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RNA-seq reveals unique and diverse effects of evolved imipenem-relebactam resistance in P. aeruginosa

ABSTRACT: Imipenem-relebactam (Imi/Rel) is a β-lactam/β-lactamase inhibitor combination that is used for the treatment of multidrug-resistant Pseudomonas aeruginosa infections. We previously reported that treatment-emergent resistance is associated with mutations in AmpC, MexAB-OprM and MexEF-OprN efflux operons; however the impact of these mutations on bacterial gene expression have not been explored extensively, particularly among patients treated with Imi/Rel. To determine the effect of treatment-emergent Imi/Rel resistance on P. aeruginosa global transcription, we performed RNA sequencing on 12 paired P. aeruginosa clinical isolates collected from six patients before and after Imi/Rel treatment. Transcriptional responses varied substantially across patients, with no conserved changes in gene expression identified across all six patients. Three isolate pairs showed significant upregulation of previously characterized Imi/Rel resistance-associated genes in the treatment-emergent resistant isolate, while two resistant isolates displayed significant downregulation of ampC. Comparisons of the top 10 differentially regulated genes in the resistant isolate from each patient revealed only one gene that was shared among all six patients. Gene set enrichment analysis using Clusters of Orthologous Group (COGs) suggested that Imi/Rel exposure may impact transcription of genes involved in translation and metabolism , and that these changes are highly variable between patients and isolates. Overall, the transcriptional response of clinical P. aeruginosa to Imi/Rel exposure appears to be highly diverse and likely dependent on the genetic background of the infecting P. aeruginosa strain.

ORGANISM(S): Pseudomonas aeruginosa

PROVIDER: GSE343731 | GEO | 2026/09/11

REPOSITORIES: GEO

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Project description:We compared the dynamics and mechanisms of resistance development to ceftazidime, meropenem, ciprofloxacin, and ceftolozane-tazobactam in wild-type (PAO1) and mutator (PAOMS, ∆mutS) 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 ug/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 ug/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.

Project description:We compared the dynamics and mechanisms of resistance development to ceftazidime, meropenem, ciprofloxacin, and ceftolozane-tazobactam in wild-type (PAO1) and mutator (PAOMS, M-bM-^HM-^FmutS) P. aeruginosa. The strains were incubated for 24 h with 0.5 to 64M-CM-^W 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 64M-CM-^W 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 ug/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 ug/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. Mutants of Pseudomonas aeroginosa PAO1 and PAO1 M-bM-^HM-^FmutS against Ceftolozane-tazobactam were generated and analysed using RNA-Seq

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RNA-seq reveals unique and diverse effects of evolved imipenem-relebactam resistance in P. aeruginosa

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