New Delhi Metallo-ß-lactamase and other mechanisms of carbapenemases among Enterobacteriaceae in rural South India.

OBJECTIVES: The emergence and dissemination of carbapenem-resistant Enterobacteriaceae (CRE) is an important public health problem. This study aimed to understand the prevalence and mechanisms of carbapenem resistance in clinically important members of Enterobacteriaceae in rural South India. METHODS: Routine clinical isolates of Escherichia coli and Klebsiella spp. were tested for Ertapenem (ETP) non-susceptibility by the disk diffusion method over a 3-year period (2012-2014). The ETP non-susceptible isolates were preserved, and tested for the MIC of carbapenems and the carriage of major carbapenemase-encoding genes. Representative genes were sequenced and selective isolates were tested for the production of carbapenemase by carbapenem inactivation method. RESULTS: A total of 444 ETP non-susceptible isolates were identified in increasing incidence over the study period. Among them, MIC50 and MIC90 of carbapenems (excluding ETP) were 0.25-0.5µg/mL and 8-16µg/mL, respectively, and the prevalence of non-ETP carbapenem resistance was estimated as 3%. Among the 177 tested isolates, 65 (37%) had one or more carbapenemase-encoding genes with a predominance of New Delhi Metallo-ß-lactamase (NDM; 32 of 65; 49.2%). CONCLUSIONS: This study documented the MIC range for carbapenems, prevalence and mechanisms of carbapenem resistance among Enterobacteriaceae in rural South India. It substantiated NDM as a leading mechanism of carbapenem resistance and highlighted the importance of MIC testing in patient management.

Carbapenem-resistant Enterobacteriaceae in Pediatric Bloodstream Infections in Rural Southern India.

OBJECTIVE: To measure the frequency of antimicrobial resistance in pediatric blood culture isolates of Escherichia coli and Klebsiella spp. with focus on carbapenem resistance. METHODS: Over a period of three years, pediatric blood culture isolates were tested for antimicrobial susceptibility, including molecular investigations for carbapenem resistance. RESULTS: Amikacin, carbapenems, colistin and tigecycline had an antimicrobial efficacy of >70% (n=140). 7 of the 15 randomly selected isolates were positive for carbapenemase gene; among them, five were New Delhi Metallo b-lactamase (NDM). CONCLUSION: There was a high prevalence of Klebsiella spp. in pediatric bacteremia and dissemination of NDM-mediated carbapenem resistance in pediatric wards.

Validation of novel cost-effective antimicrobial gradient strips for the determination of carbapenem susceptibility in Enterobacteriaceae.

OBJECTIVES: The aim of this study was to validate the use of LML antimicrobial gradient strips for quantitative determination of carbapenem susceptibility in Enterobacteriaceae. METHODS: A total of 95 non-redundant Enterobacteriaceae strains isolated during 2012-2014 were used for this validation study. Initially, LML antimicrobial gradient strips were validated for their performance in comparison with the agar dilution method. The test strip was then validated in comparison with broth microdilution (BMD) and Etest with 24 selected strains using the same inocula and other laboratory parameters. RESULTS: The LML strip showed 83%, 68% and 86% essential agreement (within ±1 log2 dilution) with the reference methods of agar dilution, BMD and Etest, respectively; furthermore, essential agreement was >90% within ±2 log2 dilution. Categorical agreement was ≥87% with all reference methods according to Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints. However, the meropenem strip requires performance improvements to fulfil US Food and Drug Administration (FDA) and International Organization for Standardization (ISO) requirements for an antimicrobial susceptibility test device. CONCLUSIONS: In LML antimicrobial gradient strip minimum inhibitory concentrations (MICs) were comparable with Etest MICs and it might serve as a reasonable, cost-effective alternative to Etest for quantitative determination of carbapenem susceptibility in Enterobacteriaceae.

Dissemination of Trimethoprim-Sulfamethoxazole Drug Resistance Genes Associated with Class 1 and Class 2 Integrons Among Gram-Negative Bacteria from HIV Patients in South India.

The antibiotic, trimethoprim-sulfamethoxazole (TMP-SMX), is generally used for prophylaxis in HIV individuals to protect them from Pneumocystis jiroveci infection. Long-term use of TMP-SMX develops drug resistance among bacteria in HIV patients. The study was aimed to detect the TMP-SMX resistance genes among gram-negative bacteria from HIV patients. TMP-SMX-resistant isolates were detected by the Kirby-Bauer disc diffusion method. While TMP resistance genes such as dfrA1, dfrA5, dfrA7, and dfrA17 and SMX resistance genes such as sul1 and sul2 were detected by multiplex PCR, class 1 and class 2 integrons were detected by standard monoplex PCR. Of the 151 TMP-SMX-resistant bacterial isolates, 3 were positive for sul1 alone, 48 for sul2 alone, 11 for dfrA7 alone, 21 for sul1 and sul2, 1 for sul1 and dfrA7, 23 for sul2 and dfrA7, 2 for sul2 and dfrA5, 41 for sul1, sul2, and dfrA7, and 1 for sul2, dfrA5, and dfrA7. Of 60 TMP-SMX-resistant isolates positive for integrons, 44 had class 1 and 16 had class 2 integrons. It was found that the prevalence of sul genes (n = 202; p < 0.001) was higher compared with dfr genes (n = 80; p < 0.001), and 87.4% (n = 132; p < 0.001) of TMP-SMX-resistant isolates also were positive for ß-lactamase production. This type of study is reported for the first time from HIV patients in India. Therefore, this study indicates that dissemination of TMP-SMX resistance genes and class 1 and class 2 integrons along with ß-lactamase production among gram-negative bacteria in HIV patients will certainly make their treatment to bacterial infections more complicated in clinical settings.

Prevalence of antimicrobial resistance in Escherichia coli and Klebsiella spp. in rural South India.

The emergence and dissemination of antimicrobial resistance (AMR) is an important public health problem as resistant organisms cause difficult-to-treat infections. In this study, the prevalence of AMR in Escherichia coli and Klebsiella spp. in rural South India was examined in order to aid empirical therapy. A cross-sectional prospective study was conducted during the period from January 2012 to December 2014. Routine clinical isolates of E. coli and Klebsiella spp. were tested for antimicrobial susceptibility to ß-lactams, aminoglycosides, fluoroquinolones, tetracyclines, colistin and nitrofurantoin by the Kirby-Bauer disk diffusion method and the data were documented and analyzed with one per patient analysis using WHONET software. A total of 2292 non-duplicate clinical isolates were recovered during the study period, including 1338 E. coli and 954 Klebsiella spp. The prevalence of AMR in the total isolates was as follows: amikacin, 17.3%; ertapenem, 14.4%; doripenem, 4.5%; colistin, 13.2%; and tigecycline, 4.1%. The study results indicate a high prevalence of carbapenem resistance in Klebsiella spp. especially from pus and urinary isolates, whilst the prevalence of aztreonam and fluoroquinolone resistance was very high in E. coli.