Study On The Identification,Typing And Establishment Of The Tracing Database Of Pseudomonas Cocovenenans Subsp.farinofermentans

Pseudomonas cocovenenans subsp. farinofermentans is a foodborne pathogen discovered by Chinese scholars in 1977,used to termed Flavobacterium farinofermentans nov sp .It produces two highly toxic compounds: toxoflavin and bongkrekic acid. There were many studies focus on the culture, identification,category of the species and the producing of the two toxins, but less studies on genotyping. The systemic study of Pseudomonas cocovenenans subsp.farinofermentans including the identification by API 20NE, VITEK 2 and 16S rDNA sequence analysis, typing by pulsed-field gel electrophoresis and fluorescent amplification fragment length polymorphism, and the establishment of the tracing database.Part ? Study on the biochemical identification, culture characteristics and toxicity test78 isolates and 4 reference strains of Burkholderia were identified and typed by two commercial identification systems respectively. All the strains could be divided into eight biotypes by API 20NE and VITEK 2 GN, respectively. Most isolates strains were identified as Burkholderia gladioli by API 20NE with two supplementary tests and also by VITEK 2 GN. Contrast to API 20NE, there were more tests in VITEK 2 GN, which would result in more biotype diversity. The biochemical test was not suitable for the typing of bacteria because of the variable results.The study referenced with GB4789.29-2003, simply described the culture characteristics and the toxicity of the isolates. In the experiment, it was feasible to enrich the pure isolate either with BHI, tremella broth or potato dextrose broth, but BHI is not applied for the contaminated isolates which the contaminates will cover the Burkholderias . Pseudomonas cocovenenans subsp. farinofermentans has stable culture characteristics in the PDA and obvious rainbow around the colony in the tremella yolk agar which would be better to identify isolates. The SS agar was not appropriate for culturing Pseudomonas cocovenenans subsp. farinofermentans for its variable characteristics. Toxicity test results showed 18 in the 25 strains were toxic while 7 strains were not. Some strains isolated from food poisoning samples were not toxic, and the toxicity of some strains was different from the results of the same strains tested before. It's conferred that the toxin producing ability of the isolates is not unstable because either the multiple passages or the changing of the medium. The mechanism of the biosynthesis of the toxins of Pseudomonas cocovenenans subsp.farinofermentans was not described well remain uncertain.Part II Analysis of the 16S rDNA sequence of Pseudomonas cocovenenans subsp. farinofermentansThe 16S rDNA sequences of 78 isolates of Pseudomonas cocovenenans subsp.farinofermentans were analyzed, and sequences were compared to the NR database of NCBI by BLAST. The phylogenetic tree was constructed by Maximum Parsimony based on multiple alignments. The BLAST results showed that 74 strains were recognized as B.gladioli, and 2 strains as B.cepacia complex, other 2 strains as Bacillus pumilus. The phylogenetic tree of 16S rDNA sequences of species about the related strains showed that Pseudomonas cocovenenans subsp.farinofermentans ,B.glacdioli, B.cocovenenans, P.antimicrobica, B.glumae belonged to the same branch of the tree. which indicate Pseudomonas cocovenenans subsp.farinofermentans and B.gladioli are highly homologous.Analysis of 16S rDNA sequence made a better identification of Pseudomonas cocovenenans subsp. farinofermentans than phenotypic identification, but not so good in the typing of the isolates.Part III Study on the genotyping of Pseudomonas cocovenenans subsp.farinofermenlansTo analyze the fingerprint of Pseudomonas cocovenenans subsp.farinofermentans, 4 reference strains and 76 isolates were digested with ,Spe I and analyzed by pulsed-field gel electrophoreses (PFGE). All the strains gave 67 different kinds of PFGE patterns, and divided into 10 groups with the condition of 71%similarity. The results showed that PFGE methods had a high discriminatory power,and could be used in the molecular typing and tracing of Pseudomonas cocovenenans subsp. farinofermentans. According to the epidemiological information, all the strains having the same patterns were isolated from the same regions or the same food poisoning sample, while some isolates from different regions with high similarity more the 94.7%, such as BG-07-BG-19, and BG-76-BG 79. In order to get different results, an accurate conclusion should combine with other typing methods and the epidemiological data.Four Low-frequency restriction endonucleases and two high-frequency restriction endonucleases were tested in Fluorescent Amplified Fragment Length Polymorphism,FAFLP, and Apa I -G/Taq I -G and Pst I -T/Taq I -G were selected based on the gel electrophoresis profiles. The fingerprint of 76 isolates and 4 reference strains gave 77 patterns, and divided into 20 groups with the condition of 92% similarity; by Apa I -G/Taq I -G, 78 patterns and divided into 13 groups with 87% similarity of Pst I -T/Taq I -G, which Simpson Diveresty Index was 0.9987 and 0.9994,respectively. The results showed that the both programs of FAFLP methods had a high discriminatory power, and could type molecular and trace Pseudomonas cocovenenans subsp. farinofermentans. Except BG 65-BG 66 isolated from the same food poisoning cases, there was some difference in the fingerprint of other isolates.Parts of the isolates from the same poisoning region had similarity more than 94%,while most isolates from different regions had a low similarity, showing that FAFLP could be used in the tracing of Pseudomonas cocovenenans subsp.farinofermentans.Part IV Establishment of tracing database of Pseudomonas cocovenenans subsp.farinofermentansBy database software BioNumerics, the database of Pseudomonas cocovenenans subsp. farinofermentans (including of phenotying, 16S rDNA sequence and molecular typing) was established. The methods and increasing data in this database would provide effective technical warranty for the tracing of outbreaks, sporadic cases and routine surveillance of Pseudomonas cocovenenans subsp. farinofermentans. The study analysised 50 isolates with epidemiological information in the database, which proved that there was significant relationship among BG 65-BG 66?BG41-BG 42-BG 43?BG 13-BG 14?BG 58-BG 59?BG 68-BG 75. To get an accurate conclusion,the accurate data of experiment and the epidemiological information was necessary.