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Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance
Published on: May 2, 2018
Pseudomonas aeruginosa ceftolozane-tazobactam resistance development requires multiple mutations leading to
Gabriel Cabot1, Sebastian Bruchmann2, Xavier Mulet1
1Servicio de Microbiología and Unidad de Investigación Hospital Universitario Son Espases, Instituto de Investigación Sanitaria de Palma (IdISPa), Palma de Mallorca, Spain.
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.
Insights
High-level resistance to ceftolozane-tazobactam in Pseudomonas aeruginosa develops slowly and primarily in mutator strains. This resistance involves AmpC overexpression and mutations, unlike resistance to other antibiotics tested.
Area of Science:
- Microbiology
- Genetics
- Antimicrobial Resistance
Background:
- Pseudomonas aeruginosa is an opportunistic pathogen known for developing antibiotic resistance.
- Understanding resistance mechanisms is crucial for effective treatment strategies.
Purpose of the Study:
- To compare resistance development dynamics and mechanisms to four antibiotics in wild-type and mutator P. aeruginosa strains.
- To investigate the genetic basis of ceftolozane-tazobactam resistance.
Main Methods:
- Serial passage of P. aeruginosa (wild-type and mutator strains) in increasing antibiotic concentrations (0.5-64x MIC) for 7 days.
- Susceptibility profiling and resistance mechanism assessment of isolated colonies.
- Whole-genome and RNA sequencing for ceftolozane-tazobactam resistant mutants.
Main Results:
- High-level resistance developed fastest to ceftazidime, meropenem, and ciprofloxacin; no cross-resistance to ceftolozane-tazobactam was observed.
- Ceftolozane-tazobactam resistance developed slowly, with high-level resistance only in mutator strains, involving 45-53 mutations.
- Mutants showed AmpC overexpression (mutator strains) and mutations in conserved AmpC residues, altering susceptibility profiles.
Conclusions:
- High-level ceftolozane-tazobactam resistance efficiently emerges only in P. aeruginosa mutator strains.
- Resistance involves multiple mutations leading to AmpC overexpression and structural changes.
- These modifications confer resistance to ceftolozane-tazobactam and ceftazidime but reduce susceptibility to other beta-lactams.
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