Longitudinal study of the short- and long-term effects of hospitalisation and oral trimethoprim-sulfadiazine administration on the equine faecal microbiome and resistome

Background: Hospitalisation and antimicrobial treatment are common in horses and significantly impact the intestinal microbiota. This may affect host health as horses depend on hindgut fermentation. Antimicrobial treatment might also increase levels of resistant bacteria in faeces, which could spread to other ecological compartments. In this study, we aimed to characterize the short- and long-term effects of transportation, hospitalisation, and trimethoprim-sulfadiazine (TMS) administration on the faecal microbiota and resistome of healthy equids. Methods: In a longitudinal experimental study design, in which the ponies served as their own control, faecal samples were collected from six healthy Welsh ponies at the farm (D0 – D13-1), immediately following transportation to the hospital (D13-2), during seven days of hospitalisation without treatment (D14 – D21), during five days of oral TMS treatment (D22 – D26) and after discharge from the hospital up to six months later (D27 – D211). 16S rRNA gene sequencing was performed on all samples. For resistome analysis, shotgun metagenomic sequencing was performed on selected samples. Results: Hospitalisation without antimicrobial treatment did not affect microbiota composition. Oral TMS treatment reduced alpha-diversity significantly. Spirochaetes, Kiritimatiellaeota, Fibrobacteres and Verrucomicrobia decreased in relative abundance, whereas Firmicutes increased. A gradual and partial recovery of the faecal microbiota composition was observed two weeks after discontinuation of TMS treatment and discharge from the hospital. Long term effects were, however, apparent. The relative abundance of Spirochaetes and Verrucomicrobia remained lower and microbiota composition still differed significantly from that at the start of the study. TMS administration led to a significant (up to 32-fold) and rapid increase in the relative abundance of resistance genes sul2, tetQ, ant6-1a and aph(3”)-lb. lnuC significantly decreased directly after treatment. Resistance genes sul2 (15-fold) and tetQ (six-fold) remained significantly increased six months later. Conclusions: Oral treatment with TMS has a rapid and long-lasting effect on faecal microbiota composition and resistome, making the equine hindgut a reservoir and potential source of resistant bacteria posing a risk to animal and human health through transmission. These findings support the judicious use of antimicrobials to minimize long-term faecal presence, excretion and the spread of antimicrobial resistance in the environment.