Limited and transient effects of intramuscular benzylpenicillin on the equine gut microbiota and resistome

The equine hindgut microbiota is essential for digestion, metabolism and host health. Antibiotic administration may disrupt microbial communities and select antimicrobial resistance genes (ARGs), but the impact of intramuscular benzylpenicillin (PEN) formulations on the equine gut microbiota and resistome remains poorly characterized. This study investigated longitudinal phenotypic and genotypic changes in the fecal microbiota of healthy horses following administration of three intramuscular PEN formulations with different absorption characteristics. Six healthy adult horses were enrolled in a randomized three period crossover study and received procaine benzylpenicillin, procaine/benzathine benzylpenicillin, or penethamate hydriodide, separated by 4–5-month washout periods. Fecal samples were collected before, during and after treatment. Phenotypic analyses included enumeration of total and β-lactam-resistant Enterobacterales and Enterococcus spp., antimicrobial susceptibility testing and detection of extended-spectrum β-lactamase (ESBL)-producing isolates. Genotypic analyses combined 16S rRNA gene sequencing and high-throughput quantitative PCR targeting 22 ARGs and mobile genetic elements (MGEs). No horse developed diarrhoea or clinically relevant adverse effect. Overall, intramuscular PEN administration induced only limited and transient microbiological changes. Penethamate hydriodide was associated with a short-lived increase in total, ampicillin-resistant and cefotaxime-resistant Enterobacterales, as well as transient detection of ampicillin- and penicillin-resistant enterococci. Six ESBL-producing Enterobacterales isolates were recovered from two horses on day 5. Most resistant isolates displayed multidrug-resistant phenotypes. However, neither treatment nor treatment-by-time interaction significantly affected ARG/MGE abundance or composition after correction for multiple testing. Tetracycline resistance genes were the most abundant ARGs detected. Multivariate analyses showed no major treatment-related restructuring of the microbiota or resistome. In contrast, sex-associated management groups, probably because of differences in male and female diets, was the principal factor associated with variation in bacterial counts and ARG/MGE profiles. Short-term intramuscular administration of three benzylpenicillin formulations caused only minor and transient alterations in the fecal microbiota of healthy horses and no detectable long-term impact on the resistome. Although temporary selection of multidrug-resistant and ESBL-producing Enterobacterales occurred after penethamate administration, these effects remained limited and were not reflected at the community resistome level. These findings support the community stability of the equine gut microbiota and resistome following benzylpenicillin treatment.