Incidence of carbapenem-resistant Escherichia coli ST2437 of clinical origin harbouring blaOXA-144 gene: a report from India.

AIMS: Carbapenem-resistant Escherichia coli has been categorized as a pathogen of critical priority by the World Health Organization as it is highly infectious with high mortality and morbidity rates and widespread transmission potential. Carbapenem resistance is primarily mediated by carbapenemase-encoding genes and, additionally, through intrinsic factors. In India, over the years, carbapenemase-encoding genes have been reported from diverse clinically significant pathogens. The present study identifies E. coli of clinical origin that harbours blaOXA-144. METHODS AND RESULTS: The study isolate was obtained from a tertiary referral hospital in northeast India. Carbapenemase production was investigated through culture on chromogenic agar and Rapidec Carba NP test as per manufacturer's instructions. Susceptibility of the isolate was performed by the Kirby-Bauer disc diffusion method and agar dilution method following CLSI guidelines. PCR targeting carbapenemase-encoding genes was performed, followed by transformation and conjugation experiments. Whole-genome sequencing of the isolate was done through the Illumina sequencing platform and the data were analysed using the Centre for Genomic Epidemiology database. BJD_EC180 is 6 919 180 bp in length and consists of six rRNA operons, 111 tRNA, and 6849 predicted protein-coding sequences. BJD_EC180 belonged to ST2437 and harboured the carbapenemase-encoding gene blaOXA-144 with ISAba1 upstream, along with multiple antibiotic resistance genes conferring clinical resistance towards beta-lactams, aminoglycosides, amphenicols, sulphonamides, tetracyclines, trimethoprim, and rifampin. CONCLUSIONS: Carbapenem-resistant E. coli harbouring blaOXA-144 associated with insertion sequence pose a serious health threat as their mobilization into carbapenem non-susceptible strains that will contribute to the resistance burden and therefore, needs urgent monitoring.

Evaluation of a novel chromogenic screening method for detection of carbapenem-resistant Escherichia coli.

Introduction. Early detection of carbapenem-resistant Escherichia coli (CREco), categorized as a critical priority pathogen by the World Health Organization (WHO), is crucial in optimizing therapeutic options and to thwart outbreaks in clinical settings.Gap statement. The need of the hour is a diagnostic method that can detect carbapenem resistance conferred by intrinsic or acquired carbapenem resistance mechanisms or both.Aim. The study investigates the performance of a novel screening chromogenic method for detection of CREco.Methodology. Carbapenem-susceptible (n=23) and non-susceptible (n=90) E. coli were used to investigate the efficiency of the blue chromogenic test. All of the isolates were received from a tertiary referral hospital in Silchar, India and subjected to the blue chromogenic test and observed for colour change. A colour change from colourless to blue is interpreted as a positive result. The test results were further compared with available methods for detection of carbapenem resistance conferred by carbapenemase production or other carbapenem resistance mechanisms.Results. The blue chromogenic test generated 100 % (CI: 95.98-100 %) sensitive and 100 % (CI: 85.75-100 %) specific results for the detection of CREco with no false-positive or false-negative results. Within 3 h after incubation, the test detects all CREco with carbapenemase activity. Additionally, the blue chromogenic test also positively detected E. coli harbouring carbapenemase variants and with efflux and porin activity, compared to other phenotypic-based approaches.Conclusion. The study highlights a novel method that is highly sensitive and specific, inexpensive, rapid and user-friendly for the detection of CREco. With the surge and expansion of CREco, this sensitive, specific, user-friendly and inexpensive method can be used in laboratories with limited facilities for early detection of CREco, thereby improving infection control along with averting future outbreaks.

Imipenem and meropenem influence the Las/Rhl quorum-sensing systems in clinical isolates of Pseudomonas aeruginosa.

The present study was conducted to study the influence of imipenem and meropenem at subinhibitory concentration on the transcriptional response of Las/Rhl quorum-sensing systems in isolates of Pseudomonas aeruginosa. In the present study, six representative carbapenem nonsusceptible clinical isolates of P. aeruginosa were obtained. The agar dilution method was used to determine the minimum inhibitory concentration against imipenem and meropenem. The bacterial isolates were then cultured up to the early log phase in fresh Luria Bertani (LB) broths at 37°C with and without 2 µg mL-1 imipenem and meropenem, respectively. mRNA was then isolated from the bacterial isolates and was immediately reverse-transcribed to cDNA. The relative quantity of the expression of the lasI, lasR, rhlI, and rhlR genes was assessed by quantitative real-time Polymerase Chain Reaction (PCR) using the ΔΔCt method. The transcriptional response of the lasI and lasR genes was upregulated at subinhibitory concentration of meropenem. In contrast, the transcriptional response of the lasI, lasR, and rhlR genes was downregulated at subinhibitory concentration of imipenem as compared to the expression in untreated isolates. The data obtained in the current study showcased the ability of imipenem and meropenem to influence the response of the quorum-sensing genes at subinhibitory concentration.

Tn5406, a staphylococcal transposon associated with macrolide-lincosamide-streptograminb resistance in clinical isolates of Staphylococcus aureus.

PURPOSE: In this study, we aimed to investigate the occurrence of MLSb resistance in clinical isolates of Staphylococcus aureus with respect to their association with transposons. METHODS: The present study was performed with clinical isolates of S. aureus. The MLSb resistant phenotypes in the obtained isolates were determined by D zone test or double disc diffusion test as per CLSI 2020 guidelines. MLSb resistance encoding genes were detected by PCR. The genes tested were ermA, ermB, ermC, msrA, mphC, vga, vgb and lnuB. The MLSb resistant Staphylococcal isolates were selected to analyze the association of the genes with mobile genetic elements Tn554, Tn5406, Tn917, Tn6133, Tn551 by PCR based method. Primer pairs were designed using sequences from transposons and the resistance genes, respectively. RESULTS: During this study, 268 isolates of S. aureus were obtained of which 233 (86.94%) isolates exhibited different MLSb resistant phenotypes. The predominant gene among the MLSb resistant isolates was msrA followed by vgaA and mphC genes. PCR assay was employed to determine whether the genes msrA, mphC and vgaA were carried by Tn554, Tn5406, Tn917, Tn6133, Tn551 transposons. PCR amplification with the designed primer pairs revealed vgaA gene being part of Tn5406. CONCLUSION: The presence of Tn5406 in all the vgaA harboring isolates highlights its potential of spread across isolates. Moreover, the co-existence of different MLSb resistance encoding genes observed in the study shows that the combination of genes involved in different mechanism mediated the nature of MLSb resistance.

Molecular characterization of extended spectrum beta lactamases in clinical isolates of Escherichia coli and Klebsiella spp from a tertiary care hospital of South Eastern Assam.

Molecular characterization of ESBLs in clinical isolates of Escherichia coli and Klebsiella spp from a tertiary care hospital of South Eastern Assam was done by detection of blaTEM, blaSHV, blaCTX-M, blaOXA-2, blaOXA-10, blaPER, blaVEB and blaGES by Multiplex PCR. One hundred isolates yielded 44 bands of ESBL genes. CTX-M was most frequently isolated gene (36/44). Two isolates carried a combination of 2 genes CTX-M and OXA-2. Apart from CTX-M, the study isolates were also found to harbour TEM(n â€‹= â€‹3), OXA-2 (n â€‹= â€‹6) OXA-10 (n â€‹= â€‹1),GES(n â€‹= â€‹2) genes.

Distribution of virulence genes and SCCmec types among methicillin-resistant Staphylococcus aureus of clinical and environmental origin: a study from community of Assam, India.

OBJECTIVE: This study was designed to discover the dissemination of virulence genes in Methicillin-resistant Staphylococcus aureus from clinical, community and environmental settings. RESULTS: This study includes 1165 isolates collected from hospital, community and environmental settings. Among them sixty three were confirmed as MRSA with varied SCCmec types viz; type I, type II, type III, type IV, type V, type VI, type VII, type VIII and type XII. The virulence gene such as sea (n = 54), seb (n = 21), eta (n = 27), etb (n = 2), cna (n = 24), ica (n = 2) and tst (n = 30) was also revealed from this study. The study underscores coexistence of resistance cassette and virulence genes among clinical and environment isolates which is first of its kind from this part of the world.

Adaptation of blaNDMthrough IncP plasmid within broad host range.

Introduction: It was also known that the IncP-1 plasmids are ubiquitous in environmental bacteria and those reside in soil, sewage, marine sediments and in manure. The blaNDMis associated with resistance determinants along with various mobile elements such as plasmid, insertion sequences and transposons, which facilitates its horizontal dissemination. These plasmids, if tracked, can be a starting point for the control of infection due to multidrug-resistant pathogens. The aim of the study was to investigate that IncP-type plasmids carrying blaNDMis adapted in different hosts. Materials and Methods: Thirteen of the isolates were harbouring IncP-type plasmid and they all were Escherichia coli isolated from hospitalised patients of Silchar Medical College and Hospital, India. The isolates were checked for susceptibility test, and the stability was assessed by a serial passage. These isolates were further subjected to transcriptional analysis of NDM gene as well as plasmid copy number alteration. Results: The study isolates were highly stable, and the resistance gene (blaNDM) was retained within isolates till 55th subsequent serial passages. Plasmid copy number alteration was random in isolates when exposed to carbapenem antibiotics, whereas increasing trend in transcriptional expression was observed with the increase in imipenem concentration. Conclusion: This study was able to underscore the presence of IncP plasmid that was harbouring blaNDMand was maintained within diverse host. The finding also highlights the adaptation of the broad-host-range plasmid that responds in terms of transcriptional expression under antibiotic exposure.

Occurrence of blaCMY-42 on an IncI1 plasmid in multidrug-resistant Escherichia coli from a tertiary referral hospital in 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 ß-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 ß-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.

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.

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

BACKGROUND & OBJECTIVES: Pseudomonas aeruginosa possessing chromosomally inducible blaPDCalong 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 blaPDCin 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 ß-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 ß-lactamase genes was performed by PCR assay. Antimicrobial susceptibility and minimum inhibitory concentrations (MICs) were determined, and repetitive extragenic palindromic-PCR of all blaPDCharbouring 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 blaPDCgene, 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 blaPDCshowed 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 ß-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 ß-lactamase amongst P. aeruginosa.

An insight into selection specificity of quinolone resistance determinants within Enterobacteriaceae family.

OBJECTIVES: Quinolone antimicrobials are frequently misused due to self-medication and suboptimal dose administration, leading to the development of resistance as well as treatment failure. The present study aimed to characterise plasmid-mediated quinolone resistance (PMQR) determinants and their genetic selection in the presence of quinolone stress within members of the Enterobacteriaceae. METHODS: A total of 209 non-duplicate Enterobacteriaceae isolates were collected from hospital and community health centres over the period July 2013-June 2014. Molecular characterisation of phenotypically screened quinolone-resistant isolates was done by multiplex PCR. Plasmids bearing the qnr and aac(6')-Ib-cr genes were transformed into Escherichia coli DH5α and were selected on Muller-Hinton agar plates containing 0.25µg/mL and 0.5µg/mL ciprofloxacin, norfloxacin, ofloxacin, levofloxacin and moxifloxacin. Conjugation experiments were performed to determine whether the aac(6')-Ib-cr- and qnr-carrying plasmids were self-transferable. RESULTS: The transformation assay revealed that transformants carrying qnrA could be selected in media containing norfloxacin, ciprofloxacin and levofloxacin, whereas qnrB and aac(6')-Ib-cr were selected on media containing norfloxacin and ciprofloxacin. Transformed qnrD could be selected in media containing norfloxacin and ofloxacin, and qnrS was selected only in the presence of levofloxacin. CONCLUSIONS: The presence of qnr genes has been associated with an increase in quinolone minimum inhibitory concentrations (MICs) and therefore leads to treatment failure when quinolones are used as selective therapeutic drugs. Since PMQR determinants have a high prevalence, effective measures should be taken and surveillance should be performed in order to avoid treatment failures using this group of antimicrobials.

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-ß-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.

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.

Diverse Genetic Array of blaCTXM-15 in Escherichia coli: A Single-Center Study from India.

CTX-M-15 is a chief contributor for expanded-spectrum cephalosporin and monobactam resistance in India, complicating treatment options. In this study, we have investigated genetic context of CTX-M-15 in Escherichia coli and their transmission dynamics in a tertiary referral hospital of India. A total of 198 isolates were collected, of which 66 were harboring blaCTXM-15. Among them, 14 isolates were carrying a single CTX-M-15 gene and 52 were harboring multiple extended-spectrum ß-lactamase genes along with blaCTX-M-15. The resistance gene was flanked by tnpA, ISEcp1, IS26, and ORF477 in 10 different arrangements. The resistance determinant was horizontally transferable through F, W, I1, and P Inc types of plasmids. Restriction mapping of plasmids showed a variable band pattern even within the same Inc types. Minimum inhibitory concentration was found above the breakpoint level against expanded-spectrum cephalosporins and monobactam while susceptible against carbapenems. blaCTX-M-15 was highly stable and sustained in the cell after 115 serial passages. In pulse-field gel electrophoresis, eight pulsotypes of E. coli were found to be responsible for the spread of blaCTX-M-15 in the tertiary referral center. We conclude that the presence of CTX-M-15 in the heterogeneous group of E. coli is highly alarming in terms of infection control and it may require regular monitoring, so as to formulate appropriate antibiotic policy to stop the spread of this resistance determinant.

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 blaKPC-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 blaKPC was confirmed by genotypic characterization followed by sequencing. Cloning of the blaKPC-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 blaKPC-2, were resistant towards aminoglycosides, quinolone and ß-lactam-ß-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 blaNDM-1. The first detection of this integron mediated blaKPC-2 coexisting with blaNDM-1 in P. aeruginosa from India is worrisome, and further investigation is required to track the gene cassette mediated blaKPC-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.

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.

Emergence of integron borne PER-1 mediated extended spectrum cephalosporin resistance among nosocomial isolates of Gram-negative bacilli.

BACKGROUND & OBJECTIVES: Pseudomonas extended resistant (PER) enzymes are rare type of extended-spectrum beta lactamases (ESBLs) that confer third generation cephalosporin resistance. These are often integron borne and laterally transmitted. The aim of the present study was to investigate the emergence of integron borne cephalosporin resistant PER-1 gene in diverse incompatibility (Inc) group plasmids among Gram-negative bacteria. METHODS: A total of 613 consecutive, non-duplicate, Gram-negative bacteria of Enterobacteriaceae family and non-fermenting Gram-negative bacteria were isolated from different clinical specimens during a period of 18 months. For amplification and detection of blaPER, multiplex PCR was done. For understanding the genetic environment of blaPER-1, integrase gene PCR and cassette PCR (59 be) was performed. Gene transferability experiment was carried out and PCR based replicon typing was performed for incompatibility group typing of plasmids using 18 pairs of primers. An inhibitor based method was used for phenotypic detection of intrinsic resistance. RESULTS: Multiplex PCR and sequencing confirmed that 45 isolates were harbouring blaPER-1. Both class 1 and class 2 integrons were observed among them. Integrase and cassette PCR (59 be) PCR results confirmed that the resistant determinant was located within class 1 integron. Transformation and conjugation experiments revealed that PER-1 was laterally transferable and disseminated through diverse Inc plasmid type. Efflux pump mediated carbapenem resistance was observed in all isolates. All isolates belonged to heterogenous groups. INTERPRETATION & CONCLUSIONS: This study demonstrates the dissemination of cephalosporins resistant, integron borne blaPER-1 in hospital setting in this part of the country and emphasizes on the rational use of third generation cephalosporins to slow down the expansion of this rare type of ESBL gene.