Background/Objectives: Klebsiella (K.) pneumoniae is a significant pathogen in both humans and animals. However, data on its occurrence in animals in Germany remain limited. This study aimed to investigate the antimicrobial resistance (AMR) phenotypes, AMR genes, and virulence traits of animal-derived K. pneumoniae isolates from Germany. Methods: A total of 59 K. pneumoniae isolates obtained in 2023 from dogs, cats, horses, cattle, and chickens across 11 German federal states were analysed. Phenotypic antimicrobial susceptibility testing (AST) was performed, and whole-genome sequencing (WGS) was used for genomic characterisation, including detection of AMR genes, virulence-associated genes, sequence types (STs), and plasmid replicons. Results: Most isolates (78%) were susceptible to all tested antibiotics, while three isolates were classified as multidrug-resistant (MDR). Resistance was most frequently observed for piperacillin (n = 8) and trimethoprim/sulfamethoxazole (n = 4). Carbapenem resistance was detected in two isolates (one from a dog and one from a cat), and phenotypic colistin resistance in one dog isolate. WGS identified 96 AMR genes across isolates, with 20–42 AMR determinants per isolate, conferring resistance to β-lactams, aminoglycosides, fluoroquinolones, sulfonamides, tetracyclines, trimethoprim, and fosfomycin. Ten extended-spectrum β-lactamase (ESBL)-producing isolates carried genes including bla_CTX-M-15, bla_SHV-2, bla_SHV-27, bla_SHV-42, bla_SHV-106, and bla_TEM-158. Although fosA was detected in all isolates, only three exhibited phenotypic resistance to fosfomycin. A total of 52 STs were identified, including high-risk clones. One hypervirulent isolate (ST60) carrying hypervirulence-associated genes rmpA and iroB was detected. Plasmid replicons were present in 70% of isolates, while plasmid-associated AMR genes were identified in nine isolates. Conclusions: This study demonstrates the genomic diversity of K. pneumoniae identified in companion animals and highlights the presence of AMR and virulence determinants relevant to a One Health context.

Klebsiella (K.) pneumoniae is a One Health pathogen that has been isolated from humans, animals, and environmental sources and is responsible for a diverse range of potentially life-threatening infections. In the present study, we analyzed the genomes of 64 presumptive K. pneumoniae strains isolated in 2023 from different companion and farm animals in Germany. Using whole-genome sequencing (WGS) data, 59 isolates (92.2%) were identified as K. pneumoniae and five (7.8%) as K. quasipneumoniae. Multilocus sequence typing (MLST) assigned 53 isolates to 46 distinct sequence types (STs). Eleven isolates could not be assigned to existing STs of the Pasteur classification scheme because they contained novel alleles not previously documented. Thus, these were considered novel and designated as ST7681-ST7689 and ST7697-ST7698. Almost all isolates in this study were assigned unique STs, and only five STs were shared among multiple isolates. This research highlights the genetic diversity among K. pneumoniae strains isolated from different companion and farm animals in Germany, provides information to help in surveillance strategies to mitigate zoonotic transmission risks, and demonstrates the value of WGS and MLST in identifying novel STs of K. pneumoniae.