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Purpose: Surgical site infections (SSIs) are common in colorectal surgery. Mechanical bowel preparation (MBP) in conjunction with oral antibiotics (OABs) have been shown to reduce SSI rates. It however is still unclear which OABs to use, and how this can be implemented in practice. Methods: This is a prospective observational study carried out in Swansea Bay University Health Board during 2019–2021, evaluating the introduction of OABs in a stepwise manner on the incidence of SSI in major colorectal surgery. A control group having MBP only was compared to two OAB groups: one group had MBP plus metronidazole only and the second MBP plus metronidazole and neomycin. A 30-day follow-up after surgery was ascertained via chart review and telephone contact. Logistic regression was performed to estimate the relation between OAB use and SSI, with adjustment for confounding. In a subset of patients, faecal samples were analysed through 16S rRNA amplicon sequencing before and after OAB treatment, depicting the impact of the gut microbiome. Results: In total 160 patients were analysed: 46 patients had MBP only, whilst 76 patients had MBP plus metronidazole only and 38 patients had MBP with metronidazole/neomycin. The SSI rate in the entire cohort was 33.8%, whilst the adjusted ORs for the single- and dual-OAB groups were 0.76 (95% CI: 0.17–1.81) and 0.50 (95% CI: 0.17–1.52). The microbial analysis demonstrated that the relative abundance for many bacterial genera was changed before and after OAB treatment, but no link with SSI development could be shown. Conclusions: Introduction of OABs in conjunction with MBP in colorectal surgery is feasible, and may potentially lead to lower rates of SSI, as well as altering the community structure of the faecal microbiome. More research is needed, especially considering different OABs and mechanistic studies of the gut microbiome in the context of colorectal surgery. [ABSTRACT FROM AUTHOR]