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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.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.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.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.

To identify the isolates of Shewanella spp. from specimens of food poisoning based on biological and biochemical analysis.Strains were obtained from the investigation on two food poisoning episodes in September and October, 2007 in Ma'anshan city, Anhui province. In accordance with the national standard protocol (GB/T 4789), all specimens were enriched and isolated on selective medium, and the suspected strains were identified by the VITEK-32 and API20E systems. For Shewanella spp. identified by the biochemical system, more characteristics were analyzed using auxiliary biochemical, growth, hemolytic and drug-resistance tests. DNAs of Shewanella spp. were extracted, 16S rDNA was PCR amplified and sequenced with universal 16S rDNA primers. Phylogenetic tree was constructed with MEGA 4.0.After enrichment, all specimens were inoculated to selective medium and Shewanella spp. strains were isolated from 8 samples with single colony on both TCBS and BP media. The characteristics of growth in the Triple Sugar Iron (TSI) agar appeared to have had hydrogen sulfide production but no gas production or positive oxidase. No Shewanella spp. strain was detected in WS, SS and EMB media. The 8 strains were identified as Shewanella algae (S. algae) or Shewanella putrefaciens (S. putrefaciens) by VITEK-32, as S. putrefaciens by API20E system. No other enteropathogenic bacteria, including Vibrio cholerae, Salmonella, Vibrio parahaemolyticus, Proteus vulgaris or Staphylococcus aureus, were detected from those 8 samples. From 16S rDNA phylogenetic trees, 7 out of 8 Shewanella spp. were identified as S. algae, 1 as S. putrefaciens.Strains of Shewanella spp. were isolated from samples of the food poisoning episodes, providing a possible clue to investigate the role of Shewanella spp. on food poisoning.

To establish a duplex nested PCR assay system which is capable for detecting O1 and O139 groups of Vibrio cholerae simultaneously, and is applicable to environmental specimens from routine cholera surveillance.Based on nucleic acid sequences available in GenBank, six sets of primers were designed by PrimerSelect program of DNAStar, targeting the rfb gene that encodes the O antigens of O1 and O139 V. cholerae, respectively. The specificity of several primer combinations was tested. A duplex nested PCR assay system for simultaneously detecting O1 and O139 V. cholerae was established, subsequently, its sensitivity, specificity, reproducibility and field evaluation were tested. The sensitivity of this assay was evaluated by comparing detection limits of nested PCR and conventional PCR. Its reproducibility was tested by 32 positive samples (11 samples positive for O1, 21 samples positive for O139) from environmental surveillance. In addition, the selected amplicons from positive samples were sequenced and analyzed with relevant sequences.This newly-established duplex nested PCR assay might distinguish O1 V. cholerae from O139 V. cholerae, based on fragment lengths of amplicons, with reliable reproducibility, and no specific amplification was observed as compared with other vibrio species. The sensitivity of this nested PCR was (15 000) higher than conventional PCR, and there was no interference observed with multiple primers and complicated templates in the same vial. In its field evaluation, 32 positive DNA samples were detected and be further confirmed with double or triple tests, implying reliable reproducibility and consistency of this system. These results indicated that this assay had reliable reproducibility. No amplification was observed in all negative specimens and also suggested the acceptable specificity of this assay. Sequence analysis of the selected amplification products revealed 100% homogeneous with relevant genes from V.cholerae, indicating that these amplicons were originated from V. cholerae.This duplex nested PCR assay system should be rapid, sensitive and especially applicable to small laboratories, and be suitable for dynamic environmental surveillance.

To understand the biochemical characteristics and 16S rDNA genetic sequence evolution of strains isolated from diarrhea specimens so as to provide basis for classification and identification of Klebsiella pneumoniae.Specimens were cultured using MacConkey and SS medium. All isolates were identified as K. pneumoniae by automated biochemical tests. DNA was extracted, 1500 bp fragments of the 16S rDNA gene were by amplified PCR and sequenced with K. pneumoniae 16S rDNA primer, after being cut. Fragments of 1000 bp overlapping sequences were analyzed by Blast to confirm the identity of the isolates. A phylogenetic tree was constructed by PHYLIP process to analyze the 16S rDNA sequence of the isolated strain with other relative bacteria species in the GenBank databases.Among 113 specimens of infectious diarrhea, 25 K. pneumoniae strains were identified by biochemical tests, of which 21 subsp. pneumoniae and 4 subsp. ozaenae, no subsp. of rhinoscleroma were isolated. Strains of subsp. pneumoniae were found having nature of resistance. All isolates were resistant to penicillin G and susceptible to polymyxin with some strains were resistant to Nitrofurantoin, Cephalothin, Kanamycin, Tobramycin. After searching in GenBank of 16S rDNA, strains biochemical identified as subsp. ozaenae shared high similarity with Salmonella strains and other intestinal bacteria. 16S rDNA phylogenetic analysis could be used to confirm subsp. pneumoniae, but could not separate other subspecies of K. pneumoniae completely.16S rDNA phylogenetic analysis useful in identifying and classifying K. pneumoniae.

To investigate the serologic type, phage-biotype and toxic factor of Vibrio cholerae isolated from different sea products, analyze the relation between the Vibrio cholerae in sea products and cholera epidemiology, and provide references for forecasting cholera epidemic situation and drawing out a preventing plan.The biotype of strains isolated was analyzed by using type and phage-biotype serological methods. The toxic gene was detected by PCR.The constituent ratio of V. cholerae O139, Ogawa and Inaba were, respectively, 48.44%, 20.31% and 31.25% in 64 strains of V. cholerae. The result of phage-biotype showed that the 26 strains of V. cholerae O1 were all non-epidemic strains. The result of toxic gene detecting showed that positive rate of V. cholerae O139 was higher than those of Ogawa and Inaba.The positive rate of toxic gene in V. cholerae O139 was high and the V. cholerae O139 was mainly in turtle, breed aquatics water and crustacean, so these sea products were the important sectors in cholera prevention and control.