Virulence genes in environmental strains of Vibrio cholerae.

The virulence of a pathogen is dependent on a discrete set of genetic determinants and their well-regulated expression. The ctxAB and tcpA genes are known to play a cardinal role in maintaining virulence in Vibrio cholerae, and these genes are believed to be exclusively associated with clinical strains of O1 and O139 serogroups. In this study, we examined the presence of five virulence genes, including ctxAB and tcpA, as well as toxR and toxT, which are involved in the regulation of virulence, in environmental strains of V. cholerae cultured from three different freshwater lakes and ponds in the eastern part of Calcutta, India. PCR analysis revealed the presence of these virulence genes or their homologues among diverse serotypes and ribotypes of environmental V. cholerae strains. Sequencing of a part of the tcpA gene carried by an environmental strain showed 97.7% homology to the tcpA gene of the classical biotype of V. cholerae O1. Strains carrying the tcpA gene expressed the toxin-coregulated pilus (TCP), demonstrated by both autoagglutination analysis and electron microscopy of the TCP pili. Strains carrying ctxAB genes also produced cholera toxin, determined by monosialoganglioside enzyme-linked immunosorbent assay and by passage in the ileal loops of rabbits. Thus, this study demonstrates the presence and expression of critical virulence genes or their homologues in diverse environmental strains of V. cholerae, which appear to constitute an environmental reservoir of virulence genes, thereby providing new insights into the ecology of V. cholerae.

Genomic diversity among Vibrio cholerae O139 strains isolated in Bangladesh and India between 1992 and 1998.

In order to assess the extent of genomic diversity among Vibrio cholerae O139 strains, restriction fragment length polymorphisms in two genetic loci, rrn and ctx, were studied. Analysis of 144 strains isolated from different regions of Bangladesh and India between 1992 and 1998 revealed the presence of at least six distinct ribotypes (B-I through B-VI) of which three were new ribotypes, and one of these was represented by a nontoxigenic O139 strain. Strains of ribotypes B-I through B-V shared 11 different CTX genotypes (A through K). Antimicrobial resistance patterns of the strains varied independently of their ribotypes and CTX genotypes. Results of this study suggest that V. cholerae O139 is undergoing rapid genetic changes leading to the origination of new variants, and temporal changes in antimicrobial resistance patterns may be contributing to the selection of different variants.

Clonal diversity among recently emerged strains of Vibrio parahaemolyticus O3:K6 associated with pandemic spread.

The genomes of the O3:K6 strains of Vibrio parahaemolyticus which abruptly emerged in Calcutta, India, in February 1996 and which demonstrated an unusual potential to spread and an enhanced propensity to cause infections were examined by different molecular techniques to determine clonality. No restriction fragment length polymorphism (RFLP) in the gene encoding the thermostable direct hemolysin was observed among the O3:K6 isolates of V. parahaemolyticus. Clonal diversity among the O3:K6 strains became evident by examining the RFLPs of the rrn operons and by the use of pulsed-field gel electrophoresis. Five ribotypes were distinguished among the O3:K6 strains examined, with ribotype R4 constituting the major type. Strains of O3:K6 isolated between June and August 1996 showed different pulsotypes compared to the pulsotypes of strains isolated before and after this period, indicating genetic reassortment among these strains, but those isolated between August 1996 and March 1998 showed identical or nearly similar pulsotypes. It is clear that there is a certain degree of genomic reassortment among the O3:K6 clones but that these strains are predominantly one clone.

Resurgent Vibrio cholerae O139: rearrangement of cholera toxin genetic elements and amplification of rrn operon.

The unprecedented genesis of a novel non-O1 Vibrio cholerae strain belonging to serogroup O139, which caused an epidemic in late 1992 in the Indian subcontinent, and its subsequent displacement by El Tor O1 vibrios after 18 months initiated a renewed investigation of the aspects of the organism that are related to pathogenesis. The reappearance of V. cholerae O139 with altered antibiotic sensitivity compared to O139 Bengal (O139B) in late 1996 has complicated the epidemiological scenario of V. cholerae and has necessitated an examination of possible rearrangements in the genome underlying such rapid changes in the phenotypic traits. With a view to investigating whether the phenotypic changes that have occurred are associated with alteration in the genome, the genome of the resurgent V. cholerae O139 (O139R) strains were examined. Pulsed-field gel electrophoresis analysis of NotI- and SfiI-digested genomic DNA of O139R isolates showed restriction fragment length polymorphism including in the cholera toxin (CTX) genetic element locus and with O139B isolates. Analyses of the organization of the CTX genetic elements in O139R strains showed that in contrast to two copies of the elements connected by two direct-repeat sequences (RS) in most of the genomes of O139B isolates, the genomes of all O139R strains examined, except strain AS192, have three such elements connected by a single RS. While the RS present in the upstream of the CTX genetic elements in the genome of O139R is of O139B origin, the RS connecting the cores of the elements has several new restriction sites and has lost the BglII site which is supposed to be conserved in all O1 strains and O139B. The endonuclease I-CeuI, which has sites only in the rrn operons in the genomes of all organisms examined so far, has 10 sites in the genomes of O139R strains, compared to 9 in the genomes of O139B strains. The recent isolates of V. cholerae O139 have thus gained one rrn operon. This variation in the number of rrn operons within a serogroup has not been reported for any other organism. The results presented in this report suggest that like the pathogenic El Tor O1 strains, the genomes of O139 strains are undergoing rapid alterations.

Vibrio cholerae O139 Bengal: combined physical and genetic map and comparative analysis with the genome of V. cholerae O1.

A combined physical and genetic map of the genome of strain SG24 of Vibrio cholerae O139 Bengal, a novel non-O1 strain having epidemic potential, has been constructed by using the enzymes NotI, SfiI, and CeuI. The genome of SG24 is circular, and the genome size is about 3. 57 Mb. The linkages between 47 NotI and 32 SfiI fragments of V. cholerae SG24 genomic DNA were determined by combining two approaches: (i) identification of fragments produced by enzyme I in fragments produced by enzyme II by the method of fragment excision, redigestion, and end labeling and (ii) use of the linking clone libraries generated from the genome of classical O1 strain 569B. The linkages between nine CeuI fragments were determined primarily by analyses of partial fragments of the CeuI-digested genome. More than 80 cloned homologous and heterologous genes, including several operons, have been positioned on the physical map. The map of the SG24 genome represents the second map of a V. cholerae genome, and a comparison of this map with that of classical O1 strain 569B revealed considerable diversity in DNA restriction sites and allowed identification of hypervariable regions. Several genetic markers, including virulence determinant genes, are in different positions in the SG24 and 569B genomes.

Rearrangements in the genomes of Vibrio cholerae strains belonging to different serovars and biovars.

The intron-encoded enzyme I-CeuI provides an excellent tool for rapidly examining the organization of genomes of related species of bacteria. Vibrio cholerae strains belonging to serovars O1 and O139 have 9 I-ceuI sites in their genomes, and V. cholerae strains belonging to serovars non-O1 and non-O139 have 10 I-ceuI sites in their genomes. This information can be used as a criterion to differentiate O1 strains from non-O1 and non-O139 strains. To our knowledge, intraspecies variation in the number of rrn operons has not been reported in any other organism. Our data revealed extensive restriction fragment length polymorphism based on a comparison of the I-ceuI digestion profiles of strains belonging to different serovars and biovars. From the analysis of partial digestion products, I-CeuI macrorestriction maps of several classical, El Tor, and O139 strains were constructed. While the linkage maps are conserved within biovars, linkage maps vary substantially between biovars.

Physical map of the genome of Vibrio cholerae 569B and localization of genetic markers.

A combined physical and genetic map of the genome of the classical O1 hypertoxinogenic strain 569B of Vibrio cholerae has been constructed. The enzymes NotI, SfiI and CeuI generated DNA fragments of suitable size distribution that could be resolved by pulsed-field gel electrophoresis. The digests produced 37, 22, and 7 fragments, respectively. The CeuI maps of the genomes of strains 569B and O395, constructed by partial restriction digestion, were identical, and the data are consistent with the concept of circular chromosomes. The genome size of each of the strains was estimated to be about 3.2 Mb. The NotI and SfiI digestion profiles of the genomic DNAs of strains 569B and O395 exhibited distinct restriction fragment length polymorphism. The linkages between the 37 NotI fragments of the genome of strain 569B were determined by combining three approaches: isolation of linking clones, analysis of partial digestion fragments, and identification of NotI fragments in isolated CeuI and SfiI fragments. To align linked fragments precisely, NotI-digested genomic DNA was end labeled and separated in the same gel with the NotI-digested DNA to be probed with linking clones. This also allowed the identification of smaller restriction fragments that are not visible in ethidium bromide-stained gels. The presence of repetitive DNA sequences in the V. cholerae 569B genome has been demonstrated. Twenty cloned homologous and heterologous genes and seven rrn operons have been positioned on the physical map. The two copies of the Ctx genetic element in the genome of strain 569B are located about 1,000 kb apart.

Cholera vaccine: developmental strategies and problems.

Over a hundred years have elapsed since Vibrio cholerae, the etiological agent for the disease cholera, was discovered by Robert Koch. Ever since then serious efforts have been made to develop prophylactic measures to combat the disease without much success. Seven pandemics have so far been reported and cholera still remains a public health problem in developing countries. Several strategies have been adopted to develop vaccines against the disease and many of these vaccines have undergone field trials. During the last two decades, an enormous amount of information has accumulated regarding the organism V. cholerae, its virulence factors, including cholera toxin, and the molecular basis of its pathogenicity. In recent years, with the advent of recombinant DNA technology and major breakthroughs in molecular biology and immunology, a new dimension has been given to the design of vaccine strains. The second generation live oral vaccines will perhaps soon replace the long-used first generation parenterally administered killed whole cell vaccines which offered protection for not more than three months. All the recombinant vaccines tested so far produced adverse reactions in volunteers, although they provided varying degrees of protection upto about one year of surveillance. Parallel to the trials of live oral vaccines, combination vaccines comprising killed whole cells and purified B subunit of cholera toxin was also tried. These vaccines had minimal side-effects but the efficacy was not upto expectations. From the failure of each vaccine strain, new information had emerged and improved strategies were adopted.(ABSTRACT TRUNCATED AT 250 WORDS)

Recombinant derivative of a naturally occurring non-toxinogenic Vibrio cholerae 01 expressing the B subunit of cholera toxin: a potential oral vaccine strain.

A clinical isolate of Vibrio cholerae 01 was identified which did not possess the heat-labile (CT), the heat-stable (ST) or the zonula occludens (Zot) toxin genes. Rabbit ileal loop assays showed that no other CT-like toxin was produced by this strain. The partly deleted cholera toxin gene which carries the intact gene for the B subunit was cloned and the recombinant plasmid, pURD110, was introduced into this non-toxinogenic natural human isolate. The transformed cells (strain URD2) secreted the B subunit gene product which competed with the holotoxin secreted by the hypertoxinogenic strain 569B of V. cholerae for the GM1 ganglioside binding sites in vivo. This strain can colonize the rabbit intestine as detected by the removable intestinal tie adult rabbit diarrhoea (RITARD) model. This construct has an advantage over other live oral attenuated V. cholerae strains used as vaccines in that the latter strains were made non-toxinogenic by only deleting part of the gene coding for the A subunit of cholera toxin while the strain described here is naturally non-toxinogenic.

Genome size and restriction fragment length polymorphism analysis of Vibrio cholerae strains belonging to different serovars and biotypes.

The genome size of Vibrio cholerae has been determined by pulsed field gel electrophoresis following digestion of chromosomal DNA with endonucleases. The genome size of all the classical strains examined was about 3000 kb and that of El Tor biotype was 2500 kb. The NotI and SfiI digestion patterns of the genomes of several V. cholerae strains belonging to different serovars and biotypes showed distinct restriction fragment length polymorphism (RFLP). RFLP analysis together with the genome size can be used to differentiate strains of different serovars and biotypes of V. cholerae.

Cell surface characteristics of environmental and clinical isolates of Vibrio cholerae non-O1.

The cell surfaces of several toxigenic and nontoxigenic environmental and clinical isolates of Vibrio cholerae non-O1 have been examined. The environmental strains, irrespective of toxigenicity, are significantly more resistant to antibiotics and detergents than are V. cholerae O1 strains. The clinical isolates of non-O1 vibrios are as sensitive to a wide variety of chemicals as the O1 vibrios. The environmental non-O1 strains are also less susceptible to lysis when treated with protein denaturants or neutral and anionic detergents than are O1 vibrios and the clinical non-O1 strains. In contrast to O1 vibrios, the environmental non-O1 vibrios do not have exposed phospholipids in their outer membranes. These features of the cell surfaces of environmental non-O1 vibrios might have a role in the better survival of these organisms under environmental fluctuations.

Reassessment of the prevalence of heat-stable enterotoxin (NAG-ST) among environmental Vibrio cholerae non-O1 strains isolated from Calcutta, India, by using a NAG-ST DNA probe.

A collection of 521 environmental isolates of Vibrio cholerae which were previously examined by the suckling mouse assay and found to be negative for the heat-stable enterotoxin NAG-ST were reassessed by a recently developed DNA probe for NAG-ST. A total of 12 (2.3%) of the isolates hybridized with the NAG-ST probe. By using a cholera toxin (CT) DNA probe, the CT gene was detected in six of the strains in the collection, although none of the isolates of V. cholerae non-O1 hybridized with both of the toxin probes. All of the NAG-ST and CT probe-positive strains were hemolysin positive. Thirty-fold-concentrated supernatants of the three representative NAG-ST DNA probe-positive V. cholerae non-O1 strains gave positive fluid accumulation ratios in the suckling mouse assay even after heating (100 degrees C for 5 min) and also inhibited the binding of a NAG-ST monoclonal antibody to the bound NAG-ST in a competitive enzyme-linked immunosorbent assay (ELISA). Likewise, all six CT probe-positive V. cholerae non-O1 strains produced in vitro CT when examined by the CT bead ELISA. HindIII digest patterns of chromosomal DNA from the representative NAG-ST gene-positive strains were visually indistinguishable. Between the groups of NAG-ST probe-positive strains examined, there was a variation in the hybridizable fragments, with one group of strains exhibiting a hybridizable fragment similar to that of the NRT 36 reference strain; a smaller HindIII fragment hybridized with the NAG-ST probe in the other group of strains.(ABSTRACT TRUNCATED AT 250 WORDS)

Campylobacter species as a cause of diarrhoea in children in Calcutta.

From 1985 to 1988, 857 children (aged between 1 day and 60 months) admitted to hospital with diarrhoea and 241 controls (aged between 5 days and 60 months) were examined for campylobacters and other enteric pathogens by means of conventional methods. The difference between the isolation rates of campylobacters in those cases in which no other enteric pathogen was found (4.8%) and controls (6.2%) was not significant (P greater than 0.05). Strains of Campylobacter jejuni/coli were isolated throughout the year with higher isolation rates during the summer and monsoon months. Mixed infections were very common. Watery diarrhoea (97.6% cases) was the most common clinical presentation of patients found to be infected solely by C. jejuni/coli. Most patients infected with campylobacters were mildly to moderately dehydrated. Biotype I of C. jejuni and C. coli was the dominant biotype associated with cases and controls. All strains of C. jejuni/coli, regardless of their source, were found to be sensitive to erythromycin. From this study, it appears that enteric infections with campylobacters among children in Calcutta are common but often asymptomatic.

A polyclonal-monoclonal antibody based sensitive sandwich enzyme linked immunosorbent assay for specific detection of cholera toxin.

A sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for specific detection of prototype cholera toxin (CT) elaborated by Vibrio cholerae serovar O1 has been developed. The use of a high affinity monoclonal antibody (MAb) for capturing of CT epitopes permitted a high efficiency. Using this ELISA, we sought in vitro production of CT from clinical strains of V. cholerae O1, Non-O1 and from LT-producing E. coli. All culture supernatants of V. cholerae O1 were positive for CT whereas V. cholerae non-O1 and LT producing E. coli were found negative for CT. This ELISA will be particularly useful in specifically designed studies where detection of CT and not of related labile toxins is mandatory.

Simplified method for the detection of DNA hydrolysis by enteric campylobacters.

A simplified medium was developed for the detection of DNase produced by enteric campylobacters. Sensitivity and reproducibility of the test were similar to that of the improved toluidine blue DNA agar method. Logistically, the simplified DNA hydrolysis test was cheaper (5.5 times) than the earlier medium. Based on this study we recommend the routine use of the simplified medium to perform the DNase test for biotyping enteric campylobacters.

Comparison of selective medium supplemented with blood & a charcoal-based blood-free medium for primary isolation of campylobacters from human faeces.

A recently developed charcoal-based, blood-free selective medium-charcoal cefoperazone deoxycholate agar (CCDA) was compared with the conventional Butzler's agar (BA) for primary isolation of enteric campylobacters from human stool specimens. CCDA yielded higher percentage (93.6%) of positive isolations as compared to BA (76.6%). The rate of isolation on CCDA was significantly higher (P less than 0.0001) than on BA. Cost-wise also, the CCDA medium was two times cheaper than BA. In addition to Campylobacter jejuni and C. coli, CCDA permitted the recovery of a recently described catalase-negative campylobacters which did not grow on BA. Based on these results the routine use of CCDA is recommended, particularly in developing countries, as blood is not required for this medium.

Serotypes & biotypes of Campylobacter jejuni & C. coli from diverse sources in Calcutta.

C. jejuni and C. coli isolates from diverse sources in Calcutta were serotyped and biotyped according to the Lior scheme. Of the 55 strains examined, 85.5 per cent reacted with one or another of the 73 antisera available. This included the formation of two new serogroups, LIO 67 and LIO 76. C. coli serogroup LIO 46 biotype II was the most frequently encountered strains (14.5%), followed by C. coli serogroup LIO 29, 55 biotype II (10.5%) and C. jejuni serogroup LIO 54, biotype I (5.5%). Serogroups recovered from animals and birds were also found to be prevalent in strains isolated from clinical sources, confirming the zoonotic implications of the disease.

Serogroups of Aeromonas species from clinical and environmental sources in Calcutta, India.

Using a recently developed somatic O-antigen serogrouping scheme, 118 isolates of Aeromonas spp. were serogrouped, 68 of which were isolated from diarrhoea patients admitted to the Infectious Diseases Hospital and B.C. Roy Children's Hospital, Calcutta, from March 1987 to February 1988 and 50 randomly selected from an environmental study conducted during the same period. The isolates were typed into 23 different serogroups which represent 35.9% of the total 64 O-serogroups in the typing scheme. Only 12 strains were not typable by the available O-antisera. The dominant serogroups recovered from clinical and environmental isolates were O34, O16 and O11. It appears from this study that virulence of individual isolates is not linked to serological identity.

Growth of Aeromonas spp. on Butzler Campylobacter selective agar and evaluation of the agar for the primary isolation of Aeromonas spp. from clinical specimens.

The fortuitous finding that Aeromonas spp. grew well on Butzler Campylobacter selective medium (BCSA) in a microaerobic atmosphere at 42 degrees C prompted us to evaluate the performance of BCSA for selective isolation of aeromonads in comparison with ampicillin (30 micrograms/ml) sheep blood agar (ASBA30). Overall recovery rates of aeromonads from 563 stool samples from patients with acute diarrhea were higher on ASBA30 (70.4%) than on BCSA (56.3%); however, 21 (29.5%) grew only on BCSA. The three human-associated Aeromonas spp. could be recovered on BCSA and ASBA30. We recommend the use of BCSA to laboratories reluctant to include a specific selective medium for aeromonads.