Note Diagnostic utility of combination of inducer and inhibitor based assay in detection of Pseudomonas aeruginosa producing AmpC β-lactamase Supriya Upadhyay a , Malay Ranjan Sen a, ⁎, Amitabha Bhattacharjee b , Pradyot Prakash a , Ram Chandra Bajpai c , Shampa Anupurba a a Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, 221005, India b Department of Microbiology, Assam University, Silchar-788011, India c Division of Biostatistics, Institute of Medical Sciences, Banaras Hindu University, Varanasi, 221005, India abstract article info Article history: Received 22 April 2011 Received in revised form 9 July 2011 Accepted 14 July 2011 Available online 23 July 2011 Keywords: AmpC β-lactamases Inducer Inhibitor Boronic acid P. aeruginosa The diagnostic utility of inducer and inhibitor based assays among 214 AmpC positive isolates of Pseudomonas aeruginosa were evaluated. Of different methods, combination of ceftazidime–imipenem antagonism and boronic acid inhibition tests came up with maximum sensitivity (76%) and specificity (100%). This combination showed reliability for both inducible and non-inducible AmpC producers. © 2011 Elsevier B.V. All rights reserved. Pseudomonas aeruginosa is one of the major pathogens causing acute nosocomial infections (Vincent, 2003). The infections often become critical due to treatment failure as the microorganism harbour antibiotic resistance determinant especially to third generation cephalosporins by production of AmpC beta-lactamases which are chromosomally mutated and sometimes acquired through plasmid (Carmeli et al., 1999; Cavallo et al., 2000; Livermore, 2002). This resistance mechanism frequently remains undetected in routine antimicrobial susceptibility testing and more complex in their detection (Thomson, 2001). Several inhibitor-based and inducer-based methods were used for their detection but none of them could be able to detect all the variants of AmpC enzyme of clinical importance (Jacoby, 2009). A modified three-dimensional test (M3DT) is considered to be optimal but it is a time consuming and labour intensive method (Tan et al., 2009). PCR detection method cannot be taken as gold standard as only previously defined enzymes can be detected. The present study was undertaken to evaluate combination of both inducer and inhibitor based rapid method for detection of AmpCs which are clinically significant among clinical isolates of P. aeruginosa. In this study a total of 329 non-repeat, consecutive clinical isolates of P. aeruginosa were isolated from inpatients of SS Hospital, Banaras Hindu University, Varanasi, from April 2008 to September 2009. The isolates were identified by conventional methods (Colee et al., 1996). A previously confirmed clinical isolate of Escherichia coli harbouring CIT type AmpC (EC-SU 422) was taken as positive control and E. coli ATCC 25922 as negative control. MIC of all the isolates were carried out by agar dilution method according to CLSI recommendation (CLSI, 2005) with cefotaxime, ceftazidime, ceftriaxone (Hi Media, Mumbai, India), cefepime (Alembic Ltd, Vadodra, India), aztreonam (Aristo Pharmaceuticals Ltd., Mumbai, India), imipenem (United Biotech, Solan, India) and meropenem (Astra Zeneca Pharmaceuticals Ltd, Bangalore, India). Screening of AmpC β-lactamase was performed by Cefoxitin disc test. Isolates that yielded a zone diameter less than 18 mm were further subjected to confirmatory tests. All the screened positive isolates of AmpC production were tested by M3DT (Coudron et al., 2000). Inducible AmpC was detected by using disc antagonism test (Sanders et al., 1982) and Ceftazidime–Imipenem antagonism test (CIAT) (Cantarelli et al., 2007). The cloxacillin inhibition test was performed by the standard disc diffusion method using 500 μg of cloxacillin (Tan et al., 2009). The boronic acid inhibition test was performed by standard disc diffusion method (Coudron, 2005). Any isolate that was positive in at least any of the AmpC phenotypic tests were subjected to genotypic Journal of Microbiological Methods 87 (2011) 116–118 ⁎ Corresponding author at: Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, 221005, India. Tel.: + 91 9415820675; fax: + 91 5422367568. E-mail address: mr_senbhu@yahoo.com (M.R. Sen). 0167-7012/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2011.07.012 Contents lists available at ScienceDirect Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth