Genetic and mutational analysis of virulence traits and their modulation in an environmental toxigenic Vibrio cholerae non-O1/non-O139 strain, VCE232.

Vibrio cholerae O1 and O139 isolates deploy cholera toxin (CT) and toxin-coregulated pilus (TCP) to cause the diarrhoeal disease cholera. The ctxAB and tcpA genes encoding CT and TCP are part of two acquired genetic elements, the CTX phage and Vibrio pathogenicity island-1 (VPI-1), respectively. ToxR and ToxT proteins are the key regulators of virulence genes of V. cholerae O1 and O139. V. cholerae isolates 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 toxigenic V. cholerae non-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 of V. cholerae O1 strains, including organization of the VPI-1 locus, ctxAB, tcpA and toxT genes, 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, ΔtoxR and ΔtoxTΔtoxR deletion mutants of VCE232. Extensive genetic analyses of these mutants indicated that the toxT and toxR genes of VCE232 are crucial for CT and TCP production. However, unlike O1 isolates, the presence of either toxT or toxR gene is sufficient for optimal CT production in VCE232. In addition, the VCE232 ΔtoxR mutant 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 toxigenic V. cholerae non-O1/non-O139 strain.

Type III secretion system confers enhanced virulence in clinical non-O1/non-O139 Vibrio cholerae.

Vibrio cholerae O1 infections mainly are responsible for significant mortality and morbidity amongst children, however, non-O1/non-O139 V. cholerae have also been reported to cause mild to severe infections because of their virulence potential. The pathogenic mechanisms of non-O1, non-O139 isolates are not as clearly understood as for that of O1 and O139 isolates. Type three secretion system (TTSS) is also considered one of the important virulent factors and during the current study, we investigated the role of TTSS in association with non-O1/non-O139 clinical isolates. We report that the presence of TTSS in non-O1/non-O139 V. cholerae clinical isolate (D13) from a child confers more virulence compared to the one lacking it (D15) in another clinical case during the small cholera epidemic. Moreover, the antibiotic susceptibility profiles of D13 and D15 indicate that they are multiple drug resistance (MDR) isolates. The sequence analysis for TTSS cluster was carried out for D13 and compared with the TTSS positive reference Vibrio parahaemolyticus RIMD2210633 and V. cholerae AM19226 non-O1/non-O139. Furthermore, the pathogenic potential of D13 & D15 was also explored in simple and economical invertebrate host model, Galleria mellonella and the results revealed that TTSS+ve isolate (D13) was more virulent compared to TTSS-ve isolate (D15). We suggest that this distinct genetic difference, seen in natural variants D13 and D15, is also reflected by the clinical picture of the former in contributing towards the severity of disease symptoms and this finding was further validated by assessing virulence potential of both isolates using inexpensive G. mellonella infection model.

Virulence-associated genes and molecular characteristics of non-O1/non-O139 Vibrio cholerae isolated from hepatitis B cirrhosis patients in China.

OBJECTIVES: We aimed to report virulence-associated genes and molecular characteristics of non-O1/non-O139 Vibrio cholerae isolated from hepatitis B cirrhosis patients in China. METHODS: Patient clinical data including course of disease, laboratory tests, antibiotic treatment and outcomes were collected. Antimicrobial susceptibility testing was performed and virulence-associated genes were detected by PCR. Genetic relatedness among non-O1/non-O139 V. cholerae strains was investigated by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). RESULTS: All three strains in this study harbored pathogenicity related genes like rtxA, rtxC, toxR, hapA, hlyA and ompW whereas they lacked ctxA, ctxB, tcpA, ompU and zot genes. None of them showed resistance to any antibiotic detected. A new allele of gyrB was submitted to the MLST database and designated as 97. Two novel sequence types (ST518 and ST519) and ST271 were identified by multilocus sequence typing (MLST). PFGE indicated considerable diversity among three non-O1/non-O139 V. cholerae strains. CONCLUSIONS: Three sporadic cases highlight that non-O1/non-O139 V. cholerae can cause opportunistic invasiveness infection in cirrhosis patients. Pathogenicity may be related to virulence-associated genes. Timely detection and antibiotic therapy should be paid more attention to in clinic.

Type III secretion is essential for the rapidly fatal diarrheal disease caused by non-O1, non-O139 Vibrio cholerae.

Cholera is a severe diarrheal disease typically caused by O1 serogroup strains of Vibrio cholerae. The pathogenicity of all pandemic V. cholerae O1 strains relies on two critical virulence factors: cholera toxin, a potent enterotoxin, and toxin coregulated pilus (TCP), an intestinal colonization factor. However, certain non-O1, non-O139 V. cholerae strains, such as AM-19226, do not produce cholera toxin or TCP, yet they still cause severe diarrhea. The molecular basis for the pathogenicity of non-O1, non-O139 V. cholerae has not been extensively characterized, but many of these strains encode related type III secretion systems (TTSSs). Here, we used infant rabbits to assess the contribution of the TTSS to non-O1, non-O139 V. cholerae pathogenicity. We found that all animals infected with wild-type AM-19226 developed severe diarrhea even more rapidly than rabbits infected with V. cholerae O1. Unlike V. cholerae O1 strains, which do not damage the intestinal epithelium in rabbits or humans, AM-19226 caused marked disruptions of the epithelial surface in the rabbit small intestine. TTSS proved to be essential for AM-19226 virulence in infant rabbits; an AM-19226 derivative deficient for TTSS did not elicit diarrhea, colonize the intestine, or induce pathological changes in the intestine. Deletion of either one of the two previously identified or two newly identified AM-19226 TTSS effectors reduced but did not eliminate AM-19226 pathogenicity, suggesting that at least four effectors contribute to this strain's virulence. In aggregate, our results suggest that the TTSS-dependent virulence in non-O1, non-O139 V. cholerae represents a new type of diarrheagenic mechanism.

[Role of galactose-specific receptor--lectin in bactericidal activity of hemolysin of Vibrio cholerae non O1/O139].

AIM: To study the role lectin galactose-specific receptor of Vibrio cholerae hemolysin in receptor mechanisms of bacterial cells lysis. MATERIALS AND METHODS: Five strains of V. cholerae eltor ctx- Hly+ and 5 strains V. cholerae eltorctx+ Hly(-) isolated from patients and environment as well as 6 strains from Shigella genus, 3 strains of Escherichia coli, 2 strains of V. cholerae eltor, and 1 strain of V. cholerae non O1 were used in the study. Preparation P-11702 obtained from strain of V. cholerae non O1 was used for the study of bactericidal activity of hemolysin. For neutralization of bactericidal effect antihemolytic serum prepared against P-11702 as well as 1% solutions of carbohydrates (galactose, sucrose, glucose, and N-acetyl-D-galactosamine) were used. RESULTS: It was shown that lytic activity of hemolysin against cholera vibrios and indicator cultures for detection of vibriocins was neutralized by galactose as well as hemolysis of erythrocytes was neutralized by specific antihemolytic serum. Sucrose, glucose, and N-acetyl-D-galactosamine did not have effect on bactericidal and hemolytic activity of hemolysin. CONCLUSION: Protein-carbohydrate receptor binding of galactose-specific lectin has an important role in realization of bactericidal effect of V. cholerae hemolysin.

VopF, a type III effector protein from a non-O1, non-O139 Vibrio cholerae strain, demonstrates toxicity in a Saccharomyces cerevisiae model.

VopF, a type III effector protein, has been identified as a contributory factor to the intestinal colonization of type III secretion system-positive, non-O1, non-O139 Vibrio cholerae strains. To gain more insight into the function of VopF, a yeast model was developed. Using this model, it was found that ectopic expression of VopF conferred toxicity in yeast.

Severe diarrhea caused by cholera toxin-producing vibrio cholerae serogroup O75 infections acquired in the southeastern United States.

BACKGROUND: From 2003 through 2007, Vibrio cholerae serogroup O75 strains possessing the cholera toxin gene were isolated from 6 patients with severe diarrhea, including 3 in Georgia, 2 in Alabama, and 1 in South Carolina. These reports represent the first identification of V. cholerae O75 as a cause of illness in the United States. V. cholerae O75 was isolated from a water sample collected from a pond in Louisiana in 2004. Subsequently, 3 V. cholerae isolates from Louisiana (2 from patients with diarrhea in 2000 and 1 from a water sample collected in 1978) that had been previously reported as serogroup O141 were also discovered to be serogroup O75. RESULTS: All 8 patients who were infected with V. cholerae O75 were adults who became ill after consuming seafood; 2 had eaten raw oysters traced back to the Gulf Coast of the United States. All 10 isolates possessed the cholera toxin gene and were susceptible to 10 antimicrobials. One clinical isolate and 1 environmental (water) isolate had the same pulsed-field gel electrophoresis pattern; 4 clinical isolates shared a common pulsed-field gel electrophoresis pattern. CONCLUSIONS: The occurrence of these cases over many years and the concurrent identification of V. cholerae O75 in water from a Gulf Coast state suggest that these strains may survive for long periods in this environment. The patients' exposure histories suggest that infection can be acquired from consumption of raw oysters from the Gulf Coast. Clinicians and public health authorities should be vigilant for the occurrence of new toxigenic serogroups of V. cholerae that are capable of causing severe diarrhea.

Rapid growth of planktonic Vibrio cholerae non-O1/non-O139 strains in a large alkaline lake in Austria: dependence on temperature and dissolved organic carbon quality.

Vibrio cholerae non-O1/non-O139 strains have caused several cases of ear, wound, and blood infections, including one lethal case of septicemia in Austria, during recent years. All of these cases had a history of local recreational activities in the large eastern Austrian lake Neusiedler See. Thus, a monitoring program was started to investigate the prevalence of V. cholerae strains in the lake over several years. Genetic analyses of isolated strains revealed the presence of a variety of pathogenic genes, but in no case did we detect the cholera toxin gene or the toxin-coregulated pilus gene, both of which are prerequisites for the pathogen to be able to cause cholera. In addition, experiments were performed to elucidate the preferred ecological niche of this pathogen. As size filtration experiments indicated and laboratory microcosms showed, endemic V. cholerae could rapidly grow in a free-living state in natural lake water at growth rates similar to those of the bulk natural bacterial population. Temperature and the quality of dissolved organic carbon had a highly significant influence on V. cholerae growth. Specific growth rates, growth yield, and enzyme activity decreased markedly with increasing concentrations of high-molecular-weight substances, indicating that the humic substances originating from the extensive reed belt in the lake can inhibit V. cholerae growth.

[Biofilm formation and response to oxidative stress in Pseudomonas aeruginosa and Vibrio cholerae non-O1 depending on culture media]. / Tvorba biofilmu a odpoved' na oxidacný stres u kmenov Pseudomonas aeruginosa a Vibrio cholerae non-O1 v závislosti na kultivacných médiách.

OBJECTIVE: To evaluate the effect of six culture media (five complex and one mineral) on biofilm formation and response to oxidative stress in Pseudomonas aeruginosa (3 strains) and Vibrio cholerae non-O1 (3 strains). METHODS: Biofilm formation was quantitatively determined by a crystal violet absorption assay. The bacterial response to oxidative stress evoked by hydrogen peroxide was visualized as a zone of clearing around the disc after 24 h incubation at 37 degrees C. RESULTS: For both of the bacterial species studied, biofilm formation was the highest after cultivation in tryptone soya broth (TSM) or in TSM supplemented with 8% glucose (TSM+GL), being the lowest in mineral medium (MM). V. cholerae non O1 strains were 1.4 to 4.3 times more responsive on average to oxidative stress depending on culture medium as compared with P. aeruginosa strains. The culture medium had no significant effect on H2O2 evoked by response to oxidative stress in vibrios in contrast to P. aeruginosa. In P. aeruginosa, the highest mean resistance to H2O2 was observed after cultivation in peptone water while the most sensitive cells were found after incubation in TSM+GL and MM. CONCLUSION: The culture medium composition influnced biofilm formation in both of the bacterial species tested and had a considerable effect on response to oxidative stress in P. aeruginosa.

Molecular cloning and characterization of all RND-type efflux transporters in Vibrio cholerae non-O1.

Resistance Nodulation cell Division (RND) efflux transporters are thought to be involved in mediating multidrug resistance in Gram-negative bacteria, including Vibrio cholerae non-O1. There are six operons for putative RND-type efflux transporters present in the chromosome of V. cholerae O1 including two operons, vexAB and vexCD, which had already been identified. All of the six operons were cloned from V. cholerae non-O1, NCTC4716 by the PCR method, introduced, and expressed in cells of drug hypersusceptible Escherichia coli KAM33 (DeltaacrAB, DeltaydhE). Only vexEF conferred elevated minimum inhibitory concentrations (MICs) of some antimicrobial agents in the E. coli cells. However, VexEF did not confer increased MIC of any drug tested in tolC-deficient E. coli KAM43 cells. On the other hand, when E. coli KAM43 was transformed with vexAB, vexCD or vexEF together with tolC(Vc) of V. cholerae NCTC4716, we observed elevated MICs of various antimicrobial agents. Among them, E. coli KAM43 expressing both VexEF and TolC(Vc) showed much higher MICs and much broader substrate specificity than the other two. We also observed ethidium efflux activity via VexEF-TolC(Vc), and the activity required Na(+). Thus, VexEF-TolC (Vc) is either a Na(+)-activated or a Na(+)-coupled transporter. To our knowledge, this is the first report on the requirement of Na(+) for an RND-type efflux transporter.

The capsule polysaccharide structure and biogenesis for non-O1 Vibrio cholerae NRT36S: genes are embedded in the LPS region.

BACKGROUND: In V. cholerae, the biogenesis of capsule polysaccharide is poorly understood. The elucidation of capsule structure and biogenesis is critical to understanding the evolution of surface polysaccharide and the internal relationship between the capsule and LPS in this species. V. cholerae serogroup O31 NRT36S, a human pathogen that produces a heat-stable enterotoxin (NAG-ST), is encapsulated. Here, we report the covalent structure and studies of the biogenesis of the capsule in V. cholerae NRT36S. RESULTS: The structure of the capsular (CPS) polysaccharide was determined by high resolution NMR spectroscopy and shown to be a complex structure with four residues in the repeating subunit. The gene cluster of capsule biogenesis was identified by transposon mutagenesis combined with whole genome sequencing data (GenBank accession DQ915177). The capsule gene cluster shared the same genetic locus as that of the O-antigen of lipopolysaccharide (LPS) biogenesis gene cluster. Other than V. cholerae O139, this is the first V. cholerae CPS for which a structure has been fully elucidated and the genetic locus responsible for biosynthesis identified. CONCLUSION: The co-location of CPS and LPS biosynthesis genes was unexpected, and would provide a mechanism for simultaneous emergence of new O and K antigens in a single strain. This, in turn, may be a key element for V. cholerae to evolve new strains that can escape immunologic detection by host populations.

[Comparative characteristics of the preparations of hemolysin of ctx+ and ctx- Vibrio cholerae eltor and Vibrio cholerae O139 strains].

Hemolysin of ctx+ Vibrio cholerae strains was obtained and studied. Ctx+ Vibrio cholerae O1 and O139 strains produced the hemolysin during cultivation in triptone medium without FeCl3. Mol.wt. and the spectrum of lytic activities of hemolysins of ctx+ Vibrio cholerae did not differ from hemolysins of ctx- strains.

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

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

Toxin(s), other than cholera toxin, produced by environmental non O1 non O139 Vibrio cholerae.

A total of 39 Vibrio cholerae non O1 non O139 strains were isolated from surface waters of different parts of Dhaka City, Bangladesh. All these strains showed lack of ctx or zot gene, as demonstrated by the PCR analysis. Eighteen representative strains were tested for enterotoxin production using a rabbit ileal loop model, of which live cells of 8 strains and culture filtrates of 6 strains produced fluid accumulation in ileal loops. However, none of them produced heat stable toxin (ST), as detected by suckling mouse assay. On the other hand, 15% of isolates produced cytotoxin as detected by the Chinese Hamster Ovary (CHO) cell assay. Fifty times concentrated culture filtrates of the representative strains did not give any precipitin band against the anti-cholera toxin, suggesting the strains produced an enterotoxin, which is antigenically different from known cholera toxin (CT). Eighty percent of the total isolates were found to be positive for heat labile haemolysin detected by tube method, whereas, 39% were found positive by the Christie-Atkins-Munch-Petersen (CAMP) method. However, 87% of the isolates were positive for haemagglutinin/protease and all of the strains were positive for mannose-sensitive-haemagglutinin assay.

Gene cloning and characterization of four MATE family multidrug efflux pumps from Vibrio cholerae non-O1.

There are six putative genes for multidrug and toxic compound extrusion (MATE) family multidrug efflux pumps in the chromosome of Vibrio cholerae. We have so far analyzed two MATE family pumps in V. cholerae non-O1 NCTC4716. Here we cloned four remaining genes for putative MATE family efflux pumps by the PCR method from this microorganism and designated them as vcmB, vcmD, vcmH and vcmN. Each one of the four genes was introduced and expressed in the drug hypersusceptible host Escherichia coli KAM32 cells. We observed elevated MICs of multiple antimicrobial agents, such as fluoroquinolones, aminoglycosides, ethidium bromide and Hoechst 33342 in the transformants. Energydependent efflux of substrate was observed with the transformed cells. We found that efflux activities of VcmB, VcmD and VcmH were Na+-dependent, but that of VcmN was Na+-independent. Thus, all six of the MATE family multidrug efflux pumps of V. cholerae non-O1 have been characterized. We also found that all six genes were expressed in cells of V. cholerae non-O1.

Comparative study of different methods for detection of toxic and other enzymatic factors in Vibrio cholerae strains.

The purpose of this work was to characterize the toxin profile and the presence of other virulence factors involved in the pathogenesis and biology of 13 V. cholerae O1 (11 clinical cases and 2 waters) and 6 V. cholerae non O1 strains (4 clinical cases and 2 waters) using genetic (PCR), immunological (RPLA), biochemical (NAD degradation, haemolysis, Kanagawa phenomenon, caseinase, lecithinase, mucinase, amylase, esculine hydrolysis) and cell culture (Vero E6, HEp-2) assays. The results indicated a concordance between PCR-RPLA (84%), PCR-NAD (73%) and RPLA-NAD (84%) methods. The sensitivity of RPLA and NAD degradation methods were comparable to PCR in detecting CT in Vibrio cholerae O1 strains. Although NAD degradation method was not exclusively specific for the CT detection, it proved its usefulness in screening certain virulent, CT-negative clones of V. cholerae. The cytotoxic effect on Vero E6 cells, enzyme production (Kanagawa haemolysins, lecithinase, caseinase, esculine hydrolysis) as well as adherence ability on inert substrate proved to be much more constant in V. cholerae non O1 (CT- negative) than in V. cholerae O1 (CT-positive). All V. cholerae non O1 strains isolated in diarrheal cases were Kanagawa positive. This complex of virulence factors detected in V. cholerae non O1 strains could probably contribute during interepidemic periods to human-to-human transmission and to greater resistance as compared to O1 strains in the environment.

Translocation of protein kinase-C with IP3-induced calcium mobilization by heat-stable enterotoxin of Vibrio cholerae non-O1 in isolated rat enterocytes.

The activity of the calcium- and phospholipid-dependent enzyme protein kinase C (PKC) in response to heat-stable enterotoxin (NAG-ST) of Vibrio cholerae non-O1 was examined in isolated rat enterocytes. Optimal stimulation of the membrane-bound PKC activity (about 4.3-fold) was observed after 1 min of incubation of cells with 10 ng/ml toxin; and the effects were dose dependent. Following NAG-ST treatment an increase in PKC activity in the membrane fraction was found with a concomitant decrease in the cytosolic fraction suggesting the redistribution of the enzyme. The pronounced enzyme activity in presence of a classical pseudosubstrate and its complete inhibition by Gö 6976 suggested the involvement of a calcium-dependent isoform of PKC (PKC-alpha). A time course study employing an immunoblot assay provided evidence that NAG-ST led to almost complete translocation of PKC-alpha to the membrane. A 65% inhibition of enzyme activity in the membrane fraction and inhibition of its translocation to some extent by dantrolene treatment further suggested that the enzyme was translocated with the rise of intracellular calcium ([Ca2+]i). The phosphorylation of three membrane proteins by toxin-induced PKC in vitro and abolition of this phosphorylation by Gö 6976 demonstrated that phosphorylation of these membrane proteins was PKC-alpha mediated and might be involved in the alteration of membrane functions.