Molecular Epidemiology And Biofilm Formation By Streptococcus Agalactiae Causing Infections In China

Background:Streptococcus agalactiae(also known as group B Streptococcus,GBS),a com-mensal of human gastrointestinal and genitourinary flora,is now the major cause of sepsis and meningitis in infants.GBS has become more prevalent in pregnant women,as well as non-pregnant adults,especially in the immuno-compromised and the elder-ly with significant morbidity.Understanding molecular characteristics of infective GBS is crucial for the treatment and prevention of clinical infections due to GBS.However,clinical data describing epidemiological feature of GBS in China are larg-ely incomplete and mainly focus on Beijing and Shanghai.Relative to planktonic states,biofilm is another states for bacterial pathogens to adapt to changing environmental conditions.Biofilm may enhance resistance to extre-me pH,antimicrobial agents and immune cells and lead to persistent colonization and infection.Therefore,understanding the molecular mechanism underlying biofilm for-mation and regulation in pathogens is essential to the control and treatment of GBS infections and establishment of suitable methods for biofilm formation is necessary for fundamental studies of GBS biofilm formation.Objectives:1.To characterize the antibiotic resistance,serotypes,virulence,STs,and CCs in GBS isolates and to compare the clinical features of invasive and non-invasive GBS isolates.2.To clarify the whole genome feature of an invasive GBS isolate and these genomic data may provide a useful basis for the fully understanding of the patho-genicity mechanisms of GBS at molecular level.To describe the characteristic of genetice volution and variability of GBS in china through comparative genome analysis.3.To establish a suitable protocol for GBS biofilm formation which is necessary for fundamental studies about it.To investigated the influences of environmental and genotypic factors on biofilm formation by clinically isolated GBS.To study the virulent factors involved in biofilm formation by hyper-virulent lineage ST-17 strains and to gain insight into the underlying molecular mechanism.Methods:1.We collected 113 GBS isolates causing invasive and non-invasive diseases from seven tertiary hospitals in Shenzhen,Guangzhou and Wuhan from October 2014 to September 2016.Electronic medical records were retrospectively reviewed and correlated.2.All isolates were confirmed to be GBS using CAMP testing and the Vitek-2 Compact Microbiology System.The antibiotic susceptibility of isolates was assessed using the disk diffusion test according to CLSI guidelines(CLSI,2015).Macrolide and tetracycline resistant isolates,as defined by phenotypic methods,were tested for the presence of the ermA,ermB,mefA/E,tetK,tetL,tetM and tetO genes by multiplex PCR using previously described primers.3.GBS capsular serotypes(?a,?Ib,?-?)were determined by multiplex PCR.The isolates that failed to type were listed as non-typeable(NT).Multiplex PCR assays were also performed to detect the PI-1,PI-2a or PI-2b genes and the presence of alp genes(bca,eps,rib,alp2/3 and alp4)and six other virulence genes(bac,lmb,hylB,cylE,hvgA and scpB)were identified by ordinary PCR.4.MLST was conducted by sequence analysis of seven genes,the sequences of the seven loci obtained were concatenated and used to build a phylogenetic tree using the MEGA6 program with the UPGMA method.5.The whole genome was sequenced using the PacBio RSII and Illumina HiSeq 4000 platform.11 databases were used for gene annotation and protein classification.Comparative genomics analysis between our strain and reference strains was performed.6.Biofilm formation by 111 clinical GBS isolates was assessed under batch and fed-batch growth conditions at different pH values(5.0 and 7.8)and in the presence or absence of glucose.GBS biofilms were visualized by confocal laser scanning microscopy(CLSM)and cell viability in biofilm was obtained with the XTT cell viability assay.7.qRT-PCR was used to compare the expressions of the adhesin encoding genes in biofilm and their planktonic counterparts and the expression of adhensin encoding genes at acidic and neutral pHs was also compared.Results:1.Sepsis was the most common clinical manifestation among patients with invasive infection,while urinary tract infections were the most prevalent manifest-tation among patients with non-invasive infection.The C-reactive protein(CRP)and procalcitonin(PCT)levels as well as absolute neutrophil counts(ANC)were significantly higher in patients with invasive infections.2.MLST analysis resulted in 24 individual STs including 7 new ones(ST897,ST898,ST917,ST918,ST919,ST938 and ST939).We found that 17 STs comprising 84%of the isolates clustered into five CCs(1,12,17,19 and 23)and the remaining seven were singletons.CC17 were significantly associated with invasive infections,especially with late-onset disease(LOD)occurs in infants.3.We also identified six serotypes(?a,?b,?,?,? and ?)encompassing 109 isolates with the remaining four non-typeable,Serotype III was the most prevalent serotype(54.0%),followed by serotypes Ib and la(20.4 and 13.3%,respectively).4.The virulence genes cylE,Imb,scpB and hylB were detected in almost all GBS isolates,All except three non-invasive isolates were positive for the presence of only one alp gene.The most frequent alp gene was rib(46.9%),followed by the eps(27.4%),bca(21.2%)and alp2/3(1.8%)genes.The bac gene occurred in 14.2%(16/113)of isolates primarily in association with bca(13/16)and this association was limited to CC12.All the isolates harbored at least one pilus variant(PI-1,PI-2a or PI-2b).PI-1,PI-2a and PI-2b were detected in 38.9%,56.6%and 41.6%of isolates,respectively.5.Significant associations between serotypes,CCs and alp gene or PI profiles were found,such as Ia/CC23 and eps,Ib/CC12 and bca,?/CC17 or CC19 and rib,PI-2b and III/CC17,PI-2a and Ia/CC23 and PI-1+PI-2a and Ib/CC12.6.All GBS strains were susceptible to penicillin and vancomycin.However,95(84.1%)isolates were resistant to tetracycline,91(80.5%)to erythromycin,81(71.7%)to clindamycin,27(23.9%)to levofloxacin and 8(7.0%)to chloramphenicol.Among the erythromycin resistant isolates,a predominance of the cMLSB phenotype mediated by the ermB genes was observed and the most common determinants of the ubiquitous tetracycline resistance were tetM and tetO.1.The genome of GBS32790 was 2,148,904 bp and were predicted to have genes encoding 2102 proteins,representing 87.4%of the whole sequence.Further-more,three prophages and one types of CRISPR were predicted in the genome and 2099(99.28%)genes were annotated with databases.8.Comparative genomic analysis of GBS32790,GBSCHO1 and other three clinical GBS strains isolated from China,revealed that extensive similar predicted proteins existed in these isolates.The results of synteny showing the genomes of GBS32790,GBSCHOl were similar,even there were some strain specific genes among them.The lack of PI-1 locus in GBS32790 strains was due to a fragment(16 kb)deletion.9.Fed-batch mode enhanced biofilm formation by GBS and the mass increase of biofilm formation by strong biofilm producers was higher than that of by weak bio-film producer.The biofilms produced by the fed-batch mode not only show a higher biovolume but also contain a higher percentage of viable cells,as suggested by the results obtained with CLSM analysis and the XTT cell viability assay.10.We observed a significant increase in the expression of bsaB,hvgA,fbsA,scpB and srr-2 in ST-17 GBS stains in biofilm than in planktonic.fbsB was unaffect-ed and rib was down-regulated in biofilm compared to planktonic.The expression of hylB and lmb was strains dependent.11.A significant decrease in the expression of bsaB hvgA,fbsB,fbsA,scpB,hylB and rib in ST-17 GBS stains was observed in pH 5.0 compared to neutral pH.The regulatory of srr-2 and lmb was different among strong-,moderate-and weak bioflim formers.Conclusions:1.CG17 and its genetic characteristics may provide alternative molecular biomarkers for diagnosis and prognosis of GBS infections.2.The inflammatory markers CRP,PCT and ANC may have future roles in the diagnosis of invasive infections caused by GBS.3.Penicillin could be used as a first-line agent for intrapartum prophylaxis and treatment of GBS infections,but prophylaxis or treatment with erythromycin and clindamycin should be considered after confirmation of susceptibility.4.Whole genome of a ST-17 GBS strains was sequenced and assembled completely which will provide a basis for further research in pathogenicity mechanisms of this hyper-virulent lineage.5.The GBS strain genomes were similar and the lack of PI-llocus in GBS32790 strains was due to a deletion of 16 kb fragment that normally contains the PI-1 operon.6.The fed-bath mode and acidic pH strongly enhanced GBS biofilm formation in vitro.A correlation between the hyper-virulent clonal CC17 and a strong biofilm phenotype was observed.7.Many adhesins such as bsaB,hvgA,fbsA,scpB and srr-2 mighit be involved in ST-17 GBS biofilm formation.8.Environmental pH regulated the biofilm formation by ST-17 GBS,probably not through the modulation of expression of adhesins.Further study is needed to confirm the underlying mechanism of this regulatory effect.