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Objectives: To determine the prevalence of Escherichia coli STs and associated resistance mechanisms carried by the community in North-East India.Methods: E. coli (108) were isolated from sewage collected from 19 sites across the city of Silchar by plating on MacConkey agar with/without selection (50 mg/L cefotaxime). Species identification was confirmed by MALDI-TOF MS for 82 isolates. Common resistance mechanisms were determined by WGS of pooled E. coli isolates. PFGE combined with specific probes determined the presence of common resistance mechanisms in all isolates. Phylotypes, multilocus STs, core-genome multilocus STs, resistance genes and virulence genes were determined by in silico analysis of 38 genomes.Results and Conclusions: Analysis of isolates collected without selection (n=33) indicated that cefotaxime resistance in E. coli was 42% (14/33) and estimated meropenem resistance at 9%. The remaining 58% (19/33) were additionally susceptible to ampicillin, trimethoprim, ciprofloxacin and aminoglycosides. The most common ST among the cefotaxime-resistant E. coli was ST167 (29%), followed by ST410 (17%) and ST648 (10%). E. coli ST131 was absent from the collection. Sixty-three isolates were resistant to cefotaxime and harboured blaCTX-M-15 [54% (34/63)] or blaCMY-42 [46% (29/63)], of which 10% (6/63) harboured both genes. Carbapenem resistance was due to blaNDM-5, found in 10/63 cefotaxime-resistant isolates, and/or blaOXA-181, found in 4/63 isolates. NDM-5 was encoded by IncX3 and/or IncFII plasmids and CMY-42 was mostly encoded by IncI plasmids. NDM-5 appears to have replaced NDM-1 in this region and CMY-42 appears to be in the process of replacing CTX-M-15. [ABSTRACT FROM AUTHOR]

Objective: The current study investigated the resistance against cycline group of antibiotic among clinical isolates of Staphylococcus aureus and molecular characterization with special reference to phenotypic screening of tigecycline resistance. Methods: 197 coagulase producing Staphylococcus spp. were collected from private diagnostic laboratories and Silchar Medical College and Hospital, India. Phenotypic screening was done with cycline groups of antimicrobials and genotypically characterized by PCR, targeting coa gene. Detection of tet(K), tet(M), tet(O) and tet(X) genes was performed by PCR. Antibiogram profiling was performed and clonality was also determined. Results: 127 coagulase positive S. aureus were phenotypically screened with cycline group of antibiotics in which prominent resistant type showed highest non-susceptibility against tigecycline 53.5% (n=68) while lowest against minocycline 20.5% (n=26). Multiplex PCR assay showed that tet(K) gene to be the most common cycline group of antimicrobial resistance determinant in S. aureus, followed by tet(M) gene. Multiple genes were also observed. However, resistant gene tet(O) was not reported. Antibiotic susceptibility testing and MIC for therapeutic purpose showed highest susceptibility rate against linezolid. DNA fingerprinting experimented with Pulsed Field Gel Electrophoresis had shown seven pulsotypes. Conclusion: Resistance to cycline group of antibiotics with special reference to tigecycline is not frequently reported from India. Thus, this study is of clinical interest in terms of selection of proper antimicrobial chemotherapy and also using tigecycline for phenotypic screening of cycline group of antibiotic resistance in Staphylococcus aureus. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance poses a great challenge to clinicians leaving very limited treatment options available. A panel of carbapenem-resistant bacterial isolates was selected based on Rapidec Carba NP test from a total of 900 samples, which were collected from different specialities hospitals of Kanpur, India. Carba NP-positive isolates were screened for carbapenemases, extended-spectrum beta-lactamases (ESBLs), quinolone resistance, and 16s methyltransferase genes. Presence of diverse blaNDM ( blaNDM-1 and blaNDM-5) were detected and horizontal transferability was determined by transformation and conjugation assay. Elimination of blaNDM-1 and blaNDM-5 harboring plasmid was done by treating the isolates with sodium dodecyl sulfate. The transcriptional response of blaNDM-1 and blaNDM-5 under the exposure of imipenem, meropenem, and ertapenem stress was determined by quantitative real-time polymerase chain reaction. blaNDM harboring isolates were found to be horizontally transferable through IncFrepB and K type plasmid and could be successfully eliminated after the single treatment with sodium dodecyl sulfate. A distinct pattern of transcriptional response was observed for blaNDM-1 and blaNDM-5 under the pressure of carbapenem antibiotics where an upregulated expression of both blaNDM-1 and blaNDM-5 was observed. Minimum inhibitory concentration (MIC) results revealed that blaNDM harboring strains showed a high MIC range against imipenem, meropenem, ertapenem, cefepime, and aztreonam. Thus, prudent action should be taken to control the spread of these multidrug-resistant determinants or at least slowing down their lateral transfer in the hospital settings. [ABSTRACT FROM AUTHOR]

Objectives: Organisms harboring multiple plasmid mediated β-lactamases are major concerns in nosocomial infections. Among these plasmid mediated β-lactamases, ACT (EBC family) is a clinically important enzyme capable of hydrolyzing broad spectrum cephalosporins. Therefore, the present study was undertaken to determine the prevalence of ACT determinant along with other co-existing β-lactamase genes in P. aeruginosa strains. Methods: A total of 176 Pseudomonas isolates were phenotypically screened for the presence of AmpC β-lactamase by M3DET Method followed by Molecular detection using PCR assay. Transcriptional evaluation of blaACT-2 gene was analyzed by RT-PCR and its transferability was performed by transformation and conjugation. Results: Present study demonstrates the presence of ACT-2 allele among 12 strains of P. aeruginosa. Co-existence of other β-lactamase genes were encountered among ACT-2 harboring strains which includes CTX-M (n=2), SHV (n=3), TEM (n=2), VEB (n=2), OXA-10 (n=1), CIT (n=2) and DHA (n=3). Fingerprinting by REP PCR revealed the isolates harboring ACT-2 to be distinct and these isolates showed high resistance to expanded-spectrum cephalosporins and even to carbapenem group of drugs. This ACT-2 allele was encoded in the plasmid (L/M, FIA, FIB Inc. Group) and conjugatively transferable. Transcriptional analysis revealed a significant increase in ACT-2 expression (483 fold) when induced by ceftriaxone at 4 μg/ml followed by ceftazidime at 8 μg/ml (31 fold) and cefotaxime 4 μg/ml (8 fold). Conclusion: In this study detection of ACT-2 plasmid mediated AmpC ß-lactamase along with other ß-lactamase genes in clinical isolates of P. aeruginosa represents a serious therapeutic challenge. Therefore, revision in antimicrobial policy is required for effective treatment of patients infected with pathogen expressing this mechanism. [ABSTRACT FROM AUTHOR]

Background: The current study reports dissemination of highly stable blaOXA-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 β-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 blaOXA-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 blaOXA-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 blaCMY-2 was also observed. Conclusion: Multiple genetic environment of blaOXA-10 in this geographical region must be investigated to prevent dissemination of these gene cassettes within bacterial population within hospital settings. [ABSTRACT FROM AUTHOR]

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 β-lactam stress. Methods: Screening of AmpC β-lactamase phenotypically by modified three dimensional extract method followed by Antimicrobial Susceptibility and MIC determination. Genotyping screening of β-lactamase genes was performed by PCR assay followed by their sequencing. The blaDHA-1 transcriptional response was evaluated under different cephalosporin stress by RT PCR. Transferability of blaDHA 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 x 102 fold), ceftriaxone at 2 μg/ml (2.63 x 103 fold), ceftazidime at 8 μg/ml (7.06 x 103 fold) and cefoxitin at 4 μg/ml (3.60 x 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 β-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 blaDHA-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 β-lactam antibiotics. Therefore, cephalosporin treatment must be restricted for the patients infected with pathogen expressing this resistance determinant. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented on a study to characterize blaNDM-1 in clinical isolates of Pseudomonas aeruginosa and their transmission dynamics.

Background: blaVIM-2 harboring Pseudomonas aeruginosa has been reported worldwide and considered as the most prevalent metallo-β-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 blaVIM-2 in clinical isolates of P. aeruginosa harbor blaNDM-1 as well which were collected from October 2012 to September 2013. Methods: Two P. aeruginosa strains harboring blaVIM-2 along with blaNDM-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: blaVIM-2 was found to be horizontally transferable through an Inc F type plasmid of approximately 30 kb in size. blaVIM-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 blaNDM-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 blaVIM-2 and blaNDM-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. [ABSTRACT FROM AUTHOR]

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 β-lactam-β-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. [ABSTRACT FROM AUTHOR]