Molecular characterisation of Vibrio cholerae O1 strains carrying an SXT/R391-like element from cholera outbreaks in Kenya: 1994-2007

Background  

Over the last decade, cholera outbreaks in parts of Kenya have become common. Although a number of recent studies describe the epidemiology of cholera in Kenya, there is pauCity of information concerning the diversity and occurrence of mobile genetic elements in Vibrio cholerae strains implicated in these outbreaks. A total of 65 Vibrio cholerae O1 El Tor serotype Inaba isolated between 1994 and 2007 from various outbreaks in Kenya were investigated for mobile genetic elements including integrons, transposons, the integrating conjugative elements (ICEs), conjugative plasmids and for their genotypic relatedness.     

Characterization of Vibrio cholerae O1 isolates responsible for cholera outbreaks in Kenya between 1975 and 2017

Kenya is endemic for cholera with different waves of outbreaks having been documented since 1971. In recent years, new variants of Vibrio cholerae O1 have emerged and have replaced most of the traditional El Tor biotype globally. These strains also appear to have increased virulence, and it is important to describe and document their phenotypic and genotypic traits. This study characterized 146 V. cholerae O1 isolates from cholera outbreaks that occurred in Kenya between 1975 and 2017. Our study reports that the 1975–1984 strains had typical classical or El Tor biotype characters. New variants of V. cholerae O1 having traits of both classical and El Tor biotypes were observed from 2007 with all strains isolated between 2015 and 2017 being sensitive to polymyxin B and carrying both classical and El Tor type ctxB. All strains were resistant to Phage IV and harbored rstR, rtxC, hlyA, rtxA and tcpA genes specific for El Tor biotype indicating that the strains had an El Tor backbone. Pulsed field gel electrophoresis (PFGE) genotyping differentiated the isolates into 14 pulsotypes. The clustering also corresponded with the year of isolation signifying that the cholera outbreaks occurred as separate waves of different genetic fingerprints exhibiting different genotypic and phenotypic characteristics. The emergence and prevalence of V. cholerae O1 strains carrying El Tor type and classical type ctxB in Kenya are reported. These strains have replaced the typical El Tor biotype in Kenya and are potentially more virulent and easily transmitted within the population.     

Vibrio cholerae serogroups O1 and O139: susceptibility to antibiotics during 7th cholera pandemic

The review presents data on circulation of antibiotic resistant and susceptible strains of Vibrio cholerae serogroups O1 and O139 isolated from cholera patients and healthy persons as well as from the environment, in Asia, Africa, Australia, and Europe (including New Independent States) during 7th cholera pandemic.     

Conservation of cholera toxin gene in a strain of cholera toxin non-producing Vibrio cholerae O1

BT23, a Vibrio cholerae O1 El Tor isolate, possesses the cholera toxin (CT) gene as determined by PCR. However, CT was not detected in the culture medium by the reversed passive latex agglutination test, nor in the whole cell lysate as examined by Western blotting. The toxin-coregulated pilus (TCP) was not detected by Western blotting. This suggests the presence of defects in the regulatory cascade. toxR, toxS and toxT, members of the regulatory cascade, were examined by PCR. toxR and toxS were conserved but toxT was not. CT and TCP production was complemented by transformation of toxT. The lack of toxT was suspected to be the cause of the undetectable production of CT in strain BT23.     

Lysophospholipase L2 of Vibrio cholerae O1 affects cholera toxin production

Abstract The implication in cholera toxin (CT) production of the newly identified gene, lypA , that encodes the lysophospholipase L₂ of Vibrio cholerae , was investigated. Introduction of lypA into the V. cholerae O1 mutant (NF404), which has a Tn5-insertion in lypA and has lost CT as well as haemolysin production, restored the lysophospholipase activity and CT production but not the haemolytic activity. Inactivation of the lypA gene of the wild-type strain by chromosomal integration of a plasmid containing a portion of the lypA gene decreased the lysophospholipase L₂ activity and the production of CT but not the haemolytic activity. Furthermore, constructed mutants of El Tor-biotype and Classical-biotype strains which have a defective lypA failed to produce CT and exhibited decreased enterotoxicity in the ligated rabbit ileal loop test. These results suggest that lypA is possibly required for the expression of CT and may play a role in pathogenicity of V. cholerae .     

Structure and arrangement of the cholera toxin genes in Vibrio cholerae O139

Abstract The sequence of the ctxB gene encoding the B subunit of cholera toxin has been determined for a strain of Vibrio cholerae of the novel O139 serotype associated with recent outbreaks of severe cholera throughout South-East Asia and found to be identical to the ctxB gene in V. cholerae O1 of the E1 Tor biotype. Analyses by Southern hybridization and PCR showed that all strains of the O139 serotype V. cholerae tested carried cholera toxin genes and other gene associated with a virulence cassette DNA region at two loci identical or homologous to those identified in the Classical rather than the E1 Tor biotype of V. cholerae serotype O1 although these loci in O139 could reside on restriction fragments of variable size.     

Seasonal cholera caused by Vibrio cholerae serogroups O1 and O139 in the coastal aquatic environment of Bangladesh

Since Vibrio cholerae O139 first appeared in 1992, both O1 El Tor and O139 have been recognized as the epidemic serogroups, although their geographic distribution, endemicity, and reservoir are not fully understood. To address this lack of information, a study of the epidemiology and ecology of V. cholerae O1 and O139 was carried out in two coastal areas, Bakerganj and Mathbaria, Bangladesh, where cholera occurs seasonally. The results of a biweekly clinical study (January 2004 to May 2005), employing culture methods, and of an ecological study (monthly in Bakerganj and biweekly in Mathbaria from March 2004 to May 2005), employing direct and enrichment culture, colony blot hybridization, and direct fluorescent-antibody methods, showed that cholera is endemic in both Bakerganj and Mathbaria and that V. cholerae O1, O139, and non-O1/non-O139 are autochthonous to the aquatic environment. Although V. cholerae O1 and O139 were isolated from both areas, most noteworthy was the isolation of V. cholerae O139 in March, July, and September 2004 in Mathbaria, where seasonal cholera was clinically linked only to V. cholerae O1. In Mathbaria, V. cholerae O139 emerged as the sole cause of a significant outbreak of cholera in March 2005. V. cholerae O1 reemerged clinically in April 2005 and established dominance over V. cholerae O139, continuing to cause cholera in Mathbaria. In conclusion, the epidemic potential and coastal aquatic reservoir for V. cholerae O139 have been demonstrated. Based on the results of this study, the coastal ecosystem of the Bay of Bengal is concluded to be a significant reservoir for the epidemic serogroups of V. cholerae.     

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.     

Cholera toxin gene-positive Vibrio cholerae O1 Ogawa and Inaba strains produce the new cholera toxin

Abstract Two strains of cholera toxin (CT) gene-positive Vibrio cholerae O1, Ogawa, isolated from patients with diarrhoea and the hypertoxigenic V. cholerae O1, Inaba (569B), were found to produce the new cholera toxin that has earlier been demonstrated to be elaborated by CT gene-negative human and environmental isolates of V. cholerae O1. The CT gene-positive strains produce the new cholera toxin simultaneously with CT, indicating that they contain the gene coding for the new cholera toxin in addition to that of CT.     

Detection of heat-stable enterotoxin in a cholera toxin gene-positive strain of Vibrio cholerae O1.

DNA colony hybridization with a polynucleotide clonal DNA probe for heat-stable enterotoxin of Vibrio cholerae non-O1 (NAG-ST) was used to screen 197 isolates of V. cholerae O1. Under stringent hybridizing and washing conditions, one strain (GP156) reacted with the probe. The concentrated supernatant from V. cholerae O1 GP156, heated at 100 degrees C for 5 min, elicited fluid accumulation in the suckling mice and that could be completely neutralized by an anti-NAG-ST monoclonal antibody (mAb2F). The preparation from V. cholerae O1 GP156 also inhibited the binding of mAb2F to NAG-ST in a competitive ELISA. V. cholerae O1 GP156 was confirmed to possess a gene encoding cholera toxin (CT). These results indicate that a heat-stable enterotoxin is produced by certain strains of CT-producing V. cholerae O1.     

Viability of the nonculturable Vibrio cholerae O1 and O139

Vibrio cholerae is capable of transforming into a viable but nonculturable (VBNC) state, and, in doing so, undergoes alteration in cell morphology. In the study reported here, Vibrio cholerae O1 and O139 cells were maintained in laboratory microcosms prepared with 1% Instant Ocean and incubated at 4 degrees C, i.e., conditions which induce the VBNC state. Cells were fixed at different stages during entry into the VBNC state and, when no growth was detectable on solid or in liquid media, the ultrastructure of these cells was examined, using both transmission and scanning electron microscopy. As shown in earlier studies, the cells became smaller in size and changed from rod to ovoid or coccoid morphology, with the central region of the cells becoming compressed and surrounded by denser cytoplasm. Because the coccoid morphology, indicative of the VBNC state is common for Vibrio cholerae in the natural environment, as well as in starved cells (Baker et al., 1983; Hood et al., 1986) viability of the coccoid, viable but nonculturable cell was investigated. The percentage of coccoid (VBNC) cells showing metabolic activity and retention of membrane integrity was monitored using direct fluorescence staining (LIVE/DEAD BacLight Bacterial Viability kit), with 75 to 90% of the viable but nonculturable coccoid cells found to be metabolically active by this test. Furthermore, the proportion of actively respiring cells, using the redox dye, 5-cyano-2, 3-ditolyl tetrazolium chloride (CTC), relative to total cells, the latter determined by DAPI staining, ranged from 10 to 50%. VBNC coccoid cells retained the antigenic determinants of Vibrio cholerae O1 and O139, respectively, evidenced by positive reaction with monoclonal fluorescent antibody. Viability was further established by susceptibility of the VBNC cells to chlorine, copper sulfate, zinc sulfate, and formaldehyde. Since retention of cell membrane integrity is a determining characteristic of viable cells, DNA was extracted from VBNC cells in microcosms maintained for two months and for one year. Conservation of cholera toxin and toxin-associated genes, ctxA, toxR, tcpA, and zot in chromosomal DNA of VBNC cells was demonstrated using PCR and employing specific primers. It is concluded that not only do VBNC V cholerae O1 and O139 retain viability up to one year, but genes associated with pathogenicity are retained, along with chromosomal integrity.     

Phenotypic and Genotypic Characterization Vibrio cholerae O139 of Clinical and Aquatic Isolates in China

To enhance the understanding of epidemiological impact of environmental Vibrio cholerae O139 strains, we characterized 10 clinical and 20 environmental isolates collected from human clinical samples and Pear River estuary during 2006 to 2008. Isolates were tested by PCR for eight virulence genes: cholera toxin (ctxA), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), hemolysin (hlyA), NAG-specific heat-stable toxin (st), toxin-coregulated pilus (tcpA), outer membrane protein (ompU), and regulatory protein genes (tcpI). Genetic relatedness was assessed by pulsed-field gel electrophoresis (PFGE), and antibiotic susceptibility was determined using disk diffusion. Seven of eight virulence markers were detected in six clinical isolates and one environmental isolate. One clinical and one environmental isolate were positive for six virulence markers. 60% clinical isolates showed multi-drug resistance to tetracycline (TET), Nalidixic acid (NAL), chloramphenicol (CHL), and ampicillin (AMP), 70% were resistant to Trimethoprim + Sulfamethoxazole (SXT), while only 35% environmental strains were resistant to SXT. PFGE analysis revealed that the isolates in this study were formed three clusters. Cluster III was more related to strains from diarrheal patients than the strains in other clusters. Different from the clinical strains, most environmental strains lacked CTX and TCP gene clusters. Most environmental strains possess a single resistance profile, while most clinical isolates show multidrug resistant. PFGE analysis indicated the cluster III has more possibility to become a potential pathogenic clonal cluster.     

Variation in epitopes of the B subunit of El Tor and classical biotype Vibrio cholerae O1 cholera toxin

Variation in epitopes of the B subunit of cholera toxin (CT-B) produced by strains of El Tor and classical biotype Vibrio cholerae O1 was examined using monoclonal antibodies prepared to V. cholerae 569B CT. CT-B epitopes were markedly conserved for V. cholerae classical biotypes. In contrast, epitope variation was observed for El Tor biotypes, which produced both a classical-like CT-B and a unique CT-B lacking at least one epitope common to 569B CT-B. The missing epitope was located outside the GM1 ganglioside-binding site. From results of the study reported here, genetic divergence is exhibited in the El Tor biotype CT-B versus classical CT-B. Furthermore, at least five unique epitopes of V. cholerae 569B CT-B can be defined.     

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).     

Characterization of enteropathogenic Vibrio cholerae non O1/O139 isolated in Uzbekistan

The results of the serotyping of 244 V. cholerae non O1/O139 cultures isolated from patients in Uzbekistan in 2000 and 2001 are presented. All isolates were studied by the method of molecular probing and in the polymerase chain reaction for the presence of virulence genes and for sensitivity to phages ctx+, ctx- and hemolytic activity. The use of monoreceptor O-sera O2-O83 made it possible to determine vibrios of 32 serogroups with the dominating role in the etiology of acute enteric diseases belonging to serogroups O18, O62, O82, O37. Genes ctx AB were detected in none of the isolates, 5 of them contained gene tcp A. A group of cultures, sensitive to phage ctx+ and belonging mainly to enteropathogenic serogroups, was detected.     

Histopathological changes in experimental cholera with a non toxigenic non- O1 non-O139 Vibrio cholerae strain isolated from Kolkata, India

This study was conducted to understand the pathophysiological changes in experimental rabbit ileal loop model using the Vibrio cholerae strain non-O1non-O139, isolated as sole pathogen from clinically diagnosed cholera patients in Kolkata. Significant amount of haemorrhagic fluid accumulation was observed in all the test loops of rabbit model where the strain of V.cholerae was inoculated as compared to control loops. Microscopic examination of the accumulated fluid showed the presence of erythrocytes and pus cells. Histology revealed structural alteration of the villous epithelium with inflammatory cells infiltration in all the layers of the gut mucosa including the nerve plexus region. Preliminary observation with a haemagglutinin protease extracted from the non-O1 non-O139 strain, was also studied in different concentrations in the same animal model which showed similar type of macroscopic and microscopic response in the ileal loops as seen with the original strain. The results highlight that along with other pathways, inflammatory cells and the enteric neurons have an important role in the pathophysiology of diarrhoea and the isolated protease may be the probable virulence factor in initiating the disease process in this non-O1non-O139 strain induced cholera.     

Comparative Characterization of Vibrio cholerae O1 from Five Sub-Saharan African Countries Using Various Phenotypic and Genotypic Techniques

We used standardized methodologies to characterize Vibrio cholerae O1 isolates from Guinea, Democratic Republic of the Congo (DRC), Togo, Côte d’Ivoire and Mozambique. We investigated 257 human isolates collected in 2010 to 2013. DRC isolates serotyped O1 Inaba, while isolates from other countries serotyped O1 Ogawa. All isolates were biotype El Tor and positive for cholera toxin. All isolates showed multidrug resistance but lacked ciprofloxacin resistance. Antimicrobial susceptibility profiles of isolates varied between countries. In particular, the susceptibility profile of isolates from Mozambique (East-Africa) included resistance to ceftriaxone and was distinctly different to the susceptibility profiles of isolates from countries located in West- and Central-Africa. Molecular subtyping of isolates using pulsed-field gel electrophoresis (PFGE) analysis showed a complex relationship among isolates. Some PFGE patterns were unique to particular countries and clustered by country; while other PFGE patterns were shared by isolates from multiple countries, indicating that the same genetic lineage is present in multiple countries. Our data add to a better understanding of cholera epidemiology in Africa.     

Variation in epitopes of the B subunit of Vibrio cholerae non-O1 and Vibrio mimicus cholera toxins.

Monoclonal antibodies reacting with the B subunit of Vibrio cholerae O1 strain 569B cholera toxin (CT-B) were used to identify unique and common epitopes of V. cholerae non-O1 and Vibrio mimicus CT-B. Vibrio cholerae non-O1 strains produced CT-B showing three monoclonal antibody reaction patterns (epitypes), which corresponded with epitypes described previously for V. cholerae O1 classical biotype CT-B (CT1), El Tor biotype CT-B (CT2), and a unique V. cholerae non-O1 CT-B (CT3), which lacked an epitope located in or near the GM1 ganglioside binding site of 569B CT-B. Vibrio mimicus CT-B was immunologically indistinguishable from 569B CT-B. These and previous results define six epitopes on 569B CT-B, and a fourth epitope in or near the GM1 ganglioside binding site.     

Vibrio cholerae and cholera: out of the water and into the host

The facultative human pathogen Vibrio cholerae can be isolated from estuarine and aquatic environments. V. cholerae is well recognized and extensively studied as the causative agent of the human intestinal disease cholera. In former centuries cholera was a permanent threat even to the highly developed populations of Europe, North America, and the northern part of Asia. Today, cholera still remains a burden mainly for underdeveloped countries, which cannot afford to establish or to maintain necessary hygienic and medical facilities. Especially in these environments, cholera is responsible for significant mortality and economic damage. During the last three decades, intensive research has been undertaken to unravel the virulence properties and to study the epidemiology of this significant human pathogen. More recently, researchers have been elucidating the environmental lifestyle of V. cholerae. This review provides an overview of the current knowledge of both the host- and environment-specific physiological attributes of V. cholerae.