Molecular Epidemiological And Drug Resistance Of Streptococcus Agalactiae Isolated In Tilapia From Guangdong Province

Streptococcus agalactiae has a broad host range that is an important pathogenic bacteria in both human and dairy cattle,it is also one of the main cause of aquatic animals infection.Recently,S.agalactiae has become an important pathogen in tilapia aquaculture worldwide,and caused serious economic losses,which hindered the healthy development of tilapia industry.In this study,molecular epidemiology and drug resistance of S.agalactiae isolated in tilapia were focused on,in order to provide scientific basis for the prevention and control strategy in streptococcal disease isolated in tilapia.The main research contents of the study were as follows:1.Development of detection method for S.agalactiae and isolation and identification ofstrains isolated from tilapia in 2016Three pairs of specific primers were designed based on the conserved sequence of hylB,ponA,and cfb genes of S.agalactiae published in GenBank,respectively.After the optimization of the reaction conditions and reaction system of multiplex PCR,the triple PCR method had good specificity and a higher sensitivity.Combined with conventional bacterial isolation and identification method,20 strains of S.agalactiae were isolated from 188 tilapia samples collected in 2016 from Guangdong Province,and the detection rate was 10.64%.2.Molecular types of S.agalactiae isolated in tilapiaIn this study,75 strains of S.agalactiae were isolated from tilapia sampled between 2009 and 2016 from Guangdong Province.Molecular serotype(MS),multilocus sequence typing(MLST)and multi-virulence-locus sequence typing(MVLST)methods were used for molecular typing analysis.The results were as follows: these strains were identified to MS Ia and they were of sequence type 7(ST-7),75 S.agalactiae was further classified into 14(a-n)virulence sequence types by MVLST.The virulence sequence types k,l,and m strains were prevalent mainly before 2014,while virulence sequence type a strains were predominant after 2015.Thus,we speculated that the differentiation of three virulence genes(srr-1,bibA and fbsA)occurred in 2014-2015.In addition,three sets of seven-fold PCR detection techniques were constructed to detect 21 virulence genes of 75 strains of S.agalactiae,fish reference strains(ATCC 51487)and human reference strains(ATCC BAA-1138).The virulence gene profiles of the 75 strains and ATCC 51487 were both V1,while the ATCC BAA-1138 was V2.It showed that these strains isolated from tilapia between 2009 and 2016 from Guangdong Province had a small variation in molecular genetic diversity,this implied that they maybe have same origins or sources of infection.Compared with MLST,MVLST appeared to bemore discriminatory and screened out potential pathogenic strains.3.Clustering analysis of antibiograms and antibiogram types of S.agalactiae strainsAntimicrobial susceptibilities of 75 S.agalactiae strains were determined by the disc diffusion method,and cluster analyses of the antibiograms and antibiogram types were performed.All strains displayed multidrug resistance(MDR)character.The antimicrobialresistance rates were highest(> 90%)to aminoglycosides,sulfonamides,pipemidic acid,and norfloxacin,followed by penicillin,ampicillin,and ciprofloxacin(26.7%–38.7%),whereas those to fradantin,lincomycin,erythromycin,ofloxacin,tetracycline,and florfenicol were low(< 10%),and no resistance to vancomycin,cefalexin,cefoxitin,amoxicillin,medemycin,doxitard,oxytetracycline,rifampin,chloramphenicol,or thiamphenicol was detected.Cluster analyses identified that the strains had 23 antibiogram types(A–W)and clustered in five groups(Groups I–V).The strains with higher antimicrobial resistance mainly clustered in Groups I and II.The results showed that the antibiograms varied and the antibiogram types were constantly updated and expanded.Effective measures must be taken to reduce the antimicrobial resistance and spread of MDR strains.The correlation analysis between antibiogram types and MLVST showed that 3 to 7 virulence sequence types exited in Groups I–V,one virulences equence type appeared in all five groups.The evolutionary relationships of similar antibiogram types strains were uncertain similar,and strains with similar virulence sequences might exhibit diverse antibiogram types.4.Detection of resistance genes of S.agalactiaeThe minimum inhibitory concentrations of ciprofloxacin-resistant S.agalactiae strains to ciprofloxacin were determined using a broth microdilution method.There was difference between the disc diffusion and broth microdilution method to detect antimicrobial susceptibilities of S.agalactiae to ciprofloxacin.Furthermore,the target genes(gyrA and parC)were amplified by PCR.Sequencing showed that there was no mutation in the QRDR region of the target gene.Sulfonamide resistance genes(sul1,sul2,and sul3)and integrase genes(intI1 and intI2)were detected by PCR assays.As a result,the detection rates of intI1,sul1,and sul2 were 100%,61.3%,and 45.3% respectively,while sul3 and intI2 were 0.It showed that the resistance gene was not a unique mechanism of drug resistance strains.