Inés Portillo-Calderón1,2,3, Miriam Ortiz-Padilla1,2,3, Jose Manuel Rodríguez-Martínez2,3,4, Belen de Gregorio-Iaria1, Jesús Blázquez3,5, Jesús Rodríguez-Baño1,2,3,6, Alvaro Pascual1,2,3,4, Fernando Docobo-Pérez2,3,4. 1. Unidad Clínica de Enfermedades Infecciosas, Microbiología y Medicina Preventiva, Hospital Universitario Virgen Macarena, Seville, Spain. 2. Instituto de Biomedicina de Sevilla IBIS, Hospital Universitario Virgen Macarena/CSIC/Universidad de Sevilla, Seville, Spain. 3. Red Española de Investigación en Patología Infecciosa (REIPI RD16/0016), Instituto de Salud Carlos III, Madrid, Spain. 4. Departamento de Microbiología, Universidad de Sevilla, Sevilla, Spain. 5. Centro Nacional de Biotecnología (CNB), Madrid, Spain. 6. Departamento de Medicina, Universidad de Sevilla, Sevilla, Spain.
Abstract
OBJECTIVES: To explore the effect of combining defects in DNA repair systems with the presence of fosfomycin-resistant mechanisms to explain the mechanisms underlying fosfomycin heteroresistance phenotypes in Enterobacteriaceae. MATERIALS AND METHODS: We used 11 clinical Escherichia coli isolates together with isogenic single-gene deletion mutants in the E. coli DNA repair system or associated with fosfomycin resistance, combined with double-gene deletion mutants. Fosfomycin MICs were determined by gradient strip assay (GSA) and broth microdilution (BMD). Mutant frequencies for rifampicin (100 mg/L) and fosfomycin (50 and 200 mg/L) were determined. Using two starting inocula, in vitro fosfomycin activity was assessed over 24 h in growth (0.5-512 mg/L) and time-kill assays (64 and 307 mg/L). RESULTS: Strong and weak mutator clinical isolates and single-gene deletion mutants, except for ΔuhpT and ΔdnaQ, were susceptible by GSA. By BMD, the percentage of resistant clinical isolates reached 36%. Single-gene deletion mutants showed BMD MICs similar to those for subpopulations by GSA. Strong mutators showed a higher probability of selecting fosfomycin mutants at higher concentrations. By combining the two mechanisms of mutation, MICs and ranges of resistant subpopulations increased, enabling strains to survive at higher fosfomycin concentrations in growth monitoring assays. In time-kill assays, high inocula increased survival by 37.5% at 64 mg/L fosfomycin, compared with low starting inocula. CONCLUSIONS: The origin and variability of the fosfomycin heteroresistance phenotype can be partially explained by high mutation frequencies together with mechanisms of fosfomycin resistance. Subpopulations should be considered until clinical meaning is established.
OBJECTIVES: To explore the effect of combining defects in DNA repair systems with the presence of fosfomycin-resistant mechanisms to explain the mechanisms underlying fosfomycin heteroresistance phenotypes in Enterobacteriaceae. MATERIALS AND METHODS: We used 11 clinical Escherichia coli isolates together with isogenic single-gene deletion mutants in the E. coli DNA repair system or associated with fosfomycin resistance, combined with double-gene deletion mutants. Fosfomycin MICs were determined by gradient strip assay (GSA) and broth microdilution (BMD). Mutant frequencies for rifampicin (100 mg/L) and fosfomycin (50 and 200 mg/L) were determined. Using two starting inocula, in vitro fosfomycin activity was assessed over 24 h in growth (0.5-512 mg/L) and time-kill assays (64 and 307 mg/L). RESULTS: Strong and weak mutator clinical isolates and single-gene deletion mutants, except for ΔuhpT and ΔdnaQ, were susceptible by GSA. By BMD, the percentage of resistant clinical isolates reached 36%. Single-gene deletion mutants showed BMD MICs similar to those for subpopulations by GSA. Strong mutators showed a higher probability of selecting fosfomycin mutants at higher concentrations. By combining the two mechanisms of mutation, MICs and ranges of resistant subpopulations increased, enabling strains to survive at higher fosfomycin concentrations in growth monitoring assays. In time-kill assays, high inocula increased survival by 37.5% at 64 mg/L fosfomycin, compared with low starting inocula. CONCLUSIONS: The origin and variability of the fosfomycin heteroresistance phenotype can be partially explained by high mutation frequencies together with mechanisms of fosfomycin resistance. Subpopulations should be considered until clinical meaning is established.
Authors: J M Rodríguez-Martínez; F Docobo-Pérez; M Ortiz-Padilla; I Portillo-Calderón; B de Gregorio-Iaria; J Blázquez; J Rodríguez-Baño; A Pascual Journal: Antimicrob Agents Chemother Date: 2021-02-17 Impact factor: 5.191
Authors: I Portillo-Calderón; M Ortiz-Padilla; B de Gregorio-Iaria; V Merino-Bohorquez; J Blázquez; J Rodríguez-Baño; J M Rodríguez-Martínez; A Pascual; F Docobo-Pérez Journal: Antimicrob Agents Chemother Date: 2021-03-08 Impact factor: 5.191