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.

Emergence of carbapenem-resistant enterobacterales co-harboring blaOXA-78 and blaOXA-58 from India.

BACKGROUND: Carbapenem-Resistant Enterobacterales (CRE) has been categorized as pathogens of critical priority by World Health organization (WHO) as they pose significant threat to global public health. Carbapenemase production considered as the principal resistance mechanism against carbapenems and with the recent surge and expansion of carbapenemases and its variants among clinically significant bacteria in India, the present study reports expansion blaOXA-78 and blaOXA-58 of in CRE of clinical origin. METHODS: Bacterial isolates were collected from a tertiary referral hospital and identified through VITEK® 2 Compact automated System (Biomerieux, France). Rapidec® Carba NP (Biomerieux, France) was used to investigate carbapenemase production followed by antibiotic susceptibility testing through Kirby-Bauer Disc Diffusion method and agar dilution method. Class D carbapenemase genes were targeted through PCR assay followed by investigation of horizontal transmission of blaOXA-58 and blaOXA-78. Whole genome sequencing was carried out using Illumina platform to investigate the genetic context of blaOXA-58 and blaOXA-78 genes and further characterization of the CRE isolates. RESULTS: The carbapenem-resistant Escherichia coli (BJD_EC456) and Serratia marcescens (BJD_SM81) received during the study from the tertiary referral hospital were isolated from sputum and blood samples respectively. PCR assay followed by whole genome sequencing revealed that the isolates co-harbor blaOXA-58 and blaOXA-78, a variant of blaOXA-51. Horizontal transfer of blaOXA-58 and blaOXA-78 genes were unsuccessful as these genes were located on the chromosome of the study isolates. Transposon Tn6080 was linked to blaOXA-78 in the upstream region while the insertion sequences ISAba26 and ISCfr1 were identified in the upstream and downstream region of blaOXA-58 gene respectively. In addition, both the isolates were co-harboring multiple antibiotic resistance genes conferring clinical resistance towards beta-lactams, aminoglycosides, fluroquinolones, sulphonamides, tetracyclines. BJD_EC180 belonged to ST2437 while BJD_SM81 was of an unknown sequence type. The nucleotide sequences of blaOXA-78 (OQ533021) and blaOXA-58 (OQ533022) have been deposited in GenBank. CONCLUSIONS: The study provides a local epidemiological information regarding carbapenem resistance aided by transposon and insertion sequences associated blaOXA-78 and blaOXA-58 genes associated and warrants continuous monitoring to prevent their further dissemination into carbapenem non-susceptible strains thereby contributing to carbapenem resistance burden which is currently a global concern.

Transcriptional Response of vanB Operon in Staphylococcus aureus Against Vancomycin and Teicoplanin Stress.

Staphylococcus aureus is a global pathogen and is responsible for causing severe life-threatening infections. The current study was designed to investigate transcriptional expression of different core, regulatory, and accessory genes within vanB operon under differential exposure of vancomycin and teicoplanin. Four isolates selected for the study, were confirmed to harbour vanB gene in which three isolates showed MIC breakpoint above 16 µg/ml and one isolate above 8 µg/ml against vancomycin while teicoplanin showed higher MIC breakpoint as compared to vancomycin. Antibiotic susceptibility results showed that these isolates were susceptible towards imipenem and linezolid. Transcriptional expressional analysis of the core gene of vanB operon showed that expression of vanB is increased under vancomycin stress but is inversely proportional to increase in the concentration of the vancomycin while under teicoplanin stress the expression of vanB showed no significant pattern. Similar expressional pattern was found for vanH gene for both the glycopeptides. In case of vanX, expression was significantly increased at 1 µg/ml exposure of vancomycin, however, no pattern could be observed in case of teicoplanin stress. In case of regulatory gene, vanR, significant increase in expression was observed under vancomycin and teicoplanin stress of 1 µg/ml, however vanS, showed significant increase in the expression under 1 µg/ml of vancomycin. The accessory gene, vanY showed marginal increase in expression under both the antibiotic, while in case of vanW, the expressional pattern was found to be inversely proportional to the increasing antibiotic concentration.

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.

Transcriptional expression of secondary resistance genes ccdB and repA2 is enhanced in presence of cephalosporin and carbapenem in Escherichia coli.

BACKGROUND: The issue of carbapenem resistance in E.coli is very concerning and it is speculated that cumulative effect of both primary resistance genes and secondary resistance genes that act as helper to the primary resistance genes are the reason behind their aggravation. Therefore, here we attempted to find the role of two secondary resistance genes (SRG) ccdB and repA2 in carbapenem resistance in E. coli (CRE). In this context influential genes belonging to secondary resistome that act as helper to the primary resistance genes like blaNDM and blaCTX-M in aggravating ß-lactam resistance were selected from an earlier reported in silico study. Transcriptional expression of the selected genes in clinical isolates of E.coli that were discretely harboring blaNDM-1, blaNDM-4, blaNDM-5, blaNDM-7 and blaCTX-M-15 with and without carbapenem and cephalosporin stress (2 µg/ml) was determined by real time PCR. Cured mutants sets that were lacking (i) primary resistance genes, (ii) secondary resistance genes and (iii) both primary and secondary resistance genes were prepared by SDS treatment. These sets were then subjected to antibiotic susceptibility testing by Kirby Bauer disc diffusion method. RESULTS: Out of the 21 genes reported in the in silico study, 2 genes viz. repA2 and ccdB were selected for transcriptional expression analysis. repA2, coding replication regulatory protein, was downregulated in response to carbapenems and cephalosporins. ccdB, coding for plasmid maintenance protein, was also downregulated in response to carbapenems except imipenem and cephalosporins. Following plasmid elimination assay increase in diameter of zone of inhibition under stress of both antibiotics was observed as compared to uncured control hinting at the reversion of antibiotic susceptibility by the-then resistant bacteria. CONCLUSION: SRGs repA2 and ccdB help sustenance of blaNDM and blaCTX-M under carbapenem and cephalosporin stress.

Expressional Pattern of psm-mec System in Methicillin-Resistant Staphylococcus aureus Under Oxacillin Stress.

The psm-mec element and other regulatory factors such as sarA, agrA, and RNAIII are responsible for maintaining the genetic framework for enhanced virulence of MRSA. psm-mec is found predominantly in the staphylococcal cassette chromosome (SCCmec). sarA, agrA, and RNAIII control gene expression to facilitate adaptation in certain environment. Genome-wide approaches have shown that expression of virulence factors is frequently regulated at transcriptional, translational level, and mRNA degradation level. In this study, transcriptional responses of psm-mec gene in accordance with other regulatory factors sarA, agrA, and RNAIII were observed under normal conditions as well as when exposed to 2 µg/ml and 6 µg/ml of oxacillin stress. One-way t-test was carried out for analysing RQ values obtained through real-time PCR. This study showed downregulation of psm-mec gene and upregulation of other regulatory genes at lower concentration of oxacillin. However, this was reverse when exposed against higher concentration of oxacillin. It was observed from the study that the expression of virulence factors were dependent on each other under different concentration of oxacillin. Thus, this study highlights that psm-mec, sarA, agrA, and RNAIII gene are under direct control of antibiotic pressure in a concentration-dependent manner.

Subinhibitory concentration stress of colistin enhanced PhoPQ expression in Escherichia coli harboring mcr-1.

The increased and inappropriate use of colistin led to the emergence of its resistance among Gram-negative bacterial isolates and the most common mechanism of colistin resistance in Gram-negative bacteria is the modification of the lipopolysaccharide mediated by two-component regulatory systems, PhoPQ and PmrAB. The aim of the present study was to investigate the transcriptional expression of the PhoPQ system against colistin stress in clinical isolates of Escherichia coli with colistin-resistant phenotype. Six colistin-resistant E. coli isolates were obtained from Silchar Medical College and Hospital, Silchar that were of clinical origin and received for routine culture and sensitivity testing. Screening for colistin resistance was done by broth microdilution method and further screened for the presence of the different types of plasmid-mediated colistin resistance mcr genes namely, mcr-1 to mcr-10 by polymerase chain reaction (PCR). The screened positive isolates were subjected to PCR assay targeting phoP and phoQ genes and their expression was measured by quantitative real-time PCR. The results of this study revealed that two E. coli isolates (TS2 and TS4) were found to carry the mcr-1 gene. PhoP and PhoQ gene amplification was observed in all the isolates. Transcriptional analysis showed that the isolates harboring the mcr-1 gene showed an enhanced level of expression in the PhoP, PhoQ genes in the presence of a subinhibitory concentration of colistin whereas no significant expression was observed for the isolates which were devoid of the mcr gene. This study demonstrates the involvement of mcr-1 in the PhoPQ system in clinical isolates of colistin-resistant E. coli which will help in designing a molecular marker for detecting colistin-resistant E. coli and contribute to the assessment of resistance burden and infection control strategy.

Enhancement of antibacterial activity of synthesized ligand-free CdS nanocrystals due to silver doping.

Recently, different nanocrystals have been reported to be the alternative, optimistic, and novel antimicrobial agent against the many antibiotic-resistant bacteria. Here, ligand-free CdS and Ag-doped CdS (Ag/CdS) nanocrystals have been synthesized by chemical methods for the study of the antimicrobial activity on Escherichia coli and Staphylococcus aureus by Kirby-Bauer diffusion method to see the effect against Gram-positive and Gram-negative bacteria. These prepared nanocrystals have been characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and X-ray diffraction (XRD). TEM and SEM images confirm the spherical morphology of both the sample and the respective XRD patterns indicate polycrystalline nature having a cubic zinc blende structure. Antibacterial activities have been tested with CdS and Ag/CdS, considering concentrations ranging from 10 to 200 µg/ml. After 24 h of incubation, the zone of inhibition (ZOI) is measured for each concentration, which shows that both the nanocrystals are ineffective against E. coli but much effective against S. aureus at this low concentration range. Furthermore, Ag/CdS nanocrystals have been found to show much more ZOI than CdS. Differences in the antibacterial activity can be due to the presence of different cell wall in E. coli and S. aureus.

Expansion of acquired 16S rRNA methytransferases along with CTX-M-15, NDM and OXA-48 within three sequence types of Escherichia coli from northeast India.

BACKGROUND: This study aimed to identify ten different 16S rRNA methyltransferase genes (rmtA, rmtB, rmtC, rmtD, armA, rmtF, npmA, rmtH, rmtE and rmtG) and their coexisting ESBL and carbapenemase with the emergence of three E.coli clones within a single study centre. METHODS: A total of 329 non-duplicate E.coli isolates were studied to detect the presence of 16S rRNA methyltransferases along with ß-lactamases (TEM, SHV, OXA, VEB, GES, PER,CTX-M types, NDM, OXA-48,VIM, IMP and KPC) using PCR assay. Horizontal transferability were validated by transformation and conjugation analysis. Plasmid incompatibility typing and MLST analysis was also performed. RESULTS: A total of 117 isolates were found to be resistant to at least one of the aminoglycoside antibiotics. It was observed that 77 (65.8%) were positive for 16S rRNA methyltransferases. Among them thirty nine isolates were found to harbour only blaCTX-M-15, whereas combination of genes were observed in three isolates (blaVEB+ blaCTX-M-15 in 2 isolates and blaPER + blaCTX-M-15 in 1 isolate). blaNDM and blaOXA-48 like genes were found in 23 and 9 isolates, respectively. All the resistance genes were conjugatively transferable, and incompatibility typing showed multiple 16S rRNA methyltransferase genes were originated from a single Inc. I1 group. MLST analysis detected 3 clones of E.coliST4410, ST1341 and ST3906. CONCLUSION: The present study identified emergence of three clones of E.coli, resistant to aminoglycoside -cephalosporin- carbapenem. This warrants immediate measures to trace their transmission dynamics in order to slow down their spread in clinical setting.

Correction to: Expansion of acquired 16S rRNA methytransferases along with CTX-M-15, NDM and OXA-48 within three sequence types of Escherichia coli from northeast India.

An amendment to this paper has been published and can be accessed via the original article.

AcrAB-TolC efflux pump system plays a role in carbapenem non-susceptibility in Escherichia coli.

BACKGROUND: Efflux pump mediated antibiotic resistance is an unnoticed and undetected mechanism in clinical microbiology laboratory. RND efflux systems are known for aminoglycoside and tetracycline resistance whereas their role in carbapenem non-susceptibility is not established. The study was undertaken to investigate the role of efflux pump in providing resistance against carbapenems and their response against concentration gradient carbapenem stress on the transcriptional level of the AcrAB gene in the clinical isolates of Escherichia coli from a tertiary referral hospital of Northeast India. RESULTS: Out of 298 non-susceptible Escherichia coli isolates 98 isolates were found to have efflux pump mediated carbapenem non-susceptibility. Among them thirty-five were non carbapenemase producers and their expressional levels were verified using qRT-PCR under concentration gradient carbapenem stress. In this study, a strong correlation between ertapenem resistance and AcrA overexpression was observed which has not been reported previously. Further, it was observed that imipenem stress increased AcrB expression in Escherichia coli which holds the novelty of this study. Additionally, the transcription of AcrR was insistently increased which is much higher than the transcriptional level of AcrA under concentration gradient carbapenem stress condition. CONCLUSION: The study established that AcrAB pump is a relevant antibiotic resistance determinant in bacterial pathogen, has an important role in developing resistance against carbapenem group of antibiotics.

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.

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

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 ß-lactamases and metallo-ß-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.

Occurrence of bla DHA-1 mediated cephalosporin resistance in Escherichia coli and their transcriptional response against cephalosporin stress: a report from India.

BACKGROUND: Treatment alternatives for DHA-1 harboring strains are challenging as it confers resistance to broad spectrum cephalosporins and may further limit treatment option when expressed at higher levels. Therefore, this study was designed to know the prevalence of DHA genes and analyse the transcription level of DHA-1 against different ß-lactam stress. METHODS: Screening of AmpC ß-lactamase phenotypically by modified three dimensional extract method followed by Antimicrobial Susceptibility and MIC determination. Genotyping screening of ß-lactamase genes was performed by PCR assay followed by their sequencing. The bla DHA-1 transcriptional response was evaluated under different cephalosporin stress by RT PCR. Transferability of bla DHA gene was performed by transformation and conjugation and plasmid incompatibility typing, DNA fingerprinting by enterobacterial repetitive intergenic consensus sequences PCR. RESULTS: 16 DHA-1 genes were screened positive from 176 Escherichia coli isolates and primer extension analysis showed a significant increase in DHA-1 mRNA transcription in response to cefotaxime at 8 µg/ml (6.99 × 102 fold), ceftriaxone at 2 µg/ml (2.63 × 103 fold), ceftazidime at 8 µg/ml (7.06 × 103 fold) and cefoxitin at 4 µg/ml (3.60 × 104 fold) when compared with untreated strain. These transcription data were found significant when analyzed statistically using one way ANOVA. Four different ESBL genes were detected in 10 isolates which include CTX-M (n = 6), SHV (n = 4), TEM (n = 3) and OXA-10 (n = 1), whereas, carbapenemase gene (NDM) was detected only in one isolate. Other plasmid mediated AmpC ß-lactamases CIT (n = 9), EBC (n = 2) were detected in nine isolates. All DHA-1 genes detected were encoded in plasmid and incompatibility typing from the transformants indicated that the plasmid encoding bla DHA-1 was carried mostly by the FIA and L/M Inc group. CONCLUSION: This study demonstrates the prevalence of DHA-1 gene in this region and highlights high transcription of DHA-1 when induced with different ß-lactam antibiotics. Therefore, cephalosporin treatment must be restricted for the patients infected with pathogen expressing this resistance determinant.

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

BACKGROUND: New-Delhi metallo-ß-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 ß-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.

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.

Occurrence of co-existing bla VIM-2 and bla NDM-1 in clinical isolates of Pseudomonas aeruginosa from India.

BACKGROUND: bla VIM-2 harboring Pseudomonas aeruginosa has been reported worldwide and considered as the most prevalent metallo-ß-lactamase after NDM which are found horizontally transferable and mostly associated with integron gene cassettes. The present study investigates the genetic background, transmission dynamics as well as stability of bla VIM-2 in clinical isolates of P. aeruginosa harbor bla NDM-1 as well which were collected from October 2012 to September 2013. METHODS: Two P. aeruginosa strains harboring bla VIM-2 along with bla NDM-1 were isolated from Silchar Medical College and Hospital, India. Genetic environment of these resistance determinants was determined and transferability was checked by transformation and conjugation assay which was further confirmed by Southern hybridization. Replicon typing was performed to determine the incompatibility group of the resistant plasmid and their stability was checked by serial passage method. Antimicrobial susceptibility pattern of the isolates was determined and their clonal relatedness was checked by pulsed field gel electrophoresis. RESULTS: bla VIM-2 was found to be horizontally transferable through an Inc F type plasmid of approximately 30 kb in size. bla VIM-2 was found to be associated with integron gene cassette and was flanked by two different types of cassette arrays. Both the isolates were co-harboring bla NDM-1 which was carried within Inc N type of plasmid with an approximate 24 kb in size and associated with ISAba125 in their upstream region. Reduced susceptibility rate as well as high MIC range was observed in case of wild strains and transformants carrying bla VIM-2 and bla NDM-1. CONCLUSIONS: The detection of this co-existence of multiple carbapenem resistance genes in this part of world is worrisome and further investigation is required in order to trace the source and to initiate proper treatment option.

Long-term outbreak of Klebsiella pneumoniae& third generation cephalosporin use in a neonatal intensive care unit in north India.

BACKGROUND & OBJECTIVES: The indiscriminate use of third generation cephalosporin has contributed to the emergence and widespread dissemination of extended spectrum ß lactamases (ESBL) genes in Klebsiella pneumoniae. This study was undertaken to elaborate the genetic behaviour of ESBL - producing K. pneumoniae isolates in the neonatal intensive care unit (NICU) of a tertiary care hospital in north India causing successive outbreaks in context with empirical third generation cephalosporin use. METHODS: Isolates of K. pneumoniae (43 from blood, 3 from pus and endotracheal tube, 4 from environment) causing successive outbreaks in the NICU of a tertiary care university hospital were studied for two years. Antimicrobial susceptibility testing was done by disc diffusion and minimum inhibitory concentration (MIC) determination by agar dilution methods. ESBL production was determined by phenotypic and genotypic methods. Clonal relatedness among the isolates was studied by enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR). Genetic environment of these isolates was assessed by the presence of integrons and gene cassettes. Transformation experiments were done, and plasmids of these isolates were characterized by stability testing and incompatibility testing. Subsequently, a change in the ongoing antibiotic policy was adopted, and corresponding changes in the behaviour of these isolates studied. RESULTS: During the period from August 2011 to January 2013, 46 isolates of monoclonal ESBL K. pneumoniae were obtained from different neonates and four similar environmental isolates were studied. Multidrug-resistant ESBL isolates harboured both blaCTXM-15 and bla SHV-5. The dfr and aac-6 ' resistant genes were found in gene cassettes. A 50 kb plasmid belonging to IncFIIA group was detected in all the isolates which was transferable and stable. The emergence and regression of the outbreaks coincided with antibiotic usage in the NICU, with widespread empirical use of cefotaxime being responsible for their persistence in the environment. INTERPRETATION & CONCLUSIONS: The study indicates that empirical use of third generation cephalosporins may promote the emergence, persistence, and dissemination of resistant isolates in the hospital environment. Periodic review of antibiotic policy is necessary for rationalized use of antibiotics.