aac（6’）-aph（2”）基因与HLGRE多重耐药性的相关性研究
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摘要
【目的】检测高水平庆大霉素耐药肠球菌(high-level gentamicin resistantenterococcus,HLGRE)的耐药性和耐药基因aac(6')-aph(2”)的阳性率,探讨该基因同HLGRE多重耐药的相关性。
【方法】对临床标本中所分离的的HLGRE,采用MIC法测定耐药情况,通过聚合酶链反应(PCR)扩增HLGRE的耐药基因aac(6')-aph(2”),并对该基因进行测序。
【结果】临床分离的89株肠球菌中,屎肠球菌(E.feacium)占59.6%(53/89),粪肠球菌(E.feacalis)占32.6%(29/89),鸟肠球菌(E.avium)和铅黄肠球菌(E.casseliflavus)占7.8%(7/89);HLGRE占75.6%(62/82),其中,屎肠球菌50%(41/82),粪肠球菌25.6%(21/82)。粪肠球菌对青霉素G、氨苄西林、环丙沙星、奎奴普丁/达福普汀的耐药率分别为37.9%、37.9%、27.6%、96.6%;而屎肠球菌的耐药率分别为94.3%、94.3%、90.6%、0%。屎肠球菌与粪肠球菌对以上四种抗菌素的耐药率经过x~2检验有显著性差异(P＜0.05)。HLGR屎肠球菌对青霉素、氨卞西林、红霉素、环丙沙星、氯霉素、利福平、呋喃妥因、低水平的庆大霉素和高水平的庆大霉素9种抗菌素的耐药率比非HLGR屎肠球菌高,但只有对环丙沙星、低水平的庆大霉素和高水平的庆大霉素3种抗菌素的耐药率有显著性差异(P＜0.05)。HLGR粪肠球菌对红霉素、环丙沙星、利福平、低水平的庆大霉素和高水平的庆大霉素5种抗菌素耐药率高于非HLGR粪肠球菌。
62株HLGRE中,aac(6')-aph(2”)基因检测的阳性率为96.8%(60/62),其中,屎肠球菌占66.1%(41/62),粪肠球菌占30.7%(19/62)。20株非HLGRE中,该基因阳性率为50%(10株)。8株HLGRE的PCR产物经测序与GenBank的序列99%～100%一致。aac(6')-aph(2”)阳性HLGRE菌株与阴性非HLGRE菌株对低水平庆大霉素、红霉素、环丙沙星3种抗菌药物耐药率有显著性差异(P＜0.05)。aac(6')-aph(2”)基因同HLGRE菌株之间,和HLGRE多重耐药之间都有较好的相关性(r=0.5683;r=0.7013)。
【结论】HLGRE较非HLGRE分离率高且多重耐药现象严重。sac(6')-aph(2”)基因是导致肠球菌对庆大霉素高水平耐药的主要基因;该基因阳性菌株与阴性菌株的多重耐药率有显著性差异并与HLGRE多重耐药性之间有较好的相关性。
Objective To investigate the positive ratio of the drug resistance gene aac (6')-aph(2")in high-level gentamicin-resistant Enterococcus.To discuss the relationship between the drug resistance gene and the multi-resistance in high-level gentamicin-resistant enterococcus.
Methods MIC tests were performed to detect the antibiotic resistance status in the clinic high-level gentamicin-resistant Enterococcus isolates that had been idengtified by Vitek-2.The drug resistance gene aac(6')-aph(2")was detected by PCR method and confirmed by sequencing.The statistical dependence between drug resistance gene and the multi-resistance was analyzed with exact propability.
Results Totally 89 clinical strains of enterococcus were isolated,the isolation ratio of E.faecium,E.faecalis and other kind of enterococcus were 59.6%,32.6%.7.8%.The ratio of HLGRE was 75.6%(62/82).Among the HLGRE,there were 41 faeciums and 21 faecalises.There were striking dissimilarity between the resistance ratios of E.faecuim and that of E.faecalis to penicillin,ampcillin,ciprofloxacin (P＜0.05).Resistance ratios of E.faecalis to penicillin,ampcillin,ciprofloxacin were 37.9%,37.9%,27.6%,respectively.They were lower than E.faecium (94.3%,94.3%,90.6%).Resistance ratio of E.faecium to quinupristin-dalfopristin was 0%.It was lower than E.faecalis(96.6%).The resistance ratios of HLGR E.faecium were higher than the non-HLGR E.faecium to penicillin,ampcillin,ciprofloxacin,erythromycin,chloramphenicol,rifampin,nitrofurant oin,gentamicin、high-level gentamicin.There were marked difference between the resistance ratios of HLGR E.faecium and that of non-HLGR E.faecium to ciprofloxacin,gentamicin,high-level gentamicin(P＜0.05).The resistance ratios of HLGR E.faecalis were higher than the non-HLGR E.faecalis to erythromycin,ciprofloxacin,rifampin,gentamicin,high-level gentamicin.
The positive ratio of aac(6')-aph(2")was 96.8%(60/62).The gene was detected in 66.1%(41/62)E.faecium and30.7%(21/62)E.faecalis.Among the non-HLGR enterococcus,the positive ratio of aac(6')-aph(2")was 50%.The DNA sequencing of 8 PCR products were consistent with it in the Genbank(99%-100%). Resistance ratio of aac(6')-aph(2")positive HLGR enterococcus was higher than that of aac(6')-aph(2")negative non-HLGR enterococcus(P＜0.05).aac (6')-aph(2")correlated with HLGRE and the multi-resistance of HLGRE (r=0.5683;r=0.7013).
Conclusion The multi-resistant status of HLGRE in the first affiliated hospital of Kunming medical college is severe.The multi-resistant ratio of aac (6')-aph(2")positive strains and aac(6')-aph(2")negative strains had significant difference.There was statistical dependence between the aac(6')-aph(2")gene and the multi-resistance of HLGRE.
【方法】对临床标本中所分离的的HLGRE,采用MIC法测定耐药情况,通过聚合酶链反应(PCR)扩增HLGRE的耐药基因aac(6')-aph(2”),并对该基因进行测序。
【结果】临床分离的89株肠球菌中,屎肠球菌(E.feacium)占59.6%(53/89),粪肠球菌(E.feacalis)占32.6%(29/89),鸟肠球菌(E.avium)和铅黄肠球菌(E.casseliflavus)占7.8%(7/89);HLGRE占75.6%(62/82),其中,屎肠球菌50%(41/82),粪肠球菌25.6%(21/82)。粪肠球菌对青霉素G、氨苄西林、环丙沙星、奎奴普丁/达福普汀的耐药率分别为37.9%、37.9%、27.6%、96.6%;而屎肠球菌的耐药率分别为94.3%、94.3%、90.6%、0%。屎肠球菌与粪肠球菌对以上四种抗菌素的耐药率经过x~2检验有显著性差异(P＜0.05)。HLGR屎肠球菌对青霉素、氨卞西林、红霉素、环丙沙星、氯霉素、利福平、呋喃妥因、低水平的庆大霉素和高水平的庆大霉素9种抗菌素的耐药率比非HLGR屎肠球菌高,但只有对环丙沙星、低水平的庆大霉素和高水平的庆大霉素3种抗菌素的耐药率有显著性差异(P＜0.05)。HLGR粪肠球菌对红霉素、环丙沙星、利福平、低水平的庆大霉素和高水平的庆大霉素5种抗菌素耐药率高于非HLGR粪肠球菌。
62株HLGRE中,aac(6')-aph(2”)基因检测的阳性率为96.8%(60/62),其中,屎肠球菌占66.1%(41/62),粪肠球菌占30.7%(19/62)。20株非HLGRE中,该基因阳性率为50%(10株)。8株HLGRE的PCR产物经测序与GenBank的序列99%～100%一致。aac(6')-aph(2”)阳性HLGRE菌株与阴性非HLGRE菌株对低水平庆大霉素、红霉素、环丙沙星3种抗菌药物耐药率有显著性差异(P＜0.05)。aac(6')-aph(2”)基因同HLGRE菌株之间,和HLGRE多重耐药之间都有较好的相关性(r=0.5683;r=0.7013)。
【结论】HLGRE较非HLGRE分离率高且多重耐药现象严重。sac(6')-aph(2”)基因是导致肠球菌对庆大霉素高水平耐药的主要基因;该基因阳性菌株与阴性菌株的多重耐药率有显著性差异并与HLGRE多重耐药性之间有较好的相关性。
Objective To investigate the positive ratio of the drug resistance gene aac (6')-aph(2")in high-level gentamicin-resistant Enterococcus.To discuss the relationship between the drug resistance gene and the multi-resistance in high-level gentamicin-resistant enterococcus.
Methods MIC tests were performed to detect the antibiotic resistance status in the clinic high-level gentamicin-resistant Enterococcus isolates that had been idengtified by Vitek-2.The drug resistance gene aac(6')-aph(2")was detected by PCR method and confirmed by sequencing.The statistical dependence between drug resistance gene and the multi-resistance was analyzed with exact propability.
Results Totally 89 clinical strains of enterococcus were isolated,the isolation ratio of E.faecium,E.faecalis and other kind of enterococcus were 59.6%,32.6%.7.8%.The ratio of HLGRE was 75.6%(62/82).Among the HLGRE,there were 41 faeciums and 21 faecalises.There were striking dissimilarity between the resistance ratios of E.faecuim and that of E.faecalis to penicillin,ampcillin,ciprofloxacin (P＜0.05).Resistance ratios of E.faecalis to penicillin,ampcillin,ciprofloxacin were 37.9%,37.9%,27.6%,respectively.They were lower than E.faecium (94.3%,94.3%,90.6%).Resistance ratio of E.faecium to quinupristin-dalfopristin was 0%.It was lower than E.faecalis(96.6%).The resistance ratios of HLGR E.faecium were higher than the non-HLGR E.faecium to penicillin,ampcillin,ciprofloxacin,erythromycin,chloramphenicol,rifampin,nitrofurant oin,gentamicin、high-level gentamicin.There were marked difference between the resistance ratios of HLGR E.faecium and that of non-HLGR E.faecium to ciprofloxacin,gentamicin,high-level gentamicin(P＜0.05).The resistance ratios of HLGR E.faecalis were higher than the non-HLGR E.faecalis to erythromycin,ciprofloxacin,rifampin,gentamicin,high-level gentamicin.
The positive ratio of aac(6')-aph(2")was 96.8%(60/62).The gene was detected in 66.1%(41/62)E.faecium and30.7%(21/62)E.faecalis.Among the non-HLGR enterococcus,the positive ratio of aac(6')-aph(2")was 50%.The DNA sequencing of 8 PCR products were consistent with it in the Genbank(99%-100%). Resistance ratio of aac(6')-aph(2")positive HLGR enterococcus was higher than that of aac(6')-aph(2")negative non-HLGR enterococcus(P＜0.05).aac (6')-aph(2")correlated with HLGRE and the multi-resistance of HLGRE (r=0.5683;r=0.7013).
Conclusion The multi-resistant status of HLGRE in the first affiliated hospital of Kunming medical college is severe.The multi-resistant ratio of aac (6')-aph(2")positive strains and aac(6')-aph(2")negative strains had significant difference.There was statistical dependence between the aac(6')-aph(2")gene and the multi-resistance of HLGRE.
引文
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