Epidemiological Characteristics And Drug Resistance Of Streptococcus Agalactiae Isolated From Tilapia In China

Tilapia is an important aquatic species that is extensively cultured in southern China.In fact,the tilapia farming industry has become one of the pillar industries of aquaculture in southern China.However,the recent outbreak of tilapia streptococcosis in China has caused huge economic losses in the tilapia industry.The main causative agent of tilapia streptococcosis is Streptococcus agalactiae(Group B Streptococcus,GBS).Traditionally,the use of antibiotics for controlling tilapia streptococcosis has been limited because of many problems such as bacterial resistance,aquaculture environmental pollution,and drug residues.Thus,prevention and control of streptococcus outbreaks in tilapia is a major problem in the tilapia farming industry.In this study,300 strains of GBS from tilapia sampled between 2007 and 2016 from southern China were isolated and purified,and their molecular types were classified using molecular biology methods.The agglutination titers of serum antibodies with WC1535 anti-rabbit serum were determined in 77 strains of GBS inactivated using formaldehyde.169 representative strains were detected by 24 antibiotics and 10 possible drug resistance genes.The main contents and conclusions of the study were as follows:1.Molecular epidemiology of GBS isolated from tilapia in ChinaThe genomic DNA of GBS was extracted from 300 isolated strains of tilapia.Multilocus sequence typing(MLST),molecular serotype,prophage genes,pilus island genes,and related virulence genes were detected using PCR.According to the results,the 300 strains of GBS from tilapia could be classified into three genotypes: Ib-ST-261,Ia-ST-7-A,and Ia-ST-7-B subtypes.The Ib-ST-261 GBS strain was ?b multilocus sequence typing,261 MLST type and V2 virulence genes,which neither carried the prophage gene nor had the complete pilus island.It was popular only in southern China for a short time.The Ia-ST-7 GBS strain was ?a multilocus sequence typing,7 MLST type and V1 virulence genes,and was a major epidemic strain of GBS of tilapia in China.The Ia-ST-7 strains were divided into the Ia-ST-7-A and Ia-ST-7-B subtypes.In the Ia-ST-7-A subtype strain,the prophage genotype was Phage-1,and the pilus island was PI-2b.The Ia-ST-7 strains were prevalent mainly during 2007-2011.In the Ia-ST-7-B subtype strain,the prophage genotype was Phage-2,and pilus island was PI-1 + PI-2b.It was first discovered in 2010,and was popular in southern China from 2011 to 2016.The Ia-ST-7 GBS was the main subtype.Thus,we speculated that a genetic change occurred in the GBS subtype strain of tilapia in 2010-2011.2.Analysis of serum antibody agglutination titers in GBS of tilapiaIn this study,77 GBS strains of tilapia sampled between 2007 and 2016 were selected.These strains were cultured and then inactivated by treatment with formaldehyde.The agglutination reaction titers of the inactivated bacterial antigens and the WC1535 stain anti-rabbit serum were determined using the 96-well microtiter plate dilution method.Serum agglutination reactions of 46 strains of inactivated bacterial antigens of GBS from tilapia with WC1535 anti-rabbit serum were positive,accounting for 59.7% of the all strains tested.All positive strains reached the same agglutination titer of 1: 10240.Twenty strains showed negative reactions,accounting for 48.8%,and the lowest serum antibody agglutination titer was 1: 5.Among the strains tested between 2007 and 2011,88% were negative,and 86.2% of the strains tested between 2012 and 2016 were positive.We speculated that the most popular strains of GBS of tilapia changed around 2011.3.Determination of drug susceptibility and drug resistance genes in Streptococcus agalactiae in tilapia from ChinaIn this study,the disc diffusion method was used to determine the drug susceptibility of 169 GBS strains of tilapia.The results showed that GBS in tilapia cultured in China was resistant to multiple drugs,and that there were no significant differences in the resistance types of strains of each prevailing time.One hundred and sixty-nine strains of GBS were sensitive to 12 types of drugs,including macrolides(roxithromycin and acetyl spiramycin),?-lactam drugs(cephalexin,cefalotin,and ampicillin),lincomycin,tetracycline drugs(tetracycline and doxycycline),ofloxacin,rifampicin,chloramphenicol,and florfenicol.The drug sensitivity rate was 100%.These strains showed high drug resistance against aminoglycosides and sulfonamides,with resistance rates of more than 85%,while the resistance rate against spectinomycin was low.Resistance to quinolone drugs was significantly different between the different strains.The resistance rates of these strains to ofloxacin and enrofloxacin,norfloxacin,lomefloxacin,and enoxacin were 0,14.2%,82.8%,and 98.2%,respectively.No penicillin-resistant strains were found.These results could provide a scientific basis for developing clinical drugs against GBS of tilapia.In this study,10 resistance genes from 60 GBS strains isolated in 2016 were found to be negative.No resistance genes were detected.Therefore,the association between resistance phenotype and drug resistance genes of GBS of tilapia should be further studied.