One of the most striking features of Pseudomonas aeruginosa is its outstanding capacity for developing antimicrobial resistance to nearly all available antipseudomonal agents through the selection of chromosomal mutations, leading to the failure of the treatment of severe hospital-acquired or chronic infections. Recent whole-genome sequencing (WGS) data obtained from in vitro assays on the evolution of antibiotic resistance, in vivo monitoring of antimicrobial resistance development, analysis of sequential cystic fibrosis isolates, and characterization of widespread epidemic high-risk clones have provided new insights into the evolutionary dynamics and mechanisms of P. aeruginosa antibiotic resistance, thus motivating this review. Indeed, the analysis of the WGS mutational resistome has proven to be useful for understanding the evolutionary dynamics of classical resistance pathways and to describe new mechanisms for the majority of antipseudomonal classes, including β-lactams, aminoglycosides, fluoroquinolones, or polymixins. Beyond addressing a relevant scientific question, the analysis of the P. aeruginosa mutational resistome is expected to be useful, together with the analysis of the horizontally-acquired resistance determinants, for establishing the antibiotic resistance genotype, which should correlate with the antibiotic resistance phenotype and as such, it should be useful for the design of therapeutic strategies and for monitoring the efficacy of administered antibiotic treatments. However, further experimental research and new bioinformatics tools are still needed to overcome the interpretation limitations imposed by the complex interactions (including those leading to collateral resistance or susceptibility) between the 100s of genes involved in the mutational resistome, as well as the frequent difficulties for differentiating relevant mutations from simple natural polymorphisms.

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This study assessed the molecular epidemiology, resistance mechanisms, and susceptibility profiles of a collection of 150 extensively drug-resistant (XDR) clinical isolates obtained from a 2015 Spanish multicenter study, with a particular focus on resistome analysis in relation to ceftolozane-tazobactam susceptibility. Broth microdilution MICs revealed that nearly all (>95%) of the isolates were nonsusceptible to piperacillin-tazobactam, ceftazidime, cefepime, aztreonam, imipenem, meropenem, and ciprofloxacin. Most of them were also resistant to tobramycin (77%), whereas nonsusceptibility rates were lower for ceftolozane-tazobactam (31%), amikacin (7%), and colistin (2%). Pulsed-field gel electrophoresis-multilocus sequence typing (PFGE-MLST) analysis revealed that nearly all of the isolates belonged to previously described high-risk clones. Sequence type 175 (ST175) was detected in all 9 participating hospitals and accounted for 68% ( = 101) of the XDR isolates, distantly followed by ST244 ( = 16), ST253 ( = 12), ST235 ( = 8), and ST111 ( = 2), which were detected only in 1 to 2 hospitals. Through phenotypic and molecular methods, the presence of horizontally acquired carbapenemases was detected in 21% of the isolates, mostly VIM (17%) and GES enzymes (4%). At least two representative isolates from each clone and hospital ( = 44) were fully sequenced on an Illumina MiSeq. Classical mutational mechanisms, such as those leading to the overexpression of the β-lactamase AmpC or efflux pumps, OprD inactivation, and/or quinolone resistance-determining regions (QRDR) mutations, were confirmed in most isolates and correlated well with the resistance phenotypes in the absence of horizontally acquired determinants. Ceftolozane-tazobactam resistance was not detected in carbapenemase-negative isolates, in agreement with sequencing data showing the absence of mutations. The unique set of mutations responsible for the XDR phenotype of ST175 clone documented 7 years earlier were found to be conserved, denoting the long-term persistence of this specific XDR lineage in Spanish hospitals. Finally, other potentially relevant mutations were evidenced, including those in penicillin-binding protein 3 (PBP3), which is involved in β-lactam (including ceftolozane-tazobactam) resistance, and FusA1, which is linked to aminoglycoside resistance.

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Resistance development to novel cephalosporin-␤-lactamase inhibitor combinations during ceftazidime treatment of a surgical infection by Pseudomonas aeruginosa was investigated. Both initial (97C2) and final (98G1) isolates belonged to the high-risk clone sequence type (ST) 235 and were resistant to carbapenems (oprD), fluoroquinolones (GyrA-T83I, ParC-S87L), and aminoglycosides (aacA7/aacA8/aadA6). 98G1 also showed resistance to ceftazidime, ceftazidime-avibactam, and ceftolozanetazobactam. Sequencing identified bla OXA-2 in 97C2, but 98G1 contained a 3-bp insertion leading to the duplication of the key residue D149 (designated OXA-539). Evaluation of PAO1 transformants producing cloned OXA-2 or OXA-539 confirmed that D149 duplication was the cause of resistance. Active surveillance of the emergence of resistance to these new valuable agents is warranted.KEYWORDS extended-spectrum OXA, Pseudomonas aeruginosa, multidrug resistance, ceftolozane-tazobactam, ceftazidime-avibactam T he increasing prevalence of nosocomial infections produced by multidrug-resistant (MDR) or extensively drug-resistant (XDR) Pseudomonas aeruginosa strains severely compromises the selection of appropriate treatments and is therefore associated with significant morbidity and mortality (1-3). This growing threat results from the extraordinary capacity of this pathogen to develop resistance to nearly all available antibiotics by the selection of mutations in chromosomal genes and from the increasing prevalence of transferable resistance determinants, particularly those encoding class B carbapenemases (metallo-␤-lactamases [MBLs]) or extended-spectrum ␤-lactamases, frequently cotransferred with genes encoding aminoglycoside-modifying enzymes (4, 5). The emergence of MDR/XDR global clones, deemed high-risk clones, disseminated in several hospitals worldwide adds further concern (6, 7).The recent introduction of novel ␤-lactam-␤-lactamase inhibitor combinations, namely, ceftolozane-tazobactam and ceftazidime-avibactam, which are stable against most mutational resistance mechanisms, including the overexpression of the chromosomal cephalosporinase AmpC, partially alleviates the urgent clinical need for new agents that combat infections by MDR/XDR P. aeruginosa (8-10). Thus, the potential emergence of resistance to these antibiotics is of particular concern. Therefore, we report here the characterization of the resistance mechanisms of a P. aeruginosa clinical In vivo emergence of resistance to novel cephalosporin-β-lactamase inhibitor combinations through the duplication of amino acid D149 from OXA-2 β-lactamase (OXA-539) in sequence type 235 Pseudomonas aeruginosa. Antimicrob Agents Chemother

It is well established that antibiotic treatment selects for resistance, but the dynamics of this process during infections are poorly understood. Here we map the responses of Pseudomonas aeruginosa to treatment in high definition during a lung infection of a single ICU patient. Host immunity and antibiotic therapy with meropenem suppressed P. aeruginosa, but a second wave of infection emerged due to the growth of oprD and wbpM meropenem resistant mutants that evolved in situ. Selection then led to a loss of resistance by decreasing the prevalence of low fitness oprD mutants, increasing the frequency of high fitness mutants lacking the MexAB-OprM efflux pump, and decreasing the copy number of a multidrug resistance plasmid. Ultimately, host immunity suppressed wbpM mutants with high meropenem resistance and fitness. Our study highlights how natural selection and host immunity interact to drive both the rapid rise, and fall, of resistance during infection.

A ceftolozane/tazobactam and ceftazime/avibactam resistant Pseudomonas aeruginosa isolate was recovered after treatment (including azithromycin, meropenem and ceftolozane/tazobactam) from a patient that had developed ventilator associated pneumonia after Covid-19 infection. Whole genome sequencing revealed that the strain, belonging to ST274, had acquired a nonsense mutation leading to a truncated carbapenem porin OprD (W277X), a 7-bp deletion (nt213Δ7) in NfxB (negative regulator of the efflux pump MexCD-OprJ), and two missense mutations (Q178R, S133G) located within the first Large Periplasmic Loop of MexD. Through the construction of mexD mutants and complementation assays with wild-type nfxB , it was evidenced that resistance to the novel cephalosporin-β-lactamase inhibitor combinations was caused by the modification of MexD substrate specificity.

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