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PSEUDOMONAS AERUGINOSA MUTATOR ISOLATES FROM CF PATIENTS SHOW AMPC ß-LACTAMASE VARIANTS WITH HIGH HYDROLYTIC EFFICIENCIES THAT CONFER COMPETITIVE ADVANTAGE TO CEPHALOSPORINS AND MONOBACTAMS

CIQUIBIC-CONICET, UNIVERSIDAD NACIONAL DE CÓRDOBA, FACULTAD DE CIENCIAS QUÍMICAS 1; INSTITUTO DE BIOLOGÍA MOLECULAR Y CELULAR DE ROSARIO, IBR, CONICET-UNR 2; CIQUIBIC-CONICET, UNIVERSIDAD NACIONAL DE CÓRDOBA, FACULTAD DE CIENCIAS QUÍMICAS / IRNASUS-CONICET 3; DEPARTMENT OF CLINICAL MICROBIOLOGY, RIGSHOSPITALET 4; DEPARTMENT OF CLINICAL MICROBIOLOGY, RIGSHOSPITALET 5; NOVO NORDISK FOUNDATION CENTRE FOR BIOSUSTAINABILITY, TECHNICAL UNIVERSITY OF DENMARK 6; INSTITUTO DE BIOLOGÍA MOLECULAR Y CELULAR DE ROSARIO, IBR, CONICET-UNR 7

Introduction and objectives: Pseudomonas aeruginosa has evolved a myriad of intrinsic and acquired resistance mechanisms to counter nearly all antibiotics used for its treatment. Common mechanisms of resistance include the selection of mutations in chromosomal genes leading to the inactivation of the carbapenem porin OprD, the upregulation of efflux pumps and the hyperproduction of AmpC mediated by mutation-dependent mechanisms. We previously showed that in patients treated with high and long-dose ßlactam therapy, P. aeruginosa is able to easily adapt and that accumulation of mutations within ampC gene is strongly selected during long-term evolution in chronic CF infection. Moreover, we showed that hypermutability favors the emergence of ampC variants consisting of several mutations differentially combined to lead for diversified alleles. When expressed in an AmpC-deficient PAO1 strain and compared to PDC-3, some of these ampC variants were associated with high resistance towards cephalosporins and monobactams including the new combination ceftolozane-tazobactam. Here, we further assessed whether the combinations of mutations affect adaptation and persistence of P. aeruginosa to a given antibiotic and ß-lactamase hydrolytic activity. Materials and methods: We performed competition experiments between lacZ+/lacZ- PAO1 strains expressing each of the ampC variants by growing each co-culture in the presence and absence of ceftazidime and aztreonam. Afterward, mature most competitive AmpC variants were expressed and purified, and their ßlactam hydrolysis capability against ceftazidime, piperacillin, and imipenem was determined by enzyme-kinetic measurements. Results: Our results show that adaptation to ceftazidime antibiotic is based on a three-mutations A89V, V213A and Q120K core, being the latter residues localized either into or spatially close to the omega-loop, respectively, suggesting possible interactions. Addition of N321S to this mutations core did not affect competitiveness in ceftazidime, unlike the addition of the H189Y mutation, which concurrently with a decrease in ceftazidime, improved competitiveness in aztreonam. We found out that AmpC variants were 10- to 30-fold more active against ceftazidime than PDC-3 whereas imipenem hydrolysis was not affected. On the other hand, AmpC variants were 10- to 100- fold less active against piperacillin, suggesting that the evolved resistance against ceftazidime simultaneously led to an increased susceptibility to piperacillin probably by collateral sensitivity processes. Conclusions: Our results demonstrate how the interplay of AmpC spontaneous mutations plays a pivotal role in the development of genetic ß-lactam antibiotic resistance and the pathogenic fitness of P. aeruginosa.