杀白细胞毒素基因阴性金黄色葡萄球菌的基因分型、耐药性及其生物学特征

Genotyping,antibiotic resistance,and biological characteristics of panton-valentine leukocidin gene-negative Staphylococcus aureus

目的:研究杀白细胞毒素(pvl)基因阴性金黄色葡萄球菌对多种抗菌药物的耐药性、基因分型和生物膜形成能力。方法:收集于内蒙古医科大学附属医院住院患者送检标本中分离出的100株金黄色葡萄球菌,包括耐甲氧西林金黄色葡萄球菌(MRSA)和甲氧西林敏感金黄色葡萄球菌(MSSA),进行细菌鉴定和12种抗菌药物的药物敏感性试验(药敏试验),采用辅助基因调控子(agr)分型方法进行基因分型,采用PCR法检测pvl基因及其他31种毒力基因,结晶紫染色法检测菌株的生物膜形成能力。结果:100株pvl基因阴性金黄色葡萄球菌临床分离株中,38株为MSSA,62株为MRSA;金黄色葡萄球菌对万古霉素、利奈唑胺和奎奴普丁/达福普丁全部敏感,除红霉素以外,MRSA对其他产生耐药抗生素的耐药率均明显高于MSSA(P<0.01)。agr基因分型,agr-Ⅰ~Ⅳ均在金黄色葡萄球菌中发现,其中agr-Ⅰ为主要基因型(79.0%),其次为agr-Ⅱ基因型(16.0%);MSSA的agr-Ⅱ基因携带率(81.3%)明显高于MRSA(19.7%)(P<0.01);MRSA的agr-Ⅰ基因携带率(72.2%)明显高于MSSA(27.8%)(P<0.01)。32个毒力基因中,hla和hld基因阳性率均为100%,而edin、etb和pvl基因均阴性。MRSA中sdrE、sea、seb、seh、tsst、lukDE、lukM、hlb和mpHLG2-1基因携带率高于MSSA,其中sea和seh基因携带率(56.5%和79.0%)明显高于MSSA(7.9%和60.5%)(P<0.05或P<0.01),而其他18个基因携带率均低于MSSA,其中mpHLG、seo、sed和sej基因的携带率明显低于MSSA(P<0.05或P<0.01),MSSA中23.7%(9/38)同时携带≥15个毒力基因,明显高于MRSA[14.5%(9/62)]。MSSA和MRSA产膜株分别占100%(38/38)和85.5%(53/62)(P<0.05)。结论:pvl基因阴性金黄色葡萄球菌主要以agr-Ⅰ型为主,MRSA对多种抗菌药物耐药率高于MSSA,而MSSA整体毒力基因携带率明显高于MRSA,且MSSA生物膜形成能力高于MRSA。

Objective:To investigate the antibiotic resistance,genotyping and biofilm formation ability of panton-valentine leukocidin(pvl)negative Staphyloccocus aureus.Methods:A total of 100 strains of Staphylococcus.aureus were isolated from patients in the Affiliated Hospital of Inner Mongolia Medical University[methicillin resistant Staphylococcus aureus(MRSA)and methicillin sensitive Staphylococcus aureus(MSSA)],and the bacterial identification and antibiotic susceptibility test to 12 kinds of antibiotics were performed.The gene typing was performed by adopting accessory gene regulator(agr)typing method.The pvl gene and 31 virulence genes were analyzed by PCR method.The biofilm-forming ability was assessed using the crystal violet staining method.Results:Among 100 pvl negative Staphylococcus aureus clinical isolates,38 isolates were MSSA,while 62 isolates were MRSA.The Staphylococcus aureus isolates were all sensitive to vancomycin,linezolid and quinupristin/dalfopristin,but the resistance rates of MRSA to the other antimicrobials were higher than those of MSSA isolates.The agr genotyping results showed that agr-Ⅰ to agr-Ⅳtypes were all found among the Staphylococus aureus isolates,and agr-Ⅰ type was found to be the dominant type,accounting for 79.0%,followed by agr-Ⅱ type(16.0%).The carriage rate of agr-Ⅱ type among MSSA isolates was significantly higher than that of MRSA isolates(81.3%vs 19.7%,P<0.05).Moreover,the carriage rate of agr-Ⅰtype among MRSA isolates was significantly higher than that of MSSA isolates(72.2%vs 27.8%,P<0.05).Among 32 virulence genes detected in this study,the positive rates of hla and hld genes were all 100%,while no isolate carried edin,etb and pvl gene.The carrige rates of sdrE,sea,seb,seH,tsst,lukDE,lukM,hlb,mpHLG2-1 among MRSA isolated were higher than those of MSSA isolates,when carrige rates of sea and seh(56.5%and 79.0%)were higher than those of MSSA(7.9 and 60.5%)(P<0.05 or P<0.01);the carriage rates of the other 18 virulence related genes of MRSA were all lower than those of MSSA isolates,and the carriage rates of mpHLG,seo,sed and sej among MRSA isolates were significantly lower than those of MSSA isolates(P<0.05 or P<0.01);23.7%(9/38)MSSA isolates carried at least 15 virulence genes,which was significantly higher that of MRSA isolates[14.5%(9/62)].Furthermore,the biofilm producing isolates among MSSA and MRSA isolates accounted for 100%(38/38)and 85.5%(53/62)(P<0.05),respectively.Conclusion:Among 100 pvl-negative Staphylococcus aureus clinical isolates,agr-Ⅰtype is the most predominant.The MRSA isolates show higher prevalence of resistance to multiple antimicrobials,while the carrying rates of virulence genes among MSSA isolates are higher than those of MRSA isolates.The biofilm forming ability of MSSA isolates is higher than that of MRSA isolates.