| Staphylococcus aureus is a Gram positive and coagulase positive conditional pathogen,which has the highest clinical detection rate worldwide.Compared with methicillin-sensitive Staphylococcus aureus,methicillin-resistant Staphylococcus aureus(MRSA)is resistant to multiple antibiotics,resulting in increased difficulty in clinical treatment,increased treatment costs,and easy to cause adverse prognosis for infected persons.Therefore,MRSA is one of the multi-drug-resistant bacteria that has received much attention from researchers.In recent years,the incidence rate of MRSA has increased in all regions of the world.It is one of the common pathogens of hospital and community acquired infections in clinical practice,which has brought huge public health burden to society and seriously threatened the life and health of the public.Because MRSA can lead to systemic multi-system infections with a high mortality rate,it is of great practical significance to carry out genome sequencing and pathogenic mechanism research on commonly isolated bacteria in clinical practice for developing new treatment strategies in the context of increasingly difficult clinical treatment of multi-drug resistant bacterial infections.Our research team has been engaged in research on the molecular epidemiology,pathogenicity,and pathogenesis of MRSA strains in Yunnan Province,and has established a resource database and molecular typing database of MRSA strains in Yunnan Province.The previous research showed that ST239-t030 strain is mainly prevalent in the hospital,and is one of the most important pathogenic bacteria causing hospital-acquired infections;The ST59-t437 strain is mainly prevalent in community settings and can infect children with severe systemic infections such as pneumonia.Why do the two main MRSA strains form dominant clones in different niche environments? What are the characteristics of their respective genomes and pathogenicity? In order to solve the above problems,the following researches have been carried out in this topic: Firstly,the dominant strains of MRSA from different sources in the region have been selected for genome sequencing and analysis to study the genomic characteristics of the strains,the distribution of resistance and virulence genes,homologous relationships,and phylogeny.Secondly,on the basis of the above studies,we selected dominant clonal strains of ST239-t030 and ST59-t437 to conduct in vitro pathogenicity studies,mainly involving the growth and replication of the strains,adhesion,expression of virulence genes,and cytotoxicity.Thirdly,using the dominant clonal group MRSA strains of ST239-t030 and ST59-t437 as research objects,we conducted in vivo pathogenicity studies on experimental animals to further explore the differences in pathogenicity between different types of dominant clonal group strains in vitro and in vivo.Finally,RNA-seq research was conducted on the dominant MRSA clones selected in the experiment to explore the signal pathways of differential expression,laying a foundation for subsequent research.The main results were shown as follows:(1)We selected 60 MRSA strains for whole-genome sequencing,of which 40 were isolated from patients while the rests were isolated from food.Of the patient isolates,16 were community-acquired(CA)and 24 were hospital-acquired(HA).Most cases of HA-MRSA infection are elderly patients with underlying diseases,while CA-MRSA is prone to infect children under 5 years of age,with more severe clinical symptoms.(2)The average genome size of the 60 isolates was 2.79±0.05 Mbp,the GC content was 33%,and the coding rate was 84.50±0.20%.The core genome size of these isolates was 1,593 genes.Pan-based genome and SNP phylogenetic analysis indicated that all sequenced MRSA generated five clusters.Cluster ST239-t030 contained all the HA-MRSA cases in this study;cluster ST59-t437 covered both community and hospital acquired infection cases.The results of virulence gene annotation showed that the number of genes annotated by virulence factors such as capsule,enterocolin,hemolysin,and cytolysin of all MRSA strains was relatively high.Compared with the 24 reference strains from different regions,the ST239-t030 strain in this study was closely related to the T0131 strain in Tianjin,China,and belonged to the "Turkish clade" of Eastern Europe;ST59-t437 strains clustered into two groups of MRSA clades belonged to the “Asia Pacific”(AP)clone and the “Taiwan”(TW)clone,respectively.(3)ST239-t030-SCCmecⅢ(YNSA163),ST59-t437-SCCmecⅣa(YNSA7)and ST59-t437-SCCmecⅤb(YNSA53)were representative dominant clones of HA-MRSA and CA-MRSA in the region respectively and were used for subsequent experimental analysis of pathogenic mechanisms.(4)Compared with ST239-t030,ST59-t437 strain showed faster growth ability,higher survival rate against human blood,higher toxin secretion level and cytotoxicity.The virulence and regulatory genes of hld,psm-α,RNAIII,agr A and crt N were highly expressed in CA-MRSA isolates,especially for the ST59-t437-SCCmecⅣa subclone.Among these MRSA bacteria,ST239-t030 strain had the strongest adhesion and biofilm formation ability.(5)Animal experiments revealed that the mortality rate of BALB/c mice infected with YNSA7 strain was the highest(83%),followed by YNSA53 strain(50%),and YNSA163 infection did not cause mouse death.Histopathological analysis of infected animals showed that the lung was the most severely damaged organ,followed by the spleen and kidney,especially for ST59-t437 MRSA.Compared with YNSA53 survival cases and YNSA163 cases,interleukin 6(IL-6)and IL-10 were highly expressed in both the spleen and lung of YNSA7 and YNSA53 dead cases.The animal experiment results of YNSA7 infection group and IL-6 intervention group showed that IL-6 monoclonal antibody intervention significantly improved the survival rate of mice infected with YNSA7.The pathological results showed that the inflammatory cell exudation and tissue necrosis in the lungs of experimental animals in the IL-6 intervention group were significantly improved compared to those in the YNSA7 infected group.(6)RNA-seq results of three MRSA isolates revealed that most of the differentially expressed genes were involved in bacterial cellular processes,metabolism and genetic information processing.Specifically,clp P,chp chemotactic inhibitor,fnb B,pathogenicity island protein and virulence-related protein E were highly expressed in the YNSA7 strain.Summary: ST239-t030 and ST59-t437 were the two major MRSA clones in the clinic.The ST239-t030 isolate has established a dominant epidemic clone in hospital acquired infections in the region for many years,while ST59-t437 showed the characterstics of multi-origins.ST59-t437 strains exhibited higher pathogenicity than ST239-t030 isolates,and ST239-t030 MRSA revealed features prevalent in hospital settings,particularly for adhesion and biofilm-forming abilities.Cytokine storm induced by CA-MRSA strains,especially for ST59-t439-SCCmec IVa clone,resulted in severe infection and lethality in BALB/c mice.High expression of IL-6 was the cytokine storm pattern of CA-MRSA infection.Some virulence-related genes(clp P,chp,fnb B,pathogenicity island protein and virulence-related protein E)were highly up-regulated in ST59-t439-SCCmecⅣa clone,suggesting that the relevant virulence genes may play an important role in the pathogenesis of highly virulent strains,which may provide clues for the subsequent study of the pathogenesis of highly virulent MRSA strains. |
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