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Functional genomics to discover antibiotic resistance genes: The paradigm of resistance to colistin mediated by ethanolamine phosphotransferase in Shewanella algae MARS 14

Author:
Telke, Amar A., Rolain, Jean-Marc
Source:
International journal of antimicrobial agents 2015 v.46 no.6 pp. 648-652
ISSN:
0924-8579
Subject:
Escherichia coli, Shewanella algae, agar, algae, amino acid sequences, antibiotic resistance genes, clones, colistin, databases, ethanolamine, genomics, lipopolysaccharides, patients, reverse transcriptase polymerase chain reaction, screening, sequence analysis, transferases
Abstract:
Shewanella algae MARS 14 is a colistin-resistant clinical isolate retrieved from bronchoalveolar lavage of a hospitalised patient. A functional genomics strategy was employed to discover the molecular support for colistin resistance in S. algae MARS 14. A pZE21 MCS-1 plasmid-based genomic expression library was constructed in Escherichia coli TOP10. The estimated library size was 1.30×10⁸bp. Functional screening of colistin-resistant clones was carried out on Luria–Bertani agar containing 8mg/L colistin. Five colistin-resistant clones were obtained after complete screening of the genomic expression library. Analysis of DNA sequencing results found a unique gene in all selected clones. Amino acid sequence analysis of this unique gene using the Integrated Microbial Genomes (IMG) and KEGG databases revealed that this gene encodes ethanolamine phosphotransferase (EptA, or so-called PmrC). Reverse transcription PCR analysis indicated that resistance to colistin in S. algae MARS 14 was associated with overexpression of EptA (27-fold increase), which plays a crucial role in the arrangement of outer membrane lipopolysaccharide.
Agid:
6048996