In vivo evolution to hypermucoviscosity and ceftazidime/avibactam resistance in a liver abscess caused by Klebsiella pneumoniae sequence type 512

Abstract

Carbapenemase-producing Klebsiella pneumoniae represents a major public health issue globally. Isolates with resistance to the newest drugs, like ceftazidime/avibactam (CZA), are increasingly reported. In this study, we analyzed the evolution of KPC-3-producing sequence type (ST) 512 K. pneumoniae strains isolated at three different times (hospitalization days 45, 56, and 78) from the same patient, two of which were observed in a pericholecystic liver abscess. The three K. pneumoniae isolates (295Kp, 304Kp, and hmv-318Kp) from the same patient were subjected to antimicrobial susceptibility testing, whole-genome sequencing, sedimentation assay, biofilm measurement, serum resistance assay, macrophage phagocytosis, and adhesion assays. KPC-producing isolate hmv-318Kp exhibited carbapenem susceptibility, hypermucoviscous (hmv) colony phenotype and CZA resistance. Virulence markers of hypervirulent Klebsiella were absent. Two non-synonymous mutations were identified in the hmv-318Kp genome comparing with isogenic strains: a single-nucleotide polymorphism (SNP) occurred in the pKpQIL plasmid, changing bla_KPC-3 in the bla_KPC-31 gene variant, conferring CZA resistance; and a second SNP occurred in the wzc gene of the capsular biosynthesis cluster, encoding a tyrosine kinase, resulting in the F557S Wzc protein mutation. The Klebsiella pneumoniae strain exhibiting an hmv phenotype (hmv-Kp) phenotype has been previously associated with amino acid substitutions occurring in the Wzc tyrosin kinase protein. We observed in vivo evolution of the ST512 strain to CZA resistance and acquisition of hypermucoviscosity. The pathogenetic role of the detected Wzc substitution is not fully elucidated, but other Wzc mutations were previously reported in hmv K. pneumoniae. Wzc mutants may be more frequent than expected and an underreported cause of hypermucoviscosity in K. pneumoniae clinical isolates.

Importance: Here we describe the evolution of KPC-3-producing ST512 K. pneumoniae isolated at three different times from the same patient of which the last one, from a biliary abscess, showed CZA resistance by KPC-31 production and manifested hmv colony phenotype. Hypervirulent Klebsiella pneumoniae (hv-Kp) isolates are increasingly reported worldwide. Their hypervirulent traits are associated with the presence of rmpA/A2 genes and an hmv. In this study, we identified an hmv-Kp that lacked the rmpA-D cluster but showed an amino acid substitution in the Wzc tyrosin kinase protein, involved in the capsular biosynthesis. This hmv-Kp strain emerged in vivo and evolved resistance to ceftazidime/avibactam resistance in a liver abscess of a patient. Our findings suggest that wzc mutations may be underreported, making it challenging to distinguish hv-Kp from "classic" K. pneumoniae with an hmv phenotype.

Keywords: KPC-31; ST512; antibiotic resistance; capsule; hmv; hypermucoid; hypervirulence; mucoviscous; rmpA negative; serum susceptibility; string test; wzc.

Fig 1

Phenotypic characterization of Klebsiella pneumoniae sequence type 512 analyzed in this study. (A) Quantitative sedimentation assay. Isolates, grown overnight in brain heart infusion, were normalized to OD₆₀₀ = 1.0 centrifuged at 1,000 × g for 5 min, and the OD₆₀₀ of the upper 900 µL was measured. Data are shown as means + SDs (n = 6). (B) Serum resistance assay. Tolerance to human sera pool was evaluated by exposing bacteria (5 × 10⁵ CFU/mL) to 100% human serum and incubating at 37°C for 2 h. CFU/mL were enumerated at T0, after 1 and 2 h. Data are shown as means + SDs (n = 6). (C) Quantitative assessment of biofilm formation. Biofilm formation was measured after 24-h incubation under static conditions and reported as the OD₅₇₀:OD₆₀₀ ratio to normalize the amount of biofilm formed to the total bacterial content. Data are shown as means + SDs (n = 48). (D) Adhesion assay to human bladder epithelial cells. Confluent HTB-9 cell monolayers were infected with each isolate using a multiplicity of infection of 100. Cell surface-adherent bacteria were enumerated after 3-h incubation. The uropathogenic Escherichia coli strain CFT073 was included as a positive control. Data are shown as means + SDs (n = 9) and expressed as percentage considering arbitrarily strain 295Kp as the reference. Asterisks above bars represent P values evaluated by one-way analysis of variance: *P < 0.05, **P < 0.01.

Fig 2

Evasion from phagocytosis by human macrophages of Klebsiella pneumoniae sequence type 512 strains isolated in this study. (A) Macrophage internalization assay. Confluent THP-1 cell monolayers were infected with each isolate using a multiplicity of infection of 100. Two hours after infection, chloramphenicol at 60 µg/mL was added to the medium to kill extracellular bacteria. Internalized bacteria were enumerated after an additional 1 h of incubation. Data are shown as means + SDs (n = 9). Asterisks above bars represent P values evaluated by one-way analysis of variance: *P < 0.05, **P < 0.01. (B) Representative microscopy images of infected cells. THP-1 cell monolayers infected in parallel were acquired in bright and fluorescence fields 4′,6-diamidino-2-phenylindole (DAPI). Scale bar = 10 µm.