Debadatta Dhar

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.

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.

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.

Occurrence of Acquired 16S rRNA Methyltransferase-Mediated Aminoglycoside Resistance in Clinical Isolates of Enterobacteriaceae within a Tertiary Referral Hospital of Northeast India.

ABSTRACT The methylation of a ribosomal target leads to a high level of resistance to all clinically relevant aminoglycoside antibiotics, so early detection of these resistance determinants will help to reduce the incidence of treatment failures as well as lessen the dissemination rate. Here, we characterized different 16S rRNA methyltransferases responsible for aminoglycoside resistance and their epidemiological background in clinical isolates of Enterobacteriaceae in a tertiary referral hospital in India. All aminoglycoside-resistant isolates were screened for different 16S rRNA methyltransferases by PCR assay, and incompatibility typing of the conjugable plasmid harboring resistance genes was performed by PCR-based replicon typing. An assay for the stability and elimination of these resistance plasmids was performed. The coexistence of extended-spectrum β-lactamases and metallo-β-lactamases was also detected, and the heterogeneity of these isolates was determined by enterobacterial repetitive intergenic consensus PCR. The PCR assay revealed the presence of armA, rmtA, rmtB, rmtC, and rmtD in single and multiple combinations, and these were carried by a diverse group of Inc plasmids. Plasmids harboring these resistance determinants were highly stable and maintained until the 55th serial passage, but SDS treatment could easily eliminate the plasmids harboring the resistance determinants. The coexistence of blaTEM, blaPER, blaGES, and blaSHV, as well as blaVIM and blaNDM, within these isolates was also detected. Strains with different clonal patterns of aminoglycoside resistance were found to spread in this hospital setting. We observed that the 16S rRNA methyltransferase genes were encoded within different Inc plasmid types, suggesting diverse origins and sources of acquisition. Therefore, the present study is of epidemiological importance and can have a role in infection control policy in hospital settings.

Genetic environment of OXA-2 beta-lactamase producing Gram-negative bacilli from a tertiary referral hospital

Sir, OXA-2 type beta lactamses belong to Ambler molecular class D and functional Group 2d. These types of beta lactamases are characterized by their high hydrolytic spectrum of activity against cloxacillin and oxacillin, and are poorly inhibited by clavulanic acid. Presence of this gene wasfirst reported in Pseudomonas in France1, in Escherichia coli from Israel2, and in India it was reported in E. coli in 20073. However, there is no knowledge regarding genetic environment and gene location of this resistant determinant from this part of the world. Our study reports presence of blaOXA-2 within IncF plasmid in a tertiary referral hospital of north-east India. This study was conducted in the department of Microbiology, Assam University, Silchar. A total number of 476 consecutive, non-duplicates, Gram-negative rods consisting of members of Enterobacteriaceae family and non-fermenting Gram-negative rods were isolated from different clinical specimens spanning a period of 12 months (March 2012 to February 2013) from different Wards/Clinics of Silchar Medical College and Hospital, Assam, India (Table). Screening and confirmation for extended spectrum beta lactamases (ESBLs) was done as per Clinical Laboratory Standards Institute (CLSI) guidelines4. Multiplex PCR was performed to characterize ESBL genes1. Reactions were run under the following conditions: initial denaturation 94°C for 5 min, 33 cycles of 94 °C for 35 sec, 51°C for one min, 72°C for one min and final extension at 72°C for seven min. PCR product was purified (Gene Jet Purification kit, Lithuania) and sequencing was done. For detection of class 1 and class 2 integron, integrase gene PCR was performed5. Two PCR reactions were carried out, one with HS287 and blaOXA-2 reverse, another with HS286 and blaOXA-2 forward1,6. The amplified products were further sequenced. Plasmids were purified by Gene Jet plasmid Miniprep kit (Thermo scientific, Lithuania). Transformation was carried out using Escherichia coli JM107 as recipient. Transformants were selected on cefotaxime (0.5 mg/l) containing Luria-Bertani agar (Hi-Media, Mumbai, India) plates. Conjugation experiments were carried out between clinical isolates as donors and a streptomycin resistant E. coli recipient strain B (Genei, Bangalore), transconjugants were selected on cefotaxime (0.5 mg/l) and streptomycin (800 mg/l) agar plates. For plasmid profiling, 1.5 μl of each sample was used and analyzed by agarose gel electrophoresis (1% agarose, Hi-Media, Mumbai, India), gel was run at 40V for 8 h at 18°C. PCR based replicon typing was carried out targeting 18 different replicon types, to perform five multiplex and three simplex PCRs as described previously7. Antimicrobial susceptibility was determined by Kirby Bauer disc diffusion and minimum inhibitory concentration (MIC) method4. Typing of isolates was done by enterobacterial repetitive intergenic consensus (ERIC) PCR8. Table Details of blaOXA-2 harbouring isolates A total of 15 isolates were harbouring OXA-2 gene which was further confirmed by sequencing. Co-existence of other ESBL genes was also noticed in all 15 isolates (Table). Class 1 integron was found in 13 isolates whereas one isolate carried class 2 integron and the remaining isolate carried class 1 and 2 both (Table). Sequencing results confimed that blaOXA-2 was found to be located within class I integron in 14 isolates while presence of this gene in class2 integron could not be established. Transformation results disclosed that in 13 isolates blaOXA-2 was located within the 20 kb plasmid which was also conjugatively transferable in E. coli strain B. Incompatibility typing of plasmids demonstrated that diverse Inc group types namely I1/Iγ, FIA, FIB, FIC, Y, FrepB, K and B/o were present in all blaOXA-2 harbouring isolates. But plasmid IncF was found to be common in all isolates as well as in their transformants and transconjugants. Tigecycline (n= 13; 86.66%) was the most effective antibiotics followed by imipenem (n=12; 80%) and meropenem (n=12; 80%). High MICs was observed against different groups of cephalosporins (≥256 μg/ml; n =15) and monobactam (≥256 μg/ml; n=15). All the OXA-2 producing isolates were clonally unrelated. This study indicates propagation of the blaOXA-2 by horizontal gene transfer additionally facilitated by integron mediated gene capture mechanism. Presence of this rare type of ESBL gene in diverse group of organisms and its carriage in integrons may restrict therapeutic options.

Contribution of efflux pumps in fluroquinolone resistance in multi-drug resistant nosocomial isolates of Pseudomanas aeruginosa from a tertiary referral hospital in north east India.

Background: Pseudomonas aeruginosa is one of the leading opportunistic pathogen and its ability to acquire resistance against series of antimicrobial agents confine treatment option for nosocomial infections. Increasing resistance to fluroquinolone (FQ) agents has further worsened the scenario. The major mechanism of resistance to FQs includes mutation in FQs target genes in bacteria (DNA gyrase and/or topoisomerases) and overexpression of antibiotic efflux pumps. Objective: We have investigated the role of efflux pump mediated FQ resistance in nosocomial isolates of P. aeruginosa from a tertiary referral hospital in north eastern part of India. Materials and Methods: A total of 234 non-duplicate, consecutive clinical isolates of P. aeruginosa were obtained from a tertiary referral hospital of north-east India. An efflux pump inhibitor (EPI), carbonyl cyanide m-chlorophenylhydrazone (CCCP) based method was used for determination of efflux pump activity and multiplex polymerase chain reaction (PCR) was performed for molecular characterisation of efflux pump. Minimum inhibitory concentration (MIC) reduction assay was also performed for all the isolates. Results and Conclusion: A total number of 56 (23%) have shown efflux mediated FQ resistance. MexAB-OprM efflux system was predominant type. This is the first report of efflux pump mediated FQ resistance from this part of the world and the continued emergence of these mutants with such high MIC range from this part of the world demands serious awareness, diagnostic intervention, and proper therapeutic option.

Occurrence of bla CMY-42 through IncI1 plasmid within multidrug-resistant Escherichia coli from a tertiary referral hospital of India

OBJECTIVES Plasmids of different replicon types are believed to be associated with the carriage and transmission of antimicrobial resistance genes. The present study was undertaken to examine the association of blaCIT with particular plasmid types and to identify Escherichia coli strains involve in the maintenance of this resistance determinant in the plasmid. METHODS Phenotypic screening of AmpC β-lactamases was performed by the modified three-dimensional extract method, followed by antimicrobial susceptibility testing and determination of minimum inhibitory concentrations (MICs). Genotyping screening of β-lactamase genes was performed by PCR assay, followed by sequencing. Transferability of the blaCMY gene was performed by transformation and conjugation experiments. Plasmid incompatibility typing and DNA fingerprinting by enterobacterial repetitive intergenic consensus (ERIC)-PCR were performed. RESULTS Among 203 E. coli obtained from different clinical specimens (pus, urine, stool and sputum), 37 were detected as harbouring the blaCIT gene and sequencing of this gene showed nucleotide sequence similarity with the blaCMY-42 variant. This study revealed IncI1-type plasmids as carriers of blaCMY-42 and its propagation within E. coli ST5377, ST361 and ST672. According to the stability results, the blaCMY-42-encoding plasmid can be maintained in E. coli strains for a longer duration without any antimicrobial pressure. CONCLUSIONS These finding document blaCMY-42 on IncI1-type plasmids, which are considered to be the main vehicles for the spread of blaCMY-42 in this hospital setting. Thus, a proper strategy should be developed to curb the expansion of IncI1-type plasmids in the hospital and community environment.

Role of inducers in detection of blaPDC-mediated oxyimino-cephalosporin resistance in Pseudomonas aeruginosa

Background & objectives: Pseudomonas aeruginosa possessing chromosomally inducible blaPDC along with other intrinsic mechanism causes infection with high mortality rate. It is difficult to detect inducible AmpC enzymes in this organism and is usually overlooked by routine testing that may lead to therapeutic failure. Therefore, three different inducers were evaluated in the present study to assess their ability of induction of blaPDC in P. aeruginosa. Methods: A total of 189 consecutive Pseudomonas isolates recovered from different clinical specimens (November 2011-April 2013) were selected for the study. Isolates were screened with cefoxitin for AmpC β-lactamases and confirmed by modified three-dimensional extract test (M3DET). Inductions were checked using three inducers, namely, clavulanic acid, cefoxitin and imipenem along with ceftazidime. Molecular screening of AmpC β-lactamase genes was performed by PCR assay. Antimicrobial susceptibility and minimum inhibitory concentrations (MICs) were determined, and repetitive extragenic palindromic-PCR of all blaPDC harbouring isolates was performed. Results: Inducible phenotype was observed in 42 (24.3%) of 97 (56%) isolates confirmed by M3DET. Among these, 22 isolates harboured chromosomal blaPDC gene, and cocarriage of both chromosomal and plasmid-mediated blaAmpC genes was observed in seven isolates. Cefoxitin-ceftazidime-based test gave good sensitivity and specificity for detecting inducible AmpC enzymes. Isolates harbouring blaPDC showed high MIC against all tested cephalosporins and monobactam. DNA fingerprinting of these isolates showed 22 different clones of P. aeruginosa. Interpretation & conclusions: P. aeruginosa harbouring inducible (chromosomal) and plasmid-mediated AmpC β-lactamase is a matter of concern as it may limit therapeutic option. Using cefoxitin-ceftazidime-based test is simple and may be used for detecting inducible AmpC β-lactamase amongst P. aeruginosa.