In Vitro Investigation of the Antibacterial Activity of Nine Commercial Water Disinfectants, Acidifiers, and Glyceride Blends against the Most Important Poultry Zoonotic Bacteria

**Background:** The poultry industry faces increasing pressure to reduce antibiotic use following EU and US bans on antibiotic growth promoters. Water sanitation and acidification have emerged as promising intervention strategies to control zoonotic pathogens at the farm level. However, knowledge gaps exist regarding the antibacterial activity of commercial water additives and the potential for bacteria to develop resistance to these alternative biocides. This study aimed to determine the in vitro minimal inhibitory concentrations (MICs) of nine commercial water disinfectants, acidifiers, and glyceride blends against the most important poultry zoonotic bacteria.

**Methods:** Thirteen bacterial strains were tested, including seven Gram-negative (Campylobacter jejuni S1 and S2, Campylobacter coli S1, Escherichia coli ATCC 11303 and ATCC 25922, Salmonella Typhimurium DT 120 and U292) and six Gram-positive (Staphylococcus aureus DSM 25629, DSM 102262, and S1; Listeria monocytogenes Scott A and S1; Listeria innocua ATCC 33090). Nine commercial products were evaluated: water disinfectants (Cid 2000™, Aqua-clean®, Virkon® S), water acidifiers (Agrocid Super™Oligo, Premium acid, Ultimate acid), and water glyceride blends (CFC Floramix, FRA® LAC34, FRA® Gut Balance). MIC determination was performed using the broth microdilution method according to CLSI standards M07-A10 and M100-S28, with concentrations ranging from 0.002% to 1.136% v/v (or w/v for Virkon® S). Each product was tested in triplicate. For Campylobacter spp., incubation was at 42°C under microaerophilic conditions for 48 hours; all other strains were incubated at 37°C for 24 hours. Statistical analysis used non-parametric Mann–Whitney tests with significance set at p ≤ 0.05.

**Key Results:** Water disinfectants showed the highest antimicrobial activity. Cid 2000™ exhibited MICs of 0.004% v/v for S. aureus (DSM 25629 and DSM 102262) and Campylobacter (C. jejuni S1 and C. coli S1), 0.018–0.036% v/v for E. coli and Salmonella, 0.071% v/v for Listeria strains, and 0.142% v/v for C. jejuni S2. Aqua-clean® showed the lowest MICs overall: 0.002% v/v for S. aureus (DSM 25629 and DSM 102262) and Campylobacter (C. jejuni S1 and C. coli S1), 0.009–0.018% v/v for E. coli and Salmonella, and 0.071% v/v for C. jejuni S2. Virkon® S recorded MICs ranging from 0.013% w/v (S. aureus DSM 25629) to 0.409% w/v (S. Typhimurium DT 120). Water acidifiers showed MICs of 0.036–0.284% v/v for Agrocid Super™Oligo, 0.071–0.568% v/v for Premium acid, and 0.036–0.142% v/v for Ultimate acid. Glyceride blends required higher concentrations: CFC Floramix MICs ranged from 0.142% v/v (Campylobacter and L. monocytogenes) to 0.568% v/v (L. innocua); FRA® LAC34 MICs ranged from 0.036% v/v (C. jejuni S1) to 0.568% v/v (E. coli ATCC 25922, S. Typhimurium DT 120, S. aureus DSM 25629 and S1); FRA® Gut Balance MICs ranged from 0.071% v/v (C. jejuni S2) to 1.136% v/v (E. coli ATCC 25922 and L. innocua ATCC 33090). Notably, C. jejuni S2 (isolated from a slaughterhouse using hydrogen peroxide sanitation) required 20-fold higher concentrations of hydrogen peroxide-based products compared to C. jejuni S1. A correlation was observed between water acidifier activity and pH reduction of the culture medium to approximately pH 5.

**Clinical Implications:** Most tested products inhibited bacterial growth at manufacturer-recommended dosages, supporting their potential as alternatives to antibiotic growth promoters in poultry production. Hydrogen peroxide-based disinfectants (Cid 2000™ and Aqua-clean®) demonstrated the highest antibacterial capacity, followed by water acidifiers, Virkon® S, and glyceride blends. The finding that C. jejuni S2 required substantially higher concentrations of hydrogen peroxide-based products raises concerns about potential adaptation or resistance development in field settings. The authors note that MIC values represent only direct antibacterial effects and do not account for indirect effects on intestinal microbiota, feed ingredients, immune system, or intestinal digesta. They caution that in vitro results may under- or overestimate field effectiveness due to the absence of interfering substances. Further in vivo and in-field studies are needed to establish optimal concentrations for pathogen control and to determine the actual impact on bird health, welfare, and productivity.