[Correlation between pulsed-field gel electrophoresis profiles and Salmonella serotypes].

OBJECTIVE: To analyze the relationship between pulsed-field gel electrophoresis (PFGE)subtyping and serotyping of Salmonella(S.). METHODS: PFGE was performed and profiles were analyzed on 1230 Salmonella isolates which comprising the top five serotypes including Typhimurium, Enteritidis, Derby, Agona and Senftenberg identified in China. The potential predictive relationship between PFGE banding patterns and particular serotypes was compared and the discriminatory consensus band class markers of individual serotypes were identified. RESULTS: Among all the 1230 Salmonella strains, 1149 strains were found assistant with serotyping through PFGE cluster analysis, providing the matching accuracy reaching 93.4%. For the five serotypes, the positive prediction rate appeared more than 90.0% and the negative prediction rate was over 95.0% on serotype cluster prediction. CONCLUSION: Results presented in this study were representatives of the top 5 Salmonella serovars, showing that PFGE cluster analysis could provide clues to identity and confirmation of serotypes.

[Molecular analysis on non-O1 and non-O139 Vibrio cholerae isolates].

OBJECTIVE: According to results from the two-month consecutive surveillance program in Maanshan, six suspected cases of non-O1 non-O139 Vibrio (V.) cholerae infection, were found that called for identification of pathogens as well as molecular-epidemiological analysis to determine the aggregation of the epidemic situation. METHODS: Biochemical and serotype identification, hemolysis test, and drug sensitive test were used to detect the drug resistance spectrum. Real-time PCR and conventional PCR were used to detect the presence of V. cholerae specific genes, virulent genes and its related genes, including ompW, ctx, tcpA, toxR, hlyA, zot, ace, rstR and gIII(CTX). Pulsed-field gel electrophoresis (PFGE) was used to analyze the molecular type of strains. RESULTS: All the six isolates of non-O1 non-O139 V. cholerae were identified by biochemical and serologic tests, and appeared to be ß hemolytic. Twelve out of the 14 kinds of drugs showed 100% sensitive. All isolates were positive of ompW gene by real-time PCR, but negative for ctx, tcpA, zot, ace, rstR and gIII(CTX). Five of the six isolates were positive for toxR and hlyA, except for strain 1001434446. All strains had different PFGE types, but two strains had similar types. All strains had a low similarity compared to the toxigenic V. cholerae. CONCLUSION: Six cases of non-O1 and non-O139 nontoxigenic V. cholerae infection appeared in the same period. Along with epidemiological information, we noticed that these cases had a sporadic nature, but frequently appeared in the same area. We got the impression that public health measurements should be strengthened, with special attention paid to those diarrhea outbreaks caused by non-O1/non-O139 strains since V. cholerae had appeared in low incidence.

[Molecular subtyping of Vibrio cholerae isolates from outbreaks of cholera by pulsed-field gel electrophoresis in Hainan in 2008].

OBJECTIVE: To analyze the molecular characteristics and genetic correlations of Vibrio cholerae isolates in Hainan in 2008, so as to provide pathogenic proof to diagnose the plague. METHODS: Seventy six cholera strains were isolated from this cholera epidemic.69 strains were obtained from patients, 7 were isolated from external environment, among which, one was from patient's toilet, one from water sample, three were isolated from fish pond near patient's home, one came from swab of the patient vomit on the ground of health center and one from swab of kitchen knife from Hainan University canteen respectively. With conventional aetiological methods, pulse-field gel electrophoresis was conducted and the patterns of the 76 isolates were analyzed. The PFGE image was analyzed using BioNumerics (Version4.0, Applied Maths BVBA, Belium). Image bands were identified and similarity coefficient was automatically generated. RESULTS: Seventy six strains were isolated from Vibrio cholerae outbreaks in Hainan in 2008.5 PFGE patterns of patient's isolates in June were the same, sharing a similarity coefficient of 100%. 70 PFGE patterns of patients and water in October and November were completely same, the similarity coefficient being 100%. But they were not same as that of June. 1 PFGE pattern of isolate from the sample in Hainan University was different, only sharing a similarity coefficient of 79.7%, which showed no correlation with the outbreak. CONCLUSION: Different outbreaks of Vibrio cholera occurred in Hainan in 2008. The epidemic in October and November at different counties was one outbreak. The pollution of water in environment was an important factor for outbreak.

[Molecular characterization of Vibrio cholerae phage-type 6b epidemic isolates from 1998 to 2001 in Sichuan province].

OBJECTIVE: To investigate the molecular characteristics of phage-type 6b isolates emerging in 1998-2001 cholera epidemics in Sichuan province. METHODS: Isolates were analyzed by phage-typing, pulsed field gel electrophoresis (PFGE) and ompW gene sequencing. RESULTS: All phage-type 1b and 6b isolates in Sichuan province from 1998 to 2001 were toxigenic. A total of 24 patterns were identified after PFGE analysis, and one predominant pattern consisted of 13 isolates. Several 1b and 6b isolates from Sichuan and isolates of the 1b from other provinces showed the same PFGE pattern. Mutation in ompW gene was found in 6b isolates. CONCLUSION: V.cholerae O1 6b isolates in Sichuan province from 1998 to 2001 have special genetic markers, and might genetically correlate with contemporaneous 1b isolates.

[Study on the epidemiological characteristics and molecular typing of Salmonella enterica subsp. enterica serovar Senftenberg in Shanghai].

OBJECTIVE: To study the molecular epidemiological characteristics of Salmonella enterica subsp. enterica serovar Senftenberg (Salmonella senftenberg) in Shanghai, from 2006 to 2007. METHODS: A retrospective analysis in 2006 and 2007 was performed to explore the source of food-borne Salmonella senftenberg. The isolates from diarrhea patients between 2006 and 2007 were identified, including biochemical test, hilA and invA gene phenotyping, drug susceptibility, Riboprinter((R)) (RP) and pulsed-field gel electrophoresis (PFGE). RESULTS: Of the diarrhea patients isolates in the monitoring program on non-typhi Salmonella infection in the year of 2006 in Shanghai, number of patients caused by Salmonella senftenberg ranked the third. The stock of Salmonella senftenberg food-born isolates were derived from swine and beef products between 2003 and 2005. All of the strains from diarrhea patients were susceptible to antibiotics except tetracylina (75.6%). With RP and PFGE molecular typing, the two groups (with hydrogen sulfide and hilA, invA gene or without) could be divided into two different independent clone cluster in genetics. 34 strains of diarrhea were divided into 16 PFGE typing-pattern, and among them 12 strains including type 4 (4 strains), type 5 (1 strains), type 6 (6 strains), type 7 (1 strains) and 13 strains including type 11 (3 strains), type 17 (5 strains), type 23 (5 strains) were two different dominant clone cluster. CONCLUSION: The epidemic of Salmonella senftenberg within 2006 might have been the result of a long period of case occurrence in Shanghai. This rare outbreak belonged to a cluster of outbreaks caused by two different PFGE clone clusters. Data suggested that the genetic clone of Salmonella senftenberg might have been unstable and the source of contamination were complicated, with the characteristics as the obvious decreasing number of patients, with no food-borne isolates in 2007.

[Molecular typing of Neisseria meningitidis serogroup C strains with pulsed field gel electrophoresis in China].

OBJECTIVE: To study the characteristics of epidemiology and molecular typing on Neisseria meningitidis serogroup C strains associated with outbreaks of Anhui province and sporadic cases in China, using pulsed field gel electrophoresis (PFGE). METHODS: 212 Neisseria meningitidis serogroup C strains were isolated from invasive meningococcal cases, close contacts and healthy carriers, including 48 strains from Anhui province with 38 strains associated with serogroup C outbreaks. PFGE were performed by genomic DNA digestion with Nhe I restriction enzyme. The results of PFGE were analyzed by BioNumerics software (Version 4.0, Applied Maths BVBA, Belgium). RESULTS: A total number of 212 Neisseria meningitidis serogroup C isolates were typed by 43 patterns, named AH1 to AH43. In China, AH1 pattern was the major PFGE pattern with 69.3% (n = 147) of all strains, distributed in 11 provinces. Three types of PFGE patterns (AH1 to AH3) were found in 48 strains from Anhui province, in which, 93.8% (n = 45) belonged to AH1. 97.4% (n = 37) of 38 strains associated with serogroup C outbreaks in Anhui province showed AH1 pattern. A total of 53 serogroup C strains were isolated from invasive meningococcal cases with 67.9% (36/53) of AH pattern. 71.9% (87/121) of serogroup C strains isolated from contacts of invasive meningococcal cases was AH1 pattern and 63.2% (24/38) of the strains from healthy carriers showed AH1 pattern. CONCLUSION: By PFGE typing and analysis, AH1 pattern of Neisseria meningitidis serogroup C strains was proved to be the main clone which causing the outbreaks in Anhui province and might be responsible for the sporadic serogroup C meningococcal disease epidemics else where in the country.

[Establishment of fluorescent amplified fragment length polymorphism in Vibrio cholerae and evaluation in molecular typing].

OBJECTIVE: To develop fluorescent amplified fragment length polymorphism (AFLP) method and to evaluate the its typing capability with pulsed-field gel electrophoresis (PFGE) in molecular typing of Vibrio cholerae. METHODS: Forty-seven strains of V. cholerae, with different PFGE patterns, were selected as the reference group to optimize the selective primers of AFLP analysis. Eighty-three strains including 20 strains from one epidemic episode, isolated from different provinces during 1961 and 2005, were used to compare the typing abilities of AFLP and PFGE. LI-COR4300 DNA sequencing system was used for AFLP electrophoresis. The images were recorded by Saga(MX) software and transferred to BioNumerics for clustering analysis. A standard protocol for V. cholerae from PulseNet was used in PFGE. RESULTS: When comparison was made with different selective primers on AFLP based on the 47 strains, results showed that the optimized selective primer pair was EcoR I-G/Mse I-T, and the reproducibility of the tests was 99.2%. Eighty-three isolates showed 52 AFLP patterns and 44 PFGE patterns, with D values as 0.9545 (AFLP) and 0.9251 (PFGE) respectively. CONCLUSION: The protocol of fluorescent AFLP on V. cholerae typing was established. AFLP was higher than PFGE in discrimination of V. cholerae which could be used for molecular typing. When combined with PFGE, AFLP became a more insightful tool to identify genome difference of different isolates.

[Study on the application of pulsed-field gel electrophoresis regarding infection sources identification during an outbreak of Vibrio cholerae in Jiangxi Province].

OBJECTIVE: To study the correlation between Vibrio cholerae strains isolated from natural enviroment and fishery products and the source of infection during V. cholerae outbreaks. METHODS: Cholera toxin gene was detected by polymerase chain reaction (PCR) amplification. Pulsed-field gel electrophoresis (PFGE) was used to subtype the isolates. Results of PFGE were analyzed and clustered by BioNumerics software (Version 4.0). RESULTS: During the outbreaks, a total number of thirty O139 V. cholerae related serogroups were collected from patients, carriers, sewage and fishery products were identified and proved to be toxigenic. They could be clustered into four PFGE patterns when digested by Not I. These two V. cholerae outbreaks were caused by the same source of infection because of the following reasons: (1) PFGE patterns of the predominant strains isolated from two outbreaks were identical; (2) they were identical to the PFGE patterns of the strains isolated from the green turtle and rana catesbiana which were bought from the same wholesale store. CONCLUSION: Green turtle and rana catesbiana that were contaminated by toxigenic O139 V. cholerae strains seemed to be the source of infection causing the O139 V. cholerae outbreaks in Jiangxi province. Rapid laboratory surveillance and epidemiologic investigation were important in identifying the source of infection during the outbreaks of V. cholera.