Struggle To Survive: the Choir of Target Alteration, Hydrolyzing Enzyme, and Plasmid Expression as a Novel Aztreonam-Avibactam Resistance Mechanism

Abstract

Aztreonam-avibactam is a promising antimicrobial combination against multidrug-resistant organisms, such as carbapenemase-producing Enterobacterales. Resistance to aztreonam-avibactam has been found, but the resistance mechanism remains poorly studied. We recovered three Escherichia coli isolates of an almost identical genome but exhibiting varied aztreonam-avibactam resistance. The isolates carried a cephalosporinase gene, blaCMY-42, on IncIγ plasmids with a single-nucleotide variation in an antisense RNA-encoding gene, inc, of the replicon. The isolates also had four extra amino acids (YRIK) in penicillin-binding protein 3 (PBP3) due to a duplication of a 12-nucleotide (TATCGAATTAAC) stretch in pbp3. By cloning and plasmid-curing experiments, we found that elevated CMY-42 cephalosporinase production or amino acid insertions in PBP3 alone mediated slightly reduced susceptibility to aztreonam-avibactam, but their combination conferred aztreonam-avibactam resistance. We show that the elevated CMY-42 production results from increased plasmid copy numbers due to mutations in inc. We also verified the findings using in vitro mutation assays, in which aztreonam-avibactam-resistant mutants also had mutations in inc and elevated CMY-42 production compared with the parental strain. This choir of target modification, hydrolyzing enzyme, and plasmid expression represents a novel, coordinated, complex antimicrobial resistance mechanism and also reflects the struggle of bacteria to survive under selection pressure imposed by antimicrobial agents. IMPORTANCE Carbapenemase-producing Enterobacterales (CPE) is a serious global challenge with limited therapeutic options. Aztreonam-avibactam is a promising antimicrobial combination with activity against CPE producing serine-based carbapenemases and metallo-β-lactamases and has the potential to be a major option for combatting CPE. Aztreonam-avibactam resistance has been found, but resistance mechanisms remain largely unknown. Understanding resistance mechanisms is essential for optimizing treatment and developing alternative therapies. Here, we found that either penicillin-binding protein 3 modification or the elevated expression of cephalosporinase CMY-42 due to increased plasmid copy numbers does not confer resistance to aztreonam-avibactam, but their combination does. We demonstrate that increased plasmid copy numbers result from mutations in antisense RNA-encoding inc of the IncIγ replicon. The findings reveal that antimicrobial resistance may be due to concerted combinatorial effects of target alteration, hydrolyzing enzyme, and plasmid expression and also highlight that resistance to any antimicrobial combination will inevitably emerge.

Therapeutic Options for Metallo-β-Lactamase-Producing Enterobacterales

“…68,69 MBL-producing Enterobacterales that coharbor an AmpC, such as bla CMY , may be particularly prone to developing ATM-AVI resistance as mutations in the gene encoding for this enzyme have also been shown to cause ATM-AVI resistance. 70,71 The insertion is not associated with MBL β-lactamases and appears limited to E. coli isolates.…”

New Delhi Metallo-β-Lactamase–Producing Enterobacterales Bacteria, Switzerland, 2019–2020

“…Similar 4-aa insertions have been reported among NDM-5–producing E. coli as a cause of elevated MICs of ATM, related to impeding accessibility to the binding site of PBP-3, and therefore were involved in ATM/AVI resistance in addition to CMY production ( 28 ). Ma et al reported that the insertion alone has a minor effect on ATM/AVI resistance levels ( 29 ), but resistance could be achieved when combined with CMY production ( 29 ). Other studies reported that CMY variants with a glycine residue at position 231 exhibited enhanced hydrolysis against ATM ( 30 , 31 ).…”

“…The high level of association of carbapenemases to the NDM-5 and RMTase genes will limit the choice of therapeutics available for treating infections because of those multidrug-resistant bacteria. Several isolates were identified that were resistant to the β-lactam/inhibitor combination ATM/AVI, a potential future treatment for infections caused by NDM-producing bacteria ( 28 , 29 ). The transmission of successful plasmids, both within and between species, was identified as a major factor in the increasing prevalence of NDM-producing Enterobacterales .…”

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

“…For the other remaining isolates, co-production and/or hyperexpression of ESBL and AmpC-type determinants could have contributed to the high MIC values of ATM in combination with AVI. Notably, the association of CMY-42 and the YRIK insertion in PBP3 has been demonstrated to confer resistance to ATM-AVI in E. coli by mutagenesis experiments [ 77 ]. ATM-AVI showed low antimicrobial activity against 15% of S. maltophilia isolates, even if potential resistance mechanisms were not investigated.…”