Role of non-O157 VTEC

Non-O157 VTEC are typical Escherichia coli that differ only in their ability to produce verocytotoxins (VT). The transmission of VTEC is discussed in relation to the transmission of commensal E. coli. The emergence over the last few decades of a great variety of VTEC serotypes from healthy and diseased humans and animals is described. Particular attention is given to the distribution of the more important serogroups pathogenic for humans that have been described from around the world, particularly serogroups O26, O111, O128 and O103. The possible role of ruminants as reservoirs is discussed. The problems of laboratory diagnosis of non-O157 VTEC are considered and various laboratory methods are assessed. Evidence is presented that the particular E. coli serotypes now known to be VTEC were present in humans and animals many years ago, but have acquired the ability to produce VT and probably other virulence factors. Finally, predictions are made of the possible increase in problems associated with these emerging pathogens.     

Adult non-biting midges: possible windborne carriers of Vibrio cholerae non-O1 non-O139

Vibrio cholerae is a waterborne bacterium native to the aquatic environment. There are over 200 known serogroups yet only two cause cholera pandemics in humans. Direct contact of human sewage with drinking water, sea-born currents and marine transportation, represent modes of dissemination of the bacteria and thus the disease. The simultaneous cholera outbreaks that occur sometimes in distant localities within continental landmasses are puzzling. Here we present evidence that flying, non-biting midges (Diptera; Chironomidae), collected in the air, carry viable non-O1 non-O139 serogroups of V. cholerae. The association of V. cholerae with chironomid egg masses, which serve as a V. cholerae reservoir, was further confirmed. In simulated field experiments, we recorded the transfer of environmental V. cholerae by adult midges from the aquatic environment into bacteria-free water-pools. In laboratory experiments, flying adult midges that emerged from V. cholerae (O1 or O139) contaminated water transferred the green fluorescent protein (GFP)-tagged pathogenic bacteria from one laboratory flasks to another. Our findings show that aerial transfer by flying chironomids may play a role in the dissemination of V. cholerae in nature.     

Genetic characterization of toxigenic Vibrio cholerae non-O1/non-O139 strains, isolated in the Middle Asia

Here, we report on the characterization of 22 clinical toxigenic V. cholerae non-O1/non-O139 strains isolated in the Middle Asia (Uzbekistan) in 1971–1990. PCR analysis has revealed that these strains contain the main virulence genes such as ctxA, zot, ace (CTXφ); rstC (RS1φ); tcpA, toxT, aldA (pathogenicity island VPI), but they lack both pandemic islands VSP-I and VSP-II specific to epidemic strains of O1 serogroup of El Tor biotype and O139 serogroup. Only two of the twenty two toxigenic strains have tcpA gene of El Tor type, one strain has tcpA gene of classical type, while nineteen other strains carry a new variant of this gene, designated as tcpA ^uzb. Nucleotide sequences analysis of virulence genes in toxigenic V. cholerae non-O1/non-O139 strains from Uzbekistan showed that they differ significantly from the sequences of these genes in epidemic O1 and O139 strain indicating that they belong to a separate line of evolution of virulent V. cholerae strains. For the first time it is shown that V. cholerae non-O1/non-O139 toxigenic strains of different serogroups may belong to the same clone.     

Differentiation among the Vibrio cholerae serotypes O1, O139, O141 and non-O1, non-O139, non-O141 using specific monoclonal antibodies with dot blotting

Seven different monoclonal antibodies (MAbs) specific to only Vibrio cholerae were produced using a combination of five representative serotypes of V. cholerae for immunization. The first three MAbs (VC-93, VC-82 and VC-223) were specific to the V. cholerae serogroup O1 with different avidity for the serotypes O1 Inaba and O1 Ogawa. The fourth and the fifth MAbs were specific to V. cholerae O139 (VC-812) or O141 (VC-191) serogroups, respectively. The sixth MAb (VC-26) bound to all three serogroups of V. cholerae. The seventh MAb (VC-63) bound to all twenty five isolates of V. cholerae used in this study. None of the seven MAbs showed cross-reactivity with other Vibrio spp. or closely-related V. cholerae species, V. mimicus or other gram-negative bacteria. The eighth MAbs (VC-201) specific to almost all Vibrio spp. was also obtained. In dot blotting, these MAbs can be used to detect a diluted pure culture of V. cholerae in solution with a sensitivity range of from 10⁵ to 10⁷ CFU ml^− 1. However, the detection capability could be improved equivalent to that of PCR technique after preincubation of samples in alkaline peptone water (APW). Thus, these MAbs constitute convenient immunological tools that can be used for simple, rapid and simultaneous direct detection and differentiation of the individual serotypes of V. cholerae in complex samples, such as food and infected animals, without the requirement for bacterial isolation or biochemical characterization.     

Characterization of a 20-kDa pilus protein expressed by a diarrheogenic strain of non-O1/non-O139 Vibrio cholerae

A diarrheogenic strain of non-O1/non-O139 Vibrio cholerae (10325) belonging to serogroup O34 was earlier shown to express a new type of pilus composed of a 20-kDa subunit protein. Amino-terminal sequence data (determined up to 20 amino acid residues) of this protein showed it to be different from the subunit proteins of other known types of pili of V. cholerae. On the other hand, it showed complete homology with the corresponding sequence of a 22-kDa outer membrane protein (OmpW) of V. cholerae. Expression of 10325 pili was favored in AKI rather than in NB medium and at 30°C rather than at 37°C. Further, cultural conditions favoring pilus expression also enhanced autoagglutination and adherence properties of strain 10325. An antiserum to the 20-kDa protein induced passive protection against challenge with the parent organism 10325, but not against V. cholerae O1 strains. Such protection was shown to be mediated by inhibition of intestinal colonization in vivo.     

An aberrant hemolysin of Vibrio cholerae non-O1.

An aberrant hemolysin produced by a Vibrio cholerae non-O1 strain N037 (N037-hly) was purified and characterized. N037-Hly was antigenically very similar to El Tor hemolysin but differed in molecular weight (48,000 vs. 60,000), interaction with glucose, and hemolytic activity. Of 100 V. cholerae non-O1 strains other than the N037 strain examined, none produced this aberrant hemolysin. The N-terminal amino acid sequence of N037-hly was highly homologous to that of El Tor hemolysin.     

R plasmids in environmental Vibrio cholerae non-O1 strains

The occurrence of drug resistance and its plasmid-mediated transferability was investigated in 140 environmental strains of Vibrio cholerae non-O1 and 6 strains of Vibrio cholerae, both O1 and non-O1, of clinical origin. Of the 146 strains tested, 93% were resistant to at least one drug and 74% were resistant to two or more antibiotics. The O1 strains were susceptible to all antibiotics used. A total of 26 of 28 selected resistant wild strains carried R plasmids that were transferable by intraspecific and intergeneric matings. The most common transmissible R factor determined resistance to ampicillin, amoxicillin, and sulfanilamide (30%), followed by resistance to ampicillin and amoxicillin (13%) and resistance to ampicillin, amoxicillin, phosphomycin, and sulfanilamide (9%). Comparison of the three methods of plasmid analysis showed that the method of Birnboim and Doly (Nucleic Acids Res. 7:1513-1523, 1979) without EDTA and lysozyme was optimal for isolation of both large and small plasmids in environmental V. cholerae strains. Most strains harbored more than one plasmid, and the molecular sizes ranged from 1.1 to 74.8 megadaltons. The plasmids of high molecular size (around 74 megadaltons) were responsible for the resistance pattern transferred and were maintained with high stability in the hosts.     

R plasmids in environmental Vibrio cholerae non-O1 strains.

The occurrence of drug resistance and its plasmid-mediated transferability was investigated in 140 environmental strains of Vibrio cholerae non-O1 and 6 strains of Vibrio cholerae, both O1 and non-O1, of clinical origin. Of the 146 strains tested, 93% were resistant to at least one drug and 74% were resistant to two or more antibiotics. The O1 strains were susceptible to all antibiotics used. A total of 26 of 28 selected resistant wild strains carried R plasmids that were transferable by intraspecific and intergeneric matings. The most common transmissible R factor determined resistance to ampicillin, amoxicillin, and sulfanilamide (30%), followed by resistance to ampicillin and amoxicillin (13%) and resistance to ampicillin, amoxicillin, phosphomycin, and sulfanilamide (9%). Comparison of the three methods of plasmid analysis showed that the method of Birnboim and Doly (Nucleic Acids Res. 7:1513-1523, 1979) without EDTA and lysozyme was optimal for isolation of both large and small plasmids in environmental V. cholerae strains. Most strains harbored more than one plasmid, and the molecular sizes ranged from 1.1 to 74.8 megadaltons. The plasmids of high molecular size (around 74 megadaltons) were responsible for the resistance pattern transferred and were maintained with high stability in the hosts.     

Molecular analysis of the rstR and orfU genes of the CTX prophages integrated in the small chromosomes of environmental Vibrio cholerae non-O1, non-O139 strains

The ctxAB genes encoding cholera toxin, reside in the genome of a filamentous bacteriophage CTXphi. The presence of CTX prophage in non-epidemic environmental Vibrio cholerae strains is rare. The CTX prophage, the lysogenic form of CTXphi 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.     

Molecular analysis of Vibrio cholerae O1, O139, non-O1, and non-O139 strains: clonal relationships between clinical and environmental isolates

A total of 26 strains of Vibrio cholerae, including members of the O1, O139, and non-O1, non-O139 serogroups from both clinical and environmental sources, were examined for the presence of genes encoding cholera toxin (ctxA), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), hemolysin (hlyA), NAG-specific heat-stable toxin (st), toxin-coregulated pilus (tcpA), and outer membrane protein (ompU), for genomic organization, and for the presence of the regulatory protein genes tcpI and toxR in order to determine relationships between epidemic serotypes and sources of isolation. While 22 of the 26 strains were hemolytic on 5% sheep blood nutrient agar, all strains were PCR positive for hlyA, the hemolysin gene. When multiplex PCR was used, all serogroup O1 and O139 strains were positive for tcpA, ompU, and tcpI. All O1 and O139 strains except one O1 strain and one O139 strain were positive for the ctxA, zot, and ace genes. Also, O1 strain VO3 was negative for the zot gene. All of the non-O1, non-O139 strains were negative for the ctxA, zot, ace, tcpA, and tcpI genes, and all of the non-O1, non-O139 strains except strain VO26 were negative for ompU. All of the strains except non-O1, non-O139 strain VO22 were PCR positive for the gene encoding the central regulatory protein, toxR. All V. cholerae strains were negative for the NAG-specific st gene. Of the nine non-ctx-producing strains of V. cholerae, only one, non-O1, non-O139 strain VO24, caused fluid accumulation in the rabbit ileal loop assay. The other eight strains, including an O1 strain, an O139 strain, and six non-O1, non-O139 strains, regardless of the source of isolation, caused fluid accumulation after two to five serial passages through the rabbit gut. Culture filtrates of all non-cholera-toxigenic strains grown in AKI media also caused fluid accumulation, suggesting that a new toxin was produced in AKI medium by these strains. Studies of clonality performed by using enterobacterial repetitive intergenic consensus sequence PCR, Box element PCR, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE) collectively indicated that the V. cholerae O1 and O139 strains had a clonal origin, whereas the non-O1, non-O139 strains belonged to different clones. The clinical isolates closely resembled environmental isolates in their genomic patterns. Overall, there was an excellent correlation among the results of the PCR, AFLP, and PFGE analyses, and individual strains derived from clinical and environmental sources produced similar fingerprint patterns. From the results of this study, we concluded that the non-cholera-toxin-producing strains of V. cholerae, whether of clinical or environmental origin, possess the ability to produce a new secretogenic toxin that is entirely different from the toxin produced by toxigenic V. cholerae O1 and O139 strains. We also concluded that the aquatic environment is a reservoir for V. cholerae O1, O139, non-O1, and non-O139 serogroup strains.     

Molecular Analysis of Vibrio cholerae O1, O139, non-O1, and non-O139 Strains: Clonal Relationships between Clinical and Environmental Isolates

A total of 26 strains of Vibrio cholerae, including members of the O1, O139, and non-O1, non-O139 serogroups from both clinical and environmental sources, were examined for the presence of genes encoding cholera toxin (ctxA), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), hemolysin (hlyA), NAG-specific heat-stable toxin (st), toxin-coregulated pilus (tcpA), and outer membrane protein (ompU), for genomic organization, and for the presence of the regulatory protein genes tcpI and toxR in order to determine relationships between epidemic serotypes and sources of isolation. While 22 of the 26 strains were hemolytic on 5% sheep blood nutrient agar, all strains were PCR positive for hlyA, the hemolysin gene. When multiplex PCR was used, all serogroup O1 and O139 strains were positive for tcpA, ompU, and tcpI. All O1 and O139 strains except one O1 strain and one O139 strain were positive for the ctxA, zot, and ace genes. Also, O1 strain VO3 was negative for the zot gene. All of the non-O1, non-O139 strains were negative for the ctxA, zot, ace, tcpA, and tcpI genes, and all of the non-O1, non-O139 strains except strain VO26 were negative for ompU. All of the strains except non-O1, non-O139 strain VO22 were PCR positive for the gene encoding the central regulatory protein, toxR. All V. cholerae strains were negative for the NAG-specific st gene. Of the nine non-ctx-producing strains of V. cholerae, only one, non-O1, non-O139 strain VO24, caused fluid accumulation in the rabbit ileal loop assay. The other eight strains, including an O1 strain, an O139 strain, and six non-O1, non-O139 strains, regardless of the source of isolation, caused fluid accumulation after two to five serial passages through the rabbit gut. Culture filtrates of all non-cholera-toxigenic strains grown in AKI media also caused fluid accumulation, suggesting that a new toxin was produced in AKI medium by these strains. Studies of clonality performed by using enterobacterial repetitive intergenic consensus sequence PCR, Box element PCR, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE) collectively indicated that the V. cholerae O1 and O139 strains had a clonal origin, whereas the non-O1, non-O139 strains belonged to different clones. The clinical isolates closely resembled environmental isolates in their genomic patterns. Overall, there was an excellent correlation among the results of the PCR, AFLP, and PFGE analyses, and individual strains derived from clinical and environmental sources produced similar fingerprint patterns. From the results of this study, we concluded that the non-cholera-toxin-producing strains of V. cholerae, whether of clinical or environmental origin, possess the ability to produce a new secretogenic toxin that is entirely different from the toxin produced by toxigenic V. cholerae O1 and O139 strains. We also concluded that the aquatic environment is a reservoir for V. cholerae O1, O139, non-O1, and non-O139 serogroup strains.     

Comparative analysis of cytotoxin, hemolysin, hemagglutinin and exocellular enzymes among clinical and environmental isolates of Vibrio cholerae O139 and non-O1, non-O139

The presence of three major virulence genes toxR, tcpA and ctxA as well as expression of several putative virulence factors were compared in 12 Vibrio cholerae O139 and non-O1,non-O139 strains of clinical and environmental origin. All the strains possessed the gene encoding the regulatory protein TOXR. None of the non-O1, non-O139 strains as well as one of the O139 environmental strains carried the genes for ctxA and tcpA. Statistically significant differences in hemagglutinin and hemolysin production were observed amongst the strains depending on the source of their isolation. Expression of extracellular enzymes such as protease, elastase, neuraminidase, phospholipase A and phospholipase C, however, did not vary significantly from the groups of strains isolated from different sources.     

Incidence and survival of non-O157 verocytotoxigenic Escherichia coli in soil

Aims: This study estimated the incidence of non‐O157 verocytotoxigenic Escherichia coli (VTEC) in farm pasture soils and investigated the survival of non‐O157 VTEC in clay and sandy loam soils. Methods and Results: Twenty farms were tested over a 12‐month period by sample enrichment in tryptone soya broth plus vancomycin, followed by PCR screening for the presence of vt1 and vt2 genes. Of the 600 soil samples, 162 (27%), across all farms, were found to contain vt1 and/or vt2 genes. The enrichment cultures from the 162 PCR‐positive samples were plated onto Chromocult tryptone bile X‐glucuronide agar (TBX), presumptive VTEC colonies recovered, confirmed as VTEC by PCR and serotyped. Samples of the two predominant soil types in Ireland (clay and sandy) were homogenized, characterized in terms of pH, boron, cobalt, copper, potassium, magnesium, manganese, phosphorus, zinc and organic matter content, inoculated with washed suspensions of eight non‐O157:H7 soil isolates and six bovine faecal isolates and stored at 10°C for up to 201 days. Inoculum survival rates were determined at regular intervals by recovering and plating soil samples on TBX. All inoculated non‐O157 serotypes had highest D‐values in the sandy loam soil with D‐values ranging from 50·26 to 75·60 days. The corresponding range in clay loam soils was 31·60–48·25 days. Conclusions: This study shows that non‐O157 VTEC occur widely and frequently in pasture soils and can persist in such environments for several months, with considerable opportunity for recycling through farm environments, and cattle, with clear potential for subsequent transmission into the human food chain. Significance and Impact of the Study: This is the first such study of non‐O157 VTEC in farm soils and found that these VTEC are frequent and persistent contaminants in farm soils. In light of recent epidemiological data, non‐O157 VTEC should be seen as an emerging risk to be controlled within the food chain.     

In vitro induction of transmissive resistance to tetracycline, chloramphenicol and ampicillin chloramphenicol in Vibrio cholera non-O1/non-O139 serogroups isolated within 1990-2005

Inducible character of resistance to tetracycline, chloramphenicol and ampicillin was investigated in 20 strains of Vibrio cholera non-O1/non-O139 serogroups isolated from inhabitants of Uzbekistan in 1990 (10 strains, ctx+) and in 2001 (5 strains, ctx-) and from inhabitants of Kalmykiya within 2003-2005 (5 strains, ctx-). Eight of the 20 isolates showed not only capacity for induction of the antibiotic resistance, but also its possible self transfer to Escherichia coli and reverse crosses in El Tor V. cholerae P-5879. It was shown that the effect of the antibacterial on the isolates phenotypic susceptibility could increase the resistance markers expression, when the genomes contained sites responsible for their expression, that required constant bacteriological control of the treatment efficacy and the use of the isolates antibioticograms for early replace of the inefficient drug by the efficient one. The prevalence of V. cholerae O1 and non-O1/non-O13 serogroups with multiple resistance to the antibacterial and the genetic potency for the antibiotic resistance development in the pathogen made difficult the choice of efficient drugs for prophylaxis and treatment of diseases caused by V. cholerae.     

Susceptibility of Escherichia coli O157 and non-O157 isolates to lactate

AIMS: To examine the effect of temperature on the antimicrobial efficacy of lactate and propionate against O157 and non-O157 Escherichia coli isolates. METHODS AND RESULTS: Lactate and, to a lesser extent, propionate effectively reduced viability at 37 degrees C. Ethanol enhanced this effect. Reducing the temperature to 20 or 5 degrees C caused an increase in survival in the presence of these organic acids with or without ethanol. At 20 degrees C the deltapH, membrane potential and intracellular lactate anion concentration were less than at 37 degrees C. CONCLUSIONS: The efficacy of lactate and propionate against E. coli O157 and non-O157 isolates is reduced at lower temperatures, perhaps due to the reduction in the deltapH, membrane potential and intracellular lactate anion concentration. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings suggest that the usefulness of organic acids as decontaminants for E. coli O157 is temperature dependent.     

TaqMan PCR for detection of Vibrio cholerae O1, O139, non-O1, and non-O139 in pure cultures, raw oysters, and synthetic seawater

Vibrio cholerae is recognized as a leading human waterborne pathogen. Traditional diagnostic testing for Vibrio is not always reliable, because this bacterium can enter a viable but nonculturable state. Therefore, nucleic acid-based tests have emerged as a useful alternative to traditional enrichment testing. In this article, a TaqMan PCR assay is presented for quantitative detection of V. cholerae in pure cultures, oysters, and synthetic seawater. Primers and probe were designed from the nonclassical hemolysin (hlyA) sequence of V. cholerae strains. This probe was applied to DNA from 60 bacterial strains comprising 21 genera. The TaqMan PCR assay was positive for all of the strains of V. cholerae tested and negative for all other species of Vibrio tested. In addition, none of the other genera tested was amplified with the TaqMan primers and probe used in this study. The results of the TaqMan PCR with raw oysters and spiked with V. cholerae serotypes O1 and O139 were comparable to those of pure cultures. The sensitivity of the assay was in the range of 6 to 8 CFU g(-1) and 10 CFU ml(-1) in spiked raw oyster and synthetic seawater samples, respectively. The total assay could be completed in 3 h. Quantification of the Vibrio cells was linear over at least 6 log units. The TaqMan probe and primer set developed in this study can be used as a rapid screening tool for the presence of V. cholerae in oysters and seawater without prior isolation and characterization of the bacteria by traditional microbiological methods.     

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.     

Isolation and characterization of a cytolysin produced by Vibrio cholerae serogroup non-O1

A thermolabile toxin (molecular weight, 52 711; isoelectric point, 8.65) produced by a clinical isolate of Vibrio cholerae serogroup non-O1 was cytotoxic for Y-1 mouse adrenal cells and Chinese hamster ovary cells. The toxin lysed rabbit red blood cells and produced a hemorrhagic zone in rabbit skin. When injected intravenously into adult mice, the cytolysin was rapidly lethal and caused fluid accumulation in both 5- and 18-h rabbit ileal loops. Strains of V. cholerae that produced cytolysin but no cholerae enterotoxin were able to cause fluid accumulation in rabbit intestinal loops.