MLST Typing And Mutation Analysing Of Penicillin-binding Protein Gene Of Penicillin Resistant Streptococcus Pneumoniae In Children Infected

Objective Through the MLST typing of penicillin resistant Streptococcus pneumoniae children infected,to understand the genetic background of resistant strains,at the mean time to analyze the mutations of penicillin-binding protein gene in resistant strains,to know the relationship between penicillin-binding protein gene mutation and penicillin resistance.Method(1)A total of 60 strains of SP strains the diagnosis of infectious pneumonia hospitalized children were randomly selected from penicillin MIC=2.0?g/m L,4.0?g/m L and ?8.0?g/m L each of 20 strains,as experimental strains of this study.Use the Phoenix-100 automatic microbiological identification/drug sensitivity analysis system and its accessory to do the clinical separation of SP strains of penicillin and other antimicrobial drugs sensitive test.The results showed that the MIC value of penicillin was checked again by E-test method.(2)Use Bio Fast soil genomic DNA extraction kit to extract the total DNA of Streptococcus pneumoniae.(3)Multilocus sequence typing: PCR amplification of seven housekeeping genes(aro E,gdh,gki,rec P,spi,xpt and ddl)of Streptococcus pneumoniae were carried out according to the primer sequence provided by MLST website.The amplified products were purified and sequenced.The sequencing results were modified,and then to compare with standard sequences using Meg Align software.Intercepting the standard length of the analysis sequence submitted to the site to determine the allele and ST(Sequence type)typing,and the genetic relationship between strains was analyzed using e BURST V3 software.(4)PBPs gene PCR amplification: Using Streptococcus pneumoniae DNA as template,PCR was performed on 6 genes(pbp1a,pbp1 b,pbp2a,pbp2 b,pbp2x,pbp3)of PRSP penicillin-binding protein by using BOX-PCR Mix kit.The amplified products were purified and sequenced,the results using MEGA 7.0 software to compare with penicillin-sensitive Streptococcus pneumoniae R6(DDBJ/EMBL/Gene Bank accession number NC003098).Focusing on the current internationally accepted PBPs conservative sequence for comparison analysis.Result(1)10525 cases of sputum specimens were detected 5872 strains of pathogens,the positive rate of 55.8%,which detected 1317 strains of Streptococcus pneumoniae,the detection rate of 12.5%,accounting for the total number of pathogens accounted for 22.4%,ranking first.(2)The resistance rates of SP to erythromycin,clindamycin and tetracycline were 98.2%,95.4% and 95.3% respectively,and the resistance rate of penicillin was 64.2%(MIC?2?g/m L),and other ?-lactam antimicrobial agents exhibit varying degrees of resistance.(3)There were 24 ST type and 6 new ST types in 60 PRSPs.There was a dominant type of ST271,accounting for 31.7%(19/60),using e BURST for homology analysis,then find 4 clonal complexes and 20 single clone.CC271 is one of the prevalent clonal complexes,it is belonging to the Pneumococcal molecular Epidemiology Network(PMEN)complex(Taiwan19F-14 complex).The PMEN14 clone has 25 strains,accounting for the total strain of 41.7%.(4)PBP3,PBP1 b sequences were not found mutations,PBP2 b,PBP1a,PBP2 x,PBP2a gene conserved or conserved zone attachments were found amino acid mutation.Conclusion(1)Streptococcus pneumoniae is an important pathogen in children with infectious pneumonia,drug resistance is serious,amoxicillin and penicillin injection is more sensitive for the treatment of preferred medication.(2)PRSP advantage type is ST271,PMEN14 clonal complexes is the main reason of resistance in this region.(3)High levels of resistance to Streptococcus pneumoniae are closely related to the variation of PBP2 b,PBP2a and PBP1 a.(4)Variation of PBP1 a is a supplement to high levels of resistance to Streptococcus pneumoniae rather than a necessary factor.(5)When Streptococcus pneumoniae occurs in the middle or high level of resistance,the vast majority of them mergering 4-6 amino acid replacement mutation in different PBP sequences,but the merger of multiple amino acid replacement mutation is not necessarily lead to a corresponding increase in drug resistance levels,the Ser461 Ala substitution of PBP2 a sequence and the Thr451 Ala / Ser substitution of the PBP2 b sequence may play a dominant role.(6)PBP3 and PBP1 b of PRSP were highly conserved in this region,there was no gene mutation was found in this two sequences.