The coexistence between humans and animals has been widely acknowledged since ancient times, gaining particular attention in recent years due to various factors that pose potential risks to public health. (1) With their greater mobility, cats face higher exposure to zoonotic pathogens than other pets. (2−4) The gut microbiota significantly influences the cat’s health, impacting the pathogen’s transmission and antibiotic resistance. (5,6)

Researchers have identified bacterial (7) and viral (8) zoonotic pathogens in cats. Notably, bacteriological studies show that 75% of cat bite incidents reveal Pasteurella species, which pose health risks to humans. (9) Urban environments are critical in the spread of these pathogens, exemplified by bacteria like Bartonella henselae, (10) presenting significant concerns for public health and urban ecology. Moreover, the diversification of Clostridioides difficile and Clostridium perfringens, categorized as zoonotic pathogens, has been documented in cats and other animals. (11,12) Urban cat populations, notably free-roaming cats, are a significant source of pathogens and their dispersion. (13)

Additionally, studies on antibiotic resistance in cats and dogs indicate the presence of ARGs and mobile genetic elements (MGEs), underscoring the risk of transmitting resistant bacteria from pets to humans. (14,15) The growing population of pet animals highlights the importance of understanding the epidemiology of bacteria in cats, particularly concerning the spread of antibiotic-resistant bacteria and zoonotic transmission. (16,17) Research has focused on the genetic mechanisms of antibiotic resistance, revealing MGEs within the feline gut that complicate the issue, although knowledge gaps persist. (18,19) Identifying gut microbial taxa linked to antibiotic resistance remains limited. Nonetheless, some bacterial groups have been referenced as potential contributors to antibiotic resistance. (20)

The misuse of antibiotics has become prevalent in both human and veterinary medicine, leading to the proliferation of antibiotic-resistant bacteria. (21) Improper antibiotic administration to urban animals can disrupt the natural balance of their microbiome, increasing susceptibility to diseases. (22,23) Contamination of environments due to antibiotic residues further complicates wildlife health and contributes to antibiotic resistance. (24) It is crucial to emphasize responsible antibiotic use in veterinary medicine and to raise awareness of its potential impacts on urban wildlife. (25)

Based on the established grouping criteria, we propose the following hypothesis: (1) The unstable food sources and interaction with a free-range environment have increased the potential health risks associated with the microbiome in the gut of cats, (2) Food serves as the primary environmental filtering factor compared to living conditions, leading to a more clustered microbiome composition in FRD cats when compared to D cats, as opposed to FR cats...

Figure 1. Taxonomic composition of Archaea and Bacteria domains. (A) Class Venn Diagram with RPKM abundance calculation method; (B) Shannon and Simpson indices calculated at the class level, two-way ANOVA group difference test (Shannon mean: D 1.3974, FR 1.4646, FRD 1.6964; Simpson mean: D 0.3928, FR 0.3633, FRD 0.2526; Inverse Simpson mean: D 2.6540, FR 3.3083, FRD 4.1620); (C) genus Venn diagram with RPKM Abundance Calculation Method; (D) Shannon and Simpson indices calculated at the genus level, two-way ANOVA group difference test (Shannon mean: D 2.8878, FR 2.8095, FRD 2.7646; Simpson mean: D 0.1746, FR 0.1736, FRD 0.1734; Inverse Simpson mean: D 6.8260, FR 10.8550, FRD 7.7240); (E) PcoA Graph, RPKM abundance calculation method, ANOSIM group difference test, Bray–Curtis distance algorithm, class classification level; (F) Pearson’s correlation with a 95% confidence interval was calculated based on the abundance of classes in each group; (G) heatmap of the abundance of classes in each group, RPKM abundance calculation method; (H) relative abundance (%) of taxonomic classes across groups, analyzed by two-way ANOVA; (I–N) relative abundance of species within the most abundant genera, one-way ANOVA group difference test. Significance thresholds (Tukey-corrected): ns (p greater than or equal to 0.1234), *(p less than 0.0332), **(p less than 0.0021).

Analyses of the taxonomic composition of intestinal prokaryotes in cats have revealed distinctive patterns that vary markedly among different groups, depending on the urban biome. (38,39) The environment plays a crucial role in gut microbiome diversity, as evidenced by comparisons between Malayan tigers in wild and captive habitats, where notable differences in microbial composition were observed. (40) FRD cats exhibit the highest gut microbial diversity, driven by extensive environmental exposure to heterogeneous urban ecosystems. This promotes the acquisition of diverse bacterial taxa from soil, prey, and environmental reservoirs, leading to taxonomic enrichment of their microbiome. (41) As observed in urban rodent models, soil-derived microbes may colonize the gut via adhesion proteins that enable mucosal binding. (42) Urban wildlife can act as a microbial vector; cats in the FRD group that consume prey likely acquire Proteobacteria from their guts. Proteobacteria’s facultative anaerobic members, such as Escherichia, thrive in oxygen-rich gut niches postprey consumption, a dynamic documented in wild carnivores with protein-rich diets. (43) This aligns with island biogeography theory, where fragmented urban habitats act as “islands,” promoting microbial endemism and host-specific adaptation. (44) This potential transmission route reflects zoonotic mechanisms observed in wild felines, highlighting parallels between urban and natural environments in microbial exchange. (45)

Our results demonstrate that the FRD cats showed a higher microbial diversity, suggesting that this type of urban environment may favor more varied and balanced microbial communities, potentially supporting local ecological balance. However, the significant presence of ARGs, especially in FRD cats, indicates that these microbial populations may act as reservoirs of ARGs, potentially spreading them in the environment. This carries the risk of HGT, compromising the efficacy of antimicrobial treatments used in public and veterinary health. Furthermore, identifying unique metabolic pathways and VFs in FRD cats also highlights how microbial microcosms can influence the health of urban ecosystems. The ability of these microorganisms to interact and adapt, which is associated with antibiotic resistance, demands rigorous attention in managing public health, biodiversity, and environmental quality in urban areas to prevent adverse effects.