Combination of Amino Acid Substitutions Leading to CTX-M-15-Mediated Resistance to the Ceftazidime-Avibactam Combination

Abstract

Single amino acid substitutions in the Ω loop of KPC β-lactamases are known to lead to resistance to the ceftazidime-avibactam combination. Here, we investigate this mechanism of resistance in CTX-M enzymes, which are the most widely spread extended-spectrum β-lactamases worldwide. Nine single amino acid polymorphisms were identified in the Ω loop of the 172 CTX-M sequences present in the Lahey database of β-lactamases. The corresponding modifications were introduced in CTX-M-15 by site-directed mutagenesis. None of the nine substitutions was associated with ceftazidime-avibactam resistance in TOP10. However, two substitutions led to 4-fold (PS) and 16-fold (LQ) increases in the MIC of ceftazidime. We determined whether these substitutions favor the selection of mutants resistant to ceftazidime-avibactam. The selection provided mutants for the LQ substitution but not for the PS substitution or for the parental enzyme CTX-M-15. Resistance to the drug combination (MIC of ceftazidime, 16 μg/ml in the presence of 4 μg/ml of avibactam) resulted from the acquisition of the SG substitution by CTX-M-15 LQ. Purified CTX-M-15 with the two substitutions, LQ and SG, was only partially inhibited by avibactam at concentrations as high as 50,000 μM but retained ceftazidime hydrolysis activity with partially compensatory decreases in and These results indicate that emergence of resistance to the ceftazidime-avibactam combination requires more than one mutation in most CTX-M-encoding genes. Acquisition of resistance could be restricted to rare variants harboring predisposing polymorphisms such as Q at position 169 detected in a single naturally occurring CTX-M enzyme (CTX-M-93).

Ceftazidime-Avibactam Resistance Mediated by the N 346 Y Substitution in Various AmpC β-Lactamases

“…The four highly divergent class C ␤-lactamases evaluated in this study included cAmpC from E. cloacae P99 (designated AmpC cloacae ), cAmpC PDC-5 from P. aeruginosa ATCC 27853, and the pAmpCs DHA-1 and FOX-3, which derive from chromosomally encoded enzymes from Morganella morganii and Aeromonas spp. For phenotypic analyses, the genes were amplified using primers depicted in Table S1 and cloned under the control of the isopropyl ␤-D-1thiogalactopyranoside (IPTG)-inducible promoter of the plasmid vector pTRC-99k (16). Recombinant plasmids were introduced by electroporation into E. coli TOP10 with selection for resistance to kanamycin (50 g/ml) conveyed by the vector pTRC-99k.…”

“…Recombinant plasmids were introduced by electroporation into E. coli TOP10 with selection for resistance to kanamycin (50 g/ml) conveyed by the vector pTRC-99k. For protein purification, gene fragments encoding soluble forms of the enzymes were amplified (see Table S1 for the sequence of the primers) and cloned under the control of the IPTG-inducible promoter of the plasmid vector pET-TEV (16). Site-directed mutagenesis was performed using the mutagenic primers depicted in Table S1, except for AmpC cloacae that was obtained by selection of a spontaneous mutant of pTRC-99k⍀ampC cloacae on agar containing ceftazidime (4 g/ml) and avibactam (4 g/ml).…”

“…Purification of ␤-lactamases. Production and purification of AmpC ␤-lactamases was performed as previously described (16). Briefly, fragments of ␤-lactamase genes, including soluble fragments of AmpC cloacae (residues 21 to 381), PDC-5 (residues 27 to 397), DHA-1 (residues 25 to 37), and FOX-3 (residues 24 to 382) were translationally fused to the sequence of the pET-TEV vector coding for an N-terminal 6ϫHis tag followed by a cleavage site for the TEV protease (GSSHHHHHHSSGENLYFQG).…”

“…The relevance of Asn 346 for acquisition of resistance to the combination arises from its critical role for efficacious cephalosporinase inactivation by avibactam but not for hydrolysis of ceftazidime and, to a certain extent, of other ␤-lactams. This appears to be a remarkable property of Asn 346 in AmpC cephalosporinases, as analyses of class A ␤-lactamases have revealed that amino acid substitutions reducing carbamoylation efficacy often lead to large decreases in hydrolysis efficacy and, in certain instances, to hyper-susceptibility to certain ␤-lactams (15,16).…”

New Carbapenemase Inhibitors: Clearing the Way for the β-Lactams

“…Modification of either KPC-2 and KPC-3 variants is most frequently associated with this particular behaviour [54]. Other mechanisms involved in the emergence of resistance to ceftazidime/avibactam in Enterobacterales include the structural modification of CTX-M-like or AmpC enzymes [93], increased KPC expression in combination with inactivation of porins and enhanced AcrAB-TolC efflux [94]. Similar to observations in Enterobacterales, development of resistance to ceftazidime/avibactam during treatment of P. aeruginosa infections has mainly been associated with selection of variants of PDC-enzymes [95].…”