RESUMEN
OBJECTIVES: To investigate the phenotypic effect of expression of selected acquired macrolide resistance genes (AMRGs) in non-typeable Haemophilus influenzae (NTHi). METHODS: The AMRGs erm(A), erm(B) and erm(C) were cloned into Escherichia coli JM109 using the shuttle vector pLS88; constructed plasmids extracted from suitable clones were used to transform H. influenzae Rd by electroporation. Erythromycin and azithromycin MICs for suitable transformants were determined by broth microdilution. AMRG expression was determined using quantitative PCR on transformant cDNA with locked nucleic acid dual-labelled hydrolysis probes. RESULTS: Expression of all AMRGs was observed in the transformants. Some variation in expression between the AMRGs was apparent, but expression of all genes was associated with a notable increase in erythromycin and azithromycin MICs compared with untransformed H. influenzae Rd. CONCLUSIONS: While the establishment of erm genes within WT populations of NTHi remains contentious, H. influenzae is capable of expression of erm. Expression may be associated with a subsequent decreased susceptibility to macrolides in isolates and future monitoring of these genes in NTHi isolates is warranted.
Asunto(s)
Antibacterianos/farmacología , Farmacorresistencia Bacteriana , Expresión Génica , Genes Bacterianos , Haemophilus influenzae/efectos de los fármacos , Haemophilus influenzae/genética , Macrólidos/farmacología , Azitromicina/farmacología , Clonación Molecular , Electroporación , Eritromicina/farmacología , Escherichia coli/genética , Vectores Genéticos , Metiltransferasas/biosíntesis , Metiltransferasas/genética , Pruebas de Sensibilidad Microbiana , Plásmidos , Transformación BacterianaRESUMEN
OBJECTIVES: The objective of this study was to determine the prevalence of specific acquired macrolide resistance genes previously reported as present in clinical isolates of Haemophilus influenzae. METHODS: A collection of 172 clinical respiratory isolates of H. influenzae, including 59 isolates from cystic fibrosis patients and 27 from non-cystic fibrosis bronchiectasis patients with significant prior macrolide use, was established. This collection was tested for azithromycin susceptibility using Etest and screened for the presence of erm(A), erm(B), erm(C), erm(F), mef(A) and mef(E) using locked nucleic acid dual-labelled hydrolysis probes. RESULTS: The azithromycin MICs ranged from 0.09 to >256 mg/L, with 2 (1.2%) isolates susceptible, 163 (94.8%) intermediate and 7 (4%) resistant according to EUCAST breakpoints (susceptible, ≤0.12 mg/L; resistant, >4 mg/L). None of the acquired macrolide resistance genes erm(A), erm(B), erm(C), erm(F), mef(A) or mef(E) was detected in any of the isolates. CONCLUSIONS: The specific acquired macrolide resistance genes are not widespread in H. influenzae and the high prevalence of these genes previously reported might be unique to the specific circumstances of that study.