Molecular and biochemical insights into the in vivo evolution of AmpC-mediated resistance to ceftolozane/tazobactam during treatment of an MDR Pseudomonas aeruginosa infection

Abstract

Background Pseudomonas aeruginosa may develop resistance to novel cephalosporin/β-lactamase inhibitor combinations during therapy through the acquisition of structural mutations in AmpC. Objectives To describe the molecular and biochemical mechanisms involved in the development of resistance to ceftolozane/tazobactam in vivo through the selection and overproduction of a novel AmpC variant, designated PDC-315. Methods Paired susceptible/resistant isolates obtained before and during ceftolozane/tazobactam treatment were evaluated. MICs were determined by broth microdilution. Mutational changes were investigated through WGS. Characterization of the novel PDC-315 variant was performed through genotypic and biochemical studies. The effects at the molecular level of the Asp245Asn change were analysed by molecular dynamics simulations using Amber. Results WGS identified mutations leading to modification (Asp245Asn) and overproduction of AmpC. Susceptibility testing revealed that PAOΔC producing PDC-315 displayed increased MICs of ceftolozane/tazobactam, decreased MICs of piperacillin/tazobactam and imipenem and similar susceptibility to ceftazidime/avibactam compared with WT PDCs. The catalytic efficiency of PDC-315 for ceftolozane was 10-fold higher in relation to the WT PDCs, but 3.5- and 5-fold lower for piperacillin and imipenem. IC50 values indicated strong inhibition of PDC-315 by avibactam, but resistance to cloxacillin inhibition. Analysis at the atomic level explained that the particular behaviour of PDC-315 is linked to conformational changes in the H10 helix that favour the approximation of key catalytic residues to the active site. Conclusions We deciphered the precise mechanisms that led to the in vivo emergence of resistance to ceftolozane/tazobactam in P. aeruginosa through the selection of the novel PDC-315 enzyme. The characterization of this new variant expands our knowledge about AmpC-mediated resistance to cephalosporin/β-lactamase inhibitors in P. aeruginosa.

The Revival of Aztreonam in Combination with Avibactam against Metallo-β-Lactamase-Producing Gram-Negatives: A Systematic Review of In Vitro Studies and Clinical Cases

“…In particular, it is well-known that AVI may show a potent inhibitory activity against the basal AmpC produced by P. aeruginosa , even though extended-spectrum OXA enzymes can escape its wide spectrum of activity [ 71 ]. Selection of extended-spectrum OXA-2 or OXA-10 variants such as OXA-539, OXA-681 and OXA-14 has previously been associated with in vivo acquisition of high-level CZA resistance [ 72 , 73 , 74 ], while OXA-10 and OXA-18 enzymes have been associated with ATM’s high MIC values [ 75 ]. Moreover, previously reported development of resistance to CZA during treatment of P. aeruginosa infections has mainly been associated with selection of variants of PDC-enzymes [ 38 , 73 ].…”

“…Selection of extended-spectrum OXA-2 or OXA-10 variants such as OXA-539, OXA-681 and OXA-14 has previously been associated with in vivo acquisition of high-level CZA resistance [ 72 , 73 , 74 ], while OXA-10 and OXA-18 enzymes have been associated with ATM’s high MIC values [ 75 ]. Moreover, previously reported development of resistance to CZA during treatment of P. aeruginosa infections has mainly been associated with selection of variants of PDC-enzymes [ 38 , 73 ]. Notably, co-production of PDC and OXA determinants conferring resistance to ATM in P. aeruginosa isolates has also previously been reported [ 76 ].…”

Activity of ceftolozane/tazobactam against Gram-negative isolates among different infections in Hong Kong: SMART 2017–2019

“…aeruginosa has been associated with ampC (PDC, Pseudomonas derived cephalosporinase) derepression, OprD loss (imipenem >meropenem), and/or hyperexpression of efflux pumps (meropenem, not imipenem) [13, 28]. OprD porin loss and the majority of known efflux pumps do not alter ceftolozane/tazobactam MICs [13, 18, 28, 29]. Rarely, P.…”

Development of antibiotic resistance reveals diverse evolutionary pathways to face the complex and dynamic environment of a long-term treated patient

“…Resistance to ceftolozane has previously been observed in P. aeruginosa infected patients when treated with this antibiotic (Munita et al, 2017; Fraile-Ribot et al, 2018), and other studies have demonstrated that expression of a PDC-3 variant carrying a single E221K mutation can confer high MICs of ceftolozane in E. coli (Barnes et al, 2018). It has also been shown that a substitution of Asp219 at the Ω-loop 219, selected after treating a multi-drug resistant P. aeruginosa strain with ceftolozane/tazobactam, enhances hydrolysis of this cephalosporin/β-lactamase inhibitor combination in vivo (Arca-Suárez et al, 2020). However, in vitro long-term experiments of wild-type and mutator strains of P. aeruginosa exposed to increasing concentrations of ceftolozane/tazobactam, showed that only mutator strains were able to develop high-levels of resistance, by acquiring multiple mutations that led to overexpression and structural modifications of PDC (Cabot et al, 2014).…”

“…This impressive number of allelic variants accounts for a highly polymorphic enzyme with a great capacity of tolerating amino acid substitutions, insertions and deletions (Oliver, 2020). Recent studies have shown that clinical resistance to β-lactams is primarily based on specific changes in conserved motifs of PDC, which lead to conformational rearrangements enhancing the catalytic efficiency of the enzyme (Raimondi et al, 2001; Jacoby, 2009; Lahiri et al, 2015; Barnes et al, 2018; Arca-Suárez et al, 2020).…”