Klebsiella pneumoniae Carbapenemase-2 (KPC-2), Substitutions at Ambler Position Asp179, and Resistance to Ceftazidime-Avibactam: Unique Antibiotic-Resistant Phenotypes Emerge from β-Lactamase Protein Engineering

Abstract

The emergence of Klebsiella pneumoniae carbapenemases (KPCs), β-lactamases that inactivate “last-line” antibiotics such as imipenem, represents a major challenge to contemporary antibiotic therapies. The combination of ceftazidime (CAZ) and avibactam (AVI), a potent β-lactamase inhibitor, represents an attempt to overcome this formidable threat and to restore the efficacy of the antibiotic against Gram-negative bacteria bearing KPCs. CAZ-AVI-resistant clinical strains expressing KPC variants with substitutions in the Ω-loop are emerging. We engineered 19 KPC-2 variants bearing targeted mutations at amino acid residue Ambler position 179 in Escherichia coli and identified a unique antibiotic resistance phenotype. We focus particularly on the CAZ-AVI resistance of the clinically relevant Asp179Asn variant. Although this variant demonstrated less hydrolytic activity, we demonstrated that there was a prolonged period during which an acyl-enzyme intermediate was present. Using mass spectrometry and transient kinetic analysis, we demonstrated that Asp179Asn “traps” β-lactams, preferentially binding β-lactams longer than AVI owing to a decreased rate of deacylation. Molecular dynamics simulations predict that (i) the Asp179Asn variant confers more flexibility to the Ω-loop and expands the active site significantly; (ii) the catalytic nucleophile, S70, is shifted more than 1.5 Å and rotated more than 90°, altering the hydrogen bond networks; and (iii) E166 is displaced by 2 Å when complexed with ceftazidime. These analyses explain the increased hydrolytic profile of KPC-2 and suggest that the Asp179Asn substitution results in an alternative complex mechanism leading to CAZ-AVI resistance. The future design of novel β-lactams and β-lactamase inhibitors must consider the mechanistic basis of resistance of this and other threatening carbapenemases.

Pharmacodynamics of Ceftazidime plus Avibactam against KPC-2-Bearing Isolates of Klebsiella pneumoniae in a Hollow Fiber Infection Model

“…Resistance emergence during CAZ/AVI has been described during therapy (9)(10)(11) in Klebsiella pneumoniae due to alterations to the ␤-lactamase (KPC-2 and KPC-3), leading to high-level resistance (11). Mutations in ompK36 have also been described to shift CAZ/AVI MIC's in Klebsiella.…”

Meropenem-Vaborbactam as Salvage Therapy for Ceftazidime-Avibactam-Resistant Klebsiella pneumoniae Bacteremia and Abscess in a Liver Transplant Recipient

“…NG056170), featured a tyrosine for aspartic acid substitution at Ambler amino acid position 179 (D179Y) within the KPC ⍀-loop compared to the wild type. This alteration is known to codify resistance to ceftazidime-avibactam (15,16).…”

Nacubactam Enhances Meropenem Activity against Carbapenem-Resistant Klebsiella pneumoniae Producing KPC

“…Escherichia coli containing bla KPC-2 in the pBR322-catI vector was a gift from Fred Tenover, previously of the Centers for Disease Control and Prevention (43). The pBC SK and pBR322-catI vectors yield reasonable expression levels of KPC (32).…”

“…Within the ⍀ loop, E166 and N170 position the deacylation water molecule for hydrolysis. Moreover, single amino acid substitutions in the ⍀-loop residues notoriously contribute to ceftazidime-avibactam resistance (32,33). The amide backbone of T237 and S70 forms the oxyanion hole for ␤-lactam and ␤-lactamase inhibitor binding.…”

“…3C). The sulfate group of the DBO is predicted to anchor the open-ring form of the DBO, thus blocking deacylation; however, the desulfation of avibactam by KPC-2 was previously observed (32,38). One proposed mechanism of desulfation occurs upon addition of a proton transferred through a water from a base, resulting in a hydroxylamine avibactam intermediate (38).…”