains and cholera stools. Toxigenic V. cholerae strains analyzed in this study to investigate the induction of lysogenic CTX included a total of 125 clinical isolates and 16 environmental isolates belonging to O1 or O139 serogroups. Clinical isolates and stools that were culture positive for cholera were obtained from patients who attended the treatment center of the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B) located in Dhaka. The clinical strains consisted of 78 strains from the culture collection of ICDDR,B (Table 1) and 47 strains isolated from freshly collected culture-positive stools, which were also analyzed in this study (Table 2). The environmental strains were obtained from surface waters in Dhaka. Strains were stored either in lyophilized form or in sealed deep nutrient agar at room temperature. Before use, the identities of the V. cholerae cultures were confirmed by biochemical reactions and serological test (27), and the presence of the CTX element was ascertained by using DNA probes (6). The relevant characteristics of V. cholerae strains used as controls or as a recipient of CTX are listed in Table 3.

View this table: [in this window] [in a new window]   TABLE 1.   Analysis of toxigenic V. cholerae O1 and O139 strains isolated between 1969 and 1997 in Bangladesh for the induction of CTX

View this table: [in this window] [in a new window]   TABLE 2.   Analysis of 47 culture-positive stools collected during June and July 1997 in Bangladesh for the presence of cell-free CTXa

Probes and hybridization. The gene probes used in this study to detect the CTX genome were a 0.5-kb EcoRI fragment of pCVD27 (14), containing part of the ctxA gene, and an 840-bp region internal to the zot gene amplified by PCR from the recombinant plasmid pBB241 as described previously (8). Strand-specific oligonucleotide probes with the sequences 5'-TCTATCTCTGTAGCCCCTATTACG and 5'-CTCAGACGGGATTTGTTAGGCACG for probing the plus and minus strands, respectively, were also used to detect the presence of single-stranded DNA of CTX. Colony blots or Southern blots were prepared with nylon filters (Hybond; Amersham International plc., Ayelesbury, United Kingdom) and processed by standard methods (17). The polynucleotide probes were labeled by random priming (10) with a random-primer DNA-labeling kit (Bethesda Research Laboratories, Gaithersburg, Md.) and [-³²P]dCTP (3,000 Ci/mmol; Amersham), and oligonucleotide probes were labeled by 3' tailing with terminal deoxynucleotide transferase and [-³²P]dCTP. Southern blots and colony blots were hybridized with the labeled probes and autoradiographed as described by us previously (5-7).

Induction of CTX lysogens. Toxigenic V. cholerae strains were grown in Luria broth (LB) at 30°C to an absorbance at 540 nm of 0.2. The cells were collected by centrifugation, washed, and resuspended in fresh LB. The suspension was divided into aliquots, to which mitomycin C (Sigma Chemical Co., St. Louis, Mo.) was added at 20 ng/ml and incubated overnight at 30°C. The culture supernatants were analyzed for extracellular phage carrying the CTX element as described in the following section. Strains grown in a similar way but without mitomycin C were used as controls.

Screening of stools and bacterial cultures for CTX. Freshly collected culture-positive stools and V. cholerae strains either isolated from the same stools (Table 2) or obtained from the culture collection (Table 1) were used. Aliquots of watery stools or cultures were centrifuged at 6,000 × g to precipitate solid particles and suspended bacteria, and the supernatants were sterilized by filtration through 0.22-µm-pore-size filters (Millipore Corp., Bedford, Mass.). To confirm that the filtrates from stools as well as the culture supernatants did not contain any bacterial cell, aliquots of the filtrates were streaked on Luria agar (LA) plates and incubated overnight at 37°C. The sterile filtrates were mixed with one-fourth volumes of a solution containing 20% polyethylene glycol 6000 and 10% NaCl and centrifuged at 12,000 × g to precipitate the phage particles. The precipitate was dissolved in a solution containing 20 mM Tris-Cl (pH 7.5), 60 mM KCl, 10 mM MgCl, and 10 mM NaCl and digested with pancreatic DNase I (100 U/ml) and RNase A (50 µg/ml) at 37°C for 1 h to remove possible nucleic acids carried over from lysed bacterial cells. The solution was extracted with phenol-chloroform to disrupt possible phage particles, and the total nucleic acids were precipitated with ethanol. Preparations containing CTX DNA were identified by Southern blot hybridization (22). Aliquots of watery stools mixed with serial dilutions of CTX-Km (10¹ to 10⁴ particles/ml) isolated from a mitomycin C-induced culture of strain SM44 were used as positive controls for the assay of phage in stools, for both in vitro and in vivo assays. CTX-Km derived from SM44 was quantified by incubating serially diluted filter-sterilized culture supernatants with strain RV508 and then determining the number of Km^r colonies, as described previously (24).

View this table: [in this window] [in a new window]   TABLE 4.   Infection of strain CVD103 by extracellular CTX isolated from the supernatant fluids of mitomycin C-induced cultures of clinical and environmental V. cholerae strains

	RESULTS AND DISCUSSION

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Induction and transmission of CTX. To investigate whether lysogenic CTX present in toxigenic V. cholerae strains could be induced to produce extracellular infectious phage particles, we cultured the strains in the presence of a DNA-damaging agent, mitomycin C, and then screened the culture supernatant fluids for the presence of CTX by using DNA probes. Similarly, freshly collected culture-positive stools were analyzed for the presence of CTX to understand whether the phage is excreted in the stools of cholera patients. Another strategy for detecting the presence of infectious CTX in culture supernatants as well as in culture-positive stools was to expose a suitable recipient strain, CVD103, to the phage preparations and then look for infection of the bacterial cells. None of the culture-positive stools analyzed was positive for the presence of a detectable level of CTX either in the DNA probe assay or in the in vivo assay in infant mice. To determine the detection limits of our assays, we used reconstituted stools containing a known number of phage particles. This was done by using a genetically marked phage, CTX-Km (derived from strain SM44), since CTX-Km could be quantified by titration with a recipient strain RV508, which constitutively expresses toxin-coregulated pili (TCP) and hence is readily infected by the phage (24). In the case of stools mixed with exogenous CTX-Km and used as positive controls, a minimum of 10³ phage particles in 5 ml of stool could be distinctly detected by Southern blot hybridization (Fig. 1). When assayed in infant mice, the presence of 10² particles of CTX-Km in 5 ml of reconstituted stools could be detected and the corresponding stool extract produced between one and nine colonies (recovered from five different mice) of Km^r transductants of CVD103.

View larger version (87K): [in this window] [in a new window]   FIG. 1.   Southern blot hybridization of bacteriophage DNA isolated from reconstituted culture-positive stools containing serial dilutions of the genetically marked phage CTX-Km and probed with the zot probe. Lanes 1 through 7 correspond to extracts from 5 ml of stools containing 1 × 102, 5 × 102, 1 × 103, 1.5 × 103, 2 × 103, 2.5 × 103, and 5 × 103 phage particles, respectively. Numbers indicating the molecular sizes of bands correspond to the supercoiled DNA ladder (Bethesda Research Laboratories).

View larger version (82K): [in this window] [in a new window]   FIG. 2.   Supernatant fluids of toxigenic V. cholerae O1 or O139 strains grown in the presence of mitomycin C (20 ng/ml) were sterilized by filtration through a 0.22-µm filter and were used to precipitate possible bacteriophage particles. The precipitates were dissolved in appropriate buffer and treated with DNase I and RNase A to remove contaminating exogenous DNA or RNA. Total phage nucleic acids were isolated as described in Materials and Methods and analyzed with the ctxA probe. Southern blot hybridization analyses of total phage nucleic acids derived from six different V. cholerae strains (indicated by strain numbers) are shown. Numbers indicating the molecular sizes of bands correspond to supercoiled DNA ladder (Bethesda Research Laboratories).

Stability of the CTX genome in the recipient strain. CTX is unusual among filamentous phages because it can either replicate as a plasmid or integrate into the V. cholerae chromosome at a specific attachment site (24). Analysis of representative probe-positive colonies for the presence of CTX genome revealed the presence of the RF of the CTX genome in freshly infected colonies. However, the concentration of the RF DNA was too low to be visible in ethidium bromide-stained gels, but it could be faintly detected by Southern blot hybridization (data not shown). The phage genome was rapidly lost from infected cells when the infected cells were cultured under laboratory conditions, and only 0.9% of the colonies recovered after 24 h of culture hybridized with the ctxA probe. The CTX genome did not integrate into the chromosome of CVD103, as shown by Southern blot analysis of genomic DNA with the ctxA probe (data not shown) as well as presumed from the rapid loss of CTX genome from infected cells. When CVD103 was infected with CTX-Km derived from strain SM44, the infected cells retained the RF of the phage when cells were grown in the presence of kanamycin. However, when the infected cells were cultured in the absence of kanamycin, the RF of CTX-Km was rapidly lost and 60.3% of viable cells recovered after 12 h of culture became susceptible to kanamycin (50 µg/ml). Previously, the CTX genome was shown to integrate into the chromosome when an El Tor strain carrying a resident attRS sequence was used as the recipient (24). In the previous study, the phage was marked with kanamycin resistance and the transfectants were maintained in the presence of kanamycin. Similarly, in the present study, CVD103 cells infected with the CTX-Km retained the RF DNA of the phage when the cells were grown in the presence of kanamycin but rapidly lost the RF of CTX-Km in the absence of kanamycin. This indicated that there was a requirement for appropriate selection pressure for the retention of the extrachromosomal CTX element in V. cholerae. The lack of selection of cells carrying the RF of CTX could account for the subsequent loss of the phage DNA. However, once the phage genome integrates into the chromosome of the recipient cell, it is likely to be more stable. There have been several studies which suggested that the gastrointestinal environment can cause a selective enrichment of toxigenic V. cholerae strains compared to nontoxigenic strains (2, 11, 13). It therefore seems possible that in addition to adequate expression of TCP, which is the receptor for the phage, the intestinal environment also contributes to selecting V. cholerae cells harboring the CTX genome. Since the CTX element itself carries the necessary genes for its own integration at resident attRS sites of the host chromosome (26), the origination of novel toxigenic strains of V. cholerae and their selective enrichment are likely to occur in the intestinal environment.

Propagation of CTX. The existence of CTX was discovered by using a genetically modified V. cholerae strain, SM44, in which the CTX element was marked with a kanamycin resistance (Km^r) determinant (12, 24). The presence of the Km^r marker facilitated the detection of the induction and transmission of the phage. The present study has investigated for the first time the prevalence of inducible lysogens of CTX among naturally occurring strains of toxigenic V. cholerae and the potential of the extracellular phage particles to infect a CT-negative recipient strain of V. cholerae. This study is also the first to examine clinical materials for the presence of CTX and to investigate whether cholera pathogenesis is normally associated with the excretion of the phage in stools. Since the CTX element was not marked with a phenotypically detectable marker in native strains, detection of the induction of CTX and its transmission in vitro and in infant mice were studied with specific DNA probes. Although V. cholerae is known to be primarily a human pathogen, it has been suggested that the bacterium can persist in the aquatic environment in unexplained ecological associations (3). The natural habitat of toxigenic V. cholerae therefore seems to consist of two compartments: the gastrointestinal tract of the host and the aquatic environment outside the host. The host compartment is known to provide signals necessary for the expression of most ToxR-regulated virulence-associated factors, including CT and TCP (1, 4). To investigate whether induction of the CTX prophage is also mediated by possible host factors in the human gastrointestinal tract, we screened culture-positive stools for the presence of the phage. That all the stools were negative for the phage suggested that induction of lysogenic CTX was possibly not associated with cholera pathogenesis in humans, unless we assume that the CTX particles were very unstable in the human intestine and rapidly degraded beyond the detection limit of our assays. However, the present study showed that when extracellular CTX isolated from naturally occurring strains and the control strain SM44 were used to infect the recipient strain in vivo, CTX particles were stable and remained infectious in the gastrointestinal environment of infant mice. It may be mentioned that induction of the CTX prophage in infant mice has been reported recently based on observed transduction of a recipient strain by a CTX lysogen inside the intestines of infant mice (16). The present study, however, did not provide any evidence in favor of the induction of lysogenic CTX inside the human intestine in association with cholera pathogenesis.