Phenotypic & molecular characterization of AmpC β-lactamases among Escherichia coli, Klebsiella spp. & Enterobacter spp. from five Indian Medical Centers.

Background & objectives: AmpC β-lactamases which are often plasmid mediated hydrolyze all β-lactam antibiotics except cefepime and carbapenems. We evaluated the presence of AmpC β-lactamases among Enterobacteriaceae strains recovered prospectively from patients at five Indian tertiary care centres. Methods: The study included 909 consecutive Gram-negative isolates recovered from clinically significant specimens during June 2007 - May 2008 as part of an ICMR-ESBL study. Among the study isolates, 312 were found to be cefoxitin resistant by disc diffusion test (DDT). Minimum inhibitory concentration (MIC) determination by E test was done against amikacin, levofloxacin, impinem, meropenem, ertapenem, tigecycline and piperacillin-tazobactam. Combined DDT using phenyl boronic acid as inhibitor with cefoxitin was used for phenotypic confirmation of AmpC phenotype. The common Amp C genotypes ACC, FOX, MOX, DHA, CIT and EBC were detected by multiplex PCR. Results: Plasmid mediated Amp C phenotype was confirmed in 114 of the 312 (36.5%) cefoxitin resistant isolates with 255 (81.7%) showing multidrug resistance. Susceptibility to tigecycline was highest (99%) followed by imipenem, meropenem (97%), ertapenem (89%), amikacin (85%), and piperacillin-tazobactam (74.6%). Levofloxacin resistance was 82 per cent. ESBL co carriage was observed among 92 per cent of Amp C producers. Among 114 Amp C producers, 48 could be assigned a genotype, this included CIT- FOX (n=25), EBC (n=10), FOX (n = 4), CIT (n=3), EBC-ACC (n=2) and one each of DHA, EBC-DHA, FOX -DHA and FOX-EBC-DHA. Interpretation & Conclusions: Overall, AmpC phenotypes were found in 12.5 per cent isolates, multidrug resistance and ESBL co-carriage among them was high suggesting plasmid mediated spread. The study results have implications in rational antimicrobial therapy and continued surveillance of mechanisms of resistance among nosocomial pathogens.

Evaluation of tigecycline activity in clinical isolates among Indian medical centers.

Background: Resistance to multiple antibiotics among Gram-positive cocci (GPC) and Gram negative bacilli (GNB) is high in India. Tigecycline, a glycylcycline antibiotic is a newer treatment option for emerging single or multidrug-resistant (MDR) GPC and GNB. Material and Method: We evaluated the in vitro activity of tigecycline and compared it against other antimicrobials. Between 2005-2007, seven Indian medical centers from diverse geographic regions forwarded 727 isolates [Escherichia coli (166), Staphylococcus aureus (125), Klebsiella spp (120), Streptococcus pneumoniae (102), Enterococcus spp. (100), Pseudomonas aeruginosa (50), Acinetobacter spp. (50) and Enterobacter spp. (14)] from patients with blood stream (BSI), skin and soft tissue (SSTI) including surgical site, urinary tract and respiratory infections to our reference laboratory. Susceptibility to 11 antimicrobials besides tigecycline included: vancomycin, linezolid, teicoplanin, quinopristin-dalfopristin, daptomycin, amikacin, imipenem, levofloxacin, meropenem, and piperacillin/tazobactam was determined by agar dilution and Etest method. Result: Tigecycline was active against all GPC (MIC 90 < 0.25 μg/ml), E. coli and Klebsiella spp. (MIC 90 ≤1 μg/ml). MDR Acinetobacter spp. showed lower susceptibility (70.6%) to tigecycline. Tigecycline MIC 90 values were not influenced by oxacillin resistance among S. aureus, S. pneumoniae, vancomycin resistance in Enterococci (VRE) and ESBL producing E. coli, Klebsiella spp. and Enterobacter spp. Increased resistance was seen to other antimicrobials among ESBL producing E. coli, Klebsiella spp., Metallo Beta Lactamase (MBL) producing P. aeruginosa and VRE. Conclusion: Tigecycline is an alternative option for emerging multidrug-resistant (MDR) pathogens exhibiting promising spectrum/potency exceeding currently available agents seen in India.

Comparison of disc diffusion & E test methods with agar dilution for antimicrobial susceptibility testing of Haemophilus influenzae.

BACKGROUND & OBJECTIVES: Reliable methods of detection of antimicrobial resistance are of paramount importance in the treatment and management of infections caused by Haemophilus influenzae. The objective of the present study was to compare and evaluate the performance of disc diffusion and E test (Epsilometer test) with agar dilution method for antimicrobial susceptibility testing of H. influenzae. METHODS: A total of 46 isolates of H. influenzae from various invasive sites were included as test strains. Antimicrobial susceptibility testing was performed by Kirby-Bauer disc diffusion method for ampicillin, chloramphenicol, trimethoprim-sulphamethoxazole (TMP-SMZ) and cefotaxime. Minimum Inhibitory Concentration (MIC) determination was performed by E test and agar dilution for the same set of antimicrobials. All tests were performed on Haemophilus test medium (HTM). RESULTS: Disc diffusion showed a very major (2%) and minor (4%) interpretative error with TMP-SMZ and minor interpretative errors to ampicillin (13%) and chloramphenicol (24%) when compared to agar dilution method. E test produced only minor interpretative errors to chloramphenicol (7%) and TMP-SMZ (2%) and no interpretative errors with ampicillin and cefotaxime as against agar dilution. E test showed good agreement with agar dilution for each of the antimicrobial tested. INTERPRETATION & CONCLUSION: Disc diffusion test may be used as a preliminary screen for susceptibility testing of H. influenzae. E test is simple, easy to perform and a reliable method for determination of resistance in H. influenzae. However its cost and limited availability in India may limit its use. The reference agar dilution method can be used reliably in routine susceptibility testing of H. influenzae.