Short communicationContribution of mutations in DNA gyrase and topoisomerase IV genes to ciprofloxacin resistance in Escherichia coli clinical isolates
Introduction
Quinolones are a group of synthetic antimicrobial agents including nalidixic acid and the fluorinated quinolones. Different mechanisms are involved in the development of resistance to fluoroquinolones. Some are the consequence of mutations involving genes encoding DNA gyrase and topoisomerase IV [1]. These enzymes are necessary for cell viability as they play vital roles in DNA replication, chromosome segregation and DNA compaction [2], [3]. Few microorganisms are as versatile as E. coli, a member of the family Enterobacteriaceae that is a common inhabitant of the human and animal gut. However, E. coli is more than just a laboratory workhorse or a harmless intestinal inhabitant; it can also be a highly versatile, and frequently deadly, pathogen [4]. It is the most common cause of Gram-negative nosocomial and community-acquired infections. Uropathogenic E. coli are the predominant cause of urinary tract infections and are one of the most frequently isolated organisms in neonatal meningitis and nosocomial bacteraemia [5]. In E. coli, the gyrA quinolone resistance-determining region (QRDR) spans amino acids 67–106, with alteration at positions 83 and 87 often associated with clinical resistance. Substitutions at Ser-83 or Asp-87 in gyrA and at Ser-80 and Glu-84 in parC are the most common mutations in E. coli [6]. Substitutions at other positions are rare in clinical isolates. Although a single mutation in gyrA confers resistance to nalidixic acid, additional mutations in gyrA and/or in parC genes, occurring in a step-wise manner, are required to achieve high-level fluoroquinolone resistance.
According to previous reports, mutations in the gyrB and parE genes confer low-level resistance and are less frequently observed [7], [8]. The objective of this study was to investigate the presence of mutations in regions that code for quinolone resistance in the genes gyrA, gyrB, parC and parE in clinical isolates of E. coli from a hospital in Delhi, India. An increase in resistance to the existing quinolones is spreading rapidly all over India and is giving rise to novel mutations and accumulation of a high number of mutations in bacterial genes.
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Materials
Ciprofloxacin was purchased from Sigma (St. Louis, MO) and Mueller–Hinton broth (MHB) was purchased from HiMedia Laboratories Pvt. Ltd. (Mumbai, India).
Isolation and identification of bacterial strains
A total of 54 consecutive clinical isolates of E. coli (from urine, blood, sputum and vaginal swab) were collected from 1 January to 30 June 2009 from Safdarjung Hospital (New Delhi, India). Isolates were identified to genus and species level by standard biochemical tests [9] as well as using a commercially available kit (HiMedia E. coli
Results and discussion
Alteration of a single amino acid at Ser-83 in gyrA is sufficient to generate decreased susceptibility to ciprofloxacin. Moreover, accumulation of amino acid changes in gyrA and the simultaneous presence of parC alterations play a central role in developing high-level resistance to ciprofloxacin.
This study focused on 54 clinical isolates of E. coli from Delhi, 53 (98.1%) and 48 (88.9%) of which possessed mutations at positions 83 and 87 of gyrA, respectively. These positions lie within the QRDR
Acknowledgments
The authors are grateful to the Director, South Campus, University of Delhi (Delhi, India) for allowing use of the facilities for DNA sequencing. They are also thankful to Institute of Pathology [Indian Council of Medical Research (ICMR), Delhi, India] for providing resistant strains for the studies.
Funding: This work was funded by the Department of Science and Technology, Department of Biotechnology and Council for Scientific and Industrial Research (India). SB received a Junior Research
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