肺炎链球菌临床分离株耐药机制和常见血清型及毒力基因研究
发布时间：2018-11-29 10:04
【摘要】：目的调查天津医科大学总医院临床分离肺炎链球菌对常用抗菌药物的敏感性,检测其常见血清型及毒力基因的分布情况,并对其耐药机制进行初步分析,为合理使用抗生素治疗肺炎链球菌感染提供依据同时控制该耐药菌株在医院内的传播。方法收集2012年10月至2014年4月天津医科大学总医院临床分离的非重复肺炎链球菌72株,应用Vitek-2 compact全自动细菌分析系统及配套鉴定卡和相应微量肉汤稀释试验分别进行菌种鉴定和药敏试验;应用聚合酶链反应(PCR)方法检测大环内酯类抗菌药物耐药基因,包括红霉素核糖体甲基化酶基因(erm B)、主动外排基因(mef A、mef E),四环素耐药基因(tet M)以及转座子整合酶基因(int Tn);常见血清型基因(3、6A/B、14、15A、19A、19F、23F)及毒力基因自溶素基因(lyt A)、溶血素基因(ply)及神经氨酸酶基因(nan A)。PCR扩增的基因阳性产物进行测序并与Gen Bank数据库比对。结果72株肺炎链球菌药敏结果显示,对万古霉素、利福平均敏感,对左氧氟沙星、头孢噻肟、氯霉素、阿莫西林、青霉素敏感率大于76%,其中对青霉素耐药率PRSP为1.4%,中介率PISP为22.2%、敏感率PSSP为76.4%。对红霉素、克林霉素、四环素敏感率低于10%。72株肺炎链球菌携带红霉素耐药基因erm B 65株(90.3%)、mef E 17株(23.6%)、mef A 9株(12.5%)、四环素耐药基因tet M 63株(87.5%),转座子整合酶基因int Tn 62株(86.1%),其中erm B+tet M+int Tn 38株(52.8%)。对四环素耐药的肺炎链球菌tet M基因阳性率是93.8%,高于四环素敏感菌株(20%);对四环素耐药的菌株,其对红霉素和克林霉素的耐药率亦高于四环素敏感的菌株;int Tn基因阳性组的红霉素、克林霉素和四环素的耐药率较int Tn基因阴性组高。72株肺炎链球菌常见血清型依次为19F(15,20.8%)、3(12,16.7%)、23F(9,12.5%)、6A/B(8,11.1%)、14(6,8.3%)、19A(4,5.6%)和15A(2,2.8%)。毒力基因检测结果为lyt A 65株(90.3%)、ply 67株(93.1%)、nan A 52株(72.2%),其中51株(70.8%)同时携带lyt A、ply和nan A三种基因,12株(16.7%)同时携带lyt A和ply基因,1株(1.4%)同时携带ply和nan A基因。PCR扩增产物经测序并与Gen Bank数据库比对后均为目的基因。结论本研究的临床分离肺炎链球菌对万古霉素、利福平敏感率均为100%,左氧氟沙星、氯霉素、头孢噻肟敏感率较高,对红霉素、克林霉素和四环素耐药率较高。临床分离的肺炎链球菌同时携带多种耐药基因,其中最常见的耐药基因组合方式为erm B+tet M+int Tn,erm B基因和tet M基因分别是引起大环内酯类抗菌药物和四环素类抗菌药物耐药的主要机制,int Tn基因可能是介导肺炎链球菌多重耐药的重要机制,临床医师应注意合理用药。血清型19F、3和23F是主要常见的血清型,本地区人群接种肺炎链球菌疫苗时应首选含有这三种血清型的PCV13荚膜多糖抗原疫苗。毒力基因lyt A和ply检出率较高,为进一步研究毒力因子的作用机制提供一定基础,并为研制抗肺炎链球菌新药和预防肺炎链球菌感染的新型疫苗提供帮助。
[Abstract]:Objective To investigate the sensitivity of Streptococcus pneumoniae to the common antibiotics in the general hospital of Tianjin Medical University, to detect the distribution of the common serotypes and virulence genes, and to analyze the mechanism of drug resistance. To provide a basis for the rational use of antibiotics for the treatment of Streptococcus pneumoniae infection, the transmission of the drug-resistant strain within the hospital is also controlled. Methods 72 strains of non-repeated Streptococcus pneumoniae were collected from October, 2012 to April, 2014 in the general hospital of Tianjin Medical University, and the strain identification and drug sensitivity test were carried out by using the Vitk-2 compact full-automatic bacterial analysis system and the matched identification card and the corresponding micro-broth dilution test. The drug-resistant genes of macrolides are detected by polymerase chain reaction (PCR) method, including the erythromycin ribosomal methylase gene (erm B), the active efflux gene (mef A, mef E), the tetracycline resistance gene (tet M) and the transposon integrase gene (int Tn); Common serotype genes (3, 6A/ B, 14, 15A, 19A, 19F, 23F) and virulence gene autolysin gene (lyt A), hemolysin gene (ply), and neuraminidase gene (nan A) are common. The PCR-amplified gene-positive product was sequenced and compared to the Gen Bank database. Results The drug sensitivity of 72 strains of Streptococcus pneumoniae showed that both vancomycin and rifampin were sensitive, and the sensitivity of levofloxacin, cefixime, chloramphenicol, amoxicillin and penicillin was more than 76%. The resistance rate of penicillin to penicillin was 1.4% and the intermediate rate of PISP was 2.2%. The sensitivity rate of PSSP was 76.4%. The sensitivity of erythromycin, klincomycin and tetracycline was lower than 10%. 72 strains of S. pneumoniae carried the erythromycin-resistant gene erm B 65 (90.3%), mef E 17 (23. 6%), mef A 9 (12.5%) and tetracycline-resistant gene tet M 63 (87.5%). The total of the transposon integrase gene int Tn 62 (86.1%), of which the erm B + tet M + int Tn 38 (52.8%). The positive rate of the Tet M gene of the streptococcus pneumoniae resistant to tetracycline is 93.8%, which is higher than that of the tetracycline-sensitive strain (20%); the resistance rate of the tetracycline-resistant strain is higher than that of the tetracycline-sensitive strain; and the erythromycin of the int Tn gene positive group, The common serotypes of the 72 strains of S. pneumoniae were 19F (15, 20. 8%), 3 (12, 16. 7%), 23F (9, 12.5%), 6A/ B (8, 11.1%), 14 (6, 8. 3%), 19A (4, 5.6%) and 15A (2, 2.8%). The results of virulence gene detection were lyt A 65 (90.3%), pldy 67 (93.1%), and nan A 52 (72.2%), of which 51 (72.8%) simultaneously carried lyt A, The three genes of ply and nan A, 12 (16. 7%) carried the lyt A and the ply genes, and 1 (1.4%) simultaneously carried the ply and nan A genes. The PCR products were sequenced and compared with the Gen Bank database. Conclusion The sensitivity of the clinical isolates of Streptococcus pneumoniae to vancomycin and rifampin is 100%, levofloxacin, chloromycetin and cemedoxime are more sensitive to vancomycin, and the resistance to erythromycin, klincomycin and tetracycline is high. The clinically isolated streptococcus pneumoniae carries a plurality of drug resistance genes at the same time, wherein the most common drug resistance gene combination mode is the erm B + tet M + int Tn, the erm B gene and the tet M gene are the main mechanisms for causing the drug resistance of the macrolide antibiotics and the tetracycline antibiotics, The int Tn gene may be an important mechanism to mediate the multiple drug resistance of S. pneumoniae, and the clinician should pay attention to the rational use of the drug. Serotypes 19F, 3, and 23F are the main common serotypes, and the population of the region is vaccinated with a Streptococcus pneumoniae vaccine that is preferred to contain the three serotypes of the PCV13 membrane polysaccharide antigen vaccine. The detection rate of the virulence genes lyt A and ply is high, which provides a basis for the further study of the mechanism of the virulence factors, and is a new type of vaccine for the development of a new drug for the streptococcus pneumoniae and the prevention of the infection of the streptococcus pneumoniae.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.5
本文编号：2364699
[Abstract]:Objective To investigate the sensitivity of Streptococcus pneumoniae to the common antibiotics in the general hospital of Tianjin Medical University, to detect the distribution of the common serotypes and virulence genes, and to analyze the mechanism of drug resistance. To provide a basis for the rational use of antibiotics for the treatment of Streptococcus pneumoniae infection, the transmission of the drug-resistant strain within the hospital is also controlled. Methods 72 strains of non-repeated Streptococcus pneumoniae were collected from October, 2012 to April, 2014 in the general hospital of Tianjin Medical University, and the strain identification and drug sensitivity test were carried out by using the Vitk-2 compact full-automatic bacterial analysis system and the matched identification card and the corresponding micro-broth dilution test. The drug-resistant genes of macrolides are detected by polymerase chain reaction (PCR) method, including the erythromycin ribosomal methylase gene (erm B), the active efflux gene (mef A, mef E), the tetracycline resistance gene (tet M) and the transposon integrase gene (int Tn); Common serotype genes (3, 6A/ B, 14, 15A, 19A, 19F, 23F) and virulence gene autolysin gene (lyt A), hemolysin gene (ply), and neuraminidase gene (nan A) are common. The PCR-amplified gene-positive product was sequenced and compared to the Gen Bank database. Results The drug sensitivity of 72 strains of Streptococcus pneumoniae showed that both vancomycin and rifampin were sensitive, and the sensitivity of levofloxacin, cefixime, chloramphenicol, amoxicillin and penicillin was more than 76%. The resistance rate of penicillin to penicillin was 1.4% and the intermediate rate of PISP was 2.2%. The sensitivity rate of PSSP was 76.4%. The sensitivity of erythromycin, klincomycin and tetracycline was lower than 10%. 72 strains of S. pneumoniae carried the erythromycin-resistant gene erm B 65 (90.3%), mef E 17 (23. 6%), mef A 9 (12.5%) and tetracycline-resistant gene tet M 63 (87.5%). The total of the transposon integrase gene int Tn 62 (86.1%), of which the erm B + tet M + int Tn 38 (52.8%). The positive rate of the Tet M gene of the streptococcus pneumoniae resistant to tetracycline is 93.8%, which is higher than that of the tetracycline-sensitive strain (20%); the resistance rate of the tetracycline-resistant strain is higher than that of the tetracycline-sensitive strain; and the erythromycin of the int Tn gene positive group, The common serotypes of the 72 strains of S. pneumoniae were 19F (15, 20. 8%), 3 (12, 16. 7%), 23F (9, 12.5%), 6A/ B (8, 11.1%), 14 (6, 8. 3%), 19A (4, 5.6%) and 15A (2, 2.8%). The results of virulence gene detection were lyt A 65 (90.3%), pldy 67 (93.1%), and nan A 52 (72.2%), of which 51 (72.8%) simultaneously carried lyt A, The three genes of ply and nan A, 12 (16. 7%) carried the lyt A and the ply genes, and 1 (1.4%) simultaneously carried the ply and nan A genes. The PCR products were sequenced and compared with the Gen Bank database. Conclusion The sensitivity of the clinical isolates of Streptococcus pneumoniae to vancomycin and rifampin is 100%, levofloxacin, chloromycetin and cemedoxime are more sensitive to vancomycin, and the resistance to erythromycin, klincomycin and tetracycline is high. The clinically isolated streptococcus pneumoniae carries a plurality of drug resistance genes at the same time, wherein the most common drug resistance gene combination mode is the erm B + tet M + int Tn, the erm B gene and the tet M gene are the main mechanisms for causing the drug resistance of the macrolide antibiotics and the tetracycline antibiotics, The int Tn gene may be an important mechanism to mediate the multiple drug resistance of S. pneumoniae, and the clinician should pay attention to the rational use of the drug. Serotypes 19F, 3, and 23F are the main common serotypes, and the population of the region is vaccinated with a Streptococcus pneumoniae vaccine that is preferred to contain the three serotypes of the PCV13 membrane polysaccharide antigen vaccine. The detection rate of the virulence genes lyt A and ply is high, which provides a basis for the further study of the mechanism of the virulence factors, and is a new type of vaccine for the development of a new drug for the streptococcus pneumoniae and the prevention of the infection of the streptococcus pneumoniae.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.5
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2 郭慧芳;张燕军;;肺炎链球菌102株的耐药性分析[J];山西医药杂志;2012年12期
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