title: Pseudomonas aeruginosa ceftolozane-tazobactam resistance development requires multiple mutations leading to overexpression and structural modification of AmpC.
abstract: We compared the dynamics and mechanisms of resistance development to ceftazidime, meropenem, ciprofloxacin, and ceftolozane-tazobactam in wild-type (PAO1) and mutator (PAOMS, ΔmutS) P. aeruginosa. The strains were incubated for 24 h with 0.5 to 64× MICs of each antibiotic in triplicate experiments. The tubes from the highest antibiotic concentration showing growth were reinoculated in fresh medium containing concentrations up to 64× MIC for 7 consecutive days. The susceptibility profiles and resistance mechanisms were assessed in two isolated colonies from each step, antibiotic, and strain. Ceftolozane-tazobactam-resistant mutants were further characterized by whole-genome analysis through RNA sequencing (RNA-seq). The development of high-level resistance was fastest for ceftazidime, followed by meropenem and ciprofloxacin. None of the mutants selected with these antibiotics showed cross-resistance to ceftolozane-tazobactam. On the other hand, ceftolozane-tazobactam resistance development was much slower, and high-level resistance was observed for the mutator strain only. PAO1 derivatives that were moderately resistant (MICs, 4 to 8 μg/ml) to ceftolozane-tazobactam showed only 2 to 4 mutations, which determined global pleiotropic effects associated with a severe fitness cost. High-level-resistant (MICs, 32 to 128 μg/ml) PAOMS derivatives showed 45 to 53 mutations. Major changes in the global gene expression profiles were detected in all mutants, but only PAOMS mutants showed ampC overexpression, which was caused by dacB or ampR mutations. Moreover, all PAOMS mutants contained 1 to 4 mutations in the conserved residues of AmpC (F147L, Q157R, G183D, E247K, or V356I). Complementation studies revealed that these mutations greatly increased ceftolozane-tazobactam and ceftazidime MICs but reduced those of piperacillin-tazobactam and imipenem, compared to those in wild-type ampC. Therefore, the development of high-level resistance to ceftolozane-tazobactam appears to occur efficiently only in a P. aeruginosa mutator background, in which multiple mutations lead to overexpression and structural modifications of AmpC.
authors.affiliation: Servicio de Microbiología and Unidad de Investigación Hospital Universitario Son Espases, Instituto de Investigación Sanitaria de Palma (IdISPa), Palma de Mallorca, Spain.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Cabot
authors.affiliation: Helmholtz Center for Infection Research, Braunschweig, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Bruchmann
authors.affiliation: Servicio de Microbiología and Unidad de Investigación Hospital Universitario Son Espases, Instituto de Investigación Sanitaria de Palma (IdISPa), Palma de Mallorca, Spain.
authors.forename: Xavier
authors.initials: X
authors.lastname: Mulet
authors.affiliation: Servicio de Microbiología and Unidad de Investigación Hospital Universitario Son Espases, Instituto de Investigación Sanitaria de Palma (IdISPa), Palma de Mallorca, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Zamorano
authors.affiliation: Servicio de Microbiología and Unidad de Investigación Hospital Universitario Son Espases, Instituto de Investigación Sanitaria de Palma (IdISPa), Palma de Mallorca, Spain.
authors.forename: Bartolomé
authors.initials: B
authors.lastname: Moyà
authors.affiliation: Servicio de Microbiología and Unidad de Investigación Hospital Universitario Son Espases, Instituto de Investigación Sanitaria de Palma (IdISPa), Palma de Mallorca, Spain.
authors.forename: Carlos
authors.initials: C
authors.lastname: Juan
authors.affiliation: Helmholtz Center for Infection Research, Braunschweig, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Haussler
authors.affiliation: Servicio de Microbiología and Unidad de Investigación Hospital Universitario Son Espases, Instituto de Investigación Sanitaria de Palma (IdISPa), Palma de Mallorca, Spain antonio.oliver@ssib.es.
authors.forename: Antonio
authors.initials: A
authors.lastname: Oliver
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002442:Ceftazidime / Q000494:pharmacology; D002511:Cephalosporins / Q000494:pharmacology; D015378:Imipenem / Q000494:pharmacology; D000077731:Meropenem; D008826:Microbial Sensitivity Tests; D009154:Mutation; D010397:Penicillanic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D010878:Piperacillin / Q000494:pharmacology; D000077725:Piperacillin, Tazobactam Drug Combination; D011552:Pseudomonas Infections / Q000382:microbiology*; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000235:genetics*; D000078142:Tazobactam; D013845:Thienamycins / Q000494:pharmacology; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002511:Cephalosporins; D013845:Thienamycins; D000077725:Piperacillin, Tazobactam Drug Combination; C519491:ceftolozane; D015378:Imipenem; D010397:Penicillanic Acid; D002442:Ceftazidime; C048795:AmpC beta-lactamases; D001618:beta-Lactamases; D000077731:Meropenem; D000078142:Tazobactam; D010878:Piperacillin