ABSTRACT
Background and Objectives: This study was designed to determine the in vitro efficacy of mecillinam against extended spectrum beta lactamse producing Enterobacterales. Materials and Methods: After proper permission from Ethical Review Committee of the Institute, all samples yielding growth of ESBL producing Enterobacterales were part of the study and were processed according to routine microbiological procedures. Routine antibiotic sensitivity testing was done on Muller Hinton Agar by Modified Kirby Bauer Method. All Gram negative isolates were subjected to concomitant detection of ESBL production by double disc synergy method. All ESBL producers were then subjected to the mecillinam Minimum Inhibitory Concentration (MIC) determination by E test. The results were interpreted as per CLSI Guidelines. Results: A total of 120 ESBL producing Enterobacterales isolates were included in the study. The mean age of patients with ESBL infection was 45 ± 18.7 years. There were 44% male and 55% female patients. Majority of the ESBL producing Enterobacterales were isolated from urine samples (56%), followed by pus. Among the isolated organisms, Escherichia coli (45%) was the most frequently isolated organism followed by Klebsiella spp. (22%). Overall 83% of the isolates turned out to be sensitive to mecillinam. MIC50 of mecillinam against ESBL producing Gram negative rods (GNR) turned out to be 1 ug/ml and MIC90 turned out to be 2 ug/ml. Conclusion: Mecillinam shows good in vitro efficacy against ESBL producing Enterobacterales in our study. Further studies with more sample size and from diverse areas across the country should be done to evaluate its efficacy.
ABSTRACT
A multiplex PCR (mPCR) assay using previously known genetic markers of Shigella, Escherichia coli and Shiga-toxic Esch. coli was standardized. uidA gene was targeted for the common detection of Esch. coli and Shigella, whereas ipaH and stx1 genes were used as markers for the detection of Shigella and shiga-toxin producing strains, respectively. The standardized assays detected the target organism specifically and selectively. The mPCR developed by combining all the three reactions generated specific products. The inclusivity and exclusivity tests depicted the precise specificity of the mPCR assay. Results were interpreted on the basis of the pattern of amplicons generated: amplifications of the ipaH and uidA gene fragments indicated the presence of Shigella spp., amplification of uidA alone revealed the presence of Esch. coli and additional presence of verotoxin gene amplicon indicated verotoxinogenic nature of the strain. Specific patterns of bands were obtained when different strains of Esch. coli and Shigella spp. were subjected to this assay. The reactions, individually as well as in the mPCR, could detect approximately 1 cell per 20-microl PCR assay. The protocols were validated by analyzing the coded samples of full fat milk spiked with different pathogens. In naturally contaminated raw milk samples (n=100), Esch. coli were detected in all samples and verotoxinogenic Esch. coli in 15 samples. Shigella, however, was not detected in any of the samples. When DNA purified from the samples found positive for Shiga-toxic Esch. coli was directly used as template for the mPCR, the results showed agreement with the enrichment based detection. The mPCR assay, standardized in this study, may be used for rapid microbiological evaluation of milk samples. Further, the study emphasizes the need for better hygienic conditions in dairies.
