Impact of mixed biofilm formation with environmental microorganisms on E. coli O157:H7 survival against sanitization

Abstract

Biofilm formation by foodborne pathogens is a serious threat to food safety and public health. Meat processing plants may harbor various microorganisms and occasional foodborne pathogens; thus, the environmental microbial community might impact pathogen survival via mixed biofilm formation. We collected floor drain samples from two beef plants with different E. coli O157:H7 prevalence history and investigated the effects of the environmental microorganisms on pathogen sanitizer tolerance. The results showed that biofilm forming ability and bacterial species composition varied considerably based on the plants and drain locations. E. coli O157:H7 cells obtained significantly higher sanitizer tolerance in mixed biofilms by samples from the plant with recurrent E. coli O157:H7 prevalence than those mixed with samples from the other plant. The mixed biofilm that best protected E. coli O157:H7 also had the highest species diversity. The percentages of the species were altered significantly after sanitization, suggesting that the community composition affects the role and tolerance level of each individual species. Therefore, the unique environmental microbial community, their ability to form biofilms on contact surfaces and the interspecies interactions all play roles in E. coli O157:H7 persistence by either enhancing or reducing pathogen survival within the biofilm community.

Keywords: Biofilms; Industrial microbiology.

Fig. 1. Viable total bacteria or E. coli O157:H7 cells in biofilms treated with sterile water.

a Viable total bacteria in mixed biofilms formed by floor drain samples. b Viable E. coli O157:H7 cells in mixed biofilms or single-strain biofilm. Viable total bacteria and E. coli O157:H7 cells in sterile water-treated biofilms were harvested and quantified on TSA and O157 Chromagar plates, respectively. Data are shown as log₁₀ CFU/chip. Error bars represent mean ± standard error of the mean. Statistical analysis was performed using ANOVA followed by post Turkey’s multiple comparisons test. P-values lower than 0.05 were considered statistically significant. Values labeled with the same letter are not statistically different.

Fig. 2. CLSM 3D images of mixed biofilms on a stainless steel surface.

Mixed biofilms developed by the drain microorganisms, with or without the addition of the E. coli O157:H7 strain, were stained with FM 1–43 dye and the structural organization of the biofilms was analyzed with CLSM and the software package. Calibration XY: 0.21 μm, Z: 0.75 μm. Resolution: 1024 × 1024 × 29.

Fig. 3. Viable total bacteria or E. coli O157:H7 cells in biofilms treated with 300 ppm QAC.

a Viable total bacteria in mixed biofilms by floor drain samples. b Viable E. coli O157:H7 cells in mixed biofilms or single-strain biofilm. Viable total bacteria and E. coli O157:H7 cells in QAC-treated biofilms were harvested and quantified on TSA and O157 Chromagar plates, respectively. Data are shown as log₁₀ CFU/chip. Error bars represent mean ± standard error of the mean. Statistical analysis was performed using ANOVA followed by post Turkey’s multiple comparisons test. P-values lower than 0.05 were considered statistically significant. Values labeled with the same letter are not statistically different.

Fig. 4. Impact of QAC treatment on species relative abundance changes of the mixed biofilms.

For each drain biofilm sample, one pre-treatment sample (A-C1–B-H2), one post-treatment sample without E. coli O157:H7 (A-C1_300–B-H2_300), and one post-treatment sample with the addition of the E. coli O157:H7 strain (A-C1_O157_300–B-H2_O157_300) were analyzed with 16S rRNA gene amplicon sequencing to demonstrate the changes in the relative abundance of microorganisms in mixed biofilms before and after QAC treatment.

Fig. 5. Proportion of relative abundance of the bacterial species in the O157 protector and non-protector groups.

Heatmap is created by normalizing the OUT table counts, that are converted to the proportions of relative abundance of the bacterial families present in the microbiome grouped by E. coli O157:H7 protector status before and after QAC treatment.

Fig. 6. Log₂ fold differences in the relative abundance of bacterial families between the O157 protector and non-protector groups.

Log₂ fold change differences between the sample groups at the family level were calculated on log₂ transformed CSS normalized counts. Only bacterial families present in more than 1% of the total microbiome and differing by more than 1.0 log between the sample groups before (a) and after (b) QAC treatment are presented.