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Enterococci, which naturally colonize the gastrointestinal tract of humans and animals, have emerged as significant healthcare-acquired pathogens (1). In veterinary medicine, Enterococcus spp. represents the third most prevalent pathogen in dogs and cats according to the China Antimicrobial Resistance Surveillance Network for Pets (CARPet) (2). These bacteria serve as reservoirs of antimicrobial resistance (AMR), presenting a public health concern through potential human transmission (3-4). The emergence of linezolid-resistant enterococci is particularly concerning as it limits therapeutic options. This study revealed enterococci in 14.39% of clinical samples from dogs and cats across China, with Enterococcus faecium (E. faecium) and E. faecalis identified as the predominant species. E. faecium exhibited higher resistance rates to daptomycin, rifampin, doxycycline, and amoxicillin-clavulanate compared to E. faecalis. Multidrug-resistant enterococcal infections were significantly associated with urinary tract catheterization history and extended hospitalization duration. Whole genome sequencing (WGS) analysis demonstrated genetic similarities between linezolid-resistant E. faecium isolates from this study and other pet isolates, while E. faecalis showed broader genetic relationships across various sources. To mitigate infection risks, particularly considering the potential for pet-to-human transmission of linezolid-resistant E. faecalis, enhanced aseptic practices and reduced hospitalization periods in veterinary clinics are recommended.
This study collected Enterococcus spp. from clinical samples of dogs and cats across 20 Chinese provinces and municipalities (2018–2021) through the CARPet surveillance system. Clinical samples and medical records were obtained from diseased animals at regional veterinary hospitals, with veterinarians collecting site-specific infected specimens. Antimicrobial susceptibility testing was conducted following CLSI guidelines (CLSI, VET01) using broth microdilution, with E. faecalis ATCC 29212 as the quality control strain. The antimicrobial panel included amoxicillin-clavulanate, doxycycline, azithromycin, rifampin, florfenicol, enrofloxacin, linezolid, vancomycin, and daptomycin. Results were interpreted according to CLSI VET01S and M100 breakpoints. Multidrug-resistant (MDR) isolates were defined as those resistant to three or more antimicrobial classes (5). Risk factors for MDR infections were evaluated using univariate analysis and logistic regression SPSS (version 22.0, International Business Machines Corporation, Armonk, USA). WGS of linezolid-resistant Enterococcus spp. isolates was performed using the Illumina HiSeq X Ten platform. Draft genomes were assembled using SPAdes and analyzed for sequence types, antimicrobial resistance genes, and virulence genes using SRST2. Phylogenetic analysis compared pet-derived isolates with human, animal, and environmental isolates from China available in the NCBI database (2018–2021). Phylogenetic clusters were determined using fastBAPS software (https://github.com/gtonkinhill/fastbaps). The genome assemblies were deposited under BioProject accession no. PRJNA1039340.
From 2018 to 2021, surveillance across 20 Chinese provinces identified Enterococcus spp. in 460 (14.39%) of 3,197 clinical samples (2,247 canine and 950 feline). The highest detection rates were observed in ascites (24.00%), hepatobiliary system (22.00%), and urinary tract (18.68%) specimens. Feline samples exhibited a significantly higher detection rate (22.53%) compared to canine samples (10.95%) (
Supplementary Figure S1 ). Among the 477 Enterococcus spp. isolates recovered, E. faecium (45.49%) and E. faecalis (43.40%) were predominant, with E. gallinarum, E. avium and others comprising the remainder (Figure 1). While most isolates originated from Beijing (n=413), others were distributed across Shanghai, Inner Mongolia, Liaoning, Fujian, Jiangsu, and 13 additional provinces (Supplementary Table S1 ).
Figure 1.Distribution of Enterococcus (E.) spp. isolates in various samples. (A) From dogs; (B) From cats.
Antimicrobial susceptibility testing revealed significantly higher resistance rates in E. faecium compared to E. faecalis for amoxicillin-clavulanate (86.75% vs. 1.14%), doxycycline (81.33% vs. 57.14%), rifampin (77.11% vs. 63.43%), and daptomycin (7.83% vs. 1.14%) (P<0.01) (Tables 1–2). Linezolid resistance was detected in 6.29% of E. faecalis and 5.42% of E. faecium isolates. All isolates were vancomycin-susceptible except for one intermediate E. faecium (Tables 1–2). Multidrug resistance was more prevalent among feline isolates (48.84%) than canine isolates (26.92%), with E. faecium exhibiting a substantially higher MDR rate (78.31%) compared to E. faecalis (4.57%).
Antimicrobial agents Number of Enterococcal faecalis corresponding to MIC (mg/L) No. of resistant isolates (%) 0.064 0.125 0.25 0.5 1 2 4 8 16 32 64 Amoxicillin-clavulanate (2:1) 6 22 126 16 3 − − 2 2 (1.14) Doxycycline 12 8 4 2 1 4 44 76 24 100 (57.14) Azithromycin 1 − − 3 14 30 8 119 − Florfenicol 6 42 93 20 − − 14 − Enrofloxacin − 1 32 91 12 − 1 38 − Rifampin 6 4 18 36 54 49 8 111 (63.43) Vancomycin 2 121 49 3 − − − − 0 (0) Linezolid 4 40 111 9 11 − − 11 (6.29) Daptomycin 27 74 52 20 1 1 − 2 (1.14) Note: The gray-shaded areas indicate untested antimicrobial concentrations. For isolates showing no growth at any concentration, the lowest MIC value was assigned. For isolates exhibiting growth at all tested concentrations, the next higher MIC value above the highest tested concentration was assigned (black numbers on gray background). MIC values for amoxicillin-clavulanic acid (2:1) are reported as amoxicillin MIC values. Color coding indicates susceptibility categories: susceptible (green), intermediate (yellow), and resistant (red).
Abbreviation: MIC=minimum inhibitory concentration.Table 1. Minimal inhibitory concentrations and resistance rates of canine and feline Enterococcus faecalis isolates.
Antimicrobial agents Number of Enterococcal faecium corresponding to MIC (mg/L) No. of resistant isolates (%) 0.064 0.125 0.25 0.5 1 2 4 8 16 32 64 Amoxicillin-clavulanate (2:1) 2 8 5 1 4 2 2 142 144 (86.75) Doxycycline 12 16 1 1 2 − − 39 95 134 (80.72） Azithromycin 1 1 − − 3 10 10 141 − Florfenicol 1 14 94 38 2 4 13 − Enrofloxacin − − − 5 6 3 6 146 − Rifampin 22 1 3 12 45 58 25 128 (77.11) Vancomycin 24 117 13 11 1 − − − 0 (0) Linezolid − 19 118 21 3 5 8 (4.82) Daptomycin 9 15 60 69 8 5 13 (1.81) Note: The gray-shaded areas indicate untested antimicrobial concentrations. For isolates showing no growth at any concentration, the lowest MIC value was assigned. For isolates exhibiting growth at all tested concentrations, the next higher MIC value above the highest tested concentration was assigned (black numbers on gray background). MIC values for amoxicillin-clavulanic acid (2:1) are reported as amoxicillin MIC values. Color coding indicates susceptibility categories: susceptible (green), intermediate (yellow), and resistant (red).
Abbreviation: MIC=minimum inhibitory concentration.Table 2. Minimal inhibitory concentrations and resistance rates of canine and feline Enterococcus faecium isolates.
Among 203 enterococcal isolates with complete case information, 76 were classified as MDR. Multivariate analysis initially considered four variables (P<0.05): duration of hospitalization, pet species, pet sex, and history of urinary tract catheterization. The final model, after backward selection, retained only two significant predictors: history of urinary tract catheterization (P=0.03) and duration of hospitalization (P=0.04). These findings indicate that pets with previous urinary tract catheterization or extended hospitalization periods had significantly higher risks of MDR Enterococcus infection (
Supplementary Figure S2 ).Analysis of 19 linezolid-resistant isolates, comprising 11 E. faecalis (LREfs) and 8 E. faecium (LREfm), revealed the presence of the optrA gene in all specimens. The LREfs represented eight distinct sequence types (STs). Phylogenetic analysis, incorporating 81 LREfs from the NCBI database, revealed distribution across six lineages with mixed-source isolates. Notably, seven LREfs showed close genetic relationships (19–94 SNPs) with isolates from humans, pets, farm animals, and plants, suggesting cross-species transmission potential (Figure 2A). The eight LREfm belonged to four known STs, with ST80 being predominant (n=4). Phylogenetic analysis of 56 isolates (including 48 from NCBI) identified three distinct lineages, with seven studied isolates clustering in lineage 3. Two isolates (20928 and 21196) from a cat and dog at the same hospital exhibited remarkable genetic similarity, differing by only five SNPs (Figure 2B), indicating clonal spread among pet isolates distinct from other sources.
Figure 2.Phylogenetic tree of (A) linezolid-resistant Enterococcus faecalis and (B) linezolid-resistant Enterococcus faecium from pets in this study and humans, pets, farm animals, food, environments, and plants available from NCBI based on the core genome analysis.
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This study provides a comprehensive analysis of enterococcal infections in Chinese veterinary clinics, encompassing diverse clinical samples from dogs and cats. Our findings, supported by CARPet surveillance data, revealed that Enterococcus spp. were present in 14.39% of clinical samples, establishing them as the third most prevalent bacterial pathogens in companion animals (2). This prevalence aligns with Korean data (19.3%) (6) and parallels reports from European countries, where Enterococcus spp. rank among the top five clinical pathogens in Spain (7), Portugal (8), and the Iberian Peninsula (9), comprising 5.6%–15.0% of isolates. Similarly, in Chinese human clinical settings, Enterococcus spp. account for 8.89% of isolates, ranking fourth (10). These parallel findings underscore the significance of Enterococcus as a pathogen in both veterinary and human medicine, emphasizing the critical need for enhanced surveillance protocols.
The emergence of antimicrobial resistance in Enterococcus spp., whether through genetic mutations or mobile genetic elements, presents significant therapeutic challenges. Our study revealed that over 50% of isolates demonstrated resistance to doxycycline or exhibited elevated MICs for azithromycin and enrofloxacin, consistent with findings (43.2%–99.0%) from Japan (11), Australia (12), the UK (13), and Sweden (14). While enrofloxacin remains exclusive in veterinary medicine, both doxycycline and azithromycin are crucial antimicrobials in human and veterinary treatment of urinary and respiratory infections. Of particular concern, 86.75% of E. faecium isolates showed resistance to amoxicillin-clavulanate, a primary therapeutic option in both veterinary and human medicine. Encouragingly, resistance rates to last-resort antimicrobials — vancomycin, linezolid, and daptomycin — remained low (0–6.29%), preserving their efficacy against multidrug-resistant infections. These findings emphasize the necessity for systematic antimicrobial susceptibility testing and resistance monitoring in veterinary practice.
Extended hospitalization significantly increases the risk of MDR enterococcal infections in companion animals, potentially through environmental transmission within hospital settings. Previous studies have demonstrated the potential for resistant Enterococcus spp. clone transmission within veterinary facilities through multiple vectors, including infected dogs, their owners, veterinary personnel, and hospital environmental surfaces (6). Similarly, pets requiring urinary tract catheterization exhibit elevated risks of MDR infections, potentially due to suboptimal aseptic technique during catheter placement and maintenance. The increased risk of healthcare-associated infections through invasive procedures is well-documented in veterinary medicine, emphasizing the critical importance of implementing rigorous cleaning and disinfection protocols to prevent MDR pathogen transmission (15).
The presence of MDR bacteria in companion animals, including colistin-resistant and ESBL-producing E. coli (16) and methicillin-resistant staphylococci (17-18), represents a substantial transmission risk to pet owners. Companion animals can serve as reservoirs for antibiotic-resistant enterococci, facilitating their dissemination across human, animal, and environmental interfaces. Our genomic analyses revealed that LREfs demonstrate high genetic similarity across human, animal, and environmental isolates, whereas LREfm exhibits closer genetic relationships primarily among pet isolates. This pattern suggests that LREfs have achieved broader host adaptation and dissemination compared to LREfm, indicating potentially higher transmission risks between pets and their owners.
Study limitations include the restricted whole genome sequencing analysis of only linezolid-resistant isolates, which constrains our ability to comprehensively evaluate transmission patterns. Furthermore, the geographical distribution of samples was notably skewed, with 86.58% of pet-derived enterococci originating from Beijing, highlighting the need for broader surveillance across China to accurately assess national prevalence patterns.
This investigation establishes E. faecium and E. faecalis as the predominant enterococcal species in Chinese veterinary clinical settings. The study demonstrates that multidrug-resistant enterococcal infections correlate significantly with urinary catheterization procedures and extended hospitalization periods. To mitigate MDR transmission, we recommend implementing dedicated isolation facilities for infected animals, establishing rigorous cleaning and disinfection protocols, and ensuring thorough sterilization of medical instruments. Additionally, enhanced veterinary staff training in aseptic techniques and evidence-based antimicrobial selection, guided by pathogen identification and susceptibility testing, is crucial. Sustained surveillance efforts are essential to prevent the bidirectional transmission of MDR organisms between companion animals and humans.
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Supported by the National Key Research and Development Program of China (2022YFD1800400), National Natural Science Foundation of China (81991531, 32002339), 2115 Talent Development Program of China Agricultural University.
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We are grateful to Yang Liu, Yinying Lou, Beibei Liang, Tianli Xie, and Yunke Chen at Beijing Zhongnongda Veterinary Hospital Co., Ltd for their invaluable assistance in sample collection.
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