Distribution Of Prophage Genes In Streptococcus Suis

Streptococcus suis(SS)is the main pathogen of Swine Streptococcosis,and it is also an important zoonotic pathogen.SS mainly parasitizes on the upper respiratory tract of pigs,which can not only cause meningitis,sepsis,arthritis and other diseases,but also mixed or secondary infection with other viruses or bacteria.At the same time,SS can infect human through damaged skin or digestive tract,which poses a serious threat to public health and food safety.Phage genes integrated into bacterial DNA or in the form of plasmids are called prophages.Prophage is closely related to the virulence of bacterial pathogens.It encodes a variety of virulence factors and has a certain impact on the environmental adaptability and drug resistance of host bacteria.Therefore,understanding and mastering the relationship between prophage and SS virulence,environmental adaptability and drug resistance will provide scientific basis for the pathogenesis and molecular epidemiology of SS.In this study,199 strains of SS isolated from clinically diseased pigs were used as test strains:(1)based on the serotype,virulence genotype,Multilocus sequence typing(MLST)and hemolysis identification of 199 strains of SS,the distribution of prophage in them was determined by PCR method which detecting the gene of prophage helicase and terminal enzyme.(2)According to the results of the pathogenicity test of SS prophage positive bacteria(referred to as positive bacteria)and prophage negative bacteria(referred to as negative bacteria)in Kunming mice,the LD50 of the mice was determined by the representative strains with strong lethal ability,and then the number of tissue charge bacteria and histopathological examination were carried out.(3)The biofilm forming ability of positive and negative bacteria was determined by crystal violet staining.(4)K-B disk method and micro broth dilution method were used to determine the susceptibility of positive and negative bacteria to antibiotics.(5)Transcriptome sequencing(RNA-seq)was used to compare the gene expression differences between positive and negative bacteria.The results showed that:(1)199 SS strains belonged to 16 serotypes,19 virulence genotypes,12 clones of 72 ST types.39 strains(19.59%)were positive for prophage,they were classified as 13 strains of SS2(33.33%),24 strains of SS3(61.54%)and 2 strains of amorphous strains(5.13%).The dominant virulence genotypes of 39 positive bacteria and 160 negative bacteria were epf-/mrp-/sly-/gapdh+/fbps+/orf2+;compared with the negative bacteria,the detection rates of gapdh,fbps and orf2 in the positive bacteria were not changed much,but the detection rates of epf,mrp and sly were decreased.ST117(43.5%),ST28(28.2%)and ST308(7.6%)were the dominant st types of positive bacteria,while ST1(14.3%),ST94(14.3%)and ST7(11.2%)were the dominant st types of negative bacteria,showing low homology and genetic diversity.97.4%(38/39)positive bacteria and 93.1%(149/160)negative bacteria showed ?-hemolysis on 5% rabbit blood medium.(2)Among 39 strains of positive bacteria and 7 strains of negative bacteria,11 strains and 2 strains of bacteria had a lethal rate of over 80.0%,the LD50 of 11 positive bacteria ranged from 8.4×108 to 2.36×109 CFU/piece,and that of 2 negative bacteria were 1.88×109 CFU/piece and 2.72×109 CFU/piece,respectively.There was no significant difference in LD50 between positive and negative bacteria(P>0.05).After the injection of BB15-4 with LD50 of 8.4×108 CFU/piece,the number of bacteria in various organs(lung,liver,spleen,kidney,heart and brain)was the most,the pathological changes were the most obvious.After the injection of BB14-5 with LD50 of 2.72×109 CFU/piece,the number of bacteria in each organ tissue was the least,and the pathological changes were the least.(3)The positive rate of BF formation of 39 positive bacteria was 97.4%,including 15(38.4%)with strong film forming ability(3+),14(35.8%)with medium film forming ability(2+),9(23.0%)with weak film forming ability(1+)and 1(2.5%)with negative(-).The positive rate of BF formation of 7 negative bacteria was 100%,including 5(71.4%)with strong film forming ability(3+),2 strains(28.5%)had medium(2+)film forming ability.(4)39 positive bacteria and 7 negative bacteria were multi resistant to tetracycline,erythromycin,clindamycin,azithromycin,ceftriaxone and doxycycline.The MIC values of positive bacteria BB15-4,FY13-4,BZ17-2 and negative bacteria BB14-5 to tetracycline and doxycycline were more than 256 ?g/ml,and there was no MBC value.There was no significant difference in drug resistance between positive and negative bacteria(P>0.05).(5)Compared with positive bacteria,virulence related genes pur D?SSUBM407₀143?cop A?tpx?pep T?fhs?neu C?cps E?cps B?msm K and gtf A were up-regulated in negative bacteria;BF formation related gene SSUBM407₁279 and SSUBM407₁791?Drug resistance genes tet O?bcr A?par C?par E and SSUBM407₀293 was also up-regulated in negative bacteria;At the same time,fatty acid biosynthesis pathway involved in the related genes SSUBM407₁477?SSUBM407₁676?SSUBM407₁680?SSUBM407₁672?SSUBM407₁670?SSUBM407₁671?SSUBM407₁674?SSUBM407 and the related genes involved in arginine and proline metabolism pathway SSUBM407₁304?SSUBM407₁303?SSUBM407₀427?SSUBM407₀274 also up-regulated in negative bacteria.The results showed that:(1)in 199 SS strains,the prophage genes were mainly integrated into SS3 and st117 and ST28 clones with relatively weak virulence,the distribution of SS major virulence genes,such as epf,mrp,sly,gapdh,fbps and orf2 was not correlated with that of prophage.also,there was no correlation between hemolysis and prophage.(2)There was no significant correlation between the presence of prophage and the increase or decrease of SS virulence;there was no significant correlation between the presence of prophages and the high or low resistance of SS;there was no significant correlation between the presence or absence of prophages and the formation of BF.(3)The presence of prophages can lead to down-regulation of genes associated with SS fatty acid biosynthesis and arginine and proline metabolism,thus,the metabolic level of fatty acids and amino acids is reduced,and the physiological activity of SS is affected.