| Staphylococcus aureus(S.aureus)is not only a most important pathogen of hospital infections,but also a major reason of skin and soft tissue infections in the community.S.aureus can cause a variety of infections ranging from benign superficial skin eruptions to life-threatening bacteremia,endocarditis,pneumonia,and toxic shock syndrome and has been a serious challenge for human health.In addition to the expression of a variety of virulence factors,its ability of rapid formation of antibiotic resistance is more dangerous.With the spread use of antibiotics,the drug resistance of S.aureus is more and more serious.The previous studies mostly focus on methicillin-resistant S.aureus(MRSA).However,many infections caused by methicillin-susceptible S.aureus(MSSA)still exist,approximately 40% of staphylococcal infections in China were caused by MSSA,but little is known about its molecular characterization.In contrast to those of MRSA,MSSA infectious isolates often show wide genetic diversity without clear geographical or temporal clustering.MSSA reportedly exhibits high prevalence of some virulence genes like Panton-Valentine Leukocidin(pvl),hemolysins,toxic shock syndrome toxin,and staphylococcal enterotoxin.Comparative genomics and other analysis have found that MRSA evolves from MSSA by acquiring the staphylococcal cassette chromosome mec(SCCmec)mobile element.MSSA is reportedly susceptible to most antibiotic agents except penicillin in the Europe and the USA,but the drug resistance of S.aureus is more and more serious in recent studies.One key step to control the infections and spread of S.aureus is by determining the molecular characteristics of S.aureus clones and the genetic relatedness of strains.Despite that MSSA causes a large burden of diseases in both nosocomial and community-associated infections,the distribution of MSSA strains across China remains poorly understood.In this study,115 S.aureus isolates collected from Chongqing and Urumqi between June 2012 and December 2013 were analysed.We utilized molecular typing methods in combination with antibiotic susceptibility testing and PCR-based profiling of pvl and resistance genes to characterize a collection of MSSA isolates from clinical specimens to understand the molecular characterizations and genotype/phenotype correlation of MSSA in Western China.Our results may help to guide the rational use of antibiotics,and also to provide experimental evidence for prevention and control of S.aureus infections.The main results are as following.1.Molecular typing(1)spa typing: The entire X region of spa gene was amplified by PCR.The products were sequenced to obtain the tandem repeats of 24 bp,and each isolate was assigned a specific spa type based on the spa database web site(http://www.ridom.de/spaserver/).Forty-five spa types were determined with the major prevalent types of t11413(8.7%,10/115),t571(6.1%,7/115),and t7164(6.1%,7/115)accounting for 20.9% of the MSSA strains.Eleven non-typable strains that have no assigned spa types in the database may be new spa types.(2)MLST typing: Seven housekeeping genes(arc C,aro E,glp,gmk,pta,tpi,and yqi L)of all MSSA strains of interest were amplified and sequenced.The allelic profiles and sequence types(STs)were assigned according to the MLST web site(http://saureus.mlst.net/).Twentysix sequence types(STs)were identified for the 115 MSSA isolates,and the most frequently STs were ST121(11.3%,13/115),ST15(11.3%,13/115),ST7(10.4%,12/115),ST22(10.4%,12/115),ST398(7.0%,8/115),ST188(6.1%%,7/115),and ST88(5.2%,6/115),which accounted for 61.7% of the tested MSSA strains.(3)agr typing: The variable region of agr was composed of the sequence encoding Agr D,two thirds of the C-terminal region of sequence encoding Agr B,and the N-terminal region of sequence encoding Agr C.The isolates could be divided into four agr types according to the polymorphism of the variable regionof agr.agr type I was identified as the most prevalent,accounting for 61.7%(71/115)of the MSSA isolates,followed by type II(14.8%,17/115),type IV(12.2%,14/115),and type III(11.3%,13/115).(4)PFGE typing: In PFGE for S.aureus,the chromosomal DNA is digested with the restriction enzyme Sma I,and the resulting DNA fragments are separated by agarose gelelectrophoresis in an electric field with an alternating voltage gradient.Among the 10 PFGE groups(A to J)identified,groups A(26.2%,28/107),B(26.2%,28/107),and F(23.4%,25/107)accounted for 75.8% of the typable MSSA isolates.8 ST398 strains were non-typable possibly due to DNA methylation.(5)pvl genes detection: The pvl genes of all isolates were detected by PCR and the presence of pvl genes was 28.7%(33/115).2.Antimicrobial susceptibility testingAntibiotic susceptibility testing showed that 83.4%(96/115)of the MSSA isolates were resistant to penicillin,60.0%(69/115)isolates were resistant to erythromycin,51.3%(59/115)isolates were resistant to clindamycin,28.7%(33/115)isolates were resistant to trimethoprim/ sulfamethoxazole,18.3%(21/115)isolates were resistant to tetracycline,7.8%(9/115)isolates were resistant to gentamicin,5.2%(6/115)isolates were resistant to ciprofloxacin,and 2.6%(3/115)isolates were resistant to levofloxacin.Only one isolate was intermediate-resistant to rifampicin and all tested MSSA isolates were susceptible to oxacillin,vancomycin,linezolid,and teicoplanin.The resistance genes carried by MSSA strains were detected.Results demonstrated that 91.3%(105/115)of the MSSA strains harbored bla Z,which is a major determinant for penicillin resistance.The erm C gene that is primarily responsible for erythromycin and clindamycin resistances was carried by 92.2%(106/115)MSSA strains.Moreover,30.4%(35/115)MSSA isolates carried the erm A gene.An isolate was considered multidrug resistant(MDR)when the isolate was resistant to at least one agent in three or more antimicrobial categories.Based on the definitions,47.8%(55/115)of the tested MSSA were MDR isolates,most of which(89.1%,49/55)were at least resistant to penicillin,erythromycin,and clindamycin.For MSSA STs,all 4 ST943 strains were MDR with varied ARPs;9 out of 13 ST121 strains(69.2%)were MDR,whereas most ST121 strains(88.9%,8/9)were resistant to penicillin,erythromycin,and clindamycin.Three ST398 MDR isolates seemed to resist at least 4 categories of antimicrobial agents.Approximately 76.0%(19/25)of PFGE group F strains were MDR MSSA,including one strain(ST45-t1081)that was resistant to 7 antimicrobial agents.These data indicate the need for MDR MSSA infection control.In conclusion,the present study reveals the population structure of MSSA and a high prevalence of MDR MSSA in Chongqing and Urumqi of China.High diversity was found in MSSA strains,and two novel prevalent spa types,t11413 and t7164,were also the most frequent types besides t571 which is a common type in China.ST121 with high virulence level and resistance level to multiple antibiotics,which were rarely reported in China,were found to be the major clones in our study.Our data provide important support for understanding the dynamic structure of MSSA.Although MRSA was still the major problem of S.aureus infections,the high-level prevalence of MDR MSSA strains may indicate that efforts to fight infections caused by MSSA need to be intensified. |
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