| Streptococcus agalactiae(group B streptococcus)is a?-hemolytic Gram-positive streptococcus.It often colonized in the vagina and rectum of women and can vertically infect the fetus.S.agalactiae is the leading cause of neonatal sepsis and meningitis in many countries.It is also an important pathogen among pregnant women,immunocompromised adults,and the elderly.The pregnant women with GBS colonization can selectively perform intrapartumantibiotic prophylaxis(IAP),usually by intravenous penicillin and ampicillin,and if the patient is allergic to?-lactam antibiotics,cephalosporin,erythromycin and vancomycin can be an alternative choice.However,with the wide use of antibiotics,drug resistance in GBS have gradually emerged.According to the drug resistance data of 2017 in China reported by China Antimicrobial Resistance Surveillance System,the resistance problem of erythromycin,clindamycin and levofloxacin resistance were serious in GBS.The prevalence of erythromycin,clindamycin and levofloxacin resistance in S.agalactiae was 73.7%,66%and 53.6%,respectively.Therefore,we should focus on the monitoring of the molecular characteristics and drug resistance of S.agalactiae,the investigation of the resistance mechanism and the genetic context of resistance genes and the exploration of possible resistance gene dissemination modes since all the aspects have the clinical significances for prevention and treatment of nosocomial infections and drug-resistant bacteria.In the first part of this study,we collected 6 strains of S.agalactiae from different specimens,including blood samples of the infants and vaginal secretion of their mothers.Antimicrobial susceptibility tests were performed by disk diffusion method,and multilocus sequence typing(MLST)and pulse field gel electrophoresis typing(PFGE)were also carried out to determine the epidemiology of the strains and whether it was nosocomial infection.Determinants of virulence factors were detected by PCR,including capsular polysaccharide,surface proteins,and pilus-like structures.Sag7/Sag13/Sag37/Sag66 are of the same results of antimicrobial susceptibility tests,while Sag142 and Sag158 are the same but different from the other 4 strains.These four strains were of the same clone type,and Sag142 and Sag158 were of another clone type.So we arrived to the conclusion that No.66mother was colonized by Streptococcus agalactiae and vertically infected No.37 newborn.No.37 newborn horizontally transmitted to No.7 and No.13 newborn.The strains of Sag7/Sag13/Sag37/Sag66 were serotype Ib,and contain bac,bca,alp2/3 and alp4 genes,while Sag142 and Sag158 are serotype III and contain?gene.All the six strains contain PI-1 and PI-2a genes.Among all the 6 strains,Sag7/Sag13/Sag37/Sag66 are of the same clone type and were horizontally transmission.Sag142 and Sag158 was of another clone type.All the 6 strains contained all the major virulence factors of S.agalctiae,including capsular polysaccharide,surface proteins and pilus-like structures,which play important roles in bacterial adhesion,immune evasion and invasiveness.In the second part of this study,Sag37 and Sag158,isolated from blood samples of new-borns with bacteremia,were further investigated.Sag37 was highly resistant to erythromycin and tetracycline,and susceptible to levofloxacin and penicillin,while Sag158was resistant to tetracycline and levofloxacin,and susceptible to erythromycin according to the Etest.Conjugal transfer experiments were performed using the strain Sag37(erythromycin-resistant and levofloxacin-susceptible)as the donor and the strain Sag158(erythromycin-susceptible and levofloxacin-resistant)as the recipient.Through mating experiments,the erythromycin resistance gene was found to be transferable from Sag37 to Sag158.Sma I-PFGE revealed a new Sma I fragment,confirming the transfer of the fragment containing the erythromycin resistance gene.Whole genome sequencing and sequence analysis revealed a mobile element,ICESag37,which was characterized using several molecular methods and in silico analyses.ICESag37 is a novel integrative and conjugative element carrying multidrug resistance and potential virulence factors.ICESag37 was excised to generate a covalent circular intermediate,which was transferable to S.agalactiae.Inverse PCR was performed to detect the circular form.A serine family integrase mediated its chromosomal integration into rumA,which is a known hotspot for the integration of streptococcal ICEs.The integration site was confirmed using PCR.ICESag37 carried genes for resistance to multiple antibiotics,including erythromycin[erm(B)],tetracycline[tet(O)],and aminoglycosides[aadE,aphA,and ant(6)].Potential virulence factors,including a two-component signal transduction system(nisK/nisR),were also observed in ICESag37.S1-PFGE analysis ruled out the existence of plasmids.ICESag37 is the first ICESa2603 family-like element identified in S.agalactiae carrying both resistance and potential virulence determinants.It might act as a vehicle for the dissemination of multidrug resistance and pathogenicity among S.agalactiae.In the third part of this study,we investigated a rare phenotype in S.agalactiae,the L phenotype which was of lincosamide resistance and macrolide susceptibility.Clindamycin is a lincosamide antibiotic used to treat staphylococcal and streptococcal infections.Reports of clinical Streptococcus agalactiae isolates with the rare lincosamide resistance/macrolide susceptibility(L~R/M~S)phenotype are increasing worldwide.However,the phenotype has not been reported in China.In this study,we characterized two clinical S.agalactiae strains with the unusual L phenotype in China.Sag3 and Sag27,susceptible to erythromycin(MIC?0.25 mg/L)but resistant to clindamycin(MIC?1 mg/L),were identified among 186 S.agalactiae isolates.Using a whole genome sequencing approach,the lnu(B)gene was found to be responsible for the L phenotype and the genetic context of this gene was nearly identical in the two strains.It was chromosomally located in an aadE-spw-lsa(E)-lnu(B)resistance gene cluster adjacent to an upstream 7-kb tet(L)-cat resistance gene cluster.The two clusters were flanked by the IS6-like element IS1216.Efforts to transfer lincomycin resistance by conjugation and to identify the circular form by inverse PCR were unsuccessful.These results establish the presence of the L~R/M~S phenotype associated with the lnu(B)gene in clinical S.agalactiae isolates in China.The lnu(B)-containing multi-resistance gene cluster possibly acts as a composite transposon flanked by IS1216 and as a vehicle for the dissemination of multidrug resistance among S.agalactiae.In conclusion,S.agalactiae can cause serious neonatal infections,and especially those that are multi-resistant,are an increasing major problem of anti-infective treatment.Moreover,S.agalactiae often acquires pre-existing resistance determinants from the bacterial gene pool by means of mobile genetic elements,including insertion sequences,transposons and ICE.Together these elements play a central role in facilitating horizontal genetic exchange and therefore promote the acquisition and spread of resistance genes.This aggravated the drug resistance problem of S.agalactiae,and increased the difficulty of clinical treatment.Therefore,we should choose appropriate antibiotics for the anti-infective treatment and monitor of the molecular characteristics of multi-resistant strains.We also need to explore the mechanism of resistance dissemination and pay attention to the prevention and control of nosocomial infections. |
PDF Full Text Request