title: AmpC β-lactamases in nosocomial isolates of Klebsiella pneumoniae from India.
abstract: OBJECTIVE
  AmpC β-lactamases are clinically significant since these confer resistance to cephalosporins in the oxyimino group, 7-α methoxycephalosporins and are not affected by available β-lactamase inhibitors. In this study we looked for both extended spectrum β-lactamases (ESBL) and AmpC β-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 β-lactamases and also in confirmation of ESBL production. The detection of AmpC β-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 β-lactamases. The subsets of isolates phenotypically AmpC β-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.
  
  
  CONCLUSIONS
  Tazobactam was the best β-lactamase inhibitor for detecting ESBL in presence of AmpC β-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 β-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 β-lactamase in the routine diagnostic microbiology laboratories.
authors.affiliation: Department of Microbiology, Government Medical College & Hospital, Chandigarh, India. varshagupta_99@yahoo.com
authors.forename: Varsha
authors.initials: V
authors.lastname: Gupta
authors.forename: Karthikeyan
authors.initials: K
authors.lastname: Kumarasamy
authors.forename: Neelam
authors.initials: N
authors.lastname: Gulati
authors.forename: Ritu
authors.initials: R
authors.lastname: Garg
authors.forename: Padma
authors.initials: P
authors.lastname: Krishnan
authors.forename: Jagdish
authors.initials: J
authors.lastname: Chander
mesh_terms: D000900:Anti-Bacterial Agents / Q000008:administration & dosage / Q000037:antagonists & inhibitors; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D000077723:Cefepime; D002511:Cephalosporins / Q000008:administration & dosage*; D003428:Cross Infection / Q000175:diagnosis / Q000201:enzymology / Q000382:microbiology; D024881:Drug Resistance, Bacterial; D006801:Humans; D007194:India; D007711:Klebsiella pneumoniae / Q000201:enzymology / Q000302:isolation & purification / Q000472:pathogenicity; D008826:Microbial Sensitivity Tests / Q000379:methods; D010397:Penicillanic Acid / Q000031:analogs & derivatives; D000078142:Tazobactam; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002511:Cephalosporins; D065093:beta-Lactamase Inhibitors; D000077723:Cefepime; D010397:Penicillanic Acid; C048795:AmpC beta-lactamases; D001618:beta-Lactamases; D000078142:Tazobactam