RESUMO

Aquaculture is a fast growing industry with its development hampered by bacterial diseases. Vibriosis caused by Harveyi clade strains is known for causing heavy loss especially in shrimp aquaculture farms. For farm treatment and pathogen spread management, veterinarians and researchers need reliable bacterial identification tools. A range of identification methods have been presented for Vibrio spp. in recent literature but little feedback on their performance have been made available to this day. This study aims at comparing Vibrio spp. identification methods and providing guidance on their use. Fifty farms were sampled and bacterial colonies were isolated using specific culture media before microscopic analysis and genomic profiling using ERIC-PCR. A preliminary identification step was carried out using MALDI-ToF mass spectrometry. Four methods were compared for strain identification on 14 newly isolated Harveyi clade Vibrio spp. strains: whole genome sequencing (digital DNA DNA Hybridization (dDDH)), 5 MLSA schemes, ferric uptake regulation (fur) and lecithin-dependent haemolysin (ldh) single gene based identification methods. Apart from dDDH which is a reference method, no technique could identify all the isolates to the species level. The other tested techniques allowed a faster, cheaper but sub genus clade identification which can be interesting when absolute precision is not required. In this regard, MALDI-ToF and fur based identification seemed especially promising.

Assuntos

Aquicultura , Técnicas Bacteriológicas/métodos , Vibrioses/diagnóstico , Vibrioses/microbiologia , Vibrio/genética , Vibrio/isolamento & purificação , Animais , Brasil , DNA Bacteriano/genética , Genes Bacterianos/genética , Proteínas Hemolisinas/genética , Filogenia , Reação em Cadeia da Polimerase/métodos , Vibrio/classificação , Vibrioses/veterinária , Sequenciamento Completo do Genoma

RESUMO

We investigated the antimicrobial susceptibility of 50 environmental isolates of Vibrio cholerae non-O1/non-O139 collected in surface waters in Haiti in July 2012, during an active cholera outbreak. A panel of 16 antibiotics was tested on the isolates using the disk diffusion method and PCR detection of seven resistance-associated genes (strA/B, sul1/2, ermA/B, and mefA). All isolates were susceptible to amoxicillin-clavulanic acid, cefotaxime, imipenem, ciprofloxacin, norfloxacin, amikacin, and gentamicin. Nearly a quarter (22.0%) of the isolates were susceptible to all 16 antimicrobials tested and only 8.0% of the isolates (n = 4) were multidrug-resistant. The highest proportions of resistant isolates were observed for sulfonamide (70.0%), amoxicillin (12.0%), and trimethoprim-sulfamethoxazole (10.0%). One strain was resistant to erythromycin and one to doxycycline, two antibiotics used to treat cholera in Haiti. Among the 50 isolates, 78% possessed at least two resistance-associated genes, and the genes sul1, ermA, and strB were detected in all four multidrug-resistant isolates. Our results clearly indicate that the autochthonous population of V. cholerae non-O1/non-O139 found in surface waters in Haiti shows antimicrobial patterns different from that of the outbreak strain. The presence in the Haitian aquatic environment of V. cholerae non-O1/non-O139 with reduced susceptibility or resistance to antibiotics used in human medicine may constitute a mild public health threat.

RESUMO

BACKGROUND: On October 21, 2010, Haiti was struck by a cholera epidemic for the first time in over a century. Epidemiological and molecular genetic data have clearly demonstrated that the bacterium was imported. Nevertheless, the persistence of the epidemic for more than two years, the high incidence rates in some coastal areas and the seasonal exacerbations of the epidemic during the rainy seasons have prompted us to examine the levels of toxigenic Vibrio cholerae in the Haitian aquatic environment. METHODS: In July 2012, during the warm and rainy season, 36 aquatic stations were sampled to search for toxigenic V. cholerae. These stations included fresh, brackish and saline surface waters as well as waste water; the sampling sites were located in both rural and urban areas (around Port-au-Prince and Gonaïves) located in the West and Artibonite Departments. V. cholerae bacteria were detected in enrichment cultures of water samples (sample volumes included 1 L, 100 mL, 10 mL, 1 mL, 0.1 mL, 0.01 mL and 0.001 mL depending on the context). Detection methods included both culture on selective agar (for strain isolation) and PCR assays targeting the genes ompW (V. cholerae species), O1-rfb and O139-rfb (O1 and O139 V. cholerae serogroups, respectively), and the cholera toxin gene ctxA, which is present exclusively in toxigenic cholera strains. RESULTS: A total of 411 culturable V. cholerae isolates from 29 stations were obtained via selective culture; however, only one of these isolates displayed a late positive reaction with polyvalent anti-O1 serum. Positive V. cholerae PCR results were obtained from each of the 32 tested stations (a total of 77 enrichments out of 107 yielded a positive result); only one sample yielded a positive V. cholerae O1 PCR result. The cholera toxin gene ctxA was never detected via PCR with either primer pair, which includes samples derived from the two stations yielding positive O1 culture or positive O1 PCR results. Therefore, we could not demonstrate the presence of toxigenic V. cholerae O1 among the 36 stations sampled. This suggests that all water samples analyzed contained less than 10 toxigenic V. cholerae O1 bacteria per liter, a level 1000-fold below the dose that has been shown to provoke cholera in healthy adults. CONCLUSIONS: Currently, there is no evidence of a significant level of contamination of the aquatic environment in Haiti by the imported toxigenic V. cholerae O1 strain. The reemergence of cholera outbreaks in Haiti during rainy seasons is therefore more likely due to persisting outbreaks insufficiently tackled during the dry periods rather than the commonly suspected aquatic reservoir of toxigenic bacteria.