P-1263. Clinical carbapenem-resistant Klebsiella pneumoniae (CRKP) strains readily develop β-lactam/β-lactamase resistance and are hypervirulent

TL;DR

Carbapenem-resistant Klebsiella pneumoniae strains with DNA repair defects become more resistant to antibiotics and more harmful in a lung transplant patient.

Contribution

Identification of hypermutator CRKP strains with MMR defects that show increased antibiotic resistance and virulence.

Findings

CRKP strains with mutHV76G mutations showed increased SNP accumulation and antibiotic resistance.

mutHV76G strains had higher virulence in mice and greater plasmid transferability.

M/V treatment reduced tissue burdens in mice infected with CRKP strains.

Abstract

Hypermutator strains with DNA mismatch repair (MMR) defects are well-recognized among E. coli but rarely described for other Enterobacterales.Figure 1.Heat map of 11 serial strains of CRKP recovered from a lung transplant recipient.Figure 2.Genotypic and phenotypic characteristics of the 11 CRKP strains

Heat map of 11 serial strains of CRKP recovered from a lung transplant recipient.

Genotypic and phenotypic characteristics of the 11 CRKP strains

Eleven CRKP (ST258) strains causing disease (D) or colonization (C) in a lung transplant recipient over ∼4 yrs underwent whole genome sequencing. Clinical and lab-created isogenic mutant strains were tested for hypermutator and other phenotypes.Figure 3.Characteristics of mutH mutantsFigure 4.Tissue burdens of WT and mutH V76G mutant strains.

Characteristics of mutH mutants

Tissue burdens of WT and mutH V76G mutant strains.

6 early (C1-C4, D1-D2) strains were from 0-2.5 mos post-transplant (Fig 1). 5 late strains were D3 (recurrent bacteremia, 40 mos) and C5, D4, C6 and D5 (41-43 mos). Early strains and D3 were meropenem (MEM)-resistant (R), ceftazidime-avibactam (C/A)-susceptible (S) KPC-3-carriers that differed by 2-7 and 12-15 single nucleotide polymorphisms (SNPs), respectively (Fig 1). C5, D4, C6 and D5 differed from early strains by 119-129, 151-161, 167-177 and 251-261 SNPs, respectively, and carried a mutHV76G (MMR gene) mutation. C5, D4 and C6 were MEM-S, C/A-R KPC-46-carriers (KPC-3-D179Y); D5 was a MEM-R, C/A-R KPC-61-carrier (S171P) (Fig 2). mutHV76G strains had significantly reduced mutH expression (RT-PCR), higher rifampin mutational frequencies (p-values< 0.0001), and greater MEM-vaborbactam (M/V) R during passage in vitro (Fig 3). Isogenic mutHΔ and mutHV76G mutants in D3 had significantly reduced mutH expression, higher rifampin mutational frequencies (p< 0.0001), greater C/A and M/V R during passage in vitro, and enhanced transfer and acceptance of IncX3 plasmid carrying NDM-5 to and from E. coli-074, respectively. In IV mouse infections (5x104 CFU/mouse), isogenic mutHV76G caused higher liver, kidney and spleen burdens than D3 (Fig 4). In mice treated with humanized M/V (0.3 mg/g intraperitoneal Q8hrs, starting 4hrs post-infection), mutHV76G and D3 tissue burdens were each reduced by ≥70%. However, mean mutational frequency (CFU/mL M/V plates/CFU/mL M/V-free plates) of mutHV76G was significantly greater than D3 (3x10-2 vs. < 1x10-8).

Hypermutator CRKP emerged due to mutHV76G, which conferred increased propensity to SNPs, transference and acceptance of antibiotic-R-conferring plasmids, heightened virulence and β-lactam/β-lactamase R in vitro and in vivo. MMR-defective hypermutators merit further investigation among antibiotic-R Enterobacterales.

Cornelius J. Clancy, MD, Merck: Grant/Research Support|Shionogi: Advisor/Consultant M Hong Nguyen, MD, Basilea: Advisor/Consultant|BioMerieux: Grant/Research Support|Melinta: Grant/Research Support|Pulmocide: Advisor/Consultant|Pulmocide: Grant/Research Support

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