Amitabha Bhattacharjee

Dissemination of the New Delhi metallo-β-lactamase-1 (NDM-1) among Enterobacteriaceae in a tertiary referral hospital in north India

Sir, Of the different classes of carbapenemases, class B enzymes are clinically the most significant; they possess the widest substrate hydrolysis range, including penicillins, cephalosporins and carbapenems, but not monobactams. In recent years, metallo-b-lactamase (MBL) genes have spread from Pseudomonas aeruginosa to members of the Enterobacteriaceae. An alarming report by the HPA UK in 2009 warned of a rapidly proliferating novel carbapenem-hydrolysing b-lactamase (carbapenemase) designated New Delhi MBL-1 (NDM-1) among the Enterobacteriaceae family, identified in UK hospital patients. Moreover, the common presence of these b-lactamase genes in transferable mobile elements means that these genes could reach virtually any Gram-negative bacterium and this provides an added risk of dissemination in the community. Our present work was undertaken with the objective of detecting the blaNDM-1 gene among clinical isolates of the Enterobacteriaceae family in a tertiary referral hospital in north India. A total of 780 consecutive, non-duplicate isolates of Escherichia coli (n1⁄4528), Klebsiella pneumoniae (n1⁄4126), Citrobacter species (n1⁄484), Enterobacter aerogenes (n1⁄422), Proteus mirabilis (n1⁄411) and Morganella morganii (n1⁄49) were recovered from different clinical specimens from patients who were admitted to different wards, as well as from those who attended the outpatient departments of S.S. Hospital, BHU, Varanasi, India. The study was conducted from February 2010 to July 2010. The work was approved by the Ethics Committee. An imipenem/EDTA disc potentiation test was performed for phenotypic detection of MBLs. Antimicrobial susceptibility tests were performed using the Kirby–Bauer disc diffusion method and results were interpreted according to the CLSI recommendations. For partial gene PCR amplification, primers specific for the blaNDM-1 gene were used for reaction with bacterial DNA as template. PCR was performed as described previously. Random amplified polymorphic DNA (RAPD) was performed using primer 7 (5′-GTGGATGCGA-3′) and isolates were typed according to their band patterns. Sixty-four isolates were phenotypically found to be MBL producers. On performing PCR for all of the MBL-producing isolates the presence of the gene encoding NDM-1 was confirmed among 54 isolates, consisting of E. coli (n1⁄430), Citrobacter species (n1⁄412) and K. pneumoniae (n1⁄412), with an overall occurrence of 6.9% (54/780). Similar to this study, Deshpande et al. reported 22 NDM-1-producing Enterobacteriaceae in a short span of 3 months, while in a previous multicentre study, the occurrence was reported to be 2% from this centre. Most often, NDM producers were recovered from intensive care unit patients (35.1%). The age of the patients ranged from 1 day to 85 years, with 32 male patients and 22 female patients (Table S1, available as Supplementary data at JAC Online). The presence of class 1 integrons was demonstrated in all of the NDM-1-harbouring isolates. On typing the NDM-1-harbouring isolates, 18 patterns of E. coli, 8 patterns of K. pneumoniae and 5 patterns of Citrobacter species were found by RAPD. These results suggest horizontal transmission of the gene at both the intraspecies and interspecies level. Disc diffusion susceptibility testing showed that 46 (85.1%) of the NDM-1-producing isolates were susceptible to polymyxin B and 25 (46.2%) were susceptible to tigecycline. For other antimicrobials, 51.8% showed susceptibility to piperacillin/tazobactam and 22.2%, 5.5% and 1.8% showed susceptibility to amikacin, gentamicin and tobramycin, respectively. Among 23 urinary tract isolates, 8 showed susceptibility to nitrofurantoin. As many as 27 (50%), 26 (48.1%) and 16 (29.6%) isolates were found to be susceptible to ertapenem, imipenem and meropenem, respectively. A previous study has demonstrated 32.4% similarity of NDM-1 to VIM-1/VIM-2-type MBL and it Research letters

Evaluation of the RAPIDEC CARBA NP Test Kit for detection of Carbapenemase Producing Gram Negative Bacteria

ABSTRACT Recently, bioMérieux, France, introduced the Rapidec Carba NP test kit for rapid detection of carbapenemase-producing Gram-negative bacteria. This kit was evaluated in this study, and we report sensitivity, specificity, and positive and negative predictive values of 92.6%, 96.2%, 95.83%, and 92.6%, respectively. The test was easy to perform and interpret and relatively inexpensive (

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

5/Rs 300 per test) and provides a practical solution for early detection of carbapenemase-producing, multidrug-resistant Gram-negative bacteria.

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

Transcriptional Analysis of MexAB-OprM Efflux Pumps System of Pseudomonas aeruginosa and Its Role in Carbapenem Resistance 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.

Genetic Environment of Plasmid Mediated CTX-M-15 Extended Spectrum Beta-Lactamases from Clinical and Food Borne Bacteria in North-Eastern 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

Background The study investigated the presence of CTX-M-15 type extended spectrum beta-lactamases (ESBL), compared their genetic arrangements and plasmid types in gram negative isolates of hospital and food origin in north-east India. From September 2013 to April 2014, a total of 252 consecutive, non-duplicate clinical isolates and 88 gram negative food isolates were selected. Phenotypic and molecular characterization of ESBL genes was performed. Presence of integrons and gene cassettes were analyzed by integrase and 59 base-element PCR respectively. The molecular environments surrounding bla CTX-M and plasmid types were investigated by PCR and PCR-based replicon typing respectively. Transformation was carried out to assess plasmid transfer. Southern blotting was conducted to localize the bla CTX-M-15 genes. DNA fingerprinting was performed by ERIC-PCR. Results Prevalence of ESBL was found to be 40.8% (103/252) in clinical and 31.8% (28/88) in food-borne isolates. Molecular characterization revealed the presence of 56.3% (58/103) and 53.5% (15/28) bla CTX-M-15 in clinical and food isolates respectively. Strains of clinical and food origin were non-clonal. Replicon typing revealed that IncI1 and IncFII plasmid were carrying bla CTX-M-15 in clinical and food isolates and were horizontally transferable. The ISEcp1 element was associated with bla CTX-M-15 in both clinical and food isolates. Conclusions The simultaneous presence of resistance determinants in non-clonal isolates of two different groups thus suggests that the microbiota of common food products consumed may serve as a reservoir for some of the drug resistance genes prevalent in human pathogens.

Screening of Natural Products and Derivatives for the Identification of RND Efflux Pump Inhibitors

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.

Computational validation of 3-ammonio-3-(4-oxido- 1H-imidazol-1-ium-5-yl) propane-1, 1-bis (olate) as a potent anti-tubercular drug against mt-MetAP.

AIM AND OBJECTIVE Overexpression of efflux pumps belonging to the Resistance Nodulation cell Division (RND) family is the most important intrinsic resistance mechanism of Pseudomonas aeruginosa. Hence, it is imperative to identify suitable efflux pump inhibitors (EPI) that can lead to increased intracellular concentration of antibiotics by blocking the pump. This study was undertaken to identify a putative plant based efflux pump inhibitor for RND efflux pump of P. aeruginosa. MATERIAL AND METHOD Using molecular docking approach, 328 secondary plant metabolites have been screened for their inhibitory activity against cytoplasmic exporter protein MexB of MexAB-OprM efflux pump of P. aeruginosa. After the initial in silico screening, the shortlisted compounds were subjected to in vitro test for efflux pump inhibitory activity using double disc synergy test. A combinatorial library of 1000 molecules was generated from active p-coumaric acid and docked with MexB protein to find a suitable EPI with better binding efficacy compared to the p-coumaric acid. RESULTS Preliminary screening resulted in five plant-based natural products with significant docking score and were subsequently subjected to double disc synergy test. p-Coumaric acid , amongst the five, was found to potentiate activity of ciprofloxacin in MexAB-OprM overexpressing P. aeruginosa strain. Library compound 482, i.e 4-(4-((Z)-2-carboxy-2-((Z)-2,3-dihydrobenzo[e][1,4]diazepin-1-yl)-1-(4- hydroxyphenyl)vinylamino) phenylsulfonamido)-2-hydroxybenzoic acid, a derivative of p-coumaric acid exhibited the highest docking score of -42.1030 Kcal/mol, which was much higher than parent compound (-17.9403 Kcal/mol) and also known EPI, MC-207,110 (-28.0960 Kcal/mol). CONCLUSION p-Coumaric acid and its derivative, 4-(4-((Z)-2-carboxy-2-((Z)-2,3-dihydrobenzo[e][1,4] diazepin-1-yl)-1-(4-hydroxyphenyl)vinylamino)phenylsulfonamido)-2-hydroxybenzoic acid may be used as potential lead molecules for effective RND efflux pump inhibition in P. aeruginosa.

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

The advent of Multi Drug Resistant (MDR) strain of Mycobacterium tuberculosis (TB) necessitated search for new drug targets for the bacterium. It is reported that 3.3% of all new tuberculosis cases had multidrug resistance (MDR-TB) in 2009 and each year, about 0.44 million MDR-TB cases are estimated to emerge and 0.15 million people with MDR-TB die. Keeping such an alarming situation under consideration we wanted to design suitable anti tubercular molecules for new target using computational tools. In the work Methionine aminopeptidase (MetAP) of Mycobacterium tuberculosis was considered as target and three non-toxic phenolic=ketonic compounds were considered as ligands. Docking was done with Flex X and AutoDock 4.2 separately. Ten proven inhibitors of MetAP were collected from literature with their IC50 and were correlated using EasyQSAR to generate QSAR model. Activity of ligands in question was predicted from QSAR. Pharmacophore for each docking was generated using Ligandscout 3.0. Toxicity of the ligands in question was predicted on Mobyle@rpbs portal and Actelion property explorer. Molecular docking with target showed that of all three ligands, 3-ammonio-3-(4-oxido-1H-imidazol-1-ium-5-yl) propane-1, 1-bis (olate) has highest affinity (- 37.5096) and lowest IC50 (4.46 µM). We therefore, propose that -3-ammonio-3-(4-oxido-1H-imidazol-1-ium-5-yl) propane-1,1- bis(olate) as a potent MetAP inhibitor may be a new anti-tubercular drug particularly in the context of Multi Drug Resistant Tuberculosis (MDR-TB).