Emergence of Haitian variant genotype and altered drug susceptibility in Vibrio cholerae O1 El Tor-associated cholera outbreaks in Solapur, India.

It is evident from previous cholera epidemics/outbreaks in India, Africa and America that isolates of the seventh pandemic Vibrio cholerae El Tor (7PET) with Haitian cholera toxin (HCT) genotype were associated with increased mortality. The present study highlights the emergence of 7PET-HCT isolates causing two cholera outbreaks in Walsang and Wagdari (Solapur, India) in 2016. Molecular analyses revealed that 7PET strains from earlier outbreaks (2010 and 2012) were progenitors of the current 7PET-HCT isolates. Isolates from the 2016 outbreaks carried qnrVC and floR genes and showed reduced susceptibility to tetracycline, ciprofloxacin and azithromycin, drugs recommended by the World Health Organization (WHO) for the treatment of cholera. Remarkably, protein profiling and mass spectrometry revealed disappearance of the outer membrane protein U (OmpU) porin in 7PET-HCT isolates from the second outbreak in 2016. Downregulation of ompU gene expression was also confirmed at the transcriptional level. Strains with downregulated OmpU showed reduced minimum inhibitory concentrations (MICs) for polymyxin B, which is a pore-forming antimicrobial agent. A multipronged approach is of utmost importance to prevent further spread of circulating 7PET-HCT strains. There is a pressing need for the formulation and implementation of international policies to closely monitor the effective use of antibiotics in order to prevent the further rise and spread of antimicrobial resistance.

Dissemination of newly emerged polymyxin B sensitive Vibrio cholerae O1 containing Haitian-like genetic traits in different parts of India.

PURPOSE: Two natural epidemic biotypes of Vibrio cholerae O1, classical and El Tor, exhibit different patterns of sensitivity against the antimicrobial peptide polymyxin B. This difference in sensitivity has been one of the major markers in biotype classification system for several decades. A recent report regarding the emergence of polymyxin B-sensitive El Tor V. cholerae O1 in Kolkata has motivated us to track the spread of the strains containing this important trait, along with Haitian-like genetic content, in different parts of India. METHODOLOGY: We have collected 260 clinical V. cholerae O1 strains from 12 states in India and screened them for polymyxin B susceptibility. Genetic characterization was also performed to study the tcpA, ctxB and rtxA genotypes by allele-specific polymerase chain reaction (PCR) and nucleotide sequencing. RESULTS: Interestingly, 88.85 % of the isolates were found to be sensitive to polymyxin B. All of the states, with the exception of Assam, had polymyxin B-sensitive V. cholerae strains and complete replacement with this strain was found in eight of the states. However, from 2016 onwards, all the strains tested showed sensitivity to polymyxin B. Allele-specific PCR and sequencing confirmed that all strains possessed Haitian-like genetic traits. CONCLUSION: Polymyxin B-sensitive strains have begun to spread throughout India and may lead to the revision of the biotype classification. The dissemination of these new variant strains needs to be carefully monitored in different endemic populations through active holistic surveillance to understand their clinical and epidemiological consequences.

Catalases and PhoB/PhoR system independently contribute to oxidative stress resistance in Vibrio cholerae O1.

All cells are subjected to oxidative stress, a condition under which reactive oxygen species (ROS) production exceeds elimination. Bacterial defences against ROS include synthesis of antioxidant enzymes like peroxidases and catalases. Vibrio cholerae can produce two distinct catalases, KatB and KatG, which contribute to ROS homeostasis. In this study, we analysed the mechanism behind katG and katB expression in two V. cholerae O1 pandemic strains, O395 and N16961, of classical and El Tor biotypes, respectively. Both strains express these genes, especially at stationary phase. However, El Tor N16961 produces higher KatB and KatG levels and is much more resistant to peroxide challenge than the classical strain, confirming a direct relationship between catalase activity and oxidative stress resistance. Moreover, we showed that katG and katB expression levels depend on inorganic phosphate (Pi) availability, in contrast to other bacterial species. In N16961, katB and katG expression is reduced under Pi limitation relative to Pi abundance. Total catalase activity in N16961 and its phoB mutant cells was similar, independently of growth conditions, indicating that the PhoB/PhoR system is not required for katB and katG expression. However, N16961 cells from Pi-limited cultures were 50-100-fold more resistant to H2O2 challenge and accumulated less ROS than phoB mutant cells. Together, these findings suggest that, besides KatB and KatG, the PhoB/PhoR system is an important protective factor against ROS in V. cholerae N16961. They also corroborate previous results from our and other groups, suggesting that the PhoB/PhoR system is fundamental for V. cholerae biology.

Unique Clones of Vibrio cholerae O1 El Tor with Haitian Type ctxB Allele Implicated in the Recent Cholera Epidemics from Nigeria, Africa.

BACKGROUND AND OBJECTIVES: The antimicrobial susceptibility patterns and genetic characteristics of Vibrio cholerae O1, which is responsible for several cholera epidemics in Nigeria, are not reported in detail since 2007. In this study, we screened V. cholerae O1 El Tor biotype isolates from cholera cases and water samples from different states to investigate their phenotypic and genetic attributes with special reference to their clonality. RESULTS: All the V. cholerae O1 biotype El Tor isolates isolated during 2007-2013 were susceptible to fluoroquinolones and tetracycline, the drugs currently used in the treatment of cholera cases in Nigeria. Emergence of CT genotype 7 (Haitian type of ctxB allele) was predominantly seen among Ogawa serotype and the CT genotype 1 (classical ctxB allele) was mostly found in Inaba serotype. Overall, V. cholerae O1 from clinical and water samples were found to be closely related as determined by the pulsed-field gel electrophoresis. V. cholerae isolates from Abia, Kano and Bauchi were found to be genetically distinct from the other states of Nigeria. CONCLUSION: Fecal contamination of the water sources may be the possible source of the cholera infection. Combined prevalence of Haitian and classical ctxB alleles were detected in Ogawa and Inaba serotypes, respectively. This study further demonstrated that V. cholerae O1 with the ctxB has been emerged similar to the isolates reported in Haiti. Our findings suggest that the use of fluoroquinolones or tetracycline/doxycycline may help in the effective management of acute cholera in the affected Nigerian states. In addition, strengthening the existing surveillance in the hospitals of all the states and supply of clean drinking water may control cholera outbreaks in the future.

Increased isolation frequency of toxigenic Vibrio cholerae O1 from environmental monitoring sites in Haiti.

Since the identification of the first cholera case in 2010, the disease has spread in epidemic form throughout the island nation of Haiti; as of 2014, about 700,000 cholera cases have been reported, with over 8,000 deaths. While case numbers have declined, the more fundamental question of whether the causative bacterium, Vibrio cholerae has established an environmental reservoir in the surface waters of Haiti remains to be elucidated. In a previous study conducted between April 2012 and March 2013, we reported the isolation of toxigenic V. cholerae O1 from surface waters in the Ouest Department. After a second year of surveillance (April 2013 to March 2014) using identical methodology, we observed a more than five-fold increase in the number of water samples containing culturable V. cholerae O1 compared to the previous year (1.7% vs 8.6%), with double the number of sites having at least one positive sample (58% vs 20%). Both seasonal water temperatures and precipitation were significantly related to the frequency of isolation. Our data suggest that toxigenic V. cholerae O1 are becoming more common in surface waters in Haiti; while the basis for this increase is uncertain, our findings raise concerns that environmental reservoirs are being established.

Haitian variant ctxB producing Vibrio cholerae O1 with reduced susceptibility to ciprofloxacin is persistent in Yavatmal, Maharashtra, India, after causing a cholera outbreak.

Vibrio cholerae O1 biotype El Tor producing Haitian variant Cholera Toxin (HCT) and showing reduced susceptibility to ciprofloxacin caused a cholera outbreak associated with a high case fatality rate (4.5) in India. HCT-secreting strains responsible for severe cholera epidemics in Orissa (India), Western Africa and Haiti were associated with increased mortality. There is a pressing need for an integrated multidisciplinary approach to combat further spread of newly emerging variant strains. The therapeutic effect of ciprofloxacin was diminished whereas use of doxycycline in moderate to severe cholera patients was found to be effective in outbreak management.

Clinical isolates of Vibrio cholerae O1 El Tor Ogawa of 2009 from Kolkata, India: preponderance of SXT element and presence of Haitian ctxB variant.

BACKGROUND: Increase in the number of multidrug resistant pathogens and the accompanied rise in case fatality rates has hampered the treatment of many infectious diseases including cholera. Unraveling the mechanisms responsible for multidrug resistance in the clinical isolates of Vibrio cholerae would help in understanding evolution of these pathogenic bacteria and their epidemic potential. This study was carried out to identify genetic factors responsible for multiple drug resistance in clinical isolates of Vibrio cholerae O1, serotype Ogawa, biotype El Tor isolated from the patients admitted to the Infectious Diseases Hospital, Kolkata, India, in 2009. METHODOLOGY/PRINCIPAL FINDINGS: One hundred and nineteen clinical isolates of V. cholerae were analysed for their antibiotic resistance phenotypes. Antibiogram analysis revealed that majority of the isolates showed resistance to co-trimoxazole, nalidixic acid, polymixin B and streptomycin. In PCR, SXT integrase was detected in 117 isolates and its sequence showed 99% identity notably to ICEVchInd5 from Sevagram, India, ICEVchBan5 from Bangladesh and VC1786ICE sequence from Haiti outbreak among others. Antibiotic resistance traits corresponding to SXT element were transferred from the parent Vibrio isolate to the recipient E. coli XL-1 Blue cells during conjugation. Double-mismatch-amplification mutation assay (DMAMA) revealed the presence of Haitian type ctxB allele of genotype 7 in 55 isolates and the classical ctxB allele of genotype 1 in 59 isolates. Analysis of topoisomerase sequences revealed the presence of mutation Ser83 → Ile in gyrA and Ser85→ Leu in parC. This clearly showed the circulation of SXT-containing V. cholerae as causative agent for cholera in Kolkata. CONCLUSIONS: There was predominance of SXT element in these clinical isolates from Kolkata region which also accounted for their antibiotic resistance phenotype typical of this element. DMAMA PCR showed them to be a mixture of isolates with different ctxB alleles like classical, El Tor and Haitian variants.

Antibacterial activity of Sonoran propolis and some of its constituents against clinically significant Vibrio species.

The aim of the present study was to evaluate the anti-Vibrio activity of propolis collected from three different areas of the Sonoran Desert in northwestern, Mexico [Pueblo de Alamos (PAP), Ures (UP), and Caborca (CP)]. The anti-Vibrio spp. activity of Sonoran propolis was determined by the broth microdilution method. UP propolis showed the highest antibacterial activity [minimal inhibitory concentration (MIC(50))<50 µg mL(-1)] against Vibrio spp. (UP>CP>PAP). UP propolis significantly inhibited the growth of Vibrio cholerae O1 serotype Inaba (MIC(50)<50 µg mL(-1)), V. cholerae non-O1 (MIC(50)<50 µg mL(-1)), V. vulnificus (MIC(50)<50 µg mL(-1)), and V. cholerae O1 serotype Ogawa (MIC(50) 100 µg mL(-1)), in a concentration-dependent manner. The UP propolis constituents, galangin and caffeic acid phenethyl ester (CAPE), exhibited a potent growth inhibitory activity (MIC(50) 0.05-0.1 mmol l(-1)) against V. cholerae strains (non-O1 and serotype Ogawa). The strong anti-Vibrio activity of Sonoran propolis and some of its chemical constituents (galangin and CAPE) support further studies on the clinical applications of this natural bee product against different Vibrio spp., mainly V. cholerae.

Drug-resistance mechanisms in Vibrio cholerae O1 outbreak strain, Haiti, 2010.

To increase understanding of drug-resistant Vibrio cholerae, we studied selected molecular mechanisms of antimicrobial drug resistance in the 2010 Haiti V. cholerae outbreak strain. Most resistance resulted from acquired genes located on an integrating conjugative element showing high homology to an integrating conjugative element identified in a V. cholerae isolate from India.

[Cholera in Rio Grande do Norte State--Brazil: sorology and sensitivity of Vibrio cholerae to different antimicrobials]. / A cólera no Estado do Rio Grande do Norte--Brasil--perfil sorológico e de sensibilidade do Vibrio cholerae a diferentes antimicrobianos.

PURPOSE: The emergence of multiple resistance to antimicrobials in Vibrio cholerae isolated in the state of Ceará, Brazil, alerted researchers in this area to the sensitivity to antimicrobials of strains isolated in Rio Grande do Norte (RN), Brazil. METHODS: One hundred and four strains of V. cholerae of human origin, isolated by Laboratório Central de Saúde Pública Dr. Almino Fernandes, were serologically typified and evaluated for in vitro sensitivity to eight antibiotics belonging to different groups (polymyxine, tetracycline, chloramphenicol, nitrofurantoin, sulphazotrin, pefloxacine, erythromycine, ampicillin). The strains were collected from patients suspected of contracting choleric diarrhea in the year 1999, in Natal/RN/Brazil. RESULTS: From the sample total, 100 were identified as V. cholerae, serogroup O:1, biotype El Tor, with 99 (95.3%) belonging to serovar Ogawa and only 1 (0.9%) to serovar Inaba. The 4 remaining were characterized as non O:1 V. cholerae, with 3 (2.9%) biochemically identified as Heiberg type I and 1 (0.9%) as type II. All the V. cholerae serogroup O:1 strains were sensitive to tetracycline, chloramphenicol, sulphazotrin, pefloxacine, erythromycine and resistant to polymyxine. In relation to nitrofurantoin, only 1 was sensitive. Only 1 was resistant to ampicillin. The non O:1 V. cholerae strains were resistant to polymyxine. CONCLUSIONS: The results showed sensitivity in 100% of the V. cholerae serogroup O:1 strains to tetracycline, an elective drug in the treatment of cholera, and an absence of multiple resistant strains in our environment. An interesting finding was the frequency of serovar Ogawa in 1999, considering the greater incidence of serovar Inaba in other years of cholera outbreaks in RN.

A cólera no Estado do Rio Grande do Norte--Brasil--perfil sorológico e de sensibilidade do Vibrio cholerae a diferentes antimicrobianos / Cholera in Rio Grande do Norte State--Brazil: sorology and sensitivity of Vibrio cholerae to different antimicrobials

PURPOSE: The emergence of multiple resistance to antimicrobials in Vibrio cholerae isolated in the state of Ceará, Brazil, alerted researchers in this area to the sensitivity to antimicrobials of strains isolated in Rio Grande do Norte (RN), Brazil. METHODS: One hundred and four strains of V. cholerae of human origin, isolated by Laboratório Central de Saúde Pública Dr. Almino Fernandes, were serologically typified and evaluated for in vitro sensitivity to eight antibiotics belonging to different groups (polymyxine, tetracycline, chloramphenicol, nitrofurantoin, sulphazotrin, pefloxacine, erythromycine, ampicillin). The strains were collected from patients suspected of contracting choleric diarrhea in the year 1999, in Natal/RN/Brazil. RESULTS: From the sample total, 100 were identified as V. cholerae, serogroup O:1, biotype El Tor, with 99 (95.3%) belonging to serovar Ogawa and only 1 (0.9%) to serovar Inaba. The 4 remaining were characterized as non O:1 V. cholerae, with 3 (2.9%) biochemically identified as Heiberg type I and 1 (0.9%) as type II. All the V. cholerae serogroup O:1 strains were sensitive to tetracycline, chloramphenicol, sulphazotrin, pefloxacine, erythromycine and resistant to polymyxine. In relation to nitrofurantoin, only 1 was sensitive. Only 1 was resistant to ampicillin. The non O:1 V. cholerae strains were resistant to polymyxine. CONCLUSIONS: The results showed sensitivity in 100% of the V. cholerae serogroup O:1 strains to tetracycline, an elective drug in the treatment of cholera, and an absence of multiple resistant strains in our environment. An interesting finding was the frequency of serovar Ogawa in 1999, considering the greater incidence of serovar Inaba in other years of cholera outbreaks in RN.