PSEUDOMONAS AERUGINOSA MUTATOR ISOLATES FROM CYSTIC FIBROSIS SHOW AMPC β-LACTAMASE VARIANTS WITH HIGH HYDROLYTIC EFFICIENCIES THAT CONFER COMPETITIVE ADVANTAGE TO CEPHALOSPORINS AND MONOBACTAMS

COLQUE CA; TOMATIS PE; ALBARRACÍN ORIO A; HEDEMANN LG; HICKMAN RA; KROGH JOHANSEN H; MOLIN S; VILA AJ; SMANIA AM

Buenos Aires

Congreso; XIV Congreso Argentino de Microbiología General (SAMIGE); 2019

{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 β-lactam 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 β-lactamase hydrolytic activity. 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. 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 Ω-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. Afterward, mature most competitive AmpC variants were expressed and purified, and their β-lactam hydrolysis capability against ceftazidime, piperacillin, and imipenem was determined by enzyme-kinetic measurements. 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. Our results demonstrate how the interplay of AmpC spontaneous mutations plays a pivotal role in the development of genetic β-lactam antibiotic resistance and the pathogenic fitness of {P. aeruginosa}.