Comparative Genomic Study Of Mechanisms Of Cross Host Infection In Streptococcus Agalactiae

Streptococcus agalactiae,also known as group B Streptococcus(GBS),can infect a variety of hosts including humans,cows,fish,etc.It has been recognized as one of the major causes of preterm delivery,sepsis and meningitis in neonates,soft tissue infection and meningitis in adults.In cow,mastitis is the disease associated with GBS infection,which causes severe decrease in milk quality and quantity.GBS is one of the major causes of meningoencephalitis in fish and causes mortality of cultured fish in large areas.Milk and fish are the important source of food protein for human.The large-scale breeding of cows and fish is closely related to humans,and the carrying rate of GBS is very high in human,which cause the cross host infection risk of GBS among humans,cattle,and fish is extremely high.But so far,all is indecisiveness:is there any clinical cross infection between human and bovine or human and fish;are cattle and fish reservoirs of GBS infecting human;does human GBS transmit in cattle and fish.Therefore,it is necessary to evaluate the risk of GBS cross host infection and to study the mechanism of cross host infection.Horizontal gene transfer promotes rapid evolution of bacterial genomes and may be involved in the formation of bacterial species.Recombination occurs frequently and can involve a large areas of the GBS genome,which is related to genome mosaic organization and is a major driver of GBS genetic diversity.Mobile genetic elements(MGEs)include integrated plasmids,phages,transposons,and integrating conjugative elements(ICEs),etc.Horizontal gene transfer causes genome diversification and the emergence of virulent clones within the species,which affects the niche and host adaptability of GBS.Comparative genome analysis between bacterial strains that have different host specificity or virulence may help to rapidly screen for dispensable genes,gene deletions or mutations,and differentially-expressed proteins;it is also an effective way of studying the mechanisms of cross host infection,pathogenicity,and immunogenic ity of GBS.Therefore,this study was the first to analyze and study 92 pathogenic GBS strains isolated from humans in Guangxi by means of capsular serotyping,multilocus sequence typing(MLST),pulsed-field gel electrophoresis(PFGE),antibiotic susceptibility test,and virulence-associated genes assay.Animal infection tests and histopathological sections were utilized to investigate the pathogenicity of the invasive isolates to tilapia.Then targeted studying human GBS with different ability of cross host infection by comparative genomic research,including genome sequencing,pan genome,phylogenetic analysis based on whole-genome,genome sequence variation analysis,CRISPRs and prophage analysis,etc,and investigated the mechanism of cross host infection of GBS.The results of research were as follows:1.92 strains were isolated from human with 19 different diseases and 75%of them were isolated from females.The MLST revealed that the proportion(20 strains,21.74%)of CC103 strains increased significantly,especially that of ST485 strains(13 strains,14.13%)isolated from multiple diseases including neonatal infection.Three CC67 strains,which were considered as the CC strains specifically infecting dairy cows,were found to carry the gbs2018C gene specific to the CC17 strains.Genomic evolution analysis showed that both bovine and human CC 103 strains alternately form an independent evolutionary branch.Three human CC67 strains were in the same evolutionary branch together with both ST61 and ST67 strains that specifically infect dairy cows.2.The interspecies transmission of GBS to tilapia made the diseased fish characteristic of blackish body color,bleeding,circling movement,etc.And the analysis of their histopathological sections showed that streptococci could break through the blood-brain barrier into the brain tissue and proliferate largely there.The results showed that 21/92(22.83%)strains including all ST23 strains were highly pathogenic to tilapia.The pathogenicity of ST 19 strains was closely related to the serotype,and 17 CC19 strains of serotype III had weak or no pathogenicity to tilapia.By contrast,11 CC19 strains of serotype V and 1 CC19 strain of serotype II had strong pathogenicity to tilapia.The isolates of two human pathogenic ST1 strains had quite different pathogenicity to tilapia.The pathogenicity of NNA048 to tilapia was not different from that of tilapia GBS,while NNA038 does not have pathogenicity to tilapia.3.Comparative genomics studies show that bovine and human sourced GBS CC103 strains had very close genetic relationship.CC103 strains propagated across humans and cows,and evolved into several evolutionary branches.Among them,the strains from ST485 flora with high pathogenicity could specifically infect human.Compared to other CC103 strains,these strains all acquired Lac.2 deletion and CadDX gain in their genomes.4.The genome comparison and prophage analysis showed that the major genome difference between virulent and nonvirulent ST23 GBS to tilapia was attributed to the different prophage sequences.The prophage in the P1 region contained about 43%GC and encoded 28-39 proteins,which can mediate the acquisition of YafQ/DinJ structure for GBS by phage recombination.The ST23 GBS strains with this prophage were not pathogenic to tilapia,but the strains without the prophage or the phage that had gene mutation or deletion,especially the deletion of YafQ/DinJ structure were highly pathogenic to tilapia.Genome comparison showed that the major genome difference between ST1 GBS strains NNA048 and NNA038 was attributed to the different phage sequences,and there was a 49.8 kb length,intact phage sequence encoding 68 proteins in NNA048 genome.There were a total of 96 SNVs and 6 indels between the two genomes.5.The proportion of core genes in GBS ST19 serotype III isolates(67.09%)was lower than that of serotype V isolates(70.59%).Phylogenetic analysis showed that the evolutionary distance among III isolates was remoter than that among type V isolates.The CRISPRs structure of serotype III strains was more complex than that of serotype V strains,and the number and types of spacers were more than those of V strains.Genome comparison of the serotype III and V strains showed that large fragment recombinations were occurred in the capsular polysaccharide region(about 111 kb)and the adjacent ICEs region(about 106 kb).The capsular polysaccharide region comprised the entire capsular polysaccharide operon.The ICEs region of the serotype III strains contained ICE with mega element and the variation among strains was large,whereas the ICEs region of the serotype V strains contained ICEs with IQ element or erm(TR)and was very conservative among strains.In summary,this study firstly clarified the pathogenicity of human GBS with different serotypes,CC or ST on tilapia and the relationship between some strains and cattle strains.Then explored the mechanism of the cross host infection of different groups of GBS by the comparative genomics,and found that the genome structures or related genes including ICEs,phages,capsular polysaccharide,YafQ/DinJ,Lac.2 and CadDX,etc,played an important role in the cross host infection of GBS,which had an important significance to the prevention and control of epidemic disease and vaccine development of GBS,and also to laid the foundation for further exploring the mechanism of GBS cross host infection.