Coşkun Serpil, Altanlar Nurten
Ankara University Faculty of Pharmacy, Department of Pharmaceutical Microbiology, Ankara, Turkey.
Mikrobiyol Bul. 2012 Jul;46(3):375-85.
Extended spectrum beta-lactamases (ESBL) or AmpC beta-lactamases may be associated with false cephalosporin susceptibility results. Although there are well-established methods for screening and confirmation of class A (ESBL) and class B metallobeta-lactamases (MBL), there is no current guideline for the detection of AmpC beta-lactamases. The aim of this study was to evaluate the performances of three different phenotypic tests [boronic acid (BA) - klavulanic acid (CA) inhibition test, AmpC disk test (TRISEDTA impregnanted), modified 3-dimensional test (M3DT)] for the detection of AmpC beta-lactamases. A total of 50 (42 were Escherichia coli and eight were Klebsiella pneumoniae) cefoxitin-insusceptible strains were collected during June-September 2009. Multiplex PCR was used as the genotypic confirmation test by using DHA, CIT, MOX, FOX and EBC primers. Twenty-five (50%) of 50 cefoxitin-insusceptible strains yielded positive result by BA-CA test. Twenty two (88%) of 25 BA-CA positive strains yielded positive result by multiplex PCR and all of them belonged to CIT family (CMY-2 to CMY-7, LAT-1 to LAT-4 and BIL-1 type) of AmpC beta-lactamases. One strain harboured both CIT and EBC family of AmpC beta- lactamases, one strain harboured CIT + MOX + ESBL. DHA and FOX family of AmpC beta-lactamases were not detected in this study. Both AmpC disk test and M3DT yielded positive test with 11 (52.3%) of 21 AmpC enzyme producing strains (except for one of the PCR positive strain which couldn't be screened by M3DT and AmpC disk test). When BA was added to CA inhibition test, the number of positive isolates of ESBL increased from 13 to 14 (63.6%) due to inhibition of AmpC with BA. Hovewer, the strains which yielded negative results by 3-aminophenylboronic acid (3-APB) were tested again by benzeneboronic acid. Three strains were also found to be positive with benzeneboronic acid. The results of this study indicated that BA test with benzene boronic acid or 3-APB warranted further study. In conclusion, for the phenotypic detection of AmpC beta-lactamases, BA-CA test is simple to perform and easy to interpret. Briefly, in order to prevent the masking effect of ESBL, CA must be added to the BA inhibition test. Also in order to prevent the masking effect of AmpC beta-lactamases on ESBL detection, BA must be added to the CA inhibition test.
超广谱β-内酰胺酶（ESBL）或AmpCβ-内酰胺酶可能与头孢菌素药敏结果假阳性有关。虽然已有成熟的方法用于筛查和确认A类（ESBL）和B类金属β-内酰胺酶（MBL），但目前尚无检测AmpCβ-内酰胺酶的指南。本研究的目的是评估三种不同表型试验[硼酸（BA）-克拉维酸（CA）抑制试验、AmpC纸片试验（含三乙醇胺浸渍）、改良三维试验（M3DT）]检测AmpCβ-内酰胺酶的性能。2009年6月至9月期间共收集了50株（42株大肠埃希菌和8株肺炎克雷伯菌）对头孢西丁耐药的菌株。采用DHA、CIT、MOX、FOX和EBC引物的多重PCR作为基因型确认试验。50株对头孢西丁耐药的菌株中，25株（50%）通过BA-CA试验呈阳性结果。25株BA-CA阳性菌株中的22株（88%）通过多重PCR呈阳性结果，且均属于AmpCβ-内酰胺酶的CIT家族（CMY-2至CMY-7、LAT-1至LAT-4和BIL-1型）。1株同时携带CIT和EBC家族的AmpCβ-内酰胺酶，1株携带CIT + MOX + ESBL。本研究未检测到DHA和FOX家族的AmpCβ-内酰胺酶。在21株产AmpC酶的菌株中（除1株PCR阳性菌株不能通过M3DT和AmpC纸片试验筛查外），AmpC纸片试验和M3DT的阳性检出率均为11株（52.3%）。当在CA抑制试验中加入BA时，由于BA对AmpC的抑制作用，ESBL阳性分离株的数量从13株增加到14株（63.6%）。然而，对3-氨基苯硼酸（3-APB）试验结果为阴性的菌株，用苯硼酸再次进行检测。发现3株用苯硼酸检测也呈阳性。本研究结果表明，用苯硼酸或3-APB进行的BA试验值得进一步研究。总之，对于AmpCβ-内酰胺酶的表型检测，BA-CA试验操作简单且易于解读。简而言之，为防止ESBL的掩盖效应，必须在BA抑制试验中加入CA。同样，为防止AmpCβ-内酰胺酶对ESBL检测的掩盖效应，必须在CA抑制试验中加入BA。
