In recent years, the escalating public concerns over the evolution and spread of bacteria resistance between livestock and humans have underscored the need for surveillance on the judicious use of antibiotics in food-producing animals (1–3). However, the aggressive use of prophylactic and therapeutic antimicrobials during intensive rabbit production may facilitate population-level transfer of antibiotic resistance in farming environments (4, 5). These inadvertent consequences could potentially expedite the acquisition of antibiotic resistance and virulence among commensal bacteria in gastrointestinal tract (6), such as Enterobacter spp. and Enterococcus spp. (7), thereby contributing to gut microbiota dysbiosis and intestinal barrier impairments (8). Several genotypes of opportunistic E. coli or Enterococci strains have been confirmed as causative agents in newborn or weaning rabbits with diarrhea (9, 10), which urge us to explore the emergence and distribution of antibiotic resistance among the bioindicators derived from healthy rabbit population.

Although the restriction on the use of antibiotic feed additives as growth promoter in food-producing animals has significantly reduced the burden of bacterial antimicrobial resistance (AMR) on food-chains and ecosystems (11, 12), the inappropriate use of therapeutic antibiotics, such as tetracyclines, chloramphenicols and β-lactams (4, 5), remains an overlooked attention to the transferable antibiotic resistance resulting from antibiotics stress or bacteria contamination in food animal production systems. Since the emerging “superbugs” that exhibit resistance to critically important antimicrobials (CIAs), such as extended spectrum beta-lactamases (ESBL) (like blaTEM, blaCTX-M, blaSHV), as well as phosphoethanolamine (pEtN) transferase (like mcr-1and mcr-2) producing E. coli strains, high-level gentamicin-resistant (HLGR) and vancomycin-resistant Enterococcus spp. strains, have been frequently reported in domestic animals (13–17), the relationship between antibiotic use and antibiotic resistance distribution should be continously monitored, particularly in the case of neglected healthy farmed rabbits.

Limited epidemiological studies have reported the presence of antibiotic-resistant E. coli and Enterococcus spp. isolates in domestic or wild rabbit populations (18–21), exhibiting diverse AMR spectrums and distinctive ARG distribution across different geographical areas. However, there is a lack of knowledge regarding updated epidemiological data in Chengdu City, which is one of largest markets for meat-rabbit production and consumption in Southwest China. Therefore, this investigation aims to characterize the antibiotic resistance in indicator E. coli and Enterococcus spp. strains derived from healthy meat-rabbits. The findings will enhance our understanding of the emergence of antibiotic resistance conferred by routinely used therapeutic antibiotics in a context of reduced antibiotic use and residues in food animals, thereby raising awareness about rational antibiotic use to combat the rising threats posed by antibiotic resistance in rabbit farming practices.

It is widely recognized that symbiotic E. coli and Enterococcus spp. play a crucial role in maintaining intestinal homeostasis and integrity (28). However, they are also prone to acquiring antibiotic resistance and harboring antimicrobial resistance genes (ARGs) when repeatedly exposed to antibiotics, particularly in intensive food-animal husbandry (6, 7). Moreover, these antibiotic-resistant commensal bacteria have been reported as opportunistic pathogens causing diarrhea among rabbits (9, 10), and they even act as negative mediators to shield the antibiotic-sensitive pathogens during antibiotic treatment (29). Therefore, it is imperative to conduct surveillance on these bioindicators to assess the potential challenge posed by transferable antibiotic resistance for animal health.

Considering that commercial meat-rabbits may receive prophylactic or therapeutic antimicrobials to address suspected bacterial infection, such as respiratory or gastrointestinal infectious diseases, either through direct contact or environmental contamination (5, 30), which may significantly changes the composition of gastrointestinal microbiota (31) and antimicrobial resistance (AMR) phenotypes of commensal E. coli and Enterocuccus spp. strains (32). In this study, we successfully isolated two bacteria species from rabbit feces with a higher identification rate for Enterococcus spp. strains (210/247, 85%) compared to E. coli strains (75/247, 30.4%), indicating the dominant presence of Enterococcus spp. in the fecal flora under local farming conditions. However, this lower recovery rate, possibly attributed to the reduced bioactivity of E. coli during feces processing and cell culture, should also be considered when compared with high isolation rates (57.1~100%) observed in other rabbit populations (19–21). Despite this discrepancy, an adequate number of E. coli and Enterococcus spp. strains were obtained for subsequent analysis.

We conducted an investigation on antibiotic resistance and relevant ARG distribution among these bacteria, and evaluated their susceptibility to seven available antimicrobials in two rabbit farms. A significant proportion of E. coli strains exhibited resistance to most antimicrobials (6/7) with diverse AMP spectrums, in contrast to Enterococcus spp. strains (3/7). This observation supports the previous finding that Enterobacter spp. harbor more mobile genetic elements (MGEs), facilitating robustly horizontal transfer of ARGs and acquisition of antibiotic resistance against specific antimicrobials (33). Notably, most strains of E. coli demonstrated reduced susceptibility to these antimicrobials, suggesting the need to consider alternative antibiotic prescription in light of the prevalence of DOX and AMP-resistant phenotypes. In addition, it is worth mentioning that no E. coli strain demonstrated resistance to PMB, which may be associated with less frequent medication. Although oral administration of ampicillin and amoxicillin/clavulanate may pose a risk of antibiotic-associated dysbiosis in certain rabbit populations (5), parenteral administration is still recommended for susceptible pathogens (18, 20). Moreover, antibiotics from the penicillin family appear to be more readily available for veterinary use in local rabbit farming practices, which may also influence the choice of antibiotic prescription and the prevalence of relevant antimicrobial resistance. Similarly, the frequent use of DOX and the sporadical use of PMB as informed by farm owners may influence the antibiotic susceptibility of E. coli strains.

Interestingly, most Enterococcus spp. strains only exhibited resistance to DOX and GEN, while no resistant strains against other antimicrobials were found. This disparity between Enterobacter spp. and Enterococcus spp. may be associated with their distinctive evolutionary pathways in antibiotic resistance against specific antimicrobials (34, 35). In particular, the difference in AMR spectrums between two farms was evident, possibly reflecting the preference of antibiotic prescriptions for veterinary technicians in farming practices, although the detailed medical history of antibiotic use was not comprehensively evaluated. Furthermore, bacteria of young rabbits origin exhibited a higher prevalence of antibiotic resistance compared to their adult counterparts, indicating that early-life exposure to antibiotics may robustly contribute to the emergence of antibiotic resistance among commensal bacteria (36). Therefore, reducing the inappropriate use of antimicrobials and medication overload will be a sustainable meat-rabbit farming practice.

According to the MIC values tested for resistant bacteria, increased levels of antibiotic resistance was prevalent, which may be inferred to the substantial exposure to antibiotics for rabbits in farming environments (30, 37). Furthermore, all GEN-resistant Enterococcus spp. strains showed high-level gentamicin resistance, whereas this characteristic was absent in GEN-resistant E. coli strains. This implies that Enterococcus spp. may serve as an active reservoir for acquired GEN resistance, which has also been detected in European wild rabbits (19). Previous research studies have indicated that patients with HLGR Enterococcus spp. strains of hospital origin could increase the risk of nosocomial infections, particularly the E. faecalis strains that typically harbor the aac[6']-Ie-aph[2”]-Ia gene (38, 39), and even produce cross-resistance with other critical important antibiotics (CIAs), such as vancomycin (40). It is important to note that the vancomycin-resistant Enterococcus spp. have been confirmed in food-producing animals (14, 15). Thus, increasing public attention should be paid toward these opportunistic pathogens of animal origin.

Several surveillance studies have reported a high prevalence of multi-resistant E. coli strains in farmed rabbits (10, 19–21). However, a similar occurrence (35/66, 53.0%) was still observed in our healthy rabbits. Additionally, we also identified a proportion of double-resistant Enterococcus spp. strains (40/164, 24.4%). As expected, most of multi-resistant E. coli strains carried ARGs associated with β-lactams (blaTEM, 34/35, 97.1%), tetracyclines (tetA, 33/35, 94.3%), fluroquinoloncs (qnrS, 25/35, 71.4%), and chloramphenicols (floR, 20/35, 57.4%) resistance. On the other hand, double-resistant Enterococcus spp. strains predominantly harbored the aac[6']-Ie-aph[2”]-Ia gene (31/40, 77.5%). Similar ARG distribution among resistant E. coli or Enterococcus spp. has been also found in farmed or wild rabbits, which may be attributed to environmental antibiotics co-selection (9, 15, 19, 41). Particularly, some resistant bacteria only carried a small number of ARGs (for example, fexA, qnrD, tetB, tetM, and aac[6′]-lb) or none at all, suggesting that certain ARG-independent mechanisms for antimicrobial resistance may be implicated for these bacteria (34, 35). It is well known that co-harboring various ARGs is one of the most important mechanisms to acquire antibiotic resistance (1, 12, 13); thus, the wide spread of antibiotic-specific ARGs among commensal E. coli and Enterococcus spp. strains may confer their resistance against commonly used antimicrobials, although other strain-level mobile genetic elements, including transposons, insertion sequences, and integrons (42), have not been evaluated in current study. In general, the commensal bacteria bearing abundant ARGs may serve as a high-risk reservoir of transferable antibiotic resistance, which pose unintended threats to rabbit welfare, farming biosecurity, and public health in a “One Health” perspective.

One limitation of the current study is the unexpanded AMR spectrum analysis for other groups of antibiotics, such as cephalosporins (e.g., ceftiofur), macrolides (e.g., azithromycin), sulfonamides (e.g., sulfadiazine/trimethoprim), and tricyclic glycopeptides (e.g., vancomycin), although they are rarely or never used in local rabbit farming practices. Second, the influence determinants involving antibiotic usage and consumption, bacteria genetic lineages, and biosecurity measures (15, 30, 31, 37) should be correlated with the emergence and enhancement of antibiotic resistance in further study. Even so, our findings provided a novel perspective on the impact of commonly used antimicrobials in shaping distinctive AMR spectrums and the increasing level of antibiotic resistance among indicator bacteria in meat-rabbit farming practices.

Overall, the distribution of AMR and ARGs is prevalent in healthy meat-rabbits, and the use of therapeutic antimicrobials in farming practices may promote the antibiotic resistance transmission among indicator bacteria. Therefore, periodic surveillance of antibiotic resistance in different geographic locations and supervisory measures for rational antibiotic use are considered to be imperative strategies for combating the rising threats posed by antibiotic resistance and maintaining rabbit welfare and public health.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

CS: Writing – original draft, Writing – review & editing, Formal analysis. ZW: Investigation, Writing – original draft, Writing – review & editing. YL: Formal analysis, Methodology, Writing – review & editing. JH: Conceptualization, Funding acquisition, Writing – review & editing.