Transcriptional Analysis of IncFrepB-Mediated blaOXA-48-Positive Plasmid Characterized from Escherichia coli ST448.

Objectives: To investigate the transcriptional response of blaOXA-48 and the copy number alteration of IncFrepB plasmid carrying blaOXA-48 under an antibiotic concentration gradient. Methods: Escherichia coli strains harboring blaOXA-48 on an IncFrepB plasmid were isolated from Silchar Medical College and Hospital, Silchar, India. Sequence type and common resistance determinants were determined by PCR assay. Plasmid copy number alteration and the transcriptional expression of blaOXA-48 under different antibiotic pressures were determined by quantitative real-time PCR, and the relative fold change was measured by the ΔΔCT method. Results and Conclusion: The plasmid that carried blaOXA-48 in E. coli ST448 was characterized as IncFrepB and found to be conjugatively transferable. The isolates were found to coexist with blaNDM-1 within the IncX3-type plasmid. It was observed that the copy number and transcriptional response of blaOXA-48 were directly proportional to the increasing concentration of meropenem and ertapenem, whereas in the case of imipenem, it was reversed. The identification of blaOXA-48 through IncFrepB-type plasmid in this study indicates the potential route of spread of this resistance determinant in this area and also the insights we gained from the transcriptional changes of blaOXA-48 in response to different antibiotic pressures could also facilitate the development of novel or alternative therapeutic options needed for multidrug-resistant infections.

Transcriptional response of mar, sox and rob regulon against concentration gradient carbapenem stress within Escherichia coli isolated from hospital acquired infection.

OBJECTIVE: The present study was carried out to investigate the transcriptional response of marA (Multiple antibiotic resistance A gene), soxS (Superoxide S gene) and rob (Right-origin-binding gene) under carbapenem stress. RESULTS: 12 isolates were found over-expressing AcrAB-TolC efflux pump system and showed reduced expression of OmpF (Outer membrane porin) gene were selected for further study. Among them, over expression of marA and rob was observed in 7 isolates. Increasing pattern of expression of marA and rob against meropenem was observed. The clones of marA and rob showed reduced susceptibility towards carbapenems.

An insight into analysis and elimination of plasmids encoding metallo-ß-lactamases in Pseudomonas aeruginosa.

OBJECTIVES: The aim of this study was to characterise metallo-ß-lactamase (MBL)-harbouring plasmids, their change in copy number in respect to different antibiotic pressure, and the efficiency of different curing agents in eliminating these resistance plasmids from nosocomial Pseudomonas aeruginosa isolates. METHODS: Plasmids were extracted from four isolates harbouring blaNDM-1 or blaVIM-2 under four different concentrations of imipenem, meropenem, ertapenem, aztreonam and cefotaxime. Quantitative real-time PCR was performed to analyse the change in plasmid copy number under these different conditions. The effect of different physical and chemical curing agents in elimination of plasmids carrying blaNDM-1 and blaVIM-2 was examined, with meropenem resistance used as a selectable marker. RESULTS: Conjugatively transferable MBL genes (blaNDM-1 and blaVIM-2) carried on plasmids were found to be highly stable. Sodium dodecyl sulfate (SDS) was the most effective agent in eliminating these resistance plasmids. The change in copy number of the blaNDM-1-encoding plasmid was found to be similar to the blaVIM-2-encoding plasmid, with a single exception under cefotaxime pressure. CONCLUSION: The spread of multidrug resistance plasmids has been noted as a key factor associated with increasing carbapenem resistance. Successful curing of resistance plasmids can reverse the bacterial phenotype back to susceptible. This study revealed that different antibiotic pressure induces a change in copy number of MBL-encoding plasmids. SDS can be successfully used as an eliminating agent for these resistance determinants, although therapeutic application of this agent is not possible due to its high toxicity and mutagenic nature.

Occurrence of diverse aminoglycoside modifying enzymes with co-existing extended-spectrum-ß-lactamases within Enterobacteriaceae isolated in India.

OBJECTIVE: The present study describes aminoglycoside modifying enzymes (AMEs) among clinical isolates with coexisting extended spectrum beta-lactamases. METHODOLOGY: A total of 227 non duplicate enterobacterial isolates were collected and identified from patients who were admitted to different wards or attended OPD of a tertiary referral hospital of North-East India. Isolates were initially screened for antimicrobial susceptibility testing followed by PCR based screening of aminoglycosides modifying enzymes and co-existing ESBLs and carbapenemases. Horizontal transferability, incompatibility typing and stability of plasmids were also analyzed. RESULTS: Diverse types of AMEs were observed namely; ant(3″)-I, ant(4')-Ia, aac(3)-IIc, ant(3')-I, aac(6')-Ib, ant(2″)-Ia and aac(6'). Majority of the AME positive isolates harboured blaTEM followed by blaCTX-M-15 and a combination of blaTEM and blaCTX-M-15 were also observed. Nine isolates were found to harbour carbapenemases genes. AME genes were found to be located within a self conjugative plasmid of Inc FIA, IncY, IncN, IncFIB and IncA/C incompatibility types. It was observed that most AME genes were stable over 50 days of serial passages whereas aph(3')-Via and aph(3')-IIb were completely lost within 50 days. CONCLUSION: This study underscores the co-existence of AMEs and ESBLs within enterobacteriaceae which emphasize a reassessment of combination therapy in the health settings.

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.

Effect of concentration gradient carbapenem exposure on expression of blaNDM-1 and acrA in carbapenem resistant Escherichia coli.

Escherichia coli, one of the major pathogens, frequently exhibits carbapenem resistance. It would be of interest to investigate which of the mechanisms responds when a strain that carries both blaNDM-1 and over expressed AcrAB-TolC efflux pump system is exposed against carbapenem under differential concentration gradient stress. Four different sets of strains were used in the study; (i) Strain that have blaNDM-1 and over expressed AcrAB-TolC system (ii) Strain that harbour blaNDM-1 and express AcrAB-TolC at basal level (iii) the strain that is devoid of blaNDM-1 but having over expressed AcrAB-TolC systems and (iv) E. coli AG100A (ΔAcrAB) and E. coli HUE 1 (ΔAcrAB-TolC) where blaNDM-1was cloned. The Quantitative Real time PCR showed blaNDM-1 was over expressed under meropenem and imipenem stress irrespective of concentration gradient. In case of ertapenem, at lower concentration AcrA were over expressed whereas, at higher concentration blaNDM-1 showed elevated expression. A consistent elevated expression of AcrA and AcrB was observed against all carbapenems in the strains devoid of blaNDM-1 where as in case of the strain with basal level expression of AcrA, no significant over expression could be observed for blaNDM-1. In case of clones in group IV, expression of blaNDM-1 was elevated in the presence of carbapenem stress.

Transcriptional response of OmpC and OmpF in Escherichia coli against differential gradient of carbapenem stress.

OBJECTIVE: This study was designed to investigate the transcriptional response of OmpF and OmpC along with an antisense RNA, MicF under concentration gradient carbapenem exposure. RESULT: An elevation in the expression of OmpF gene under concentration gradient imipenem stress from a particular concentration was observed. For OmpC gene a significant decrease in the expression was noticed under concentration gradient imipenem and meropenem stress. The study showed reduction in the expression of OmpC gene against imipenem and meropenem possibly preventing the entry of carbapenem antibiotic inside the cell indicating a possible role in carbapenem resistance.

Association of glycerol kinase gene with class 3 integrons: A novel cassette array within Escherichia coli.

BACKGROUND: Integrons are genetic elements which are known for their role in capturing and spreading of antibiotic resistance determinants among Gram-negative bacilli. So far, there is no study regarding Class 3 integron and their genetic organisation in India. OBJECTIVE: This study investigates the occurrence of Class 3 integron and their gene cassette array among Escherichia coli. MATERIALS AND METHODS: In this study, a total of 200 E. coli isolates were collected from indoor and outdoor patients from Silchar Medical College and Hospital during September 2015 to February 2016. Detection of the integrase genes and gene cassettes within the Class 3 integron was performed by polymerase chain reaction which was further analysed by sequencing. RESULTS: Twenty-seven isolates were found to harbour Class 3 integron. Sequencing of the gene cassettes and whole Class 3 integron revealed the presence of nine different types of cassettes array, out of which the arrangement with glycerol kinase gene cassette was found to be the most prevalent. Arrangement with blaCTX-Mgene cassette was also detected in few isolates. CONCLUSION: This study provides epidemiological profiling of Class 3 integrons in this geographical area. The data generated in this study are helpful in infection control programme, anti-infective research and search for epidemiological markers.

Observation of a new pattern of mutations in gyrA and parC within Escherichia coli exhibiting fluroquinolone resistance.

Therapeutic options with quinolones are severely compromised in infections caused by members of Enterobacteriaceae family. Mutations in chromosomal region are one of the major reasons for bacterial resistance towards this group of antibiotic. The aim of the study is to detect the mutations in gyr A and par C responsible for quinolone resistance among clinical isolates of Escherichia coli. A total of 96 quinolone-resistant clinical isolates of E. coli were collected from a tertiary care hospital of North-east India during March 2015 to August 2015. All the quinolone-resistant E. coli strains were investigated for mutations in the topoisomerases genes gyrA and parC by amplifying and sequencing the quinolone resistance determining regions. Among the 96 E. coli isolates, 83.3% were resistant to nalidixic acid and 80.2%, 66.6%, 23.9% and 50% to ciprofloxacin, norfloxacin, levofloxacin and ofloxacin, respectively. Several alterations were detected in gyrA and parC genes. Three new patterns of amino acid substitution are reported in E. coli isolates. The findings of this study warrant a review in quinolone-based therapy in this region of the world to stop or slow down the irrational use this drug.

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.

Sub-inhibitory concentration of ertapenem induces overexpression of regulator of antibiotic resistance A in Escherichia coli.

AcrAB-TolC is a tripartite efflux pump system constitutively expressed which functions as an intrinsic-resistant mechanism found to be responsible for conferring resistance towards dyes, detergents and different compounds including various classes of antibiotics. One global regulator belonging to AraC-type regulator family, regulator of antibiotic resistance A (RarA) up-regulates the expression of AcrAB-TolC encoded in Klebsiella pneumoniae, Enterobacter sp. 638, Serratia proteamaculans 568 and Enterobacter cloacae resulting in multidrug-resistant phenotypes. The present work was initiated to find out the transcriptional response of RarA in clinical isolates of Escherichia coli against concentration gradient carbapenem stress. A total of 22 clinical isolates of E. coli and expression level of regulators were analysed via quantitative real-time polymerase chain reaction with and without carbapenem stress. As a result, a strong correlation between the expressional levels of RarA in AcrAB overexpressed isolates of E. coli and elevated expression was observed when exposed under concentration gradient ertapenem stress. The clones containing pRar showed reduction in the zone of inhibition towards carbapenem, indicating the active participation of RarA in AcrAB overexpressed isolates of E. coli conferring resistance towards carbapenems.

Transcriptional response of AcrEF-TolC against fluoroquinolone and carbapenem in Escherichia coli of clinical origin.

INTRODUCTION: Efflux pump systems constitute a major means of intrinsic resistance in Escherichia coli. AcrEF-TolC pump is known to exhibit higher expression level in quinolone resistant isolates. However, the transcriptional response of this pump is yet to be known when exposed to quinolone and other group of antibiotics. OBJECTIVE: The present study analyses the transcriptional response of AcrEF-TolC in the presence of quinolones and carbapenems. METHODOLOGY: A total of 167 non-duplicate clinical isolates from Silchar medical college and Hospital, Silchar, India were included in this study. Of which 27 were devoid of any carbapenemase activity and among them 13 isolates showed overexpression of AcrE and AcrF gene. Transcriptional response of AcrE was directly proportional to increasing concentration of levofloxacin and ofloxacin. However, the response of AcrE and AcrF was inconsistent with carbapenems. RESULT: The study isolates showed susceptibility towards amikacin (68.4%), gentamicin (59.6%), cefepime (52.7%) and pipercillin/tazobactam (48.3%). The present investigation highlights that apart from qnr genes and mutational changes in gyr region, AcrEF-TolC plays a major role in fluoroquinolone resistance in this part of the world. CONCLUSION: Upregulation of AcrE in the presence of levofloxacin and ofloxacin warrants further investigation to establish their active role in efflux of this drug.

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.

Expansion of highly stable bla OXA-10 ß-lactamase family within diverse host range among nosocomial isolates of Gram-negative bacilli within a tertiary referral hospital of Northeast India.

BACKGROUND: The current study reports dissemination of highly stable bla OXA-10 family of beta lactamases among diverse group of nosocomial isolates of Gram-negative bacilli within a tertiary referral hospital of the northern part of India. METHODS: In the current study, a total number of 590 Gram negative isolates were selected for a period of 1 year (i.e. 1st November 2011-31st October 2012). Members of Enterobacteriaceae and non fermenting Gram negative rods were obtained from Silchar Medical College and Hospital, Silchar, India. Screening and molecular characterization of ß-lactamase genes was done. Integrase gene PCR was performed for detection and characterization of integrons and cassette PCR was performed for study of the variable regions of integron gene cassettes carrying bla OXA-10. Gene transferability, stability and replicon typing was also carried out. Isolates were typed by ERIC as well as REP PCR. RESULTS: Twenty-four isolates of Gram-negative bacilli that were harboring bla OXA-10 family (OXA-14, and OXA16) with fact that resistance was to the extended cephalosporins. The resistance determinant was located within class I integron in five diverse genetic contexts and horizontally transferable in Enterobacteriaceae, was carried through IncY type plasmid. MIC values were above break point for all the tested cephalosporins. Furthermore, co-carriage of bla CMY-2 was also observed. CONCLUSION: Multiple genetic environment of bla OXA-10 in this geographical region must be investigated to prevent dissemination of these gene cassettes within bacterial population within hospital settings.

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 ß-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 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 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.

Transcriptional analysis of blaNDM-1 and copy number alteration under carbapenem stress.

BACKGROUND: New Delhi metallo beta-lactamase is known to compromise carbapenem therapy and leading to treatment failure. However, their response to carbapenem stress is not clearly known. Here, we have investigated the transcriptional response of blaNDM-1 and plasmid copy number alteration under carbapenem exposure. METHODS: Three blaNDM-1 harboring plasmids representing three incompatibility types (IncFIC, IncA/C and IncK) were inoculated in LB broth with and without imipenem, meropenem and ertapenem. After each 1 h total RNA was isolated, immediately reverse transcribed into cDNA and quantitative real time PCR was used for transcriptional expression of blaNDM-1. Horizontal transferability and stability of the plasmids encoding blaNDM-1 were also determined. Changes in copy number of blaNDM-1 harboring plasmids under the exposure of different carbapenems were determined by real time PCR. Clonal relatedness among the isolates was determined by pulsed field gel electrophoresis. RESULTS: Under carbapenem stress over an interval of time there was a sharp variation in the transcriptional expression of blaNDM-1 although it did not follow a specific pattern. All blaNDM-1 carrying plasmids were transferable by conjugation. These plasmids were highly stable and complete loss was observed between 92nd to 96th serial passages when antibiotic pressure was withdrawn. High copy number of blaNDM-1 was found for IncF type plasmids compared to the other replicon types. CONCLUSION: This study suggests that the single dose of carbapenem pressure does not significantly influence the expression of blaNDM-1 and also focus on the stability of this gene as well as the change in copy number with respect to the incompatible type of plasmid harboring resistance determinant.