| Summary: | Shiga toxin-producing <i>Escherichia coli</i> (STEC) is a major concern in the food industry and requires effective control measures to prevent foodborne illnesses. Previous studies have demonstrated increased difficulty in the control of biofilm-forming STEC. Desiccation, achieved through osmotic stress and water removal, has emerged as a potential antimicrobial hurdle. This study focused on 254 genetically diverse <i>E. coli</i> strains collected from cattle, carcass hides, hide-off carcasses, and processing equipment. Of these, 141 (55.51%) were STEC and 113 (44.48%) were generic <i>E. coli</i>. The biofilm-forming capabilities of these isolates were assessed, and their desiccation tolerance was investigated to understand the relationships between growth temperature, relative humidity (RH), and bacterial survival. Only 28% of the STEC isolates had the ability to form biofilms, compared to 60% of the generic <i>E. coli</i>. Stainless steel surfaces were exposed to different combinations of temperature (0 °C or 35 °C) and relative humidity (75% or 100%), and the bacterial attachment and survival rates were measured over 72 h and compared to controls. The results revealed that all the strains exposed to 75% relative humidity (RH) at any temperature had reduced growth (<i>p</i> < 0.001). In contrast, 35 °C and 100% RH supported bacterial proliferation, except for isolates forming the strongest biofilms. The ability of <i>E. coli</i> to form a biofilm did not impact growth reduction at 75% RH. Therefore, desiccation treatment at 75% RH at temperatures of 0 °C or 35 °C holds promise as a novel antimicrobial hurdle for the removal of biofilm-forming <i>E. coli</i> from challenging-to-clean surfaces and equipment within food processing facilities.
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