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？ < ？0.05). All Gram？negative isolates were positive for？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.
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