To study the characteristics of molecular typing and phylogenic relationship among the Vibrio cholerae serogroup O139 strains isolated from environment and sea food samples during cholera outbreaks, in Sichuan province in 2004 and to trace the source of infections so as to support the ascertainment of epidemic control strategy.
Cholera toxin gene was detected by polymerase chain reaction amplification. Pulsed-field gel electrophoresis (PFGE) was used for subtyping of isolates and clustering of patterns was analysed with the software BioNumerics.
In all the 72 strains under analysis, 68 appeared to be toxigenic while 4 from river water derived isolates were toxin gene negative. Sixty-seven strains were clustered into 16 PFGE patterns when digested with Not I. The patterns of toxigeinc O139 strains isolated from turtles in the markets were identical with the patterns of strains appeared in the outbreaks respectively. The PFGE patterns of isolates from different outbreaks were inconsistent.
The sources of infection causing these outbreaks were complicated. Contaminated turtles might also be one of the major sources of outbreaks when being served at the dinner parties in Sichuan in 2004.
[Show abstract][Hide abstract] ABSTRACT: Molecular typing of Vibrio cholerae strains is a powerful tool for the surveillance of cholera. Amplified fragment length polymorphism (AFLP) is considered to be a powerful subtyping technique to distinguish bacterial strains at the genetic level. Optimization and standardization of AFLP protocol is required to allow data comparisons across different laboratories in a surveillance network. Here, we performed AFLP using different restriction enzymes and primer pairs for subtyping of V. cholerae serogroups O1 and O139 and compared the optimized AFLP protocol with pulsed-field gel electrophoresis (PFGE) to evaluate the applicability of AFLP for conducting epidemiological surveillance of cholera. The discriminatory index (D-value) of PFGE for serogroup O1 strains was similar when digested with NotI and SfiI, whereas that for O139 strains was higher for NotI digestion than for SfiI. EcoRI-G/MseI-T was the restriction enzyme and primer combination with highest discriminatory index used in the AFLP analysis. Capillary electrophoresis-based AFLP showed higher discriminatory power than that of polyacrylamide gel electrophoresis-based AFLP. When the two methods were compared using 72 epidemiologically unrelated serogroup O1 El Tor isolates, AFLP had a lower D-value than PFGE with NotI and SfiI digestions, respectively. For 54 epidemiologically unrelated serogroup O139 isolates, NotI PFGE had the highest discriminatory power, and SfiI PFGE and AFLP yielded almost the same but lower discriminatory power. We conclude that NotI and SfiI are both suitable for the PFGE of V. cholerae serogroup O1, whereas NotI should be defined as the primary enzyme for serogroup O139. The applicability of AFLP in V. cholerae subtyping and outbreak investigations is limited.
[Show abstract][Hide abstract] ABSTRACT: The aerobic oral and cloacal bacterial microbiota and their antimicrobial resistance were characterized for 64 apparently healthy sea turtles captured at their foraging grounds in Ojo de Liebre Lagoon (OLL), Baja California Sur (BCS), Mexico (Pacific Ocean) and the lagoon system of Navachiste (LSN) and Marine Area of Influence (MAI), Guasave, Sinaloa (Gulf of California). A total of 34 black turtles (Chelonia mydas agassizii) were sampled in OLL and eight black turtles and 22 olive ridley turtles (Lepidochelys olivacea) were sampled in LSN and MAI, respectively from January to December 2012. We isolated 13 different species of Gram-negative bacteria. The most frequently isolated bacteria were Vibrio alginolyticus in 39/64 (60%), V. parahaemolyticus in 17/64 (26%), and V. cholerae in 6/64 (9%). However, V. cholerae was isolated only from turtles captured from the Gulf of California (MAI). Among V. parahaemolyticus strains, six O serogroups and eight serovars were identified from which 5/17 (29.4%) belonged to the pathogenic strains (tdh + gene) and 2/17 (11.7%) had the pandemic clone (tdh + and toxRS/new +). Among V. cholerae strains, all were identified as non-O1/non-O139, and in 4/6 (66%) the accessory cholera enterotoxin gene (ace) was identified but without virulence gene zot, ctxA, and ctxB. Of the isolated V. parahaemolyticus, V. cholerae, and V. alginolyticus strains, 94.1, 33.4, and 100% demonstrated resistance to at least one commonly prescribed antibiotic (primarily to ampicillin), respectively. In conclusion, the presence of several potential (toxigenic) human pathogens in sea turtles may represent transmission of environmental microbes and a high-risk of food-borne disease. Therefore, based on the fact that it is illegal and unhealthy, we discourage the consumption of sea turtle meat or eggs in northwestern Mexico.
Frontiers in Microbiology 07/2015; 1(6). DOI:10.3389/fmicb.2015.00635 · 3.99 Impact Factor
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