DOI: 10.5061/dryad.zpc866tgh
title: Disinfectant efficacy on mixed biofilms comprising Escherichia coli and spoilage microorganisms

description: 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 &lt; 0.0001). Lactobacillus and
 Carnobacterium were more susceptible than the rest of the tested bacteria
 (P &lt; 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 &gt; Quats &gt; HyP
 &gt; SHyd &gt; 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 &lt; 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
 &lt; 0.0001). O157:H7-R508 strains were less tolerant to Quats and
 Shypo when combined with T2 and T3 (P &lt; 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 &lt; 0.0001). The
 study shows that STEC within multi-species biofilms’ are more tolerant to
 disinfectants.
description: 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 <em>Escherichia coli</em> (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 [<em>E. coli</em> O145 (75-83),
 <em>E. coli</em> O157:H7 (1934), <em>E. coli</em>
 O103:H2 (99-2076), and <em>E. coli</em> 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:
 <strong>Bacterial Strain Selection and Culture
 Conditions</strong>: 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 10<sup>6</sup> CFU/ml.
 <strong>Culture Medium Used</strong>: 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 <em>Pseudomonas</em>
 agar. <strong>Biocide Solutions</strong>:
 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.
 <strong>Sanitizers along with abbreviations</strong>:
 Tested biocides included quaternary ammonium compounds (Quats), sodium
 hypochlorite (Shypo), sodium hydroxide (SHyd), hydrogen peroxide (HyP),
 and BioDestroy®-organic peroxyacetic acid (PAA).
 <strong>Minimum Bactericidal Concentration
 (MBC)</strong>: 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. <strong>Biofilm Eradication
 Concentration (BEC)</strong>: The efficacy of biocides against
 single-species and multispecies biofilms was evaluated by determining the
 lowest biocide concentration that prevented bacterial growth after
 exposure. <strong>Statistical
 Analysis</strong>: Statistical analyses, including frequency
 analysis, chi-square tests, and factorial models, were performed to
 analyze data from biofilm formation, biocide susceptibility, and bacterial
 counts.
description: # Disinfectant efficacy on mixed biofilms comprising *Escherichia coli*
 and spoilage microorganisms
 [https://doi.org/10.5061/dryad.zpc866tgh](https://doi.org/10.5061/dryad.zpc866tgh) ## Description of the data and file structure The provided data files are mostly optical density data collected from 96-well microplates containing STEC biofilms. Another set of data also contains an enumeration of the bacterial strains used in this study within multispecies and single-species biofilms. The data used in the study was gathered in Canada in a Biosafety level 2 laboratory (BSL2) from 2021 to 2023. This study aimed to determine Shiga toxigenic *E. coli* (STEC) strains' capacity to form multispecies biofilms and to test the biofilm's susceptibility to different biocide. ### Data variables: * Label: unique identifier * REP: replicate number * Sanitizer: Tested biocides included quaternary ammonium compounds (Quats), sodium hypochlorite (Shypo), sodium hydroxide (SHyd), hydrogen peroxide (HyP), and BioDestroy®-organic peroxyacetic acid (PAA). * Bacteria: strain of bacteria (STEC = Shiga toxigenic *E. coli*, SP = spoilage bacteria, LAB = lactic acid bacteria) * combination: multispecies biofilms T1: *Carnobacterium piscicola* + *Lactobacillus bulgaricus*, and two SP T2: *Comamonas koreensis* + *Raoultella terrigena;* T3: *Pseudomonas aeruginosa* + *C. koreensis*. STEC strains: O157 (R508), O145, O103, O157 (1934). Each STEC strain was added to each biofilm combination as a factorial design. * Group: microplates were divided into groups for storage at different temperatures (10° & 25° C) each group was exposed the the different biocides * BEC PPM: Biofilm eradication concentration (ppm) * MBC (PPM): Minimum bactericidal concentration (ppm) * E. coli: 0 = absent, 1 = present * Spoilage: 0 = absent, 1 = present * Day: 0 = defined as the day when microplates were washed 3 times after incubation. * Temperature: in degrees Celcius ## Code/Software Data were analyzed using SAS, Version 9.4, SAS Institute INC., Cary, NC (SAS, 2012).