This study aimed to investigate the impact of temperature and the presence of other microorganisms on the susceptibility of STEC to biocides. Mature biofilms were formed at both 10°C and 25°C. An inoculum of planktonic bacteria comprising 10⁶ CFU/ml of spoilage bacteria and 10³ CFU/ml of a single E. coli strain (O157, O111, O103, and O12) was used to form mixed biofilms. The following bacterial combinations were tested: T1: Carnobacterium piscicola + Lactobacillus bulgaricus +STEC, T2: Comamonas koreensis + Raoultella terrigena + STEC, and T3: Pseudomonas aeruginosa + C. koreensis + STEC. Tested biocides included quaternary ammonium compounds (Quats), sodium hypochlorite (Shypo), sodium hydroxide (SHyd), hydrogen peroxide (HyP), and BioDestroy®-organic peroxyacetic acid (PAA). Biocides were applied to 6-day-old biofilms. Minimum Bactericidal Concentrations (MBC) and Biofilm Eradication Concentrations (BEC) were determined. Planktonic cells and single-species biofilms exhibited greater susceptibility to sanitizers (P < 0.0001). Lactobacillus and Carnobacterium were more susceptible than the rest of the tested bacteria (P < 0.0001). Single species biofilms formed by E. coli O111, O121, O157, and O45 showed resistance (100%) to Shypo sanitizer (200 ppm) at 25°C. From the most effective to the least effective, sanitizer performance on single-species biofilms was PAA > Quats > HyP > SHyd > Shypo. In multi-species biofilms, spoilage bacteria within T1, T2, and T3 biofilms showed elevated resistance to SHyd (30%), followed by quats (23.25%), HyP (15.41%), SHypo (9.70%), and BioDestroy® (3.42%) (P < 0.0001). Within T1, T2, and T3, the combined STEC strains exhibited superior survival to Quats (23.91%), followed by HyP (19.57%), SHypo (18.12%), SHyd (16.67%), and BioDestroy® (4.35%) (P < 0.0001). O157:H7-R508 strains were less tolerant to Quats and Shypo when combined with T2 and T3 (P < 0.0001). O157:H7 and O103:H2 strains in mixed biofilms T1, T2, and T3 exhibited higher biocide resistance than the weak biofilm former, O145:H2 (P < 0.0001). The study shows that STEC within multi-species biofilms’ are more tolerant to disinfectants.

The dataset was collected through a series of experiments designed to evaluate the efficacy of various disinfectants against different bacterial strains, including Shiga toxin-producing Escherichia coli (STEC), spoilage bacteria (SP), and lactic acid bacteria (LAB).

A total of nine STEC, four spoilage bacteria (SP), and six lactic acid bacteria (LAB) strains were included in this study. 

Six disinfectants commonly employed in the food industry were evaluated. Disinfectants were prepared according to the manufacturer's specified concentrations. Stock solutions were prepared in sterile hot water (50°C) and maintained at 40° to 50°C. Solutions were used within 30 min after preparation. Before initiating testing, the active chlorine concentration in the sodium hypochlorite solution was tested using a chlorine analysis method (Total) Test Kit (HACH, Model CN-65, Ontario, Canada).

STEC strains [E. coli O145 (75-83), E. coli O157:H7 (1934), E. coli O103:H2 (99-2076), and E. coli O157:H7 (R508)] were included in the multispecies biofilms section.

Biofilm Assessment Using the Crystal Violet Method

To assess biofilm development, 200 µL of methanol was pipetted into wells containing pre-formed biofilms and allowed to stand for 15 min. Methanol was then aspirated using a microplate washer (405 LS, BioTek, Winooski, VT, United States), and 200 µL of 0.1% crystal violet (CV) (Sigma Aldrich) was added to each well and allowed to stand for an additional 15 min. Microplates were then washed three times with 300 µL BPB per well. Residual crystal violet was solubilized in 200 µL of 85% ethanol. Biofilm forming ability was determined indirectly by measuring the level of residual chromophore using a microplate reader at 630 nm (BioTek ELx800; BioTek Instruments Inc., Winooski, VT, United States). The experiment was repeated three times in duplicate for each strain combination. Biofilm-forming ability was estimated using optical density cutoffs (ODc) as described by Adator et al. (2018)

Additional Information:

Bacterial Strain Selection and Culture Conditions: Nine STEC strains, four spoilage (SP) bacteria strains, and six LAB (Lactic acid bacteria) strains were included. Cultures were maintained in Trypticase Soy Broth (TSB) with glycerol, plated on appropriate media, and incubated to achieve a bacterial suspension of 10⁶ CFU/ml.

Culture Medium Used: STEC strains were cultured on MacConkey agar overlayed with TSA (Trypticase Soy Agar) while SP (spoilage) and LAB (Lactic Acid Bacteria) were cultured on TSA, Lactobacilli MRS agar (Difco) and Pseudomonas agar.

Biocide Solutions: Six disinfectants commonly used in the food industry were prepared according to manufacturer specifications and tested within 30 minutes of preparation. The concentration of active chlorine in sodium hypochlorite solution was verified before testing.

Sanitizers along with abbreviations: Tested biocides included quaternary ammonium compounds (Quats), sodium hypochlorite (Shypo), sodium hydroxide (SHyd), hydrogen peroxide (HyP), and BioDestroy®-organic peroxyacetic acid (PAA).

Minimum Bactericidal Concentration (MBC): MBC values were determined using 96-well microplates to establish the lowest concentration of biocide required to kill 99.9% of the bacterial population over a fixed contact time. Each well contained a diluted antimicrobial agent and bacterial suspension with appropriate controls. MBC was determined by assessing bacterial growth after exposure to biocides.

Biofilm Eradication Concentration (BEC): The efficacy of biocides against single-species and multispecies biofilms was evaluated by determining the lowest biocide concentration that prevented bacterial growth after exposure.

Statistical Analysis: Statistical analyses, including frequency analysis, chi-square tests, and factorial models, were performed to analyze data from biofilm formation, biocide susceptibility, and bacterial counts.