临床分离金黄色葡萄球菌生物膜动态观察及药物敏感性研究

Dynamic observation and antibiotic susceptibility analysis of biofilm of Staphylococcus aureus isolated from clinical specimens

目的分析临床分离金黄色葡萄球菌生物膜与耐药性间的关系。方法参照《临床微生物检验标准化操作》收集广西医科大学第一附属医院检验科于2018年1月自住院患者送检的血液、尿液、痰液及脓性分泌物等临床样本中分离的非重复致病性金黄色葡萄球菌58株,使用刚果红平板法与结晶紫染色法鉴定菌株产膜能力,通过纸片扩散法和VITEK-2全自动微生物药敏试验系统进行药敏分析,采用96孔培养法与结晶紫染色法观察生物膜形成规律及形态。卡方检验用于比较产膜株与非产膜株耐药率的差异,产膜阳性菌株与产膜阴性菌株抑菌圈直径的比较采用t检验,生物膜形成能力不同的菌株间耐药种类的差异采用非参数秩和检验进行分析。结果耐甲氧西林菌株产膜率(68.42%)较甲氧西林敏感株高(20.00%)(χ^2=12.304,P=0.001),产膜菌株对多种抗菌药物耐药率均高于非产膜菌株,生物膜阳性菌株对苯唑西林与克林霉素耐药率分别为73.33%与53.33%,且生物膜形成能力越强的菌株其耐药种类越多(χ^2=9.099,P=0.008)。细菌生物膜的黏附量随着培养时间的推移逐渐增加,72h后生物膜发展至成熟,成熟的生物膜呈片状复层生长,结构紧密。结论对于临床分离的金黄色葡萄球菌,生物膜阳性株耐药率显著高于生物膜阴性株,产膜能力越强的菌株其耐药种类越多,生物膜的粘附量随着培养时间的推移逐渐增加,72h后生物膜发展至成熟。

Objective To analyze the correlation between antibiotic susceptibility andbiofilm formation of Staphylococcus aureus.MethodsAccording to Standardized Operational Guidance for Clinical Microbiological Testing,fifty-eight non-repetitive pathogenic Staphylococcus aureus isolates were collected from blood,urine,sputum and purulent secretions of inpatients in the Laboratory Department of the First Affiliated Hospital of Guangxi Medical University in January 2018.The antibiotics susceptibility of strains was characterized by disk diffusion method and VITEK-2.96-hole culture.The biofilm formation ability was detected bythe crystal violet assay and Congo redplate methods.The in vitro dynamic forming process of S.aureus′sbiofilm was analyzed by crystal violet staining combined with bacterial culture in 96 wells.Besides,the rate differences of drug resistance between biofilm producers and non-producers was performed by Chi-square test.The diameter of bacteriostasis zone of biofilm producers and non-producers was compared by t test.The drug resistance among strains with different biofilm formation ability was analyzed withnonparametric rank sum test.ResultsThe positive ratio of biofilm producing S.aureus was higher in MRSA(68.42%)than in MSSA(20.00%)(χ^2=12.304,P=0.001.Antibiotic resistance rates of biofilm producers were higher than non-producers.The resistance rates of biofilm positive strains to oxacillin and clindamycin were 73.33%and 53.33%,respectively.The antibiotic resistance of the strain was higher along with the biofilm forming ability was increasing.(χ^2=9.099,P=0.008).Depths of the S.aureus′s biofilm on the 96-well plates increased significantly over time and reached biofilm maturation after 72 hours′incubation.Light microscopic observation revealed that the mature biofilm was compact and growing with many layers.ConclusionsFor the clinical isolates of S.aureus,the antibiotic resistance of biofilm producers is significantly higher than non-producers.Strains with stronger biofilm forming ability had higher antibiotics resistance.Depths of the S.aureus′s biofilm on the 96-well plates increased significantly over time and reached biofilm maturation after 72 hours′incubation.