| Cronobacter sakazakii is a kind of harmful foodborne pathogens.It can infect babies,and cause bacteremia,meningitis and necrotizing enterocolitis.Recently,some researches show that immunocompromised people,especially the elderly,were infected by Cronobacter sakazakii,and this thing may cause diarrhea,wound infection and urinary tract infection.The rapid tracing of pollution after the outbreak is the key to reduce the risk of damage.However,according to the traditional typing methods of phenotype and serotype,it is difficult to separate pathogenic bacteria from non pathogenic bacteria.PFGE technology has a good classification ability of pathogens.By analysizing whole genome DNA of strains,the differences between the pathogen and its subtypes were found.more rapid and accurate judgment and traceability of Cronobacter sakazakii.Identification and traceability of Cronobacter sakazakii is faster and more accurate.This study explores Cronobacter sakazakii genotypes.Cronobacter sakazakii was analyzed by pulsed field gel electrophoresis with 4 restriction enzymes.More useful information for PFGE type of Cronobacter sakazakii was provided,PFGE type effect and reliability were improved.At the same time,Cronobacter sakazakii was typing in gene level by using a multilocus sequence typing technique,and this made that the analysis on the strain of genetic evolution and population structure was more accurate.By combining many genotyping methods of Cronobacter sakazakii,Cronobacter sakazakii can be quickly traceable after the outbreak of the pollution source.The pathogenicity of the clinical isolates and the relationship between virulence and genotype can be analyzed and judged.The results are as follows:(1)Pulsed field electrophoresis of Bacillus Craunot was carried out with four restriction endonucleases which were Xba I,Spe I,Nhe I and Bln I.The results showed that the Nhe I was the best in the single enzyme segmentation,followed by Spe I,Bln I,and Xba I finally.In the typing of Nhe I,Cronobacter sakazakii was digested into 17 ~ 23 effective bands,the34 strains were divided into 21 genotypes.The percentage of the strains,the average number of bundles,the Simpson index and the ratio of the knot and the strains were 38%,3.25,0.964 and 0.79.(2)When Cronobacter sakazakii was decomposed by two kinds of enzyme,the best result can be achieved by using Nhe I and Spe I.The 34 strains were divided into 22 genotypes.The indexes were 56%?3.17?0.955?0.76.When Cronobacter sakazakii was decomposed by three kinds of enzyme,the best result can be achieved by using Xba?,Spe?and Nhe?.The indexes were 38%,3.25,0.964,0.79.(3)When Cronobacter sakazakii was decomposed by four kinds of enzyme,the experimental effect was the best.The indexes were improved obviously.27 genotypes were produced.The indexes were 32%,2.75,0.977,0.85.The results show that when the strain is sub – Classified,if the more types of restriction enzymes were used,the typing effect will be better,and the result will be more accurate.(4)The 34 strains were amplified by using ERIC-PCR method.Electrophoresis patterns were analyzed by using the UPGMA cluster method.The similarity coefficient 90% is used as the critical value to distinguish the gene types.The 34 strains were divided into 13 genotypes.The indexes were 2.38,91%,0.910,0.32.PFGE was better than ERIC-PCR on typing Cronobacter sakazakii,but the ERIC-PCR Simpson index was more than 0.90.So ERIC-PCR has good resolution.(5)According to the results of PFGE,selecte 25 strains from the experimental strains for typing by MLST.Three types of ST are obtained.The clustering tree and the minimum spanning tree are obtained by analyzing.The typing result was in agreement with that of PFGE.In the same strain ST,the similarity coefficient of PFGE was more than 90%. |
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