Research On Serotyping And Molecular Typing Of S.Agalactiae From Different Hosts

Streptococcusagalactiae is an important pathogen that can infect human,livestock and kinds of fish.Streptococcus agalactiae not only seriously threat the development prospect of aquaculture,but also harm human health.These different hosts exist the possibility of cross infection whether because of these different source of Streptococcus agalactiae having the same genetic model is unknown.Therefore,the aim of this study is to understand the serotype diversity and molecular typing population structure of streptococcus agalactiae isolated from human,bovine,rabbit and fish.On the basis of PCR dentification of S.agalactiae with cfb gene,physiology and biochemistry,identifying Capsular polysaccharide serotyping(CPS),Multilocus sequence typing(MLST)and Pulsed field gel electrophoresis(PFGE)were used to confirmed genotypes and molecular characteristic diversity of 56 S.agalactiae strains isolated from human,bovine,rabbit and fish between 2006 and 2015 in Sichuan province.The results revealed that 56 of recoveried strains could form a smooth surface,micro uplift,edge neat milky circular colony in BHI tablet after incubation at 37? for 32h.And gram staining was spherical,single,double or catenulate G+ cocci.The cfb gene PCR identification results of 56 recoveried strains were consistent with the ATCC51487 standard strain,and a length of about 600bp of specific stripe could be obtained,then 56 recoveried strains were S.agalactiae combined with morphology and PCR detection.19 primers were synthesized for identification of capsular polysaccharide serotype in this exqeriment.The specific amplification was performed with multiple PCR method based on genomic DNA of 56 S.agalactiae isolated from human,bovine,rabbit and fish.The capsular polysaccharide serotype of strains was identificated according to the size and composition of specific bands of the genome.Electrophoresis results showed that there were two serotypes(la and ?),the serotype la could amplificated band of 688bp and 272bp at the same time,and the serotype ? could amplified band of 688bp and 352bp at the same time.The predominant serotype was la(85.7%)in those two serotypes,and distributed in strains from different hosts.Both bovine and rabbit S.agalactiae only had la serotype,human S.agalactiae existed la and III two serotypes.7 pairs of specific primers was synthesized according to 7 housekeeping genes(adhP?pheS?atr?glnA?sdhA?glcK?tkt)of S.agalacrtiae.The specific amplification was performed with PCR method based on genomic DNA of 56 S,agalactiae isolated from human,bovine,rabbit and fish.Sequence type determined by BLSAT in MLST database(http://pubmlst.org/sagalactiae/to).Results showed that MLST typing received nine STs sequence types(ST-7?ST-10?ST-19?ST-61?ST-103?ST-199?ST-486?ST-651?ST-891)and six clonal complexes CCs(CC-7?CC-10?CC-19?CC-23?CC-61?CC-103?CC-891),among which ST-891 a new sequence type discovered from fish S.agalactiae in this study and the most common STs of isolates was ST-891(35.7%).3 kinds of sequence types(ST-19?ST-103?ST-891)of 26 of fish S.agalactiae were identified,2 sequence types(ST-103.ST-486)of 12 of bovine S.agalactiae were identified,and one sequence type(ST-7)of 2 of rabbit S.agalactiae was identified in the test.However,16 of human S.agalactae possessed of the largest number of sequence types(ST-10?ST-19?ST-61?ST-199?ST-651)and clonal complexes(CC-10?CC-19?CC-23?CC-61?CC-103),speculating that human S.agalactiae may have more abundant genetic diversity.56 strains were buried in the plastic pieces,after protease K and cell lysis liquid relasing genomic DNA digested with Sma I restriction enzymes,then observated the electrophoresis images after 22 h under alternating current in the electrophoresis tank,took pictures and clusteried analysis with the Quantity One software.Results showed that PFGE clustering analysis was divided into 18 banding patterns(cluster A-R),among which 2 of rabbit S.agalactiae mainly distributed in the cluster I?J;12 of bovine S.agalactiae mainly distributed in the cluster L.P.Q;S.agalactiae from Schizopygopsis pylzovi Kessler and tilapia mainly distributed in the cluster D.E?F in 26 of fish S.agalactiae,but Schizothorax prenati S.agalactiae distributed in the cluster M?N.Similar to MLST classification results,the genetic typing distribution of human S.agalactiae was the most extensive in PFGE clustering analysis,mainly distributed in the cluster A?B?C?G?H?K?O?P?It was found that a large genotyping-diversity difference existed among S.agalactiae isolated from varous hosts,only cluster P was in possession of human and bovine isolates simultaneously.The genotyping of S.agalactiae from different hosts has not previously been reported in China and found that serotyping,MLST types,PFGE types and host's sources had no obvious correlation with each other.Different sources of strains could belong to serotype la similarily and all serotype Ia strains could distribut in varous STs and PFGE pattens.However Ia/ST-651 typing of human S.agalactiae were the clonal derivative of Ia/ST-103 typing of bovine S.agalactiae in dividing clonal complexes of MLST,which belonged to clonal complexes CC-103 similarily.Ia/ST651was single-locusvariant(SLV)ofla/ST-103,which suggested the existence of the possibility of cross infection at the genetic level.