β-内酰胺酶同步检测方法的建立与临床应用

Establishment of β-lactamase synchronous detection method and its clinical application

目的建立同步检测超广谱β-内酰胺酶（ESBLs）和AmpC β-内酰胺酶（AmpCs）的头孢噻肟平板试验（CTX-AM），并研究β-内酰胺酶在肠杆菌科细菌中的流行情况及对其耐药性的影响。方法应用琼脂稀释法和CTX-AM分别测定分离菌的最低抑菌浓度（MIC）和产酶情况，并对产与不产酶菌株的耐药性进行比较。以肺炎克雷伯菌ATCC 700603、阴沟肠杆菌029M和大肠埃希菌ATCC 25922作质控，并与双纸片增效试验（DDET）和酶粗提物三维试验（TDEM）进行比对。结果CTX-AM检出的产AmpCs菌株与TDEM检测结果完全一致，同时产ESBLs和AmpCs的11株菌中，DDET检出8株ESBLS阴性；且CTX-AM识别的产耐酶抑制剂β-内酰胺酶和产其他广谱酶的菌株，DDET未能识别。183株菌中，33．9％、3．3％、6．6％和14．8％分别单产ESBI。S、AmpCs，同时产ESBLs／AmpCs和其他广谱酶。亚胺培南抑菌活性最高（MIC90=0．5mg／L），72%以上单产ESBLS菌株对哌拉西林／他唑巴坦、头孢西丁、头孢吡肟和头孢哌酮／舒巴坦敏感，66％以上单产AmpCs菌株对头孢吡肟敏感，而同时产生ESBLs／AmpCs菌株对除亚胺培南外的抗生素敏感性均低于20％。结论CTXAM操作简便，易于判读，成本低廉，在一块平板上即可完成对ESBLs和AmpCs的检测。

Objective To establish an easy, rapid and reproducible cefotaxime-agar medium （CTX-AM） for phenotypic detection of extended-spectrum β-lactamases （ESBLs） and AmpC β-lactamases （AmpCs） synchronously, and to investigate the prevalence of ESBLs in Enterobacteriaceae and its impact on antibiotic susceptibility. Methods 183 clinical Enterobacteriaceae isolates were enrolled in the study. The minimal inhibitory concentration （MIC） was determined by standard agar dilution method, and the statue of β-1actamase production by cefotaxime-agar medium（CTX-AM） method. The resistance of β-1actamase producting strains and nowproducting strains was compared. A total 183 isolates of Enterobacteriaceae,as identified by the double-disk enhancement test （DDET） and the threedimensional extract method（TDEM）, were used to evaluate the CTX-AM method. K. pneumoniae ATCC700603,E. cloacae 029M and E. coli ATCC25922 were set as quality controls. Results The de tected AmpCs-producing strains by CTX-AM were coincident with those by TDEM. In both ESBLs and AmpCs-producing strains （n =11 ）, 8 strains showed ESBI,s negative by DDET. In addition, beta lactamase and other broad-spectrum enzyme-producing strains identified by CTX-AM weren＇t detected by DDET method. Among 183 isolates, 33.9% produced only ESBLs, 3. 30% produced only AmpCs, 6.6% produced both ESBLs and AmpCs, and 14. 8% produced other β- lactamases. Imipenem showed the highest inhibitory activity against Enterobacteriaceae strains （MIC90= 0.5 mg/L）. More than 72% of the isolates producing only ESBLs were susceptible to piperacillin/tazobactam, cefoxitin, cefoperazone/sulbactam and cefepime. More than 66% of the isolates producing only AmpCs were susceptible to cefepime. The antibiotic sensitivities of the isolates producing both ESBLs and AmpCs to tested β- lactam antibiotics other than imipenem were all below 20%. Conclusion The news method described above is easier to perform and interpret and cost-effective,allows for testing of ESBL and AmpCs on a single plate. Clinical laboratories can use this technique to detect the presence of ESBL and AmpCs. Imipenem is the first choice to cure the infection by ESBLs and/or AmpCs-producing Enterobacteriaceae.