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Applied and Environmental Microbiology, February 2007, p. 1256-1265, Vol. 73, No. 4
0099-2240/07/$08.00+0     doi:10.1128/AEM.01766-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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

Hoikyung Kim,1 Jee-Hoon Ryu,2* and Larry R. Beuchat1*

Center for Food Safety and Department of Food Science and Technology, University of Georgia, 1109 Experiment Street, Griffin, Georgia,1 Graduate School of Life Sciences and Biotechnology, Korea University, Anam-dong, Sungbuk-ku, Seoul 136-791, Republic of Korea2

Received 26 July 2006/ Accepted 5 December 2006


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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.


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INTRODUCTION
 
Enterobacter sakazakii causes meningitis (7, 19), sepsis (47), bacteremia (40), and necrotizing enterocolitis (51) in preterm neonates and infants. Powdered infant formula has been implicated as a source of E. sakazakii in outbreaks of infections (2, 5, 6, 21, 39, 40, 47, 51). In surveys done to determine the presence of E. sakazakii in powdered infant formula, the organism was detected in 2.4 to 14.2% of the products tested (22, 38).

The presence of E. sakazakii on the surface of utensils and equipment used for infant formula preparation has been reported to occur in clinical settings where neonatal infections have been documented (2, 10, 40, 47). The ability of bacteria to form biofilms on abiotic surfaces (22, 27, 30) raises the possibility that infections may occur following cross-contamination of freshly prepared infant formulas upon contact with soiled surfaces in formula preparation areas in hospitals, day-care centers, food service kitchens, and the home. Food-borne pathogens, e.g., Escherichia coli O157:H7 and Listeria monocytogenes, and spoilage bacteria such as Pseudomonas spp. have enhanced resistance to antibiotics or sanitizers when cells are in biofilms (13, 17, 18, 43), thus increasing the potential for survival on these surfaces.

Surface disinfection is routinely carried out in formula preparation areas in hospitals, food service kitchens, and day-care centers by applying liquid chemical disinfectants to food contact and non-food contact surfaces. The microbicidal activity of commercial surface cleaners and disinfectants is largely based on quaternary ammonium compounds, phenolic compounds, organic acids, alcohols, chlorine, and iodophors. Various commercial hard-surface cleaners and disinfectants have been evaluated for their efficacy in killing bacteria capable of causing food-borne infections (15, 45, 49, 53). During infant formula preparation and feeding, reconstituted formula containing E. sakazakii may contaminate abiotic surfaces. These surfaces may be treated with disinfectants immediately after contamination occurs, after the formula remains on the surface and dries, or after growth of E. sakazakii and the formation of biofilm. The efficacy of commercial disinfectants used in formula preparation areas in hospitals and child day-care centers in killing E. sakazakii in dried infant formula and biofilm has not been described.

We undertook studies to determine the effectiveness of disinfectants in killing E. sakazakii in suspension, dried on the surface of stainless steel, and embedded in biofilm on stainless steel. Quaternary ammonium and phenolic disinfectants commonly used in infant formula preparation areas, laboratories, and hospital, food service, and child day-care settings were evaluated. The effects of time elapsed after drying cells on stainless steel as well as the age of biofilms on resistance of cells to disinfectants were determined.


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MATERIALS AND METHODS
 
Bacterial strains and preparation of cells for treatment with disinfectants.
E. sakazakii strains 3231, isolated from cerebral spinal fluid of an infant, and 3439, isolated from a commercially manufactured powdered infant formula, were grown in tryptic soy broth (BBL/Difco, Sparks, Md.) at 37°C for 24 h. Cultures were transferred by loop inoculum (ca. 10 µl) three times at 24-h intervals. Each culture was centrifuged at 4,000 x g for 15 min at 4°C. Cells resuspended in two carriers, sterile synthetic hard water (400 µg of CaCO3/ml) and reconstituted infant formula, were used in various experiments.

Media in which cells were suspended.
Standard synthetic hard water was prepared according to the AOAC International (1) official methods of analysis. Phosphate-buffered saline (PBS) (pH 7.4) containing (per liter of distilled water) NaCl (8 g), KCl (0.2 g), Na2HPO4 (1.44 g), and KH2PO4 (0.24 g) was used as a medium to suspend cells for attachment to stainless steel. Reconstituted infant formula was made by combining Similac Neosure Advance powdered infant formula (Ross Pediatrics, Abbott Laboratories, Columbus, Ohio) with distilled water at a ratio of 1:10 (wt/vol), dissolving by heating at 50 to 60°C, and autoclaving at 121°C for 15 min.

Preparation of stainless steel coupons for spot inoculation and biofilm studies.
Stainless steel coupons (type 304; 5 cm by 2 cm) with no. 4 finish were used. The coupons were sonicated in 15% phosphoric acid solution at 80°C for 20 min, rinsed with distilled water, sonicated in alkali detergent solution (FS Pro-Chlor; Zep, Atlanta, Ga.) at 80°C for 20 min, and rinsed again with distilled water. The washed stainless steel coupons were dried and sterilized by autoclaving before use.

Preparation of disinfectant solutions.
Descriptions of disinfectants evaluated in the study are shown in Table 1. All disinfectants were tested at minimum concentrations recommended by the manufacturers. Thirteen products were evaluated for their efficacy in killing planktonic cells of E. sakazakii. Disinfectants 1 to 9 were prepared at double the strength (2x) of the desired treatment concentrations. Equal volumes of cell suspensions and 2x disinfectant solutions were combined to form the treatment mixture. Disinfectants 10 to 13, intended to be used without dilution, were tested at concentrations received from the manufacturers. For experiments involving spot inoculation of cells on stainless steel and cells in biofilms, disinfectants 2, 5, 6, 7, and 9 were diluted in sterile hard water to obtain the minimum treatment concentrations recommended; undiluted disinfectant 11 was also tested.


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  		TABLE 1. Disinfectants evaluated for lethality to E. sakazakii

Efficacy of disinfectants in killing planktonic cells.
Cells in pellets obtained as described above were resuspended in 100 ml of sterile hard water or reconstituted infant formula to give populations of ca. 7 log CFU/ml. Ten milliliters of cell suspension was deposited in a sterile 25- by 150-mm test tube containing 10 ml of 2x disinfectants 1 to 9 or sterile hard water (control) at 22 ± 2°C and thoroughly mixed. For evaluation of disinfectants 10 to 13, cells from pellets were resuspended in 1 ml of sterile hard water or reconstituted infant formula to give a population of ca. 9 log CFU/ml. Cell suspensions (0.1 ml) were added to sterile 25- by 150-mm test tubes containing 20 ml of disinfectants 10 to 13 or sterile hard water (control) and mixed thoroughly.

At time zero (within 10 s after combining the cell suspension with sterile hard water) and after holding the treatment mixtures (suspension to which water [control] or disinfectants were added) for 1, 5, and 10 min at 22 ± 2°C, 2 ml of the suspension was withdrawn and combined with 2 ml of 2x Dey-Engley (DE) neutralizing broth (BBL/Difco). According to Sutton et al. (48), at concentrations of quaternary ammonium and phenolic disinfectants used in our study, DE broth neutralizes components otherwise lethal to Enterobacteriaceae, such as Salmonella and Escherichia coli. Preliminary experiments showed that viability of E. sakazakii is unaffected when cells are suspended in DE broth to which disinfectants evaluated in our study were added. Undiluted suspensions (0.25 ml in quadruplicate and 0.1 ml in duplicate) and suspensions (0.1 ml in duplicate) serially diluted in 0.1% peptone water were surface plated on tryptic soy agar (TSA; BBL/Difco). Plates were incubated at 37°C for 48 h before colonies were counted.

Efficacy of disinfectants in killing E. sakazakii spot inoculated and dried on stainless steel.
Sterile stainless steel coupons were placed on a wire screen elevated 7 cm above the work surface in a laminar-flow biosafety cabinet. Suspensions (100 µl) of E. sakazakii in sterile hard water or reconstituted infant formula, prepared as described above, were deposited on each coupon to give ca. 8 log CFU/coupon. The inoculum was dried for 20 h (45% ± 7% relative humidity) at 22 ± 2°C in a laminar-flow biosafety cabinet. Inoculated coupons were immersed in sterile 25- by 150-mm test tubes containing 25 ml of disinfectant 2, 5, 6, 7, or 9 prepared at minimum treatment concentrations recommended by manufacturers, 25 ml of undiluted (full-strength) disinfectant 11, or sterile hard water (control) at 22 ± 2°C and thoroughly mixed. After treatment for 0 min (within 10 s after immersing coupons in sterile water) and after treatment for 1, 5, and 10 min in water or disinfectant solution, each coupon was transferred to a 50-ml tube containing 30 ml of DE broth and 3 g of sterile glass beads. The tube containing DE broth, coupon, and glass beads was vortexed at maximum speed for 1 min. Immediately after vortexing, undiluted samples (0.25 ml in quadruplicate and 0.1 ml in duplicate) and samples (0.1 ml in duplicate) serially diluted in 0.1% peptone water were surface plated on TSA and incubated at 37°C for 48 h. Colonies were counted, and populations (log CFU/coupon) of E. sakazakii remaining on stainless steel coupons before and after treatment with disinfectants were calculated.

Efficacy of disinfectants in killing E. sakazakii in biofilm on stainless steel.
Each sterile stainless steel coupon was immersed in a sterile 25- by 150-mm test tube containing 25 ml of a suspension of E. sakazakii in PBS (ca. 7 log CFU/ml) and incubated at 4°C for 24 h to facilitate attachment of cells. The coupons were transferred to 50-ml tubes containing 30 ml of sterile reconstituted infant formula prepared as described above and incubated at 25°C for 6 or 12 days. Coupons were removed from the formula and washed in 400 ml of sterile water (22 ± 2°C) with agitation for 15 s to remove most of the cells not present in or firmly attached to the biofilm matrix. The washed coupons were transferred to sterile 25- by 150-mm test tubes containing 25 ml of disinfectants 2, 5, 6, 7, and 9 prepared at minimum treatment concentrations recommended by manufacturers, undiluted disinfectant 11, or sterile hard water (control). After treatment for 0 min (within 10 s after immersing coupons in sterile water) and after treatment for 1, 5, and 10 min in water or disinfectant solution, each coupon was transferred to a 50-ml tube containing 30 ml of DE broth and 3 g of sterile glass beads and vortexed at maximum speed for 1 min to dislodge cells from the biofilms. The undiluted suspensions (0.25 ml in quadruplicate and 0.1 ml in duplicate) and suspensions (0.1 ml in duplicate) serially diluted in 0.1% peptone water were surface plated on TSA. Plates were incubated at 37°C for 48 h before the colonies were counted, and the number of cells (log CFU/coupon) surviving treatment was calculated.

Statistical analysis.
All experiments were replicated three times. In experiments involving stainless steel coupons, two coupons were examined at each sampling time. Data were analyzed using the general linear model of the Statistical Analysis Systems procedure (SAS; SAS Institute, Cary, N.C.). Statistically significant differences in populations of planktonic cells, spot-inoculated cells, dried cells, and cells of E. sakazakii in biofilms caused by treatment with disinfectants were determined. The influence of the presence of infant formula on efficacy of disinfectants in reducing populations of planktonic cells and spot-inoculated dried cells and the efficacy of disinfectants in killing cells in biofilms as affected by the maturation period (age) were determined using Fisher's least significant difference test. Significant differences are presented at a 95% confidence level (P ≤ 0.05).


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RESULTS AND DISCUSSION
 
Resistance of planktonic cells to disinfectants.
Numbers of E. sakazakii strain 3231 (Table 2) and strain 3439 (Table 3) suspended in hard water and reconstituted infant formula not containing disinfectants did not change significantly (P > 0.05) within 10 min at 22°C. Treatment of both strains suspended in water containing disinfectants for 1 min resulted in significant reductions (P ≤ 0.05) in populations compared to the number of cells recovered from water not containing disinfectants. For disinfectants 1 to 9, with the exceptions of planktonic cells in water treated with disinfectants 1, 5, and 6, populations of E. sakazakii strain 3231 (Table 2) decreased to <0.30 log CFU/ml within 1 min; with the exceptions of disinfectants 5 and 6, populations of strain 3439 (Table 3) were reduced to <0.30 log CFU/ml within 1 min. This indicates that disinfectants 1, 5, and 6, at the concentrations tested, have the lowest lethality among disinfectants 1 to 9 to E. sakazakii suspended in water. Disinfectants 1, 5, and 6 contain alkyl (50% C14, 40% C12, and 10% C16) dimethyl benzyl ammonium chloride as a major active ingredient (3 to 8%). After treatment of cells in water with disinfectants 1 to 9 for 5 min, populations of strains 3231 and 3439 were reduced from initial populations of 7.01 and 7.60 log CFU/ml, respectively, to <0.30 log CFU/ml, indicating that with sufficient exposure time, disinfectants 1 to 9 are equivalent in lethality to E. sakazakii. Populations of strains 3231 and 3439 suspended in infant formula were decreased significantly (P ≤ 0.05) by treatment with disinfectants 3 and 7 for 1 to 5 min, while treatment with the other disinfectants for 10 min did not decrease the populations.


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  		TABLE 2. Survival of planktonic cells of E. sakazakii strain 3231 as affected by treatment with disinfectants


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  		TABLE 3. Survival of planktonic cells of E. sakazakii strain 3439 as affected by treatment with disinfectants

Disinfectants 10 to 13 are spray products which are ready to use without dilution or additional preparation. Treatment of cells of E. sakazakii strains 3231 (Table 2) and 3439 (Table 3) suspended in water containing disinfectants 10 to 13 reduced initial populations of 7.01 and 7.60 log CFU/ml, respectively, to ≤0.30 log CFU/ml within 1 min. Of the ready-to-use products tested, disinfectant 10 had the lowest initial lethality to E. sakazakii, regardless of carrier composition.

Disinfectants 1 to 6 and 10 to 12 are quaternary ammonium-based disinfectants. At the concentrations tested, all contain alkyl dimethyl benzyl ammonium chloride (benzalkonium) at concentrations of 0.006 to 0.105%. The time required to achieve the same level of lethality to E. sakazakii in water, however, differed among these disinfectants, indicating that pH and constituents other than benzalkonium chloride contribute to bactericidal activity. Disinfectants 3 and 11, containing alkyl (60% C14, 30% C16, 5% C12, and 5% C18) dimethyl benzyl ammonium chloride, showed the greatest lethality, whereas disinfectants 1, 5, 6, and 10, which had lowest lethality, contain alkyl (50% C14, 40% C12, and 10% C16) dimethyl benzyl ammonium chloride. These differences may in part be responsible for differences in efficacy of these two groups of quaternary ammonium compounds in killing E. sakazakii suspended in water. Benzalkonium chloride is known to have different bactericidal activities depending on the length of its hydrophobic chain (24). The general order of antibacterial activity, which depends on the length of the alkyl chain, has been reported to be C14 > C16 > C12 ≥ C18 > C10 > C8 (24, 29, 46). Merianos (35) reported that C12, C14, and C16 homologues are most effective in inactivating yeasts and molds, gram-positive bacteria, and gram-negative bacteria, respectively. Observations in our study that quaternary ammonium compounds with the highest alkyl C14 and C16 content cause higher lethality to E. sakazakii are in agreement with these findings.

The type of cell carrier, i.e., water or infant formula, in which E. sakazakii was suspended significantly (P ≤ 0.05) affected the efficacy of disinfectants 1 to 9 in killing both test strains (Tables 2 and 3). The lethality of all disinfectants was markedly decreased in the presence of infant formula. The microbicidal activity of chlorine, quaternary ammonium compounds, peroxyacetic acid, hydrogen peroxide, and phenolic compounds is known to be reduced upon contact with organic materials (4, 25, 28, 33, 41, 50). In addition, upon combining infant formula (pH 6.6) with disinfectant solutions, the pH shifted toward 7.0. Disinfectants 6 and 8, for example, had the lowest pH (2.72 and 2.76, respectively) when combined with cells suspended in water, while respective pH values were 5.64 and 5.77 when combined with infant formula. The pHs of disinfectants 3, 5, and 9 ranged from 9.30 to 10.71 when combined with water containing cells but was reduced to 7.61 to 9.69 when combined with infant formula. Exposure of E. sakazakii to low pH (<4.0) is known to cause reductions in populations (14, 26). Exposure of E. sakazakii to environments at pHs 9.30 to 10.71, however, may not cause immediate death. Gurtler and Beuchat (20) reported that populations of E. sakazakii decreased by 0.5 log CFU/ml when cells were exposed to an environment at pH 11.25 for 5 min. Considering disinfectants 1 to 9, only in inoculated formulas treated with disinfectants 3 and 7 did significant (P ≤ 0.05) reductions in populations of both strains occur within 10 min. Disinfectant 3 contains n-alkyl (60% C14, 30% C16, 5% C12, and 5% C18) dimethyl benzyl ammonium chloride and n-alkyl dimethyl ethylbenzyl ammonium chloride, whereas disinfectant 7 contains peroxyacetic acid and hydrogen peroxide as active ingredients.

In contrast to the decreased effect infant formula has on lethality of disinfectants 1 to 9 to planktonic E. sakazakii, the type of carrier had no effect on lethality of disinfectants 10 to 13. This is attributed to the low concentration of organic material in the reaction mixture, which was 100-fold less than the amount introduced by the infant formula in experiments involving the evaluation of disinfectants 1 to 9.

Resistance of spot-inoculated, dried cells to disinfectants.
Table 4 shows populations of E. sakazakii strains 3231 and 3439 recovered from the surface of stainless steel coupons on which cells in water and infant formula were dried and treated with disinfectants 2, 5, 6, 7, 9, and 11. These disinfectants were selected for evaluation because they had different levels of lethality to E. sakazakii cells in suspension, represent a wide range of recommended applications, and are based on various types of microbicides, viz., quaternary ammonium compounds, phenolic compounds, and a combination of peroxyacetic acid and hydrogen peroxide. Treatment of coupons that had been inoculated with cells suspended in water with disinfectant 7, which contains peroxyacetic acid and hydrogen peroxide, and disinfectant 9, which contains phenolic compounds, reduced initial populations of 7.98 and 8.01 log CFU/coupon for strains 3231 and 3439, respectively, to <1.48 log CFU/coupon within 5 min. Both strains were reduced to populations <1.48 log CFU/coupon by treating coupons for 1 min with disinfectant 11, a quaternary ammonium compound product. Studies have shown that hydrogen peroxide (42), peroxyacetic acid (16), and phenolic compounds (52) are effective in reducing microbial populations on abiotic surfaces. We have observed a peroxyacetic acid-based sanitizer to be effective in killing E. sakazakii on produce (28). Cells of strains 3231 and 3439 applied to stainless steel using water as a carrier and initially at populations of 7.98 and 8.01 log CFU/coupon, respectively, survived 10-min treatments with disinfectants 2, 5, and 6, all quaternary ammonium products containing alkyl (50% C14, 40% C12, and 10% C16) dimethyl benzyl ammonium chloride as the major microbicide. Disinfectant 11, the most effective among the six disinfectants tested, contains alkyl (60% C14, 30% C16, 5% C12, and 5% C18) dimethyl benzyl ammonium chloride and n-alkyl dimethyl ethylbenzyl ammonium chloride.


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  		TABLE 4. Survival of E. sakazakii strains 3231 and 3439 spot inoculated and dried on the surface of stainless steel coupons as affected by the type of carrier (water or infant formula) in which cells were suspended

As with planktonic cells treated with disinfectants 1 to 9 (Tables 2 and 3), the composition of the carrier used to suspend cells significantly (P ≤ 0.05) affected the efficacy of disinfectants in killing cells of E. sakazakii strains 3231 and 3439 dried on the surface of stainless steel (Table 4). Cells of both strains of E. sakazakii in infant formula dried on stainless steel showed significantly (P ≤ 0.05) higher resistance to all disinfectants compared to the resistance of cells applied to stainless steel using water as a carrier (Tables 4). With the exceptions of disinfectants 7 and 11, ≥7.40 and ≥8.04 log CFU/coupon inoculated with strains 3231 and 3439, respectively, survived in infant formula initially containing 8.74 and 8.65 log CFU/coupon, respectively, after treatment with disinfectants for 10 min. Treatment with disinfectant 7 for 10 min or disinfectant 11 for 5 min reduced the number of E. sakazakii cells in dried infant formula to <1.48 log CFU/coupon.

The greatest reductions in populations of spot-inoculated, dried cells were achieved by treatment with disinfectant 11, regardless of type of cell carrier and strain (Table 4). In addition to its unique alkyl ammonium chloride composition, among the disinfectants tested, disinfectant 11 has the highest pH (12.04). The alkaline pH of disinfectant 11 may in part contribute additively to reductions in populations. Highly alkaline pH environments can cause disruption of the cell membrane of gram-negative bacteria, resulting in leakage of cytoplasm and death (34).

Compared to planktonic cells, a lower percentage of cells dried on stainless steel coupons were killed upon treatment with disinfectants. Although a portion of the dried cells would be expected to be injured by desiccation, thereby potentially increasing sensitivity to disinfectants, organic material (in the case of the infant formula carrier) and cells at or near the surface of the dried inoculum would provide protective barriers against contact with disinfectants. Some of the cells dried on the surface of stainless steel may have undergone starvation during drying for 20 h, a condition also known to increase the resistance of bacteria to sanitizers (3, 23, 54). Mosley et al. (36) reported that bacteria inoculated on stainless-steel strips were more resistant than planktonic cells to sanitizers. Treatment with various sanitizers, including quaternary ammonium compounds and peroxyacetic acid, was reported to be effective in killing Pseudomonas fluorescens and Yersinia enterocolitica in liquid suspension but relatively ineffective in killing cells attached to surfaces (37). Our observations on the behavior of E. sakazakii are in agreement with these reports.

Efficacy of disinfectants in killing E. sakazakii in biofilm.
Biofilms formed by E. sakazakii on stainless steel immersed in reconstituted infant formula for 6 or 12 days were treated with water and disinfectants 2, 5, 6, 7, 9, and 11 for 1, 5, and 10 min. Populations of strains 3231 and 3439 surviving treatments are shown in Table 5. Treatment with disinfectants 2, 5, and 6 did not significantly (P > 0.05) reduce the population of strain 3231 (7.68 log CFU/coupon) in 6-day-old biofilm compared to treatment with water (control). Treatment with disinfectants 7 and 9 for 10 min significantly (P ≤ 0.05) reduced populations but only by 2.45 and 0.81 log CFU/coupon, respectively. Treatment with disinfectant 11 decreased the population of strain 3231 in 6-day-old biofilm by 0.77 log CFU/coupon within 1 min and subsequently caused decreases to an undetectable level (<1.48 log CFU/coupon) at 5 min. As with strain 3231, treatment with disinfectant 11 for 5 min resulted in significant reductions in populations of strain 3439 in 6-day-old biofilms to an undetectable level (<1.48 log CFU/coupon). Exposure of biofilms to disinfectants 2, 5, 6, and 9 for 5 to 10 min caused significant reductions in populations of strain 3439 in 6-day-old biofilms, but ≥6.35 log CFU/coupon survived after treatment for 10 min.


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  		TABLE 5. Survival of E. sakazakii strains 3231 and 3439 in biofilm formed on the surface of stainless steel coupons immersed in infant formula at 25°C for 6 or 12 days as affected by treatment with disinfectants

The behavior of E. sakazakii in 12-day-old biofilms was similar to that in 6-day-old biofilms, indicating that the age of the biofilms did not have a major effect on the resistance of cells to disinfectants. Significant (P ≤ 0.05) reductions in numbers of both strains of E. sakazakii in 12-day-old biofilms to <1.48 log CFU/coupon occurred only upon treatment with disinfectant 11 for 5 min. Treatment with all other disinfectants significantly reduced populations of strains 3231 and 3439 in 12-day-old biofilms; however, reductions in populations were ≤2.89 log CFU/coupon upon treatment for 10 min. Regardless of strain or age of biofilms, disinfectant 11 had the greatest lethality to E. sakazakii; disinfectant 7 had the second greatest lethality.

At the concentrations tested, the lethality of all test disinfectants to E. sakazakii in biofilms was lower than that observed for planktonic cells or spot-inoculated, dried cells on the surface of stainless steel. The overall order of efficacy of all disinfectants in killing E. sakazakii was planktonic cells > spot-inoculated, dried cells > cells in biofilms. E. sakazakii has been observed to form biofilms on the surfaces of stainless steel, silicon, latex, polycarbonate, glass, and polyvinyl chloride (22, 27, 30); however, inactivation of the bacterium in biofilms by the disinfectants examined in our study was not reported.

Mechanisms that enhance the resistance of bacteria in biofilms to environmental stresses have been proposed. Extracellular polymeric substances produced by microorganisms during biofilm formation behave as protective barriers against exposure to environmental stresses (11, 31, 32). Ryu and Beuchat (43) reported that exopolysaccharide is a major factor enhancing resistance of E. coli O157:H7 in biofilms to sanitizers. Oxidizing sanitizers, including hydrogen peroxide, can be neutralized and lose bactericidal activity upon contact with the surface of biofilms (9, 12, 32, 55). Some strains of E. sakazakii are reported to produce extracellular polysaccharide (30, 44), the amount produced being affected by nutrient availability and temperature. The production of exopolysaccharide by E. sakazakii during biofilm formation would likely provide a protective barrier against disinfectants.

While E. sakazakii cells at or near the surface of biofilms would utilize nutrients and oxygen from the surrounding environment, cells deeply within the biofilm matrix may have undergone starvation, which may increase their resistance to stress. Pseudomonas aeruginosa in biofilms showed higher resistance than non-biofilm formers to several antibiotics (13). Resistance of E. coli O157:H7 (43) and L. monocytogenes (17, 18) to sanitizers is significantly greater in biofilms on abiotic surfaces compared to resistance of planktonic cells. Peracetic acid, mercuric chloride, and formaldehyde at otherwise lethal concentrations were shown to be ineffective in killing microorganisms in biofilms (8).

In summary, the disinfectants evaluated in this study exhibited various levels of lethality to E. sakazakii, depending on the composition of the carrier used to suspend cells and treatment time. The two test strains, one isolated from a clinical specimen and the other from a food source, behaved similarly upon exposure to experimental test parameters. The presence of infant formula enhanced the resistance of planktonic cells and cells spot inoculated and dried on the surface of stainless steel to the disinfectants. The overall order of resistance of E. sakazakii to disinfectants was planktonic cells < cells spot inoculated and dried on stainless steel < cells in biofilms on stainless steel.

The results emphasize the importance of proper cleaning of surfaces soiled by rehydrated infant formula and other foods. Otherwise, infant formula remaining on these surfaces protects E. sakazakii against the lethality of disinfectants or may serve as a source of nutrients, resulting in growth and production of biofilm. The results provide information useful in assessing the efficacy of disinfectants in killing E. sakazakii embedded in organic matrices on surfaces in formula preparation and feeding areas in hospitals, day-care centers, food service kitchens, and the home.


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ACKNOWLEDGMENTS
 
We are grateful to ZEP Manufacturing Co., Atlanta, Ga., and Steris Co., St. Louis, Mo., for supplying gratis samples of disinfectants for evaluation.


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FOOTNOTES
 
* Corresponding author. Mailing address for Larry R. Beuchat: Center for Food Safety, University of Georgia, 1109 Experiment Street, Griffin, GA 30223-1797. Phone: (770) 412-4740. Fax: (770) 229-3216. E-mail: lbeuchat{at}uga.edu. Mailing address for Jee-Hoon Ryu: Graduate School of Life Sciences and Biotechnology, Korea University, Anam-dong, Sungbuk-ku, Seoul 136-791, Republic of Korea. Phone: 82 2 3290 3409. Fax: 82 2 3290 3918. E-mail address: escheri{at}korea.ac.kr. Back

{triangledown} Published ahead of print on 15 December 2006. Back


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Applied and Environmental Microbiology, February 2007, p. 1256-1265, Vol. 73, No. 4
0099-2240/07/$08.00+0     doi:10.1128/AEM.01766-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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