Disinfection alternatives for contact surfaces and toys at child care centers.

Child care surfaces are vehicles for disease-causing organisms. Disinfectant procedures prevent microbial dispersion. This study reports the effectiveness of CITRUS Farm Edition® (CFE), Clorox® GreenWorks™ (CGW) and Clorox® Anywhere® (CA) against Salmonella Typhimurium and Staphylococcus aureus inoculated (1 ml of 9Log₁₀ CFU/ml) on a high chair and ball toy. Disinfectants were sprayed and bacteria recovered from surfaces by sponge method. Exposing an inoculated high chair to CA resulted in the highest reduction of S. aureus (3.92 Log₁₀) and S. Typhimurium (3.22 Log₁₀). CGW reduced S. aureus and S. Typhimurium by 2.84 and 2.12 Log₁₀ from the inoculated high chair, while the inoculated ball toy showed a 2.50 and 1.80 Log₁₀ reduction, respectively. CFE showed the lowest reduction with 1.42 and 1.53 Log₁₀ of S. aureus and S. Typhimurium from the inoculated ball toy. CA was the best disinfectant no matter which bacteria or surface was analyzed. Emphasis on the effectiveness of disinfectant products is needed to be included in child care center infection control programs.

Keywords: surface disinfection; Salmonella Typhimurium; Staphylococcus aureus; child care centers

Child care centers (CCCs) have acquired an important role in the work system. Nowadays, women are participating in work-related activities which increase the need for CCCs for children to attend (Alonzo and Rodríguez [2]). Children at CCCs improve socialization and early development skills; however, interaction with other children might increase the risk of microbial dissemination among them and their environment (Villasusa et al. [28]). CCCs are known to be high risk areas to trigger acute infant infectious diseases, regardless of social status or geographical zone (Ochoa et al. [22]). Apparently, infants at CCCs are exposed to different infectious agents than those existing in home environments (Nesti and Goldbaum [21]).

Epidemiological studies have indicated that microorganisms transmitted at CCCs are mostly associated with enteric and respiratory diseases (Young and Edet [29]; Villasusa et al. [28]; Nesti and Goldbaum [21]). Significant variations have been described in diarrhea episodes of children attending CCCs to those staying at home; Bartlett et al. ([4]) reported that 42 cases per 100 CCC users occurred in the same period versus 27 cases per 100 child-months in households not using CCC. Hand to mouth events, sharing toys, eliminating nasal secretions and changing diapers have been confirmed as the most common habits that might help microbial dissemination, especially when inadequate hand-washing occurs (Merriman et al. [18]; Kotch et al. [16]; Cosby et al. [8]).

Direct contact with contaminated surfaces is considered the principal mean of pediatric infectious transmission and surfaces from children's playground equipment and daycare centers were the most frequently contaminated compared with other public surfaces (Reynolds et al. [24]). Good hand-washing technique is the single but most important process to prevent microbial transmission after toilet use, diapering or coughing (Delpiano et al. [12]; Kotch et al. [16]). Also, cleaning and disinfecting toys and contact surfaces at CCCs prevent microbial dispersion (Cosby et al. [8]).

Cleaning and disinfecting practices in CCCs are regulated by national and local regulations which establish the necessary conditions for healthcare, feeding, education and recreation of the minors. In Mexico, the norm IMSS-3000-001-009 establishes the dispositions for the operation and service of CCCs, but do not contain information about toys and disinfection of contact surfaces (IMSS [14]). North Carolina Child Care Sanitation Rules recommend chlorine-based solutions to disinfect food-contact surfaces and toys. These solutions are proved to be the most used and efficient disinfectants; however, special care must be taken when preparing the solution concentration to avoid toxic effects if children are exposed at the CCCs (North Carolina Department of Environment and Natural Resources [NC DENR] [20]). Home-made chlorine solutions from household bleach are frequently used; nevertheless, solutions should be prepared daily to make sure of the concentration and effectiveness (Parnes [23]; Rutala et al. [25]).

Also, commercial "ready-to-use" products for cleaning or disinfecting surfaces are available, their formulations are stable, non-hazardous and non-toxic and some of them are easy to use as sprays (Bronson-Lowe [6]; Layton [17]). In addition, some of these products contain biodegradable compounds approved by the US Environmental Protection Agency (US EPA [27]) (CITRUS Fact Sheet [9]). The efficacy information generated by ready-to-use products for disinfection can be used to elaborate Infection Control Education Programs (ICEP) to reduce the presence of disease-causing organisms in CCCs (Ackerman et al. [1]). Efficacy of ICEP depends on factors such as training, emphasis on hand-washing and compliance on monitoring; but the most important factor is the appropriate application of disinfecting products to ensure the effectiveness of them (Kotch et al. [15]). Therefore, the objective of this study was to evaluate the effectiveness of two organic disinfecting products (CITRUS Farm Edition® CFE and Clorox® Green Works™ CGW) and one chlorine-based product (Clorox® Anywhere® CA) against Salmonella Typhimurium and Staphylococcus aureus on contact surfaces and toys in CCCs.

A pure culture of Salmonella enterica serovar Typhimurium (ATCC 19585) and Staphylococcus aureus (strain provided by Public Health State Laboratory, Sinaloa, Mexico) were selected and subjected to evaluation. Bacterial culture was grown in tryptic soy broth (TSB; Bioxon, Mexico) for 24 h at 37°C and harvested by centrifugation at 13,080 g for 10 min at 4°C (Chaidez et al. [7]). The cellular pellet was washed three times in a 0.1 M phosphate buffer saline (PBS) solution to minimize the concentration of non-cell associated constituent's solution. Appropriate 10-fold dilutions in PBS were made to enumerate final concentration of viable bacteria in suspension (9 Log10 CFU/ml approximately). These bacterial suspensions were used in the inoculation procedure.

A non-porous plastic high chair and rough plastic ball toy were selected for the study. Plastic high chairs are commonly used for serving foods at CCCs (Figure 1). These chairs have a surface area of 40 × 20 cm, where the disinfectant products were applied. The ball toy was approximately 10 cm in diameter and the whole surface was used to evaluate the disinfectant products (Figure 1). Both surfaces were disinfected before each test procedure by immersion in a 1000 ppm chlorine solution for 10 min; residual chlorine was neutralized with 10% sodium thiosulphate solution (American Public Health Association [APHA] [3]). Surfaces were washed twice with sterile distilled water and paper dried; finally they were aseptically stored until test procedure.

Graph: Figure 1. High chair and ball toy surfaces (not scaled).

One milliliter of 9 Log10 CFU/ml of bacterial suspension was placed over an area of 45 × 25 cm of high chair or a ball toy surface of 10 cm diameter, extended with a sterile cotton swab and allowed to dry under ambient conditions. At the end of the inoculation procedure, bacterial sponge recovery procedure was applied to determine initial counts of microorganism on surfaces.

Bacteria were recovered with a sterile sponge (Nazco Whirl-Pak® 4 oz) by surface scrubbing. Sponge was immersed onto 25 ml of sterile purified water, and then surfaces were sponge swabbed. Finally, the sponge was vigorously washed for 1 min and strong tightened to allow bacteria release. One ml of bacteria recovered from the sponge was serially diluted to countable numbers and spread-plated per triplicate onto selective agars, Hektoen agar for S. Typhimurium and Mannitol Salt agar for S. aureus (Bioxon, Edo de Mexico). Agar plates were incubated at 37°C for 24 h prior to enumeration and bacterial mean counts were reported as CFU/ml.

CITRUS Farm Edition® is a biodegradable product with EPA registration No. 83030-1; this product contains a mix of organic acids (citric, ascorbic and lactic acids) as active ingredients. The product was tested as manufacture recommendation diluting a proportion of 1:1000 with sterile distilled water and pH adjusted to 5.0 with citric acid (CITRUS Fact Sheet [9]).

Clorox® Green Works™ Natural All-Purpose Cleaner is a product 99.93% natural formulated with filtered water, coconut-based cleaning agent (alkyl polyglucoside), corn-based ethanol, glycerin, essential lemon oil, biodegradable preservative and colorants. It was tested directly from the source as spray. Green Works is included at the Design for Environment Program at the US EPA, identified by DfE logo (http://www.epa.gov/dfe/).

Clorox® Anywhere® Hard Surface ™ Daily Sanitizing Spray is a product which contains 0.095% of sodium hypochlorite as active ingredient (95 ppm); this product is recommended for direct use as spray. It is an EPA registered sanitizer No. 5813-85 (Clorox® Company [11]).

Disinfectants were sprayed onto inoculated surfaces during 5 sec and left acting for 5 min at room temperature. Distilled water was used as test control and identically applied as the test products. After contact time, final counts of microorganisms were determined by the bacterial sponge procedure described above.

Three observations were made for each test procedure and bacterial counts were logarithmically transformed in order to calculate bacterial Log10 reduction using the equation:

Graph

Analysis of variance (ANOVA) was performed using Log10 reduction as response variable. Dunnett's test was performed in order to compare test products with a control level (purified water) with alpha ≤ 0.05 significance level. In addition, Tukey test was used to determine differences between disinfectants. Data were subjected to MINITAB (2004) for statistical analysis.

Initial counts of S. Typhimurium were recovered from the high chair and ball toy with 5.27 and 6.37 Log10, respectively. S. aureus was recovered from the high chair and ball toy reaching 5.66 and 6.55 Log10, respectively (Table 1).

Table 1. Bacterial initial counts on surfaces.

Clorox® Anywhere® was the best product evaluated; the inoculated high chair showed a bacterial reduction of 3.92 and 3.22 Log10 of S. aureus and S. Typhimurium, respectively, while the inoculated ball toy showed 2.99 and 2.69 Log10 reduction of S. aureus and S. Typhimurium, respectively. Clorox® Green Works™ showed higher reduction for S. aureus with 2.85 and 2.50 Log10 reductions on the high chair and ball toy, than those obtained with S. Typhimurium that reached a reduction of 2.12 and 1.80 Log10 on the high chair and ball toy, respectively. CITRUS Farm Edition® was the less effective product with 1.76 and 1.53 Log10 reductions of S. Typhimurium over the high chair and ball toy, respectively, while S. aureus reduction reached 2.37 and 1.42 Log10 over the high chair and ball toy, respectively. Control test (purified sterile water) reduction for S. Typhimurium were 1.10 and 0.83 Log10 over the high chair and ball toy, respectively, while S. aureus showed 0.91 and 0.79 Log10 reduction over the high chair and ball toy surfaces, respectively. All these results are shown in Figure 2.

Graph: Figure 2. Log10 mean reduction of bacteria on surfaces with test products.

Statistical analysis showed no interaction effects between variables (bacteria, surfaces and test products) indicating similar behavior when evaluating in combination for each test procedure (p = 0.719). However, when variables were individually analyzed, significant differences were observed between products tested (p = 0.000) (Table 2). Therefore, Dunnett's test was conducted to compare each product with the control test (purified sterile water). Results showed that Clorox® Anywhere® and Clorox® Green Works™ were significantly different with the control with p values of 0.0000 and 0.0085, respectively, while CITRUS Farm Edition® had no significant differences with the control test (p = 0.1491). Also, the Tukey test was conducted to compare disinfectant products between them including control test. Results of this analysis confirmed that CITRUS Farm Edition® had no significant differences with the control test (p = 0.2334). Also, this test showed significant differences between control test and Clorox® Green Works™ (p = 0.0156) and Clorox® Anywhere® (p = 0.000). Finally, the results of the test did not exhibit differences between Clorox® Green Works™ and Clorox® Anywhere®.

Table 2. Log reduction comparison between sources, using ANOVA test.

The disinfectant test procedure used in this study is an easy protocol that can be applied to prove the microbicidal effect of disinfectant products. Study results suggested that commercial ready-to-use products can be effective in reducing microbial contamination from contact surfaces. Clorox® Anywhere® showed the highest Log10 reduction when used over high chair surface; however, the ball surface showed a lower reduction for both bacteria (Figure 1). Merriman et al. ([18]) concluded that hard surfaces or non-porous surfaces are easier to clean and disinfect than porous surfaces by hypochlorite solution, since they evaluated 2500 ppm. The same pattern was observed when Clorox® Green Works™ and CITRUS Farm Edition® were evaluated.

Clorox® Green Works™ reduced more than 2 Log10 in almost all the treatments obtaining results statistically similar to those obtained with Clorox® Anywhere®. Clorox® Green Works™ is designed as a cleaning product, not as sanitizer; however, bioethanol might have the microbicidal effect to bacterial surrogates used in the present study and many other lipid-containing outer membrane pathogens; however, further studies are needed to prove it.

Our results coincide with Rutala et al. ([26]) who evaluated an alcohol-based product (Lysol® Disinfectant Spray) over different surfaces, with more than 3 Log10 reduction of adenovirus.

CITRUS Farm Edition® had the lowest microbial reduction (1.42 mean Log10 reduction from Figure 1). This pattern was observed in both bacterial surrogates and might be related to pH and light effect over CITRUS Farm Edition® activity, a fact that could be included in futures studies (CITRUS Fact Sheet [9]). Home prepared disinfecting solutions might have some microbial effect; however, their activity is affected over time. The effect of time over freshly prepared sodium hypochlorite solutions and free available chlorine (FAC) was studied by Rutala et al. ([25]). This type of approach should be performed to CITRUS Farm Edition®. They found that the antimicrobial activity of fresh solutions was maintained for 30 days; even so, initial concentrations of FAC was 10,000 ppm and after a 30-day period it was only 225 ppm of the active ingredient.

Evaluation of ready-to-use commercial products is increasing. Its practicability and stable concentration ensure high levels of microbial reduction (Layton [17]; Rutala et al. [26]; CITRUS Fact Sheet [9]; Clorox® Company [11]).

Bronson-Lowe ([6]) examined the effect of ready-to-use commercial products used in sanitizing protocols to prevent disease incidence in children at CCCs, but no significant differences were detected due to multifactor variables affecting the study. Thus, the effectiveness of intervention programs to reduce microbial presence at CCCs should be permanently evaluated and the efficacy of each factor must be controlled. Special care on antimicrobial activity of disinfectants must be considered before the interpretation of the results (Herman [13]).

Some others studies have demonstrated that great emphasis in improving hygienic conditions at CCCs should be established, especially on training and the development of surfaces sanitization procedures to avoid the spread of microorganisms (Boone and Gerba [5]; Cosby et al. [8]).

In conclusion, the use of ready-to-use chlorine-based disinfecting products such as Clorox® Anywhere® can be considered an effective disinfectant of contact surfaces and toys from CCCs; Clorox® Green Works™ can also be used to reduce microbial populations but not enough to be considered as an effective disinfectant. CITRUS Farm Edition® showed a lower reduction than Clorox® products; however, further evaluation of its preparation can be conducted to understand its activity.

The authors thank Yesenia Leal at the Centro de Investigacion en Alimentacion y Desarrollo, Unidad Culiacan, for technical assistance in this research work.