Abstract
Background
The emergence of ESBL/AmpC-producing K. pneumoniae is a significant concern in humans and veterinary medicine. This study aims to ascertain the antimicrobial resistance profiles and molecular characteristics of ESBL/AmpC-producing K. pneumoniae isolated from diseased companion animals during 2018–2023 in South Korea.
Methods
The obtained isolates (dogs, n = 130 and cats, n = 30) from urine, genital organs, diarrheal feces, skin/ear, and respiratory tract were assessed for antimicrobial susceptibility by broth microdilution. Molecular characteristics were determined by polymerase chain reaction (PCR), multi-locus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE).
Results
Among the tested antimicrobials, the highest resistance rates were demonstrated for tetracycline, followed by cefazolin. In the sample levels, isolates from non-digestive tract showed overall higher antimicrobial resistance rates than digestive tract samples for both dogs and cats. In general, 25% (40/160) of the K. pneumoniae isolates harbored ESBL and/or AmpC genes. Of them, ESBL was identified in 30 isolates, with blaCTX−M−15, blaCTX−M−65, and blaCTX−M−55 being the predominant, while AmpC was detected in 20 isolates, with blaDHA−1 and blaCMY−2. Noticeably, co-occurrence of blaCTX−M and blaDHA was found in 7 isolates. Virulence factors were identified in 40% of the isolates, mostly comprising terB (56.3%) and irp2 (43.8%). MLST analysis revealed that sequence types (ST)307 and ST15 were predominant among 18 STs. Furthermore, the identical PFGE pattern was detected in different hospitals, suggesting the clonal spread of K. pneumoniae.
Conclusion
Taken together, the findings emphasize the role of dogs and cats as reservoirs of antimicrobial-resistant K. pneumoniae that could be transmitted to humans. Therefore, it is necessary to conduct continuous surveillance and ensure the judicious use of antimicrobials in companion animals.
Supplementary Information
The online version contains supplementary material available at 10.1186/s12917-025-05102-2.
Keywords: Companion animals, blaCTX−M−15, Virulence factors, Sequence types
Introduction
Klebsiella pneumoniae are Gram-negative opportunistic bacteria that inhabit the skin, upper respiratory, and gastrointestinal tract of humans and other animals. It can infect humans, e.g., resulting in pneumonia, meningitis, and sepsis, especially in individuals with an impaired immune system [1]. Clinical treatment is further complicated by the emergence and dissemination of multidrug-resistant (MDR) K. pneumoniae [2]. Overuse and/or misuse of antimicrobials has led to the development of multidrug resistance in K. pneumoniae strains from humans and companion animals [3].
β-lactam antimicrobials are frequently used to treat the bacterial infections caused by Enterobacteriaceae in humans and the veterinary practice, triggering the selection and spread of extended-spectrum β-lactamase/AmpC β-lactamase (ESBL/AmpC)-producing K. pneumoniae [4, 5]. ESBL/AmpC enzymes can deactivate a wide range of β-lactam antimicrobials, including several broad-spectrum cephalosporins, making it challenging to treat bacterial infections [6]. It was observed that K. pneumoniae isolates from companion animals such as dogs and cats in many countries, including Japan [7], Korea [8], Taiwan [6], Portugal [4], and Brazil [9], demonstrated resistance to critically important antimicrobials, including extended-spectrum cephalosporins. Antimicrobial-resistant bacteria can be transmitted from companion animals to humans either directly or indirectly due to their shared environment, close contact, and exposure to antimicrobials frequently used in human treatment [10]. For example, Hong et al. [11] showed that ESBL/AmpC-carrying K. pneumoniae was spread between companion animals and humans.
In South Korea, the popularity of pets is on the rise, increasing a potential reservoir for antimicrobial-resistant bacteria that can pose human health hazards. K. pneumoniae is the third most prevalent bacteria in diarrhea samples and is also observed in respiratory and urine samples in dogs and cats [12, 13]. Despite this, nationwide surveys on antimicrobial resistance and characterization studies on resistant bacteria have been very limited. Moreover, these investigations concentrated on a limited number of isolates collected from a few locations, over a short period [8, 9, 11]. Third-generation cephalosporins are frequently used to treat K. pneumoniae infections in both humans and companion animals, leading to increasing resistance issues and necessitating molecular genetic analysis to understand the potential for transmission between humans and animals. Thus, this study aimed to investigate the antimicrobial resistance and characterization of ESBL/AmpC-carrying K. pneumoniae isolated from companion animals nationwide in South Korea between 2018 and 2023.
Materials and methods
Klebsiella pneumoniae isolates
Sampling, isolation, and identification of K. pneumoniae were performed based on the previously described method [4]. K. pneumoniae was obtained from urine, genital organs, diarrheal feces, skin/ear, and respiratory systems of dogs and cats from 10 laboratories/centers that participated in the Korean Veterinary Antimicrobial Resistance Monitoring system between 2018 and 2023 (Supplementary Tables 1 and 2). The isolation procedure commences with the inoculation of swab samples onto MacConkey agar (Spark, Baltimore, USA) for 24 h at 37 °C, followed by lactose-fermented colonies re-inoculation onto tryptic soy agar (Becton Dickinson, NV, USA) overnight at 37 °C. The selected colonies were subsequently confirmed as K. pneumoniae by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI Biotyper® Sirius, Bruker Corporation, MA, USA). One isolate from each sample, with no repetition from the same animal, was considered for subsequent analysis. We do not have information on the usage of antimicrobials in dogs and cats included in this study. A total of 160 K. pneumoniae isolates were collected from dogs (n = 130) and cats (n = 30) (Table 1).
Table 1.
Klebsiella pneumoniae isolates obtained from diseased dogs and cats during 2018–2023 in South Korea
| Year | Dogs | Cats | Total | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| No. of Hospital |
Digestive disease | Respiratory disease | Reproductive disease | Skin/ear | Urine | Subtotal | No. of Hospital |
Digestive disease | Urine | Respiratory disease | Reproductive disease | Skin/ear | Subtotal | ||
| 2018 | 14 | 15 | 3 | 5 | 4 | 0 | 27 | 3 | 4 | 1 | 0 | 0 | 0 | 5 | 32 |
| 2019 | 28 | 41 | 1 | 5 | 7 | 1 | 55 | 6 | 5 | 1 | 1 | 1 | 1 | 9 | 64 |
| 2020 | 16 | 23 | 10 | 6 | 4 | 1 | 44 | 9 | 4 | 4 | 3 | 2 | 2 | 15 | 59 |
| 2021 | 1 | 0 | 1 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 |
| 2022 | 1 | 0 | 2 | 0 | 0 | 0 | 2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 2 |
| 2023 | 1 | 0 | 1 | 0 | 0 | 0 | 1 | 1 | 1 | 0 | 0 | 0 | 0 | 1 | 2 |
| Total | 44* | 79 | 18 | 16 | 15 | 2 | 130 | 16* | 14 | 6 | 4 | 3 | 3 | 30 | 160 |
*Duplicate hospitals were counted once
Antimicrobial susceptibility testing
We used the broth microdilution method to assess the isolates’ susceptibility to antimicrobial agents (amikacin, amoxicillin/clavulanic acid, cefazolin, cefovecin, cefoxitin, chloramphenicol, doxycycline, enrofloxacin, gentamicin, marbofloxacin, piperacillin/tazobactam, tetracycline, and trimethoprim/sulfamethoxazole) of different classes using a commercially available Sensititre® KRNVF panel (Thermo Fisher, Waltham, USA). The minimum inhibitory concentration (MIC) values were interpreted based on the breakpoints for human and veterinary medicine provided by the Clinical and Laboratory Standards Institute (CLSI) 2023 (Supplementary Table 3) [14, 15]. K. pneumoniae ATCC 43,816 served as a quality reference strain. The multidrug resistance (MDR) was defined as resistance to three or more classes of antimicrobial agents in one isolate [16]. The double-disk synergy test was conducted to identify ESBL using cefotaxime-cefotaxime/clavulanic acid discs, as per the CLSI 2023 guidelines [14, 15].
Detection of ESBL and AmpC genes
The polymerase chain reaction (PCR) was used to detect ESBL/AmpC genes (blaCTX−M, blaNDM−1, blaCMY−2, blaDHA−1, and blaSHV) following the previously delineated primers and methods [17–21]. These β-lactamase (bla) genes were determined by sequencing PCR products produced using the primers and reaction conditions mentioned in Supplementary Table 4. The DNA sequencing was conducted by Intron Biotechnology (Seongnam, South Korea), and the homolog sequences were explored against the GenBank database with the BLAST software from the National Center for Biotechnology Information website (https://www.ncbi.nlm.nih.gov/BLAST).
Detection of virulence genes
The virulence genes in K. pneumoniae were identified by PCR: aerobactin siderophore biosynthesis (iucA), tellurite resistance (terB), and iron uptake (irp2). The primers, annealing temperature, and PCR product size are described in Supplementary Table 3.
Multi-locus sequence typing
The multi-locus sequence typing (MLST) approach was used to determine the sequence types (STs) of ESBL/AmpC-carrying K. pneumoniae based on the allelic profiles of seven housekeeping genes [22]. PCR was performed with primers for the following genes: gapA, infB, mdh, pgi, phoE, rpoB, and tonB. The allelic profiling and sequence type determination were conducted using the web-based MLST website (https://pubmlst.org/organisms/Klebsiella/).
Pulsed-field gel electrophoresis analysis
The pulsed-field gel electrophoresis (PFGE) of restriction enzyme XbaI (Takara Bio, Shiga, Japan)-digested genomic DNA of K. pneumoniae was performed in accordance with the previously delineated method [8]. The similarities among fragments were evaluated utilizing GelCompar II software (version 6.5; Applied Maths, Sint-Martens-Latem, Belgium) to generate the dendrogram. The cluster analysis was carried out using the unweighted pair group method with average linkage (UPGMA) and the Dice similarity index.
Results
Antimicrobial resistance
The antimicrobial resistance in 160 K. pneumoniae strains to the tested antimicrobial agents is presented in Fig. 1; Table 2. Differences in antimicrobial resistance were observed across sample types in dogs. Overall, resistance in digestive tract samples from dogs was significantly lower. In canine digestive tract samples, resistance to all but two antibiotics was 20% or less. However, in non-digestive tract samples from dogs and all samples from cats, resistance to all but two antibiotics exceeded 20%. In dogs, resistance to tetracyclines (doxycycline and tetracycline) was the highest, exceeding 50%, while in cat samples, resistance to fluoroquinolones and trimethoprim/sulfamethoxazole was the highest (50% and 56.3%, respectively). Regarding specific antimicrobials, amikacin resistance was low (≤ 15%) across all sample types in both dogs and cats. Resistance to β-lactam/inhibitor combinations was very low (≤ 5%) in canine digestive tract samples but higher in other samples (17.6%–43.8%). Resistance to third-generation cephalosporins (cefovecin) and cephamycins (cefoxitin) was low (12.7% vs. 11.4%) in canine digestive samples but high (21.6%–43.8%) in other canine samples and cats. Fluoroquinolone resistance (enrofloxacin and marbofloxacin) was approximately 10% or less in canine digestive samples, but high (28.6%–50.0%) in all other samples. Resistance to tetracyclines ranged from 20% to 50%, with particularly high levels (approximately 50%) observed in non-digestive samples from dogs.
Fig. 1.
Antimicrobial resistance profiles of Klebsiella pneumoniae isolates obtained from diseased dogs and cats in South Korea. p < 0.05 was regarded as a statistically significant change in the antimicrobial resistance rate. AMK, amikacin; AMC, amoxicillin/clavulanic acid; CFZ, cefazolin; VEC; cefovecin; FOX, cefoxitin.; CHL, chloramphenicol; DOX, doxycycline; ENO, enrofloxacin; GEN, gentamicin; MAR, marbofloxacin; PTZ, piperacillin/tazobactam; TET, tetracycline; SXT, trimethoprim/sulfamethoxazole
Table 2.
Antimicrobial resistance in Klebsiella pneumoniae isolates obtained from diseased dogs and cats during 2018–2023 in South Korea [14, 15]
| Antimicrobials agents | Break-points(µg/mL) | Dogs (n = 130) | p-value | Cats (n = 30) | p-value | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Digestive tract (n = 79) | Non-digestive tract (n = 51) | Digestive tract (n = 14) | Non-digestive tract (n = 16) | ||||||||||||
| MIC50 | MIC90 | % Resist. (n) | MIC50 | MIC90 | % Resist. (n) | MIC50 | MIC90 | % Resist. (n) | MIC50 | MIC90 | % Resist. (n) | ||||
| Aminoglycosides | |||||||||||||||
| Amikacin | ≥16 | 4 | 4 | 6.3 (5) | 4 | 64 | 13.7 (7) | 0.15 | 4 | 4 | 14.3 (2) | 4 | 4 | 12.5 (2) | 0.88 |
| Gentamicin | ≥8 | 0.25 | 0.5 | 8.9 (7) | 0.25 | 16 | 27.5 (14) | <0.01 | 0.25 | 0.5 | 21.4 (3) | 0.25 | 16 | 50.0 (8) | 0.1 |
| β-lactam/β-lactamase inhibitors | |||||||||||||||
| Amoxicillin/clavulanic acid | ≥32 | 8 | 8 | 3.8 (3) | 8 | 128 | 25.5 (13) | <0.01 | 8 | 128 | 35.7 (5) | 8 | 128 | 43.8 (7) | 0.65 |
| Piperacillin/tazobactam | ≥32 | 8 | 8 | 2.5 (2) | 8 | 32 | 17.6 (9) | <0.01 | 8 | 64 | 21.4 (3) | 8 | 64 | 31.3 (5) | 0.54 |
| Cephalosporin I | |||||||||||||||
| Cefazolin | ≥8 | 2 | 64 | 16.5 (13) | 2 | 64 | 41.2 (21) | <0.01 | 4 | 64 | 42.9 (6) | 2 | 64 | 50.0 (8) | 0.7 |
| Cephalosporin Ⅲ | |||||||||||||||
| Cefovecin | ≥8 | 1 | 16 | 12.7 (10) | 2 | 16 | 41.2 (21) | <0.01 | 1 | 16 | 35.7 (5) | 2 | 16 | 43.8 (7) | 0.65 |
| Cephamycin | |||||||||||||||
| Cefoxitin | ≥32 | 16 | 16 | 11.4 (9) | 16 | 128 | 21.6 (11) | 0.11 | 16 | 256 | 35.7 (5) | 16 | 256 | 37.5 (6) | 0.91 |
| Fluoroquinolones | |||||||||||||||
| Enrofloxacin | ≥4 | 0.12 | 2 | 10.1 (8) | 1 | 8 | 43.1 (22) | <0.01 | 0.12 | 8 | 35.7 (5) | 1 | 8 | 50.0 (8) | 0.43 |
| Marbofloxacin | ≥4 | 0.12 | 1 | 7.6 (6) | 0.25 | 8 | 35.3 (18) | <0.01 | 0.12 | 4 | 28.6 (4) | 1 | 8 | 50.0 (8) | 0.23 |
| Phenicols | |||||||||||||||
| Chloramphenicol | ≥32 | 8 | 64 | 13.9 (11) | 8 | 64 | 39.2 (20) | <0.01 | 8 | 64 | 28.6 (4) | 8 | 64 | 31.3 (5) | 0.87 |
| Tetracyclines | |||||||||||||||
| Doxycycline | ≥16 | 2 | 16 | 19.0 (15) | 8 | 16 | 51.0 (26) | <0.01 | 2 | 16 | 42.9 (6) | 4 | 16 | 37.5 (6) | 0.76 |
| Tetracycline | ≥16 | 4 | 32 | 24.1 (19) | 16 | 32 | 56.9 (29) | <0.01 | 4 | 32 | 42.9 (6) | 4 | 32 | 37.5 (6) | 0.91 |
| Folate pathway inhibitors | |||||||||||||||
| Trimethoprim/sulfamethoxazole | ≥4 | 0.5 | 8 | 21.5 (17) | 0.5 | 8 | 43.1 (22) | <0.01 | 0.5 | 1 | 21.4 (3) | 8 | 8 | 56.3 (9) | 0.05 |
| MDR | 25.3 (20) | 52.9 (27) | <0.01 | 42.9 (6) | 50.0 (8) | 0.69 | |||||||||
Minimum inhibition concentration (MIC). MIC50 and MIC90 are the concentrations (µg/mL) of antimicrobials at which 50% and 90% of the isolates were inhibited, respectively. MDR multidrug resistance
p <0.05 indicates a statistically significant change in antimicrobial resistance rates between digestive tract and non-digestive tract isolates
Multidrug resistance (MDR) and resistance patterns
We found that 50% of the K. pneumoniae isolates exhibited resistance to one or more antimicrobial agents (Tables 3 and 4). Moreover, a total of 38.1% of the isolates demonstrated multidrug resistance, comprising 33.1% from dogs and 43.3% from cats. Resistance to five or more antimicrobials comprising at least three classes was observed in 25.4% of dog isolates, while 30% (9/30) of the cat isolates showed resistance to nine or more antimicrobials.
Table 3.
Antimicrobial resistance patterns in Klebsiella pneumoniae isolates obtained from diseased dogs (n = 130) during 2018–2023 in South Korea [14, 15]
| No. of antimicrobial | % (No. of resistant isolates) | Most common resistance pattern (No. of isolates) |
|---|---|---|
| 0 | 54.6 (71) | – |
| 1 | 5.4 (7) | FOX (n = 3) |
| 2 | 5.4 (7) | DOX TET (n = 3) |
| 3 | 3.8 (5) | CFZ VEC FOX (n = 1) |
| CFZ VEC SXT (n = 1) | ||
| CHL DOX TET (n = 1) | ||
| DOX TET SXT (n = 1) | ||
| FOX TET SXT (n = 1) | ||
| 4 | 5.4 (7) | CHL DOX TET SXT (n = 4) |
| 5 | 4.6 (6) | FOX CHL DOX TET SXT (n = 2) |
| 6 | 1.5 (2) | AMC CFZ VEC DOX TET SXT (n = 1) |
| CFZ VEC FOX DOX ENO TET (n = 1) | ||
| 7 | 1.5 (2) | CFZ VEC CHL ENO MAR TET SXT (n = 2) |
| 8 | 1.5 (2) | CFZ VEC DOX ENO GEN MAR TET SXT (n = 2) |
| 9 | 5.4 (7) | AMC CFZ VEC DOX ENO GEN MAR TET SXT (n = 4) |
| 10 | 3.1 (4) | AMC CFZ VEC CHL DOX ENO MAR PTZ TET SXT (n = 2) |
| 11 | 2.3(3) | AMK CFZ VEC FOX CHL DOX ENO GEN MAR TET SXT (n = 2) |
| 12 | 3.8 (5) | AMK CFZ VEC FOX CHL DOX ENO GEN MAR PTZ TET SXT (n = 2) |
| 13 | 1.5 (2) | AMK AMC CFZ VEC FOX CHL DOX ENO GEN MAR PTZ TET SXT (n = 2) |
| MDR | 33.1 (43) | – |
AMC amoxicillin/clavulanic acid, AMK amikacin, CFZ cefazolin, CHL chloramphenicol, DOX doxycycline, ENO enrofloxacin, FOX cefoxitin, GEN gentamicin, MAR marbofloxacin, PTZ piperacillin/tazobactam, SXT trimethoprim/sulfamethoxazole, TET tetracycline, VEC cefovecin, MDR multidrug resistance
Table 4.
Antimicrobial resistance patterns in Klebsiella pneumoniae isolates obtained from diseased cats (n = 30) during 2018–2023 in South Korea [14, 15]
| No. of antimicrobials | % (No. of resistant isolates) | Most common resistance pattern (No. of isolates) |
|---|---|---|
| 0 | 30.0 (9) | – |
| 1 | 13.3 (4) | FOX (n = 2) |
| 2 | 10.0 (3) | DOX TET (n = 1) |
| AMC FOX (n = 1) | ||
| CHL DOX (n = 1) | ||
| 3 | 3.3 (1) | CHL DOX TET (n = 1) |
| 6 | 3.3 (1) | AMC CFZ VEC FOX ENO PTZ (n = 1) |
| 8 | 10.0 (3) | AMC CFZ VEC FOX DOX ENO MAR TET (n = 1) |
| AMC CFZ VEC ENO GEN MAR PTZ SXT (n = 1) | ||
| AMC CFZ VEC CHL ENO GEN MAR SXT (n = 1) | ||
| 9 | 6.7 (2) | AMC CFZ CHL DOX ENO GEN MAR TET SXT (n = 1) |
| AMC CFZ VEC FOX ENO GEN MAR PTZ SXT (n = 1) | ||
| 10 | 6.7 (2) | AMK CFZ VEC FOX DOX ENO GEN MAR TET SXT (n = 1) |
| AMC CFZ VEC CHL DOX ENO GEN MAR TET SXT (n = 1) | ||
| 11 | 3.3 (1) | CFZ VEC FOX CHL DOX ENO GEN MAR PTZ TET SXT (n = 1) |
| 12 | 10.0 (3) | AMK AMC CFZ VEC FOX DOX ENO GEN MAR PTZ TET SXT (n = 1) |
| AMK AMC CFZ VEC CHL DOX ENO GEN MAR PTZ TET SXT (n = 1) | ||
| AMC CFZ VEC FOX CHL DOX ENO GEN MAR PTZ TET SXT (n = 1) | ||
| 13 | 3.3 (1) | AMK AMC CFZ VEC FOX CHL DOX ENO GEN MAR PTZ TET SXT (n = 1) |
| MDR | 43.3 (13) | – |
AMC amoxicillin/clavulanic acid, AMK amikacin, CFZ cefazolin, CHL chloramphenicol, DOX doxycycline, ENO enrofloxacin, FOX cefoxitin, GEN gentamicin, MAR marbofloxacin, PTZ piperacillin/tazobactam, SXT trimethoprim/sulfamethoxazole, TET tetracycline, VEC cefovecin, MDR multidrug resistance
The isolates were shown to possess a total of 36 resistant patterns, 18 each from dogs and cats. Interestingly, about 17.7% (23/130) and 43.3% (13/30) isolates were resistant to all classes in dogs and cats, respectively.
Molecular characterization
A total of 40 ESBL/AmpC β-lactamase-producing K. pneumoniae isolates were detected in dogs and cats from 38.6% (17/44) and 43.8% (7/16) hospitals located in six provinces, respectively (Table 5). Among the animal species, the prevalence of β-lactamase-carrying isolates was similar, with 23.1% (30/130) in dogs and 33.3% (10/30) in cats. However, the prevalence of β-lactamase-harboring K. pneumoniae differed at sample levels, with the highest occurrence observed in samples from the respiratory tract (61.1%, 11/18), followed by skin (33.3%, 5/15), urine (18.8%, 3/16), and reproductive tract (13.9%, 11/79) in dogs. In cats, 83.3% (5/6) was in urine, 28.6% (4/14) in the digestive tract, and 25% (1/4) in the respiratory tract.
Table 5.
Characterization of ESBL/AmpC-carrying Klebsiella pneumoniae isolates obtained from diseased dogs and cats during 2018–2023 in South Korea [14, 15]
| Isolates | Animals | Sample | Age (year) |
Province | Animal ID |
Hospital ID |
Year | MIC (µg/mL) | Non-β-lactam resistance | ESBL/ AmpC gene |
Virulence factor | ST | PFGE | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| CTX | FOX | |||||||||||||
| P11-017-002 | Dog | Respiratory tract | < 1 | Seoul | A | B | 2018 | 4 | >128 | CHL DOX ENO MAR TET SXT | bla DHA−1 | – | 709 | P3 |
| D11-030-002 | Dog | Skin | < 1 | Incheon | B | H | 2018 | >16 | ≤ 8 | DOX ENO GEN MAR TET SXT | bla CTX−M−15 | – | 307 | P16 |
| E11-030-001 | Dog | Digestive tract | 6–10 | Incheon | C | A | 2018 | >16 | >128 | AMK CHL DOX ENO GEN MAR PTZ TET SXT |
bla CTX−M−27 bla DHA−1 |
– | 37 | P28 |
| G11-030-003 | Dog | Reproductive tract | 1–5 | Daejeon | F | T | 2018 | >16 | 16 | CHL DOX ENO GEN MAR PTZ TET SXT | bla CTX−M−15 | irp2 | 307 | P18 |
| D11-025-005 | Dog | Skin | 16–20 | Seoul | G | D | 2019 | >16 | 64 | CHL DOX ENO GEN MAR PTZ TET SXT |
bla CTX−M−15 bla SHV−28 |
irp2 | 307 | P17 |
| G11-030-001 | Dog | Reproductive tract | 11–15 | Daegu | H | V | 2019 | >16 | >128 | AMK CHL DOX ENO GEN MAR TET SXT |
bla CTX−M−65 bla DHA−1 |
– | 1128 | P10 |
| A11-030-001 | Dog | Digestive tract | Unknown | Incheon | I | F | 2019 | 2 | 128 | AMK CHL DOX ENO GEN MAR TET SXT | bla DHA−1 | terB | 1121 | P29 |
| E11-030-001 | Dog | Digestive tract | Unknown | Incheon | J | A | 2019 | 2 | >128 | AMK CHL DOX ENO GEN TET SXT | bla DHA−1 | terB | 359 | P5 |
| E11-030-002 | Dog | Digestive tract | 11–15 | Incheon | K | A | 2019 | >16 | >128 | AMK CHL DOX ENO GEN MAR TET SXT |
bla CTX−M−27 bla DHA−1 |
– | 37 | P22 |
| E11-032-019 | Dog | Digestive tract | < 1 | Daejeon | L | R | 2019 | 8 | 64 | – | bla CMY−2 | – | 35 | P25 |
| H12-E1-7 | Dog | Digestive tract | < 1 | Seoul | AA | J | 2019 | >16 | ≤ 8 | AMK CHL DOX ENO GEN MAR TET SXT | bla CTX−M−55 | – | 8213 | P8 |
| H15-D2-4 | Dog | Skin | 14 | Seoul | AB | I | 2019 | >16 | 16 | CHL DOX ENO MAR PTZ TET SXT | bla CTX−M−15 | irp2 | 307 | P11 |
| H9-E2-7 | Dog | Digestive tract | 14 | Seoul | AC | G | 2019 | >16 | 16 | DOX ENO GEN MAR TET SXT | bla CTX−M−15 | – | 392 | P2 |
| H8-E6-9 | Dog | Digestive tract | 9 | Seoul | AE | C | 2019 | >16 | ≤ 8 | DOX ENO GEN MAR TET SXT | bla CTX−M−15 | – | 307 | P15 |
| H1-E89-7 | Dog | Digestive tract | 8 | Seoul | AF | E | 2019 | >16 | >128 | PTZ | bla CMY−2 | – | 8215 | P9 |
| H15-U3-1 | Dog | Reproductive tract | 6 | Seoul | AG | I | 2019 | >16 | 16 | CHL DOX ENO MAR PTZ TET SXT | bla CTX−M−15 | irp2 | 3368 | P11 |
| H9-E18-9 | Dog | Digestive tract | < 1 | Seoul | AH | G | 2019 | >16 | ≤ 8 | ENO TET | bla CTX−M−15 | – | 307 | P14 |
| H2-D28-3 | Dog | Skin | 7 | Seoul | AI | N | 2019 | 16 | >128 | AMK DOX ENO GEN MAR PTZ TET SXT | bla DHA−1 | terB | 392 | P6 |
| P11-030-001 | Dog | Respiratory tract | ≤ 1 | Seoul | N | M | 2020 | >16 | ≤ 8 | CHL ENO MAR TET SXT |
bla CTX−M−15 bla CTX−M−65 |
– | 15 | P27 |
| P11-030-002 | Dog | Respiratory tract | ≤ 1 | Incheon | O | M | 2020 | >16 | ≤ 8 | CHL ENO MAR TET SXT |
bla CTX−M−15 bla CTX−M−65 |
– | 15 | P26 |
| P11-030-003 | Dog | Respiratory tract | ≤ 1 | Seoul | P | M | 2020 | >16 | >128 | AMK CHL DOX ENO GEN MAR PTZ TET SXT |
bla CTX−M−15 bla CTX−M−65 bla DHA−1 |
terB | 15 | P26 |
| P11-030-004 | Dog | Respiratory tract | ≤ 1 | Seoul | Q | M | 2020 | >16 | ≤ 8 | CHL DOX ENO GEN MAR TET SXT |
bla CTX−M−15 bla CTX−M−65 |
– | 15 | P27 |
| P11-030-008 | Dog | Respiratory tract | ≤ 1 | Seoul | R | M | 2020 | >16 | >128 | AMK CHL DOX ENO GEN MAR PTZ TET SXT |
bla CTX−M−15 bla DHA−1 |
terB | 15 | P26 |
| P11-030-011 | Dog | Respiratory tract | ≤ 1 | Seoul | S | M | 2020 | >16 | >128 | AMK CHL DOX ENO GEN MAR PTZ TET SXT |
bla CTX−M−3 bla NDM−5 bla SHV−27 |
– | 967 | P31 |
| P11-030-001 | Dog | Respiratory tract | 7 | Daegu | T | Q | 2020 | >16 | 16 | DOX ENO GEN MAR TET SXT | bla CTX−M−15 | terB | 392 | P1 |
| D11-030-001 | Dog | Skin | 4 | Ulsan | V | O | 2020 | 4 | 32 | DOX ENO TET | bla CTX−M−15 | irp2 | 307 | P12 |
| H4-E14-8 | Dog | Digestive tract | 6 | Seoul | AJ | S | 2020 | >16 | ≤ 8 | SXT | bla CTX−M−15 | – | 8216 | P4 |
| P11-030-001 | Dog | Respiratory tract | 10 | Gwangju | X | P | 2021 | >16 | ≤ 8 | DOX TET SXT | bla CTX−M−15 | irp2 | 792 | P7 |
| P11-030-001 | Dog | Respiratory tract | Unknown | Gyeonggi | AM | W | 2022 | >16 | ≤ 8 | AMK CHL DOX GEN TET SXT | bla CTX−M−55 | – | 1530 | P30 |
| P11-030-002 | Dog | Respiratory tract | Unknown | Gyeonggi | AN | W | 2022 | >16 | ≤ 8 | AMK CHL DOX GEN TET SXT | bla CTX−M−55 | - | 1530 | P30 |
| E12-030-001 | Cat | Digestive tract | Unknown | Incheon | D | A | 2018 | >16 | >128 | AMK DOX ENO GEN MAR PTZ TET SXT | bla CMY−2 | terB | 15 | P23 |
| E12-030-002 | Cat | Digestive tract | Unknown | Incheon | E | A | 2018 | >16 | >128 | AMK CHL DOX ENO GEN MAR PTZ TET SXT |
bla CTX−M−27 bla DHA−1 |
– | 37 | P28 |
| H14-U5C-1 | Cat | Urine | 10 | Seoul | Y | K | 2018 | >16 | >128 | ENO GEN MAR PTZ SXT |
bla CTX−M−15 bla CMY−2 |
– | 307 | P13 |
| U12-030-001 | Cat | Urine | 6–10 | Ulsan | M | O | 2019 | >16 | >128 | CHL DOX ENO GEN MAR PTZ TET SXT | bla DHA−1 | iucA | 709 | P24 |
| H9-E5C-7 | Cat | Digestive tract | 2 | Seoul | AD | G | 2019 | 16 | 128 | DOX ENO MAR TET | bla CMY−2 | terB | 8214 | P20 |
| P12-030-001 | Cat | Respiratory trsct | < 1 | Daegu | U | U | 2020 | >16 | >128 | AMK DOX ENO GEN MAR TET SXT |
bla CTX−M−15 bla DHA−1 |
irp2, terB | 15 | P23 |
| U12-030-001 | Cat | Urine | 7 | Ulsan | W | O | 2020 | >16 | >128 | CHL DOX ENO GEN MAR PTZ TET SXT | bla DHA−1 | – | 709 | P24 |
| H3-U295C-3 | Cat | Urine | 5 | Seoul | AK | L | 2020 | >16 | ≤ 8 | ENO GEN MAR PTZ SXT | bla CTX−M−15 | – | 8217 | P14 |
| H3-U307C-1 | Cat | Urine | 1 | Seoul | AL | L | 2020 | >16 | ≤ 8 | CHL ENO GEN MAR SXT | bla CTX−M−15 | – | 307 | P14 |
| 23D81 | Cat | Feces | 8 | Daejeon | AO | X | 2023 | >16 | 16 | CHL DOX ENO GEN MAR TET SXT | bla CTX−M−27 | – | 395 | P21 |
AMK amikacin, CHL, chloramphenicol, CTX cefotaxime, DOX doxycycline, ENO enrofloxacin, GEN gentamicin, MAR marbofloxacin, PTZ piperacillin/tazobactam, SXT trimethoprim/sulfamethoxazole, TET tetracycline, MIC minimum inhibitory concentration, ESBL/AmpC extended-spectrum β-lactamase/AmpC β-lactamase, ST sequence type, PFGE pulsed-field gel electrophoresis
Moreover, ESBL/AmpC β-lactamase-harboring K. pneumoniae was found in different proportions of various age groups. Compared with the sample level, more than half of K. pneumoniae (57.1%, 12/21) from the young age group (< 1 year) carried ESBL/AmpC β-lactamase, while other groups contained the remains: 6–10 years (33.3%, 12/36), > 11 years (15.6%, 5/32), and 1–5 years (8.9%, 5/56).
In total, four different blaCTX−M and two AmpC genes were detected. Among the blaCTX−M, blaCTX−M−15 (n = 20) was predominantly detected, followed by blaCTX−M−65 (n = 5) and blaCTX−M−55 (n = 3). Of the AmpC-producing isolates, both blaDHA−1 and blaCMY−2 were mostly detected in the diarrheal fecal samples of dogs and cats. Interestingly, the co-occurrence of different blaCTX−M genes (n = 4) and blaCTX−M and blaDHA−1 genes (n = 7) was also identified. Moreover, most ESBL and/or AmpC producers were resistant to other non-β-lactam antimicrobials, including fluoroquinolones, tetracyclines, and chloramphenicol.
The virulence factors iucA, terB, and irp2 were detected in 40% (16/40) of K. pneumoniae isolates (Table 5). In particular, biomarkers for hypervirulent iucA and irp2 were detected in all kinds of samples except digestive organs. The prevalence of virulence factors was different by the samples. In dogs, 80% (4/5) of isolates from skin carried virulence factors; however, only 18.2% (2/11) of isolates from digestive samples possessed them. Isolates from digestive and respiratory samples carried mainly terB, while skin isolates carried irp2. Of note, both terB and irp2 were identified in one cat respiratory isolate.
The MLST analysis showed that a total of 18 STs were observed among 40 K. pneumoniae isolates. Of them, two STs comprised about 40%, with ST307 (22.5%) predominantly detected, followed by ST15 (17.5%), found at 15 hospitals in five megacities. Moreover, ST709, ST37, and ST392 were identified in 17.5% of the isolates. These five STs comprised 57.5% of isolates. ST307, ST15, ST709, and ST37 were detected in both dogs and cats. However, ST395, ST8214, and ST8217 were detected in cats, and others were detected in dogs. Notably, among the ST307, 44.4% (4/9) carried virulence factor irp2.
The PFGE analysis indicated that 30 distinct patterns were present among the isolates (Table 5 and Supplementary Fig. 1). Moreover, identical PFGE and STs (ST15-P26 and ST15-P27) were observed in the same (M) hospitals. Interestingly, similar PFGE and STs (ST307-P14) were also observed in dogs and cats from different hospitals across various provinces in different years.
Discussion
Our findings showed that a significant portion of the K. pneumoniae isolates demonstrated multiple drug resistance and carried ESBL/AmpC genes, mostly blaCTX−M−15 and blaDHA−1. Moreover, specific clones, ST307 and ST15, and the virulence factors, terB and irp2, were predominantly detected.
K. pneumoniae isolates obtained from companion animals often exhibited resistance to different antimicrobials. We found tetracycline resistance was detected in 37.5% of the isolates. This finding is consistent with previous investigations in China (50%) [23], South Africa (35%) [24], and Portugal (88%) [25], showing that a significant proportion of K. pneumoniae strains isolated from companion animals demonstrated tetracycline resistance. Similarly, resistance rates for cefazolin (30%) concurred with previous investigations conducted in Korea (57.1%) [26] and Portugal (53%) [25]. Moreover, ≤ 30% of K. pneumoniae isolates from dogs and cats showed resistance to most of the antimicrobials, including some critically important cephalosporins and quinolones, aligned with studies from China [27], India [28], and Germany [29]. On the contrary, resistance rates for amikacin and amoxicillin/clavulanic acid were relatively meager (≤ 15%), concordant with the previous reports in Korea [26] and Spain [30]. Nonetheless, resistance to these antimicrobials may present a considerable health risk to both humans and animals [31].
Antimicrobial resistance in K. pneumoniae isolates varied according to their different origin sites. Our findings demonstrated that resistance to most antimicrobials was much higher in dog isolates of non-digestive system origin, including urine, reproductive system, skin/ear, and respiratory tracts, than in digestive system-origin isolates. Similarly, resistance rates of these antimicrobials were higher in non-digestive compared to digestive isolates in cats. This finding aligned with prior investigations, demonstrating that urogenital, skin/ear, and respiratory system isolates from companion animals showed significant resistance to various antimicrobials [27, 32]. The reasons might be the collective excessive use or misuse of antimicrobials for treating infections in these systems caused by K. pneumoniae in dogs and cats [33].
MDR was detected in a significant proportion of K. pneumoniae isolates in the current investigation. Our findings concur with those of the previously published reports, revealing that K. pneumoniae isolated from dogs and cats showed MDR features [27]. Moreover, diverse MDR patterns were observed that encompass different classes of antimicrobials, including critically important ones, consistent with the previous study [33]. Particularly, a considerable portion of the K. pneumoniae isolates demonstrated resistance to five or more antimicrobials. Hence, the development and spread of MDR K. pneumoniae in dogs and cats are increasing concerns, restricting treatment options and complicating the control of antimicrobial resistance.
In this study, four distinct types of blaCTX−M genes were detected: blaCTX−M−15 was the most prevalent, followed by blaCTX−M−65 and blaCTX−M−55. blaCTX−M−15 is among the most frequently identified ESBL types in K. pneumoniae isolates from companion animals, documented worldwide, including in Korea [26], Finland [18], and Germany [29]. Moreover, blaCTX−M−15-carrying K. pneumoniae has frequently been identified in human clinical isolates [34, 35]. Similarly, blaCTX−M−65-bearing K. pneumoniae has increased in dogs and cats [33]. In addition, recently, it was found that in humans and animals, blaCTX−M−55-harboring K. pneumonia has augmented globally [29, 32, 36]. Of the AmpC-producing isolates, the blaDHA−1 gene comprised the predominant portion, followed by blaCMY−2. These genes confer resistance to many β-lactam antimicrobials, including third-generation cephalosporins, frequently detected in humans and companion animals in different geographical locations [10, 26, 37]. Furthermore, the co-occurrence of blaCTX−M with AmpC genes blaDHA−1 or blaCMY−2 was detected in K. pneumoniae isolates, corroborating with previous studies [7, 38]. In addition, it has been demonstrated that ESBL/AmpC-producing Enterobacterales can be transmitted from companion animals to humans via direct contact, complicating treatments [39]. These findings underscore the necessity of coordinated management of ESBL and/or AmpC-producing K. pneumoniae in humans and other animals.
Our findings indicated that the majority of the ESBL and/or AmpC-producing isolates showed resistance to additional non-β-lactam antimicrobials, including fluoroquinolone, tetracycline, and chloramphenicol, corroborating prior research that demonstrated K. pneumoniae can co-exist various antimicrobial resistances with bla genes [27]. It was found that ESBL or AmpC genes can co-occur with the floR gene (which imparts chloramphenicol resistance), the tetA gene (which confers tetracycline resistance), and the gyrA (gene for fluoroquinolone resistance), correlating with an increased level of resistance to these antimicrobials [40].
In this study, virulence gene terB was predominantly detected in digestive and respiratory K. pneumoniae isolates, and irp2 in skin and respiratory. terB is among the important hypervirulence genes widely detected in respiratory K. pneumoniae from humans and companion animals, contributing to its pathogenicity [41, 42]. Moreover, the presence of the irp2 gene, which is associated with iron metabolism, also facilitates the synthesis of enterobactin, mediating the iron acquisition in Enterobacterales [43]. This factor can also be linked to the high pathogenicity of some K. pneumoniae [44]. Furthermore, specific virulence factors in a specific clone could trigger the incidence of their infection. Hyeon et al. [45] showed that K. pneumoniae ST307 possessed the gene for an iron uptake virulence factor (e.g., irp2), making it hypervirulent, which causes bacteremia in dogs by triggering its gastrointestinal translocation to the bloodstream.
In our investigation, siderophore aerobactin, iucA, was identified in one K. pneumoniae isolate from urine. Aerobactin, a crucial virulence factor frequently found in K. pneumoniae and regarded as an efficient marker for this siderophore in hypervirulent K. pneumoniae [46]. This has been identified in K. pneumoniae isolated from various disorders, including renal disease [47]. In a study conducted in Korea, hypervirulent K. pneumoniae exhibited the presence of iucA associated with renal abscesses [48]. An investigation in Spain reported a high prevalence of the iucA gene in K. pneumoniae isolated from urine [49]. Zhang et al. [50] identified the iucA gene in 5.7% of K. pneumoniae isolates from cats and dogs in China. In India, iucA was detected in human clinical K. pneumoniae isolates in a tertiary care hospital [51].
We found various STs in K. pneumoniae, suggesting the acquired resistance individually. Nonetheless, among different STs, ST307 and ST15 were predominantly detected in five megacities. Especially, blaCTX−M-carrying K. pneumoniae ST307 has been identified as a prevalent clone in dogs and cats, indicating its widespread dissemination in animals [10]. The predominant prevalence of K. pneumoniae ST307, harboring the blaCTX−M−15 gene, was also reported in Korean companion animals [45]. Moreover, this clone is frequently detected in human clinical isolates in different countries [52, 53]. In Korea, ST307 was also increased in human isolates [54]. The predominant presence of specific clones indicates that resistant K. pneumoniae is spreading within different regions of South Korea and globally, attributed to the expansion and dissemination of particular lineages with similar genetic characteristics, as well as the enhanced mobility of animals and humans across geographical boundaries. However, epidemiological studies are still needed to clarify their transmission between humans and companion animals. Notably, multidrug-resistant K. pneumoniae ST307, harboring the blaCTX−M−15 and irp2, was mainly detected in isolates from skin infections, sampled in three cities. K. pneumoniae ST15 carrying the blaCTX−M−15 gene is recurrently detected in companion animals, consistent with our investigation [55]. Furthermore, K. pneumoniae from the other three STs, ST709, ST37, and ST392, were identified in isolates from dogs and cats harboring ESBL or AmpC genes [26, 27]. Among them, K. pneumoniae ST392, a global clone, has been predominantly identified in humans and companion animals [56–58]. Identical MLST types and PFGE patterns from different dogs were observed in the same hospital. Additionally, certain identical PFGE and STs were identified in dogs and cats in different hospitals, suggesting the introduction of these K. pneumoniae to the hospitals with the animals.
Conclusion
In conclusion, this investigation demonstrates significant insight into the occurrence and characteristics of K. pneumoniae isolates obtained from diseased dogs and cats in South Korea. The prevalent presence of ESBL/AmpC genes and virulence factors was found among the isolates. Moreover, specific clones, including ST307 and ST15, were predominantly detected, which may facilitate the transmission of particular K. pneumoniae strains among the companion animals. These findings indicate that companion animals can act as a reservoir of antimicrobial-resistant K. pneumoniae that could be spread to humans or other animals, which warrants further assessment. In addition, strict regulation of antimicrobial usage in companion animals and regular surveillance are necessary to prevent the dissemination of antimicrobial-resistant K. pneumoniae between companion animals and humans.
Limitations and future perspectives
It is noted that there are some shortcomings in this study. The small sample size and proportion of isolated K. pneumoniae from dogs (n = 130) outweigh those from cats (n = 30), which makes it challenging to compare the results between them. Furthermore, the lack of antimicrobial usage history in the animals incorporated for this investigation makes them prone to reporting bias, which should be carefully considered in future studies. In addition, whole-genome sequence analysis remains to be performed to compare the genetic characteristics among the isolates, including the presence of various virulence factors.
Supplementary Information
Additional file 1: Number of recovered Klebsiella pneumoniae isolates from four targeted samples of different age groups of dogs.
Additional file 2: Number of recovered Klebsiella pneumoniae isolates from four targeted samples of different age groups of cats.
Additional file 3: Tested range and breakpoint of the assessed antimicrobials against Klebsiella pneumoniae isolated from dogs and cats during 2018–2023 in South Korea.
Additional file 4: List of primer sequences and polymerase chain reaction (PCR) conditions.
Additional file 5: XbaI-digested pulsed-field gel electrophoresis band profiles of Klebsiella pneumoniae isolates (n = 40) obtained from diseased dogs and cats during 2018–2023 in South Korea.
Acknowledgements
Not applicable.
Authors’ contributions
S.-K.L., B.-Y. M., Y.-J. H. and Y.-H.L.: Conceptualization; M.S.A., Y.-J.H., Y.-H.L., J.-H.C., Y.-K.L., H.-S.K., J.-I.K., B.-Y. M., and M.Y.K.: Methodology; M.S.A., B.-Y. M., and S.-K.L.: Data curation; B.-Y. M., Y.-J. H., Y.-H.L., and M.S.A.: Writing – original draft; B.-Y.M., S.-K.L., and J.-M. K.: Writing – review & editing; S.-K.L.: Fund acquisition. All authors read and approved the final version of the manuscript for publication.
Funding
This study was supported by the Animal and Plant Quarantine Agency, Ministry of Agriculture, Food, and Rural Affairs, Republic of Korea [Grant number: B-1543081-2024-26-01].
Data availability
The data produced for this study are within the article/supplementary materials.
Declarations
Ethics approval and consent to participate
No ethical approval was deemed necessary for this study as it did not involve direct experimentation on animals. Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Yu-Jeong Hwang and Yeon-Hee Lee contributed equally to this work.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Additional file 1: Number of recovered Klebsiella pneumoniae isolates from four targeted samples of different age groups of dogs.
Additional file 2: Number of recovered Klebsiella pneumoniae isolates from four targeted samples of different age groups of cats.
Additional file 3: Tested range and breakpoint of the assessed antimicrobials against Klebsiella pneumoniae isolated from dogs and cats during 2018–2023 in South Korea.
Additional file 4: List of primer sequences and polymerase chain reaction (PCR) conditions.
Additional file 5: XbaI-digested pulsed-field gel electrophoresis band profiles of Klebsiella pneumoniae isolates (n = 40) obtained from diseased dogs and cats during 2018–2023 in South Korea.
Data Availability Statement
The data produced for this study are within the article/supplementary materials.