Genotypic Evolution of Klebsiella pneumoniae Sequence Type 512 during Ceftazidime/Avibactam, Meropenem/Vaborbactam, and Cefiderocol Treatment, Italy

Abstract

In February 2022, a critically ill patient colonized with a carbapenem-resistant K. pneumoniae producing KPC-3 and VIM-1 carbapenemases was hospitalized for SARS-CoV-2 in the intensive care unit of Policlinico Umberto I hospital in Rome, Italy. During 95 days of hospitalization, ceftazidime/avibactam, meropenem/vaborbactam, and cefiderocol were administered consecutively to treat 3 respiratory tract infections sustained by different bacterial agents. Those therapies altered the resistome of K. pneumoniae sequence type 512 colonizing or infecting the patient during the hospitalization period. In vivo evolution of the K. pneumoniae sequence type 512 resistome occurred through plasmid loss, outer membrane porin alteration, and a nonsense mutation in the cirA siderophore gene, resulting in high levels of cefiderocol resistance. Cross-selection can occur between K. pneumoniae and treatments prescribed for other infective agents. K. pneumoniae can stably colonize a patient, and antimicrobial-selective pressure can promote progressive K. pneumoniae resistome evolution, indicating a substantial public health threat.

Keywords: Acinetobacter baumannii; CirA; Italy; KPC-154; KPC-3; KPC-31; Klebsiella pneumoniae; Providencia stuartii; ST512; VIM-1; antimicrobial resistance; avibactam; bacteria; carbapenemase; carbapenemase co-production; cefiderocol; ceftazidime; colistin resistance; fosfomycin resistance; inhibitor-resistant variants; meropenem; outer membrane porin disruption; respiratory infections; sequence type 512; siderophore disruption; vaborbactam.

Figure 1

Timeline of colonization and infection of 1 patient by Klebsiella pneumoniae clones in study of genotypic evolution of K. pneumoniae sequence type 512 during ceftazidime/avibactam, meropenem/vaborbactam, and cefiderocol treatment, Italy. A) Schematic diagram of carbapenemase genes and plasmid content for K. pneumoniae strains 6379, 1186, 6099, and 0296; strain 1186 comprised 2 phenotypes: W and T colonies. Isolate collection day is indicated during 95 hospitalization days in either the COVID-19 (purple scale) or general (yellow scale) intensive care unit. Asterisk indicates the premature stop codon at position 133 (E133) in the catecholate iron outer membrane transporter CirA. B) Timeline of colonization and infection by K. pneumoniae, Providencia stuartii, and Acinetobacter baumannii as well as β-lactam therapies. C) Phylogenetic analysis used to compare the 5 K. pneumoniae strains isolated from the same patient. Core genome alignments were conducted for 5,215 core genes. KPC variants KPC-154, KPC-3, and KPC-31 are shown according to each strain; the bla_VIM-1 gene was present in strains 0296 and 6379. Nonsense mutation in cirA was found in strain 0296, producing a premature stop codon (E133), indicated by an asterisk, in the protein. FDC MICs (mg/L) for each strain are shown. Scale bar indicates number of single-nucleotide polymorphisms in the core genome. FDC, cefiderocol; ICU, intensive care unit; KPC, K. pneumoniae carbapenemase; MDR, multidrug resistant; OmpK36, outer membrane porin K36; T, transparent; VIM, Verona integron-encoded metallo-β-lactamase; W, white; WGS, whole-genome sequencing.

Figure 2

Phylogenetic analysis and genetic features of Klebsiella pneumoniae ST512 in study of the strain’s genotypic evolution during ceftazidime/avibactam, meropenem/vaborbactam, and cefiderocol treatment, Italy. Phylogenetic tree was constructed from 4,654 core gene alignments from a total of 133 K. pneumoniae ST512 genome sequences: 5 genomes sequenced in this study (pink shading), 12 genomes from the same hospital (pale blue shading), and 116 genomes downloaded from the Pasteur Institute BIGSdb database (https://bigsdb.pasteur.fr/klebsiella). Colors indicate resistance and acquired-resistance genes (or corresponding proteins) associated with carbapenemases, yersiniabactin, and chromosomal mutations within the different strains. The same color in the legend on the left indicates the expected resistance phenotype. Asterisk after CirA E133 indicates this mutation produced a premature stop codon. Scale bar indicates number of single-nucleotide polymorphisms in the core genome. Del, deletion; KPC, K. pneumoniae carbapenemase; PUI, Policlinico Umberto I; ST, sequence type; VIM, Verona integron-encoded metallo-β-lactamase.

Figure 3

In silico 3-dimensional structure predictions for mutated OmpK36 porin in Klebsiella pneumoniae sequence type 512 strain 0296 in study of K. pneumoniae genotypic evolution during ceftazidime/avibactam, meropenem/vaborbactam, and cefiderocol treatment, Italy. The outer membrane porin OmpK36 from strain 0296 (blue) containing a 26 aa deletion from residue Thr263 through residue Tyr289 was modeled and compared with the model of reference OmpK36 chain B crystal structure from the Protein Data Bank (no. 6RCP; https://www.rcsb.org) (30). Both ribbon cartoon (top) and surface (bottom) models are shown. Structures for strain 0296 were obtained by using Alphafold2 on the European Galaxy server (https://usegalaxy.eu). Spatial arrangements of the porins in lipid bilayers were visualized by using the positioning of proteins in membranes web server in the Orientations of Proteins in Membranes database (31).