Klebsiella pneumoniae (Kp) has gained prominence in the last two decades due to its global spread as a multidrug-resistant (MDR) pathogen. Further, carbapenem-resistant Kp are emerging at an alarming rate. The objective of this study was (1) to evaluate the prevalence of β-lactamases, especially carbapenemases, in Kp isolates from India, and (2) determine the most prevalent sequence type (ST) and plasmids, and their association with β-lactamases. Clinical samples of K. pneumoniae (n = 65) were collected from various pathology labs, and drug susceptibility and minimum inhibitory concentrations (MIC) were detected. Whole genome sequencing (WGS) was performed for n = 22 resistant isolates, including multidrug-resistant (MDR) (n = 4), extensively drug-resistant (XDR) (n = 15), and pandrug-resistant (PDR) (n = 3) categories, and genomic analysis was performed using various bioinformatics tools. Additional Indian MDRKp genomes (n = 187) were retrieved using the Pathosystems Resource Integration Center (PATRIC) database. Detection of β-lactamase genes, location (on chromosome or plasmid), plasmid replicons, and ST of genomes was carried out using CARD, mlplasmids, PlasmidFinder, and PubMLST, respectively. All data were analyzed and summarized using the iTOL tool. ST231 was highest, followed by ST147, ST2096, and ST14, among Indian isolates. bla_ampH was detected as the most prevalent gene, followed by bla_CTX-M-15 and bla_TEM-1. Among carbapenemase genes, bla_OXA-232 was prevalent and associated with ST231, ST2096, and ST14, which was followed by bla_NDM-5, which was observed to be prevalent in ST147, ST395, and ST437. ST231 genomes were most commonly found to carry Col440I and ColKP3 plasmids. ST16 carried mainly ColKP3, and Col(BS512) was abundantly present in ST147 genomes. One Kp isolate with a novel MLST profile was identified, which carried bla_CTX-M-15, bla_OXA-1, and bla_TEM-1. ST16 and ST14 are mostly dual-producers of carbapenem and ESBL genes and could be emerging high-risk clones in India.

Introduction and Aim: Carbapenem Resistant Enterobacteriaceae (CRE) have emerged at an alarming rate. Multi locus sequence typing (MLST) is an important parameter for identifying drug resistant organisms. The present study was carried out for elucidating the mechanisms of CRE and MLSTs associated with CRE. Materials and Methods: CRE (n=14) were obtained from various clinical samples and subjected to Rapidec Carba NP (CNP) test and multiplex polymerase chain reaction (M-PCR) and five isolates proceeded for whole genome sequencing (WGS). ?-lactamase (bla) genes were analysed using Resfinder and CARD tool. Bioinformatics tools: mlplasmids, plasmid finder, mobile element finder, and Center for Genomic Epidemiology (CGE) toolbox were used. Results: All isolates (n=14) were positive for CNP and bla genes using M-PCR. Isolates (J21, J22, J23, J27) were identified as Escherichia coli while (J34) was Enterobacter hormaechei. MLST showed E. coli isolates (J21& J22) as ST648; E. coli (J23) was ST940; E. coli (J27) was ST 2851, and E. hormaechei (J34) was closest to ST1325. Genes blaTEM, blaNDM & blaampC were found to be present in all isolates; blaCTX-M was present in all E. coli isolates but not in E. hormaechei. blaOXA was present in E. coli (J23) and in E. hormaechei (J34); while ESBL blaSFO-1 in E. hormaechei (J34). Conclusion: ESBLs (blaTEM & blaCTXM) and metallo beta-lactamase -MBL (blaNDM) cause carbapenem resistance in rare sequence types of E. coli while; ESBL (blaSFO-1) and MBL (blaNDM) cause carbapenem resistance in E. hormaechei.

Klebsiella pneumoniae (Kp) has gained prominence in the last two decades due to its global spread as a multi-drug resistant (MDR) pathogen. Further, Carbapenem-Resistant Kp are emerging at an alarming rate. The objective of this study was (1) to evaluate the prevalence of β-lactamases, especially carbapenemases in Kp isolates from India, (2) determine the most prevalent sequence type (ST) & plasmids, and their association with β-lactamases. Clinical samples of K. pneumoniae (n=65) were collected from various pathology lab, drug susceptibility and minimum inhibitory concentrations (MIC) were detected. Whole genome sequencing (WGS) was done for (n=22) resistant isolates and WGS analysis was performed using various bioinformatics tools. Additional Indian MDR Kp genomes (n=187) were retrieved using Pathosystems Resource Integration Center (PATRIC) database. Detection of β-lactamase genes, location, plasmid replicons, and ST type of genomes were carried out using CARD, mlplasmids, PlasmidFinder, and PubMLST respectively. All data were analyzed and summarized using iTOL tool. ST231 was highest, followed by ST147, ST2096 & ST14 among Indian isolates. blaAmpH was detected as the most prevalent gene followed by blaCTX-M-15, blaTEM-1. Among carbapenemase genes, blaOXA-232 was prevalent and associated with ST231, ST2096 and ST14, which was followed by blaNDM-5 which was observed to prevalent in ST147, ST395 &ST437. ST231 genomes were most commonly found to carry Col440I and ColKP3 plasmids. ST16 carried mainly ColKP3, and Col (BS512) was abundantly present in ST147 genomes. One Kp isolate with novel MLST profile was identified, which carried blaCTX-M-15, blaOXA-1 and blaTEM-1. ST16 &ST14 from this study, which is mostly dual producer of carbapenem and ESBL genes, could be emerging high-risk clones in India.

Due to an increase in serious infections and a lack of efficient therapies, Klebsiella pneumoniae has recently gained more recognition. The production of carbapenemases is one of the most common strategies by which K. pneumoniae acquire resistance to carbapenems which is considered the last resort of antibiotics. Previously collected isolates from different clinical settings and on the basis of their genetic profile, mainly the absence and presence of single or dual carbapenemases (OXA-181, OXA-232, NDM-1, NDM-5, NDM-5+OXA-181, and NDM-1+OXA-232), mutations in porins, and efflux pumps, seven isolates (M40, M52, M39, J20, M53, M49, and M17B) were selected. Its phenotypic resistance against two carbapenem drugs (ertapenem and meropenem) was checked and we found NDM-5 followed by OXA-181 and NDM-5+OXA-181 carrying isolates showed high MIC values. Further, no significant differences were observed either in the presence of efflux pumps or mutations in porins among isolates. By molecular docking, among single amino acid differences between OXA-181 and OXA-232 and with two amino acids differences between NDM-1 and NDM-5, OXA-232 and NDM-5 showed a higher binding affinity than OXA-181 and NDM-1 with both antibiotics. It is concluded that the presence of specific carbapenemases or combinations of the same can drastically increase MIC values. The presence of NDM-5, and OXA-181, or their combinations is more fatal than NDM-1+OXA-232.