RESUMEN
Purpose: The aim of the present study was to perform molecular characterisation of the blaNDM plasmids and to understand the mechanism of its spread among pathogenic bacteria. Materials and Methods: Seventy-six non-repetitive carbapenem-resistant isolates which were collected during Nov 2011 to April 2013 from four hospitals in Chennai were analyzed for the presence of the blaNDM gene by PCR. Further, the genetic context of the blaNDM gene was analyzed by PCR specifi c to ISAba125 and bleMBL gene. One of the blaNDM plasmid was completely sequenced in the Illumina HiSeq platform. Results: Twenty-three isolates consisting of 8 Escherichia coli, 8 Klebsiella pneumoniae, 3 Klebsiella oxytoca, 3 Acinetobacter baumanii and 1 Pseudomonas aeruginosa were found to carry the blaNDM gene. In 18 isolates the blaNDM gene was associated with a bleMBL gene and the ISAba125 element. The complete sequencing of pNDM-MGR194 revealed an IncX3 replication type plasmid, with a length of 46,253 bp, an average GC content of 47% and 59 putative ORFs. The iteron region contained the blaNDM5 gene and the bleMBL, trpF and dsbC genes downstream and an IS5 inserted within the ISAba125 element upstream. Conclusion: This is the fi rst report where the blaNDM gene insertion in a plasmid is not accompanied by other resistance gene determinants. These observations suggest that the IncX3 plasmid pNDM-MGR194 is an early stage in the dissemination of the blaNDM.
RESUMEN
Purpose: Resistance to fluoroquinolones, a commonly prescribed antimicrobial for Gram-negative and Gram-positive microorganisms, is of importance in therapy. The purpose of this study was to screen for the presence of Plasmid-Mediated Quinolone Resistance (PMQR) determinants in clinical isolates of Klebsiella pneumoniae. Materials and Methods: Extended-Spectrum Beta-Lactamase (ESBL) isolates of K. pneumoniae collected during October 2009 were screened by the antimicrobial susceptibility test. The plasmids from these isolates were analysed by specific Polymerase chain Reaction (PCR) for qnrA, qnrB and aac(6')-1b. The amplified products were sequenced to confirm the allele. Results: Our analysis showed that 61% out of the 23 ESBL K. pneumoniae isolates were resistant to ciprofloxacin and 56% to levofloxacin. The PMQR was demonstrated by transforming the plasmids from two isolates P12 and P13 into E. coli JM109. The PMQR gene qnrA was found in 16 isolates and qnrB in 11 isolates. The plasmid pKNMGR13 which conferred an minimum inhibitory concentration (MIC) of more than 240 ΅g/ml in sensitive E. coli was found to harbour the qnrA1 and qnrB1 allele. Furthermore, the gene aac(6')-1b-cr encoding a variant aminoglycoside 6'-N Acetyl transferase which confers resistance to fluoroquinolones was found in the same plasmid. Conclusions: Our report shows the prevalence of PMQR mediated by qnrA and qnrB in multidrug-resistant K. pneumoniae isolates from Chennai. A multidrug-resistant plasmid conferring high resistance to ciprofloxacin was found to harbour another PMQR gene, aac(6')-1b-cr mutant gene. This is the first report screening for PMQR in K. pneumoniae isolates from India.
RESUMEN
Purpose: Resistance to fluoroquinolones, a commonly prescribed antimicrobial for Gram-negative and Gram-positive microorganisms, is of importance in therapy. The purpose of this study was to screen for the presence of Plasmid-Mediated Quinolone Resistance (PMQR) determinants in clinical isolates of Klebsiella pneumoniae. Materials and Methods: Extended-Spectrum Beta-Lactamase (ESBL) isolates of K. pneumoniae collected during October 2009 were screened by the antimicrobial susceptibility test. The plasmids from these isolates were analysed by specific Polymerase chain Reaction (PCR) for qnrA, qnrB and aac(6')-1b. The amplified products were sequenced to confirm the allele. Results: Our analysis showed that 61% out of the 23 ESBL K. pneumoniae isolates were resistant to ciprofloxacin and 56% to levofloxacin. The PMQR was demonstrated by transforming the plasmids from two isolates P12 and P13 into E. coli JM109. The PMQR gene qnrA was found in 16 isolates and qnrB in 11 isolates. The plasmid pKNMGR13 which conferred an minimum inhibitory concentration (MIC) of more than 240 ΅g/ml in sensitive E. coli was found to harbour the qnrA1 and qnrB1 allele. Furthermore, the gene aac(6')-1b-cr encoding a variant aminoglycoside 6'-N Acetyl transferase which confers resistance to fluoroquinolones was found in the same plasmid. Conclusions: Our report shows the prevalence of PMQR mediated by qnrA and qnrB in multidrug-resistant K. pneumoniae isolates from Chennai. A multidrug-resistant plasmid conferring high resistance to ciprofloxacin was found to harbour another PMQR gene, aac(6')-1b-cr mutant gene. This is the first report screening for PMQR in K. pneumoniae isolates from India.