改良麦康凯平板筛查肠道定植耐碳青霉烯类肠杆菌科细菌的性能评价

Performance evaluation of modified MacConkey plate for screening intestinal colonization of carbapenem-resistant Enterobacteriaceae

目的建立一种运用改良麦康凯平板快速筛查肠道定植耐碳青霉烯类肠杆菌科细菌(CRE)的方法,并进行性能评价。方法分别制备含有终浓度为4μg/mL亚胺培南、4μg/mL美罗培南、2μg/mL厄他培南的改良麦康凯平板。选用临床连续分离45株CRE及30株非CRE菌株,检测改良麦康凯平板的敏感性及特异性,采用其中20株CRE菌株对改良平板的最低检测限进行评估。PCR方法检测CRE菌株耐药基因(TEM、SHV、NDM、KPC、OXA-48、VIM、IMP)。收集142例粪便样本评价改良平板的筛查性能,并与肉汤增菌法及CHROMagar KPC显色培养基进行比较。结果45株CRE菌株中TEM、SHV检出率分别为62.22%(28/45)和24.44%(11/45),NDM、KPC、OXA-48的检出率分别为62.22%(28/45)、15.56%(7/45)和13.33%(6/45),未检出VIM和IMP。45株CRE菌株在厄他培南改良平板上均生长,亚胺培南改良平板生长44株,美罗培南改良平板生长43株,敏感性分别为100%(45/45)、97.78%(44/45)和95.56%(43/45)。30株非CRE菌株在亚胺培南与美罗培南改良平板上均未生长,在厄他培南改良平板上生长2株,特异性分别为100%(30/30)、100%(30/30)和93.33%(28/30)。20株CRE中,接种菌液浓度为10^(5)~10^(8) CFU/mL时,亚胺培南、美罗培南和厄他培南改良平板的检出率分别为70%(14/20)、65%(13/20)和100%(20/20);接种浓度为10^(5) CFU/mL以下时亚胺培南、美罗培南、厄他培南改良平板的检出率分别为10%(2/20)、10%(2/20)和95%(19/20)。亚胺培南、美罗培南改良麦康凯平板最低检测限大多分布在10^(5)~10^(8) CFU/mL接种水平,而厄他培南改良麦康凯平板最低检测限集中分布于10^(2)~10^(3) CFU/mL。142份粪便样本中,亚胺培南、美罗培南、厄他培南改良麦康凯平板分别筛查出3株、2株、4株CRE,均未检出非CRE菌株;肉汤增菌法检出5株CRE及2株非CRE;CHROMagar KPC显色培养基检出5株CRE及1株非CRE。3种改良平板与肉汤增菌法及CHROMagar KPC显色培养基筛查结果间差异均无统计学意义(P>0.05)。与亚胺培南和美罗培南相比,厄他培南平板筛查结果与肉汤增菌法、CHROMagar KPC显色培养基一致性更好,Kappa值分别为0.717和0.885。结论改良麦康凯平板筛查肠道CRE菌株敏感性和特异性高,且操作简便,价格低廉,对操作人员技术要求低。厄他培南改良麦康凯平板具有更低的检测限,且与肉汤增菌法及CHROMagar KPC显色培养基筛查结果有更好的一致性,更适合推广使用。

Objectives To establish a method to rapidly screen the carbapenem-resistant Enterobacteriaceae(CRE)bacteria colonized in intestine by using modified MacConkey plate,and evaluate the performance.Methods The modified MacConkey plates with final concentrations of 4μg/mL imipenem,4μg/mL meropenem and 2μg/mL ertapenem were prepared respectively.The 45 strains of CRE and 30 strains of non-CRE were selected and continuously isolated from clinical samples to detect the sensitivity and specificity of the modified MacConkey plate.Twenty stains of CRE among the above strains were used to evaluate the minimum detection limit of the modified plate.PCR method was used to detect drug resistance genes of CRE strains,i.e.,TEM,SHV,NDM,KPC,OXA-48,VIM and IMP.A total of 142 stool samples were collected to evaluate the screening performance of the modified plate and compared with the results of broth-enhancing bacteria method and CHROMagar KPC chromogenic medium.Results Among the 45 strains,the detection rates of TEM and SHV were 62.22%(28/45)and 24.44%(11/45).The detection rates of NDM,KPC and OXA-48 were 62.22%(28/45),15.56%(7/45)and 13.33%(6/45)respectively.No VIM and IMP were detectable.All the 45 CRE strains were grown on modified ertapenem plates,44 strains were grown on modified imipenem plates,and 43 strains were grown on modified meropenem plates.The sensitivities were 100%(45/45),97.78%(44/45)and 95.56%(43/45)respectively.The 30 non-CRE strains did not grow on modified imipenem and meropenem plates,but 2 strains were grown on modified ertapenem plates with specificities of 100%(30/30),100%(30/30)and 93.33%(28/30)for the 3 antibiotics respectively.Among the 20 CRE strains,when the inoculation concentration was 10^(5) to 10^(8) CFU/mL,the detection rates of modified plates of imipenem,meropenem and ertapenem were 70%(14/20),65%(13/20)and 100%(20/20)respectively;when the inoculation concentration was less than 105 CFU/mL,the detection rates of modified plates of imipenem,meropenem and ertapenem were 10%(2/20),10%(2/20)and 95%(19/20)respectively.The minimum detection limits of modified MacConkey plates of imipenem and meropenem were mostly distributed at the inoculation levels of 10^(5) to10^(8) CFU/mL,while the minimum detection limits of modified ertapenem MacConkey plates were concentrated at the distribution between 10^(2) and 10^(3) CFU/mL.Among the 142 stool samples,3,2 and 4 stains of CRE were screened on modified imipenem,meropenem and ertapenem MacConkey plates respectively,and no non-CRE strains were detectable.Five CRE and 2 non-CRE strains were screened by broth-enhancing bacteria method,and 5 CRE strains and 1 non-CRE strain were screened by CHROMagar KPC chromogenic medium.There was no significant difference among the screening results of the three modified plate and broth-enhancing bacteria method and CHROMagar KPC chromogenic medium(P>0.05).Compared with imipenem and meropenem,the results of ertapenem plate screening were more consistent with broth-enhancing bacteria method and CHROMagar KPC chromogenic medium with Kappa values of 0.717 and 0.885 respectively.Conclusion The modified MacConkey plate designed in this study showed high sensitivity and specificity,simple operation,low price and low technical requirements for operators.Among the improved modified MacConkey plates,the ertapenem plate showed lower detection limit and better consistency with the broth-enhancing bacteria method and CHROMagar KPC chromogenic medium for the screening results,and should be more suitable for popularized application.