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Vibrio choleraeO1 and O139 isolates deploy cholera toxin (CT) and toxin-coregulated pilus (TCP) to cause the diarrhoeal disease cholera. ThectxABandtcpAgenes encoding CT and TCP are part of two acquired genetic elements, the CTX phage andVibriopathogenicity island-1 (VPI-1), respectively. ToxR and ToxT proteins are the key regulators of virulence genes ofV. choleraeO1 and O139.V. choleraeisolates belonging to serogroups other than O1/O139, called non-O1/non-O139, are usually devoid of virulence-related elements and are non-pathogenic. Here, we have analysed the available whole genome sequence of an environmental toxigenicV. choleraenon-O1/non-O139 strain, VCE232, carrying the CTX phage and VPI-1. Extensive bioinformatics and phylogenetic analyses indicated high similarity of the VCE232 genome sequence with the genome ofV. choleraeO1 strains, including organization of the VPI-1 locus,ctxAB, tcpAandtoxTgenes, and promoters. We established that the VCE232 strain produces an optimal amount of CT at 30 °C under AKI conditions. To investigate the role of ToxT and ToxR in the regulation of virulence factors, we constructed ΔtoxT, ΔtoxRand ΔtoxTΔtoxRdeletion mutants of VCE232. Extensive genetic analyses of these mutants indicated that thetoxTandtoxRgenes of VCE232 are crucial for CT and TCP production. However, unlike O1 isolates, the presence of eithertoxTortoxRgene is sufficient for optimal CT production in VCE232. In addition, the VCE232 ΔtoxRmutant showed differential regulation of the major outer membrane proteins, OmpT and OmpU. This is the first attempt to explore the regulation of expression of major virulence genes and regulators in an environmental toxigenicV. choleraenon-O1/non-O139 strain.

Bacterial species are hosts to horizontally acquired mobile genetic elements (MGEs), which encode virulence, toxin, antimicrobial resistance, and other metabolic functions. The bipartite genome of Vibrio cholerae harbors sporadic and conserved MGEs that contribute in the disease development and survival of the pathogens. For a comprehensive understanding of dynamics of MGEs in the bacterial genome, we engineered the genome of V. cholerae and examined in vitro and in vivo stability of genomic islands (GIs), integrative conjugative elements (ICEs), and prophages. Recombinant vectors carrying the integration module of these GIs, ICE and CTXΦ, helped us to understand the efficiency of integrations of MGEs in the V. cholerae chromosome. We have deleted more than 250 acquired genes from 6 different loci in the V. cholerae chromosome and showed contribution of CTX prophage in the essentiality of SOS response master regulator LexA, which is otherwise not essential for viability in other bacteria, including Escherichia coli In addition, we observed that the core genome-encoded RecA helps CTXΦ to bypass V. cholerae immunity and allow it to replicate in the host bacterium in the presence of similar prophage in the chromosome. Finally, our proteomics analysis reveals the importance of MGEs in modulating the levels of cellular proteome. This study engineered the genome of V. cholerae to remove all of the GIs, ICEs, and prophages and revealed important interactions between core and acquired genomes.

Expression of cholera toxin (CT), the principal virulence factor of the cholera pathogen Vibrio cholerae, is positively modulated by the RNA polymerase binding unusual transcription factor DksA (DksAVc) of the stringent response pathway. Here we report that even though CT (encoded by the genes ctxAB) production is downregulated in the V. cholerae ΔdksA (ΔdksAVc) mutant, the expression of the ctxA gene as well as the genes encoding different virulence regulators, namely, AphA, TcpP and ToxT, were also upregulated. Since DksAVc positively regulates HapR, a known negative regulator of CT production, the increased expression of different virulence genes in ΔdksAVc was due most probably to downregulation of HapR. There was no secretion/transport-related defect in ΔdksAVc cells because whole cell lysates of the mutant showed a negligible amount of CT accumulation similar to WT cells. To understand further, the hapR gene was deleted in ΔdksAVc background, however, the double mutant failed to rescue the CT production defect suggesting strongly towards post-transcriptional/translational regulation by DksAVc. This hypothesis was further confirmed when the site-directed mutagenesis of each or both of the conserved aspartic acid residues at positions 68 and 71 of DksAVc, which are essential for transcription initiation during the stringent response, had no effect in the regulation of CT expression. Interestingly, progressive deletion analysis indicated that the C4-type Zn finger motif present in the C-terminus of DksAVc is essential for optimal CT production. Since this motif plays important roles in DNA/RNA binding, the present study indicates a novel complex post-transcriptional regulation of CT expression by DksAVc.

Single cell microorganisms including pathogens relentlessly face myriads of physicochemical stresses in their living environment. In order to survive and multiply under such unfavorable conditions, microbes have evolved with complex genetic networks, which allow them to sense and respond against these stresses. Stringent response is one such adaptive mechanism where bacteria can survive under nutrient starvation and other related stresses. The effector molecules for the stringent response are guanosine-5'-triphosphate 3'-diphosphate (pppGpp) and guanosine-3', 5'-bis(diphosphate) (ppGpp), together called (p)ppGpp. These effector molecules are now emerging as master regulators for several physiological processes of bacteria including virulence, persistence, and antimicrobial resistance. (p)ppGpp may work independently or along with its cofactor DksA to modulate the activities of its prime target RNA polymerase and other metabolic enzymes, which are involved in different biosynthetic pathways. Enzymes involved in (p)ppGpp metabolisms are ubiquitously present in bacteria and categorized them into three classes, i.e., canonical (p)ppGpp synthetase (RelA), (p)ppGpp hydrolase/synthetase (SpoT/Rel/RSH), and small alarmone synthetases (SAS). While RelA gets activated in response to amino acid starvation, enzymes belonging to SpoT/Rel/RSH and SAS family can synthesize (p)ppGpp in response to glucose starvation and several other stress conditions. In this review, we will discuss about the current status of the following aspects: (i) diversity of (p)ppGpp biosynthetic enzymes among different bacterial species including enteropathogens, (ii) signals that modulate the activity of (p)ppGpp synthetase and hydrolase, (iii) effect of (p)ppGpp in the production of antibiotics, and (iv) role of (p)ppGpp in the emergence of antibiotic resistant pathogens. Emphasis has been given to the cholera pathogen Vibrio cholerae due to its sophisticated and complex (p)ppGpp metabolic pathways, rapid mutational rate, and acquisition of antimicrobial resistance determinants through horizontal gene transfer. Finally, we discuss the prospect of (p)ppGpp metabolic enzymes as potential targets for developing antibiotic adjuvants and tackling persistence of infections.

Bacterial species are hosts to horizontally acquired mobile genetic elements (MGEs), which encode virulence, toxin, antimicrobial resistance, and other metabolic functions. The bipartite genome of Vibrio cholerae harbors sporadic and conserved MGEs that contribute in the disease development and survival of the pathogens. For a comprehensive understanding of dynamics of MGEs in the bacterial genome, we engineered the genome of V. cholerae and examined in vitro and in vivo stability of genomic islands (GIs), integrative conjugative elements (ICEs), and prophages. Recombinant vectors carrying the integration module of these GIs, ICE and CTXΦ, helped us to understand the efficiency of integrations of MGEs in the V. cholerae chromosome. We have deleted more than 250 acquired genes from 6 different loci in the V. cholerae chromosome and showed contribution of CTX prophage in the essentiality of SOS response master regulator LexA, which is otherwise not essential for viability in other bacteria, including Escherichia coli. In addition, we observed that the core genome-encoded RecA helps CTXΦ to bypass V. cholerae immunity and allow it to replicate in the host bacterium in the presence of similar prophage in the chromosome. Finally, our proteomics analysis reveals the importance of MGEs in modulating the levels of cellular proteome. This study engineered the genome of V. cholerae to remove all of the GIs, ICEs, and prophages and revealed important interactions between core and acquired genomes.

The stringent response, an adaptive response to nutrient limitation and exposure to xenobiotics in bacteria, is mediated by two intracellular signaling molecules, pppGpp and ppGpp, together called (p)ppGpp. The cellular level of (p)ppGpp in bacterial cells is controlled by the Rel/Spo family of proteins. In the cholera pathogen, Vibrio cholerae, (p)ppGpp metabolism is regulated by the products of at least three genes relA, spoT, and relV. In this study, we identify and characterize the function of the guanosine-5'-triphosphate 3'-diphosphate pyrophosphatase A (GppA) encoding gene gppA of V. cholerae. Genomic analysis indicates that the gppA locus is conserved in vibrios and organized as a bicistronic operon along with the rhlB gene. We engineered the genome of V. cholerae to develop different mutants devoid of GppA and/or other phosphate metabolic enzymes. Our findings indicate that in V. cholerae, GppA plays an important role in the conversion of pppGpp to ppGpp during amino acid deprivation but not during glucose starvation. Quantitative analyses of the gppA transcript level reveal its differential expression pattern at different growth phases and starvation conditions. It has been observed that the GppA deficiency during amino acid starvation condition could be complemented by overexpressing the exopolyphosphatase coding gene ppx of V. cholerae. By deletion analysis, we further demonstrate that the amino and carboxy terminal sequences flanking the Ppx-GppA motif of the GppA protein of V. cholerae are also important for its enzymatic function.

Emergence of antimicrobial resistant Gram-negative bacteria has created a serious global health crisis and threatens the effectiveness of most, if not all, antibiotics commonly used to prevent and treat bacterial infections. There is a dearth of detailed studies on the prevalence of antimicrobial resistance (AMR) patterns in India. Here, we have isolated and examined AMR patterns of 654 enteric pathogens and investigated complete genome sequences of isolates from six representative genera, which in aggregate encode resistance against 22 antibiotics representing nine distinct drug classes. This study revealed that ~97% isolates are resistant against ≥2 antibiotics, ~24% isolates are resistant against ≥10 antibiotics and ~3% isolates are resistant against ≥15 antibiotics. Analyses of whole genome sequences of six extensive drug resistant enteric pathogens revealed presence of multiple mobile genetic elements, which are physically linked with resistance traits. These elements are therefore appearing to be responsible for disseminating drug resistance among bacteria through horizontal gene transfer. The present study provides insights into the linkages between the resistance patterns to certain antibiotics and their usage in India. The findings would be useful to understand the genetics of resistance traits and severity of and difficulty in tackling AMR enteric pathogens.

The Bay of Bengal is known as the epicenter for seeding several devastating cholera outbreaks across the globe. Vibrio cholerae , the etiological agent of cholera, has extraordinary competency to acquire exogenous DNA by horizontal gene transfer (HGT) and adapt them into its genome for structuring metabolic processes, developing drug resistance, and colonizing the human intestine. Antimicrobial resistance (AMR) in V. cholerae has become a global concern. However, little is known about the identity of the resistance traits, source of AMR genes, acquisition process, and stability of the genetic elements linked with resistance genes in V. cholerae . Here we present details of AMR profiles of 443 V. cholerae strains isolated from the stool samples of diarrheal patients from two regions of India. We sequenced the whole genome of multidrug-resistant (MDR) and extensively drug-resistant (XDR) V. cholerae to identify AMR genes and genomic elements that harbor the resistance traits. Our genomic findings were further confirmed by proteome analysis. We also engineered the genome of V. cholerae to monitor the importance of the autonomously replicating plasmid and core genome in the resistance profile. Our findings provided insights into the genomes of recent cholera isolates and identified several acquired traits including plasmids, transposons, integrative conjugative elements (ICEs), pathogenicity islands (PIs), prophages, and gene cassettes that confer fitness to the pathogen. The knowledge generated from this study would help in better understanding of V. cholerae evolution and management of cholera disease by providing clinical guidance on preferred treatment regimens.

How Salmonella enterica serovar Typhi (S. Typhi), an important human pathogen, survives the stressful microenvironments inside the gastrointestinal tract and within macrophages remains poorly understood. We report here that S. Typhi has a bonafide stringent response (SR) system, which is mediated by (p)ppGpp and regulates multiple virulence-associated traits and the pathogenicity of the S. Typhi Ty2 strain. In an iron overload mouse model of S. Typhi infection, the (p)ppGpp0 (Ty2ΔRelAΔSpoT) strain showed minimal systemic spread and no mortality, as opposed to 100% death of the mice challenged with the isogenic wild-type strain. Ty2ΔRelAΔSpoT had markedly elongated morphology with incomplete septa formation and demonstrated severely attenuated motility and chemotaxis due to the loss of flagella. Absence of the Vi-polysaccharide capsule rendered the mutant strain highly susceptible to complement-mediated lysis. The phenotypes of Ty2ΔRelAΔSpoT was contributed by transcriptional repression of several genes, including fliC, tviA, and ftsZ, as found by reverse transcriptase quantitative polymerase chain reaction and gene complementation studies. Finally, Ty2ΔRelAΔSpoT had markedly reduced invasion into intestinal epithelial cells and significantly attenuated survival within macrophages. To the best of our knowledge, this was the first study that addressed SR in S. Typhi and showed that (p)ppGpp was essential for optimal pathogenic fitness of the organism.

The bipartite genome of Vibrio cholerae is divided into two circular non-homologous chromosomes, which harbor several genetic elements like phages, plasmids, transposons, integrative conjugative elements, and pathogenic islands that encode functions responsible for disease development, antimicrobial resistance, and subsistence in hostile environments. These elements are highly heterogeneous, mobile in nature, and encode their own mobility functions or exploit host-encoded enzymes for intra- and inter-cellular movements. The key toxin of V. cholerae responsible for the life-threatening diarrheal disease cholera, the cholera toxin, is coded by part of the genome of a filamentous phage, CTXϕ. The replicative genome of CTXϕ is divided into two distinct modular structures and has adopted a unique strategy for its irreversible integration into the V. cholerae chromosomes. CTXϕ exploits two host-encoded tyrosine recombinases, XerC and XerD, for its integration in the highly conserved dimer resolution site (dif) of V. cholerae chromosomes. CTXϕ can replicate only in the limited number of Vibrio species. In contrast, the phage integration into the bacterial chromosome does not rely on its replication and could integrate to the dif site of large numbers of gram-negative bacteria. Recent pangenomic analysis revealed that like CTXϕ, the bacterial dif site is the integration spot for several other mobile genetic elements such as plasmids and genomic islands. In this review we discuss about current molecular insights into CTXϕ genomics and its replication and integration mechanisms into hosts. Particular emphasis has been given on the exploitation of CTXϕ genomics knowledge in developing genetic tools and designing environmentally safe recombinant live oral cholera vaccine strains.

DksA acts as a co-factor for the intracellular small signalling molecule ppGpp during the stringent response. We recently reported that the expression of the haemagglutinin protease (HAP), which is needed for shedding of the cholera pathogen Vibrio cholerae during the late phase of infection, is significantly downregulated in V. cholerae ∆dksA mutant (∆dksAVc) cells. So far, it has been shown that HAP production by V. cholerae cells is critically regulated by HapR and also by RpoS. Here, we provide evidence that V. cholerae DksA (DksAVc) positively regulates HapR at both the transcriptional and post-transcriptional levels. We show that in ∆dksAVc cells the CsrB/C/D sRNAs, required for the maintenance of intracellular levels of hapR transcripts during the stationary growth, are distinctly downregulated. Moreover, the expression of exponential phase regulatory protein Fis, a known negative regulator of HapR, was found to continue even during the stationary phase in ∆dksAVc cells compared to that of wild-type strain, suggesting another layer of complex regulation of HapR by DksAVc. Extensive reporter construct-based and quantitative reverse-transcriptase PCR (qRT-PCR) analyses supported that RpoS is distinctly downregulated at the post-transcriptional/translational levels in stationary phase-grown ∆dksAVc cells. Since HAP expression through HapR and RpoS is stationary phase-specific in V. cholerae, it appears that DksAVc is also a critical stationary phase regulator for fine tuning of the expression of HAP. Moreover, experimental evidence provided in this study clearly supports that DksAVc is sitting at the top of the hierarchy of regulation of expression of HAP in V. cholerae.

Rel(Mtb), a GTP pyrophosphokinase encoded by the Mycobacterium tuberculosis (Mtb) genome, catalyzes synthesis of (p)ppGpp from ATP and GDP(GTP) and its hydrolysis to GDP(GTP) and pyrophosphate to mediate stringent response, which helps bacteria to survive during nutrient limitation. Like other members of Rel_Spo homologs, Rel(Mtb) has four distinct domains: HD, Rel_Spo (RSD), TGS and ACT. The N-terminal HD and RSD are responsible for (p)ppGpp hydrolysis and synthesis, respectively. In this study, we have dissected the rel(Mtb) gene function and determined the minimal region essential for (p)ppGpp synthetic activity. The Rel(Mtb) and its truncated derivatives were expressed from an arabinose inducible promoter (P(BAD)), and in vivo functional analyses were done in a (p)ppGpp null Escherichia coli strain. Our results indicate that only 243 amino acids (188-430 residues) containing fragment are sufficient for Rel(Mtb) (p)ppGpp synthetic activity. The results were further confirmed by in vitro assays using purified proteins. We further characterized the RSD of Rel(Mtb) by substituting several conserved amino acids with structurally related residues and identified six such residues, which appeared to be critical for maintaining its catalytic activity. Furthermore, we have also extended our analysis to an RSD encoding gene rv1366 of Mtb, and experimental results indicated that the encoded protein Rv1366 is unable to synthesize (p)ppGpp.

Fused tricyclic oxazaquinolinium salts were synthesized using Amberlite IRA 402(OH) in water and assessed for their sperm-immobilizing efficacy in vitro by modified Sander–Cramer test followed by double fluoroprobe staining of rat sperm. Out of 11 derivatives 3 showed potent spermicidal effect. The mode of spermicidal action was assessed by (a) Hypoosmotic swelling tests, (b) Lipid peroxidation studies, and (c) scanning electron microscopy. Antimicrobial efficacies of the potent derivatives, assessed by disc diffusion as well as by agar and broth dilution methods using human isolates of bacteria and fungi, revealed their moderate antimicrobial potential. The compounds, found to be nontoxic to rabbit erythrocytes by haemolytic assay, may be exploited as microbicidal spermicides.

Background & objectives: The four species of the genus Shigella, namely, S. dysenteriae , S. flexneri, S. boydii and S. sonnei cause a wide spectrum of illness from watery diarrhoea to severe dysentery. Genomes of these four species show great diversity. In this study, NotI, XbaI or I-CeuI restriction enzyme digested genomes of two Shigella dysenteriae isolates belonging to the serotypes 2 and 7 were extensively analyzed to find their relatedness, if any, with the whole genome sequenced strains of S. dysenteriae type 1 and S. flexneri type 2a. Methods: Pulsed-field gel electrophoresis (PFGE) technique was used to determine the diversity of Shigella genomes by rapid construction of physical maps. DNA end labelling, Southern hybridization and PCR techniques were also applied for mapping purposes. Results: The intron-coded enzyme I-CeuI cuts the bacterial genome specifically at its rrn operon. PFGE of I-CeuI digested S. dysenteriae genomes were found to carry seven rrn operons. However, I-CeuI profiles showed distinct restriction fragment polymorphism (RFLP) between the isolates as well as with the whole genome sequenced isolates. Further studies revealed that the genome sizes and I-CeuI linkage maps of the S. dysenteriae type 7 and type 2 isolates were similar to that of S. dysenteriae type 1 and S. flexneri type 2a genomes, respectively. Interpretation & conclusions: Our findings indicate that the type 7 and type 1 isolates of S. dysenteriae were probably evolved from a same precursor, while the type 2 and S. flexneri type 2a were probably evolved and diversified from a common progenitor.

Vibrio cholerae causes cholera outbreaks in endemic regions where the water quality and sanitation facilities remain poor. Apart from biotype and serotype changes, V. cholerae undergoes phase variation, which results in the generation of two morphologically different variants termed smooth and rugose. In this study, 12 rugose (R-VC) and 6 smooth (S-VC) V. cholerae O1 Ogawa isolates were identified in a cholera outbreak that occurred in Hyderabad, India. Antimicrobial susceptibility results showed that all the isolates were resistant to ampicillin, furazolidone and nalidixic acid. In addition, R-VC isolates were resistant to ciprofloxacin (92 %), streptomycin (92 %), erythromycin (83 %), trimethoprim-sulfamethoxazole (75 %) and tetracycline (75 %). Based on the ctxB gene analysis, all the isolates were identified as El Tor variant with mutation in two positions of ctxB, similar to the classical biotype. The R-VC isolates specifically showed excessive biofilm formation and were comparatively less motile. In addition, the majority of these isolates (~83 %) displayed random mutations in the hapR gene, which encodes haemagglutinin protease regulatory protein. In the PFGE analysis, R-VC and S-VC were placed in distinct clusters but remained clonally related. In the ribotyping analysis, all the R-VC isolates exhibited R-III pattern, which is a prevailing type among the current El Tor isolates. A hapR deletion mutant generated using an S-VC isolate expressed rugose phenotype. To our knowledge, this is the first report on the association of rugose V. cholerae O1 in a large cholera outbreak with extended antimicrobial resistance and random mutations in the haemagglutinin protease regulatory protein encoding gene (hapR).

Incidence of epidemic Vibrio cholerae serogroup O139 has declined in cholera endemic countries. However, sporadic cholera caused by V. cholerae O139 with notable genetic changes is still reported from many regions. In the present study, 42 V. cholerae O139 strains isolated from 2001 to 2006 in Delhi, India, were retrospectively analyzed to understand their phenotype and molecular characteristics. The majority of isolates were resistant to ampicillin, furazolidone and nalidixic acid. Though the integrative conjugative element was detected in all the O139 isolates, the 2004-2006 isolates remained susceptible to co-trimoxazole, chloramphenicol, and streptomycin. Cholera toxin genotype 1 was present in the majority of the O139 isolates while few had type 3 or a novel type 4. In the cholera toxin encoding gene (ctx) restriction fragment length polymorphism, the majority of the isolates harbored three copies of CTX element, of which one was truncated. In this study, the ctx was detected for the first time in the small chromosome of V. cholerae O139 and one isolate harbored 5 copies of CTX element, of which 3 were truncated. The ribotype BII pattern was found in most of the O139 isolates. Three V. cholerae O139 isolated in 2001 had a new ribotype BVIII. Pulsed-field gel electrophoresis analysis revealed clonal variation in 2001 isolates compared to the 2004-2006 isolates. Molecular changes in V. cholerae O139 have to be closely monitored as this information may help in understanding the changing genetic features of this pathogen in relation to the epidemiology of cholera.

Background:Chronic arsenic exposure via contaminated drinking water is a global environmental health problem associated with hematological, hepatic and many serious systemic disorders. This study on adult male rats evaluated the protective effects of vitamin E (VE) and vitamin C (VC) against arsenic-mediated hematological and hepatic toxicities.Methods:Arsenic was administered orally as arsenic trioxide (3 mg/kg body weight/day), as a single dose for 30 consecutive days or along with VC/ascorbic acid (200 mg/kg body weight/day dissolved in water) and VE/α-tocopherol (400 mg/kg body weight/day dissolved in olive oil) as supplements. Multiple hematological and hepatic parameters were assessed.Results:Arsenic exposure caused significant reduction of erythrocyte counts (pConclusions:The present investigation offers strong evidence regarding the protective efficacy of co-administration of VC and VE against hematotoxicity and hepatotoxicity in adult male rats caused by chronic arsenic exposure.

Neonatal sepsis is the invasion of microbial pathogens into blood stream and is associated with a systemic inflammatory response with production and release of a wide range of inflammatory mediators. The increased serum levels of cytokines were found to correlate with the severity and mortality in course of sepsis. There have been no reports on the role of microbial proteases in stimulation of proinflammatory response in neonatal sepsis. We have identified YghJ, a secreted metalloprotease from a neonatal septicemic Escherichia coli (NSEC) isolate. The protease was partially purified from culture supernatant by successive anion and gel filtration chromatography. MS/MS peptide sequencing of the protease showed homology with YghJ. YghJ was cloned, expressed and purified in pBAD TOPO expression vector. YghJ was found to be proteolytically active against Methoxysuccinyl Ala-Ala-Pro-Met-p-nitroanilide oligopeptide substrate, but not against casein and gelatin. YghJ showed optimal activity at pH 7-8 and at temperatures 37-40°C. YghJ showed clear changes in cellular morphologies of Int407, HT-29 and HEK293 cells. YghJ stimulated the secretion of cytokines IL-1α, IL-1β and TNF-α in murine macrophages (RAW 264.7) and IL-8 from human intestinal epithelial cells (HT-29). YghJ also down-regulated the production of anti-inflammatory cytokines such as IL-10. YghJ is present in both septicemic (78%) and fecal E. coli isolates (54%). However, expression and secretion of YghJ is significantly higher among the septicemic (89%) than the fecal isolates (33%). This is the first study to show the role of a microbial protease, YghJ in triggering proinflammatory response in NSEC.

The ctxAB operon, encoding cholera toxin (CT) in Vibrio cholerae, is carried by the genome of a filamentous phage, CTXΦ. Usually, specific CTXΦ infect each of the two important biotypes, classical and El Tor, of epidemic V. cholerae strains belonging to serogroup O1, and are called CTXclassΦ and CTXETΦ, respectively. However, an unusual hybrid El Tor strain carrying CTXclassΦ caused the cholera epidemic in Mozambique in 2004. To understand the evolution of that strain, we have further analysed some representative hybrid El Tor strains isolated in Kolkata, India, in 1992, and the results indicate that both the Mozambique and the Indian strains are infected with a unique CTXclassΦ having only four copies of the tandem heptamer repeat sequence 5′-TTTTGAT-3′ present in the ctxAB promoter (P ctxAB ) region, like in CTXETΦ. Usually, the P ctxAB of the classical biotype contains seven to eight copies of such sequences. However, sequence analyses of the P ctxAB regions of several classical strains indicated that the copy number of heptamer repeat sequences might vary from four to eight copies, which was previously unknown. Since the hybrid strains analysed in this study carry four copies of the heptamer sequences, it may thus serve as a marker to trace the strain in future. Interestingly, while the Mozambique strain is devoid of an El Tor-specific free RS1 element or pre-CTX prophage, the Indian hybrid strains carry such elements. The free RS1 has been mapped, cloned and sequenced. As in pre-CTX and CTX prophages, multiple copies of free RS1 elements were found to be integrated in tandem in the large chromosomal dif site. Since Indian hybrid El Tor strains carry either free RS1 or pre-CTX prophage in their large chromosomes, it is possible that the Mozambique hybrid El Tor strain has evolved from these progenitor strains by step-wise deletion of CTX genetic elements from their large chromosomes.

RelA and SpoT of Gram-negative organisms critically regulate cellular levels of (p)ppGpp. Here, we have dissected the spoT gene function of the cholera pathogen Vibrio cholerae by extensive genetic analysis. Unlike Escherichia coli, V. choleraeDeltarelADeltaspoT cells accumulated (p)ppGpp upon fatty acid or glucose starvation. The result strongly suggests RelA-SpoT-independent (p)ppGpp synthesis in V. cholerae. By repeated subculturing of a V. choleraeDeltarelADeltaspoT mutant, a suppressor strain with (p)ppGpp(0) phenotype was isolated. Bioinformatics analysis of V. cholerae whole genome sequence allowed identification of a hypothetical gene (VC1224), which codes for a small protein (approximately 29 kDa) with a (p)ppGpp synthetase domain and the gene is highly conserved in vibrios; hence it has been named relV. Using E. coliDeltarelA or DeltarelADeltaspoT mutant we showed that relV indeed codes for a novel (p)ppGpp synthetase. Further analysis indicated that relV gene of the suppressor strain carries a point mutation at nucleotide position 676 of its coding region (DeltarelADeltaspoT relV676), which seems to be responsible for the (p)ppGpp(0) phenotype. Analysis of a V. choleraeDeltarelADeltaspoTDeltarelV triple mutant confirmed that apart from canonical relA and spoT genes, relV is a novel gene in V. cholerae responsible for (p)ppGpp synthesis.

The chitin-binding protein GbpA of Vibrio cholerae has been recently described as a common adherence factor for chitin and intestinal surface. Using an isogenic in-frame gbpA deletion mutant, we first show that V. cholerae O1 El Tor interacts with mouse intestinal mucus quickly, using GbpA in a specific manner. The gbpA mutant strain showed a significant decrease in intestinal adherence, leading to less colonization and fluid accumulation in a mouse in vivo model. Purified recombinant GbpA (rGbpA) specifically bound to N -acetyl- d -glucosamine residues of intestinal mucin in a dose-dependent, saturable manner with a dissociation constant of 11.2 μM. Histopathology results from infected mouse intestine indicated that GbpA binding resulted in a time-dependent increase in mucus secretion. We found that rGbpA increased the production of intestinal secretory mucins (MUC2, MUC3, and MUC5AC) in HT-29 cells through upregulation of corresponding genes. The upregulation of MUC2 and MUC5AC genes was dependent on NF-κB nuclear translocation. Interestingly, mucin could also increase GbpA expression in V. cholerae in a dose-dependent manner. Thus, we propose that there is a coordinated interaction between GbpA and mucin to upregulate each other in a cooperative manner, leading to increased levels of expression of both of these interactive factors and ultimately allowing successful intestinal colonization and pathogenesis by V. cholerae .

An unusual strain of Vibrio cholerae O1 biotype El Tor harbouring multiple tandem copies of classical CTX prophage caused a cholera epidemic in Mozambique in 2004. However, the location of the classical CTX prophage in the genome of the Mozambique strain was unknown. In this study, pulsed field gel electrophoresis (PFGE) of the whole genome along with Southern hybridization experiments indicated that the classical CTX prophage present in the Mozambique strain is located in the small chromosome. To determine the CTX prophage integration site in the small chromosome of Mozambique strain, the 5'and 3' junctions of the prophage and small chromosome were PCR amplified, cloned and sequenced. Sequence analysis indicated that the prophage was integrated in the conserved dif site of the replication terminus region of the Mozambique strain. While using an O1 El Tor isolate VC44 as a control strain, which carries tandem copies of CTX prophage in its small chromosome like the Mozambique strain, it was unexpectedly detected that the strain VC44 also possesses classical cholera toxin B gene allele. Since the strain VC44 was isolated in India in the year 1992, it appears that the Mozambique strain has probably originated from a VC44-like strain.

The cholera toxin (CT) is a critical determinant of the virulence of epidemic Vibrio cholerae strains. The ctxAB operon encoding CT is part of the genome of a filamentous bacteriophage CTXΦ, which may integrate as a single copy or as multiple copies in the genome of V. cholerae. The CTXΦ genome is composed of RS2 (2.4 kb) and core (4.5 kb) regions. In the present study extensive genetic mapping analyses indicated that two copies of tandemly arrayed CTX prophages are integrated in the small chromosome of an environmental V. cholerae strain, VCE232, belonging to serogroup O4. Further mapping revealed that the integration of prophages has occurred in the same genetic locus of the small chromosome of VCE232 as that of V. cholerae O1 biotype El Tor strains. Interestingly, a new type of RS2-like element 3.5 kb in size was found in the CTX prophage genome in the small chromosome of VCE232. Cloning followed by sequencing of the new RS2-like element of VCE232 revealed the presence of three ORFs, which probably encode highly divergent types of phage regulatory proteins. Furthermore, the strain VCE232 also harbours two copies of a tandemly arranged CTX prophage devoid of the ctxAB genes, called pre-CTX prophage, in its large chromosome. The presence of multiple copies of diverse CTX prophages in both the chromosomes of VCE232 suggests that toxigenic environmental V. cholerae non-O1, non-O139 strains could play a role in the emergence of new epidemic clones.

Summary The ctxAB genes encoding cholera toxin, reside in the genome of a filamentous bacteriophage CTXφ. The presence of CTX prophage in non-epidemic environmental Vibrio cholerae strains is rare. The CTX prophage, the lysogenic form of CTXφ in V. cholerae, is comprised of the ‘RS2’ and the ‘Core’. Analysis of the rstR gene present in the RS2 region of the CTX prophage revealed the presence of new alleles of the prophages in four environmental non-O1, non-O139 strains VCE22 (O36), VCE228 (O27), VCE232 (O4) and VCE233 (O27), and the CTX prophages are located in the small chromosomes. Phylogenetic analysis based on the nucleotide sequences of the rstR and orfU (present in the core) genes of these prophages placed them in a single unique cluster, which is distally located compared with that of epidemic V. cholerae O1 strains. Further analysis indicated that the genome of the prophage present in the strain VCE22 is devoid of the ctxAB genes, called pre-CTX prophage and the strain also possess the toxin-coregulated pilus protein coding gene tcpA of classical type, another important pathogenicity determining locus of the epidemic V. cholerae strains. Comparative analysis of the nucleotide sequences of the rstR and orfU genes indicated that the pre-CTX prophage of VCE22 might be the progenitor of new alleles of the CTX prophages present in these environmental strains.

InVibrio cholerae, the causative agent of cholera, products of three genes,relA,spoTandrelV, govern nutritional stress related stringent response (SR). SR in bacteria is critically regulated by two intracellular small molecules, guanosine 3′-diphosphate 5′-triphosphate (pppGpp) and guanosine 3′,5′-bis(diphosphate) (ppGpp), collectively called (p)ppGpp or alarmone. Evolution ofrelVis unique inV. choleraebecause other Gram-negative bacteria carry onlyrelAandspoTgenes. Recent reports suggest that RelV is needed for pathogenesis. RelV carries a single (p)ppGpp synthetase or RelA-SpoT domain (SYNTH/RSD) and belongs to the small alarmone synthetase (SAS) family of proteins. Here, we report extensive functional characterizations of therelVgene by constructing several deletion and site-directed mutants followed by their controlled expression in (p)ppGpp0cells ofEscherichia coliorV. cholerae. Substitution analysis indicated that the amino acid residues K107, D129, R132, L150 and E188 of the RSD region of RelV are essential for its activity. While K107, D129 and E188 are highly conserved in RelA and SAS proteins, L150 appears to be conserved in the latter group of enzymes, and the R132 residue was found to be unique in RelV. Extensive progressive deletion analysis indicated that the amino acid residues at positions 59 and 248 of the RelV protein are the functional N- and C-terminal boundaries, respectively. Since the minimal functional length of RelV was found to be 189 aa, which includes the 94 aa long RSD region, it seems that the flanking residues of the RSD are also important for maintaining the (p)ppGpp synthetase activity.

The methanolic crude and methanol-aqueous extract of Alstonia macrophylla leaves and n-butanol part of the crude extract showed antimicrobial activity against various strains of Staphylococcus aureus, Staphylococcus saprophyticus, Streptococcus faecalis, Escherichia coli, Proteus mirabilis, Trichophyton rubrum, Trichophyton mentagrophytes var. mentagrophytes and Microsporum gypseum. The minimum inhibitory concentration (MIC) values ranges from 64 to 1000 microg/ml for bacteria and 32-128 mg/ml for dermatophytes. However, the strains of Pseudomonas aeruginosa, Klebsiella sp. and Vibrio cholerae showed resistance against in vitro treatment of the extracts up to 2000 microg/ml concentration, while the two yeast species were resistant even at 128 mg/ml concentration. The stem bark extract prepared similarly was found to be less active compared to the leaves. Phytochemical study indicates that the crude extract contains tannins, flavonoids, saponins, sterols, triterpene and reducing sugars. Further fractionation and purification of n-butanol part of the extract showed the presence of beta-sitosterol, ursolic acid, beta-sitosterol glucoside and a mixture of minor compounds only detected in TLC.

Seasonal incidence ofVibrio cholerae was monitored for a year in a man-made freshwater lake, an open sewage canal, and a pond composed of rainwater accumulations, located in Calcutta.V. cholerae was found in all sites. It exhibited a distinct bimodal seasonal cycle in the lake with a primary peak in August–September and a secondary peak in May–June. Correlation with environmental parameters revealed that temperature and, to a certain extent, pH were the important factors governing the densities ofV. cholerae. In the lake, sediment samples harbored high densities ofV. cholerae immediately after months when peak counts were observed in plankton, suggesting a cycle of cells between sediment and water. At the other sampling areas, no defined seasonality was observed. Instead, high counts ofV. cholerae were observed at these severely polluted sites throughout the study period, including the winter months. All the 15 water samples passed via the ligated loop of rabbits yielded pure cultures ofV. cholerae, indicating that the rabbit intestine selects outV. cholerae from a mixed flora. Uniformly high isolation rates ofV. cholerae were observed from brackish water and freshwater species of export quality prawns.V. cholerae was found to be abundant and was represented by 32 individual Louisiana State University (LSU) serovars, including two new serovars. The 01 serovar could not be isolated from any of the samples examined in this study. It was concluded thatV. cholerae non-01 is common in the freshwater environs of Calcutta.

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 .

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.

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 Not I, Sfi I, and Ceu I. The genome of SG24 is circular, and the genome size is about 3.57 Mb. The linkages between 47 Not I and 32 Sfi I 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 Ceu I fragments were determined primarily by analyses of partial fragments of the Ceu I-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.

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-Ceu I sites in their genomes, and V. cholerae strains belonging to serovars non-O1 and non-O139 have 10 I-Ceu I 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-Ceu I digestion profiles of strains belonging to different serovars and biovars. From the analysis of partial digestion products, I-Ceu I 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.

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.

PFGE analysis of the Notl-and Sfil-digested genome of Vibrio cholerae O139 strains isolated from different epidemic regions of India showed that all the strains are of clonal origin and the genome size is about 2·2 Mb. An analysis of the electrophoretic profiles of the genome of O139 strains, the RFLP of the cholera toxin (ctx) gene and Southern blot hybridization of Notl-digested genomes of classical, El Tor and 0139 with a Notl-linking clone of classical strain 569B, suggest that these strains closely resemble V. cholerae 01 biotype El Tor, but are widely different from the classical 01 vibrios. Using restriction enzymes which cleave a single site in either the core region or in the direct repeat sequence (RS) of the CTX genetic element, it has been shown that the genome of most of the O139 strains has two copies of the ctx gene in tandem connected by two RSs. The chromosomal location of the CTX genetic element in the 0139 strain is the same as that reported for El Tor vibrios. The organization of the virulence gene cassettes in different O139 strains shows genetic heterogeneity in the population. Whilst most of the epidemic strains have two copies of the CTX genetic element, in some strains the number of elements has been amplified and in at least one strain a single copy of the element has been deleted.

In bacteria, nutrient deprivation evokes the stringent response, which is mediated by the small intracellular signaling molecule ppGpp. In Gram negatives, the RelA enzyme synthesizes and SpoT hydrolyzes ppGpp, although the latter protein also has weak synthetase activity. DksA, a recently identified RNA polymerase binding transcription factor, acts as a coregulator along with ppGpp for controlling the stringent response. Recently, we have shown that three genes, relA , spoT , and relV , govern cellular levels of ppGpp during various starvation stresses in the Gram-negative cholera pathogen Vibrio cholerae . Here we report functional characterization of the dksA gene of V. cholerae ( dksA Vc ), coding for the protein DksA Vc . Extensive genetic analyses of the Δ dksA Vc mutants suggest that DksA Vc is an important component involved in the stringent response in V. cholerae . Further analysis of mutants revealed that DksA Vc positively regulates various virulence-related processes, namely, motility, expression of the major secretory protease, called hemagglutinin protease (HAP), and production of cholera toxin (CT), under in vitro conditions. We found that DksA Vc upregulates expression of the sigma factor FliA (σ 28 ), a critical regulator of motility in V. cholerae . Altogether, it appears that apart from stringent-response regulation, DksA Vc also has important roles in fine regulation of virulence-related phenotypes of V. cholerae .

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 the 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.

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 straimns 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.

Nutritional stress elicits stringent response in bacteria involving modulation of expression of several genes. This is mainly triggered by the intracellular accumulation of two small molecules, namely, guanosine 3’-diphosphate 5’-triphosphate and guanosine 3’,5’-bis(diphosphate), collectively called (p)ppGpp. Like in other Gram-negative bacteria, the cellular level of (p)ppGpp is maintained in Vibrio cholerae, the causative bacterial pathogen of the disease cholera, by the products of two genes relA and spoT. However, apart from relA and spoT, a novel gene relV has recently been identified in V. cholerae, the product of which has been shown to be involved in (p)ppGpp synthesis under glucose or fatty acid starvation in a ΔrelA ΔspoT mutant background. Furthermore, the GTP binding essential protein CgtA and a non-DNA binding transcription factor DksA also seem to play several important roles in modulating stringent response and regulation of other genes in this pathogen. The present review briefly discusses about the role of all these genes mainly in the management of stringent response in V. cholerae.

Vibrio cholerae, the causative agent of Asiatic cholera, still remains a major public health problem in most of the developing countries. Despite tremendous effort given in developing immunotherapeutics, availability of the whole genome sequence, and transcriptional profiling data, still a safe, effective, and long-lasting cholera vaccine is not available. One probable reason could be that our knowledge about stress adaptive mechanisms of V. cholerae with special reference to its survival both under in vitro and in vivo conditions is still incomplete. Indeed, a rapidly growing number of reports indicate that various intra- and extracellular small signaling molecules play critical roles in overcoming environmental onslaughts and allowing the pathogen to survive, grow, and cause the disease. Vibrio cholerae has two most important small molecule-mediated signaling pathways, which are extracellular quorum-sensing and intracellular 3ʹ,5ʹ-cyclic diguanylic acid (c-di-GMP) signaling system. Apart from these two systems, the intracellular (p)ppGpp molecule, called alarmone, also helps the organism to survive under nutritional scarcity and other stresses. The universal second messenger cAMP, another important small molecule, and its receptor protein, called CRP, have been implicated in virulence gene expression in V. cholerae, which may be due to its well-known overall impact on bacterial metabolism. Since these small molecules are crucial for the bacterial survival, the organism has evolved with multiple backup systems. If we consider that virulence gene expression of V. cholerae in human host is an outcome of stress-combating mechanism, then convergence of different small molecule-mediated signaling pathways is highly expected, but knowing how it is achieved is a real challenging job. This review focuses on how much we have learnt about this fascinating aspect of signaling systems of V. cholerae during post-genome sequencing era.

BackgroundTwo well-characterized proteases secreted by Vibrio cholerae O1 strains are hemagglutinin protease (HAP) and V. cholerae protease (PrtV). The hapA and prtV knock out mutant, V. cholerae O1 strain CHA6.8ΔprtV, still retains residual protease activity. We initiated this study to characterize the protease present in CHA6.8ΔprtV strain and study its role in pathogenesis in rabbit ileal loop model (RIL).Methodology/principal findingsWe partially purified the residual protease secreted by strain CHA6.8ΔprtV from culture supernatant by anion-exchange chromatography. The major protein band in native PAGE was identified by MS peptide mapping and sequence analysis showed homology with a 59-kDa trypsin-like serine protease encoded by VC1649. The protease activity was partially inhibited by 25 mM PMSF and 10 mM EDTA and completely inhibited by EDTA and PMSF together. RIL assay with culture supernatants of strains C6709 (FA ratio 1.1+/-0.3 n = 3), CHA6.8 (FA ratio 1.08+/-0.2 n = 3), CHA6.8ΔprtV (FA ratio 1.02+/-0.2 n = 3) and partially purified serine protease from CHA6.8ΔprtV (FA ratio 1.2+/-0.3 n = 3) induced fluid accumulation and histopathological studies on rabbit ileum showed destruction of the villus structure with hemorrhage in all layers of the mucosa. RIL assay with culture supernatant of CHA6.8ΔprtVΔVC1649 strain (FA ratio 0.11+/-0.005 n = 3) and with protease incubated with PMSF and EDTA (FA ratio 0.3+/-0.05 n = 3) induced a significantly reduced FA ratio with almost complete normal villus structure.ConclusionOur results show the presence of a novel 59-kDa serine protease in a ΔhapAΔprtV V. cholerae O1 strain and its role in hemorrhagic response in RIL model.

Aeromonas hydrophila strain AO1 isolated from an infected fish was found to be resistant to several quinolones. A plasmid isolated from the strain AO1, termed pBRST7.6, was cloned and sequenced and shown to be 7621bp in length with a GC content of 60%. Further analysis confirmed that it contained a gene with 100% identity to qnrS2 genes described in plasmids associated with other Aeromonas species, the product of which usually confers increased resistance to quinolones. The plasmid backbone contained a replication initiation module (repA repC) belonging to the IncQ-family and two genes (mobC and mobB), the products of which are putatively involved in plasmid mobilization. Putative iteron-based origin of replication and characteristic oriT like sequences were also present in the plasmid. The result suggests that Aeromonas spp. carrying plasmids with quinolone resistance genes are potential reservoirs of antimicrobial resistance determinants in the environment.

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.

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)

Summary 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 > 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.

Retrospective analysis led to the detection of twoVibrio choleraevariant O1 strains (VC51 and VC53), which were isolated in 1992 in Kolkata from clinical cases, with identical traits to 2004 Mozambique variant O1 strains. The Mozambique O1 strains that caused a huge outbreak in 2004 have been shown to have phenotypic traits of both classical and El Tor biotypes, and thereby have been reported as variant. Our study demonstrated that two O1 strains isolated in Kolkata during 1992 were of the El Tor background as evidenced by polymyxin B (50 U ml−1) resistance, positivity in Voges–Proskauer reactions and sensitivity to biotype-specific vibrio phages. With the features of classical CTX prophage, localization in the small chromosome, and an absence of RS1 and pTLC, both Mozambique and Kolkata strains appeared to be identical. Furthermore, two Kolkata strains exhibited an identical ribotype to that of the Mozambique variant, displaying ribotype pattern RI that had been assigned to KolkataV. choleraeO1 strains isolated on or before 1992.NotI pulsotype analysis indicated that these 1992 Kolkata strains along with the Mozambique variant O1 belonged to very closely related clones. Considering the chronological events, and the typical identity at the phenotypic and the genotypic level between the two O1 strains isolated during 1992 from Kolkata and during 2004 from Mozambique, we propose that some of the 1992 Kolkata O1 strains might have acted as progenitors for Mozambique variant O1 strains.

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.

During a 6-month survey, 131 prawn samples belonging to five different species cultured in paddy fields were examined for the presence ofVibrio cholerae and related vibrios associated with human disease. Non-O1V. cholerae was the most commonVibrio species associated with the prawn samples followed byV. parahaemolyticus, V. mimicus, V. vulnificus andV. alginolyticus, in that order.V. cholerae non-O1 appeared to constitute the normal microflora of prawns and this association was not limited by salinity. Examination of a representative number ofV. cholerae andV. parahaemolyticus isolates for established virulence factors revealed that none of theV. cholerae strains possessed genes that were homologous with cholera-toxin genes and none produced a heat stable enterotoxin while none of theV. parahaemolyticus isolates were Kanagawa phenomenon-positive and most of the strains were untypeable. The presence of such high numbers of vibrios in paddy field cultured prawn samples and its impact on the bacteriological quality of prawns destined for human consumption needs critical assessment.