Molecular Characteristics Of Streptococcus Agalactiae From Fish And Comparative Study On Phenotype And Genomics Of Two Different Serotype Stains

Streptococcus agalactiae is a Gram-positive Streptococcus with a wide host range,which can cause neonatal meningitis and dairy mastitis.It also can infect various farmed fishes,causing huge losses to aquaculture.Now some study found that Ia and ? are main serotypes of human,cow and fish.However,reports about the differences in phenotypic and molecular characteristics of the different serotype Streptococcus agalactiae from fish is still lacking.Therefore,we studied a variety of molecular characteristics of 28 clinical isolates of Streptococcus agalactiae,and two serotypes were selected as Ia and ? strains for biological characteristics and genomics research,to provide the experimental basis for the study in different serotypes of phenotypic and molecular characteristics and pathogenic mechanism and prevention of contact.1.The study of serotypes,resistance and virulence gene spectrum of 28 Streptococcus agalactiae from fish.The results of serotype,resistance and virulence gene spectrum of 28 Streptococcus agalactiae strains showed that 24 strains were serotype Ia,4 strains were serotype ? and serotype Ia was the dominant in prevalence.The resistance and resistance genes of serotype ? strains were higher than serotype Ia strains.The sensitivity rates of all the strains to florfenicol,ampicillin and vancomycin were all 100%.The resistance of erythromycin was highest,up to 42.6%.The detection rates of ermB,gyrA and parC genes were 100%,tetM and pbp2x genes were 82.1%and 25%.The results about virulence genes showed that the rate of cylE,hylB and cfb were 100%,the rate of bca,lmb also was high,cfb/cylE/hylB/bca was the most popular spectrum of virulence genes accounted for 21.4%.2.The comparative study of phenotypic characteristics of two different serotypes Streptococcus agalactiae?serotype Ia S07 and serotype ? S03?.To explicit the differences between molecular characteristics of serotype Ia and ? strains from Schizothoraxs,we screen out serotype Ia S07 and serotype ? S03 strains to compare their phenotypic characteristics.We analyzed and compared diverse characteristics and capabilities of serotype Ia S07 and serotype ? S03 preserved in the lab,such as growth curve,pH curve,cultivating temperature curve,tolerance to salt,adhesive capacity and invasion,antimicrobial resistance and pathogenicity.The results indicated that the growth trend of these two strains was S03 obviously faster than S07.S07 showed poor tolerance while S03 performed a relatively strong tolerance in different temperature,pH and salinity conditions.S03 and S07 were identical in the 40 kinds of biochemical test including sucrose,leucine arylamidase,beta-hemolysis and VP test,etc.But S03 was positive in 11 kinds of biochemical test including glucose,maltose,alpha-glucosidase and phosphate,etc.while S07 was negative.S03 showed resistant to seven kinds of antibiotics,however S07 was sensitive or intermediate to all antibiotics.The adhesion ability of S07was much stronger than S03.In the pathogenicity test of Schizothorax prenanti and Danio rerio,S07 showed significantly more virulent with the LD₅₀0 reaching 8.9×10³ cfu/mL and1.88×10³cfu/mL,compared to S03 with the LD₅₀0 reaching 5.27×10⁸cfu/mL and 7.09×10⁷cfu/mL,respectively.3.The comparative study of genomics characteristics of two different serotypes Streptococcus agalactiae?serotype Ia S07 and serotype? S03?.To explicit the differences in genomics between serotype Ia S07 and serotype ? S03strains,we carried out the genome sequencing and genomics analysis of S03 and S07strains.We extracted DNA from S03 and S07 strains,and the whole genome sequencing was carried out on the Illumina Miseq platform.The genomic sequence of two strains was assembled by Denovo.The sequences length of S03 and S07 genome was 2157989 bp and1795371 bp respectively.The S03 genome was significantly larger than S07.Using COG,ARDB and VFDB databases,the genomic sequences of S03 and S07 were annotated with functional gene,resistance and virulence gene,genome circle diagram.The number of gene islands predicted by S03 was far more than that of S07,but S07 predicted 4 phage genes,while S03 did none.Both the virulence genes and the resistance genes of the S03genome were more than that of S07.The S03 genome has a higher proportion of genes in information storage and processing and cell procedures and signals than S07,while the S07genome has a significantly higher proportion in metabolism than S03.The results of phylogenetic analysis showed that S03 was clustered with human serotype ? ST19 type SAG158 in China,SG-25 in Singapore and serotype ? ST736 type H002 in China.S07was clustered alone.In summary,the results of serotype,resistance and virulence gene spectrum of 28Streptococcus agalactiae strains showed that there were two serotypes of Ia and ? in the detected strains and serotype Ia was the dominant in prevalence.The sensitivity rates of all the strains to florfenicol,ampicillin and vancomycin were all 100%.Cfb/cylE/hylB/bca was the most popular spectrum of virulence genes.Two strains of different serotypes had great differences in biological characteristics,such as growth,antimicrobial resistance,pathogenicity,and genomics,such as genome size,function,virulence genes,resistance genes.Our study speculated that S03 and S07 may have different origins and evolutionary directions and provided some foundation for the further research on the pathogenic mechanism of bacteria and the resistance mechanism and evolution mechanism of Streptococcus agalactiae.