| [Background]Yersina enterocolitica (Y. enterocolitica) is a gram-negative budless bacillus, which belongs to Yersina genus and is one of the three pathogenic bacteria for humans and animals of this genus. It distributes widely in the natural environment, and mainly infects humans by contaminated food. Y. enterocolitica infections are mainly characterized with gastrointestinal symptoms, such as vomit, abdominal pain and diarrhea, which are usually self-limiting. However, the infection could induce serious complications, such as endocarditis, reactive arthritis, abscess and fatal septicemia. Yersiniosis, which is caused by Y. enterocolitica and Y. pseudotuberculosis, is the fourth most commonly reported zoonosis in Europe. There are about117,000infections caused by Y. enterocolitica annually in America. Two outbreaks of Y. enterocolitica infection were reported in literatures in China. It has been differentiated into six biotypes (1A,1B, and2-5) and60serotypes. The pathogenicity of Y. enterocolitica is usually determined by testing five virulence genes, which are ail, ystA, ystB, yadA and virF. Y. enterocolitica is resistant to penicillins and first generation cephalosporins naturally, and has different resistant rates to other antimicrobial agents. Pulsed field gel electrophoresis (PFGE) typing is the most frequently used method for Y. enterocolitica molecular typing nowadays and is mainly used for tracing to the strain’s source.In2006, Shandong Province set up two surveillance points, Gaomi City and Wulian County, for Y. enterocolitica monitoring. Until the end of2011,237strains of Y. enterocolitica had been isolated from meat, flies and feces of chickens, swines, sheeps, cattles and other animals. The motility, biotype, serotype, virulence gene and antimicrobial agent susceptibility of these strains had not been analyzed systematically, and the molecular typing had also not been studied.[Objectives]1. To systematically analyze the motility, biotype, serotype, virulence gene and antimicrobial agent susceptibility of the Y. enterocolitica isolates and the distribution of time, geography and host for each biological characteristic.2. To classify the Y. enterocolitica isolates by PFGE, in order to explore the genetic relationship of these strains and discuss the relations between phenotyping and PFGE typing.3. To establish a Y. enterocolitica database of Shandong Province, in order to lay a foundation for the future long-term surveillance and provide technical support for the prediction and alert of yersiniosis.[Methods]1. Motility was tested by stabbing culture in semi-solid medium. Biotype was determined by the results of biochemical reactions. Serotype was determined by serum agglutination test on a glass slide. Virulence gene was tested by polymerase chain reaction. Antimicrobial agent susceptibility was tested by VITEK2COMPACT system and AST-GN16card.2. PFGE typing was operated according to the standard method recommended by PulseNet China. BioNumerics6.1was used for PFGE spectrum processing and clustering analysis.3. Excel2007was used for data entering, compilation and simple analysis. SPSS16.0was used for data statistic analysis.[Results]1. Among the strains,4.2%(10/237) had medium-active motility,3.0%(7/237) had poor-active motility, and the others all had well-active motility. The strains of medium-and poor-active motility were isolated from most kinds of hosts from the two districts every year.2. Two hundred and thirty-four of the237strains (98.7%) were biotype1A, and the other three strains were all biotype4, which were isolated from swine feces samples in Gomi City, in2006.3. One hundred and eighteen of the237strains could be classified by the antiserum, and O:5(21.9%,52/237) and0:8(21.1%,20/237) were the dominant serotypes. All the five serotypes which could be calssified were tested in the strains isolated in2011. Gaomi isolates were dominated by O:5serotype (23.9%,39/163) while Wulian isolates were dominated by O:8serotype (33.8%,25/74); isolates from each kind of hosts were all dominated by O:5and0:8serotypes.4. The positive rate of ystB gene was the highest, which was81.4%(193/237); ail and ystA gene were found only in three strains, and the positive rate was1.3%(3/237) each; yadA and virF gene were negative in all the strains. The ystB gene positive strains were dominant in each year, either district and each kind of hosts.5. Two hundred and thirty-five strains were tested for antimicrobial agent susceptibility. All the strains (235strains) were sensitive to ceftriaxone, cefepime, amikacin, gentamicin and tigecycline; the resistant rate to ampicillin was highest, which was94.5%(222/235). The multiple resistant rate was12.3%(29/235). The susceptibility to nitrofurantoin between2007and2011was significantly different (P <0.05); the resistant rate to trimethoprim/sulfamethoxazole between Gaomi City (10.4%,17/163) and Wulian County (1.4%,1/72) was significantly different (P<0.05); the resistant rate to ampicillin, amoxicillin/clavulanic acid, cefazolin and cefoxitin and the intermediate resistant rate to nitrofurantoin of virulence genotype A (ail-ystA-ystB+yadA+virF-) strains, which was99.0%(190/192),89.6%(172/192),94.8%(182/192),80.2%(154/192) and94.8%(182/192) respectively, was significantly higher than that of virulence genotype B (ail-ystA-ystB-yadA-virF-) strains (P<0.05), which was72.5%(29/40),65.0%(26/40),67.5%(27/40),42.5%(17/40) and65.0%(26/40) respectively.6. Two hundred and thirty-one of the237strains were divided into144PFGE types with no one significantly dominant, and the similarity was61.1%to100.0%. The clustering of strains isolated from Wulian County was more obvious than that from Gaomi City, and the PFGE types, which had seven or more than seven strains, were all in Wulian County. The other six strains couldn’t be classified by PFGE. Fifteen of the144PFGE types could find their same or similar types in the database of Chinese center for disease control and prevention.7. Fourteen of all the17medium-or poor-active motility strains were grouped into three clusters. Thirty-one of all the52O:5serotype strains were grouped into four clusters. Nineteen of all the50O:8serotype strains were grouped into one cluster. Twenty of all the41virulence genotype B (ail-ystA-ystB-yadA-virF-) strains were grouped into one cluster. The antimicrobial agent susceptibility types were distributed irregularly in the PFGE clustering.8. The Simpson’s diversity index of motility, biotyping, serotyping, virulence genotyping, antimicrobial agent susceptibility typing and PFGE typing was0.1362,0.0251,0.6564,0.3081,0.7214and0.9982respectively.[Conclusions]1. The Y. enterocolitica strains isolated from Gaomi City and Wulian County had motility mutant strains. Almost all the strains isolated were biotype1A. The serotypes were dominated by O:5and O:8and had a difference between the two districts. The main virulence gene that the strains had was ystB and there was no pathogenic strain traditionally defined. The isolates were resistant to many antimicrobial agents, but most of the strains were resistant to penicillins and first and second generation cephalosporins. There were multiple resistant strains. The antimicrobial agent susceptibility of ystB-positive biotype1A strains were significantly different to that of ystB-negative biotype1A strains.2. The Y. enterocolitica strains isolated from Gaomi City and Wulian County had many clones, and the gene heterogeneity was obvious. Some of the clones existed persistently. There might be relative dominant clones in Wulian County. Some of the clones might have relations with strains from other provinces.3. The PFGE types of Y. enterocolitica might have relations with motility, but no relations with biotypes, serotypes, virulence genotypes or antimicrobial agent susceptibility types were observed.4. PFGE was a typing method with high discrimination power, but a combined use with phenotyping was necessary in microbial source tracking... |
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