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OXA-48 has rapidly disseminated worldwide and become one of the most common carbapenemases in many countries with more than 45 variants reported with, in some cases, significant differences in their hydrolysis profiles. The R214 residue, located in the ß5-ß6 loop, is crucial for the carbapenemase activity, as it stabilizes carbapenems in the active site and maintains the shape of the active site through interactions with D159. In this study, we have characterized a novel variant of OXA-48, OXA-933 with a single D159N change. To evaluate the importance of this residue, point mutations were generated (D159A, D159G, D159K, and D159W), kinetic parameters of OXA-933, OXA-48 D159G, and OXA-48 D159K were determined and compared to those of OXA-48 and OXA-244. The bla OXA-933 gene was borne on Tn 2208 , a 2,696-bp composite transposon made of two IS 1 elements surrounded by 9 bp target site duplications and inserted into a non-self-transmissible plasmid pOXA-933 of 7,872 bp in size. Minimal inhibitory concentration values of E. coli expressing the bla OXA-933 gene or of its point mutant derivatives were lower for carbapenems (except for D159G) as compared to those expressing the bla OXA-48 gene. Steady-state kinetic parameters revealed lower catalytic efficiencies for expanded spectrum cephalosporins and carbapenems. A detailed structural analysis confirmed the crucial role of D159 in shaping the active site of OXA-48 enzymes by interacting with R214. Our work further illustrates the remarkable propensity of OXA-48-like carbapenemases to evolve through mutations at positions outside the β5-β6 loop, but interacting with key residues of it., Competing Interests: The authors declare no conflict of interest.

Ceftolozane-tazobactam is considered to be a last resort treatment for infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa Although, resistance to this antimicrobial have been described in vitro , development of resistance in vivo was rarely reported. Here, we described the evolution of resistance to ceftolozane-tazobactam of P. aeruginosa isolates recovered from the same patient during recurrent infections over 2.5 years.Antimicrobial susceptibility testing results showed that 24 of the 27 P. aeruginosa isolates recovered from blood (n=18), wound (n=2), pulmonary sample (n=1), bile (n=2) and stools (n=4) of the same patient were susceptible to ceftolozane-tazobactam and ceftazidime-avibactam but resistant to ceftazidime, piperacillin-tazobactam, imipenem and meropenem. Three clinical isolates acquired resistance to ceftolozane-tazobactam and ceftazidime-avibactam along with a partial restoration of piperacillin-tazobactam and carbapenems susceptibilities. Whole genome sequencing analysis reveals that all isolates were clonally related (ST-111) with a median of 24.9 single nucleotide polymorphisms (SNPs) (range 8-48). The ceftolozane-tazobactam and ceftazidime-avibactam resistance was likely linked to the same G183D substitution in the chromosome-encoded cephalosporinase.Our results suggest resistance to ceftolozane-tazobactam in P. aeruginosa might occur in vivo upon treatment through amino-acid substitution in the intrinsic AmpC leading to ceftolozane-tazobactam and ceftazidime-avibactam resistance accompanied by re-sensitization to piperacillin-tazobactam and carbapenems., (Copyright © 2019 American Society for Microbiology.)