AmpC β-lactamases in nosocomial isolates of Klebsiella pneumoniae from India

Background & objectives: AmpC beta-lactamases are clinically significant since these confer resistance to cephalosporins in the oxyimino group, 7-alpha methoxycephalosporins and are not affected by available beta-lactamase inhibitors. In this study we looked for both extended spectrum beta-lactamases (ESBL) and AmpC beta-lactamases in Klebsiella pneumoniae clinical isolates. Methods: One hundred consecutive, non-duplicate clinical isolates of K. pneumoniae collected over a period of one year (June 2008 - June 2009) were included in the study. An antibiotic susceptibility method was used with 10 antibiotics for Gram-negative infections which helped in screening for ESBL and AmpC beta-lactamases and also in confirmation of ESBL production. The detection of AmpC beta-lactamases was done based on screening and confirmatory tests. For screening, disc diffusion zones of cefoxitin <18 mm was taken as cefoxitin resistant. All cefoxitin resistant isolates were tested further by AmpC disk test and modified three dimensional test. Multiplex-PCR was performed for screening the presence of plasmid-mediated AmpC genes. Results: Of the 100 isolates of K. pneumoniae studied, 48 were resistant to cefoxitin on screening. AmpC disk test was positive in 32 (32%) isolates. This was also confirmed with modified three dimensional test. Indentation indicating strong AmpC producer was observed in 25 isolates whereas little distortion (weak AmpC) was observed in 7 isolates. ESBL detection was confirmed by a modification of double disk synergy test in 56 isolates. Cefepime was the best cephalosporin in synergy with tazobactam for detecting ESBL production in isolates co-producing AmpC beta-lactamases. The subsets of isolates phenotypically AmpC beta-lactamase positive were subjected to amplification of six different families of AmpC gene using multiplex PCR. The sequence analysis revealed 12 CMY-2 and eight DHA-1 types. Interpretation & conclusions: Tazobactam was the best beta-lactamase inhibitor for detecting ESBL in presence of AmpC beta-lactamase as this is a very poor inducer of AmpC gene. Amongst cephalosporins, cefepime was the best cephalosporin in detecting ESBL in presence of AmpC beta-lactamase as it is least hydrolyzed by AmpC enzymes. Cefepime-tazobactam combination disk test would be a simple and best method in detection of ESBLs in Enterobacteriaceae co-producing AmpC beta-lactamase in the routine diagnostic microbiology laboratories.