Biofilm Formation by Shiga Toxin-Producing Escherichia coli on Stainless Steel Coupons as Affected by Temperature and Incubation Time

Abstract

Forming biofilm is a strategy utilized by Shiga toxin-producing Escherichia coli (STEC) to survive and persist in food processing environments. We investigated the biofilm-forming potential of STEC strains from 10 clinically important serogroups on stainless steel at 22 °C or 13 °C after 24, 48, and 72 h of incubation. Results from crystal violet staining, plate counts, and scanning electron microscopy (SEM) identified a single isolate from each of the O113, O145, O91, O157, and O121 serogroups that was capable of forming strong or moderate biofilms on stainless steel at 22 °C. However, the biofilm-forming strength of these five strains was reduced when incubation time progressed. Moreover, we found that these strains formed a dense pellicle at the air-liquid interface on stainless steel, which suggests that oxygen was conducive to biofilm formation. At 13 °C, biofilm formation by these strains decreased (P < 0.05), but gradually increased over time. Overall, STEC biofilm formation was most prominent at 22 °C up to 24 h. The findings in this study identify the environmental conditions that may promote STEC biofilm formation in food processing facilities and suggest that the ability of specific strains to form biofilms contributes to their persistence within these environments.

Efficacy of Commercial Sanitizers Used in Food Processing Facilities for Inactivation of Listeria monocytogenes, E. Coli O157:H7, and Salmonella Biofilms

“…The results showing less sensitivity to the sanitizers suggests that protection might be afforded by the EPS produced by these organisms (Figure 6). Such structures have been observed by others and not only act as a 'glue' that holds the biofilm together, but also as a protective coating that restricts diffusion of nutrients and/or antimicrobials from reaching retained and embedded cells [53,54].…”

Biofilm formation by South African non-O157 Shiga toxigenic Escherichia coli on stainless steel coupons

“…Coupons were treated with 70% (v/v) ethanol, air-dried for 5 min at room temperature, and autoclaved at 121°C for 15 min prior to use. Biofilms were formed under static conditions as described previously by Ma et al (2019). Specifically, overnight STEC cultures were diluted in M9 medium to achieve a final concentration of 7 log CFU/mL.…”

“…Fixed coupons were air-dried for 2 min and stained with 0.5% (m/v) crystal violet (CV) solution (Sigma-Aldrich, St. Louis, Missouri, USA) for 15 min, followed by three washes with distilled water and airdrying. The dye bound to the biofilm was then dissolved with 25 mL of 33% glacial acetic acid (Sigma-Aldrich, St. Louis, Missouri, USA) and measured at a wavelength of 590 nm with a spectrophotometer, as described by Ma et al (2019). The optical density cutoff value (ODc) of 0.025 was determined to be three standard deviations above the mean OD of the negative controls.…”

“…Planktonic and attached bacterial cells were quantified as described previously by Ma et al (2019). Briefly, 1 mL of the cell suspension was pipetted from the Falcon tubes, serially diluted with 10 mmol/L phosphatebuffered saline (pH 7.4), plated on LB agar, and incubated aerobically at 37°C for 18 h. To quantify the attached bacterial cells, we rinsed the coupons three times with sterile water to remove loosely attached cells.…”

“…However, unlike brass, copper, and silver, stainless steel does not possess chemical properties that directly kill bacteria (Packiyam et al 2016). Therefore, potential pathogens can colonize and proliferate on stainless steel surfaces, with various STEC serotypes being capable of forming biofilms on this matrix (Lajhar et al 2018;Ma et al 2019;Vogeleer et al 2016). Stainless steel can thus serve as a potential reservoir of infection and dissemination of foodborne pathogens, including STEC (Solheim et al 2013).…”