Pseudomonas aeruginosa Ceftolozane-Tazobactam Resistance Development Requires Multiple Mutations Leading to Overexpression and Structural Modification of AmpC

Abstract

bWe 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 g/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 g/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 piperacillintazobactam 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.

Mutation-Driven β-Lactam Resistance Mechanisms among Contemporary Ceftazidime-Nonsusceptible Pseudomonas aeruginosa Isolates from U.S. Hospitals

“…Interestingly, ceftolozane-tazobactam MIC values for these mutant strains were still low (17). Six isolates displaying OprD loss and derepression of MexABOprM had modestly elevated median MIC values for cephalosporins and ␤-lactam/␤-lactamase inhibitor combinations (from 0.5 to 16 g/ml) and elevated median MIC values for imipenem and meropenem (8 g/ml for both carbapenems) ( Table 4).…”

“…However, when analyzing isolates carrying AmpC derepression with hyperexpression of one or more tripartite efflux systems, the median MIC values were elevated for ceftazidime, cefepime, and piperacillin-tazobactam (median MIC ranges, 32 to Ͼ32, 16 to Ͼ16, and 64 to Ͼ64 g/ml, respectively) but not for carbapenems (Table 4). In a recent study, Cabot et al (17) evaluated meropenem-induced mutants and observed that these strains displayed OprD deficien- (8) OprD loss, MexAB-OprM overexpression…”

“…Additionally, development of mutation experiments performed with P. aeruginosa laboratory strains exposed to elevated concentrations of ceftolozane-tazobactam and other efflux pump substrates showed that mutant strains had generally low MIC values for this cephalosporin/␤-lactamase inhibitor combination. These mutants carried a combination of alterations that included OprD loss and AmpC derepression with or without MexABOprM hyperexpression (17).…”

The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

“…However, its activity is adversely affected by increased efflux in A. baumannii and P. aeruginosa (955). Moreover, several new cephalosporin-␤-lactamase-inhibitor combinational products in clinical trials (e.g., ceftazidime-avibactam, ceftaroline-avibactam, and ceftolozane-tazobactam) (944) are still likely to be the substrates of RND pumps, as are other ␤-lactams and ␤-lactamase inhibitors (13,390,439,545), because avibactam cannot reverse efflux-mediated ceftazidime resistance (956), and both ceftaroline and ceftolozane (a new antipseudomonal cephalosporin) are still affected by efflux pump-and/or porin-related resistance mechanisms (although ceftolozane, containing multiple charged groups, appears less impacted by Mex pumps than many other ␤-lactams and did not select in vitro for pump overproducers in P. aeruginosa, unlike other agents) (417,(957)(958)(959). These observations could also illustrate their reduced or lack of synergistic activity against multidrug-resistant A. baumannii and P. aeruginosa (960)(961)(962).…”

Multidrug-resistant Gram-negative bacteria-resistant infections: epidemiology, clinical issues and therapeutic options

“…69 The development of resistance to ceftolozane tazobactam among strains of P. aeruginosa has been reported to be slower than resistance to other antipseudomonal agents, and it remained active against mutants resistant to ceftazidime, ciprofloxacin, and meropenem. 70 Spectrum of activity of ceftolozane/ tazobactam includes difficult-to-treat Gram-negative pathogens, including ESBL strains. 28,[71][72][73] Another new therapeutic option is represented by ceftazidime/avibactam, where the new b-lactam inhibitor agent avibactam improves the activity of ceftazidime against MDR P. aeruginosa.…”