Presence of <i>rpoS</i> Gene and Environmental Stress-Dependent Biofilm Formation in Gram-Negative Bacteria in Mexican Artisanal Dairy Establishments

Artisanal Mexican cheeses are frequently produced without pasteurization, favoring microbial contamination and potential biofilm formation. Aim: To characterize Gram‑negative isolates from artisanal cheese outlets in Tijuana, Mexico, evaluate their biofilm‑forming ability under different environmental conditions and detect the stress‑response gene rpoS. Methods: Seventeen Gram‑negative and 13 Gram‑positive bacteria were isolated from cheese, contact surfaces and air. Curves of growth (BHI, 37 ˚C), antimicrobial susceptibility (VITEK 2‑AST), biofilm formation in microtiter plates (four media/temperature combinations) and PCR detection of rpoS were performed. Results: Escherichia coli displayed the highest growth rate (µ = 0.28 h−1, g = 149 min), while Citrobacter freundii formed the only “strong” biofilm in all conditions. Two Pantoea agglomerans isolates were resistant to cefoxitin and one E. coli to ampicillin/sulbactam; tetracycline resistance appeared in a single airborne Staphylococcus epidermidis. Biofilm biomass (OD630) differed significantly by genus and condition (two‑way ANOVA, p rpoS, but gene presence alone did not predict biofilm intensity. Conclusions: Gram‑negative biofilm‑forming bacteria carrying rpoS are common in Tijuana artisanal cheeses, yet antimicrobial resistance remains sporadic. Targeted sanitation against mixed‑species biofilms and continued AMR surveillance are warranted to enhance food safety.