产新德里金属β-内酰胺酶-1醋酸钙-鲍曼不动杆菌复合体碳青霉烯酶、外膜孔蛋白和外排泵相关基因检测

Detection of Carbapenemase,Outer Membrane Protein and Efflux Pump Related Genes of Acinetobacter Calcoaceticus-Acinetobacter Baumannii Complex Producing New Delhi Metallo-β-lactamase-1

目的研究临床分离的产新德里金属β-内酰胺酶(NDM)醋酸钙-鲍曼不动杆菌(Acb)复合体耐碳青霉烯类药物的机制。方法通过VITEK-2 compact全自动微生物分析仪鉴定、42℃生长实验、聚合酶链反应(PCR)检测OXA-51-like基因,rpoB基因序列测定对收集的耐碳青霉烯类Acb复合体进行菌种鉴定。利用PCR筛查是否携带NDM基因,通过测序确定NDM型别并进一步检测其他碳青霉烯酶相关基因、外膜孔蛋白基因和外排泵基因。利用DiversiLab细菌DNA指纹图谱分析系统对产NDM-1的Acb复合体进行基因分型。结果共筛选出10株产NDM-1的Acb复合体,其中5株为Pittii不动杆菌,3株为Nosocomialis不动杆菌,2株为鲍曼不动杆菌。在5株Pittii不动杆菌中,2株检出OXA-58-like基因,5株检出外膜孔蛋白oprD基因和外排泵adeI,adeK基因,4株检出adeJ基因。3株Nosocomialis不动杆菌中均检出外膜孔蛋白carO和oprD基因以及外排泵adeB,adeI,adeJ和adeK基因。2株鲍曼不动杆菌中均检出外膜孔蛋白carO基因以及外排泵adeB,adeI,adeJ和adeK基因,1株检出外膜孔蛋白oprD基因,1株检出OXA-23-like基因。鲍曼不动杆菌、Pittii不动杆菌和Nosocomialis不动杆菌基因分型结果均分为两型。结论在福建省Pittii不动杆菌中同时检出NDM-1和OXA-58-like基因,在鲍曼不动杆菌中同时检出NDM-1和OXA-23-like基因。本地分离的Nosocomialis不动杆菌与鲍曼不动杆菌对碳青霉烯类抗菌药物耐药可能与AdeIJK外排泵及NDM-1酶作用有关;Pittii不动杆菌对碳青霉烯类抗菌药物耐药则可能是由外膜孔蛋白CarO缺失、AdeIJK外排泵及NDM-1酶共同作用的结果。

Objective To study carbapenems-resistant mechanisms of Acinetobacter calcoaceticus-Acinetobacter baumannii(Acb)complex strains producing New Delhi metallo-β-lactamase-1(NDM-1)collected in our hospital.Methods All the strains were identified by VITEK-2 compact system,42℃growth test,OXA-51-like gene detection,and rpoB gene sequence analysis.NDM genes were screened by PCR in carbapenem-resistant Acb strains.Types of NDM gene were further identified by sequencing.Other carbapenem-resistant related genes,outer membrane protein genes and efflux pump genes were detected by PCR for NDM-1 gene positive strains.The 10 clinical isolates were typed by DiversiLab repetitive element PCR system.Results A total of 10 strains of Acb strains producing NDM were identified,among which 5 strains were Acinetobacter pittii,3 strains were Acinetobacter nosocomialis and 2 strains were Acinetobacter baumannii.All the strains of Acinetobacter pittii carried outer membrane protein oprD,efflux pump adeI and adeK genes,among which 2 strains carried OXA-58-like gene and 4 strains carried efflux pump adeJ gene.All the strains of Acinetobacter nosocomialis carried outer membrane protein carO and oprD,efflux pump adeB,adeI,adeJ and adeK genes.2 strains of Acinetobacter baumannii carried outer membrane protein carO,efflux pump adeB,adeI,adeJ and adeK gene,among which one strain carried OXA-23-like gene and the other strain carried outer membrane protein oprD gene.5 strains of Acinetobacter pittii,3 strains of Acinetobacter nosocomialis,and 2 strains of Acinetobacter baumannii were classified into two types respectively.Conclusion It is found that Acinetobacter pittii simultaneously carries NDM-1 and OXA-58-like genes,and that Acinetobacter baumannii simultaneously carries NDM-1 and OXA-23-like genes in Fujian Province.Synergistic effect of efflux pump AdeIJK and NDM-1 enzyme might mainly be the causes of Acinetobacter Nosocomialis and Acinetobacter baumannii resistance to carbapenem.As for Acinetobacter pittii,the mechanisms of resistance to carbapenem are also possibly related to the lack of outer membrane protein CarO besides above reasons.