Whole-genome Sequencing And Virulence Characteristics Of Staphylococcus Aureus ST88

ObjectiveRecently,Staphylococcus aureus ST88 strains has been increasingly isolated in the Asia-Pacific region and African countries.Genomic studies and virulence phenotypic characterization of Staphylococcus aureus ST88 strains are relatively limited.In this study,whole-genome sequencing(WGS)and virulence phenotypic studies were performed on ST88 strains.This study investigated the genomics characteristics and potential virulence of ST88 strains collected from a tertiary hospital in Nanchang.Methods1.Non-duplicate clinical isolates of Staphylococcus aureus were isolated from a tertiary hospital in Nanchang.WGS was carried out on Illumina Nova Seq platform.Spades v3.15.2 was used to assemble the processed Illumina data.The antimicrobial susceptibility of each isolate was retrospectively investigated.By WGS,we analyzed the MLST,spa typing and SCCmec typing of all S.aureus isolates.2.We analyze the ST88 strains which were screened from the strains collected in the first part.The clinical information,antibiotic susceptibility profiles and molecular characteristics of ST88 strains were analyzed.3.Pathogenicity island,prophages,t RNA and r RNA were predicated according to sequencing data.The predicted genes were aligned to Kyoto Encyclopedia of Genes and Genomes(KEGG)and Clusters of Orthologous Groups of proteins(COG)databases for gene function annotation to understand the metabolic characteristics.Comprehensive Antibiotic Resistance Database(CARD)and Virulence Factors of Pathogenic Bacteria(VFDB)databases were used to predict the antimicrobial resistance genes and virulence factors.4.Finally,the haemolysin activity determination,biofilm semi-quantitative assay,G.mellonella infection model,and mouse skin abscess formation experiment were used to assess the virulence of ST88 strains.Results1.102 of the 176 S.aureus strains were Methicillin-resistant Staphylococcus aureus(MRSA)and 74 were Methicillin-susceptible Staphylococcal aureus(MSSA).Twenty distinct sequence types were identified in MRSA isolates,with the most common being ST59,ST630,and ST338.The major genotypes of MSSA were ST188 and ST7.Eight clinical isolates of Staphylococcus aureus ST88 named P1217,P1343,P1347,P1388,P1537,P237,P688 and P848 were respectively isolated,of 2 were MSSA isolates(P1217,P688)and the rest were MRSA isolates.2.In the ST88 strains,5 strains were isolated from wound secretion,while the others were isolated from blood,drainage fluid and mucous membrane,respectively.In terms of drug resistance,ST88 clones maintained high susceptibility to most drugs and ST88-MRSA isolates were mainly resistant to penicillin and erythromycin.3.Genome sequencing and assembly data showed that the genome size of the eight ST88 strains ranged from 2698331 bp to 2797535 bp,with 32.65 %-32.7 %GC content.The number of pathogenic islands ranged from 3 to 8.Except for P1343 strains,all strains detected the presence of prophages.4.The functional classification of COG shows that,8 strains had the highest percentage of genes encoding proteins related to amino acid transport and metabolism,followed by genes for inorganic ion transport and metabolism.KEGG annotation results show that 71.4% to 75.8 % of predicted genes were involved in cellular process,metabolism,human diseases and other six types of metabolic pathway functions,of which 68% were polysaccharose,fats,vitamins nucleotides and other metabolic pathway genes,suggesting that ST88 strains have more protein products involved in genetic material metabolism and energy production pathways.5.We detected 35 important virulence genes,but 16 of them(including eta,etb,clf A and sea)were not contained in ST88.We found one ST88-MRSA(1/6,16.7%)and one ST88-MSSA(1/2,50%)isolate carry the pvl toxin,and sep was the only enterotoxin gene detected form ST88 strains(7/8,87.5%).6.No difference of growth of S.aureus strains were found at all stages of bacterial.All ST88 strains were biofilm-forming,but the biofilm formation levels were significantly lower than that of the positive control strain ATCC35984.The skin abscesses in mice caused by P1343,P1388 and P848 strains were more serious than USA300.Notably,we found P1343 strain showed the highest level of hemolysin activity,the lowest survival rate of G.mellonella and larger abscess area.7.The hla expressions of P1343 was higher,while the agr A expression of P1217 and P237 was lower than USA300.The pvl expressions of P1217 was lower than USA300 and P848.And we found the levels of sae R expression in all ST88 strains were significantly lower than that of USA300.ConclusionThe whole-genome sequencing and genome annotation of Staphylococcus aureus ST88 strains provide us with a basic understanding of the genome composition,metabolic characteristics,resistance genes and virulence genes situation.Some of the ST88 strains exhibiting higher or similar virulence potential as the USA300 strain.Strain of ST88 lineage might be a great concern for its high virulence.It is necessary to alerted about the spread of highly virulent strains in clinic and strengthen the monitoring and management of Staphylococcus aureus.