Klebsiella pneumoniae is designated as a critical pathogen of priority by the World Health Organization (WHO) (1), and it poses a significant threat to public health. This opportunistic bacterium commonly infects immunocompromised individuals in hospital settings, leading to various severe infections such as pneumonia, sepsis, urinary tract infections (UTIs), bacteremia, meningitis, and pyogenic liver abscesses (2). The emergence of resistance to antibiotics, particularly colistin and carbapenem, further complicates the treatment of K. pneumoniae infections in both human and animal populations.

The prevalence of carbapenem resistance in K. pneumoniae can vary depending on the geographical region and the specific population being studied. Generally, carbapenem-resistant K. pneumoniae (CRKP) strains have become a significant concern for both human and animal health (3–5). The prevalence of CRKP strains can be high in human healthcare setting in some regions, leading to increased morbidity and mortality rates among infected individuals (3). In animal settings, including in livestock and companion animals, CRKP isolates also have been reported, although the prevalence tends to be lower compared to in human populations (6, 7). Nevertheless, the presence of CRKP in animals raises concerns about potential transmission to humans through direct contact, environmental contamination, or foodborne routes (5). The One Health approach recognizes the interconnections between human, animal, and environmental health and emphasizes the importance of surveillance, prevention, and control measures to mitigate the spread of carbapenem resistance between different reservoirs (6).

Phenotypic methods are typically the first step in detecting carbapenem resistance. These methods involve performing antimicrobial susceptibility testing (AST) using disk diffusion or broth microdilution methods. Carbapenem-resistant strains exhibit reduced susceptibility or resistance to carbapenem antibiotics, such as imipenem, meropenem, or ertapenem (8). Carbapenemase enzymes are the most common mechanism of carbapenem resistance in K. pneumoniae. Several methods can detect carbapenemase production, including the Modified Hodge Test (MHT), Carba NP test, and Carba-R assay (9, 10). These tests detect the presence of carbapenemase enzymes by assessing the ability of the organism to hydrolyze carbapenem antibiotics. CRKP strains often possess various carbapenemase encoding genes, such as NDM-1, OXA variants, and KPC, which contribute to their resistant phenotype (11, 12).

Carbapenem resistance in K. pneumoniae is predominantly associated with the presence of carbapenemase genes carried on large, self-conjugative plasmids, although the bla_OXA-232 gene (13) encoding resistance to carbapenems is commonly found on small ColE-like plasmids. Furthermore, the co-existence of multiple carbapenemase genes within a single isolate, such as bla_NDM-1 and bla_OXA-232 in sequence type (ST) 14, has been reported (14). Isolates of the globally prevalent K. pneumoniae ST16 also demonstrate diverse antimicrobial resistance profiles. For example, an outbreak of nosocomial infections caused by ST16 K. pneumoniae carrying bla_CTX-M-15 occurred in Sweden and Denmark (4), while the co-occurrence of bla_NDM-1 and bla_OXA-232 in ST16 K. pneumoniae was identified in Italy (15). In a study in Thailand, the two STs that were most commonly found in K. pneumoniae from clinical affected humans were ST16 (n = 15) and ST231 (n = 14) (16).

The current study aimed to enhance understanding of the occurrence of antimicrobial resistance genes in K. pneumoniae isolates from clinically affected humans and dogs in hospital settings in Thailand. Specifically, we sought to elucidate the genetic mechanisms underlying carbapenem resistance in K. pneumoniae, assess the co-occurrence of other antimicrobial resistance genes, and compare the genetic characteristics of isolates from humans and dogs to help determine the possibility of transmission. Resistance to colistin was of interest as studies have revealed the presence of the mcr-3.5 gene in the IncFII plasmid type of pig-derived Escherichia coli isolates in Thailand and other countries (17–19). To date, the prevalence of K. pneumoniae in clinically affected companion animals in Thailand has not been elucidated. In this work, we outline the prevalence, characteristics, molecular typing, and whole genome sequence data for antibiotic resistant K. pneumoniae isolates from clinically affected companion dogs and humans in Hospital settings.

The emergence and spread of carbapenem-resistant Klebsiella pneumoniae strains are of significant concern due to their limited treatment options and potential for causing severe infections (1). In this study, we characterized six CRKP isolates from humans and dogs with UTIs, focusing on their antimicrobial susceptibility, genomic features, and similarity of their plasmidome, focusing on plasmid-mediated resistance genes. The SNP tree indicated that KPH1 and KPA1 shared ST16 and were indistinguishable apart from their plasmid content (Figure 1). This suggests that these closely related isolates may be transmissible between humans and dogs. The other four isolates were dispersed across the SNP tree and were related to isolates from various sources and countries.

Antimicrobial susceptibility testing confirmed that all six isolates were phenotypically and genotypically resistant to ertapenem, with four showing additional resistance to imipenem and meropenem. The differential resistance pattern of K. pneumoniae to ertapenem compared to imipenem and meropenem can be attributed to several factors. Although all three antibiotics belong to the carbapenem class (39), there are subtle differences in their chemical structures and mechanisms of action, which can affect their effectiveness against resistant bacteria. K. pneumoniae can acquire various resistance mechanisms, including the production of carbapenemases that degrade or modify carbapenem antibiotics. Different carbapenemases may have varying affinities for different carbapenem drugs, leading to differences in resistance profiles (40). Importantly, none of the isolates exhibited tigecycline resistance, highlighting its potential as an alternative treatment option for CRKP infections (41).

Genomic characterization of the CRKP isolates revealed the presence of different carbapenemase genes, including NDM-1 and OXA variants. The presence of these genes indicates the potential for high-level carbapenem resistance in K. pneumoniae isolates and highlights the presence of specific resistance mechanisms that contribute to the spread and persistence of carbapenem resistance in isolates from animal and human patients (22).

The isolates shared common replicon types, such as InFIA and IncFIB, indicating the potential for plasmid-mediated horizontal gene transfer among them. The InFIA replicon type is associated with the plasmid family IncF, which is known for its broad host range and ability to transfer between different bacterial species. IncF plasmids often carry multiple resistance genes and are frequently found in clinical isolates of K. pneumoniae (27, 42, 43). In particular, InFIA and IFB plasmids have been associated with the dissemination of carbapenem resistance genes, such as bla_NDM-1, in K. pneumoniae strains (22). Unfortunately, their identification in K. pneumoniae is a common finding, especially among multidrug-resistant strains and those carrying carbapenemase genes. Their presence in our isolates suggests the potential for horizontal gene transfer and the dissemination of resistance genes among bacterial populations in Thailand.

Notably, a small ColKP3 plasmid was found in both human and dog isolates, suggesting a potential reservoir for the dissemination of carbapenem resistance genes between humans and animals. The ColKP3 replicon plasmid has been implicated in the dissemination of carbapenemase encoding genes such as bla_OXA-232 (44) that can hydrolyze carbapenems, leading to resistance. The presence of the ColKP3 replicon plasmid carrying carbapenemase genes could contribute to the spread of carbapenem resistance among K. pneumoniae strains. The ColKP3 replicon plasmid belongs to the group of small plasmids known as ColE-like plasmids (10) that are often self-transmissible and can replicate autonomously. Their small size facilitates their transfer between bacteria, allowing for the rapid dissemination of resistance genes (45).

Analysis of the chromosomes of the six isolates showed that they had similar characteristics, including comparable CG content, number of CDSs, rRNAs, and tRNAs. The presence of bla_SHV genes encoding β-lactam resistance and fosA genes conferring fosfomycin resistance was observed on the chromosomes of all isolates. Additionally, the combination of oqxA and oqxB genes responsible for quinolone resistance was identified. This finding suggests that these resistance determinants are part of the inherent genetic makeup of the bacteria. In this case, their presence on the chromosome may not necessarily indicate the immediate potential for horizontal gene transfer. In clinical isolates the oqxA and oqxB genes generally locate on the chromosome and/or plasmids flanked by IS26-like elements, conferring low to intermediate resistance to quinoxalines, quinolones, tigecycline, and nitrofurantoin. These have the potential to co-spread with other antimicrobial resistance genes (bla_CTX-M, rmtB and aac(6′)-Ib etc.) (46). The bla_SHV genes encode beta-lactamase enzymes that can hydrolyze and inactivate beta-lactam antibiotics, including penicillins and some cephalosporins. The presence of bla_SHV genes on the chromosome of K. pneumoniae strains may indicate the intrinsic resistance of these bacteria to beta-lactam antibiotics (47). However, the presence of plasmid-borne non-ESBL-encoding bla_SHV is a risk factor for the development of an ESBL-positive phenotype, despite the same gene being present on the chromosome (4). This can impact the choice of antibiotics for treatment, as these strains may not respond well to certain drugs.

The ST16 clonal type of K. pneumoniae has proliferated globally and is commonly associated with multidrug-resistant strains carrying enzymes that produce NDM-1, CTX-M-15, and OXA-232 that cause resistance to crucial medicines (48). The spread of these antibiotic-resistant bacteria, particularly between animals and humans, poses a serious threat to public health since it reduces treatment options and can result in treatment failures (1). The two ST16 isolates from a human and a dog in this study had highly similar profiles of resistance and virulence genes on their chromosomes, including genes encoding ESBL-generating enzymes. On the other hand, bla_NDM-1 and bla_OXA-9 genes were identified on plasmids in the human isolates, whereas bla_OXA-48 genes were found in the dog isolates. The identification of these specific resistance genes on plasmids in human and dog isolates suggests the involvement of mobile genetic elements in gene transmission. This finding emphasizes the potential for genetic exchange and the spread of resistance genes via mobile genetic components, but not chromosomal reliance (49).

The plasmid analysis revealed the presence of two to four plasmids in each CRKP isolate, with varying sizes and replicon types. Interestingly, several plasmids showed the coexistence of multiple replicon types, indicating the potential for diverse plasmid compatibility and dissemination mechanisms. The plasmids harbored various resistance genes, including bla_OXA-1, bla_OXA-9, bla_OXA-48, bla_OXA-232, and bla_NDM-1. Prior to this study, examination of the genomic characteristics of multidrug-resistant ST11 K. pneumoniae isolates and their plasmids demonstrated the existence of several plasmids of various sizes and replicon types, indicating variable plasmid compatibility and dissemination mechanisms (50). In a previous study in Thailand, 12 of 15 K. pneumoniae ST16 isolates with chromosomal clonality contained both bla_NDM-1 and bla_OXA-232, indicating that these plasmids were widely distributed in K. pneumoniae (16). In comparison, all but one of 13 ST231 isolates in that study only harbored bla_OXA-232 (16). Another study in clinically affected humans from Thailand also described K. pneumoniae ST16 as a clone that had virulence genes that were similar to those of other highly transmissible K. pneumoniae that carried bla_NDM-1 and bla_OXA-232 (51). When considering ST15, dog strain KPA3 in the current study showed resistance associated with plasmid pKPA3_1 (NDM-1) and pKPA3_2 (Figure 1). In another study in Thailand, pig strain 45 PM1 showed no plasmid-associated resistance determinants, whereas human strain Mu1413 isolated in the USA carried resistance determinants on plasmids unnamed1 (SHV-28), unnamed2, and unnamed3 (KPC-3, TEM-1) (52). For ST16, in the current study dog strain KPA1 carried resistance determinants on plasmids pKPA1_1, pKPA1_2 (OXA-48), and pKPA1_3 (OXA-232). Moreover, the closely related human strain KPH1 carried resistance determinants on plasmids pKPH1_1 (CTX-M-15, TEM1, OXA-9) and pKPH1_2 (NDM-1). In the earlier study in Thailand, human strain 1UK exhibited no resistance associated with the plasmids identified in other Thai isolates (52). These findings highlight the genetic relatedness and varied distribution of AMR genes among K. pneumoniae isolates, emphasizing the interconnectedness of human, animal, and environmental sources and transmission of resistance mechanisms across diverse hosts and environments.

Notably, the mcr-3.5 gene conferring colistin resistance was identified on an IncFII plasmid in one of the human isolates (KPH4), indicating the coexistence of multiple resistance mechanisms. The mcr-3.5 gene is a variant of the mobile colistin resistance gene (mcr) family (53). It encodes resistance to colistin, an antibiotic belonging to the polymyxin class. The mcr-3.5 gene was first identified in an Escherichia coli strain isolated from pig feces in China (54). It has been reported to be associated with IncFII plasmids, which are known to have a broad host range and can be transferred between different bacterial species (53). The presence of colistin resistance genes on IncFII plasmids supports the notion of multiple resistance mechanisms coexisting in K. pneumoniae strains. The findings will help guide the development of effective surveillance strategies and infection control measures, ultimately contributing to the preservation of public health and the improvement of patient outcomes.

In conclusion, this study offers valuable insights into the antimicrobial susceptibility, genomic characteristics, and plasmid-mediated resistance genes of CRKP isolates from unrelated humans and dogs with UTIs in Thailand. However, the limited number of isolates investigated in this study and the restricted clinical data rendered it difficult to determine whether the outbreak inside the animal and human hospital developed and evolved over time. Although human isolate KPH1 and dog isolate KPA1 both belonged to ST16 and were indistinguishable on the SNP tree, they had different plasmid types and had no known direct epidemiological connections. Nevertheless, the demonstration that human and dog isolates shared antimicrobial resistance and virulence genes on their chromosomes and had genes encoding NDM and OXA enzymes on plasmids that are identical to ColKP3 indicates the potential for interspecies transmission, and emphasizes the need for surveillance and control measures to prevent the spread of CRKP strains. Moreover, when the six isolates in the current study where compared to global isolates of K. pneumonia, these showed a variety of divergent genomes and plasmid types. There is a need for additional research into the precise mechanisms underlying the transmissibility and pathogenicity of CRKP strains and their potential effects on human and animal health.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

Ethical approval was not required for the study involving animals in accordance with the local legislation and institutional requirements because all the isolates we used were obtained from the frozen stock of the Pathogen Bank in our institute. These were clinical isolates that were submitted for diagnostic purposes as a routine procedure. Written informed consent was obtained from the owners for the participation of their animals in this study.

RP: Writing – review & editing, Writing – original draft, Validation, Software, Methodology, Investigation, Formal analysis, Data curation. NK: Writing – original draft, Resources, Methodology, Investigation, Formal analysis, Data curation. NPh: Writing – review & editing, Visualization, Methodology, Investigation, Formal analysis, Data curation. TW: Writing – review & editing, Validation, Software, Resources, Methodology. WN: Writing – review & editing, Resources, Methodology, Data curation. TC: Writing – review & editing, Resources, Methodology, Data curation. DH: Writing – review & editing, Supervision, Conceptualization. NPr: Writing – review & editing, Writing – original draft, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Data curation, Conceptualization.