Birson Ingti

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

BACKGROUND New-Delhi metallo-β-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 β-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.

Molecular and in silico analysis of a new plasmid-mediated AmpC β-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 β-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 β-lactamase gene was designated as CMH-2.

Emergence of Multidrug-Resistant Uropathogens harboring ESBL, Carbapenem, Aminoglycosides and AmpC resistant genes from Northern India

Extended-spectrum β-lactamase (ESBL) producing bacteria acts as a serious threat, and its co-existence with other antibiotic resistant gene makes the clinical scenario worse nowadays. Therefore in this study, we investigated the occurrence of ESBL genes coexisting with carbapenem, AmpC and aminoglycoside resistance gene in uropathogens. Out of 1516 urine samples, 454 showed significant bacteriuria with a prevalence rate of 29.94 %. Escherichia coli (n=340) were found to be the most predominant uropathogen followed by Klebsiella pneumoniae (n=92), Pseudomonas aeruginosa (n=10) and Proteus mirabilis (n=9). Among the total uropathogens, sixty-three ESBL-producers were identified which included blaCTX-M-15 (n=32), followed by blaCTX-M-15 + blaOXA-2 (n=15), blaCTX-M-15 + blaOXA-2 + blaTEM (n=6), blaOXA-2 (n=5), bla OXA-2 + bla SHV-76 (n=1), bla TEM+SHV-76 (n= 1) and bla TEM (n=1). All ESBL genes were found on plasmid incompatibility types: HI1, I1, FIA+FIB, FIA and Y and were horizontally transferable. Among 63 ESBL-producers, 59 isolates harboured carbapenem-resistant genes which included blaNDM-5 (n=48), blaNDM-5 + blaOXA-48 (n=5), blaNDM-5 + blaIMP (n=5) and blaNDM-5 + blaIMP + blaVIM (n=1). The ESBL producing uropathogens also harbored 16S rRNA methylase genes which included rmtB (n=9), rmtA (n=4), rmtC (n=1) and ArmA (n=1) followed by AmpC genes which includes CIT (n=8) and DHA-1 (n=1) genes. Imipenem and gentamicin were found to be more effective. We speculating, this is the first report showing the prevalence of multidrug-resistant uropathogens in this area demanding regular surveillance for such resistance mechanisms which will be useful for health personnel to treat ESBL infection and its co-existence with another antibiotic resistance gene.

Occurrence of bla CMY-42 through IncI1 plasmid within multidrug-resistant Escherichia coli from a tertiary referral hospital of 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 β-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 β-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.

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

Background & objectives: Pseudomonas aeruginosa possessing chromosomally inducible blaPDC along 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 blaPDC in 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 β-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 β-lactamase genes was performed by PCR assay. Antimicrobial susceptibility and minimum inhibitory concentrations (MICs) were determined, and repetitive extragenic palindromic-PCR of all blaPDC harbouring 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 blaPDC gene, 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 blaPDC showed 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 β-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 β-lactamase amongst P. aeruginosa.