Genomic Analysis Identifies Novel Pseudomonas aeruginosa Resistance Genes under Selection during Inhaled Aztreonam Therapy In Vivo

Abstract

Inhaled aztreonam is increasingly used for chronic Pseudomonas aeruginosa suppression in patients with cystic fibrosis (CF), but the potential for that organism to evolve aztreonam resistance remains incompletely explored. Here, we performed genomic analysis of clonally related pre- and posttreatment CF clinical isolate pairs to identify genes that are under positive selection during aztreonam therapy in vivo. We identified 16 frequently mutated genes associated with aztreonam resistance, the most prevalent being ftsI and ampC, and 13 of which increased aztreonam resistance when introduced as single gene transposon mutants. Several previously implicated aztreonam resistance genes were found to be under positive selection in clinical isolates even in the absence of inhaled aztreonam exposure, indicating that other selective pressures in the cystic fibrosis airway can promote aztreonam resistance. Given its potential to confer plasmid-mediated resistance, we further characterized mutant ampC alleles and performed artificial evolution of ampC for maximal activity against aztreonam. We found that naturally occurring ampC mutants conferred variably increased resistance to aztreonam (2- to 64-fold) and other β-lactam agents but that its maximal evolutionary capacity for hydrolyzing aztreonam was considerably higher (512- to 1,024-fold increases) and was achieved while maintaining or increasing resistance to other drugs. These studies implicate novel chromosomal aztreonam resistance determinants while highlighting that different mutations are favored during selection in vivo and in vitro, show that ampC has a high maximal potential to hydrolyze aztreonam, and provide an approach to disambiguate mutations promoting specific resistance phenotypes from those more generally increasing bacterial fitness in vivo.

Pseudomonas Aeruginosa Antibiotic Efflux Pump Variants Exhibit Increased Virulence

“…First, strain PAO1-AzEvB8 that was the focus of the present study was not an actual clinical isolate and it is unclear how frequently mutants arise that have both mexR and mexEF mutations. As mentioned above, literature suggests that mexR mutations are very common in CF, and that clinical isolates can display varying degrees of mexE gene expression [20,22,24,33,36,37], so we feel that this study still has relevance to real-world P. aeruginosa infections.…”

“…P. aeruginosa with mutations in nalD and mexR overexpress the MexAB-OprM efflux pump, rendering them multidrug resistant [17,19,21]. Moreover, mutations in these two genes have been found in anywhere from 7-47% of clinical isolates in various studies [20][21][22][23][24]. Importantly, strains with mutations in nalD and mexR evolved under aztreonam selection showed an increased virulence in vivo, challenging the longstanding theory that antibiotic resistance mutations inherently pay fitness costs that attenuate virulence (Jorth et al, 2017).…”

Quest for Novel Preventive and Therapeutic Options Against Multidrug-Resistant Pseudomonas aeruginosa

“…Being one of the top three globally most prevalent nosocomial infectious agents and equipped with virulence and resistance, P. aeruginosa’s infections require additional resources and time for patients to recover (Pires, et al 2018 ; Moradali et al 2017 ; Juan et al 2017 ). Countless studies have reported the extent of antibiotic resistance due to multiple factors ranging from over-prescription, self-medication, and abuse of antibiotics on farms, all ultimately affecting the patients’ survival and recovery (Xipell 2017 ; McLean 2019 ; Pang 2019 ; Frieri et al 2017 ; Qiao 2018 ; Karkman 2018 ). While the lack of vaccines for superbugs was not even an area of interest for academia as well as an industry until recently (Rappuoli et al 2017 ).…”

Quest for Novel Preventive and Therapeutic Options Against Multidrug-Resistant Pseudomonas aeruginosa

“…Being one of the top three globally most prevalent nosocomial infectious agents and equipped with virulence and resistance, P. aeruginosa's infections require additional resources and time for patients to recover [62][63][64] . Countless studies have reported the extent of antibiotic resistance due to multiple factors ranging from over-prescription, self-medication, and abuse of antibiotics on farms, all ultimately affecting the patients' survival and recovery [65][66][67][68][69][70] . While the lack of vaccines for superbugs was not even an area of interest for academia as well as an industry until recently 71 .…”