CHRONIC USE OF B-LACTAMS CAN SELECT AMPC MEDIATED PRERESISTANCE TO LAST GENERATION CEFTOLOZANE IN PSEUDOMONAS AERUGINOSA

COLQUE CA; TOMATIS PE; ALBARRACÍN ORIO A; DOTTA G; MORENO D; HEDEMANN LG; MOYANO AJ; BONOMO RA; KROGH JOHANSEN H; MOLIN S; VILA AJ; SMANIA AM

Salta

Congreso; LV Annual SAIB, LIV PABMB 2019; 2019

Cephalosporin resistance mediated by β-lactamase production remains one of the ultimate challenging problems in clinical settings. Among the most understudied, are the class C AmpC cephalosporinase, which confers high-level resistance. The recently introduced fifth-generation cephalosporin ceftolozane (CTZ), in combination with the β-lactamase inhibitor tazobactam (TAZ), has shown to be a promising therapeutic tool against P. aeruginosa resistant to far the most used ceftazidime, carbapenemes, and piperacillin-tazobactam. Regrettably, rapid emergence of resistants to CTZ/TAZ have been observed in patients treated with this important new therapy. We previously showed that after more than two decades of evolutionary history under long-dose of β-lactam treatment during cystic fibrosis (CF) chronic infection, P. aeruginosa hypermutator lineages were able to adapt by the accumulation of mutations within the ampC gene. Interestingly, hypermutability favored the emergence of 7 undescribed alleles consisting of differentially combined mutations (referred to as AmpC-1 to AmpC-7) shaping a highly diversified population. When expressed in an AmpC-deficient PAO1 strain and compared to wild-type β-lactamase (PDC-3), some alleles conferred 3- to 5-fold MICs increase to ceftazidime and aztreonam contributing to the high β-lactam resistance of the clinical isolates. Evenly, by enzyme-kinetic measurements, mature purified AmpC proteins displayed β-lactam hydrolysis capability 10- to 30-fold more active against ceftazidime than PDC-3. Here, we further assessed whether combinations of mutations were involved in CTZ resistance. Although the CF patient was never treated with this antibiotic, ampC alleles harboring 3 to 5 distinct mutations were intermediate or resistant to CTZ. Of notice, AmpC-2 (Q120K-P154L-V213A) and 4 (A89V-Q120K-V213A) were the less susceptible variants. Combination with TAZ, partially decrease MICs of AmpC-2 and 6 (A89V-Q120K-H189Y-V213A) whereas AmpC-4 and 5 (A89V-Q120K-V213A-N321S) were not affected by inhibitor addition. Mature AmpC-4, 5, 6 and PDC-3 were expressed and purified and their hydrolysis capability against CTZ was determined through enzyme-kinetic measurements. Importantly, kcat/Km values were between 8-150 times higher than that of PDC-3, showing robust CTZ hydrolysis. Molecular Docking simulations revealed key structural insights, indicating that mutations Q120K, V213A and N321S wide the substrate-binding pocket entrance, enabling accommodation of the ceftazidime, aztreonam and CTZ bulkier R1 side chain. Q120K and V213A also contribute to a conformational change in Y221 residue favoring stacking interaction between aromatic residues of the enzyme and β-lactam. This flip possibly facilitates substrate binding in AmpC-4 and explains its higher hydrolytic capability. A deeper understanding of the mechanisms involved in AmpC protein evolution is imperative to advance in the design of novel compounds to overcome this resistance.