title: In vitro development and analysis of Escherichia coli and Shigella boydii azithromycin-resistant mutants.
abstract: The aim of this study was to develop and analyze in vitro azithromycin (AZM)-resistant mutants of Escherichia coli and Shigella boydii. Three clinical isolates of E. coli and one S. boydii isolated from feces samples collected from children under 5 years of age with diarrhea in Lima, Peru were inoculated onto Mueller-Hinton plates containing increasing serial dilutions of AZM ranging from their specific minimal inhibitory concentration (2 or 4 mg/l) to 64 mg/l. From these plates, 16 AZM-resistant mutants were selected to determine the stability of the resistance and the presence of cross resistance with other antibiotics. The role of Phe-Arg-β-Naphthylamide (PAβN)-inhibitible efflux pumps as well as the presence of mutations in the rplV, rplD, and rrlH (23S rRNA) genes and alterations in the outer membrane profiles were determined in these 16 mutants. The rate of mutation ranged from < 2.70×10(-10) to 2.17×10(-7) for E. coli and from < 9.58×10(-10) to 1.05×10(-8) for S. boydii. E. coli mutants showed an increase in the AZM-MIC up to sixfold with one strain achieving a MIC >256 mg/l. In contrast, S. boydii only presented increases of up to twofold in MIC levels. All the strains obtained, but one showed stable AZM resistance. In the presence of PAβN, the AZM MICs decreased to parental levels in Shigella mutants, while no MIC returned to parental levels among the E. coli mutants. No cross resistance to other classes of antibiotics was found. These results show the relevance of PAβN-inhibitible efflux pumps in the basal levels and development of AZM resistance. Further studies to characterize the remaining unidentified mechanisms of AZM resistance are needed.
authors.affiliation: Barcelona Center for International Health Research (CRESIB, Hospital Clínic-Universitat de Barcelona), Barcelona, Spain.
authors.forename: Cláudia
authors.initials: C
authors.lastname: Gomes
authors.forename: Maria J
authors.initials: MJ
authors.lastname: Pons
authors.forename: Ariel
authors.initials: A
authors.lastname: Magallon-Tejada
authors.forename: David
authors.initials: D
authors.lastname: Durand
authors.forename: Angela
authors.initials: A
authors.lastname: Lluque
authors.forename: Susan
authors.initials: S
authors.lastname: Mosquito
authors.forename: Maribel
authors.initials: M
authors.lastname: Riveros
authors.forename: Erik
authors.initials: E
authors.lastname: Mercado
authors.forename: Ana
authors.initials: A
authors.lastname: Prada
authors.forename: Theresa J
authors.initials: TJ
authors.lastname: Ochoa
authors.forename: Joaquim
authors.initials: J
authors.lastname: Ruiz
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D017963:Azithromycin / Q000494:pharmacology*; D002675:Child, Preschool; D004151:Dipeptides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D024881:Drug Resistance, Bacterial / Q000187:drug effects* / Q000235:genetics; D004405:Dysentery, Bacillary / Q000188:drug therapy / Q000382:microbiology; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D004927:Escherichia coli Infections / Q000188:drug therapy / Q000382:microbiology; D006801:Humans; D026901:Membrane Transport Proteins / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D009154:Mutation; D012761:Shigella boydii / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D000900:Anti-Bacterial Agents; D004151:Dipeptides; D026901:Membrane Transport Proteins; C474925:phenylalanylarginine-naphthylamide; D017963:Azithromycin