Abstract
We evaluated a variety of methods to recover S. aureus from inanimate surfaces. Two contact agar plates and three swab sampling methods were tested on porous and non-porous surfaces and bar soap. The cost and ease of use of each method was also evaluated. S. aureus was recovered using all methods on both porous and non-porous surfaces. S. aureus could not be detected on three of four brands of soap.
Keywords: Staphylococcus aureus, environmental sampling, soap
Introduction
Staphylococcus aureus is an important and versatile pathogen with the ability to colonize individuals and cause superficial and invasive infection. S. aureus can survive on environmental surfaces for prolonged periods of time and can be transferred to skin by fomites [1, 2]. Thus, environmental surfaces are potential reservoirs for S. aureus transmission [3-5]. Bar soap that has been in contact with human skin has been demonstrated to harbor microorganisms [6]. A paucity of data exists regarding optimal sampling techniques to recover S. aureus from environmental surfaces. A recent review of studies evaluating environmental S. aureus contamination found a lack of consistency in sampling methods as well as limited information regarding specific techniques utilized in these investigations [7]. Additionally, the typical bioburden of S. aureus surface contamination has not been well described [8] and no “gold standard” method for environmental sampling exists. Thus, we performed a qualitative assessment of five sampling methods to detect serial dilutions of S. aureus applied to multiple surface types. Our primary objective was to determine effective and efficient methods to recover S. aureus from porous and non-porous surfaces in addition to multiple brands of bar soap while also considering the practicality of use and cost of sampling. Secondarily, we were interested in evaluating the ability of S. aureus to persist on bars of soap. The results from this investigation can inform future epidemiologic studies of environmental reservoirs of S. aureus.
Materials and Methods
Three surface types were tested: a laboratory countertop (Trespa Toplab, New York, NY) representing a non-porous surface; cotton washcloths representing a porous, textured surface; and four common brands of bar soap (a moisturizing bar, an antibacterial soap, and two deodorant soaps) placed in sterilized plastic boxes to mimic soap in dishes. Five sampling methods were tested: the Baird Parker Agar contact plate (Hardy, Santa Maria, CA), the RODAC (replicate organism detection and counting) trypticase soy agar (TSA) + lecithin and polysorbate 80 contact plate (Becton Dickinson [BD], Franklin Lakes, NJ), the Eswab (BD) with and without enrichment in trypticase soy broth (TSB) with 6.5% NaCl (BBL, BD), and the Enviroswab (3M, St. Paul, MN).
Suspensions of a strain of USA300 methicillin-resistant S. aureus (MRSA) recovered from a human buttock abscess at St. Louis Children's Hospital (St. Louis, Missouri, USA) were prepared to a density of 0.5 McFarland Standard in normal saline. From this, six ten-fold dilutions were prepared to create ultimate colony counts ranging from 0 to 105 colony forming units (CFU)/mL. Dilutions were verified by plating directly to TSA with sheep blood (blood agar plates, BAPs; BBL, BD) and performing colony counts after overnight incubation. Before inoculation, the countertop surface was decontaminated with ethanol and rinsed with sterile water, and the washcloths and soap dishes were autoclaved. Soap bars were new (i.e., unused) and placed into the dish in a manner which did not introduce contamination. A unique area of bench top, washcloth, or soap bar was used for each dilution and each sampling method. Each surface was cultured initially to ensure the absence of S. aureus at baseline.
After an initial pilot evaluation of different volumes for S. aureus inoculation of surfaces, dilutions were delivered to surfaces in 15 mL volumes, as this amount allowed uniform delivery of inocula to each surface. Immediately following preparation, ten-fold dilutions (from 0 to 105 CFU/mL) were applied evenly to a 6 × 12 inch (15.2 × 30.5 cm) area of laboratory countertop and 6 × 12 inch washcloths and allowed to dry overnight. After 24 hours, contact plates were stamped for five-second intervals over each surface in six non-overlapping locations. Swabs were swiped back and forth across the entire surface in two perpendicular directions. All soap bars were of approximately equal size. Dilutions (0, 103-105 CFU/mL) were applied to each bar of soap and allowed to incubate at room air overnight. Contact plates were uniformly stamped twice each on the top (dry side) of the soap bars in the location that the suspensions were applied, and again on the bottom (wet side) of each soap bar. Swabs were swiped back and forth across the entire top and bottom of each bar. The soap dishes were then sampled with a separate set of contact plates and swabs.
Contact plates were incubated overnight at 35°C in ambient air. Growth on contact plates was subcultured to BAPs. For Eswabs, 100 μL of eluate was inoculated to each of a BAP and TSB with 6.5% NaCl and incubated overnight. Following incubation, broth cultures were plated to BAPs and incubated overnight. Enviroswabs were inoculated directly onto BAPs which were subsequently incubated overnight. Beta-hemolytic colonies characteristic of our parent strain recovered on BAPs were confirmed as S. aureus with catalase and Staphaurex (Remel, Lenexa, KS) tests. The limit of detection (LOD) was defined as the lowest dilution of S. aureus (CFU/mL) applied to each surface that could be detected by each method. Three independent replicates of each experiment were performed. The ultimate goal of this investigation was to determine qualitatively whether S. aureus could be detected from the surface sampled by each method.
Results
From the non-porous surface, the limit of detection for four of five methods (i.e., all methods with the exception of the Enviroswab) was an inoculum of 102 CFU/mL (Table 1). From the porous surface, the RODAC contact plate and Eswab with broth enrichment were able to detect an inoculum of 10 CFU/mL. S. aureus was not detected at any inoculum using any of the sampling techniques from the antibacterial or deodorant soaps (or their corresponding “soap dishes”). S. aureus was detected on the moisturizing bar (and its corresponding “soap dish”) using four of five methods (i.e., all methods with the exception of the Eswab without broth enrichment), at an inoculum of 103-105 CFU/mL, dependent upon method used (Table 1). The reproducibility of S. aureus detection over three replicates of the experiment is reported in Table 1.
Table 1. Detection of S. aureus dilutions applied to environmental surfaces by sampling method.
| Sampling Method | ||||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|
| ||||||||||||||||||||||||||||||
| Hardy Baird Parker
Agar Contact Plate |
BD RODAC Contact Plate | 3M Enviroswab | BD Eswab | BD Eswab with
Broth Enrichment |
||||||||||||||||||||||||||
|
| ||||||||||||||||||||||||||||||
| Surface Type | Applied dilution (CFU/mL) | |||||||||||||||||||||||||||||
|
| ||||||||||||||||||||||||||||||
| 105 | 104 | 103 | 102 | 10 | 0 | 105 | 104 | 103 | 102 | 10 | 0 | 105 | 104 | 103 | 102 | 10 | 0 | 105 | 104 | 103 | 102 | 10 | 0 | 105 | 104 | 103 | 102 | 10 | 0 | |
| Non-porous | D3 | D3 | D3 | D2 | ND | ND | D3 | D3 | D3 | D3 | ND | ND | D3 | D2 | D3 | ND | ND | ND | D2 | D1 | D1 | D1 | ND | ND | D1 | D1 | D1 | D2 | ND | ND |
| Porous | D3 | D2 | D2 | ND | ND | ND | D3 | D1 | ND | ND | D1 | ND | D3 | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND | D3 | D1 | D1 | D2 | D1 | ND |
| Soap | ||||||||||||||||||||||||||||||
| Moisturizing bar | D2 | ND | ND | - | - | ND | D2 | D1 | ND | - | - | ND | ND | D1 | D1 | - | - | ND | ND | ND | ND | - | - | ND | D1 | D1 | D1 | - | - | ND |
| Antibacterial soap | ND | ND | ND | - | - | ND | ND | ND | ND | - | - | ND | ND | ND | ND | - | - | ND | ND | ND | ND | - | - | ND | ND | ND | ND | - | - | ND |
| Deodorant soap A | ND | ND | ND | - | - | ND | ND | ND | ND | - | - | ND | ND | ND | ND | - | - | ND | ND | ND | ND | - | - | ND | ND | ND | ND | - | - | ND |
| Deodorant soap B | ND | ND | ND | - | - | ND | ND | ND | ND | - | - | ND | ND | ND | ND | - | - | ND | ND | ND | ND | - | - | ND | ND | ND | ND | - | - | ND |
| Soap dish | ||||||||||||||||||||||||||||||
| Moisturizing bar | D3 | ND | ND | - | - | ND | ND | D2 | ND | - | - | ND | D1 | D1 | D1 | - | - | ND | ND | ND | ND | - | - | ND | ND | D1 | D1 | - | - | ND |
| Antibacterial soap | ND | ND | ND | - | - | ND | ND | ND | ND | - | - | ND | ND | ND | ND | - | - | ND | ND | ND | ND | - | - | ND | ND | ND | ND | - | - | ND |
| Deodorant soap A | ND | ND | ND | - | - | ND | ND | ND | ND | - | - | ND | ND | ND | ND | - | - | ND | ND | ND | ND | - | - | ND | ND | ND | ND | - | - | ND |
| Deodorant soap B | ND | ND | ND | - | - | ND | ND | ND | ND | - | - | ND | ND | ND | ND | - | - | ND | ND | ND | ND | - | - | ND | ND | ND | ND | - | - | ND |
Note: D1 = S. aureus detected in 1 of 3 replicates; D2 = S. aureus detected in 2 of 3 replicates; D3 = S. aureus detected in all 3 replicates; ND = S. aureus not detected in any of the replicates; - = Dilution not performed for soap surfaces; 15 mL of each dilution were applied to each surface.
In addition to recovery of S. aureus if present, other important factors including cost, ease of use, and minimum days to obtain final results were also considered (Table 2).
Table 2. Cost, ease of use, and minimum days to results for each S. aureus environmental sampling method.
| Factor | Sampling Method | ||||
|---|---|---|---|---|---|
| Hardy Baird Parker Agar Contact Plate | BD RODAC Contact Plate | 3M Enviroswab | BD Eswab | BD Eswab with Broth Enrichment | |
| Costa | $3.17 | $3.80 | $1.80 | $0.81 | $1.94 |
| Ease of useb | +++ | + | + | +++ | ++ |
| Days to results | 2 | 2 | 1 | 1 | 2 |
List price for each product from manufacturer/distributor (USD)
Ease of use scale: + = most difficult to use, +++ = easiest to use
Contact plates
The list price of the Hardy contact plate, which is supplied with a locking lid, is $3.17 USD. The list price of the RODAC contact plate is $3.80 USD and is supplied with a non-locking lid, which was more difficult to transport and could contribute to contamination (although a locking lid is available for an additional fee). Contact plates require a second day of processing (subculture to BAP and overnight incubation) prior to S. aureus verification.
Swabs
The 3M Enviroswab is $1.80 USD; the BD Eswab is $0.81 USD, and the additional step of broth enrichment results in a cost of $1.94 USD as well as an additional day of incubation/processing. Notably, direct plating of the Enviroswab to BAPs resulted in gouging and deterioration of the agar, which could compromise results. Eswabs (when plated directly to BAP) require only 1 day for processing and S. aureus verification.
Discussion
The existing literature is inconsistent and incomplete regarding the optimal method to detect S. aureus on environmental surfaces [9-13]. We employed a systematic approach to evaluate the recovery of different concentrations of MRSA from common environmental surface types using a variety of sampling techniques, including contact plates and swabs, with and without broth enrichment. Similar to other studies, while variation in S. aureus detection was noted using different sampling methods, all methods, studied qualitatively, recovered S. aureus from both porous and non-porous surfaces [9]. While several studies have demonstrated superiority of contact plates for recovery of microorganisms from environmental surfaces, the contour of the surface to be cultured is an important consideration; contact plates are limited to flat surfaces while swabs are able to sample uneven surfaces and larger surface areas [9-11]. A study by Claro and colleagues investigated Petrifilm (3M) for environmental sampling, which provided the benefit of contact methods and could adapt to the contour of surfaces [12].
Bar soap has been epidemiologically associated with MRSA transmission. In a study by Nguyen and colleagues, football players with MRSA skin or soft tissue infection (SSTI) were 15 times more likely to have shared bar soap with teammates than players without recent SSTI [14]. Thus patients with recurrent infections are often discouraged from using and sharing bar soaps. However, in a laboratory setting, this risk has not been demonstrated. In a study by Desai and colleagues, MRSA could be transferred to skin from all tested fomites (e.g. toys, towels, razors), with the exception of soap bars [1]. Bannan and colleagues determined that, though bacteria (Serratia marcescens) could be transmitted from skin to bar soap, the bacteria was not transmitted to subsequent users of the same soap bar [15]. Similarly, in our qualitative study, recovery of S. aureus from bars of soap was limited compared to other surfaces. Even with broth enrichment (which potentially dilutes soap deposited on the sampling device which could enhance organism recovery), S. aureus was detected on only one brand tested, a moisturizing bar, using multiple sampling methods that successfully recovered the same dilutions of S. aureus from other non-soap surfaces. As the potential for bar soap to harbor and transmit MRSA appears limited, clinicians may reconsider advising against the use of bar soap until additional epidemiologic studies of MRSA transmission via bar soap are performed.
While our study has several unique strengths, and represents one of the first efforts to directly compare multiple sampling methods for S. aureus simultaneously, this study is not without its limitations. First, the S. aureus contamination burden and recovery could be altered in community or hospital settings due to the presence of organic material, other microorganisms, cleaning methods, or disinfectant residues at sampling sites on hospital or community surfaces and thus our in vitro study may not accurately recapitulate all these variables [6, 12, 15]. In addition, while we only tested one MRSA strain type, we selected a strain representative of a contemporary MRSA epidemic clone that is common in both community and hospital settings.
Conclusion
We compared the performance of five sampling methods to detect MRSA in the environment. We determined that both contact plates and swabs provided adequate S. aureus recovery from porous and non-porous environmental surfaces, while MRSA was infrequently recovered from bar soap. Environmental sampling protocols for large epidemiologic studies must balance cost (which may vary by institutional contracts), time to results, ease of use, and the contour of the surface to be sampled. The importance of each of these metrics may vary depending on the objective of a particular investigation.
Acknowledgments
We thank Duha Al-Zubeidi, MD and Krista Johnson, MD for assisting with experiment preparation.
Funding Sources: Funding for this project was provided by the Children's Discovery Institute of Washington University and St. Louis Children's Hospital; National Institutes of Health grant K23-AI091690; and grant R01-HS021736 from the Agency for Healthcare Research and Quality. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the Agency for Healthcare Research and Quality.
Abbreviations
- MRSA
methicillin-resistant Staphylococcus aureus
Footnotes
This work has been presented in part at IDWeek in San Diego, California, USA on October 18th, 2012.
Conflict of Interest Statement: All authors report no conflicts of interest.
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