Joseph F. Frank

Surface-adherent growth of Listeria monocytogenes is associated with increased resistance to surfactant sanitizers and heat

Surface-adherent microcolonies of Listeria monocytogenes were obtained by growing cells on glass slides immersed in a low nutrient medium containing excess glucose. The susceptibility of the adherent populations to benzalkonium chloride (100, 400, and 800 ppm solutions), anionic acid sanitizer (200 and 400 ppm solutions), and heat (55 and 70°C) was determined. Adherent microcolony cells decreased by 2 to 3 log cycles immediately after exposure to the sanitizers. The remaining population of microcolony cells survived 20 min of exposure demonstrating resistance to both sanitizers at all concentrations. Adherent single cells exhibited an initial 3 to 5 log decline in numbers and reached undetectable levels after 12 to 16 min of exposure to the sanitizers. Planktonic cells were reduced to undetectable levels after 30 sec exposure to the lowest concentration of each sanitizer. Removing adherent cells from the surface increased their sanitizer susceptibility to near that of planktonic cells. Heating adherent microcolonies at 70°C for 5 min resulted in a less than 5-log decrease in population with a surviving population of over 10 cfu/sq cm. These results demonstrate the ability of L. monocytogenes to develop resistance to inactivating agents when exposed to specific growth environments.

Growth of Listeria monocytogenes as a biofilm on various food-processing surfaces

The objective of this research was to determine the ability of Listeria monocytogenes to grow as a biofilm on various food-processing surfaces including stainless steel, Teflon®, nylon, and polyester floor sealant. Each of these surfaces was able to support biofilm formation when incubation was at 21°C in Trypticase soy broth (TSB). Biofilm formation was greatest on polyester floor sealant (40% of surface area covered after 7 days of incubation) and least on nylon (3% coverage). The use of chemically defined minimal medium resulted in a lack of biofilm formation on polyester floor sealant, and reduced biofilm levels on stainless steel. Biofilm formation was reduced with incubation at 10°C, but Teflon® and stainless steel still allowed 23 to 24% coverage after incubation in TSB for 18 days. Biofilm growth of L. monocytogenes was sufficient to provide a substantial risk of this pathogen contaminating the food-processing plant environment if wet surfaces are not maintained in a sanitary condition.

Penetration of Escherichia coli O157:H7 into lettuce tissues as affected by inoculum size and temperature and the effect of chlorine treatment on cell viability.

Penetration of Escherichia coli O157:H7 into iceberg lettuce tissues and the effect of chlorine treatment on cell viability were evaluated. Attachment of different inoculum levels (10(9), 10(8), and 10(7) CFU/ml) was examined by determining the number of cells at the surface and the cut edge of lettuce leaves (2 by 2 cm). E. coli O157:H7 attached preferentially to cut edges at all inoculum levels, with greater attachment per cm2 of lettuce at higher inoculum levels. A longer attachment time allowed more cells to attach at both sites. Immunostaining with a fluorescein isothiocyanate-labeled antibody revealed that cells penetrated into lettuce leaves from cut edges. Cells showed greater penetration when lettuce was held at 4 degrees C compared with 7, 25, or 37 degrees C and were detected at an average of 73.5 +/- 16.0 microm below the surfaces of cut tissues. Penetrating cells were mostly found at the junction of lettuce cells. The viability of attached cells after treatment with 200 mg/liter (200 ppm) of free chlorine for 5 min was examined by plating on tryptic soy agar and by a nalidixic acid elongation method. Although chlorine treatment caused significant reduction in attachment (0.7- and 1.0-log reduction at surfaces and cut edges, respectively), cells remained attached at high numbers (7.9 and 8.1 log CFU/cm2 at surfaces and cut edges, respectively). Elongated cells were observed in stomata and within the tissues of the lettuce, indicating they were protected from contact with chlorine.

Comparison of the attachment of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and Pseudomonas fluorescens to lettuce leaves.

Attachment of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and Pseudomonas fluorescens on iceberg lettuce was evaluated by plate count and confocal scanning laser microscopy (CSLM). Attachment of each microorganism (approximately 10(8) CFU/ml) on the surface and the cut edge of lettuce leaves was determined. E. coli O157:H7 and L. monocytogenes attached preferentially to cut edges, while P. fluorescens attached preferentially to the intact surfaces. Differences in attachment at the two sites were greatest with L. monocytogenes. Salmonella Typhimurium attached equally to the two sites. At the surface, P. fluorescens attached in greatest number, followed by E. coli O157:H7, L. monocytogenes, and Salmonella Typhimurium. Attached microorganisms on lettuce were stained with fluorescein isothiocyanate and visualized by CSLM. Images at the surface and the cut edge of lettuce confirmed the plate count data. In addition, microcolony formation by P. fluorescens was observed on the lettuce surface. Some cells of each microorganism at the cut edge were located within the lettuce tissues, indicating that penetration occurred from the cut edge surface. The results of this study indicate that different species of microorganisms attach differently to lettuce structures, and CSLM can be successfully used to detect these differences.

Inactivation of Listeria monocytogenes/Pseudomonas Biofilms by Peracid Sanitizers

The ability of peracetic acid and peroctanoic acid sanitizers to inactivate mixed-culture biofilms of a Pseudomonas sp. and Listeria monocytogenes on stainless steel was investigated. Types of biofilms tested included a 4-h attachment of the mixed-cell suspension and a 48-h biofilm of mixed culture formed in skim milk or tryptic soy broth. Biofilm-containing coupons were immersed in solutions of hypochlorite, peracetic acid, and peroctanoic acid either with or without organic challenge. Organic challenge consisted of either coating the biofilms with milk that were then allowed to dry, or adding milk to the sanitizing solution to achieve a 5% concentration. Surviving cells were enumerated by pouring differential agar directly on the treated surfaces. The peracid sanitizers were more effective than chlorine for inactivating biofilm in the presence of organic challenge. The 48-h mixed-culture biofilm grown in milk was reduced to less than 3 CFU/cm2 by 160 ppm of peracid sanitizer after 1 min of exposure. Peroctanoic acid was more effective than peracetic acid against biofilm cells under conditions of organic challenge. Pseudomonas and L. monocytogenes were inactivated to similar levels by the sanitizer treatments, even though Pseudomonas predominated in the initial biofilm population.

Growth of Listeria monocytogenes at 10°C in Biofilms with Microorganisms Isolated from Meat and Dairy Processing Environments

Listeria monocytogenes was able to grow in multispecies biofilms formed on stainless steel at 10°C in the presence of bacteria isolated from dairy and meat plant environments. Biofilm L. monocytogenes increased more slowly in the presence of competing microflora than in monoculture, but none of the eight competing isolates studied were able to completely exclude L. monocytogenes from the biofilm within 25 days of incubation. Once a population of L. monocytogenes was established on the surface, it invariably increased in numbers. In biofilms containing a mixture of 4 competitive cultures, L. monocytogenes maintained itself at about 1% of the total population.

Quantitative determination of the role of lettuce leaf structures in protecting Escherichia coli O157:H7 from chlorine disinfection.

Viability of Escherichia coli O157:H7 cells on lettuce leaves after 200 mg/liter (200 ppm) chlorine treatment and the role of lettuce leaf structures in protecting cells from chlorine inactivation were evaluated by confocal scanning microscopy (CSLM). Lettuce samples (2 by 2 cm) were inoculated by immersing in a suspension containing 10(9) CFU/ml of E. coli O157: H7 for 24+/-1 h at 4 degrees C. Rinsed samples were treated with 200 mg/liter (200 ppm) chlorine for 5 min at 22 degrees C. Viability of E. coli O157:H7 cells was evaluated by CSLM observation of samples stained with Sytox green (dead cell stain) and Alexa 594 conjugated antibody against E. coli O157:H7. Quantitative microscopic observations of viability were made at intact leaf surface, stomata, and damaged tissue. Most E. coli O157:H7 cells (68.3+/-16.2%) that had penetrated 30 to 40 microm from the damaged tissue surface remained viable after chlorine treatment. Cells on the surface survived least (25.2+/-15.8% survival), while cells that penetrated 0 to 10 microm from the damaged tissue surface or entered stomata showed intermediate survival (50.8 +/-13.5 and 45.6+/-9.7% survival, respectively). Viability was associated with the depth at which E. coli O157:H7 cells were in the stomata. Although cells on the leaf surface were mostly inactivated, some viable cells were observed in cracks of cuticle and on the trichome. These results demonstrate the importance of lettuce leaf structures in the protection of E. coli O157:H7 cells from chlorine inactivation.

Inactivation of surface-adherent Listeria monocytogenes hypochlorite and heat

Inactivation by hypochlorite of Listeria monocytogenes cells adherent to stainless steel was determined. Adherent cell populations were prepared by incubating stainless steel slides with a 24 h culture of L. monocytogenes for 4 h at 21°C. Adherent microcolonies were prepared by growing L. monocytogenes on stainless steel slides submerged in a 1:15 dilution of tryptic soy broth at 21°C. The slides were then rinsed and transferred to fresh sterile broth every 2 d with a total incubation time of 8 d. Although the 4 h and 8 d adherent populations were at similar levels, 8 d adherent cells were over 100 times more resistant than the 4 h adherent cell population when exposed to 200 ppm hypochlorite for 30 s. When stainless steel slides containing adherent cells were heated at 72°C both adherent cell populations were inactivated after 1 min. Detectable numbers of L. monocytogenes remained on stainless steel slides after treatment at 65°C for 3 min when adherent 8 d cells were tested but not when adherent 4 h cells were used.

Attachment of Escherichia coli O157:H7 grown in tryptic soy broth and nutrient broth to apple and lettuce surfaces as related to cell hydrophobicity, surface charge, and capsule production

This study investigated the effect of growth in tryptic soy broth (TSB) and nutrient broth (NB) on the ability Escherichia coli O157:H7 to attach to lettuce and apple surfaces. In addition, cell surface hydrophobicity, charge and capsule production were determined on cells grown in these media. Cells grown in NB attached less to lettuce and apple surfaces than did those grown in TSB. TSB, but not NB, supported capsule production by E. coli O157:H7. Cells grown in TSB were more hydrophilic than those grown in NB. No difference was found in the electrokinetic properties of cells grown in these media. Electrostatic and hydrophobic interactions and surface proteins did not appear to play an important role in the attachment of E. coli O157:H7 to these surfaces. Of the factors studied, only capsule production was associated with attachment ability.

Effect of nutrients on biofilm formation by Listeria monocytogenes on stainless steel

The effect of nutrients on the development of biofilms by Listeria monocytogenes was investigated using semicontinuous culture in a chemically defined minimal medium (modified Welshimers broth, MWB). Inoculated slides were used for the development of biofilms for 12 days at 21°C under various nutrient conditions. Biofilms were quantified by measuring the percentage of area covered by cells, using computerized image analysis. MWB allowed slow development of Listeria monocytogenes biofilms. Glucose levels between 1 and 20 g/l did not affect biofilm development. A reduction or increase in phosphate level from that occurring in MWB (37.52 g/l) reduced biofilm development. The degree to which biofilms initially developed was associated with amino acid concentration within the range of 0.12 to 6 g/l; however, after 12 days of incubation, the amount of biofilm produced was not affected by amino acid concentration. Replacement of amino acids in MWB with tryptone initially enhanced biofilm development, but the effect was not significant after 12 days of incubation. Of five carbohydrates tested, mannose and trehalose enhanced biofilm development.