The Distribution Of Prophage And The Investigation Of Spontaneous Prophage Induction In Staphylococcus Aureus

Staphylococcus aureus is a Gram-positive bacterium and an important pathogen that can infect humans and animals.In addition,Methicillin-resistant S.aureus(MRSA)carries the mecA gene causing the resistant to β-lactams,making it difficult for S.aureus clinical treatment.Livestock-associated MRSA(LA-MRSA)is mainly isolated from livestock and slaughterhouse,which can be transmitted to humans through direct contact,posing a potential risk of zoonotic transmission.Bacteriophages are viruses that infect bacteria and can be classified into lytic and lysogenic phages depending on their infection outcomes.The bacteriophage infection process includes adsorption,nucleic acid injection,replication,assembly,and release.Phages can also integrate their genome into the host genome forming lysogens,and can be induced to the lytic cycle under certain conditions.Most S.aureus strains carry multiple prophages.Several studies have shown that prophages can carry and spread host genes in the bacterial population by transduction,which can enhance the host’s adaptability in a stressful environment.Based on epidemiological investigation of MRSA isolates,this study used whole-genome sequencing and PHASTER analysis to explore the distribution of prophages in MRSA isolates,and investigated the induction conditions of lytic phages,providing a theoretical basis for studying the interaction between prophages and hosts.This project conducted an epidemiological investigation of S.aureus in three pig farms in Jiangsu.We collected a total of 982 samples and isolated 53 MRSA isolates,with a prevalence of 5.40%,including 16 isolates from 23 fecal samples(7.17%);28 isolates from 541 anal(5.18%),four isolates from 105 nasal samples(3.80%),five from 113 environmental samples(4.42%).All 53 MRSA isolates were conducted antibiotic test for 14 antibiotics,and all isolates were resistant to penicillin,cefotaxime,ampicillin and tetracycline.According to the results of whole-genome sequencing and the analysis of resistance genes,all MRSA isolates contained mecA for β-lactam resistance.In addition,50 isolates carried cfr-mediated linezolid resistance,and 48 isolates carried fexA-mediated macrolide resistance.Multi-locus sequence analysis(MLST)identified two clone types,with 49 isolates belonging to ST398 and four isolates belonging to ST9.The spa typing analysis identified three spa types,which t034 was the predominant spa type(84.9%),four isolates were t899,and one isolates was t1793.The second part of the project investigated the occurrence of spontaneous prophage induction in S.aureus.To verify the induction effect of the quorum sensing system Agr on prophages in S.aureus.We constructed Δagr mutant on both NCTC 8325 strain and LA-MRSA CC398 strain,and measured the growth curve and phage plaque for both WT and Δagr mutant.Our results indicated that QS had no effect on the induction of prophages in S.aureus.We further investigated the prophage induction in all MRS A isolates and found 19 MRS A isolates could induce prophages in TSB and under mitomycin C treatment at 2 h and 8 h.We observed that MOI of 17 strains were significantly higher at 2 h than 8 h,which were also consistent with the results of NCTC 8325 and LA-MRSA CC398 and their Δagr mutants,indicating the phenomenon of spontaneous prophage induction occured in the early log phase during S.aureus growth.We further used PHASTER to predict prophages in 19 MRS A isolates,and used MEGA to analyze the phylogenetic relationship of all prophages,which prophages were clustered into four groups.MRSA strain YZU1852 were found to form clear plaque in both TSB and mitomycin C treatment,which were selected for the verification of spontaneous prophage induction in MRSA.With the analysis of PHASTER and RNA-seq at 2 h and 8 h,we found that YZU1852 contained two prophages.We further confirmed that only prophage YZU1852_phi1 could be circled and inducted at the attachment site attP(5’-AAAATGGCTATTGATACCATTTTGATA-3’)by PCR verification.TEM showed that YZU1852_phi1 belonged to the Siphoviridae family,with a typical long non-contractile tail sheath and a polyhedral capsid.By transcriptome and genome analysis,we confirmed the genomic structure of YZU1852_phi1,which the genome length was 43,349 bp with 65 genes for integration sites,integrases and anti-repressors for lysogenetic cycle,DNA replication and transcriptional regulation genes,lytic phage structural genes for lytic cycle.RNA-seq results at the 2 h and 8 h revealed that the head protein,tail protein,and lytic terminase genes of phage YZU1852_phi1 were significantly up-regulated in the early log phase(2 h).The expression of the agr gene cluster was consistent with the growth status of YZU 1852,which were significantly up-regulated in the late log phase(8 h).The expression of the SOS response gene recA did not show difference,and lexA and clpX in the clp cluster were up-regulated in the early log phase,while clpC was significantly up-regulated in the late log phase.qRT-PCR results verified that lexA and clpX were significantly up-regulated at 2 h,while clpC was significantly up-regulated at 8 h.Previous studies have shown that S.aureus ClpX protease played a key role in inducing the prophage by inhibiting the anti-repressor protein CI under mitomycin treatment.This study indicated that the spontaneous prophage induction could occurred at the early log phase in S.aureus,which may relate to the regulation of Clp proteins.This study explores the spontaneous prophage induction in S.aureus,and providing a theoretical basis for further research on the interaction between prophage and host.