Global DNA Methylation Level among Ciprofloxacin-Resistant Clinical Isolates of Escherichia coli DC27-DC30
Dr. Belgode Narasimha Harish,
Department of Microbiology, Second Floor, Institute Block, JIPMER, Gorimedu, Puducherry - 605006, India.
Introduction: Fluoroquinolone resistant clinical isolates belonging to the family Enterobacteriaceae, is a major public health concern in India. Data analysis in JIPMER hospital revealed 10% rise in fluoroquinolone resistance within a span of three years suggestive of the possible involvement of mechanism/s other than QRDR capable of imparting fluoroquinolone resistance. DNA methylation regulates gene expression. Moreover, methylated cytosine is a mutational hotspot. Thus, DNA methylation can alter bacterial gene expression profile as well as facilitate the bacteria in accumulating mutations possibly leading to increased antimicrobial resistance. Therefore, the present study was carried out to identify the potential involvement of DNA methylation in ciprofloxacin resistance.
Aim: To elucidate and compare the methylation level of genomic and plasmid DNA among clinical isolates of E. coli sensitive and resistant to ciprofloxacin.
Materials and Methods: The study included 40 clinical E. coli isolates of which, 30 were ciprofloxacin-resistant and 10 were sensitive to ciprofloxacin. Genomic DNA (gDNA) and plasmid DNA were extracted and quantified. Methylation levels were elucidated using 5-mC DNA ELISA kit (Zymoresearch, California, USA) as per kit protocol and guidelines. Statistical Analysis: Spearman correlation 2-tailed test was used. A p-value <0.05 was considered significant.
Results: The MIC values of sensitive and resistant strains against ciprofloxacin ranged from 0.125 µg/mL – 0.75 µg/mL and 8 µg/mL - >256 µg/mL respectively. No difference was found in plasmid DNA methylation level but, the gDNA methylation level of the resistant strains significantly differed from that of the sensitive strains. Based on Spearman correlation test gDNA methylation level of bacteria was found to be inversely proportional to its MIC against ciprofloxacin with p= -0.956 (p-value < 0.0001).
Conclusion: The influence of DNA methylation over plasmid-mediated quinolone resistance needs to be further confirmed by bisulphite DNA sequencing of the plasmid-borne genes. Extensive usage of ciprofloxacin has led to rise in ciprofloxacin resistance possibly induced by DNA methylation. Thus rational usage of ciprofloxacin in a clinical setting is essential to combat the further development of ciprofloxacin resistance. Hypomethylated genes and adenine methylation needs to be identified to fill up gaps in knowledge concerning the involvement of DNA methylation in fluoroquinolone resistance exhibited by E. coli.