改良头孢西丁平板试验检测大肠埃希菌和肺炎克雷伯菌质粒介导的AmpC酶

A modified cefoxitin-agar method for detection of plasmid-mediated AmpC β-iactamases in Escherichia coii and Kiebsieila pneumoniae

目的依据EDTA可渗透菌细胞内并促进β-内酰胺酶释放至外环境的原理,建立并评价检测大肠埃希菌和肺炎克雷伯菌质粒介导的AmpC酶的改良头孢西丁平板试验（MCAM）。方法于含有2、4、6、8μg/ml的头孢西丁平板上接种过夜培养的大肠埃希菌ATCC25922,然后用直径5mm的打孔器在琼脂表面均匀间隔打孔,于孔中分别加入0.5mol/LEDTA0、3、5、7、10μl（使孔中约含EDTA分别为0、450、750、1000、1500μg）,各孔加待检菌悬液（〉1011CFU/ml）25μl,35℃过夜培养。以孔周有细菌生长环为AmpCs阳性。以阴沟肠杆菌029M和大肠埃希菌ATCC25922作质控,用MCAM和酶粗提物三维试验（TDEM）同时检测临床分离的头孢西丁不敏感大肠埃希菌（14株）和肺炎克雷伯菌（13株）的AmpC酶,对两种方法进行比较。结果MCAM试验应用6μg/ml头孢西丁平板和750μgEDTA进行检测,其结果与TDEM试验完全一致。结论该方法操作简便、结果易于判读,在一块平板上可同时进行多株菌的检测,适于各级临床微生物实验室应用。

Objective To establish and evaluate a new modified cefoxitin-agar medium （MCAM） method to detect plasmid-mediated AmpC β-lactamases in Escherichia colt and Klebsiella pneumoniae based on property of EDTA permeabilizing a bacterial cell and releasing β-lactamases into the external environment. Methods Mueller-Hinton agar with cefoxitin concentrations of 2, 4, 6 and 8μg/ml was used. Plates were inoculated with E. colt ATCC 25922 to cover the entire surface. Circular wells with diameters of 5 mm were made in the agar and filled with 0, 3, 5, 7 and 10μl of 0.5 mol/L EDTA and 25 μl of bacterial suspension （〉 10^11 CFU/ml） from individual strains. Positive control （E. cloacae 029M） and negative control （E. colt ATCC25922） strains were included in each plate. Plates were incubated overnight aerobically at 35℃. A zone of growth presenting around the periphery of wells was considered as AmpC enzyme positive. The MACM method was compared to the previously published three-dimensional extract method （TDEM） for the detection of AmpC enzymes. Clinical isolates of cefoxitin-nonsusceptible E. colt （n= 14） and K. pneumoniae （n= 13） were tested by both methods. Results The MCAM method with 6 μg/ml of cefoxitin and 750 μg/well of EDTA were equivalent to the TDEM method for detecting AmpC production in E. colt and K. pneumoniae. Conclusion The new method is easier to perform and interpret and allows for testing of multiple isolates on a single plate. Clinical laboratories can use this technique to confirm the presence of AmpC enzyme.