Identification and validation of reference genes for reliable analysis of differential gene expression during antibiotic induced persister formation in Klebsiella pneumoniae using qPCR.

The study of differential gene expression in persister cells is compounded by ceasure of conventional cellular metabolic pathways during persistence. There is, hence, a requirement to identify and validate suitable reference genes whose expression remains stable during persistence. We evaluated the suitability of five genes viz. dnaJ, groEL, rpoB, kp751, kp4432 as references to study gene expression using real-time polymerase chain reaction (qPCR) during persister cell formation in Klebsiella pneumoniae. Results obtained showed that while dnaJ and groEL suffered from unstable expression; rpoB, kp751 and kp4432 showed stable expression. Further, it was observed that data normalization using either of the stable genes viz. rpoB, kp751, kp4432 alone, resulted in either too low expression levels or too high variation among replicates. Our study indicates the concurrent use of kp4432 and rpoB as reference genes to be the most suitable for reliable analysis of differential gene expression during antibiotic induced persister formation in K. pneumoniae. kp4432 and rpoB encode NAD-dependant phenylacetaldehyde dehydrogenase and DNA-directed RNA polymerase beta subunit respectively. The outcome of this study will increase the utility of qPCR in studying the temporal changes in gene expression during persistence. The study will also aid in understanding mechanisms underlying persister cell formation particularly in K. pneumoniae.

Retrospective study on clonal relationship of multidrug-resistant Klebsiella spp. indicates closed circulation and initiation of clonal divergence.

PURPOSE: Antibiotic resistance patterns often exhibit geographical variations. Periodic analyses of resistance spectra and phylogenetic trends are important guides for facilitating judicious use of therapeutic interventions. The present study retrospectively analysed the infection trends, resistance patterns, and clonal relationships between isolates of Klebsiella spp. from a tertiary care hospital. METHODOLOGY: Bacterial isolates were collected from January 2013 to June 2014 and their resistance profiles were identified using an automated bacterial identification system. A phylogenetic tree was constructed using housekeeping genes with Molecular Evolutionary Genetic Analysis software. The dN/dS ratio was determined by the Synonymous Non-synonymous Analysis Program while polymorphic sites, and the difference per site was calculated using DNA Sequence Polymorphism software. Statistical Package for Social Science software was used to perform all statistical analyses. KEY FINDINGS: The results of this study indicated the prevalence of community-acquired urinary tract and lower respiratory tract infections caused by Klebsiella spp. among geriatric patients. The occurrence of new allelic profiles, a low dN/dS ratio and the lack of strong evolutionary descent between isolates indicated that mutations play a major role in the evolution of the organism. CONCLUSION: The findings of this study highlight the consequences of antimicrobial agents exerting a silent and strong selective force on the evolution of Klebsiella spp. The expansion of such analyses is of great importance for addressing rapidly emerging antibiotic-resistant opportunistic pathogens.

Draft Genome Sequences of Clinical and Nonclinical Isolates of Klebsiella spp. Exhibiting Nonheritable Tolerance toward Antimicrobial Compounds.

A clinical isolate and a nonclinical isolate of Klebsiella pneumoniae were found to exhibit nonheritable tolerance in response to antimicrobial compounds. The draft genome sequences of both isolates are presented here.

Draft Genome Sequences of Nonclinical and Clinical Enterobacter cloacae Isolates Exhibiting Multiple Antibiotic Resistance and Virulence Factors.

Enterobacter spp. have been implicated as opportunistic pathogens which over the years have gained resistance toward most of the available therapeutic drugs. We sequenced two multidrug-resistant Enterobacter cloacae isolates harboring multiple efflux pump genes. These isolates exhibited strain-specific modulation of efflux pump protein expression.

Antibiotic resistance determinants and clonal relationships among multidrug-resistant isolates of Klebsiella pneumoniae.

In the present study, we performed PCR based screening to determine the presence of antibiotic resistance genes and sequencing to find mutation in QRDR region among fourteen isolates of K. pneumoniae. Association analysis was conducted to detect the co-resistance among the isolates. Multi-locus sequence analysis was carried out to determine the clonal relationship among them. All the K. pneumoniae isolates showed resistance to multiple antibiotics and exhibited cross-resistance to antibiotics. Although few isolates co-harbored variants of ß-lactamase genes, others carried qnrB on plasmid and mutations in Quinolone-Resistant Determining Region (QRDR). This study thus indicates that clonally unrelated K. pneumoniae isolates exhibited co-resistance, harboured multiple antibiotic resistance genes present on the chromosome, plasmids and/or integron Therefore, the data from this study can provide guidelines for the prudent use of antibiotics to avert the impending danger of losing out on the available antibiotics for therapeutic use.

Efflux mediated colistin resistance in diverse clones of Klebsiella pneumoniae from aquatic environment.

The peptide drug colistin is commonly used to treat carbapenem resistant gram negative bacterial infections. In the present study, we report efflux mediated colistin resistance in multidrug resistant Klebsiella pneumoniae isolates belonging to ST200 and ST1296, isolated from a fresh water environment. The isolates exhibited intermediate resistance to human serum, possessed Type 1 fimbriae and harbored blaSHV-34 and blaTEM-1 genes. Our results highlight the evolving nature of these clones in the country. These observations emphasize the need for judicious usage of antibiotics to prevent the imminent danger of losing out on currently available therapeutic options.