Biovolume and spatial distribution of foodborne Gram-negative and Gram-positive pathogenic bacteria in mono- and dual-species biofilms.

The objective of this study was to characterize the biofilms formed by Salmonella enterica serotype Agona, Listeria monocytogenes, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE) after 12, 48, 72, 120 and 240 h of incubation at 10 °C. Biofilms containing a single species, together with dual-species biofilms in which S. enterica and a Gram-positive bacterium existed in combination, were formed on polystyrene and evaluated by using confocal laser scanning microscopy (CLSM). All strains were able to form biofilm. The greatest biovolume in the observation field of 14,161 µm2 was observed for mono-species biofilms after 72 h, where biovolumes of 94,409.0 µm3 ± 2131.0 µm3 (S. enterica), 58,418.3 µm3 ± 5944.9 µm3 (L. monocytogenes), 68,020.8 µm3 ± 5812.3 µm3 (MRSA) and 59,280.0 µm3 ± 4032.9 µm3 (VRE) were obtained. In comparison with single-species biofilms, the biovolume of S. enterica was higher in the presence of MRSA or VRE after 48, 72 and 120 h. In dual-species biofilms, the bacteria showed a double-layer distribution pattern, with S. enterica in the top layer and Gram-positive bacteria in the bottom layer. This spatial disposition should be taken into account when effective strategies to eliminate biofilms are being developed.

Susceptibility of Listeria monocytogenes planktonic cultures and biofilms to sodium hypochlorite and benzalkonium chloride.

The susceptibility of four L. monocytogenes isolates from pork to sodium hypochlorite (SHY) and benzalkonium chloride (BZK) was tested. Minimum inhibitory concentration (MIC) values of 3500 ppm (SHY), or between 3 ppm and 13 ppm (BZK), were found. Minimum bactericidal concentration (MBC) values ranged from 3500 ppm to 4500 ppm (SHY), and from 3 ppm to 14 ppm (BZK). The effect of SHY and BZK on the architecture and cellular viability of 24-h-old biofilms formed by such strains on polystyrene was determined through confocal laser scanning microscopy (CLSM) in conjunction with fluorescent dyes for live cells (SYTO 9) and dead cells (propidium iodide). Strains were able to form biofilm (biovolume values in the observation field of 14,161 µm2 ranged between 103,928.3 ±â€¯6730.2 µm3 and 276,030.9 ±â€¯42,291.9 µm3). Treatment of biofilms for 10 min with SHY (1MIC or 1.5MIC) or BZK (0.5MIC, 1MIC or 1.5MIC) decreased the biovolume of live (potentially dangerous) cells. SHY reduced the cellular viability of biofilms by more than 90%. On the other hand, BZK was able to remove most biofilm mass (live and dead cells), but decreased cellular viability only to a lesser extent, this suggesting strong biofilm detachment and dissemination of live cells.

Structure and viability of 24- and 72-h-old biofilms formed by four pathogenic bacteria on polystyrene and glass contact surfaces.

The biofilms formed by Salmonella Hadar (SH174), Listeria monocytogenes (LM6), methicillin-resistant Staphylococcus aureus (MRSA125) and vancomycin-resistant Enterococcus faecium (VRE61s) on polystyrene and glass after 24 h and 72 h of incubation at 37 °C were examined by confocal laser scanning microscopy (CLSM) after staining with SYTO9 and propidium iodide (PI). A lower average biovolume (P < 0.05) was observed for SH174 biofilms (73,073.61 ±â€¯52,365.90 µm3 in the observation field of 14,161 µm2) than for LM6 (180,804.50 ±â€¯32,554.62 µm3), MRSA125 (208,605.34 ±â€¯57,534.93 µm3) and VRE61s (212,543.91 ±â€¯39,718.62 µm3) biofilms. SH174 showed the greatest (P < 0.05) biovolume on glass, as compared with polystyrene. Biofilms of LM6, MRSA125 and VRE61s were produced at comparable levels on both contact surfaces. After 24 h, SH174 formed small scattered cell clusters, and biovolume of biofilms increased (P < 0.05) after 72 h. By contrast, LM6, MRSA125 and VRE61s formed compact biofilms quickly (24 h) on both contact surfaces. Seventy-two-hour-old biofilms showed the largest biovolumes of dead or damaged (PI-stained) cells, except for MRSA125 (polystyrene) and VRE61s (polystyrene and glass). Appropriate procedures for the disinfection of food processing surfaces immediately after use are required to prevent the formation of biofilm by pathogenic bacteria.

Visualization and quantification of the cellular and extracellular components of Salmonella Agona biofilms at different stages of development.

Salmonella is a major food-borne pathogen able to persist in food processing environments because of its ability to form biofilms. A Salmonella enterica serotype Agona isolate from poultry (S24) was grown at 37°C in biofilms for up to 144 hours (H144) in attachment to polystyrene surfaces. Biofilm structures were examined at different stages in their development (H3, H24, H48, H72, H96 and H144) using confocal laser scanning microscopy (CLSM) in conjunction with fluorescent dyes for live cells (SYTO 9), dead cells (propidium iodide), proteins (fluorescein isothiocyanate isomer I), lipids (DiD'oil), α-polysaccharides (concanavalin A, tetramethylrhodamine conjugate), and ß-polysaccharides (calcofluor white M2R). Strain S24 developed a robust biofilm at H72 (biovolume of 166,852.5 ± 13,681.8 µm3 in the observation field of 16,078.2 µm2). The largest biovolume of live cells was also detected at H72 (128,110.3 ± 4,969.1 µm3), decreasing thereafter, which was probably owing to the detachment of cells prior to a new phase of colonization. The percentage of dead cells with regard to total cells in the biofilms increased throughout the incubation, ranging from 2.3 ± 1.1% (H24) to 44.2 ± 11.0% (H144). Proteins showed the greatest biovolume among the extracellular components within the biofilms, with values ranging from 1,295.1 ± 1,294.9 µm3 (H3) to 19,186.2 ± 8,536.0 µm3 (H96). Maximum biovolume values of 15,171.9 ± 660.7 µm3 (H48), 7,055.3 ± 4,415.2 µm3 (H144), and 2,548.6 ± 1,597.5 µm3 (H72) were observed for ß-polysaccharides, α-polysaccharides and lipids, respectively. A strong (P < 0.01) positive correlation was found between the total biovolume of biofilm and the biovolume of live cells, proteins and ß-polysaccharides, which may serve as useful markers of biofilm formation. The present work provides new insights into the formation of S. Agona biofilms. Our findings may contribute to the designing of reliable strategies for preventing and removing these bacterial communities.

Effect of Sub-Lethal Concentrations of Biocides on the Structural Parameters and Viability of the Biofilms Formed by Salmonella Typhimurium.

This study aimed to investigate the effect of sub-minimum inhibitory concentrations (sub-MICs) of three food-grade biocides (benzalkonium chloride -BZK-, trisodium phosphate -TSP-, and sodium hypochlorite -SHY-) on Salmonella biofilms. The structural parameters and bacterial viability of the biofilms formed by a S. Typhimurium isolate from poultry was investigated by means of confocal laser scanning microscopy after staining with SYTO9 and propidium iodide. The MIC values for Salmonella cells before exposure to subinhibitory concentrations of biocides were 8.0 µg/mL (BZK), 18.0 mg/mL (TSP), and 6.0 mg/mL (SHY). The cultures exhibited a stable acquired tolerance to BZK and SHY. The maximum concentrations of biocides that allowed bacterial growth after several passages through gradually higher concentrations of such compounds were 30.4 µg/mL (BZK) and 10.1 mg/mL (SHY). The architecture and viability of S. Typhimurium biofilms varied in response to sub-MICs of different biocides. Previous adaptation to SHY enhanced (p < 0.001) biofilm formation (average biovolume in the observed field -14,161 µm2-: 139,856.15 ± 155,213.27 µm3) relative to unexposed cells (53,779.05 ± 55,535.62 µm3) and cells previously exposed to BZK (58,216.97 ± 58,644.45 µm3) or TSP (30,052.13 ± 28,290.56 µm3). This was particularly marked when biofilm was grown in the absence of biocides or in the presence of sub-MICs of SHY. The highest percentage of dead cells was shown by biofilms formed by cultures previously exposed to TSP relative to the other conditions tested (34.08% ± 13.74% vs. 23.70% ± 16.16%; p < 0.001). The importance of maintaining higher than MICs of SHY during sanitizing procedures to fight foodborne infections by Salmonella biofilms is highlighted.

Effect of sub-inhibitory concentrations of biocides on the architecture and viability of MRSA biofilms.

The effect of sub-minimum inhibitory concentrations (sub-MICs) of three biocides (benzalkonium chloride [BZK], trisodium phosphate [TSP] and sodium hypochlorite [SHY]) upon the architecture and viability of the biofilms formed by a methicillin-resistant Staphylococcus aureus strain of food origin (MRSA 48a) was investigated. Images were examined through confocal laser scanning microscopy (CLSM) after staining with SYTO9 and propidium iodide. Sub-MICs of BZK or TSP reduced the ability of MRSA to produce biofilm. In contrast, the presence of sub-MICs of SHY enhanced the biofilm-forming ability of MRSA when cells had undergone previous adaptation to this compound (biovolume in the observation field was 137,785.31 ± 47,682.79 µm3 for biofilms formed in the presence of SHY, and 70,204.13 ± 31,603.98 µm3 in the absence of biocides; P < 0.05). The largest amount of live (green stained) cells (P < 0.05) was observed in biofilms grown in the presence of SHY relative to the other conditions tested (58,999.75 ± 55,312.37 µm3vs 31,976.29 ± 38,594.98 µm3). Findings from the present work constitute the first report of biofilm production by MRSA being induced by sub-inhibitory concentrations of SHY. The data suggest that repeated use of SHY at low concentrations could represent a public health risk.

Exposure of Escherichia coli ATCC 12806 to sublethal concentrations of food-grade biocides influences its ability to form biofilm, resistance to antimicrobials, and ultrastructure.

Escherichia coli ATCC 12806 was exposed to increasing subinhibitory concentrations of three biocides widely used in food industry facilities: trisodium phosphate (TSP), sodium nitrite (SNI), and sodium hypochlorite (SHY). The cultures exhibited an acquired tolerance to biocides (especially to SNI and SHY) after exposure to such compounds. E. coli produced biofilms (as observed by confocal laser scanning microscopy) on polystyrene microtiter plates. Previous adaptation to SNI or SHY enhanced the formation of biofilms (with an increase in biovolume and surface coverage) both in the absence and in the presence (MIC/2) of such compounds. TSP reduced the ability of E. coli to produce biofilms. The concentration of suspended cells in the culture broth in contact with the polystyrene surfaces did not influence the biofilm structure. The increase in cell surface hydrophobicity (assessed by a test of microbial adhesion to solvents) after contact with SNI or SHY appeared to be associated with a strong capacity to form biofilms. Cultures exposed to biocides displayed a stable reduced susceptibility to a range of antibiotics (mainly aminoglycosides, cephalosporins, and quinolones) compared with cultures that were not exposed. SNI caused the greatest increase in resistances (14 antibiotics [48.3% of the total tested]) compared with TSP (1 antibiotic [3.4%]) and SHY (3 antibiotics [10.3%]). Adaptation to SHY involved changes in cell morphology (as observed by scanning electron microscopy) and ultrastructure (as observed by transmission electron microscopy) which allowed this bacterium to persist in the presence of severe SHY challenges. The findings of the present study suggest that the use of biocides at subinhibitory concentrations could represent a public health risk.