**Background:** The genus Sarcina within the Clostridiaceae family comprises morphologically atypical, almost spherical cells forming packets of eight or more units. Two validly recognized taxa exist: Sarcina ventriculi (first described in 1842 from human stomach contents) and Sarcina maxima (isolated from elephant feces in 1969). Sarcina findings in the digestive tract of humans and animals are often associated with pathologies including delayed gastric emptying, nausea, vomiting, ulcers, chronic dyspepsia, and gastric dilations in monogastric animals. However, some publications have described Sarcina spp. in healthy animals and humans. The pathogenicity of Sarcina bacteria remains unclear, and most identifications have been based solely on morphology rather than molecular genetic taxonomic approaches. This study aimed to perform culture-dependent screening and proper taxonomic identification of Sarcina isolates from diverse mammalian hosts.
**Methods:** A total of 197 fresh fecal samples were collected from primates (n=65), dogs (n=70), calves of domestic cattle (n=50), elephants (n=10), and rhinoceroses (n=2) between 2015-2020 from apparently healthy animals kept in zoos or by breeders in the Czech Republic and Slovakia. Samples were collected directly after defecation and placed in anaerobically prepared medium. Cultivation was performed on modified Wilkins-Chalgren agar supplemented with soya peptone, L-cysteine, Tween 80, acetic acid, and mupirocin. Yellow-pigmented irregular colonies with typical Sarcina-like morphology were selected for identification. DNA was isolated using PrepMan Ultra™, and 16S rRNA gene sequencing was performed using primers fd1 and rP2. For 24 selected isolates, multi-locus sequence analysis (MLSA) was conducted targeting six operating genes: ileS, pheT, pyrG, rplB, rplC, and rpsC. Phylogenetic relationships were reconstructed using maximum-likelihood methods in MEGA v5.05. One human isolate of S. maxima 7 from a previous study was also included.
**Key Results:** Sarcina-like colonies were detected in 25/65 primates (38.5%), 1/70 dogs (1.4%), 2/50 calves (4%), 10/10 elephants (100%), and 2/2 rhinoceroses (100%). In elephants, potentially novel Sarcina species were detected at 10^7 CFU g^-1 of feces in Zoo Ústí nad Labem and 10^3 CFU g^-1 in Zoo Liberec. In primates, Sarcina occurrence was more common in Old World monkeys (guenons and gibbons), typically at 10^5 to 10^7 CFU g^-1. In dogs, calves, and humans, Sarcina-like cells were detected at counts <10^4 CFU g^-1. Successful 16S rRNA identification was performed for 24 of 60 DNA samples. Phylogenetic analysis grouped Sarcina strains into four clusters: S. ventriculi, S. maxima, and two potentially novel Sarcina groups. The first novel group comprised seven strains (D3/3C, K3/7B, S8/2c, S10/2a, S1/3c, K1/7A, and S2/2b) from elephants with 98.62-99.08% 16S rRNA gene similarity to type strains of S. maxima and S. ventriculi, and 92.83-94.49% pairwise identity based on the six-gene concatenate. The dog isolate N13/4e had 98.39-90.0% 16S rRNA gene identity to type strains and 91.92-94.04% based on the operating gene concatenate. S. maxima isolates were detected only in rhinoceroses and one human vegetarian host. S. ventriculi was identified in multiple primate species including guenons, gibbons, ring-tailed lemurs, and golden lion tamarins.
**Clinical Implications:** This study demonstrates that Sarcina spp. are common members of the gut microbiota in various mammals without obvious health complications, suggesting they may be opportunistic pathogens rather than obligate pathogens. The taxonomic variability of Sarcina isolates is more dependent on animal species than host location, with S. ventriculi being particularly common in captive primates, especially Old World monkeys. The identification of two potentially novel Sarcina taxonomic units (from elephants and dogs) indicates that the genus likely comprises an overlooked complex of species ranging from benign commensals to frank pathogens. These findings underscore the need for molecular genetic approaches rather than morphology-based identification for accurate Sarcina taxonomy, and highlight the importance of further genomic characterization to understand pathogenicity mechanisms. The study also validates the use of modified Wilkins-Chalgren agar (typically used for Bifidobacterium isolation) as a suitable medium for Sarcina cultivation and detection.