Jee Hoon Ryu

Biofilm Formation by Escherichia coli O157:H7 on Stainless Steel: Effect of Exopolysaccharide and Curli Production on Its Resistance to Chlorine

ABSTRACT The resistance of Escherichia coli O157:H7 strains ATCC 43895-, 43895-EPS (an exopolysaccharide [EPS]-overproducing mutant), and ATCC 43895+ (a curli-producing mutant) to chlorine, a sanitizer commonly used in the food industry, was studied. Planktonic cells of strains 43895-EPS and/or ATCC 43895+ grown under conditions supporting EPS and curli production, respectively, showed the highest resistance to chlorine, indicating that EPS and curli afford protection. Planktonic cells (ca. 9 log10 CFU/ml) of all strains, however, were killed within 10 min by treatment with 50 μg of chlorine/ml. Significantly lower numbers of strain 43895-EPS, compared to those of strain ATCC 43895-, attached to stainless steel coupons, but the growth rate of strain 43895-EPS on coupons was not significantly different from that of strain ATCC 43895-, indicating that EPS production did not affect cell growth during biofilm formation. Curli production did not affect the initial attachment of cells to coupons but did enhance biofilm production. The resistance of E. coli O157:H7 to chlorine increased significantly as cells formed biofilm on coupons; strain ATCC 43895+ was the most resistant. Population sizes of strains ATCC 43895+ and ATCC 43895- in biofilm formed at 12°C were not significantly different, but cells of strain ATCC 43895+ showed significantly higher resistance than did cells of strain ATCC 43895-. These observations support the hypothesis that the production of EPS and curli increase the resistance of E. coli O157:H7 to chlorine.

Influence of acid tolerance responses on survival, growth, and thermal cross-protection of Escherichia coli O157:H7 in acidified media and fruit juices

A study was done to determine survival and growth characteristics of acid-adapted, acid-shocked, and control cells of Escherichia coli O157:H7 inoculated into tryptic soy broth (TSB) acidified with organic acids and three commercial brands of apple cider and orange juice. The three types of cells behaved similarly in TSB acidified with acetic acid; however, in TSB (pH 3.9) acidified with lactic acid, acid-adapted cells were more tolerant than acid-shocked cells which, in turn, were more tolerant than control cells. The ability of the three types of cells to grow after inoculation into acidified TSB, then plated on tryptic soy agar containing sodium chloride was determined. Tolerance of acid-adapted cells and, less markedly, acid-shocked cells to sodium chloride was diminished, compared to control cells. The pathogen showed extraordinary tolerance to the low pH of apple cider and orange juice held at 5 or 25 degrees C for up to 42 days. Growth occurred in one brand of apple cider (pH 3.98) incubated at 25 degrees C. Regardless of test parameters, there was no indication that cell types differed in tolerance to the acidic environment in apple cider or orange juice. Survival of control, acid-adapted, and acid-shocked cells heated in apple cider and orange juice was studied. Within each apple cider or orange juice, D(52 degrees C)-values of acid-adapted cells were considerably higher than those of acid-shocked or control cells, which indicates that heat tolerance can be substantially enhanced by acid adaptation compared to acid shock.

Biofilm formation and sporulation by Bacillus cereus on a stainless steel surface and subsequent resistance of vegetative cells and spores to chlorine, chlorine dioxide, and a peroxyacetic acid-based sanitizer.

Biofilm formation by Bacillus cereus 038-2 on stainless steel coupons, sporulation in the biofilm as affected by nutrient availability, temperature, and relative humidity, and the resistance of vegetative cells and spores in biofilm to sanitizers were investigated. Total counts in biofilm formed on coupons immersed in tryptic soy broth (TSB) at 12 and 22 degrees C consisted of 99.94% of vegetative cells and 0.06% of spores. Coupons on which biofilm had formed were immersed in TSB or exposed to air with 100, 97, 93, or 85% relative humidity. Biofilm on coupons immersed in TSB at 12 degrees C for an additional 6 days or 22 degrees C for an additional 4 days contained 0.30 and 0.02% of spores, respectively, whereas biofilm exposed to air with 100 or 97% relative humidity at 22 degrees C for 4 days contained 10 and 2.5% of spores, respectively. Sporulation did not occur in biofilm exposed to 93 or 85% relative humidity at 22 degrees C. Treatment of biofilm on coupons that had been immersed in TSB at 22 degrees C with chlorine (50 microg/ml), chlorine dioxide (50 microg/ml), and a peroxyacetic acid-based sanitizer (Tsunami 200, 40 microg/ml) for 5 min reduced total cell counts (vegetative cells plus spores) by 4.7, 3.0, and 3.8 log CFU per coupon, respectively; total cell counts in biofilm exposed to air with 100% relative humidity were reduced by 1.5, 2.4, and 1.1 log CFU per coupon, respectively, reflecting the presence of lower numbers of vegetative cells. Spores that survived treatment with chlorine dioxide had reduced resistance to heat. It is concluded that exposure of biofilm formed by B. cereus exposed to air at high relative humidity (> or =97%) promotes the production of spores. Spores and, to a lesser extent, vegetative cells embedded in biofilm are protected against inactivation by sanitizers. Results provide new insights to developing strategies to achieve more effective sanitation programs to minimize risks associated with B. cereus in biofilm formed on food contact surfaces and on foods.

Inactivation of Escherichia coli O157:H7 in biofilm on stainless steel by treatment with an alkaline cleaner and a bacteriophage

Aims:  To determine the effectiveness of an alkaline cleaner used in food‐processing plants and a lytic bacteriophage specific for Escherichia coli O157:H7 in killing wild type and rpoS‐deficient cells of the pathogen in a biofilm.

Attachment of and Biofilm Formation by Enterobacter sakazakii on Stainless Steel and Enteral Feeding Tubes

ABSTRACT Enterobacter sakazakii has been reported to form biofilms, but environmental conditions affecting attachment to and biofilm formation on abiotic surfaces have not been described. We did a study to determine the effects of temperature and nutrient availability on attachment and biofilm formation by E. sakazakii on stainless steel and enteral feeding tubes. Five strains grown to stationary phase in tryptic soy broth (TSB), infant formula broth (IFB), or lettuce juice broth (LJB) at 12 and 25°C were examined for the extent to which they attach to these materials. Higher populations attached at 25°C than at 12°C. Stainless steel coupons and enteral feeding tubes were immersed for 24 h at 4°C in phosphate-buffered saline suspensions (7 log CFU/ml) to facilitate the attachment of 5.33 to 5.51 and 5.03 to 5.12 log CFU/cm2, respectively, before they were immersed in TSB, IFB, or LJB, followed by incubation at 12 or 25°C for up to 10 days. Biofilms were not produced at 12°C. The number of cells of test strains increased by 1.42 to 1.67 log CFU/cm2 and 1.16 to 1.31 log CFU/cm2 in biofilms formed on stainless steel and feeding tubes, respectively, immersed in IFB at 25°C; biofilms were not formed on TSB and LJB at 25°C, indicating that nutrient availability plays a major role in processes leading to biofilm formation on the surfaces of these inert materials. These observations emphasize the importance of temperature control in reconstituted infant formula preparation and storage areas in preventing attachment and biofilm formation by E. sakazakii.

Cronobacter sakazakii in foods and factors affecting its survival, growth, and inactivation

Cronobacter sakazakii has been isolated from a wide range of environmental sources and from several foods of animal and plant origin. While infections caused by C. sakazakii have predominantly involved neonates and infants, its presence on or in foods other than powdered infant formula raises concern about the safety risks these foods pose to immunocompromised consumers. We have done a series of studies to better understand the survival and growth characteristics of C. sakazakii in infant formula, infant cereal, fresh-cut produce, and juices made from fresh produce. Over a 12-month storage period, the pathogen survived better in dried formula and cereal at low a(w) (0.25-0.30) than at high a(w) (0.69-0.82) and at 4 degrees C compared to 30 degrees C. C. sakazakii grows in formulas and cereals reconstituted with water or milk and held at 12-30 degrees C. The composition of formulas or cereals does not markedly affect the rate of growth. C. sakazakii grows well on fresh-cut apple, cantaloupe, watermelon, cabbage, carrot, cucumber, lettuce, and tomato at 25 degrees C and in some types of produce at 12 degrees C. Treatment of fresh fruits and vegetables with sanitizers such as chlorine, chlorine dioxide, and a peroxyacetic acid-based solution causes reductions of 1.6-5.4 log CFU/apple, tomato, and lettuce. Cells of C. sakazakii in biofilms formed on stainless steel and enteral feeding tubes or dried on the surface of stainless steel have increased resistance to disinfectants. Death of cells in biofilms is affected by atmospheric relative humidity. These studies have contributed to a better understanding of the behavior of C. sakazakii in and on foods and on food-contact surfaces, thereby enabling the development of more effective strategies and interventions for its control.

Attachment and biofilm formation on stainless steel by Escherichia coli O157:H7 as affected by curli production

Aims:  The aim of this study was to determine the role of curli in attachment and biofilm formation by Escherichia coli O157:H7 on stainless steel.

Behavior of acid-adapted and unadapted Escherichia coli O157:H7 when exposed to reduced pH achieved with various organic acids.

A study was done to determine if various organic acids differ in their inhibitory or lethal activity against acid-adapted and unadapted Escherichia coli O157:H7 cells. E. coli O157:H7 strain EO139, isolated from venison jerky, was grown in tryptic soy broth (TSB) and in TSB supplemented with 1% glucose (TSBG) for 18 h at 37 degrees C, then plated on tryptic soy agar (TSA) acidified with malic, citric, lactic, or acetic acid at pH 5.4, 5.1, 4.8, 4.5, 4.2, and 3.9. Regardless of whether cells were grown in TSB or TSBG, visible colonies were not formed when plated on TSA acidified with acetic, lactic, malic, or citric acids at pH values of < or =5.4, < or =4.5, < or =4.2, or < or =4.2, respectively. Cells not adapted to reduced pH did not form colonies on TSA acidified with lactic acid (pH 3.9) or acetic acid (pH 3.9 and 4.2); however, a portion of acid-adapted cells remained viable on TSA containing lactic acid (pH 3.9) or acetic acid (pH 4.2) and could be recovered in TSB. Inactivation of acid-adapted cells was less than that of unadapted cells in TSB acidified at pH 3.9 with citric, lactic, or acetic acid and at pH 3.4 with malic acid. Significantly (P< or =0.05) higher numbers of acid-adapted cells, compared with unadapted cells, were detected 12 h after inoculation of TSB acidified with acetic acid at pH 3.9; in TSB containing lactic acid (pH 3.9), the number of acid-adapted cells was higher than the number of unadapted cells after 5 h. In TSB acidified at pH 3.9 with citric acid or pH 3.4 with malic acid, significantly higher numbers of acid-adapted cells survived. This study shows that organic acids differ in their inhibitory or lethal activity against acid-adapted and unadapted E. coli O157:H7 cells, and acid-adapted cells are more tolerant than unadapted cells when subsequently exposed to reduced pH caused by these acids.

Effectiveness of Disinfectants in Killing Enterobacter sakazakii in Suspension, Dried on the Surface of Stainless Steel, and in a Biofilm

ABSTRACT The effectiveness of 13 disinfectants used in hospitals, day-care centers, and food service kitchens in killing Enterobacter sakazakii in suspension, dried on the surface of stainless steel, and in biofilm was determined. E. sakazakii exhibited various levels of resistance to the disinfectants, depending on the composition of the disinfectants, amount and type of organic matrix surrounding cells, and exposure time. Populations of planktonic cells suspended in water (7.22 to 7.40 log CFU/ml) decreased to undetectable levels (<0.30 log CFU/ml) within 1 to 5 min upon treatment with disinfectants, while numbers of cells in reconstituted infant formula were reduced by only 0.02 to 3.69 log CFU/ml after the treatment for 10 min. The presence of infant formula also enhanced the resistance to the disinfectants of cells dried on the surface of stainless steel. The resistance of cells to disinfectants in 6-day-old and 12-day-old biofilms on the surface of stainless steel was not significantly different. The overall order of efficacy of disinfectants in killing E. sakazakii was planktonic cells > cells inoculated and dried on stainless steel > cells in biofilms on stainless steel. Findings show that disinfectants routinely used in hospital, day-care, and food service kitchen settings are ineffective in killing some cells of E. sakazakii embedded in organic matrices.

Attachment and Biofilm Formation by Escherichia coli O157:H7 on Stainless Steel as Influenced by Exopolysaccharide Production, Nutrient Availability, and Temperature

The influence of exopolysaccharide (EPS) production, nutrient availability, and temperature on attachment and biofilm formation by Escherichia coli O157:H7 strains ATCC 43895 (wild type) and 43895-EPS (extensive EPS-producing mutant) on stainless steel coupons (SSCs) was investigated. Cells grown on heated lettuce juice agar and modified tryptic soy agar were suspended in phosphate-buffered saline (PBS). SSCs were immersed in the cell suspension (10(9) CFU/ml) at 4 degrees C for 24 h. Biofilm formation by cells attached to SSCs as affected by immersing in 10% tryptic soy broth (TSB), lettuce juice broth (LJB), and minimal salts broth (MSB) at 12 and 22 degrees C was studied. A significantly lower number of strain 43895-EPS cells, compared to strain ATCC 43895 cells, attached to SSCs during a 24-h incubation (4 degrees C) period in PBS suspension. Neither strain formed a biofilm on SSCs subsequently immersed in 10% TSB or LJB, but both strains formed biofilms in MSB. Populations of attached cells and planktonic cells of strain ATCC 43895 gradually decreased during incubation for 6 days in LJB at 22 degrees C, but populations of strain 43895-EPS remained constant for 6 days at 22 degrees C, indicating that the EPS-producing mutant, compared to the wild-type strain, has a higher tolerance to the low-nutrient environment presented by LJB. It is concluded that EPS production by E. coli O157:H7 inhibits attachment to SSCs and that reduced nutrient availability enhances biofilm formation. Biofilms formed under conditions favorable for EPS production may protect E. coli O157:H7 against sanitizers used to decontaminate lettuce and produce processing environments. Studies are under way to test this hypothesis.