Factors associated with virulence and survival in environmental and clinical isolates of Vibrio cholerae O1 and non O1 in Romania

Four hundred ninety seven strains of Vibrio cholerae selected from isolates in Romania in the last decade 1990-1999 were investigated for antibiotic resistance and for classical and putative virulence factors. V. cholerae O1 strains predominated in clinical cases and non O1 strains in the environment, excepting in 1992 when non O1 strains were frequent in clinical and environmental sources. V. cholerae O1 strains previously susceptible to tetracycline acquired clinically significant resistance to this drug during 1993-1994, but this trend was reversed in 1995, following the introduction of nalidixic acid in cholera treatment in 1994. V. cholerae O1 and non O1 clinical isolates acquired simultaneous resistance to the vibriostatic agent O/129 and cotrimoxazole during 1994-1995. High levels of intrinsic resistance to multiple antibiotics were exhibited by all strains examined. The presence of cholera toxin (CT) was concentrated in clinical V. cholerae O1 strains and was substituted in clinical non O1 strains by four putative virulence markers (Kanagawa haemolysin, slime, lipase, and colonial opacity). Colonial opacity (30%) was present only in clinical isolates of V. cholerae non O1. Pigmentogenesis (11.7%) has present only in environmental sources. Antibioresistance profiles differ for V. cholerae O1 and non O1 strains with respect to their source of isolation. This aspect may imply a role in virulence and survival of V. cholerae in the natural environment where they may serve as a reservoir of virulence and multiple drug resistance genes.     

Lecithinase activity of Vibrio cholerae and of vibrios not agglutinated by O-cholera serum

The lecithinase activities of 187 V. cholerae strains differing in their virulence and 70 cultures of NAG vibrios of different origin varied in all experimental groups. The level of lecithinase activity in the groups of V. cholerae strains having low virulence, or no virulence at all, was considerably higher than in virulent strains. NAG vibrios isolated from diarrhea patients and from samples of lake and pond water did not differ in the activity of this enzyme.     

Detection of cholera toxin by a highly sensitive bead-enzyme linked immunosorbent assay.

A bead-enzyme linked immunosorbent assay (bead-ELISA) for detection and quantification of cholera toxin (CT) in broth cultures of Vibrio cholerae O1 has been developed. Under optimal buffer and pH conditions the bead-ELISA could consistently detect 40 pg/ml of CT. None of the ingredients of commonly used media for in vitro culture of V. cholerae O1 hindered the performance of the bead-ELISA. Evaluation of the sensitivity and specificity of the bead-ELISA against the commonly used reversed passive latex agglutination (RPLA) test for detection of CT was performed using a collection of 239 strains of V. cholerae O1 (including both biotypes and serotypes) which were examined by a gene probe encoding for the A1 subunit of CT. Although both the assays were highly specific, the bead-ELISA was more sensitive than the RPLA. Quantification of CT by the bead-ELISA revealed that the concentration of CT produced by the strains of V. cholerae O1 which were negative by the RPLA was lower than 1 ng/ml and therefore below the minimum detection ability of the RPLA. The bead-ELISA is a simple, specific and highly sensitive assay for routine detection of CT and is recommended for routine use in clinical microbiology laboratories.     

多重PCR方法检测霍乱弧菌的研究

霍乱弧菌是霍乱的病原体,可以分为O1群、O139群和非O1/非O139群。O1群和O139群霍乱弧菌产生的霍乱肠毒素(也称霍乱毒素)是产生霍乱的主要原因,也只有O1群和O139群霍乱弧菌可引起霍乱。其他群的霍乱弧菌毒性不高,但在食品中也不允许被检出。实验以霍乱胶原酶基因和霍乱毒素基因为目的基因,试图建立一种PCR方法对霍乱弧菌进行检测研究,结果表明此方法可以用于食品中的霍乱弧菌检测。     

Analysis of toxin genetic determinants of Vibrio cholerae virulence cassette and neuraminidase with DNA probes

V. cholerae strains of different origin have been studied for the presence of cholera toxin genes (vct), the proximal part of the virulence cassette including genes zot, ace and orfU, as well as neuraminidase genes (neu), in their genomes with the use of molecular DNA probes. The possibility, in principle, for some strains to lose only a part of their virulence cassette (gene vct), while retaining its proximal part has been shown. In most cases such strains are isolated from patients with diarrhea of different severity and may probably play some etiological role, provided that the expression of the genes of additional toxins of the virulence cassette occurs. The gene expressing neuraminidase which facilitates the penetration of cholera toxin into the epithelial cells of the intestine is always present in vct+ strains and may be absent in vct- strains. The absence of genetic relationship between neuraminidases in V. cholerae O139 and V. cholerae O1 and non-O1 (non-O139) has been confirmed. The problems in connection with the integration and deletion of genetic determinants of V. cholerae virulence factors are discussed.     

Distribution of genes for virulence and ecological fitness among diverse Vibrio cholerae population in a cholera endemic area: tracking the evolution of pathogenic strains

The pathogenic strains of Vibrio cholerae that cause acute enteric infections in humans are derived from environmental nonpathogenic strains. To track the evolution of pathogenic V. cholerae and identify potential precursors of new pathogenic strains, we analyzed 324 environmental or clinical V. cholerae isolates for the presence of diverse genes involved in virulence or ecological fitness. Of 251 environmental non-O1, non-O139 strains tested, 10 (3.9%) carried the toxin coregulated pilus (TCP) pathogenicity island encoding TCPs, and the CTX prophage encoding cholera toxin, whereas another 10 isolates carried the TCP island alone, and were susceptible to transduction with CTX phage. Most V. cholerae O1 and O139 strains carried these two major virulence determinants, as well as the Vibrio seventh pandemic islands (VSP-1 and VSP-2), whereas 23 (9.1%) non-O1, non-O139 strains carried several VSP island genes, but none carried a complete VSP island. Conversely, 30 (11.9%) non-O1, non-O139 strains carried type III secretion system (TTSS) genes, but none of 63 V. cholerae O1 or O139 strains tested were positive for TTSS. Thus, the distribution of major virulence genes in the non-O1, non-O139 serogroups of V. cholerae is largely different from that of the O1 or O139 serogroups. However, the prevalence of putative accessory virulence genes (mshA, hlyA, and RTX) was similar in all strains, with the mshA being most prevalent (98.8%) followed by RTX genes (96.2%) and hlyA (94.6%), supporting more recent assumptions that these genes imparts increased environmental fitness. Since all pathogenic strains retain these genes, the epidemiological success of the strains presumably depends on their environmental persistence in addition to the ability to produce major virulence factors. Potential precursors of new pathogenic strains would thus require to assemble a combination of genes for both ecological fitness and virulence to attain epidemiological predominance.     

Vibrio cholerae O139 produces a protease which is indistinguishable from the haemagglutinin/protease of Vibrio cholerae O1 and non-O1

Abstract Haemaglutinin/protease (HA/P) is one of the virulence factors of Vibrio cholerae O1 and pathogenic strains of V. cholerae non-O1. In this study, we examined protease activity of a new serogroup of Vibrio cholerae recently designated as O139 synonym Bengal. The protease activity was produced by all eight isolates of V. cholerae O139 from Bangladeshi patients. Purification and partial characterization of the protease from V. cholerae O139 demonstrated the purified protease (O139-P) was indistinguishable from that previously reported for HA/P of V. cholerae non-O1 (NAG-HA/P) and V. cholerae O1 (Vc-HA/P). These results prove that V. cholerae O139 produces a protease belonging to solHA/P, and suggest that the protease is another virulence factor found in newly emerged V. cholerae O139, as in V. cholerae O1.     

多重实时PCR检测产毒素性霍乱弧菌和副溶血弧菌

设计引物和探针,优化多重实时PCR条件,以同时检测霍乱弧菌霍乱毒素基因ctxA、副溶血弧菌种特异性基因gyrB和耐热肠毒素基因tdh。该多重实时PCR方法检测产毒素性的O1群(3株)和O139群(44株)霍乱弧菌菌株、不产毒素的O1群(12株)和O139群(6株)及非O1非O139群(7株)霍乱弧菌菌株的ctxA,阳性和阴性结果与普通PCR检测结果100%符合;检测副溶血弧菌种特异性gyrB,116株副溶血弧菌均阳性,而9株其它细菌和72株霍乱弧菌均阴性;检测tdh的阳性和阴性结果也与普通PCR结果完全一致。另外还建立了检测副溶血弧菌菌株trh1和trh2的单重实时PCR方法。     

Virulence genes in environmental strains of Vibrio cholerae

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

Identification of virulence factors in Vibrio cholerae isolated from Iraq during the 2007-2009 outbreak

Thousands of people were infected with Vibrio cholerae during the outbreak in Iraq in 2007-2009. Vibrio cholerae was shown to be variable in its content of virulence determinants and in its antibiotic sensitivity. This study was designed to isolate and characterize clinical and environmental V.?cholerae isolates and to determine antibiotic sensitivity, enzyme and toxin production, and the presence of virulence genes. Eighty clinical and five environmental bacterial isolates were collected and diagnosed by subjecting them to microscopic, biochemical, serological, and molecular analysis. The results revealed that 55% of clinical isolates belonged to the Inaba serotype, 32.5% to the Ogawa serotypes, and 12.5% to the Non-O1 serotype. All environmental V.?cholerae isolates belonged to the Non-O1 serotype. All environmental isolates were sensitive to all examined antimicrobial agents, while all clinical isolates showed a high sensitivity (100%) to ampicillin, gentamicin, cephalothin, tetracycline, erythromycin, and ciprofloxacin, and a high resistance (97.5%) to co-trimoxazole, nalidixic acid, and chloramphenicol. It was found that all V.?cholerae (O1) isolates were resistant to the Vibrio static O129 and all Non-O1 V.?cholerae isolates were sensitive to the Vibrio static O129. All clinical and environmental isolates produced hemolysin (100%) and lecithinase (100%), while they showed various production rates of protease (90% of clinical and 60% of environmental) and lipase (50% of clinical and 20% of environmental). The ompW gene was amplified in all the clinical and environmental V.?cholerae isolates, but not in other related and nonrelated bacteria. Multiplex PCR analysis showed that the toxR gene was amplified in all clinical and environmental isolates, while ctxA, ctxB, tcpA genes were amplified only in clinical (O1) isolates. This study indicates the differences in the production of some enzymes and toxins and in the content of virulence genes between clinical and environmental isolates in Iraq during the outbreak (2007-2009).     

Enterobacterial repetitive intergenic consensus sequences and the PCR to generate fingerprints of genomic DNAs from Vibrio cholerae O1, O139, and non-O1 strains.

Enterobacterial repetitive intergenic consensus (ERIC) sequence polymorphism was studied in Vibrio Cholerae strains isolated before and after the cholera epidemic in Brazil (in 1991), along with epidemic strains from Peru, Mexico, and India, by PCR. A total of 17 fingerprint patterns (FPs) were detected in the V. cholerae strains examined; 96.7% of the toxigenic V. cholerae O1 strains and 100% of the O139 serogroup strains were found to belong to the same FP group comprising four fragments (FP1). The nontoxigenic V. cholerae O1 also yielded four fragments but constituted a different FP group (FP2). A total of 15 different patterns were observed among the V. cholerae non-O1 strains. Two patterns were observed most frequently for V. cholerae non-01 strains, 25% of which have FP3, with five fragments, and 16.7% of which have FP4, with two fragments. Three fragments, 1.75, 0.79, and 0.5 kb, were found to be common to both toxigenic and nontoxigenic V. cholerae O1 strains as well as to group FP3, containing V. cholerae non-O1 strains. Two fragments of group FP3, 1.3 and 1.0 kb, were present in FP1 and FP2 respectively. The 0.5-kb fragment was common to all strains and serogroups of V. cholerae analyzed. It is concluded from the results of this study, based on DNA FPs of environmental isolates, that it is possible to detect an emerging virulent strain in a cholera-endemic region. ERIC-PCR constitutes a powerful tool for determination of the virulence potential of V. cholerae O1 strains isolated in surveillance programs and for molecular epidemiological investigations.     

01群与非01群0139型霍乱弧菌毒力的对比研究

非０１群０１３９型霍乱弧菌是近年引起南亚次大陆霍乱流行的新型病原体,将其与０１群霍乱弧菌的毒力特性进行对比研究对于了解其特性及研制相关的菌苗具有重要意义。本文报告了４株０１３９型霍乱弧菌与０１群霍乱弧菌菌株的对比测定结果。发现０１３９型霍乱弧菌与０１群霍乱弧菌有所不同,呈不透明的菌落形态,光学显微镜及电子显微镜检显示有荚膜的表型,在体内具有较高的繁殖能力并产生肠毒素,体内侵袭试验结果表明所有４株０     

The comparative characteristics of the pathogenic action of Vibrio cholerae of the non-O1 group O139 serovar and of the O1 group on the intestinal APUD system of suckling rabbits

The comparative study of the enteropathogenic action of V. cholerae strains of group non-O1, serovar O139, and group O1 with different virulence on the APUD system of the intestine of suckling rabbits after intraenteral infection revealed that V. cholerae of group non-O1 induced inflammatory changes in the intestine and the pronounced toxic lesion of parenchymal organs. This was accompanied by a decrease in the number of apudocytes and an increase in the functional tension of the APUD system. After the infection of the animals with V. cholerae of group O1 changes in the APUD system and internal organs directly depended on the virulence of the microbes and the infective dose.     

Attachment of Vibrio cholerae serogroup O1 to zooplankton and phytoplankton of Bangladesh waters

Vibrio cholerae serogroup O1, the causative agent of cholera, is capable of surviving in aquatic environments for extended periods and is considered an autochthonous species in estuarine and brackish waters. These environments contain numerous elements that may affect its ecology. The studies reported here examined physical interactions between V. cholerae O1 and natural plankton populations of a geographical region in Bangladesh where cholera is an endemic disease. Results showed that four of five clinical V. cholerae O1 strains and endogenous bacterial flora were attached preferentially to zooplankton molts (exuviae) rather than to whole specimens. One strain attached in approximately equal numbers to both exuviae and whole specimens. V. cholerae O1 also attached to several phytoplankton species. The results show that V. cholerae O1 can bind to diverse plankton species collected from an area where cholera is an endemic disease, with potentially significant effects on its ecology.     

A comparative analysis of genomes of virulent and avirulent strains of Vibrio cholerae O139

A comparative analysis of the genome of V. cholerae O139 strains isolated in Russia's territory from patients with cholera and from the environment showed essential differences in their structures. The genome of clinical strains possessed all tested genes associated with virulence (ctxAB, zot, ace, rstC, rtxA, hap, toxR and toxT) and the at-tRS site for the CTXp phage DNA integration. As for the O139 V. cholerae chromosome strains isolated from water, 70% of the studied genes (ctxAB, zot, ace, rstC, tcpA, and toxT) and the attRS sequence were not detected in them. A lack of the key virulence genes in O139-serogroup "water" vibrios, including genes of toxin-coregulated adhesion pili. (that are receptors for the CTXp phage), and of the attachment site of the above phage are indicative of that the O139 V. cholerae strains isolated from open water sources located in different Russia's regions are epidemically negligible.     

Survival of Vibrio cholerae and Escherichia coli in estuarine waters and sediments.

In in vitro estuarine water and sediment chambers, the survival of Vibrio cholerae and Escherichia coli was determined by plate counting and direct counting techniques. V. cholerae strains included environmental, clinical, and serotype O1 and non-O1 isolates, whereas E. coli strains included ATCC 25922 and a freshly cultured human isolate. Recovery of V. cholerae varied significantly with incubation temperature. Growth and extended periods of survival occurred in sterile sediments, sterile waters, and nonsterile waters, but not in nonsterile sediments. In contrast to V. cholerae, viable cells of E. coli decreased rapidly in both sterile and nonsterile estuarine waters. Direct counts revealed that E. coli cells were intact in the estuarine water, but attempts to resuscitate them were unsuccessful. The data suggest that V. cholerae survives better in estuarine waters than E. coli. The results may explain the recent observations that V. cholerae levels do not correlate well with fecal coliform concentrations in estuarine waters. Furthermore, the results add increasing evidence to support the theory that V. cholerae is an autochthonous bacterium in estuaries.     

Incidence and toxigenicity of Vibrio cholerae in a freshwater lake during the epidemic of cholera caused by serogroup O139 Bengal in Calcutta, India

Abstract The extent of contamination of a freshwater lake with Vibrio cholerae 0139 Bengal and the toxigenicity of all the V. cholerae isolates recovered during the period of the study were examined during and after an explosive outbreak of 0139 cholera in Calcutta. Strains biochemically characterized as V. cholerae could be isolated throughout the period of study examined from the freshwater lake samples. Most probable number of V. cholerae belonging to the 0139 serogroup in surface waters was 3 to 4 per 100 ml during major part of the study but isolation of this serogroup from sediment and plankton samples was infrequent. Of the total of 150 strains recovered, 23 (15.3%) agglutinated with the 0139 antiserum while the remaining belonged to the non-O1 non-O139 serogroups. None of the strains agglutinated with the O1 antiserum. All the 23 strains of V. cholerae O139 produced cholera toxin while 7.9% of the 127 non-O1 non-O139 strains also produced cholera toxin. Resistance to ampilicillin, furazolidone and streptomycin was encountered among strains belonging to both V. cholerae O139 and V. cholerae non-O1 non-O139 strains, but the percentage of resistant strains in the former was much higher than in the latter. During this cholera epidemic, possibly due to the introduction of large numbers of toxigenic V. cholerae such as the O139 serogroup, there was an increase in the number of toxigenic vibrios among the innocuous aquatic residents. This presumably occured through genetic exchange and, if substantiated, could play an important role in the re-emergence of epidemics.     

Electrophoretic mobility and immune response of outer membrane proteins of Vibrio cholerae O139

Abstract The outer membrane (OM) protein components of a Vibrio cholerae O1 and four V. cholerae O139 strains, collected from cholera patients, were analysed by SDS-PAGE. A protein of 69 kDa molecular mass was observed only when the OMPs were prepared from strains grown in synthetic broth. As a result of passage in the rabbit ileal loop (RIL), virulence was enhanced, and a protein component around 18 kDa of the V. cholerae O139 OM became the major protein component. On immunoblot analysis with rabbit antiserum against V. cholerae O139 OM, it was shown that, apart from the major protein component of V. cholerae O1 OM of around 45 kDa and that of V. cholerae O139 OM of around 38 kDa, all other minor protein components were cross-reactive between the two serogroups. In immunoblot assays with convalescent sera obtained from V. cholerae O139-infected patients, it was observed that in addition to the lipopolysaccharide (LPS)-induced antibody, only the 38 kDa major protein component elicited considerable levels of antibody in the pateint. Minor OM components of 18 kDa were detected in the immunoblot analysis by LPS-directed antibody, however, as the OM proteins are known to be associated with LPS.     

Attachment of Vibrio cholerae serogroup O1 to zooplankton and phytoplankton of Bangladesh waters.

Vibrio cholerae serogroup O1, the causative agent of cholera, is capable of surviving in aquatic environments for extended periods and is considered an autochthonous species in estuarine and brackish waters. These environments contain numerous elements that may affect its ecology. The studies reported here examined physical interactions between V. cholerae O1 and natural plankton populations of a geographical region in Bangladesh where cholera is an endemic disease. Results showed that four of five clinical V. cholerae O1 strains and endogenous bacterial flora were attached preferentially to zooplankton molts (exuviae) rather than to whole specimens. One strain attached in approximately equal numbers to both exuviae and whole specimens. V. cholerae O1 also attached to several phytoplankton species. The results show that V. cholerae O1 can bind to diverse plankton species collected from an area where cholera is an endemic disease, with potentially significant effects on its ecology.     

Bactericidal properties of hemo-cytolysin from Vibrio cholerae non O1 P-11702 strain in a panel of indicator cultures for detection of vibriocins

The influence of the preparation of hemo-cytolysin, obtained from V. cholerae non O1 strain P-11702 and inducing lysis of both red blood cells and V. cholerae cultures using a panel of indicator cultures for the detection of vibriocins, was studied. The set of indicator cultures contained 2 Shigella flexneri strains, 1 S. dysenteriae strain, 3 S. sonnei strains, 3 Escherichia coli strains and 2 V. cholerae strains, one of them being atypical. Hemo-cytolysin exhibited lytic activity with respect to S. dysenteriae, S. sonnei strains and 1 V. cholerae strain. i.e. to 4 out of 11 indicator strains. V. cholerae atypical strain proved to be resistant to the preparation in contrast to 33 V. cholerae typical strains, studied previously.