Debadatta Dhar Chanda

Co-Carriage of blaKPC-2 and blaNDM-1 in Clinical Isolates of Pseudomonas aeruginosa Associated with Hospital Infections from India

Global spread of KPC poses to be a serious threat complicating treatment options in hospital settings. The present study investigates the genetic environment of bla KPC-2 among clinical isolates of Pseudomonas aeruginosa from a tertiary referral hospital of India. The study isolates were collected from different wards and clinics of Silchar Medical College and Hospital, India, from 2012–2013. The presence of bla KPC was confirmed by genotypic characterization followed by sequencing. Cloning of the bla KPC-2 gene was performed and the genetic environment of this gene was characterized as well. Transferability of the resistance gene was determined by transformation assay and Southern hybridization. Additionally, restriction mapping was also carried out. Two isolates of P. aeruginosa were found to harbor bla KPC-2, were resistant towards aminoglycosides, quinolone and β-lactam-β-lactamase inhibitor combination. In both the isolates, the resistance determinant was associated with class 1 integron and horizontally transferable. Both the isolates were co-harboring bla NDM-1. The first detection of this integron mediated bla KPC-2 coexisting with bla NDM-1 in P. aeruginosa from India is worrisome, and further investigation is required to track the gene cassette mediated bla KPC-2 in terms of infection control and to prevent the spread of this gene in hospitals as well as in the community.

Premature Termination of MexR Leads to Overexpression of MexAB-OprM Efflux Pump in Pseudomonas aeruginosa in a Tertiary Referral Hospital in India.

Objectives The present study was undertaken to investigate the mutations that are present in mexR gene of multidrug resistant (MDR) isolates of Pseudomonas aeruginosa collected from a tertiary referral hospital of north east India. Methods 76 MDR clinical isolates of P. aeruginosa were obtained from the patients who were admitted to or attended the clinics of Silchar medical college and hospital. They were screened phenotypically for the presence of efflux pump activity by an inhibitor based method. Acquired resistance mechanisms were detected by multiplex PCR. Real time PCR was performed to study the expression of mexA gene of MexAB-OprM efflux pump in isolates with increase efflux pump activity. mexR gene of the isolates with overexpressed MexAB-OprM efflux pump was amplified, sequenced and analysed. Results Out of 76 MDR isolates, 24 were found to exhibit efflux pump activity phenotypically against ciprofloxacin and meropenem. Acquired resistance mechanisms were absent in 11 of them and among those isolates, 8 of them overexpressed MexAB-OprM. All the 8 isolates possessed mutation in mexR gene. 11 transversions, 4 transitions, 2 deletion mutations and 2 insertion mutations were found in all the isolates. However, the most significant observation was the formation of a termination codon at 35th position which resulted in the termination of the polypeptide and leads to overexpression of the MexAB-OprM efflux pump. Conclusions This study highlighted emergence of a novel mutation which is probably associated with multi drug resistance. Therefore, further investigations and actions are needed to prevent or at least hold back the expansion and emergence of newer mutations in nosocomial pathogens which may compromise future treatment options.

Transcriptional Analysis of MexAB-OprM Efflux Pumps System of Pseudomonas aeruginosa and Its Role in Carbapenem Resistance in a Tertiary Referral Hospital in India

Carbapenem resistance presents severe threat to the treatment of multidrug resistant Pseudomonas aeruginosa infections. The study was undertaken to investigate the role of efflux pumps in conferring meropenem resistance and effect of single dose exposure of meropenem on transcription level of mexA gene in clinical isolates of P. aeruginosa from a tertiary referral hospital of India. Further, in this investigation an effort was made to assess whether different components of MexAB-OprM operon expresses in the same manner and the extent of contributions of those components in meropenem resistance in its natural host (P. aeruginosa) and in a heterologous host (E. coli). Out of 83 meropenem nonsusceptible isolates, 22 isolates were found to possess efflux pump activity phenotypically. Modified hodge test and multiplex PCR confirmed the absence of carbapenemase genes in those isolates. All of them were of multidrug resistant phenotype and were resistant to all the carbepenem drug tested. MexAB-OprM efflux pump was found to be overexpressed in all the study isolates. It could be observed that single dose exposure meropenem could give rise to trivial increase in transcription of mexA gene. Different constructs of MexAB-OprM (mexR-mexA-mexB-OprM; mexA-mexB-OprM; mexA-mexB) could be expressed in both its natural (P. aeruginosa PAO1) and heterologous host (E. coli JM107) but transcription level of mexA gene varied in both the hosts before and after single dose exposure of meropenem. Different components of the operon failed to enhance meropenem resistance in E. coli JM107 and P. aeruginosa PAO1. This study could prove that MexAB-OprM efflux pump can significantly contribute to meropenem resistance in hospital isolates of P. aeruginosa where an acquired resistant mechanism is absent. Thus, equal importance should be given for diagnosis of intrinsic resistance mechanism so as to minimize treatment failure. As meropenem could not enhance mexA transcriptions significantly, there might be a possibility that the increase in expression of efflux pump genes does not mediated by single antibiotic but rather by a combination of antipseudomonal drugs which are used during treatments. Early detection of efflux genes will help in selection of proper therapeutic options.

Integron-Borne Transmission of VEB-1 Extended-Spectrum β-Lactamase in Pseudomonas aeruginosa in a Tertiary Care Hospital in India

ABSTRACT A total 14 clinical isolates of Pseudomonas aeruginosa that produced VEB-1 and were susceptible only to polymyxin B were recovered from hospitalized patients. VEB-1 was located within variable regions of the class 1 integron, flanked by resistant genes, and was horizontally transferable as well as carried within the IncP-type plasmid. We conclude that the IncP-type plasmid is responsible for the horizontal transmission of VEB-1-mediated expanded-spectrum cephalosporin resistance in this medical center.

IncX3 plasmid mediated occurrence of blaNDM-4 within Escherichia coli ST448 from India.

This study was designed to investigate blaNDM-4 encoded within IncX3 type plasmid and their copy number alteration under carbapenem pressure within clinical isolates of Escherichia coli. NDM-4 producing E. coli isolates were collected from an Indian hospital and transferability as well as plasmid incompatibility typing was determined. Genetic environment and antibiogram profiling was carried out. Quantitative Real Time PCR was done to determine the change in plasmid copy number under concentration gradient carbapenem stress. Multilocus sequence typing and pulsed field gel electrophoresis was performed for typing of isolates. Four multidrug resistant isolates were found to harbour transconjugable blaNDM-4 carrying within IncX3 type plasmid. The blaNDM-4 was flanked by insertion sequences ISAba125 and IS5 in the upstream region whereas bleMBL was present in the downstream area. Copy number results indicated that the blaNDM-4 gene was maintained high in plasmid under exposure of ertapenem. All the strains belonged to ST448 and PFGE analysis revealed three different pulsotypes. This is the first report of blaNDM-4 encoded IncX3 type plasmid in E. coli of ST448 and needs a systematic screening policy to rapid detection of NDM-4 poducing strains to prevent dissemination of this resistant determinant in future.

Carriage of blaNDM-1 in Pseudomonas aeruginosa through multiple Inc type plasmids in a tertiary referral hospital of northeast India.

Pseudomonas aeruginosa is known to be a predominant opportunistic pathogen and also a frequent cause of nosocomial infection in patients with compromised immune system. Treatment option becomes complicated when this type of organism harbour resistance determinants such as New Delhi metallo-β-lactamase-1 (NDM-1). The genetic vehicles carrying this gene are often responsible for their horizontal spread, dissemination and maintenance within a broad host range1. Knowledge about transmission dynamics of blaNDM-1 is a key to succeed in the effort of infection control and slowing down the spread of multidrug resistance. This study was undertaken to characterize blaNDM-1 in clinical isolates of P. aeruginosa, their transmission dynamics and plasmid Inc types responsible for their horizontal transfer in a tertiary referral hospital of northeast India.

Distribution of Class II integrons and their contribution to antibiotic resistance within Enterobacteriaceae family in India.

Background: Integrons are the main contributors to the development of multidrug resistance (MDR) among Gram-negative bacilli. There is a lack of knowledge about the molecular relation between gene cassettes and antibiotic resistance in India. Objective: In this study, we have investigated the occurrence of Class II integron and their cassette array among Enterobacteriaceae. Materials and Methods: A total of 268 MDR non-duplicate strains of Enterobacteriaceae were collected from Silchar Medical College and Hospital, Silchar, Assam, India, during June 2012 to May 2013. Polymerase chain reaction was performed for detection of the integrase genes and gene cassettes within the Class II integron which were further analysed by sequencing. Results: Class II integron was observed in 47 isolates. Four different gene cassette arrangements were detected: dfrA1-sat2-aadA1; dfrA1-sat2-aadA1-orfX-ybeA-ybfA-ybfB-ybgA; dfrA12-sat2-aadA1; and dfrA1-linF-aadA1. The most prevalent cassette combination was dfrA1-sat2-aadA1. This study has also identified a set of gene cassette associated with linF gene instead of sat2 gene. Conclusion: Further investigation is required to determine the current situation and important reservoir of Class II integron for the transmission of drug resistance among Enterobacteriaceae and their contribution to antimicrobial resistance in hospital environment .

Detection and molecular typing of methicillin-resistant Staphylococcus aureus from northeastern part of India

Background In Staphylococcus aureus, methicillin resistance is exhibited by modifications in penicillin-binding protein that minimises the binding affinity to beta-lactam antibiotics. The present study investigated the occurrence of methicillin-resistant S. aureus (MRSA) in community-acquired infections, that is, community-acquired MRSA (CA-MRSA) and in-hospital-acquired infections, that is, hospital-acquired MRSA (HA-MRSA) from Northeast India. Methods A total of 197 consecutive non-duplicate isolates were collected from Silchar Medical College and Hospital and other private diagnostic laboratories. The isolates were confirmed to be S. aureus at our centre. All isolates were subjected to antibiotic susceptibility testing and were screened for methicillin resistance using cefoxitin disc test. All MRSA were subjected to Polymerase Chain Reaction (PCR) assay for detection of mecA and mecC genes. DNA fingerprinting was performed for determining clonal diversity. Results Seventy-one isolates of 127 confirmed S. aureus were found to be methicillin resistant by screening test. mecA gene was detected in 43 isolates, and none of the isolates were positive for mecC gene. Linezolid and teicoplanin showed better activity with susceptibility pattern being 83.6% and 72.44%, respectively, whereas 66.14% were sensitive to vancomycin. Other antibiotic showed low level of activity. Pulsed Field Gel Electrophoresis (PFGE) showed 14 different banding patterns that suggest isolates were of different clonal types. Conclusion mecA was responsible for methicillin resistance in majority of strains. Polyclonal spread of MRSA infection in the study area indicates its diverse origin and possible lateral transfer. Thus, this study is of clinical interest in terms of selection of proper antimicrobial chemotherapy and infection control management.

Observation of a new pattern of mutations in gyrA and parC within Escherichia coli exhibiting fluroquinolone resistance

Therapeutic options with quinolones are severely compromised in infections caused by members of Enterobacteriaceae family. Mutations in chromosomal region are one of the major reasons for bacterial resistance towards this group of antibiotic. The aim of the study is to detect the mutations in gyr A and par C responsible for quinolone resistance among clinical isolates of Escherichia coli. A total of 96 quinolone-resistant clinical isolates of E. coli were collected from a tertiary care hospital of North-east India during March 2015 to August 2015. All the quinolone-resistant E. coli strains were investigated for mutations in the topoisomerases genes gyrA and parC by amplifying and sequencing the quinolone resistance determining regions. Among the 96 E. coli isolates, 83.3% were resistant to nalidixic acid and 80.2%, 66.6%, 23.9% and 50% to ciprofloxacin, norfloxacin, levofloxacin and ofloxacin, respectively. Several alterations were detected in gyrA and parC genes. Three new patterns of amino acid substitution are reported in E. coli isolates. The findings of this study warrant a review in quinolone-based therapy in this region of the world to stop or slow down the irrational use this drug.

Association of glycerol kinase gene with class 3 integrons: A novel cassette array within Escherichia coli

Background: Integrons are genetic elements which are known for their role in capturing and spreading of antibiotic resistance determinants among Gram-negative bacilli. So far, there is no study regarding Class 3 integron and their genetic organisation in India. Objective: This study investigates the occurrence of Class 3 integron and their gene cassette array among Escherichia coli. Materials and Methods: In this study, a total of 200 E. coli isolates were collected from indoor and outdoor patients from Silchar Medical College and Hospital during September 2015 to February 2016. Detection of the integrase genes and gene cassettes within the Class 3 integron was performed by polymerase chain reaction which was further analysed by sequencing. Results: Twenty-seven isolates were found to harbour Class 3 integron. Sequencing of the gene cassettes and whole Class 3 integron revealed the presence of nine different types of cassettes array, out of which the arrangement with glycerol kinase gene cassette was found to be the most prevalent. Arrangement with blaCTX-Mgene cassette was also detected in few isolates. Conclusion: This study provides epidemiological profiling of Class 3 integrons in this geographical area. The data generated in this study are helpful in infection control programme, anti-infective research and search for epidemiological markers.