The overuse of antimicrobials in both human and veterinary medicine has driven the emergence of multi-drug resistant bacteria. In pig production, medicated feed is a common practice to control diseases during critical periods like weaning, a time when piglets are highly susceptible to infections, including *Streptococcus suis*. *S. suis*, a common colonizer of the upper respiratory tract, can cause severe outbreaks after weaning when maternal antibodies wane. Traditionally, amoxicillin or other beta-lactams have been used to control *S. suis*, but the rise of antimicrobial resistance necessitates alternative strategies. This research aimed to evaluate the effectiveness of three different feed additive combinations in mitigating *S. suis* disease post-weaning, comparing their effects to amoxicillin treatment, examining clinical signs, fecal and nasal microbiota composition, and production parameters.

Extensive literature supports the detrimental effects of antimicrobial overuse, particularly in livestock, highlighting the need for alternatives. Studies have shown that weaning is a critical period for pig health due to immune system immaturity and environmental changes, resulting in increased susceptibility to various pathogens, including *S. suis*. The pathogen's ability to colonize the upper respiratory tract and cause severe disease, including meningitis and septicemia, is well-documented. Previous research has explored the use of antibiotics, such as amoxicillin, for controlling *S. suis* infections; however, the emergence of resistance underscores the necessity for exploring alternative approaches. Optimizing nutritional programs during post-weaning to modulate the gut microbiome and enhance the immune system has been suggested as a potential avenue to reduce disease susceptibility and antibiotic dependence. This study builds on existing knowledge by investigating the effects of specific feed additives on both clinical disease and the composition of the nasal and fecal microbiotas.

A total of 569 twenty-one-day-old piglets from 54 dams were randomly assigned to five treatment groups: lysozyme (Lys), medium-chain fatty acids plus lysozyme (FA + Lys), medium-chain fatty acids plus a natural anti-inflammatory (FA + antiinf), amoxicillin (Amox), and a negative control group. Piglet weight was recorded before weaning. Clinical signs were monitored daily by farm personnel and bi-daily by a veterinarian. Animals requiring antibiotic treatment were removed. Three animals per treatment group, some exhibiting *S. suis*-consistent clinical signs, were euthanized for post-mortem examination and *S. suis* and *Glaesserella parasuis* isolation. Nasal swabs were collected from a subset of 125 piglets at weaning (D0) and after three weeks (D21) for PCR detection of *S. suis* and *G. parasuis*. A smaller subset (12 at D0 and 10 per group at D21) was selected for fecal and nasal microbiota analysis using 16S rRNA gene sequencing. The V3-V4 region was targeted for amplicon sequencing. Alpha diversity (Shannon, Chao) and beta diversity (Bray-Curtis, Jaccard) were calculated. Analysis of Composition of Microbiomes (ANCOM) was used to identify differentially abundant genera and ASVs. Data were analyzed using R software, with statistical significance set at p ≤ 0.05.

The study revealed that the FA + antiinf and Amox groups had significantly lower prevalence of *S. suis*-like clinical signs compared to the negative control group (p=0.006 and p=0.01, respectively). However, the difference between the FA + antiinf and Amox groups was not statistically significant (p=0.93). The Lys group showed a higher prevalence (17.7%) compared to Amox (9.6%), approaching statistical significance (p=0.07). Average daily weight gain (ADWG) was not significantly different among groups (p=0.67). PCR confirmed the presence of *S. suis* in almost all piglets at weaning and after three weeks. No treatment group showed a reduction in *S. suis* colonization. Alpha diversity (Shannon index) and richness (Chao index) of both fecal and nasal microbiota increased significantly from D0 to D21. The FA + antiinf group showed significantly higher nasal microbiota alpha diversity compared to other groups (p<0.01), while the Amox group showed a trend towards higher diversity compared to the control. In fecal microbiota, only *Mitsuokella* was differentially abundant in the FA + Lys group. In nasal microbiota, *Helicobacter*, and an unclassified genus from *Weeksellaceae* were differentially abundant among treatments. Dam parity impacted microbiota composition, with piglets from gilts exhibiting lower richness and diversity than those from sows.

This study demonstrated that the feed additives, particularly FA + antiinf, could be effective alternatives to amoxicillin in controlling *S. suis* disease and improving nasal microbiota diversity, an indicator of better health. The lack of impact on *S. suis* colonization prevalence suggests the additives' effect is primarily on modulating the host's immune response or competition with *S. suis* rather than directly inhibiting bacterial growth. The higher nasal microbiota diversity in the FA + antiinf group may have contributed to better disease control. The observed effects on microbiota composition may be attributed to the immunomodulatory and antimicrobial properties of the additives. The influence of dam parity on offspring microbiota highlights the importance of considering maternal factors in livestock health management. Further research is needed to fully understand the complex interactions between feed additives, microbiota composition, and host immune responses.

This study provides evidence for feed additives, especially FA + antiinf, as a viable alternative to antibiotics in managing post-weaning *S. suis* disease. The positive effects on nasal microbiota diversity are noteworthy. Future research should focus on elucidating the mechanisms of action of these additives and exploring the potential for targeted interventions to optimize microbiota composition, particularly in piglets from gilts, to enhance disease resistance and improve overall pig health.

The study was conducted on a single farm, potentially limiting the generalizability of the findings. The sample size for microbiota analysis was relatively small. Long-term effects of the feed additives on production parameters were not assessed as animals were not followed until slaughter. The study design may not fully account for other factors that could influence the prevalence of *S. suis* or microbiota composition.