Prevalence of virulence genes (ctxA, stn, OmpW and tcpA) among non-O1 Vibrio cholerae isolated from fresh water environment

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Abstract

The virulence of a pathogen is reliant on the presence of a discrete set of genetic determinants and their expression in the host. The virulence of Vibrio spp. is regulated by the ctxAB and tcpA genes. These genes are alleged to be exclusively associated with clinical strains of O1 and O139 serogroups. In the present study, we examined the presence of virulence genes viz. stn, OmpW, ctxA and tcpA of classical and ElTor variants, in environmental strains of non-O1 Vibrio cholerae cultured seasonally from four sampling stations of the river Narmada at Jabalpur (MP), India. Unexpectedly, the PCR analysis of the strains revealed the presence of these genes among environmental V. cholerae. The strains harboring the tcpA gene also carried the ctxA gene. Sequencing of the tcpA gene and ctxA gene carried by an environmental strain showed ∼97% homology with the previously sequenced genes submitted in the GenBank. We report here the prevalence of cholera toxin gene and the gene for toxin co-regulated pilus among non-O1 V. cholerae strains isolated from fresh water environment. This study supports the idea that cholera toxin has an environmental derivation and that the intricate aquatic environment can give rise to pathogenic Vibrio organisms.

Introduction

Vibrio cholerae is an autochthonous inhabitant of aquatic systems and is the causative agent of severe dehydrating diarrheal disease, cholera. Though this organism is conscientious for the several epidemics and pandemics, it is a universal conviction that most of the environmental strains do not produce cholera toxin (CT) and are therefore of trifling magnitude in epidemic potential. In spite of copious studies over more than a century, the epidemiology of cholera remains mysterious and challenging to investigators in the field. Until the emergence of V. cholerae O139 in 1992, toxigenic strains of V. cholerae O1 were considered to be the solitary causative agents of epidemic and pandemic cholera (Faruque et al., 2000b).

The knack of pathogenic Vibrio spp. to cause disease depends on the expression of virulence factors like a potent enterotoxin (CT), a pilus colonization factor (toxin co-regulated pilus; TCP). CT is encoded by a transferable filamentous phage (CTXφ) and reports have implied the acquisition of these CT genes under conditions analogous to those in the aquatic environments (Faruque and Nair, 2002).

CtxAB operon, which encodes for the A and B subunits of CT, resides in the genomes of CTXφ. All Vibrio strains proficient of causing cholera perpetually carry genes for TCP which is an adhesin that is coordinately regulated with CT production (Taylor et al., 1987). TCP is the solitary V. cholerae pilus that has been demonstrated to date to have a role in colonization of the gut mucosa of humans (Herrington et al., 1988) and of infant mice (Taylor et al., 1987), the latter being an experimental model.

It has been presumed that CT and TCP are exclusively associated with clinical strains of V. cholerae, notably those belonging to serogroups O1 and O139. Similarly, TCP has infrequently been allied with the environmental strains of V. cholerae (Nair et al., 1988). The major structural subunit of TCP is encoded by the tcpA gene, which is a part of a gene cluster comprising at least 15 open reading frames (Kovach et al., 1996).

The NAG-ST is a 17 amino acid peptide; encoded by the stn gene, which exhibits remarkable similarity, especially in the carboxyl-terminal toxin domain, to the heat-stable enterotoxins (STs) produced by enterotoxigenic Escherichia coli (ETEC). The NAG-ST gene has been cloned and sequenced (Ogawa et al., 1990), which has enabled the construction of DNA probes for detection of NAG-ST. Taking advantage of the availability of these probes, we undertook a study to examine a large collection of V. cholerae non-O1 for the presence of a NAG-ST-like enterotoxin.

The presence of OmpW in V. cholerae strains, coupled with the fact that its nucleotide sequence remained practically unchanged among different V. cholerae strains, makes it a highly suitable genetic marker for the organism (Nandy et al., 2000).

The present investigation was undertaken on the vibrios isolated from the largest west flowing river of the Indian subcontinent. The total length of the river is 1312 km, which before draining off into the gulf of Gambay passes through two states of India viz. Madhya Pradesh (MP) and Gujarat. River Narmada is an important source of fresh water supply for Jabalpur (MP), where the sampling was carried out.

The intent of the present study was to monitor Vibrio strains isolated from the river Narmada for virulence genes (ctxA and tcpA Classical and tcpA ElTor, stn and OmpW), collected seasonally from Jabalpur (MP), an expanse of non-epidemicity.

Section snippets

Collection and processing of environmental samples

Water samples were collected from four stations (Gwarighat, Jelharighat, Lamhetaghat and Tilwaraghat), over a 2-year period from January 2002 to December 2003, at monthly intervals. Four water samples were collected from each station per month. Ca. 500 ml water samples were collected, concentrated on 0.45 μm pore diameter filters, and enriched in alkaline peptone water (APW (1% peptone, 1% NaCl, pH 8.4–8.6)) for isolation of Vibrio spp. (Kaper et al., 1979). Bacterial colonies were isolated from

Results

A total of 115 environmental isolates of the genus Vibrio were analyzed in the present study. The results show that the CT gene (ctxA) and the gene for toxin co-regulated pili (tcpA of classical variants) was present in 14 isolates (∼13%) by PCR detection (Fig. 1). The identity of the V. cholerae was confirmed by screening with the OmpW gene, which showed 100% specificity for all V. cholerae strains tested (Fig. 2). These strains constitute a reservoir of virulence genes in the environment. The

Discussion

V. cholerae is a well-defined species on the basis of biochemical test activity and DNA homology, but the species is not homogenous with regards to its pathogenic potential (Baumann et al., 1984). Among the 193 currently recognized somatic ‘O’ serogroup of V. cholerae, only O1 and O139 strains are capable of causing epidemic cholera, but these two serogroups are rarely found in the environment except during an epidemic.

TCP has been shown to be essential for colonization in the infant mouse

Acknowledgments

Authors are thankful to the Head, Department of Biosciences, R.D. University, Jabalpur, for providing lab facilities, Dr. T. Ramamurthy, Assistant Director, National Institute of Cholera and Enteric Diseases, Kolkata, India, for molecular studies and to the UGC for giving financial assistance.

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