多重PCR检测技术在耐碳青霉烯酶肺炎克雷伯菌基因型中的应用

Application value of multiplex PCR detection technology in carbapenemase-resistant Klebsiella pneumoniae genotype

目的利用多重PCR技术检测耐碳青霉烯酶肺炎克雷伯菌(Carbapenem-resistant Klebsiella pneumoniae,CRKP)基因型。方法收集2015年1月—2018年1月某院住院患者各种临床标本(痰液、血液、尿液、腹腔引流液等)中分离得到的CRKP共132株,采用Vitek-2 Compact全自动微生物鉴定仪对分离菌株进行体外药物敏感试验;利用拉丝试验筛选高粘液阳性菌株;利用改良Hodge实验对碳青霉烯酶表型进行检测;利用多重PCR法检测碳青霉烯酶耐药基因型:kpc、imp、vim、ndm、sme、ges、gim、oxa-48;常见毒力基因rmpA、magA、fimH、mrkD、kpn、entB、ybtS、iutA、aerobactin。结果 132株CRKP对头孢类抗生素耐药率较高,氨苄西林、氨曲南次之,对多粘菌素B、四环素、氯霉素、磺胺甲噁唑/甲氧苄啶敏感;改良Hodge试验阳性118株,阳性率89.39%;132株CRKP中,20株(15.15%)为黏液丝检测阳性。所有CRKP中毒力基因携带情况中,mrkD基因型比例最高,其次为fimH,未检测到magA毒力基因;132株CRKP中,130株检测到kmc基因,2株检测到imp基因,均未检测到ndm、vim、sme、ges、gim、oxa-48毒力基因。结论某院分离得到的CRKP主要毒力基因为mrkD、fimH,耐药基因主要为kpc。

Objective To detect Carbapenem-resistant Klebsiella pneumoniae(CRKP) genotype by multiplex PCR technology.Methods A total of 132 strains of CRKP isolated from clinical specimens(sputum, blood, urine, abdominal drainage fluid, etc) of inpatients in the hospital from January 2015 to January 2018 were collected, Vitek-2 Compact full-automatic microbial identifier was used to test the drug sensitivity test of isolated strains in vitro, positive high mucus strains were screened by the string test, carbapenemase phenotype was detected by modified Hodge test, and multiple PCR technology was used to detect drug-resistance genotypes of carbapenemase including kpc,imp,vim,ndm,sme,ges,gim and oxa-48 and common virulence genes including rmpA,magA,fimH,mrkD,kpn,entB,ybtS,iutA and aerobactin.Results The resistance rate of 132 strains of CRKP to cephalosporins was higher, followed by ampicillin and aztreonam, and the CRKP were susceptible to polymyxin B,tetracycline, chloramphenicol and sulfamethoxazole/trimethoprim;118 strains with a positive rate of 89.39% were found to be positive in modified Hodge test;20(15.15%) out of 132 CRKP strains were found to be positive in the mucus filament test.In all virulence genes carrying CRKP,the proportion of mrkD genotype was the highest, followed by fimH,magA virulence genes were not detected.In 132 CRKP strains, 130 strains were detected with kmc genes, imp gene was detected in 2 strains, ndm,vim,sme,ges,gim and oxa-48 virulence genes were not detected in all the strains of CRKP.Conclusion Major virulence genes of CRKP isolated in a hospital are mrkD and fimH,and drug resistance gene is mainly kpc.