Downloaded from www.microbiologyresearch.org by IP: 54.242.161.225 On: Thu, 03 Mar 2016 15:22:23 Comparison of methods for AmpC b-lactamase detection in Enterobacteriaceae Paul R. Ingram, Tim J. J. Inglis, Tessa R. Vanzetti, Barbara A. Henderson, Gerald B. Harnett and Ronan J. Murray Correspondence Tim J. J. Inglis tim.inglis@health.wa.gov.au Received 16 December 2010 Accepted 24 February 2011 Department of Microbiology, PathWest Laboratory Medicine, QEII Medical Centre, Nedlands, WA 6009, Australia AmpC b-lactamases (Bla AmpC ) are an emerging group of antimicrobial resistance determinants. The lack of an agreed Bla AmpC detection method hinders investigation of their epidemiology and understanding of their clinical significance. This study compared the sensitivity and specificity of phenotypic methods of Bla AmpC detection in a collection of 246 Enterobacteriaceae with a diverse range of b-lactam resistance profiles. The Bla AmpC screening methods evaluated were based on cephamycin, ceftazidime and cefepime susceptibility. These were compared with Bla AmpC screening using conventional ESBL detection methods. The confirmatory methods evaluated were biologically based assays, inhibitor-based assays, an AmpC Etest and a rapid chromogenic assay. A multiplex nucleic acid amplification test and the three-dimensional enzyme extraction assay were used as reference methods. Bla AmpC activity was present in 74 isolates. The majority of the enzymes were plasmid-encoded and belonged to the CMY, DHA and EBC families. The screening methods had sensitivities between 47 and 99 % and specificities of 45–95 %. The performance of confirmatory tests varied widely, ranging in sensitivity from 19 % to 97 % and in specificity from 88 % to 100 %. Only the Tris-EDTA and MAST ID D68C disc tests had a sensitivity and a specificity above 90 %. Further investigation is needed to establish the most suitable enzyme substrates, inhibitor types, inhibitor concentrations and interpretative cut-offs in order to refine the inhibitor-based methods. A simple disc-based protocol using cefoxitin non-susceptibility as a screening tool, followed by the Tris-EDTA method for confirmation, detects Bla AmpC activity with 95 % sensitivity and 98 % specificity. INTRODUCTION In spite of the recent decision by both the Clinical and Laboratory Standards Institute (CLSI) and the Euro- pean Committee on Antimicrobial Susceptibility Testing (EUCAST) to lower expanded-spectrum cephalosporin breakpoints for Enterobacteriaceae, there remains a need to detect and characterize specific resistance determinants such as extended-spectrum b-lactamases (ESBLs) and AmpC b-lactamases (Bla AmpC ) for epidemiological surveil- lance and infection control purposes (Doi & Paterson, 2007). Although less prevalent than ESBLs (Jacoby, 2009), Bla AmpC have a comparatively broader substrate range, are not inhibited by traditional b-lactamase inhibitors and bacteria producing them are more likely to become resis- tant to carbapenems (Philippon et al., 2002). Whereas standardized screening and confirmatory methods for ESBL identification are agreed upon (CLSI, 2010), no such methods for Bla AmpC detection exist. Members of the family Enterobacteriaceae such as Entero- bacter spp., Citrobacter freundii and Serratia marcescens possess chromosomally encoded Bla AmpC , obviating the need for specific Bla AmpC testing for these species (Thomson, 2010). Chromosomally encoded Bla AmpC in Escherichia coli is accompanied by negligible expression because of the presence of a transcriptional attenuator coupled with a weak promoter (Tan et al., 2009). Infrequently, hyperproduction of Bla AmpC in E. coli arises following mutations in these regulatory regions or acquisition of imported Bla AmpC genes (Mulvey et al., 2005). Most of the remaining clinically important Enterobacteriaceae, including Klebsiella spp., Proteus mirabilis and Salmonella spp., lack chromosomally encoded Bla AmpC but the emergence of plasmid-encoded Bla AmpC in the last few decades has altered the situation. The mobility afforded by transferable genetic elements has enabled rapid global dissemination reminiscent of the emergence of ESBLs (Kohner et al., 2009). For example, in China the prevalence of E. coli and Klebsiella pneumoniae expressing plasmid-encoded Bla AmpC increased from 2 % to 9 % between 2005 and 2006 (Ding et al., 2008). Abbreviations: Bla AmpC , AmpC b-lactamase(s); ESBL, extended-spectrum b-lactamase. Journal of Medical Microbiology (2011), 60, 715–721 DOI 10.1099/jmm.0.029140-0 029140 G 2011 SGM Printed in Great Britain 715