A novel method using a differential staining fluorescence microscopy (DSFM) to track the location of enteric pathogens within mixed-species biofilms

Abstract

This study developed a new tool, differential staining fluorescence microscopy (DSFM), to measure the biovolume and track the location of enteric pathogens in mixed-species biofilms which can pose a risk to food safety in beef processing facilities. DSFM was employed to examine the impact of pathogenic bacteria, Escherichia coli O157:H7 and three different Salmonella enterica strains on mixed-species biofilms of beef processing facilities. Fourteen floor drain biofilm samples from three beef processing plants were incubated with overnight BacLight stained enteric pathogens at 7 °C for 5 days on stainless steel surface then counter-stained with FM-1-43 biofilm stain and analyzed using fluorescence microscopy. Notable variations in biovolume of biofilms were observed across the fourteen samples. The introduction of E. coli O157:H7 and S. enterica strains resulted in diverse alterations of biofilm biovolume, suggesting distinct impacts on mixed-species biofilms by different enteric pathogens which were revealed to be located in the upper layer of the mixed-species biofilms. Pathogen strain growth curve comparisons and verification of BacLight Red Stain staining effectiveness were validated. The findings of this study show that the DSFM method is a promising approach to studying the location of enteric pathogens within mixed-species biofilms recovered from processing facilities. Understanding how foodborne pathogens interact with biofilms will allow for improved targeted antimicrobial interventions.

Figure 1

Summary diagram showing the key steps (1–3) in Non-invasive differential staining fluoresce microscopic method to track the location of enteric pathogens within mixed-species biofilm.

Figure 2

Fluorescence images (magnification 10 ×) of 11A drain biofilm with E. coli 110 exposed under: (left) Y5 channel showing BacLight stain only; (middle) GFP channel showing FM-1-43 stain only; and (right) overlapping image of both GFP and Y5 channels.

Figure 3

Overlapping fluorescence images (magnification 10 ×) of 11A drain biofilm with three different E. coli 110 exposed under both GFP and Y5 channel.

Figure 4

Overlapping fluorescence images (magnification 10 ×) of 11A drain biofilm with three different E. coli 138 exposed under both GFP and Y5 channel.

Figure 5

Overlapping fluorescence images (magnification 10 ×) of 11A drain biofilm with three different E. coli 141 exposed under both GFP and Y5 channel.

Figure 6

Fluorescence images (magnification 10 ×) of 11B drain biofilm with E. coli 138 exposed under: (left) Y5 channel showing BacLight stain only; (middle) GFP channel showing FM-1-43 stain only; and (right) overlapping image of both GFP and Y5 channels.

Figure 7

Overlapping fluorescence images (magnification 10 ×) of 11B drain biofilm with three different E. coli 110 exposed under both GFP and Y5 channel.

Figure 8

Overlapping fluorescence images (magnification 10 ×) of 11B drain biofilm with three different E. coli 138 exposed under both GFP and Y5 channel.

Figure 9

Overlapping fluorescence images (magnification 10 ×) of 11B drain biofilm with three different E. coli 141 exposed under both GFP and Y5 channel.

Figure 10

Fluorescence images (magnification 10 ×) of 15C drain biofilm with S. enterica serovar Montevideo exposed under: (left) Y5 channel showing BacLight stain only; (middle) exposed under GFP channel showing FM-1-43 stain only; and (right) overlapping image of both GFP and Y5 channels.

Figure 11

Overlapping fluorescence images (magnification 10 ×) of 15C drain biofilm with three different S. enterica serovar Cerro exposed under both GFP and Y5 channel.

Figure 12

Overlapping fluorescence images (magnification 10 ×) of 15C drain biofilm with three different S. enterica serovar Montevideo exposed under both GFP and Y5 channel.

Figure 13

Overlapping fluorescence images (magnification 10 ×) of 15C drain biofilm with three different S. enterica serovar Typhimurium exposed under both GFP and Y5 channel.

Figure 14

Change of biovolume for drain samples from plant A and B with (E. coli O157:H7 110, 138, and 141) and without (Control) enteric pathogen.

Figure 15

Change of biovolume for drain samples from plant C with (S. enterica C-Cerro, C-Typhimurium, and C-Montevideo) and without (Control) enteric pathogen.