Hoikyung Kim

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.

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.

Survival and growth of Enterobacter sakazakii on fresh-cut fruits and vegetables and in unpasteurized juices as affected by storage temperature.

Enterobacter sakazakii is an emerging foodborne pathogen that has caused illnesses and deaths in infants and elderly immunocompromised adults. Outbreaks of E. sakazakii infection have been associated with infant formulas, but the documented presence of this pathogen in a wide variety of ready-to-eat foods, including lettuce and other raw vegetables, makes it important to learn more about its behavior in these environments. We investigated the survival and growth characteristics of E. sakazakii on fresh-cut apple, cantaloupe, strawberry, watermelon, cabbage, carrot, cucumber, lettuce, and tomato and in juices prepared from these fruits and vegetables. Produce and juices were inoculated with E. sakazakii at 2 to 3 log CFU/g and 1 to 2 log CFU/ml, respectively, and stored at 4, 12, or 25 degrees C. Populations either did not change or gradually decreased in fresh-cut produce and juices stored at 4 degrees C but grew at 12 degrees C on fresh-cut apple, cantaloupe, watermelon, cucumber, and tomato and in all juices except apple, strawberry, cabbage, and tomato juices. All fresh-cut fruits and vegetables except strawberry supported growth of E. sakazakii at 25 degrees C. Growth occurred in all juices except apple, strawberry, and cabbage juices, followed by decreases in population to < 1 CFU/ml after 48 to 72 h, which coincided with decreases in pH and an increase in the population of lactic acid bacteria. Increases in total counts occurred in all juices except strawberry juice stored at 25 degrees C and apple and strawberry juices stored at 12 degrees C. Total counts increased in cantaloupe, carrot, cucumber, and lettuce juices stored at 4 degrees C. Populations of molds and yeasts increased in apple and tomato juices stored at 25 degrees C but decreased to <1 CFU/ml in cabbage, lettuce, and cucumber juices. Further characterization of the behavior of E. sakazakii on fresh produce and in unpasteurized juice as affected by commercial packaging and handling practices is needed.

Fate of Enterobacter sakazakii attached to or in biofilms on stainless steel upon exposure to various temperatures or relative humidities.

Survival of Enterobacter sakazakii dried on the surface of stainless steel and exposed to 43% relative humidity, as affected by temperature, was studied. Populations of E. sakazakii (7.4 to 8.6 log CFU per coupon) on coupons dried for 2 h at 22 degrees C decreased significantly (P < or = 0.05) at 4, 25, and 37 degrees C within 10, 3, and 1 day(s), respectively, but the pathogen remained viable for up to 60 days. At a given storage temperature and time, reductions were significantly greater when cells had been suspended in water rather than in infant formula before drying. Formation of biofilm by E. sakazakii on stainless steel immersed in M9 medium, which contains minimal concentrations of nutrients, and infant formula at 25 degrees C and subsequent survival of cells at 25 degrees C as affected by exposure to 23, 43, 68, 85, and 100% relative humidity were investigated. Some of the cells in these biofilms survived under all test relative humidities for up to 42 days. The overall order of survival as affected by relative humidity was 100 > 23 = 43 = 68 > 85% relative humidity, regardless of the medium in which the biofilm was formed. Reduction in viability of cells was significantly greater in biofilm that had formed in M9 medium than in biofilm formed in infant formula. Results indicate that infant formula provides protection for attached cells, as well as cells in biofilm, against lethality on exposure to desiccation. These results are useful when predicting the survival characteristics of E. sakazakii on stainless steel surfaces in processing and preparation kitchen environments.

Inactivation of Escherichia coli O157:H7 in biofilm on food-contact surfaces by sequential treatments of aqueous chlorine dioxide and drying

We investigated the efficacy of sequential treatments of aqueous chlorine and chlorine dioxide and drying in killing Escherichia coli O157:H7 in biofilms formed on stainless steel, glass, plastic, and wooden surfaces. Cells attached to and formed a biofilm on wooden surfaces at significantly (P ≤ 0.05) higher levels compared with other surface types. The lethal activities of sodium hypochlorite (NaOCl) and aqueous chlorine dioxide (ClO₂) against E. coli O157:H7 in a biofilm on various food-contact surfaces were compared. Chlorine dioxide generally showed greater lethal activity than NaOCl against E. coli O157:H7 in a biofilm on the same type of surface. The resistance of E. coli O157:H7 to both sanitizers increased in the order of wood>plastic>glass>stainless steel. The synergistic lethal effects of sequential ClO₂ and drying treatments on E. coli O157:H7 in a biofilm on wooden surfaces were evaluated. When wooden surfaces harboring E. coli O157:H7 biofilm were treated with ClO₂ (200 μg/ml, 10 min), rinsed with water, and subsequently dried at 43% relative humidity and 22 °C, the number of E. coli O157:H7 on the surface decreased by an additional 6.4 CFU/coupon within 6 h of drying. However, when the wooden surface was treated with water or NaOCl and dried under the same conditions, the pathogen decreased by only 0.4 or 1.0 log CFU/coupon, respectively, after 12 h of drying. This indicates that ClO₂ treatment of food-contact surfaces results in residual lethality to E. coli O157:H7 during the drying process. These observations will be useful when selecting an appropriate type of food-contact surfaces, determining a proper sanitizer for decontamination, and designing an effective sanitization program to eliminate E. coli O157:H7 on food-contact surfaces in food processing, distribution, and preparation environments.

Controlled fermentation of kimchi using naturally occurring antimicrobial agents.

Kimchi is a traditional Korean fermented food. Since it ferments continuously during distribution and storage, the extension of shelf life by preventing over-acidification is a major concern in the kimchi industry. One of the most frequently attempted ways to delay fermentation is to add naturally occurring antimicrobial agents. Many researchers have investigated ways to delay over-acidification by adding minor ingredients, fruits or fruit seed extracts, extracts of medicinal herbs, culinary herbs and spices, and other miscellaneous substances to kimchi. The addition of naturally occurring antimicrobial agents may enhance the acceptability of kimchi to consumers over a longer period of time but may also have a disadvantage in that it may cause changes in sensory quality, especially if added in large amounts. To avoid undesirable sensory changes, application of hurdle technologies (i.e., multifactor preservative systems) which involve using combinations of low amounts of various naturally occurring antimicrobial agents as ingredients should be explored with the goal of controlling fermentation. If synergistic or additive antimicrobial effects can be achieved using small amounts of a combination of natural agents, changes in sensory qualities will be minimized, thereby prolonging shelf life. Research findings summarized in this review provide a basis for developing effective hurdle technologies using naturally occurring antimicrobial agents to extend shelf life of kimchi and perhaps other types of traditional fermented foods.

Efficacy of gaseous chlorine dioxide in inactivating Bacillus cereus spores attached to and in a biofilm on stainless steel

We evaluated the lethal activity of gaseous chlorine dioxide (ClO2) against Bacillus cereus spores attached to and in biofilm formed on a stainless steel surface. Aqueous ClO2 was prepared by mixing sulfuric acid (5% w/v) with sodium chlorite (10mg/mL), and gaseous ClO2 was produced by vaporization of aqueous ClO2 in an air-tight container. The concentration of gaseous ClO2 in the air within the container increased rapidly at first but gradually decreased over time. The lethality of gaseous ClO2 against B. cereus spores attached to stainless steel coupons (SSCs) and in biofilm formed by the pathogen on SSCs was determined. The B. cereus spores attached to SSCs (5.3±0.1logCFU/coupon) were completely inactivated within 1h at 25°C when treated with gaseous ClO2 (peak concentration: 115.3±5.0 parts per million [ppm]). The total number of vegetative cells and spores in biofilm formed by B. cereus on SSCs was 5.9±0.3logCFU/coupon; the spore count was 5.3±0.1logCFU/coupon. The vegetative cells and spores in biofilm were completely inactivated within 6h (peak concentration: 115.3±5.0ppm). Results show that B. cereus spores in biofilms are more resistant to gaseous ClO2 than are attached spores not in biofilms. Gaseous ClO2 was, nevertheless, very effective in killing B. cereus spores in biofilm on the surface of stainless steel. Results show promise for application of gaseous ClO2 to enhance the microbiological safety of foods that may come in contact with stainless steel and possibly other hard surfaces on which B. cereus biofilms have formed.

Combined effects of chlorine dioxide, drying, and dry heat treatments in inactivating microorganisms on radish seeds

We determined the combined effectiveness of ClO(2) (200 and 500 μg/ml, 5 min), air drying [25 °C, 40% relative humidity (RH), 2 h], and mild dry heat (55 °C, 23% RH, up to 48 h) treatments in killing total aerobic bacteria (TAB), Escherichia coli O157:H7, and molds and yeasts (MY) on radish seeds. A 5.1-log reduction in the number of TAB was achieved on radish seeds treated with 200 or 500 μg/ml ClO(2) followed by air drying for 2 h and dry heat treatment for 48 h or 24 h, respectively. When radish seeds were treated with 200 and 500 μg/ml ClO(2), air dried, and heat treated for 12 h and 6 h, respectively, the initial population of E. coli O157:H7 (5.6 log CFU/g) on seeds was reduced to an undetectable level (<0.8 log CFU/g). However, the pathogen was detected in 5-day-old sprouts. The reduction of MY (1.2-1.0 log CFU/g) on radish seeds under similar experimental conditions was not changed significantly during subsequent heat treatment up to 48 h. Results show that treating radish seeds with 500 μg/ml ClO(2), followed by air dried at 25 °C for 2 h and heat treatment at 55 °C for 36 h achieved a >5-log CFU/g reduction of TAB and E. coli O157:H7. These observations will be useful when developing effective strategies and practices to enhance the microbiological safety of radish sprouts.