Deepjyoti Paul

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.

Prevalence of community acquired urinary tract infections in Silchar Medical College, Assam, India and its antimicrobial susceptibility profile.

BACKGROUND This study was conducted to know the rate of prevalence of community acquired UTI infections in both males and females, Assam, North-East India. MATERIALS AND METHODS A 50 μl of urine sample from each of the subjects was streaked on cystine lactose electrolyte-deficient agar medium. The plates were incubated overnight at 37°C. Pure, isolated colonies of each type was separately cultured and characterized. The susceptibility of the clinical isolates to routinely prescribed antibiotics in the hospital was performed against various antibiotics. RESULTS Of the total 40 patients, 26 positive samples were obtained of which 19 females (73.07%) and 7 male (26.92%) patients were shown to be urine culture positive. The most commonly isolated bacterium was Escherichia coli with a frequency rate of (33.3%) followed by Staphyloccous aureus (22.2%), Klebsiella pnemoniae (11.1%), coagulase negative Staphylococcus (CoNS) (7.4%), Pseudomonas sp. (7.4%), Proteus myxofaciens (3.7%), Proteus mirabilis (3.7%), Edwardsiella tarda (3.7%), Morganella morganii (3.7%), and Citrobacter fruendii (3.7%). Analysis of the samples showed that UTI was more common in females of younger age group as compared with males. It was also observed that the patients responded effectively to imepenem (IE), cepefime, amikacin, norfloxacin, and co-trimoxazole antimicrobial agents against Gram-negative bacilli. Furthermore, the most effective antibiotics against Gram-positive cocci was found to be ampicillin, ciprofloxacin, IE, and penicillin G (benzyl penicillin). CONCLUSIONS Antibiotics have been in use for a long period and more often the misuse of antimicrobial drugs has today led to a general rise in the emergence of resistant bacteria.

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.

An unusual occurrence of plasmid-mediated blaOXA-23 carbapenemase in clinical isolates of Escherichia coli from India

The blaOXA-23 group was considered as the first group of OXA-type β-lactamases conferring carbapenem resistance and has been reported worldwide in Acinetobacter baumannii, however their presence in Escherichia coli is very rare and unique. This study describes an unusual occurrence of blaOXA-23 in 14 clinical isolates of E. coli obtained from intensive care unit patients admitted to a tertiary referral hospital in India. The blaOXA-23 gene was found located within a self-conjugative plasmid of IncFrepB and IncK incompatibility types and simultaneously carrying blaCTX-M-15, blaVEB-1, blaPER-1 and/or blaNDM-1. The copy number of blaOXA-23 within the IncK-type plasmid was inversely proportional to increasing concentrations of imipenem, whereas in the case of the IncFrepB-type the result was variable; and increased copy number of the IncK-type plasmid was observed with increasing concentrations of meropenem. Plasmids encoding blaOXA-23 could be successfully eliminated after single treatment and were found to be not highly stable, as complete loss of plasmids was observed within 5-10 days. This study emphasises that carbapenem stress invariably altered the copy number of two different Inc type plasmids encoding the blaOXA-23 resistance gene and also highlights a potential threat of clonal expansion of this class D carbapenemase through a heterologous host in this country, which is in second incidence globally.

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.

Occurrence of blaNDM-7 within IncX3-type plasmid of Escherichia coli from India

BACKGROUND New-Delhi metallo-β-lactamase-7 with higher hydrolytic activity than its ancestor NDM-1 is emerging across the globe including India. In this study, we have investigated the genetic context of blaNDM-7 and alteration in plasmid copy number under concentration gradient carbapenem stress. MATERIALS AND METHODS Six blaNDM-7 producing Escherichia coli isolates were obtained from Silchar Medical College and Hospital and the co-existence of other β-lactamases and transferability of this resistant determinant was determined by transformation and conjugation assay followed by typing of the plasmid by PBRT method. Genetic context and plasmid stability of blaNDM-7 was also determined. The change in copy number of transconjugable plasmid carrying blaNDM-7 under exposure of different carbapenem antibiotics was determined by quantitative Real Time PCR. RESULTS All the six isolates carrying blaNDM-7 were conjugatively transferable through an IncX3-type plasmid and were also found to co-harbor blaCTX-M-15. Genetic analysis of blaNDM-7 showed an association of ISAba125, IS5 and a truncated portion of ISAba125 in the upstream region and bleMBL gene in the downstream region of blaNDM-7. Complete loss of the plasmids carrying blaNDM-7 was observed between 85th to 90th serial passages when antibiotic pressure was withdrawn. After analyzing the relative copy number it was observed that the copy number of the blaNDM-7 encoding plasmid was highly affected by the concentration of ertapenem. CONCLUSION The present study has first demonstrated presence of IncX3-type plasmid encoding blaNDM-7 within nosocomial isolates of E. coli. Measures must be taken to prevent or atleast slowdown the emergence of this resistance determinant in this country.

Effect of single-dose carbapenem exposure on transcriptional expression of blaNDM-1 and mexA in Pseudomonas aeruginosa

The therapeutic option of a carbapenem antibiotic is compromised in Pseudomonas aeruginosa owing both to acquired and intrinsic resistance mechanisms. In recent years, New Delhi metallo-β-lactamase has been the focus as a predominant carbapenem resistance determinant. However, it is unclear which of the mechanisms might be adopted by a P. aeruginosa strain possessing both blaNDM-1 and an overexpressed MexAB-OprM system during carbapenem therapy. This study investigated the interplay of both mechanisms in clinical isolates of P. aeruginosa when exposed to meropenem. Five strains were used: (i) strain overexpressing MexAB-OprM but with no blaNDM-1; (ii) strain harbouring blaNDM-1 but expressing MexAB-OprM at basal level; (iii) strain possessing blaNDM-1 and overexpressing MexAB-OprM; (iv) P. aeruginosa PAO1; and (v) P. aeruginosa K2733-PAO1 (ΔMexAB-OprMΔMexCD-OprJΔMexEF-OprNΔMexXY-OprM) into which blaNDM-1 was cloned. Strains were incubated in Luria-Bertani broth with and without 1μg/mL meropenem. Total RNA was isolated at 45-min intervals and was immediately reverse transcribed to cDNA. This was repeated for 6h. Quantitative real-time PCR was performed for both resistance mechanisms. Meropenem exposure did not significantly elevate transcription of either the blaNDM-1 or mexA gene. However, an interesting finding was that upon single-dose exposure to carbapenem, the efflux pump system played a major role in bacterial survival compared with NDM-1. This study gives an insight into the bacterial response to carbapenem antibiotic when two different resistance mechanisms coexist. This type of study would be helpful in designing future antimicrobials.

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.

Molecular and in silico analysis of a new plasmid-mediated AmpC β-lactamase (CMH-2) in clinical isolates of Klebsiella pneumoniae.

Two Klebsiella strains isolated from urine samples were positive for blaAmpC by PCR and showed sequence similarity with CMH-1 (98.6%) after sequencing. It also shares 82% similarity with ACT-1, 85% with MIR-1 and 81% with the chromosomal AmpC gene of Enterobacter cloacae. This gene was associated with the plasmid of IncK type. It has an open reading frame of 381 amino acid with four amino acid substitutions at position D144A, C189R, Q192E, and A195T as compared to CMH-1. When expressed in E.coli DH5α and E.coli strain B, this β-lactamase conferred resistance to cefotaxime, ceftriaxone and ceftazidime. In addition, both in vitro and in silico analysis revealed that this cephalosporinase was inhibited by cefepime and carbapenem group of drugs. Therefore, this new plasmid-encoded AmpC type β-lactamase gene was designated as CMH-2.