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 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.

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.

Molecular characterization of enteropathogenic Escherichia coli isolated from patients with gastroenteritis in a tertiary referral hospital of northeast India.

PURPOSE: Diarrhoeal illness accounts for a high morbidity and mortality both in paediatric as well as adult groups and diarrhoeagenic Escherichia coli occupies a top position as a causative agent of infectious diarrhoeal illness worldwide. The aim of the current investigation was to determine the virulence and pattern of antibiotic resistance of enteropathogenic, enterotoxigenic, and shiga toxigenic Escherichia coli that are linked to diarrhoea in patients of both adult and paediatric age groups. METHODS: A total of 50 consecutive, nonduplicate Escherichia coli isolates were collected from patients with gastro-enteritis who were admitted to different clinical wards Silchar Medical College and Hospital, Silchar, India. PCR was used to identify the virulence genes of EPEC (eaeA and bfpA), STEC (stx1, stx2, and eae) and ETEC (eltA, eltB, estA1 and estA2) in the isolates of E. coli. The antibiotic susceptibility pattern of virulent E. coli isolates were checked using disc diffusion method. Molecular typing of the virulent E. coli detected in the study based on enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) was also done. RESULT: Out of 50 E. coli isolates, 13 (26%) were found to carry atleast one virulence gene. 11 isolates harboured eae gene and were characterized as EPEC and two isolates carried stx1 gene of STEC. These virulent isolates showed different antibiotic susceptibility pattern and harboured single or multiple antibiotic resistance genes. ERIC PCR established 12 different clonal patterns of the virulent study isolates of E. coli harbouring. CONCLUSION: EPEC pathotypes were found to be the most detected pathotype in the stool samples. Majority of the virulent isolates were also resistant to multiple antibiotics which is a serious public health concern and therefore requires a proper surveillance and studies to track their reservoirs to contain their spread.

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.

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.

Distribution of carbapenem resistant Acinetobacter baumannii with blaADC-30 and induction of ADC-30 in response to beta-lactam antibiotics.

A wide range of intrinsic Acinetobacter-derived cephalosporinases (ADC) along with other carbapenemases has now been detected in Acinetobacter baumannii leaving clinicians with few treatment options. The present study reports the spread of ADC-30 co-producing KPC-2 along with other ß-lactamases among carbapenem resistant A. baumannii strains obtained from ICU patients in two Indian hospitals. Primer extension analysis revealed higher transcript level of the ADC gene when induced with cefoxitin at 8 µg/ml (170 fold), ceftriaxone at 8 µg/ml (136 fold), ceftazidime at 4 µg/ml (65 fold), cefepime at 8 µg/ml (77 fold) and aztreonam at 8 µg/ml (21 fold) when compared with the basal level without antibiotic pressure. Slight increase in expression of blaADC-30 when induced with imipenem and meropenem at 0.25 µg/ml (3 and 6 fold) was observed and may help in conferring resistance to carbapenem. MLST analysis revealed the circulation of A. baumannii sequence types ST188, ST386, ST583 and ST390 in these hospitals.

Prevalence of Giardia intestinalis with other co-infecting parasites in Barak Valley, Assam, India: a molecular approach.

Giardia intestinalis was included in the World Health Organization's Neglected Disease Initiative in 2004 as it may range from asymptomatic to chronic or severe diarrhoea and chronic disorders post-infection. The present study aimed to find out the rate of sole infection of G. intestinalis and co-infection of this with other protozoan parasites among the inhabitants of Barak Valley region of Southern Assam by conventional and molecular detection. A total of 1168 samples were collected from different groups of individuals, all the collected samples were subjected to microscopy after specific staining by Lugol's iodine solution, Trichrome staining and modified ZN staining procedures. Microscopically positive samples were further confirmed by PCR using specific primer sets. Of the total no. of samples, 267 (22.85%) were positive by PCR for G. intestinalis with a little higher rate of infection in female (24.06%) (OR = 1.2192, CI = 0.9262 to 1.6049) than male (21.27%). The rate of infection is comparatively higher (25.93%) in the age group of 0-5 years (OR = 1.9149, CI = 1.2558 to 2.9200). In 196 samples G. intestinalis co-existence was observed and detected by PCR with some other protozoan parasites like Entamoeba spp., Cryptosporidium spp. and Blastocystis spp. The rate of infection was higher (31.96%) among the participants who collected water from river. Least of the participants showed diarrhoeal symptoms (18.18%) but majority (28.45%) complained for having abdominal cramps (OR = 1.3402, CI = 0.8815 to 1.7855). Among the human infective assemblages, assemblage specific molecular detection revealed the rate of infection of assemblage B was comparatively higher (60.30%) than assemblage A.

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 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.

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.

Molecular and in silico analysis of a new plasmid-mediated AmpC ß-lactamase (CMH-2) in clinical isolates of Klebsiella pneumoniae.

Two Klebsiella strains isolated from urine samples were positive for blaAmpC by PCR and showed sequence similarity with CMH-1 (98.6%) after sequencing. It also shares 82% similarity with ACT-1, 85% with MIR-1 and 81% with the chromosomal AmpC gene of Enterobacter cloacae. This gene was associated with the plasmid of IncK type. It has an open reading frame of 381 amino acid with four amino acid substitutions at position D144A, C189R, Q192E, and A195T as compared to CMH-1. When expressed in E.coli DH5α and E.coli strain B, this ß-lactamase conferred resistance to cefotaxime, ceftriaxone and ceftazidime. In addition, both in vitro and in silico analysis revealed that this cephalosporinase was inhibited by cefepime and carbapenem group of drugs. Therefore, this new plasmid-encoded AmpC type ß-lactamase gene was designated as CMH-2.

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.