Functional genomics to discover antibiotic resistance genes: The paradigm of resistance to colistin mediated by ethanolamine phosphotransferase in Shewanella algae MARS 14.
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 1.30×10(8) bp. Functional screening of colistin-resistant clones was carried out on Luria-Bertani agar containing 8 mg/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.
Unité de recherche sur les maladies infectieuses et tropicales émergentes (URMITE), CNRS-IRD UMR 6236, Méditerranée Infection, Faculté de Médecine et de Pharmacie, Aix-Marseille Université, Marseille, France.
Amar A
AA
Telke
Unité de recherche sur les maladies infectieuses et tropicales émergentes (URMITE), CNRS-IRD UMR 6236, Méditerranée Infection, Faculté de Médecine et de Pharmacie, Aix-Marseille Université, Marseille, France. Electronic address: jean-marc.rolain@univ-amu.fr.
Jean-Marc
JM
Rolain
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001483:Base Sequence; D003091:Colistin / Q000494:pharmacology*; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D004982:Ethanolaminephosphotransferase / Q000235:genetics* / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D008826:Microbial Sensitivity Tests; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D020592:Shewanella / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000900:Anti-Bacterial Agents; D012336:RNA, Ribosomal, 16S; D004982:Ethanolaminephosphotransferase; D003091:Colistin
Colistin resistance; Ethanolamine phosphotransferase; Functional genomics; Lipopolysaccharide; RT-PCR; Shewanella algae MARS 14