In a previous study, we identified Congo red-binding and -nonbinding phase variants of Escherichia coli serotype O157:H7 strain ATCC 43895. The Congo red-binding variant, strain 43895OR, produced a ...dry, aggregative colony that was similar to the red, dry, and rough (rdar) phenotype characteristic of certain strains of SALMONELLA: In contrast, variant 43895OW produced a smooth and white colony morphology. In this study, we show that, similar to rdar strains of Salmonella enterica serovar Typhimurium, strain 43895OR forms large aggregates in broth cultures, firm pellicles at the air-medium interface on glass, and dense biofilms on glass and polystyrene. However, unlike S. enterica serovar Typhimurium, strain 43895OR does not stain positive for cellulose production. When strain 43895OR was fixed on agar, scanning electron microscopy showed cells expressing extracellular matrix (ECM) containing curli fibers. Strain 43895OW was devoid of any ECM or curli fibers on agar but showed expression of curli fibers during attachment to glass. Strain 43895OR produced >4-fold-larger amounts of biofilm than strain 43895OW on polystyrene, glass, stainless steel, and Teflon; formation was >3-fold higher in rich medium than in nutrient-limited medium. Biofilm-associated cells of both strains showed statistically greater resistance (P < 0.05) to hydrogen peroxide and quaternary ammonium sanitizer than their respective planktonic cells. This study shows that the rdar phenotype of E. coli O157:H7 strain 43895OR is important in multicellular growth, biofilm formation, and resistance to sanitizers. However, the lack of cellulose production by strain 43895OR indicates important differences in the ECM composition compared to that of SALMONELLA:
The ability of 71 strains of Salmonella enterica originating from produce, meat, or clinical sources to form biofilms was investigated. A crystal violet binding assay demonstrated no significant ...differences in biofilm formation by isolates from any source when tested in any of the following three media: Luria-Bertani broth supplemented with 2% glucose, tryptic soy broth (TSB), or 1/20th-strength TSB. Incubation was overnight at 30 degrees C under static conditions. Curli production and cellulose production were monitored by assessing morphotypes on Luria-Bertani agar without salt containing Congo red and by assessing fluorescence on Luria-Bertani agar containing calcofluor, respectively. One hundred percent of the clinical isolates exhibited curli biosynthesis, and 73% demonstrated cellulose production. All meat-related isolates formed curli, and 84% produced cellulose. A total of 80% of produce-related isolates produced curli, but only 52% produced cellulose. Crystal violet binding was not statistically different between isolates representing the three morphotypes when grown in TSB; however, significant differences were observed when strains were cultured in the two other media tested. These data demonstrate that the ability to form biofilms is not dependent on the source of the test isolate and suggest a relationship between crystal violet binding and morphotype, with curli- and cellulose-deficient isolates being least effective in biofilm formation.
Salmonella enterica forms biofilms that are relatively resistant to chemical sanitizing treatments. Ionizing radiation has been used to inactivate Salmonella on a variety of foods and contact ...surfaces, but the relative efficacy of the process against biofilm-associated cells versus free-living planktonic cells is not well documented. The radiation sensitivity of planktonic or biofilm-associated cells was determined for three food-borne-illness-associated isolates of SALMONELLA: Biofilms were formed on sterile glass slides in a coincubation apparatus, using inoculated tryptic soy broth, incubated at 37°C for 48 h. Resulting biofilms were 18 to 24 microm in height as determined by confocal scanning laser microscopy. The planktonic and biofilm cultures were gamma irradiated to doses of 0.0 (control), 0.5, 1.0, 1.5, 2.0 and 2.5 kGy. The D₁₀ value (the dose of radiation required to reduce a population by 1 log₁₀, or 90%) was calculated for each isolate-culture based on surviving populations at each radiation dose. The D₁₀ values of S. enterica serovar Anatum were not significantly (P < 0.05) different for biofilm-associated (0.645 kGy) and planktonic (0.677 kGy) cells. In contrast, the biofilm-associated cells of S. enterica serovar Stanley were significantly more sensitive to ionizing radiation than the respective planktonic cells, with D₁₀ values of 0.531 and 0.591 kGy, respectively. D₁₀ values of S. enterica serovar Enteritidis were similarly reduced for biofilm-associated (0.436 kGy) versus planktonic (0.535 kGy) cells. The antimicrobial efficacy of ionizing radiation is therefore preserved or enhanced in treatment of biofilm-associated bacteria.
In this study, the transmission of Escherichia coli O157:H7 to lettuce plants through spray and surface irrigation was demonstrated. For all treatments combined, the number of plants testing positive ...following a single exposure to E. coli O157: H7 through spray irrigation (29 of 32 plants) was larger than the number testing positive following surface irrigation (6 of 32 plants). E. coli O157:H7 persisted on 9 of 11 plants for 20 days following spray irrigation with contaminated water. Immersion of harvested lettuce heads for 1 min in a 200 ppm chlorine solution did not eliminate all E. coli O157:H7 cells. The results of this study suggest that regardless of the irrigation method used, crops can become contaminated; therefore, the irrigation of food crops with water of unknown microbial quality should be avoided.
Irrigation water collected at farms growing crops for human consumption was artificially contaminated with E. coli O157:H7 and used to irrigate lettuce plants. Plants in a growth chamber were spray ...irrigated either once or intermittently with water contaminated with 10(2) or 10(4) CFU of E. coli O157:H7 per ml and were then sampled over a 30-day period. Only plants exposed to 10(2) CFU/ml on day 1 did not harbor the pathogen at the end of the sampling period. All other treatments resulted in contaminated plants at harvest. Plants irrigated with 10(4) CFU/ml contained high levels (up to 5 log CFU/g) of the pathogen at harvest. The results obtained in this study underscore the assertion that spray irrigation (the application of water directly to plant leaves) is linked to the contamination of crops and suggest that repeated exposure increases the E. coli O157:H7 level on the plant.
The ability of two strains of Salmonella to form biofilms on whole cantaloupe melons was investigated. Ten microliters of bacterial suspensions was spot-inoculated onto cantaloupe melon rinds in ...pre-marked areas, and the cantaloupe melons were held at either 10 or 20C. Biofilm formation was monitored using scanning electron microscopy on excised portions of the cantaloupe melon rind at 2, 24, 48, 72 and 144 h postinoculation. Micrographs indicated that biofilm formation occurred rapidly following introduction of cells (2 h at 20C) onto the cantaloupe melon rind. A fibrillar material was visible after just 2 h at 20C, and cells were embedded in extracellular polymeric material after 24 h at either temperature. These results indicate that a human pathogen is capable of forming a biofilm on plant tissue and that biofilm formation may be responsible for the increased recalcitrance of bacteria to aqueous sanitizers.
Foodborne pathogens may exist as endophytes of growing plants. The internalization of Escherichia coli O157:H7 or other foodborne pathogens in growing lettuce plants may be independent of microbial ...factors. Mature lettuce plants were surface irrigated with E. coli O157:H7 or with FluoSpheres (fluorescent microspheres) and harvested 1, 3, and 5 days postexposure. FluoSpheres were utilized as a bacterial surrogate. Microscopic examination of root, stem, and leaf tissue sections revealed that FluoSpheres were internalized into growing plants. Laser scanning confocal microscopy revealed that FluoSpheres were present within the root tissue and leaf stem tissue. The presence of FluoSpheres in internal portions of stem and leaf tissue suggests transport of the spheres from the root upward into the edible tissue. The level of uptake of FluoSpheres and E. coli O157:H7 was quantified using filtration. Numbers of FluoSpheres and E. coli O157:H7 cells in plant tissue were similar. The entry of E. coli O157:H7 into lettuce plants may be a passive event because the concentration of FluoSpheres was similar to that of the pathogen.
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