Serotype Of Streptococcus Agalactiae And Molecular Mechanism Of Antibiotic Resistance

Objective:Group B Streptococcus(GBS),also known as Streptococcus agalactiae,is a type of bacteria that causes illness in human in all ages.GBS disease can be especially severe in newborns,most commonly causing sepsis(infection of the blood),pneumonia(infection in the lungs),and sometimes meningitis(infection of the fluid and lining around the brain and spinal cord).The most common problems caused by group Streptococcus agalactiae in adults are bloodstream infections,pneumonia,skin and soft-tissue infections,and bone and joint infections.In order to research the serotype distribution and antibiotic resistance mechanism of Streptococcus agalactiae in urinary tract infections(UTI),the first part of this study was to collect the urine bacterial culture samples from December 2015 to December2016 in the microbiology laboratory of Suzhou Traditional Chinese Medicine Hospital,and 27 strains of Streptococcus agalactiae were identified.The second part was to study the characteristics of serotype distribution of Streptococcus agalactiae in Suzhou area based on the different capsular polysaccharide(cps)genes of Streptococcus agalactiae.The third part was that we used MIC method and K-B method to detect the antibiotic resistance of Streptococcus agalactiae,designed primers of common antibiotic resistance genes to detect specific antibiotic resistance genes,and provided reference for clinical rational drug using.In the fourth part,?a,?b,?,? and ? strains were sequenced according to the results of serotype and antibiotic susceptibility of Suzhou area urinary tract infections patients who carrying Streptococcus agalactiae.Using common assembly software to assemble the genome.The prediction genes of the assembled genome using Prokka were then compared with the Antibiotic Resistance Genes Database(ARDB),and the antibiotic resistance genes were identified according to the results of the alignment.MethodsPart One.Culture and identification of Streptococcus agalactiae from urineThe urine samples received were inoculated with the blood plate and the Mac-Conkey plate,and then the Streptococcus agalactiae was identified using the CAMP test and the VITEK~?2 Compact bacterial identification equipment.Part Two.Serotype of Streptococcus agalacitaeTwenty-seven strains of Streptococcus agalactiae were inoculated with blood plate,and DNA was extracted by using Ezup column bacterial genomic DNA extraction kit.The product was amplified by multiplex PCR.We determined the serological type based on the results of agarose gel electrophoresis,and the amplified sequence sent to the Sanger sequencing to confirm.Part Three.Streptococcus agalactiae antibiotic sensitive and antibiotic resistance gene detection of Streptococcus agalactiaeThe minimum inhibitory concentration(MIC)test was performed used VITEK~?2Compact microbial identification system.The Kirby-Bauer testing(KB method)and the inducible clindamycin resistance test were performed by manual method.Streptococcus agalactiae was tested for antibiotic susceptibility,and the antibiotic resistance gene primers were designed based on the results of antibiotic susceptibility test.The antibiotic resistance gene was confirmed by Sanger sequencing.Part Four Streptococcus agalactia sequencingDNA was extracted via QIAGEN DNeasy~?Blood&Tissue kit,and sent to the sequencing center to sequencing.Raw data which was removed connector and cleaned low-quality reads was assembled with SPAdes,Velvet,Abyss,Soapdenovo and SSPACE software.Using predictive annotation software Prokka to predict and annotate gene.The predicted results were submitted to the ARDB for online comparison to obtain specific antibiotic resistance genes.Result:Part One.Culture and identification of Streptococcus agalactiae from urine5443 samples of urine bacterial culture were cultured and identified with the VITEK~?2 Compact Bacteria equipment.There were 1,400 urine cultures of bacteria,of which 27 were Streptococcus agalactiae.Part Two.Serotype of Streptococcus agalacitaeThe PCR products were identified by agarose gel electrophoresis.Serotype ?a,?b,?,? and ? were identified,accounting for 18.5%(5/27),22.2%(6/27),29.6%(8/27),22.2%(6/27)and 7.4%(2/27)respectively.The product was sent to the sequencing company for Sanger sequencing,and sequencing result was compared with the expected gene sequence by blast.Part Three.Streptococcus agalactiae antibiotic resistance and antibiotic resistance gene detection(1)Streptococcus agalactia was mainly resistant to tetracycline(TET),Erythromycin(E),Clindamycin(CLI)and Fluoroquinolones(FQNS).The proportion of the total strains respectively was 74.1%,63.0%,55.6%and 48.1%.There were 4 strains which were intermediary to nitrofurantoin(NIT),accounting for 18.5%.All strains were sensitive to Ceftriaxone(CRO),penicillin Penicillin(PEN),vancomycin(VAN),linezolid(LNZ),Quinupristin-Dalfoprintin(QDA)and tigecycline(TGC).(2)The structural MLSB phenotype(cMLSB)refers to strains that are resistant to ERY and CLI.A total of 10 strains,accounting for 37.0%(10/27).Inducible clindamycin resistance test positive is the induced MLS_B phenotype(iMLSB).Negative is the MS phenotype.Accounting for all erythromycin resistance and clindamycin sensitive strains were 42.9%(3/7),57.1%(4/7).(3)Tetracycline-resistant genes were tetM predominant,accounting for 66.7%(18/27).tetO,tetK and tetL were 11.1%(3/27),3.7%(1/27)and 3.7%(1/27)respectively.tetL was not detected.The results were consistent with antibiotic susceptibility experiments.The genes resistant to erythromycin and clindamycin were mainly ermB,mefE,lnuB and ermTR,accounting for 37.0%(10/27),33.3%(9/27),29.6%(8/27)and 11.1%(3/27).Part Four.Streptococcus agalactia sequencingAccording to the susceptibility and serotype of Streptococcus agalactiae of UTI patients in Suzhou area,DNA was extracted from Streptococcus agalactiae No.24(?a),No.8(?b),No.11(?),No.17(?)and No.20(?)strains for next generation sequencing.Compared with the results,the results used SPAdes software assembly was the best.The genomic size was 2.12M,2.20M,2.11M,2.11M and 2.06M respectively.The number of scaffold which greater than 1000bp was 24,32,42,35 and 12 respectively.GC content is 35.64%,35.71%,35.54%,35.48%and 35.39%respectively.N50 was468051bp,183219bp,107763bp,117386bp and 1656169bp respectively.The maximum scaffold's length was 712759bp,394159bp,247508 bp,265957bp and 1656169bp respectively.2(baca,aac6ie,aph3iiia,ermB,lnuB,tetL,tetM),7(ant6ia,aph3iiia,baca,ermB,lnub,mefE,tetO),3(baca,ermT,tetM),5(baca,cata16,ermA,mefA,tetM)and1(baca)antibiotic resistance genes were obtained by comparison with ARDB.Conlusions:(1)Older female kidney patients accounted for the major part in the urinary tract infection by Streptococcus agalactiae.(2)The serotype of Streptococcus agalactiae in Suzhou was mainly ?a,?b,? and ?.Serotype ? was few.In the Suzhou area,Streptococcus agalactia serotype showed a certain regional.(3)Streptococcus agalactia was resistant to tetracycline,erythromycin and clindamycin mainly in Suzhou area while a small number of fluoroquinolones resistant.In the treatment of Streptococcus agalactia infection,penicillins and cephalosporins antibiotics was the preferred drug.Critical patients or who allergic to?-lactam drugs could use vancomycin.(4)In tetracycline resistance genes,tetM was dominant.Erythromycin and clindamycin resistance genes were ermB,mefE,lnuB and ermTR mainly.(5)Streptococcus agalactiae serotype ? was sensitive to all antibiotics.None antibiotic resistance gene was detected(6)The next generation sequencing to predict antibiotic resistance genes was very convenient and fast.With the prices continuing to decline,next generation sequencing could replace the PCR to detect antibiotic resistance genes in the future.(7)The identification,serotyping and antibiotic resistance gene detection system established in this study was also suitable for the detection of other strains of Streptococcus agalactiae.