The Genome Analysis And Study Of Resistance Mechanism In Shigella Flexneri

Shigellosis, also known as bacillary dysentery, is an intestinal-infectious-disease with highly contagious and serious harmfulness. The superiority strains prevalence in China is Shigella flexneri type 2. The susceptible populations were children under 5 years old and immune disorders people. Preferred drug is ciprofloxacin, and ceftriaxone and pivmecillinam often considered to be the second choice drugs which WHO recommended for the treatment of bacillary dysentery. The drug-resistance mechanism of Shigella flexneri 301 strain at the genomic level was studied by using ciprofloxacin and ceftriaxone commonly used in clinical, and a broad-spectrum tetracycline respectively induced resistance of Shigella flexneri, and then sequenced the genome of strain.1. Multiplex PCR system that was constructed according to various characteristics of gene [ipa HM32063), gtrⅡ（AF021347）, gtrⅩ（L05001）, R002（AF250878）] of Shigella serotypes that published in Gene Bank used to validate the Shigella flexneri 301 strain. This method was able to detect Shigella flexneri 301 strain in different bacterial species with the characteristics of rapid, sensitive, specific. It not only laid a foundation to reducing pollution in the following experiment, also created a rapid and effective method to detecting and isolating of Shigella.2. The results showed that the minimum inhibitory concentration（MIC） of drugs on bacteria were 64 μg/m L, 64 μg/m L and 128 μg/m L individually, and has reached the resistance level. We selected Shigella flexneri to de novo sequencing based on growth curve of MIC of each antibiotic-induced strain and the 5th generation,7th generation and 10 th generation of bacteria that induced by ciprofloxacin and tetracycline and the 5th generation,9th generation and 10 th generation of bacteria that induced by ceftriaxone were re-sequenced.3. It was found that 732 Mb data was generated by de novo sequencing method and 4,832 genes, 78 tandem repeats, 44 small satellite sequences, 11 microsatellite sequences, 102 t RNA and 16 r RNA were obtained through the genome composition analysis after the assembly. And then annotated the gene function, named and classified the 4832 gene sequences obtained finding using ten large databases, including GO, KEGG, Swiss-Prot, COG, NR, Tremble, CAZy, ARDB, VFDB and PHI. It was found that 317 SNP loci were detected, including 74 synonymous mutation sites,237 non-synonymous mutation sites and 6 nonsense mutation sites using the comparison method between the de novo sequencing data and published data of Shigella flexneri genome（NC₀04337）.5. The study found that there were many mutation sites in different genes using the method of comparison between the re-sequencing sequence and the de novo sequence. It contained met G gene loci 284497 in the TC-2, CIP-2, CRO-3 undergone T<->G mutation,gyr B gene loci 1953446 in the CIP-2 undergone C<->A mutation,gyd A gene loci 670796 in the CIP-7 undergone A<->C mutation,rob gene loci 335049 in the CIP-10 undergone G<->A mutation,nar X gene loci 1072783 in the CRO-8 undergone A<->G mutation,gyd A gene loci 670796 in the CIP-7 undergone A<->C mutation,gyd A gene loci 670796 in the CIP-7 undergone A<->C mutation,intergenic region loci 1538933 in the CIP3、CRO3、TC3 undergone A<->T mutation,intergenic region loci 1538929 in the CIP5、CR04、TC5 undergone G<->A mutation, and intergenic region loci 1538924 in the CIP8、CRO8、TC5 undergone G<->C mutation.6. The study confirmed that met G mainly affects the activation and transfer of methionine and the activation of other amino acids through the mutant gene annotation of met G（methionyl t RNA synthetase）. Also found that met G ultimately affects protein synthesis by the method of annotation of KEGG pathway. Intergenic mutation may affect the function of phthalyl prolyl cis-trans isomerase ppi A. These findings indicate that met G and ppi A mutation not only affect protein synthesis, also may be effective targets of antibiotics.