Phenotypic characteristics of clinical isolates of Klebsiella pneumoniae & evaluation of available phenotypic techniques for detection of extended spectrum beta-lactamases

Background & objectives: Identifying organisms that harbour extended spectrum beta lactamases (ESBLs) is a major challenge for a diagnostic clinical microbiology laboratory. Wide variety of ESBLs produced and lack of a sensitive phenotypic method for their detection make the detection of ESBLs difficult and is responsible for under-recognition. The present study was undertaken to evaluate phenotypic characteristics, initial screening tests and established confirmatory phenotypic methods for detection of ESBLs Klebsiella pneumoniae isolates prevalent in a hospital in north India. Methods: One hundred, non-repeat clinical isolates of K. pneumoniae collected over a period of six months were included in the study. Susceptibilities of the isolates to 20 different antimicrobial agents were determined. Agar dilution and broth dilution methods were used to determine minimum inhibitory concentrations (MICs) of ceftazidime (CAZ) and cefotaxime (CTX). CAZ and CTX were used with and without clavulanic acid to detect ESBL harbouring isolates. Using agar dilution and broth dilution, MIC reduction of two and three doubling dilutions were evaluated as a criterion for ESBL harbouring isolates. Standard double disk synergy test (DDST) with disks placed at 30mm and modified DDST with disks placed at 16mm center-to-center distance, using at least two different third generation cephalosporins and combined disk method were also performed to detect ESBL harbouring isolates. Results: Multi-drug resistance (resistance to three or more antimicrobials of different classes) was found among 94 per cent of the isolates. Pooling the results of all the three confirmatory techniques MIC reduction of > 3 doubling dilutions using broth dilution method (using CTX and CAZ), combined disk method [(using CTX, ceftriaxone [(CRO), CAZ and aztreonam)] and standard DDST (using CTX, CRO, CAZ and aztreonam), revealed as many as 87 per cent of the isolates as ESBL producers. CTX had greater sensitivity in identifying isolates that harboured ESBLs. Modified DDST using CTX was as sensitive method as broth dilution method and combined disk method in detecting ESBL harbouring isolates. MIC reduction technique using agar dilution method and standard DDST had lowest overall sensitivity in detecting ESBLs. Interpretation & conclusion: Modified DDST using at least two different third generation cephalosporins was considered to be the best technique for detection of ESBL producing K. pneumoniae at our hospital. MIC reduction test with > 2 doubling dilution reduction in MICs was found to be a better criterion than the presently recommended > 3 doubling dilution reduction. For screening of potential ESBL producers, MIC determination using agar dilution was as good as that using broth dilution method. However, while performing MIC reduction test agar dilution method was found highly unreliable for detection of ESBL harbouring isolates.