金黄色葡萄球菌临床分离株对夫西地酸的耐药性检测及分子特征研究

Detection of fusidic acid resistance in Staphylococcus aureus and analysis of molecular characteristics

目的 调查金黄色葡萄球菌临床分离株对夫西地酸的耐药情况并对其进行相关分子特征的研究.方法 收集温州医科大学附属第一医院2013年10月—2016年3月间临床分离的金黄色葡萄球菌,共1333株,用K-B法筛选耐夫西地酸菌株,并用琼脂稀释法测定夫西地酸的最小抑菌浓度（MIC值）.PCR法检测耐药基因fusB、fusC、fusD和金黄色葡萄球菌A蛋白基因（spa基因）,同时用测序方法对fusB、fusC、fusD阴性的耐药菌株进行fusA基因突变检测.PCR法检测金黄色葡萄球菌管家基因并进行多位点序列分型.结果 1333株金黄色葡萄球菌中,共检出52株对夫西地酸耐药,占3.90％.2013—2016年的耐药率分别为：5.3％（7/132）、3.5％（20/571）、4.1％（21/510）、3.3％（4/120）.夫西地酸对金黄色葡萄球菌的MIC值为2、4、8、64和〉64μg/ml分别占26.9％（14/52）、34.6％（18/52）、13.5％（7/52）、1.9％（1/52）、23.1％（12/52）.52株夫西地酸耐药株中,多位点序列分型（MLST）分型共发现13种序列型（ST）,其中以ST630为主,占50％（26/52）,其次为ST5,占17.3％（9/52）;spa分型共19种序列型,以t4549（11/52）、t2460（8/52）、t377（8/52）为主.52株耐夫西地酸的菌株中耐药基因总携带率为17.3％（9/52）,其中fusB基因阳性菌株共7株,fusD阳性株共2株,未检测到fusC阳性菌株.另外,本研究中共检出11株fusA基因突变菌株,占21.2％,且MIC值均〉64μg/ml,分子分型以ST5-t2460为主.结论 夫西地酸对金黄色葡萄球菌表现出较好的体外抗菌活性,且保持相对稳定的耐药趋势.对夫西地酸高水平耐药的机制与fusA基因突变密切相关.

Objective To investigate fusidie acid resistance in Staphylococcus aureus （S. aureus） strains and to analyze their molecular characteristics. Methods A total of 1 333 strains of S. aureus isolated in the First Affiliated Hospital of Wenzhou Medical University from October 2013 to March 2016 were collect- ed. Fusidie acid resistance in these strains was detected by K-B method. Minimum inhibitory concentrations （MIC） of fusidie acid were measured by agar dilution method. Resistance genes offusB, fusC andfusD and S. aureus A protein-coding gene （spa） were detected by PCR. Strains that did not carry fusB, fusC or fusD were detected for mutations in fusA by sequencing. Housekeeping genes were detected by PCR and analyzed by muhilocus sequence typing （MIST）. Results Among the 1 333 strains of S. aureus, 52 were resistant to fusidic acid, accounting for 3.90%. From 2013 to 2016, fusidic acid-resistant strains accounted for 5.3% （7/132）, 3.5% （20/571）, 4.1% （21/510） and 3.3% （4/120）, respectively. The MIC values of fusidic acid against S. aureus were 2, 4, 8, 64 and 〉64 txg/ml, which inhibited the growth of 26.9% （14/52）, 34.6% （18/52）, 13.5% （7/52）, 1.9% （1/52）and23.1% （12/52）of strains, respective- ly. MIST revealed that there were 13 sequence types （STs） , among which ST630 was the predominant type accounting for 50% （26/52）, followed by ST5 accounting for 17.3% （9/52）. There were 19 spa types, among which t4549 （11/52） , t2460 （8/52） , t377 （8/52） were the predominant types. Nine strains carried drug resistance genes accounting for 17.3 % （9/52） , including seven fusB-positive and two fusD-posi-tive strains. No fusc-positive strains were detected. Mutations in fusA gene were detected in 11 strains （21.2%, 11/52） and the MIC values against them were all 〉64μg/ml. The predominant type of the strains with fusA gene mutations was ST5-t2460. Conclusion Fusidic acid shows a good and relatively stable in vitro antibacterial activity against S. aureus. The mechanism of high resistance to fusidic acid is closely related to fusA gene mutation.