Survival of Staphylococcus aureus applied to Poa pratensis, L. and Synthetic 1 Turf 2 3 4 Andrew S. McNitt1, Dianne M. Petrunak2, Thomas J. Senensits3 5 6 7 8 9 10 11 12 13 14

ABSTRACT 15 16 Methicillin resistant Staphylococcus aureus (MRSA) isolates have become more prevalent 17 among athletes. The objective of this study is to examine the survival of S. aureus on synthetic 18 turf systems and natural turf under varying environmental conditions and to evaluate the 19 effectiveness of various control agents applied to the synthetic turf. Under non-extreme 20 temperature and very limited light conditions, a very small percentage of the S. aureus applied 21 survived on both synthetic and natural turfgrass for multiple days. Applied control agents 22 reduced survival rates somewhat. When S. aureus was applied to outdoor surfaces in the 23 presence of sunlight, the bacterial survival rate was very low and effectiveness of the control 24 products were difficult to evaluate. Exposure to UV light and higher temperature seemed to be 25 the most effective disinfectant under the conditions of this experiment. Survival rates on 26 synthetic and natural turf did not differ significantly in either indoor or outdoor conditions. 27 28

ABBREVIATIONS: 29 30 CA-, community associated; CFU, colony forming units; Kentucky bluegrass, Poa pratensis, L.; 31 MRSA, Methicillin resistant Staphylococcus aureus; MSSA, Methicillin Susceptible 32 Staphylococcus aureus; S. aureus, Staphylococcus aureus 33 34 35 36 37 38 39 40

1Penn State University, 116 ASI Bldg., University Park, PA 16802, USA, asm4@psu.edu 2 Penn State University, 116 ASI Bldg., University Park, PA 16802, USA, dmp6@psu.edu 3 Penn State University, 116 ASI Bldg., University Park, PA 16802, USA, tjs204@psu.edu

41 INTRODUCTION 42

43 Staphylococcus aureus is a common bacterium found on human skin and in the nasal cavity 44 (Dukic , Lauderdale, Wilder, Daum, & David 2013). Typically, the presence of the bacterium 45 causes no problems and goes unnoticed. Occasionally, S. aureus is capable of causing diseases 46 ranging from minor soft tissue infections and food poisoning to serious medical problems such as 47 toxic shock syndrome (Marples, Richardson, & Newton 1990; Bennet & Lancette, 1998). Most 48 community associated (CA-) S. aureus infections are believed to be transmitted either via 49 person-to-person contact or through a common source or shared items (Dukic et al., 2013). 50 51 Strains of S. aureus demonstrating resistance to ß-lactam antibiotics were first noted in the 1960s 52 and are termed Methicillin Resistant Staphylococcus aureus (MRSA) (Jevons, 1961). These 53 strains spread via the same mechanisms as Methicillin Susceptible Staphylococcus aureus 54 (MSSA)(Tinelli, Monaco, Vimercati, Ceraminiello, & Pantosti, 2009). In the 1990s, new strains 55 of CA-MRSA began circulating among athletes (Turbeville, Cowan, & Greenfield, 2006). 56 Contact sports have been shown to be a clear risk factor in acquiring both CA-MRSA and CA-57 MSSA and that shaving and other activities which create breaks in the skin or abrasions 58 increases the risk of athletes acquiring CA-MRSA (Begier et al., 2004). Braun and Kahanov 59 (2018) reported that high school football players acquired MRSA at a 4 times greater rate than 60 other high school athletes. The role of surfaces in the locker rooms, training rooms, and the 61 sports fields with which these players interact has been questioned as potential sources for 62 transmission of S. aureus (McNitt, Petrunak, & Serensits, 2008). 63 64 The temperature for growth of S. aureus is 7-48 ℃, with an optimum of 30-37 ℃ (Kadariya, 65 Smith, & Thapaliya, 2014). Temperatures required to destroy S. Aureus are time dependent. For 66 example Yaniarti, Amarantini, & Budiarso (2017) reported death of S. aureus after exposure to 67 60 ℃ for 60 m or 70 ℃ for 50 m. In a study examining the survival of bacteria on various 68 synthetic surfaces including polyethylene, S. aureus was able to survive for relatively long 69 periods of time (22 to 40 days) under ideal conditions and protected from sunlight (Neely & 70 Maley, 2000). Waninger et al. (2010) reported that survival of CA-MRSA on synthetic turf was 71 not affected by the presence or absence of a biofilm but was dependent on availability of 72 nutrients. The researchers also found that mucins can act as a source of nutrients slightly 73 increasing the survivability of MRSA on synthetic turf. Keller, Turco, Gray, & Sigler, (2020) 74 examined the survivability of MRSA on various synthetic turf fiber and infill types. Although 75 MRSA remained viable on all fiber types for as long as 24 hours of incubation, no significant 76 difference in MRSA availability was observed among the 3 turf fiber types. MRSA availability 77 differed among infill types. Survival after 6 hours of incubation was greatest in sand (93%) and 78 organic infill (90%) while the lowest survival was reported for ethylene propylene diene 79 monomer (EPDM) (17%). Crumb rubber, thermoplastic elastomer (TPE) and polymer coated 80 sand or polymer coated crumb rubber was intermediate with a survival rate between 70-80% 81 after 24 hours of incubation. The authors noted that their results are applicable to indoor surfaces 82 as it has been shown that sunlight can dramatically lower the survival of bacteria (Fujioka, 83 Hashimoto, Siwak, & Young, 1981). 84 85

The objective of this study is to examine the survival of CA-MSSA, termed S. Aureus in this 86 paper, on infilled synthetic turf systems and natural turfgrass under different environmental 87 conditions and to evaluate the effectiveness of various control agents applied to the synthetic 88 turf. 89

MATERIALS & METHODS 90 91 One indoor and three outdoor studies were performed. 92 93

Experiment 1: Indoor Study 94 95 Treatments 96 97 Individual experimental units were prepared by cutting the carpet backing containing the tufted 98 upright (65 mm long) polyethylene slit film pile (Sprinturf, Danial Island, SC) to fit into 99 individual boxes. Each box was 190 by 300 mm in size. Ambient crumb rubber infill was added 100 to the boxes and worked into the pile to a depth of 24-27 mm to simulate a synthetic infill 101 system. The Kentucky bluegrass (Poa pratensis, L.) treatment was prepared approximately one 102 year in advance by seeding Kentucky bluegrass onto 5 cm of a USGA specified sand rootzone. 103 Prior to the start of the experiment, the turfgrass was kept in a greenhouse and cut weekly to a 75 104 mm height. 105 106 All experimental units were inoculated with an isolate of S. aureus except the untreated control 107 which received no bacteria. The isolate of Staphylococcus aureus subsp. aureus was obtained 108 from American Type Culture Collection (ATCC). The bacterium was originally isolated from a 109 human lesion and had no antibiotic resistance. Sufficient quantity of the inoculum was obtained 110 by transferring the S. aureus culture to petri plates containing tryptic soy agar (TSA) and 111 incubating the plates at 36℃ (Bennet & Lancette, 1998). The bacterial suspension was produced 112 by adding sterile distilled water to the TSA plates and dislodging the bacteria from the agar with 113 a sterile loop. The concentration of the bacterial suspension was determined using a 114 hemocytomer. The concentration was adjusted to 1 x 104 colony forming units (CFU) ml-1. 115 116 Individual experimental units were inoculated by applying the bacterial suspension to the infilled 117 surfaces or Kentucky bluegrass turfgrass boxes using a sterile needleless syringe and then rolling 118 the surface with a 10 cm foam paint roller to distribute the bacteria. Bacteria were applied at a 119 rate of approximately 156 CFU cm-2 for the high rate and approximately 16 CFU cm-2 for the 120 low rate. All treatments that included S. aureus application were inoculated at the higher rate 121 except the treatment labeled Low rate S. aureus which was inoculated at the rate of 16 CFU cm-2. 122 All experimental units were stored in a constant temperature room at 20 ℃. No light entered the 123 room except when treatments were being applied or data was being collected. At these times 124 light was provided by one 40 w incandescent bulb. 125 126 A cationic surfactant, SportsClean, (Coatings Specialist Group, Rochester Hills, MI) was applied 127 to two of the infilled surface treatments at a rate of approximately 1300 ml m-2 of surface. This 128 was sufficient to wet the fibers and the uppermost layer of crumb rubber with the surfactant. As 129 per label directions, the surface was wiped with a cloth after 3 minutes to further distribute the 130

surfactant and then allowed to dry. The treatment labelled Surfactant 1 hr prior One experimental 131 unit was treated with the surfactant 60 min prior to inoculation with S. aureus. Another 132 experimental unit was treated 25 days prior to the inoculation and is discussed below. 133 134 An antimicrobial product, SportsAide (Coatings Specialist Group, Rochester Hills, MI) was 135 applied to 3 infill system treatments. The antimicrobial was diluted according to the label and 136 applied at a rate of approximately 1300 ml m-2 of surface. The 3 treatments were as follows. The 137 first was applied to one of the treatments receiving the Sportsclean surfactant 25 days before 138 bacterial inoculation and is termed A+S 25 days prior. The antimicrobial was also applied alone 139 to another treatment 25 days prior to inoculation and is termed Antimicrobial 25 days prior. The 140 antimicrobial alone was also applied to third treatment 1 hr prior to inoculation and is termed 141 Antimicrobial 1hr prior. This treatment was allowed to dry prior to inoculation. The experimental 142 units treated 25 days prior to inoculation were kept undisturbed in the constant temperature room 143 away from sunlight for 25 days before being inoculated with S. aureus. 144 145 The liquid detergent Tide (Procter & Gamble Company, Cincinnati, OH) was applied to another 146 infill system treatment 90 min prior to inoculation with S. aureus at the rate of 25 ml m-2 and is 147 termed Detergent 1.5 hr prior. This liquid detergent is an anionic surfactant. A different treatment 148 consisted of the same rate of detergent applied 15 min after inoculation and lightly brushed 149 (Detergent post). For both treatments the detergent was diluted by a 1:50 ratio with water before 150 application. 151 152 Additional treatments included: the inoculated (156 CFU cm-2) infill system with no applied 153 detergent, surfactant, or antimicrobial (High rate S. aureus); an infill system with no applied 154 detergent, surfactant, or antimicrobial inoculated at the rate of 16 CFU cm-2 (Low rate S. aureus); 155 Kentucky bluegrass turfgrass inoculated at the rate of 156 CFU cm-2 (Kentucky bluegrass); and 156 an untreated uninoculated control (Untreated control). The ten treatments are shown in Table 1. 157

Table 1. Treatments for indoor study in controlled 158

environment. 159

Treatment 160 Untreated control 161 Low rate S. aureus 162 High rate S. aureus 163 Antimicrobial 1 hr 164 prior Surfactant 1 165 hr prior 166 Antimicrobial 25 167 days prior A+S 25 168 days prior 169 Kentucky bluegrass 170 Detergent post 171 Detergent 1.5 hr prior 172

173 174 175 176

Evaluation Procedures 177 178 The experimental design was a completely random design with three replications. Experimental 179 units were sampled at 1, 2, 3, and 4 hours after inoculation on Day 1 for all experimental units 180 except the Untreated control, Low rate S. aureus, and High rate S. aureus. These 3 treatments 181 were sampled at 1 and 4 hours after inoculation on Day 1. Thereafter, experimental units were 182 sampled daily for a total of 12 days unless no living bacteria were detected for at least 3 183 consecutive days. In these cases, sampling was sometimes ended in an effort to reduce sample 184 processing. Sampling was conducted in two ways. Petri plates (50 mm diameter) containing 185 Baird-Parker (BP) agar were pressed onto the surfaces of each experimental unit. While this 186 method sampled both the fibers and the surface crumb rubber, the amount of crumb rubber being 187 tested by this method was small due to the predominance of the exposed fiber canopy. So, in 188 addition to the petri plate method, at each sampling time a 0.2 g crumb rubber sample was also 189 extracted from the surface of selected treatments using a sterile test tube. Five ml sterile distilled 190 water was added to the crumb rubber in the test tube and shaken. A 0.1 ml subsample of the 191 liquid was plated onto 85 mm BP agar plates and spread using a sterile inoculating loop. All 192 plates were parafilmed and incubated at 36 ℃. After 48 hours, colonies on the plates were 193 counted. 194 195 The CFUs cm-2 was analyzed using analysis of variance and Fisher’s Least Significant 196 Difference (lsd) test at the 0.05 level. A lsd was not calculated when the F ratio was not 197 significant at the 0.05 level. 198 199

Experiments 2, 3, and 4: Outdoor Studies 200 201 Treatments 202 203 Outdoor experiments were conducted in May, July, and August 2008. Three experimental times 204 were chosen to represent varying environmental conditions (Table 2). A mild period in May 205 (Experiment 2) was selected when the temperatures were moderate and the skies were overcast 206 (lower light). A second period in July (Experiment 3) was selected during a time of higher 207 temperatures and clear skies. Experiment 3 was repeated in August under similar clear skies and 208 higher temperatures (Experiment 4). 209 210 An existing 6-year old infilled synthetic surface ForeverGreen (Levittown, Pennsylvania) was 211 used for Experiments 2-4. The pile fibers were slit-film polyethylene 57 mm in length. The infill 212 was 100% ambient crumb rubber 36 mm in depth. Individual treatment areas were 200 by 250 213 mm with a minimum 300 mm border between areas. 214 215 A treatment area of similar size was delineated on adjacent established Kentucky bluegrass 216 turfgrass. This experimental unit is termed Kentucky bluegrass. This area was approximately 10 217 m from the synthetic turf treatment areas. 218 219 220 221 222

Table 2. Maximum surface and air temperatures recorded during Experiments 2-4. 223 Maximum daily temperature 224

Date

Synthetic Turf1

Air2

Kentucky bluegrass1

Experiment 2 -------------------------oC----------------------- 27 May 40.6 26.7 31.1 28 May 37.6 18.4 26.7 29 May 43.1 22.8 28.3 30 May 42.6 27.2 32.8 31 May 43.1 26.7 30.0 1 Jun 39.6 23.9 28.9 2 Jun 31.1 24.5 28.3

Experiment 3

30 Jul 54.1 29.5 33.3 31 Jul 50.6 29.5 32.2 1 Aug 51.6 29.5 32.8 2 Aug 51.1 28.9 30.6 3 Aug 49.6 25.6 31.7 4 Aug 51.2 27.8 33.1

Experiment 4

25 Aug NA3 25.6 30.6 26 Aug 50.1 26.7 30.3 27 Aug 47.1 25.6 31.7 28 Aug 19.6 21.2 21.7 225 226 1Temperature determined using WatchDog 100 Series data loggers (Spectrum Technologies, Inc. Plainfield, ILL) 227 placed directly on surfaces. 228 2Temperature obtained from Pennsylvania State Climatologist website, PSU Department of 229 Meteorology (http://climate.met.psu.edu) 230 3Not available 231 232

The experimental design was a completely random design with three replications. All 233 experimental units were inoculated with an isolate of S. aureus using the method described in 234 Experiment 1 except the Untreated control which received no bacteria. Bacteria were applied at a 235 rate so that there were approximately 16 CFU cm-2 for each treated area. 236 237 The cationic surfactant, SportsClean, described in Experiment 1 was applied to the treatment area 238 at a rate of approximately 1300 ml m-2 surfactant 25 or 14 days prior to inoculation with bacteria 239 in Experiments 2 and 3-4 respectively. The rate of surfactant was sufficient to wet the fibers and 240 the uppermost layer of crumb rubber. As per label instructions, the surface was wiped with a 241 cloth after 3 minutes to further distribute the surfactant and then allowed to dry. The 242 antimicrobial product, Aegis AEM 5772/4.2 (Pioneer Athletics, Cleveland, OH), was then 243 applied to the same treatment receiving the surfactant. The Aegis product was diluted according 244 to the label and applied at a rate of approximately 1300 ml m-2 product. This treatment is termed 245 Antimicrobial + surfactant in Experiments 2-4. 246 247 Twenty-four hr prior to inoculation with S. aureus, liquid detergent (Tide) was applied to the 248 infill system at the rate of 5.7 ml m-2. This treatment is labeled ‘Detergent 1 day prior’ in 249 Experiments 2-4. The liquid detergent is an anionic surfactant. Three additional liquid detergent 250

treatments were included in the 3 outdoor studies. Detergent was applied to 2 different treatments 251 1.5 hours prior to inoculation (Detergent 1.5 hr prior) at two different rates, 5.7 ml m-2 and 14.3 252 ml m-2. A third treatment consisted of applying the detergent at the 5.7 ml m-2 rate 15 min after 253 inoculation and is termed Detergent post. 254 255 Similar to the detergent treatments, two fabric softener (cationic surfactant) treatments were also 256 included in the outdoor studies. Each treatment consisted of Downy fabric softener (Proctor and 257 Gamble Co., Cincinnati, OH) being applied to an area at the rate of 5.7 ml m-2. One application 258 was made 24 hr prior to inoculation (Fabric softener 1 day prior) and the other was applied 15 259 minutes after inoculation (Fabric softener post). 260 261 For all fabric softener and detergent treatments, the product was diluted by a 1:50 ratio with 262 water before application. 263 264 Additional treatments included: an inoculated infill system with no applied detergent, surfactant, 265 or antimicrobial (Positive control) and an uninoculated infill system with no applied detergent, 266 surfactant, or antimicrobial (Untreated control). 267 268 During Experiments 3 and 4 another treatment was added (Broom post). Approximately 15 269 minutes after inoculation the treatment area was broomed in an effort to simulate the dragging of 270 the surface with a stiff bristled broom. 271 272 Evaluation 273 274 Experimental units were sampled at 1, 2, 3, and 4 hours after inoculation on Day 1 for all 275 experimental units. During the August study the positive control was sampled 15 min. after 276 inoculation. Thereafter, all experimental units were sampled daily for a total of 3 days. The value 277 used to represent the number of live bacteria collected from an experimental unit at a given 278 sampling time is the average of three sub-samples taken at different locations within each 279 experimental unit. While this method sampled both the fibers and the surface crumb rubber, the 280 amount of crumb rubber being tested by this method was small. So, in addition to the petri plate 281 method, at each sampling time a 0.2 g crumb rubber sample was also taken from the surface of 282 selected treatments using a sterile test tube. Five ml sterile distilled water was added to the crumb 283 rubber in the test tube and shaken. A 0.1 ml subsample of the liquid was plated onto 85 mm BP 284 agar plates and spread using a sterile inoculating loop. All plates were parafilmed and incubated 285 at 36 ℃. After 48 hours, colonies on the plates were counted. 286 287 The number of bacteria detected per cm2 was analyzed using analysis of variance and Fisher’s 288 Least Significant Difference (lsd) test at the 0.05 level. A lsd was not calculated when the F ratio 289 was not significant at the 0.05 level. 290 291 292 293 294

RESULTS AND DISCUSSION 295 296

Experiment 1: Indoor Study 297 298 S. aureus survived for a number of hours on all inoculated treatments including the Kentucky 299 bluegrass (Tables 3 and 4). Surfaces inoculated with S. aureus where no control products were 300 applied still tested positive for living bacteria after 288 hours. This included the Kentucky 301 bluegrass treatment. Treatments where control products were applied, varied in their 302 effectiveness at reducing S. aureus survival. The treatments that resulted in the quickest 303 reduction of S. aureus were the Surfactant 1 hr prior, Detergent 1 hr prior, and the Detergent 304 post. No living S. aureus was found on these 3 treatments after 24 hours. The treatments 305 Antimicrobial 25 days prior and A+S 25 days prior did not result in as large a reduction in initial 306 bacteria survival compared to treatments where the antimicrobial, surfactant, or detergent were 307 applied close to the time of inoculation. The Antimicrobial 1 hr prior treatment did result in a 308 significant reduction in survival rate after 24 hours compared to the untreated High rate S. aureus 309 treatment; however, live bacteria were detected on all three antimicrobial treatments for at least 310 216 hours after inoculation. As a percentage of applied bacteria surviving, the Low rate S. 311 aureus had a greater percentage of applied bacteria survive compared to the High rate S. aureus 312 even though the High rate S. aureus treatment resulted in more living bacteria being detected on 313 all but two rating times. This may be due to limited nutrient availability discussed by (Waninger 314 et al. 2011) potentially limiting the larger population of bacteria applied in the High rate S. 315 aureus treatment. 316

The survival rate for the bacteria detected on the crumb rubber alone was lower than detected 317 using the petri plate method where predominantly fibers were being tested (Table 5). Although a 318 small numbers of living bacteria were found at 120 hours on the untreated positive control, with 319 few exceptions most treatments had no detectable bacteria after 24 hours. 320

Experiments 2-4: Outdoor Studies 321 322 Compared to Experiment 1, populations of detected bacteria in Experiments 2-4 decreased to 323 very low levels rapidly for all treatments (Tables 6 and 7). Typically, within 3 hours after 324 bacteria application, the number of bacteria detected was below 0.1 CFUs cm-2 for most 325 treatments. On 14 of the 20 rating times in Experiments 2-4 where both were sampled, the 326 Kentucky bluegrass treatment had bacteria counts higher than or equal to the Positive control. 327 The reasons for this are unknown but may include: 1) natural turfgrass does not reach the very 328 high surface temperatures that are achieved on the infill systems (McNitt, 2005), 2) the turfgrass 329 canopy density may provide greater protection from UV light, or 3) there may be greater nutrient 330 availability for bacteria on the turfgrass canopy compared to the infill system (Waninger et al., 331 2010). 332 333 334 335 336 337

Table 3. Colony forming units (CFU) cm-2 of S. aureus detected for Experiment 1 on indoor infill systems and Poa pratensis, L. 338 up to 288 hours (12 days) after bacterial application. 339

Hours after bacterial application 340 Treatment 1 2 3 4 24 48 72 96 120 144 170 194 216 240 264 288 341

CFU cm-2 342 343

Untreated control 0 - - 0 0 0 0 0 0 0 0 0 0 0 - - Low rate S. aureus 4.15 - - 15.44 3.94 2.87 2.18 7.94 7.08 8.84 7.81 5.05 1.21 0.24 0.52 0.60 High rate S. aureus 45.92 - - 45.92 17.21 16.58 14.39 9.35 14.76 4.30 5.17 9.32 4.30 0.97 1.25 2.10 Antimicrobial 1 hr prior1 12.76 6.17 4.29 8.88 0.92 0.24 0 0.07 0.12 0.01 0.07 0.07 0.01 0 - - Surfactant 1 hr prior1 1.53 0.51 0.05 0.04 0 0 0 0 0 0 0 0 0 0 - - Antimicrobial 25 days prior1 7.84 17.06 19.88 23.81 13.93 7.13 6.73 5.22 3.06 2.87 1.19 0.42 0.46 0.17 0.10 0 A+S 25 days prior1 12.33 10.07 11.48 7.19 1.16 0.83 0.22 0.34 0.07 0.04 0.04 0 0.02 0.02 - - Kentucky bluegrass 27.03 17.24 19.58 11.52 6.55 8.08 7.03 3.15 1.87 0.80 0.90 1.34 1.06 0.36 0.68 0.07 Detergent post2 1.65 0.41 0.41 0.05 0 0 0 - - - - - - - - - Detergent 1.5 hr prior2 0.60 0.39 0.07 0.04 0 0 0 - - - - - - - - - lsd(0.05) 7.6 7.7 5.2 5.5 4.1 4.4 6.7 5.5 9.0 3.3 2.0 NS3 NS NS NS NS 1Treatment applied at a rate equal to 1300 ml m-2 344 2Treatment applied at a rate equal to 25 ml m-2 345 3Not significant 346

347 Table 4. Percent of viable bacteria detected for Experiment 1on indoor infill systems and Poa pratensis, L. up to 288 hours 348 (12 days) after bacterial application. 349

Hours after bacterial application 350 Treatment 1 2 3 4 24 48 72 96 120 144 170 194 216 240 264 288 351

% viable bacteria 352 Untreated control 0 - - 0 0 0 0 0 0 0 0 0 0 0 - - Low rate S. aureus 25.94 - - 96.50 24.63 17.94 13.63 49.63 44.25 55.25 48.81 31.56 7.56 1.50 3.28 3.73 High rate S. aureus 29.44 - - 29.44 11.03 10.63 9.22 5.99 9.46 2.76 3.31 5.97 2.76 0.62 0.80 1.35 Antimicrobial 1 hr prior1 8.18 3.96 2.75 5.69 0.59 0.15 0 0.04 0.08 0.01 0.04 0.04 0.01 0 - - Surfactant 1 hr prior1 0.98 0.33 0.03 0.03 0 0 0 0 0 0 0 0 0 0 - - Antimicrobial 25 days prior1 5.03 10.94 12.74 15.26 8.93 4.57 4.31 3.35 1.96 1.84 0.76 0.27 0.29 0.11 0.07 0 A+S 25 days prior1 7.90 6.46 7.36 4.61 0.74 0.53 0.14 0.22 0.04 0.03 0.03 0 0.01 0.01 - - Kentucky bluegrass 17.33 11.05 12.55 7.38 4.20 5.18 4.51 2.02 1.20 0.51 0.58 0.86 0.68 0.23 0.43 0.04 Detergent post2 1.06 0.26 0.26 0.03 0 0 0 - - - - - - - - - Detergent 1.5 hr prior2 0.38 0.25 0.04 0.03 0 0 0 - - - - - - - - - 1Treatment applied at a rate equal to 1300 ml m-2 353 2Treatment applied at a rate equal to 25 ml m-2 354

10

Table 5. Colony forming units (CFU) of S. aureus detected for Experiment 1 on 0.2 g samples of ambient 355 crumb rubber. 356

Hours after bacterial 357 application 358

Treatment 1 2 3 4 24 48 72 96 120 144 359 CFU per 0.2 g sample 360 361

High rate S. aureus 23.18 - - 50.96 0 0 0 0 0.04 0 Antimicrobial 1 hr prior1 0 0 0 0 0 0 0 0 0 0 Surfactant 1 hr prior1 6.06 0.10 1.29 8.47 0 0 0 0 0 0 Antimicrobial 25 days prior1 0 0 0 0 0 0 0 0 0 0 A+S 25 days prior1 18.09 0 0 0 0 0 0.09 0.12 0 0 Detergent post2 0.10 0 0.04 0 0 0 0 0 0 0 Detergent 1.5 hr prior2 0.04 0.14 0.07 0 0 0 0 0 0 0 1Treatment applied at a rate equal to 1300 ml m-2 362 2Treatment applied at a rate equal to 25 ml m-2 363 364

S. aureus survival was greatest during Experiment 2 compared with the more rapid decline 365 measured in Experiments 3 and 4. During the entirety of Experiment 2, surface temperatures did 366 not exceed the upper limit (48℃) where S. aureus can continue to grow (Table 2)(Kadariya et 367 al., 2014). Surface temperatures of the infill system exceeded levels known to be lethal to S. 368 aureus within two hours of inoculation during Experiment 3 (Baird-Parker, 1990). The surface 369 temperature of the infill system exceeded 48℃ every day during Experiments 3 and 4. 370 371 While the survival of S. aureus is likely affected by surface temperatures in Experiments 3 and 372 4, ultraviolet (UV) radiation may play an equal or greater role in its survival as relatively short 373 exposure to UV light has been shown to inhibit or destroy S. aureus (Hardjawinata, Setiawari, 374 & Warta, 2005); (Silva, Andrade, Soares, & Ferreira, 2003). While surface temperatures during 375 Experiment 4 exceeded the upper limit for the survival of S. aureus (Table 2), a high percentage 376 of the bacteria were non-viable in the first hour after inoculation when the surface temperature 377 was still below 48℃ (Table 7). Also, the Kentucky bluegrass treatment never exceeded 48℃ in 378 any of the experiments but still measured lower in survival percentage in Experiments 2-4 379 compared to Experiment 1. The percent of applied bacteria forming CFUs one hour after 380 application was highest in Experiment 1 (Table 4) lowest in Experiments 3 and 4, and 381 intermediate in Experiment 2 (Table 7). Bright sunshine was present during daylight hours of 382 the first 48 hours of Experiments 3 and 4 while experimental units in Experiment 1 were not 383 exposed to any sunlight, and during Experiment 2, sunlight intensity was low compared to 384 Experiments 3 and 4 (Table 2). 385 386 By 72 hours after the application of bacteria, no S. aureus was detected in any treatment in 387 Experiments 3 and 4 (Table 6). 388 389 Data collected during Experiment 1 indicated that the survival rate for bacteria was generally 390 lower on the crumb rubber alone compared to the petri plate method which sampled fibers to a 391 greater degree (Table 5). It was assumed that during Experiments 2-4 sampling of the crumb 392 rubber alone would not yield useful results after one or two days. In retrospect, this data may 393 well have proven useful especially during the Experiment 3 (Table 8). The reason for the 394 variance in the amount of S. aureus survival on crumb rubber in Experiments 3 and 4 is 395 unknown. Overall survival of S. aureus on crumb rubber was low compared to polyethylene 396

11

fibers in all 4 experiments which is in general agreement with the results reported by Keller et al. 397 (2020). 398 399 Table 6. Colony forming units (CFU) cm-2 of S. aureus detected for Experiments 2, 3, and 4 on outdoor 400 infill systems and Poa pratensis, L. up to 144 hours (6 days) after bacterial application. 401

Experiment 2 Hours after bacterial application 402 Treatment 1 2 3 24 48 72 96 144 403

CFU cm-2 Untreated control 0 0 0 0 0 0 0 0 Kentucky bluegrass 0.95 0.26 0.15 0.10 0.02 0 0.05 0 Fabric softener 1 day prior 1 0.87 0.07 0 0.04 0.12 0.02 0.04 0 Detergent 1.5 hour prior2 1.22 0.04 0.02 0 0.02 0.07 0.04 0 Detergent 1.5 hour prior1 0.8 0.1 0 0.05 0.26 0.07 0.04 0 Detergent 1 day prior 1 0.47 0.02 0 0 0.04 0.02 0.02 0 Antimicrobial + surfactant3 4.05 0.17 0.04 0.09 0 0.04 0.05 0 Detergent post1 0.24 0 0 0 0.04 0.05 0.02 0 Fabric softener post1 0.04 0 0.02 0 0.09 0.02 0.07 0.02 Positive control 1.31 0.04 0.02 0.12 0.07 0.02 0.02 0 lsd(0.05) 0.80 0.11 NS4 NS NS NS NS NS

Experiment 3 Treatment

1

2

3

24

48

72

CFU cm-2 Untreated control 0 0 0 0 0 0 Kentucky bluegrass 0.36 0.05 0.68 0.02 0.07 0

Fabric softener 1 day prior1 0.04 0 0.15 0.05 0 0 Detergent 1.5 hour prior2 0.02 0.04 0.04 0 0 0 Detergent 1.5 hour prior1 0 0 0 0.07 0.02 0 Detergent 1 day prior1 0.12 0.04 0.05 0.12 0 0 Antimicrobial + surfactant3 0.22 0.02 0.04 0.05 0 0 Broom post 0 0.09 0.02 0.04 0.09 0

Detergent post1 0.12 0.02 0.1 0.18 0.02 0 Fabric softener post1 0.05 0 0.07 0.08 0.02 0 Positive control 0.29 0.05 0.09 0.15 0.07 0

lsd(0.05) NS NS NS NS NS NS

Experiment 4

Treatment 0.25 1 2 3 24 48 72

CFU cm-2 Untreated control - 0 0 0 0 0 0 Kentucky bluegrass - 0.34 0 0.15 0.05 0.22 0

Fabric softener 1 day prior1 - 0 0 0 0.09 0 0 Detergent 1.5 hour prior2 - 0.07 0.04 0 0.02 0 0 Detergent 1.5 hour prior1 - 0.02 0 0 0 0.02 0 Detergent 1 day prior 1 - 0.09 0 0 0 0.07 0 Antimicrobial + surfactant3 - 0 0.02 0 0 0 0 Broom post - 0.12 0.02 0 0 0 0

Detergent post1 - 0 0 0 0.02 0 0 Fabric softener post1 - 0.04 0 0 0.02 0 0 Positive control 1.07 0.02 0.12 0 0.04 0.02 0

lsd(0.05) - NS 0.05 0.07 NS NS NS 1 Treatment applied at a rate equal to 5.7 ml m-2 404 2 Treatment applied at a rate equal to 14.3 ml m-2 405 3Treatment was applied 25 days (Experiments 2 and 3) or 14 days (Experiment 4) prior to 406

application of bacteria at a rate equal to 1300 ml m-2 4Not significant 407

12

Table 7. Percent of viable S. aureus bacteria detected for Experiments 2, 3, and 4 on outdoor infill systems 408 and Poa pratensis, L. up to 144 hours (6 days) after bacterial application. 409

Experiment 2 Hours after bacterial application 410 Treatment 1 2 3 24 48 72 96 144 411

% of viable bacteria Untreated control 0 0 0 0 0 0 0 0 Kentucky bluegrass 5.94 1.63 0.94 0.63 0.13 0 0.31 0 Fabric softener 1 day prior 1 5.44 0.44 0 0.25 0.75 0.13 0.25 0 Detergent 1.5 hour prior2 7.63 0.25 0.13 0 0.13 0.44 0.25 0 Detergent 1.5 hour prior1 5.00 0.63 0 0.31 1.63 0.44 0.25 0 Detergent 1 day prior 1 2.94 0.13 0 0 0.25 0.13 0.13 0 Antimicrobial + surfactant3 25.31 1.06 0.25 0.56 0 0.25 0.31 0 Detergent post1 1.50 0 0 0 0.25 0.31 0.13 0 Fabric softener post1 0.25 0 0.13 0 0.56 0.13 0.44 0.13 Positive control 8.19 0.25 0.13 0.75 0.44 0.13 0.13 0

Experiment 3

Treatment 1 2 3 24 48 72

% of viable bacteria Untreated control 0 0 0 0 0 0 Kentucky bluegrass 2.25 0.31 4.25 0.13 0.44 0

Fabric softener 1 day prior1 0.25 0 0.94 0.31 0 0 Detergent 1.5 hour prior2 0.13 0.25 0.25 0 0 0 Detergent 1.5 hour prior1 0 0 0 0.44 0.13 0 Detergent 1 day prior1 0.75 0.25 0.31 0.75 0 0 Antimicrobial + surfactant3 1.38 0.13 0.25 0.31 0 0 Broom post 0 0.56 0.13 0.25 0.56 0

Detergent post1 0.75 0.13 0.63 1.13 0.13 0 Fabric softener post1 0.31 0 0.44 0.50 0.13 0

Positive control 1.81 0.31 0.56 0.94 0.44 0

Experiment 4 Treatment

0.25

1

2

3

24

48

72

% of viable bacteria Untreated control - 0 0 0 0 0 0 Kentucky bluegrass - 2.13 0 0.94 0.31 1.38 0 Fabric softener 1 day prior1 - 0 0 0 0.56 0 0 Detergent 1.5 hour prior2 - 0.44 0.25 0 0.13 0 0 Detergent 1.5 hour prior1 - 0.13 0 0 0 0.13 0 Detergent 1 day prior 1 - 0.56 0 0 0 0.44 0 Antimicrobial + surfactant3 - 0 0.13 0 0 0 0 Broom post - 0.75 0.13 0 0 0 0 Detergent post1 - 0 0 0 0.13 0 0 Fabric softener post1 - 0.25 0 0 0.13 0 0 Positive control 6.69 0.13 0.75 0 0.25 0.13 0 1 Treatment applied at a rate equal to 5.7 ml m-2 412 2 Treatment applied at a rate equal to 14.3 ml m-2 413 3Treatment was applied 25 days (May and July) or 14 days (August) prior to application 414

of bacteria at a rate equal to 1300 ml m-2 415 416 417 418 419

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Table 8. Colony forming units (CFU) cm-2 of S. aureus detected for Experiments 2, 3, and 4 per 0.2 g 420 sample of ambient crumb rubber extracted from surface of outdoor infill systems up to 48 hours after 421 bacterial application. 422

Experiment 2 Hours after bacterial application 423 Treatment 1 2 3 24 48 424

CFU per sample 425 Negative control 0 0 0 0 0 Antimicrobial + surfactant1 0 0.02 0 0.14 0.02 Positive control 0.15 0.03 0 0.02 0.02

426 Experiment 3 427 Treatment 1 2 3 24 428

---------- CFU per sample -------- 429 Negative control 0 0 0 0 Antimicrobial + surfactant1 0.59 1.04 0.10 0.53 Positive control 0.07 0.51 0.73 0.48

430 Experiment 4 431 Treatment 1 2 3 24 432

---------- CFU per sample -------- 433 Negative control 0 0 0 0 Antimicrobial + surfactant1 0 0 0 0 Positive control 0.02 0 0 0 434 1Treatment applied 25 days (Experiments 2 and 3) or 14 days (Experiment 4) prior to application 435 of bacteria at a rate equal to 1300 ml m-2. 436

437 CONCLUSIONS 438

439 At 20℃ and extremely limited light conditions, CFUs of S. aureus could be detected on both 440 synthetic and natural turfgrass for the 12 days tested. The bacteria do not appear to thrive under 441 these conditions as the percentages of surviving bacteria decreased significantly with time. S. 442 aureus survival was greatest on the fibers compared to the crumb rubber infill which is consistent 443 with the results of other researchers. Commercially available antimicrobial treatments as well as 444 detergent significantly decreased the survival rate of S. aureus present on these surfaces indoors 445 although every experimental unit inoculated tested positive for the presence of S. aureus for the 446 first 4 hours and a number were still positive 9 days after inoculation. The commercial surfactant 447 SportAid and the commercially available liquid detergent treatments had no CFUs of S. aureus 448 detected 24 hr after inoculation. These results indicate that applying the liquid detergent 1.5 449 hours prior to inoculation was as effective as applying it 15 min after inoculation. 450 451 When S. aureus was applied to outdoor surfaces under conditions of higher surface temperatures 452 in the presence of sunlight, the bacterial survival rate was much lower. After 6 days, CFUs were 453 found on only one treatment in Experiment 2 with the lower temperature and lower light 454 intensity. The higher temperatures and light intensity in Experiments 3 and 4 resulted in fewer 455 CFUs overall and no CFUs detected on any treatment 3 days after inoculation. It is difficult to 456 draw conclusions regarding the effectiveness of various treatments in the outdoor experiments 457 because the bacteria do not survive very long under these conditions whether treatments were 458 applied or not, but both detergent and fabric softener applied to the surface from 1 day prior to 15 459 minutes after inoculation reduced S. aureus survival somewhat. However, exposures to UV light 460 and higher temperature seem to be the most effective disinfectant under the conditions of this 461

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experiment. It should be noted that S. aureus survival rate on a common turfgrass species used 462 for athletic fields in the Northern United States was comparable to the survival rate on infilled 463 synthetic turf when no disinfectants were applied. 464 465 466 467

ACKNOWLEDGEMENTS 468 469 This work was supported by the USDA National Institute of Food and Federal Appropriations; 470 The Keystone Athletic Field Managers Organization, The Pennsylvania Turfgrass Council, and 471 the The Synthetic Turf Council. 472 473

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