Newly emerged multiple-antibiotic-resistant Shigella dysenteriae type 1 strains in and around Kolkata, India, are clonal.
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Anti-Bacterial Agents↗
Biomedical subjects
G Balakrish Nair
Publications and source records attributed to G Balakrish Nair.
DNA-level characterization of Helicobacter pylori strains from patients with overt disease and with benign infections in Bangladesh.
The complex relation between the genotype of Helicobacter pylori and its association with clinical outcome is not well understood. Studies in the West have showed that strains expressing certain virulence factors (vacAs1, vacAm1, and cagA) are associated with duodenal ulcer disease. However, the H. pylori genotype is known to vary with geographic region. In the present study, we compared several virulence markers (cagA, vacA, and iceA) and neutral markers (IS605, IS606, and IS608) in H. pylori strains isolated from 65 adult patients with peptic ulcer (PU) and 50 patients with nonulcer dyspepsia (NUD). PCR tests indicated that cagA is present in 75% of the strains from patients with PU compared to 55% in patients with NUD, and 80% of the isolates from patients with PU carried potentially toxigenic vacAs1 alleles of the vacuolating cytotoxin gene (vacA) compared to 60% in isolates from patients with NUD. However, no significant difference in any other virulence marker was observed in isolates from both groups. Phylogenetic analysis of the vacA middle region and the 5' end of the cagA gene indicates that Bangladeshi isolates are more closely related to H. pylori isolates from India and are different from isolates from East Asia.
Adult↗
Virulence genes and neutral DNA markers of Helicobacter pylori isolates from different ethnic communities of West Bengal, India.
Virulence-associated genes and neutral DNA markers of Helicobacter pylori strains from the Santhal and Oroan ethnic minorities of West Bengal, India, were studied. These people have traditionally been quite separate from other Indians and differ culturally, genetically, and linguistically from mainstream Bengalis, whose H. pylori strains have been characterized previously. H. pylori was found in each of 49 study participants, although none had peptic ulcer disease, and was cultured from 31 of them. All strains carried the cag pathogenicity island and potentially toxigenic s1 alleles of vacuolating cytotoxin gene (vacA) and were resistant to at least 8 micro g of metronidazole per ml. DNA sequence motifs in vacA mid-region m1 alleles, cagA, and an informative insertion or deletion motif next to cagA from these strains were similar to those of strains from ethnic Bengalis. Three mobile elements, IS605, IS607, and ISHp608, were present in 29, 19, and 10%, respectively, of Santhal and Oroan strains, which is similar to their prevalence in Bengali H. pylori. Thus, there is no evidence that the gene pools of H. pylori of these ethnic minorities differ from those of Bengalis from the same region. This relatedness of strains from persons of different ethnicities bears on our understanding of H. pylori transmission between communities and genome evolution.
Alleles↗
Use of dipsticks for rapid diagnosis of cholera caused by Vibrio cholerae O1 and O139 from rectal swabs.
We evaluated the recently developed dipsticks for the rapid detection of Vibrio cholerae serotypes O1 and O139 from rectal swabs of hospitalized diarrheal patients after enrichment for 4 h in alkaline peptone water. The sensitivity and specificity of the dipsticks were above 92 and 91%, respectively. The dipsticks represent the first rapid test which has been successfully used to diagnose cholera from rectal swabs, and this would immensely improve surveillance for cholera, especially in remote settings.
Adolescent↗
Class I integrons and SXT elements in El Tor strains isolated before and after 1992 Vibrio cholerae O139 outbreak, Calcutta, India.
We examined the distribution of class I integrons and SXT elements in Vibrio cholerae O1 El Tor strains, isolated in Calcutta, India, before and after the V. cholerae O139 outbreak in 1992. Class I integrons, with aadA1 gene cassette, were detected primarily in the pre-O139 strains; the SXT element was found mainly in the post-O139 strains.
Cholera↗
Molecular epidemiology of O139 Vibrio cholerae: mutation, lateral gene transfer, and founder flush.
Vibrio cholerae in O-group 139 was first isolated in 1992 and by 1993 had been found throughout the Indian subcontinent. This epidemic expansion probably resulted from a single source after a lateral gene transfer (LGT) event that changed the serotype of an epidemic V. cholerae O1 El Tor strain to O139. However, some studies found substantial genetic diversity, perhaps caused by multiple origins. To further explore the relatedness of O139 strains, we analyzed nine sequenced loci from 96 isolates from patients at the Infectious Diseases Hospital, Calcutta, from 1992 to 2000. We found 64 novel alleles distributed among 51 sequence types. LGT events produced three times the number of nucleotide changes compared to mutation. In contrast to the traditional concept of epidemic spread of a homogeneous clone, the establishment of variant alleles generated by LGT during the rapid expansion of a clonal bacterial population may be a paradigm in infections and epidemics.
Alleles↗
Reemergence of epidemic Vibrio cholerae O139, Bangladesh.
During March and April 2002, a resurgence of Vibrio cholerae O139 occurred in Dhaka and adjoining areas of Bangladesh with an estimated 30,000 cases of cholera. Patients infected with O139 strains were much older than those infected with O1 strains (p<0.001). The reemerged O139 strains belong to a single ribotype corresponding to one of two ribotypes that caused the initial O139 outbreak in 1993. Unlike the strains of 1993, the recent strains are susceptible to trimethoprim, sulphamethoxazole, and streptomycin but resistant to nalidixic acid. The new O139 strains carry a copy of the Calcutta type CTX(Calc) prophage in addition to the CTX(ET) prophage carried by the previous strains. Thus, the O139 strains continue to evolve, and the adult population continues to be more susceptible to O139 cholera, which suggests a lack of adequate immunity against this serogroup. These findings emphasize the need for continuous monitoring of the new epidemic strains.
Adolescent↗
Enhanced isolation of Shigella species by extended incubation of primary isolation plates.
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Bacteriological Techniques↗
A 4-year study of the epidemiology of Vibrio cholerae in four rural areas of Bangladesh.
How Vibrio cholerae spreads around the world and what determines its seasonal peaks in endemic areas are not known. These features of cholera have been hypothesized to be primarily the result of environmental factors associated with aquatic habitats that can now be identified. Since 1997, fortnightly surveillance in 4 widely separated geographic locations in Bangladesh has been performed to identify patients with cholera and to collect environmental data. A total of 5670 patients (53% <5 years of age) have been studied; 14.3% had cholera (10.4% due to V. cholerae O1 El Tor, 3.8% due to O139). Both serogroups were found in all locations; outbreaks were seasonal and often occurred simultaneously. Water-use patterns showed that bathing and washing clothes in tube-well water was significantly protective in two of the sites. These data will be correlated with environmental factors, to develop a model for prediction of cholera outbreaks.
Adolescent↗
Structure of the O-polysaccharide from the lipopolysaccharide from Vibrio cholerae O6.
The O-polysaccharide from Vibrio cholerae O6 was isolated from the LPS by mild-acid hydrolysis and has been investigated by sugar and methylation analysis and NMR spectroscopy. The polysaccharide was also depolymerized with aqueous hydrofluoric acid to give the repeating unit and multiples thereof. The O-polysaccharide had the following tetrasaccharide repeating unit. Two O-acetyl groups are present, one of them making the GlcNAc residue fully substituted and the steric crowding considerable at the branching residue.
Carbohydrate Sequence↗
Incidence and molecular analysis of Vibrio cholerae associated with cholera outbreak subsequent to the super cyclone in Orissa, India.
An epidemiological study was carried out to find out the aetiological agent for diarrhoeal disorders in the cyclone and flood affected areas of Orissa, India. Rectal swabs collected from 107 hospitalized diarrhoea patients were bacteriologically analysed to isolate and identify the various enteropathogens. Detection of toxic genes among E. coli and V. cholerae was carried out by polymerase chain reaction (PCR) assay. Of the 107 rectal swabs analysed, 72.3% were positive for V. cholerae O1 Ogawa, 7.2% for V. cholerae O139, 1.2% for E. coli (EAggEC) and 1.2% for Shigella flexneri type 6. Using multiplex PCR assay it was found that all V. cholerae isolates were ctxA positive and El Tor biotype. Strains of V. cholerae O1 were observed to be resistant to nalidixic acid, furazolidone, streptomycin, co-trimoxazole and ampicillin. Except for nalidixic acid, the resistance pattern for O139 was identical to that of O1 strains. Representative strains of V. cholerae were further characterized by randomly amplified polymorphic DNA (RAPD) analysis and ribotyping. Both O1 and O139 V. cholerae strains exhibited the R3 pattern of ribotype and belonged to a similar pattern of RAPD compared with that of Calcutta strains. Early bacteriological and epidemiological investigations have revealed the dominance of V. cholerae O1 among the hospitalized patients in cyclone affected areas of Orissa. Drinking water scarcity and poor sanitation were thought to be responsible for these diarrhoeal outbreaks. Timely reporting and implementation of appropriate control measures could contain a vital epidemic in this area.
Cholera↗
Preferential association of the heat-stable enterotoxin gene (stn) with environmental strains of Vibrio cholerae belonging to the O14 serogroup.
Toxigenic Vibrio cholerae O1 and O139 serogroups have the capacity of causing epidemic and pandemic cholera but are infrequently found in the environment. The other serogroups are abundant in aquatic environments but do not possess the virulence genes necessary for causing the disease. Of the 559 environmental strains of V. cholerae, collected during different periods from environmental samples in Calcutta, 9 (1.6%) harboured the heat-stable enterotoxin gene (stn). Six of the 9 strains belonged to the O14 serogroup. Thus, V. cholerae strains carrying the stn gene revealed preferential association with the O14 serogroup. Three of the six strains harboured the tcpA gene of the E1 Tor type, which is an unusual feature among environmental V. cholerae strains. A strain that possessed the E1 Tor type tcpA also had the CTX prophage. Pulsed field gel electrophoresis (PFGE) revealed that the stn gene positive O14 strains of V. cholerae were not clonal.
Animals↗
Molecular ecology of toxigenic Vibrio cholerae.
Toxigenic Vibrio cholerae is the etiological agent of cholera, an acute dehydrating diarrhea that occurs in epidemic form in many developing countries. Although V. cholerae is a human pathogen, aquatic ecosystems are major habitats of Vibrio species, which includes both pathogenic and nonpathogenic strains that vary in their virulence gene content. V. cholerae belonging to the 01 and 0139 serogroups is commonly known to carry a set of virulence genes necessary for pathogenesis in humans. Recent studies have indicated that virulence genes or their homologues are also dispersed among environmental strains of V. cholerae belonging to diverse serogroups, which appear to constitute an environmental reservoir of virulence genes. Although the definitive roles of the virulence-associated factors in the environment, and the environmental selection pressures for V. cholerae-carrying virulence genes or their homologues is not clear, the potential for origination of new epidemic strains from environmental progenitors seems real. It is likely that the aquatic environment harbors different virulence-associated genes scattered among environmental vibrios, which possess a lower virulence potential than the epidemic strains. The ecosystem comprising the aquatic environment, V. cholerae, genetic elements mediating gene transfer, and the mammalian host appears to support the clustering of critical virulence genes in a proper combination leading to the origination of new V. cholerae strains with epidemic potential.
Cholera↗
Role of active efflux in association with target gene mutations in fluoroquinolone resistance in clinical isolates of Vibrio cholerae.
Quinolones are among the drugs of choice in the management of cholera caused by Vibrio cholerae. In this study, we demonstrate that, in addition to mutations detected in the target genes gyrA and parC, proton motive force-dependent efflux is involved in quinolone resistance in clinical isolates of V. cholerae.
Anti-Infective Agents↗
Isolation of Shigella dysenteriae type 1 and S. flexneri strains from surface waters in Bangladesh: comparative molecular analysis of environmental Shigella isolates versus clinical strains.
Bacillary dysentery caused by Shigella species is a public health problem in developing countries including Bangladesh. Although, shigellae-contaminated food and drinks are often the source of the epidemic's spread, the possible presence of the pathogen and transmission of it through environmental waters have not been adequately examined. We analyzed surface waters collected in Dhaka, Bangladesh, for the presence of shigellae by a combination of PCR assays followed by concentration and culturing of PCR-positive samples. Analysis of 128 water samples by PCR assays for Shigella-specific virulence genes including ipaBCD, ipaH, and stx1 identified 14 (10.9%) samples which were positive for one or more of these virulence genes. Concentration of the PCR-positive samples by filtration followed by culturing identified live Shigella species in 11 of the 14 PCR-positive samples. Analysis of rRNA gene restriction patterns (ribotype) showed that the environmental isolates shared ribotypes with a collection of clinical isolates, but in contrast to the clinical isolates, 10 of the 11 environmental isolates were either negative or carried deletions in the plasmid-encoded invasion-associated genes ipaB, ipaC, and ipaD. However, all environmental Shigella isolates were positive for the chromosomal multicopy invasion-associated gene ipaH and all Shigella dysenteriae type 1 isolates were positive for the stx1 gene in addition to ipaH. This study demonstrated the presence of Shigella in the aquatic environment and dispersion of different virulence genes among these isolates which appear to constitute an environmental reservoir of Shigella-specific virulence genes. Since critical virulence genes in Shigella are carried by plasmids or mobile genetic elements, the environmental gene pool may contribute to an optimum combination of genes, causing the emergence of virulent Shigella strains which is facilitated in particular by close contact of the population with surface waters in Bangladesh.
Anti-Bacterial Agents↗
RS1 element of Vibrio cholerae can propagate horizontally as a filamentous phage exploiting the morphogenesis genes of CTXphi.
In toxigenic Vibrio cholerae, cholera toxin is encoded by the CTX prophage, which consists of a core region carrying ctxAB genes and genes required for CTXPhi morphogenesis, and an RS2 region encoding regulation, replication, and integration functions. Integrated CTXPhi is often flanked by another genetic element known as RS1 which carries all open reading frames (ORFs) found in RS2 and an additional ORF designated rstC. We identified a single-stranded circularized form of the RS1 element, in addition to the CTXPhi genome, in nucleic acids extracted from phage preparations of 32 out of 83 (38.5%) RS1-positive toxigenic V. cholerae strains analyzed. Subsequently, the corresponding double-stranded replicative form (RF) of the RS1 element was isolated from a representative strain and marked with a kanamycin resistance (Km(r)) marker in an intergenic site to construct pRS1-Km. Restriction and PCR analysis of pRS1-Km and sequencing of a 300-bp region confirmed that this RF DNA was the excised RS1 element which formed a novel junction between ig1 and rstC. Introduction of pRS1-Km into a V. cholerae O1 classical biotype strain, O395, led to the production of extracellular Km(r) transducing particles, which carried a single-stranded form of pRS1-Km, thus resembling the genome of a filamentous phage (RS1-KmPhi). Analysis of V. cholerae strains for susceptibility to RS1-KmPhi showed that classical biotype strains were more susceptible to the phage compared to El Tor and O139 strains. Nontoxigenic (CTX(-)) O1 and O139 strains which carried genes encoding the CTXPhi receptor toxin-coregulated pilus (TCP) were also more susceptible (>1,000-fold) to the phage compared to toxigenic El Tor or O139 strains. Like CTXPhi, the RS1Phi genome also integrated into the host chromosomes by using the attRS sequence. However, only transductants of RS1-KmPhi which also harbored the CTXPhi genome produced a detectable level of extracellular RS1-KmPhi. This suggested that the core genes of CTXPhi are also required for the morphogenesis of RS1Phi. The results of this study showed for the first time that RS1 element, which encodes a site-specific recombination system in V. cholerae, can propagate horizontally as a filamentous phage, exploiting the morphogenesis genes of CTXPhi.
Bacterial Proteins↗
Cloning and characterization of genes encoding homologues of the B subunit of cholera toxin and the Escherichia coli heat-labile enterotoxin from clinical isolates of Citrobacter freundii and E. coli.
We identified and characterized a gene encoding a homologue of the B subunits of cholera toxin (CTB) and heat-labile enterotoxin (LTB) of Escherichia coli from a clinical isolate of Citrobacter freundii that was found to produce a factor in the culture supernatant that cross-reacted with antibodies to CTB and LTB when assayed by enzyme-linked immunosorbent assay (ELISA). The gene encoding the ELISA-positive factor, cfxB, consisted of 375 nucleotides and was located downstream of an 852-nucleotide open reading frame, cfxA, with a 56-nucleotide intergenic space. The cfxB gene was predicted to encode a 125-amino-acid polypeptide, which had 73.8 and 72.8% identities with the amino acid sequences of LTB and CTB, respectively. However, the amino acid sequence of the deduced polypeptide CFXA had no homologies to those of the A subunits of CT or LT. DNA probes developed from the sequences of cfxA and cfxB were used to screen 67 C. freundii isolates and 152 E. coli isolates from diarrheal patients by colony blot hybridization. Two strains, C. freundii 48 and E. coli 176, reacted with both DNA probes under conditions of high stringency. We cloned homologues of the cfxA and cfxB genes from E. coli 176 and designated them ecxA and ecxB, respectively. The ecxA gene and the ecxB gene comprise 855 and 375 nucleotides, respectively, with a 50-nucleotide intergenic space, and encode a 285- and a 125-amino-acid residue polypeptides, respectively. The results of the present study may provide important clues to the origin and evolution of immunologically related factors sharing a common enterotoxin-like A and B subunit structures.
Amino Acid Sequence↗
Vibrio pathogenicity island and cholera toxin genetic element-associated virulence genes and their expression in non-O1 non-O139 strains of Vibrio cholerae.
A non-O1 non-O139 Vibrio cholerae strain, 10259, belonging to the serogroup O53 was shown to harbor genes related to the vibrio pathogenicity island (VPI) and a cholera toxin (CT) genetic element called CTX. While the nucleotide sequence of the strain 10259 tcpA gene differed significantly (26 and 28%) from those of O1 classical and El Tor biotype strains, respectively, partial sequence analysis data of certain other VPI-associated genes (aldA, tagA, tcpP/H, toxT, acfB/C, and int) and intergenic regions (tcpF to toxT and tcpH to tcpA) of the strain showed only minor variations (0.4 to 4.8%) from corresponding sequences in O1 strains. Strain 10259 also contained CTX element-associated toxin genes with sequences almost identical to those of O1 strains. Growth of the organism in Luria broth (LB) under ToxR inducing conditions (30 degrees C and pH 6.5) led to transcriptional activation of tcpP/H, toxR, toxT, and tcpA genes, but not of ctxA, as determined by reverse transcription-PCR (RT-PCR). Subsequent analysis revealed that strain 10259 possessed only two copies (instead of three or more copies found in epidemic-causing O1 or O139 strains) of the heptanucleotide (TTTTGAT) repeats in the intergenic region upstream of ctxAB. Therefore, a strain 10259 mutant was generated by replacement of this region with a homologous region (1.4 kb) derived from a V. cholerae O1 classical biotype strain (O395) that contained seven such repeats. The resultant recombinant strain (10259R) was found to be capable of coordinately regulated expression of toxT, ctxA, and tcpA when grown under the ToxR inducing conditions. Serological studies also demonstrated that the recombinant strain produced TcpA and a significantly ( approximately 1,000-fold) higher level of CT in vitro compared to that of the parent strain. Virulence gene expression in two other non-O1 non-O139 strains (serogroup O37) containing VPI and the CTX element was studied by RT-PCR and serological assay. One strain (S7, which was involved in an epidemic in Sudan in 1968) showed coordinately regulated expression of virulence genes leading to the production of both CT and TcpA in LB medium. However, the other strain, V2, produced RT-PCR-detectable transcripts of toxT, ctxA, or tcpA genes in the early phase (6 h), but not in the late phase (16 h) of growth in LB medium. These results are consistent with the low levels of production of CT and TcpA by the strain that were serologically detectable. The significance of these results is discussed in relation to the role of virulence genes and their expression to the pathogenic potential of V. cholerae strains belonging to non-O1 serogroups.
Bacterial Outer Membrane Proteins↗