Frequent use of chlorhexidine-based body wash associated with a reduction in

AAC Accepts, published online ahead of print on 24 November 2014 Antimicrob. Agents Chemother. doi:10.1128/aac.03993-14 Copyright 2014, American Society for Microbiology. All Rights Reserved. 1 2 3 Frequent use of chlorhexidine-based body wash associated with a reduction in methicillin-resistant Staphylococcus aureus (MRSA) nasal colonization among military trainees 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Eugene V. Millar 1, Wei-Ju Chen 1, Carey D. Schlett 1, Tianyuan Cui 1, Katrina B. Crawford 1, Jeffrey B. Lanier 2, David R. Tribble 1, Michael W. Ellis 3 1 Infectious Disease Clinical Research Program, Department of Preventive Medicine and Biometrics, Uniformed Services University of the Health Sciences, Bethesda, MD 2 Martin Army Community Hospital, Fort Benning, GA 3 Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD Key words: Methicillin-resistant Staphylococcus aureus (MRSA), chlorhexidine, colonization, military Running title: Chlorhexidine impact on colonization Corresponding Author: Eugene V. Millar, PhD Infectious Disease Clinical Research Program Department of Preventive Medicine and Biometrics 1

24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 Uniformed Services University of the Health Sciences 11300 Rockville Pike, Suite 1211 Rockville, MD 20852 emillar@idcrp.org Alternate Corresponding Author: Carey D. Schlett, MPH Infectious Disease Clinical Research Program Department of Preventive Medicine and Biometrics Uniformed Services University of the Health Sciences 11300 Rockville Pike, Suite 1211 Rockville, MD 20852 cschlett@idcrp.org Key points: In a field-based cluster-randomized trial among high-risk military recruits, frequent use of chlorhexidine body wash was associated with a reduction in nasal colonization with methicillin-resistant Staphylococcus aureus (MRSA). Clinical Trials Registration. NCT01105767 2

45 ABSTRACT 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 Background. In a field-based trial among military trainees, personal hygiene measures, including chlorhexidine (CHG) body wash, did not prevent overall and methicillinresistant Staphylococcus aureus (MRSA) skin and soft-tissue infections (SSTI). We conducted a secondary analysis of anterior nares cultures obtained during the trial to evaluate the impact of hygiene measures on Staphylococcus aureus colonization. Methods. A cluster-randomized trial for SSTI prevention was conducted among US Army Infantry trainees from May 2010-January 2012. There were three study groups with incrementally increasing education- and hygiene-based components: Standard (S), Enhanced Standard (ES), and CHG. Anterior nares cultures were obtained from participants to determine the prevalence of S. aureus colonization. Results. A total of 1706 participants (469 S, 597 ES, 640 CHG) without SSTI were included in the colonization analysis. Of those randomized to the CHG group, 360 (56.3%) reported frequent use of body wash. Frequent use of body wash had no effect on overall S. aureus colonization (53.3% vs. 56.8% among infrequent/non-users; p=0.25). MRSA colonization prevalence was marginally lower among frequent users (2.5% vs. 4.7%; p=0.07). In multi-variable analysis, the odds of MRSA colonization were lower among frequent users (OR=0.36, 95%CI=0.16-0.77). This CHG-associated reduction was not observed when comparing colonization with USA300 versus non- USA300 types (OR=0.59, 95%CI=0.06-5.76). 3

67 68 69 70 71 Conclusions. Frequent use of CHG body wash was associated with a reduction in MRSA nasal colonization among high-risk military trainees. Topical chlorhexidine may contribute to MRSA SSTI prevention by reducing colonization. However, further studies evaluating the pathogenesis of SSTI are needed. Downloaded from http://aac.asm.org/ on April 29, 2018 by guest 4

72 INTRODUCTION 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 Skin and soft-tissue infections (SSTI), especially those caused by methicillinresistant Staphylococcus aureus (MRSA), have become increasingly common in communities. Individuals in congregate settings (e.g. athletes, inmates, military personnel) are known to be at increased risk for SSTI (1-3). Among military personnel, infection rates are highest in trainees (4, 5). Because SSTI can interrupt training cycles and compromise operational readiness, effective SSTI prevention strategies for military populations are critically needed. In the absence of a vaccine for Staphylococcus aureus, hygiene-based measures (e.g., hand washing, appropriate wound care, environmental disinfection, and patient education) comprise the current strategy for stemming SSTI outbreaks and preventing new cases of disease (1, 3, 6). As colonization has a demonstrated role in the pathogenesis and transmission of S. aureus, elimination of the carrier state with topical and systemic agents has also been employed as a prevention strategy (7-10). Nevertheless, the optimal method to prevent SSTI in congregate settings remains unknown. To date, two field-based randomized controlled trials for SSTI prevention, both using chlorhexidine gluconate (CHG), have been conducted among US military trainees (11, 12). Among recruits attending Officer Candidate School at Marine Corps Base Quantico, Virginia, a thrice-weekly application of CHG-impregnated body cloths over a six-week period did not reduce rates of SSTI (11). With respect to colonization, however, an analysis of nasal/axillary swabs showed a significant reduction in MRSA acquisition rates among CHG-randomized participants as compared to the control 5

95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 (3.3% vs. 6.5%; p=0.004) (13). More importantly, a reduction in colonization was observed for USA300, the predominant community-associated MRSA strain (13, 14). In May 2010, a three-group, hygiene-based cluster-randomized trial was initiated among US Army Infantry trainees at Fort Benning, Georgia (12). In addition to receiving enhanced education on SSTI/MRSA awareness and prevention, participants in one arm of the trial were instructed to use a CHG-based body wash once weekly during training and were surveyed regarding the frequency of use. As a secondary objective of the trial, nasal swabs were collected to assess the impact on Staphylococcus aureus colonization. Herein we evaluate the association between frequent body wash use and S. aureus nasal colonization and consider the implications for future SSTI prevention strategies among high-risk populations. METHODS Study design. In brief, we conducted a three-group, field-based cluster-randomized trial to determine the effectiveness of hygiene-based measures in preventing SSTI among military trainees (12). We then conducted a secondary analysis of anterior nares cultures obtained during the trial to evaluate the impact of hygiene measures on Staphylococcus aureus colonization. Study Participants and Setting. The study population was comprised of US Army soldiers undergoing 14-week Infantry training at Fort Benning, Georgia. The population was all male, between 17-42 years of age, ethnically diverse, and in generally good physical condition. 6

118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 Interventions. There were three study groups. Each was comprised of two battalions of trainees (~10,000 soldiers per group). Each group (Standard, Enhanced Standard, and Chlorhexidine) was assigned an intervention consisting of incrementally increasing hygiene measures (12). A critical component of the Chlorhexidine (CHG) group was that trainees received chlorhexidine antiseptic body wash (4% chlorhexidine gluconate, Hibiclens, Mӧlnlycke Heath Care, Norcross, Georgia) to use with a wash cloth after using their personal soap during an additional once-weekly shower. Trainees were provided with verbal and written/graphic instructions for use, and were instructed to use the body wash for the entire training period. Randomization. Training battalions were the unit of randomization and platoons were the unit of analysis. Two battalions each were assigned by computer-generated random numbers to one of the three study groups. Each platoon received the intervention assigned to its respective battalion at study start. Investigators had no control over individual trainee battalion assignment or battalion training schedules. Eligibility. Enrollment began on 7 May 2010 and follow-up was completed on 20 January 2012. All trainees during this period comprised the eligible study population. All eligible trainees underwent a group-based informed consent process for the hygiene measures. 7

140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 Enrollment and Data Collection. For the colonization endpoint, we recruited a convenience sample of infantry trainees who presented to the Troop Medical Clinic (TMC) for non-infectious conditions (e.g. musculo-skeletal injury). Specifically, trainees with SSTI were excluded. After informed consent, participants completed a risk factor questionnaire and underwent anterior nares screening culture (BD BBL CultureSwabs (BD Diagnostic, Sparks, MD)). The questionnaires assessed SSTI risk factors, hygiene practices and adherence to the intervention components (i.e. frequency of use of CHG body wash). Platoon characteristics were obtained from the OSUT command. Laboratory Methods. Anterior nares specimens were placed in 5 ml of tryptic soy broth (TSB) supplemented with 6.5% NaCl (BBL, BD Diagnostic, Sparks MD), and were incubated for 18-24 hours at 37 C. After incubation, an aliquot of broth was plated onto mannitol salt agar (MSA). Mannitol fermenting colonies were isolated and plated onto trypticase soy agar with 5% sheep s blood and incubated overnight. S. aureus isolates were identified based on colony morphology, Gram stain, latex agglutination (Staphaurex, Remel, Lenexa, KS) and slide catalase testing. All S. aureus isolates underwent identification and susceptibility testing using Microscan Walk-Away -96 (Dade Behring Inc., Deerfield, Illinois), according to Clinical Laboratory Standards Institute (CLSI) methods (15). Additionally, all MRSA isolates underwent typing with pulsed-field gel electrophoresis (PFGE) (16), and PCR to assess for resistance (17) and virulence genes (18). PFGE findings were resolved and analyzed using BioNumerics (Applied Math, Austin, TX). Laboratory personnel were blinded to randomization assignment. 8

163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 Statistical analysis. Based on survey responses and for the purposes of the analysis, trainees were categorized as frequent (daily/weekly) or infrequent (every other week)/non-users of the body wash. Differences in trainee characteristics were assessed using proportions for categorical variables, and median and ranges for skewed continuous variables. Bivariate associations of potential risk factors for colonization were assessed using p<0.10 as criterion for inclusion in the final model. To assess the impact of CHG on colonization, we performed multivariate logistic regression with adjustment for within-platoon and within-class correlation using generalized estimating equations (GEE) (19). Odds ratios (OR) and 95% confidence intervals (CI) are reported. Analyses were performed using SAS Version 9.3 (SAS Institute, Cary, North Carolina). Human Subjects. The Uniformed Services University Infectious Diseases Institutional Review Board approved the investigation. RESULTS Participant Characteristics. A total of 1706 trainees (382 platoons) were included in the colonization analysis. By study group, the distribution of participants was as follows: S 469 trainees (121 platoons); ES 597 trainees (130 platoons); CHG 640 trainees (131 platoons). Reported CHG Use. Surveys were completed by all 1706 trainees. With respect to the use of body wash, all trainees in the S and ES groups reported never having used the 9

186 187 188 189 product. Among trainees in the CHG group, 210 (32.8%) reported never having used it, and 70 (10.9%) reported using it every other week. A total of 360 (56.3%) trainees in the CHG group reported weekly (n=325; 50.8%) or daily (n=35; 5.5%) use, which we categorized as frequent use for the analysis. 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 The characteristics of trainees, stratified by frequent vs. infrequent/no use of body wash, are described in Table 1. A higher proportion of those reporting frequent use of body wash were in phase I (weeks 1-9) of training (77.5% vs. 64%; p<0.001), were swabbed in the Spring/Summer months (71.1% vs. 57.6%; p<0.001), washed towels daily or several times per week (49.5% vs. 41.2%; p=0.004) and used hand sanitizer four or more times per day (83.6% vs. 77.7%; p=0.02). The groups did not differ in the reported frequency of showering (two or more times per day: 31.9% vs. 31.4%; p=0.86) or washing their uniform (62.5% vs. 58.1%; p=0.13). A higher proportion of subjects in the infrequent/no use group came from a platoon where an SSTI case was diagnosed prior to their enrollment and collection of swab (55.1% vs. 46.7%; p=0.005). Colonization. Of 1706 swabs, 956 (56%) were positive for S. aureus, and 72 (7.5%) of these were MRSA (Table 2). The prevalence of S. aureus colonization did not differ between those reporting frequent vs. infrequent/no use (53.3% vs. 56.8%, respectively; p=0.25). Prevalence estimates did not differ by the season in which the swab was collected (p=0.18), nor by frequency of use of hand sanitizers (p=0.2), frequency of showers (p=0.28), or frequency of washing of towels (p=0.09). S. aureus colonization prevalence was higher among those in a platoon where at least one case of SSTI had 10

209 210 been diagnosed prior to collection of the swab (59.4% vs. 52.2%; p=0.003), and among those reporting a higher frequency of washing their uniform (58.1% vs. 53.1%; p=0.04). 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 MRSA colonization prevalence was lower among those reporting frequent use (2.5% vs. 4.7%; p=0.07; Table 2). However, these differences were not statistically significant. Estimates of MRSA colonization prevalence did not differ across levels among the various covariates that were evaluated. Multivariate Analysis. The odds of S. aureus colonization did not differ between those reporting frequent vs. infrequent/no use of body wash (Odds Ratio [OR]: 0.91, 95% Confidence Interval [CI]: 0.66-1.27; Table 3A). The odds of S. aureus colonization were higher among those individuals in platoons where at least one SSTI case had been diagnosed prior to collection of the swab (OR: 1.28, 95% CI: 1.01-1.63). There was no association between S. aureus colonization prevalence and week of training at the time of swab (OR: 1.00, 95% CI: 0.97-1.03), frequency of hand sanitizer use (OR: 0.84, 95% CI: 0.67-1.06), frequency of washing towels (OR: 1.11, 95% CI: 0.89-1.38), or frequency of washing uniforms (OR: 1.13, 95% CI: 0.90-1.42). With regard to MRSA colonization, the odds of MRSA colonization were significantly lower among those reporting frequent vs. infrequent/no use of body wash (OR=0.36, 95%CI=0.16-0.76; Table 3B). Participants who started training in the Spring months were more likely to be colonized with MRSA than those who started in Winter (OR=2.54, 95% CI=1.19-5.45). The odds of MRSA colonization did not differ among those 11

232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 individuals in platoons where at least one SSTI case had been diagnosed prior to collection of the swab (OR: 1.34, 95% CI: 0.76-2.34). Furthermore, there was no association between MRSA colonization prevalence and week of training at the time of swab (OR: 0.96, 95% CI: 0.89-1.04), frequency of hand sanitizer use (OR: 0.83, 95% CI: 0.46-1.48), frequency of washing towels (OR: 1.19, 95% CI: 0.65-2.18), or frequency of washing uniforms (OR: 1.05, 95% CI: 0.53-2.06). Molecular Analysis. Of the 72 MRSA isolates, 60 (83.3%) were available for PFGE. Of these, 33 (55%) were USA300, 14 (23.4%) were USA800, 5 (8.3%) were USA100, and eight (13.3%) were other types (undistinguished) (Table 4A). Among USA300 isolates, three (9.1%) were from trainees reporting frequent use of body wash, whereas among non-usa300 isolates, five (18.5%) had reported frequent use (p=0.45). In multivariate analysis, the odds of MRSA USA300 colonization did not vary by frequency of body wash use (OR: 0.74, 95% CI: 0.09-6.37), season (OR-Spring: 1.52, 95% CI: 0.34-6.68), week of training (OR: 0.97, 95% CI: 0.83-1.13), frequency of hand sanitizer use (OR: 0.64, 95% CI: 0.16-2.50), frequency of washing towels (OR: 1.81, 95% CI: 0.51-6.37), or frequency of washing uniforms (OR: 0.65, 95% CI: 0.15-2.88) (Table 4B). DISCUSSION In the setting of a large-scale, field-based cluster-randomized trial for SSTI prevention, frequent use of CHG-based body wash was associated with a reduction in MRSA nasal 12

255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 colonization among military trainees at high-risk for MRSA colonization and disease. By contrast, there was no such reduction in overall S. aureus nasal colonization, which suggests that CHG and other commonly used agents for decolonization may have differential effects on methicillin-resistant as compared to methicillin-susceptible S. aureus (MSSA) strains (13). The CHG-associated reduction in MRSA nasal colonization is particularly noteworthy because, in the context of the larger trial in which this study was conducted, these same hygiene-based measures had no effect on rates of overall SSTI or MRSA SSTI (12). The observed impact of chlorhexidine on MRSA colonization is consistent with that of another SSTI prevention trial among military recruits (13). In that study, conducted in a similar close-quartered training environment, routine application of CHG-based body wipes failed to prevent overall SSTI, but appeared to delay the nasal and axillary acquisition of MRSA USA300. While the role of MRSA colonization in the pathogenesis of SSTI is still not clearly understood, these studies suggest that routine use of decolonization measures in high-risk, congregate populations may contribute to the prevention of SSTI. The ecology of S. aureus colonization at various anatomic sites is very likely dynamic and multifactorial, involving both host and microbial factors. This complex ecology has yet to be fully described. Non-nasal sites are colonized by MRSA (20-23). Significant differences in colonization patterns between MRSA and MSSA have been reported. For example, MRSA has been shown to more frequently colonize inguinal folds than the 13

278 279 280 anterior nares, while MSSA has been shown to more frequently colonize the anterior nares (20, 21). Chlorhexidine use may impact these non-nasal anatomic sites, and subsequently interfere with overall MRSA ecology. 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 The mechanism by which topical CHG body wash reduces MRSA nasal colonization is not known. It is possible that the application of CHG to body surfaces reduces the total burden of MRSA colonization in the host, and thereby reduces the likelihood of colonization of the nasal cavity. Alternatively, widespread use of CHG in a congregate setting may reduce the overall burden of MRSA colonization in the group. As person-toperson transmission is an important means by which MRSA is spread (9, 20, 22, 24, 25), CHG may confer protection by reducing the host reservoir for MRSA and thereby interrupting its transmission to non-colonized individuals. In our study, S. aureus colonization was associated with being in a platoon where a case of SSTI had recently occurred. Routine widespread use of decolonization measures over a prolonged duration might be necessary to minimize infection risk. Additional measures might also need to be applied; the application of intranasal mupirocin in conjunction with CHGbased skin cleansers has been shown to be effective in other community-based studies (8, 9, 22). The presence and persistence of MRSA on environmental surfaces has been previously described (26), and may represent an important reservoir and means of transmission of MRSA in congregate settings. While uniform cleaning of environmental surfaces with standard EPA-registered agents was a standard component of the trial (27), we did not 14

301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 collect samples from common-touch surfaces (e.g. countertops, wrestling mats, and weight benches) to assess MRSA prevalence on non-human reservoirs. The extent to which MRSA contamination of environmental surfaces contributes to the risk of SSTI is not known. The complex epidemiology of SSTI is likely explained by a dynamic interplay of host, pathogen, and environmental factors (25). Understanding these factors and their role in the pathogenesis of SSTI is critical to developing effective prevention strategies. There are several strengths to this study. First, it was conducted in the context of a cluster-randomized controlled trial in a population known to be at increased risk for MRSA colonization and disease. In a trial of this design and magnitude, the biases associated with participant recruitment and sampling were likely minimal. Second, the study was conducted in a military setting, where the supervisory structure between drill sergeants and trainees, the schedule of training activities, and the access and utilization of healthcare is uniform and highly regimented. Lastly, the reported rates of CHG adherence were similar to those of other community-based studies for SSTI prevention (8, 9, 11). There are limitations to our study. First, we defined colonization as the isolation of S. aureus from a single nasal culture. As noted, S. aureus/mrsa colonizes other body sites (e.g. axilla, groin, perineum), but we restricted sampling to the nose. Second, the frequency of use of body wash was obtained through self-administered trainee surveys. It is possible that some trainees overestimated the frequency of use either because of 15

324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 their inability to recall or to provide what they perceived to be the desirable response. That said, overestimates of CHG use would in fact underestimate its effectiveness on reducing MRSA colonization, and the true impact may be greater than what we reported. Third, due to logistical issues, colonization swabs were collected from trainees presenting to the TMC for non-infectious conditions, rather than from a random sample of trainees from all platoons. It is unlikely, however, that the epidemiology of S. aureus colonization would differ between the groups that we sampled versus the groups that we did not. Fourth, we did not collect information on antibiotic use. Finally, our estimates of colonization are derived from a cross-sectional study design. It is possible that colonization occurred below the limits of detection and/or that we failed to capture the impact of CHG on the dynamics of colonization (i.e. colonization of reduced duration). In conclusion, we report that frequent use of CHG body wash was associated with reduced MRSA nasal colonization among military recruits at high-risk for colonization and disease. Although these same measures failed to prevent SSTI, the magnitude of the impact on colonization suggests there may still be a role for CHG and other decolonizing agents in community-based strategies for SSTI prevention. Longitudinal studies are needed to better understand MRSA colonization and transmission dynamics, and to understand their relation to disease pathogenesis, particularly in congregate populations of otherwise healthy individuals. 16

344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 FUNDING The work was supported by the Infectious Disease Clinical Research Program (IDCRP), a Department of Defense (DoD) program executed through the Uniformed Services University of the Health Sciences. This project has been funded in whole, or in part, with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH), under Inter-Agency Agreement [Y1-AI-5072]. Additional funding was provided by Centers for Disease Control and Prevention, National Center for Emerging and Zoonotic Infectious Diseases, Division of Healthcare Quality Promotion (NCEZID-DHQP); Interagency Agreement [09FED914272 to MWE], and the Department of Defense Global Emerging Infections Surveillance (GEIS) program [C0366-11-HS to MWE]. CONFLICT OF INTEREST All authors. No reported conflicts. ACKNOWLEDGEMENTS We are indebted to the study team of clinical research coordinators, laboratory personnel, and data management staff for their dedication to the project. The views expressed in this paper are those of the authors and do not necessarily represent the views of the Uniformed Services University of the Health Sciences, the Department of Defense (DoD), or other federal agencies. 17

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486 487 488 TABLES Table 1. Characteristics of trainees by reported use of CHG body wash Reported frequency of body wash use Biweekly/Never (n=1346) Daily/Weekly (n=360) N (%) N (%) Number of platoons 345 114 Range of number of enrolled trainees per platoon 1-23 1-13 P-value* Mean age (SD), yrs 20.9 (3.6) 20.6 (3.6) 0.129 Week of training at time of swab 1-3 274 (20.4) 112 (31.1) <0.001 4-6 303 (22.5) 83 (23.1) 7-9 284 (21.1) 84 (23.3) 10-12 426 (31.7) 75 (20.8) 13-14 58 (4.3) 6 (1.7) Season of swab Spring 337 (25.0) 142 (39.4) <0.001 Summer 438 (32.5) 114 (31.7) Fall 431 (32.0) 47 (13.1) Winter 140 (10.4) 57 (15.8) Season of training start Spring 396 (29.4) 145 (40.3) <0.001 Summer 514 (38.2) 86 (23.9) Fall 166 (12.3) 11 (3.1) Winter 270 (20.1) 118 (32.8) 24

Whether there was SSTI case in the platoon prior to enrollment No 605 (44.9) 192 (53.3) 0.005 Yes 741 (55.1) 168 (46.7) Frequency of hand sanitizer use, per day 3 times 300 (22.3) 59 (16.4) 0.015 Four or more times 1043 (77.7) 300 (83.6) Frequency of showers, per day One 920 (68.6) 245 (68.1) 0.856 Two or more 422 (31.4) 115 (31.9) Have shared towels during training Ever 75 (5.6) 11 (3.1) 0.052 Never 1269 (94.4) 349 (96.9) Frequency of washing towels 1 time/week or never Daily/Several times per week Frequency of washing uniform 1 time/week or never Daily/Several times per week 789 (58.8) 181 (50.4) 0.004 553 (41.2) 178 (49.5) 560 (41.9) 135 (37.5) 0.131 776 (58.1) 225 (62.5) 489 Study group Standard 469 (34.8) 0 (0) <0.001 Enhanced Standard 597 (44.4) 0 (0) Chlorhexidine 280 (20.8) 360 (100) * Degree of freedom for chi-square tests: number of category of each variable minus 1. 25

490 491 492 Table 2. Univariate analysis of S. aureus and MRSA colonization Total=1706 No. S. aureus MRSA Total 956 (56.0) N (%) p-value* N (%) p-value* 72 (4.2) Use of body wash Biweekly/Never 1346 764 (56.8) 0.245 63 (4.7) 0.068 Weekly/Daily 360 192 (53.3) 9 (2.5) Week of training at time of swab 1-3 386 209 (54.1) 0.275 19 (4.9) 0.486 4-6 386 209 (54.1) 13 (3.4) 7-9 368 209 (56.8) 16 (4.3) 10-12 501 298 (59.5) 19 (3.8) 13-14 64 31 (48.4) 5 (7.8) Season of swab Spring 479 255 (53.2) 0.182 23 (4.8) 0.105 Summer 552 320 (58.0) 30 (5.4) Fall 478 279 (58.4) 15 (3.1) Winter 197 102 (51.8) 4 (2.0) Season of training start Spring 541 310 (57.3) 0.020 36 (6.7) 0.005 Summer 600 354 (59.0) 22 (3.7) Fall 177 101 (57.1) 4 (2.3) Winter 388 191 (49.2) 10 (2.6) Whether there was SSTI case in the platoon prior to enrollment 26

No 797 416 (52.2) 0.003 33 (4.1) 0.878 Yes 909 540 (59.4) 39 (4.3) Frequency of hand sanitizer use, per day 3 times 359 212 (59.1) 0.197 17 (4.7) 0.593 Four or more times 1343 742 (55.3) 55 (4.1) Frequency of showers, per day One 1165 664 (57.0) 0.278 50 (4.3) 0.852 Two or more 537 291 (54.2) 22 (4.1) Have shared towels during training Ever 86 55 (64.0) 0.129 6 (7.0) 0.193 Never 1618 900 (55.6) 66 (4.1) Frequency of washing towels 1 time/week or never 970 527 (54.3) 0.093 38 (3.9) 0.457 Daily/Several times per week Frequency of washing uniform 731 427 (58.4) 34 (4.6) 1 time/week or never 695 369 (53.1) 0.040 26 (3.7) 0.391 Daily/Several times per week 1001 582 (58.1) 46 (4.6) Study group Standard 469 266 (56.7) 0.425 28 (6.0) 0.071 Enhanced Standard 597 344 (57.6) 19 (3.2) CHG (Biweekly/never) 640 346 (54.1) 25 (3.9) 27

493 494 495 * Degree of freedom for chi-square tests: number of category of each variable minus 1; CHG: chlorhexidine 28

496 497 498 Table 3. Results of the multivariate logistic regression for S. aureus and MRSA colonization with adjustment for within-cluster correlation Overall S. aureus Colonization MRSA Colonization Characteristics Odds Ratio (95% CI) Odds Ratio (95% CI) Use of body wash Biweekly/Never 1.00 1.00 Daily/Weekly 0.91 (0.66, 1.27) 0.36 (0.16, 0.77)* Study Group Standard (Reference) 1.00 1.00 Enhanced Standard 0.95 (0.73, 1.24) 0.53 (0.27, 1.04)# Chlorhexidine 0.96 (0.69, 1.32) 0.99 (0.50, 1.97) Season of training start Winter 1.00 1.00 Spring 1.35 (1.00, 1.84) # 2.54 (1.19, 5.45)* Summer 1.32 (0.98, 1.78) # 1.27 (0.58, 2.78) Fall 1.38 (0.97, 1.95) # 0.79 (0.29, 2.16) 29

Week of training at the time of swab Week n+1 vs. n (n=1, 2,15) 1.00 (0.97, 1.03) 0.96 (0.89, 1.04) Whether there was any SSTI case in platoon prior to enrollment No 1.00 1.00 Yes 1.28 (1.01, 1.62)* 1.34 (0.76, 2.34) Frequency of hand sanitizer use, per day 3 times 1.00 1.00 Four or more times 0.84 (0.67, 1.06) 0.83 (0.46, 1.48) Frequency of washing towels 1 time/week or never 1.00 1.00 Daily/Several times per week 1.11 (0.89, 1.38) 1.19 (0.65, 2.18) Frequency of washing uniform 1 time/week or never 1.00 1.00 30

499 Daily/Several times per week 1.13 (0.90, 1.42) 1.05 (0.53, 2.06) * P-value <0.05, # P-value<0.1 and P-value>0.05 31

500 501 502 503 504 505 Table 4. Distribution of pulsed-field types among MRSA colonization isolates (A) Distribution of USA300 MRSA and non-usa300 colonization by frequency of body wash use Use of body wash MRSA isolates with PFGE type (n=60) USA300 Non-USA300 Missing USA100 USA800 Other No available isolate Never 28 5 9 5 10 Biweekly 2 0 1 2 1 Weekly 3 0 4 1 1 Biweekly/Never 30 (57.7) Weekly/Daily 3 (37.5) 5 (62.5) 32 P-value 22 (42.3) 0.448 (exact) (B) Results of the multivariate logistic regression for MRSA USA300 colonization with adjustment for within-cluster correlation Characteristics Use of body wash Odds Ratio (95% CI) Biweekly/Never 1.00

Daily/Weekly 0.74 (0.09, 6.37) Study Group Standard (Reference) 1.00 Enhanced Standard 1.20 (0.32, 4.51) Chlorhexidine 0.58 (0.13, 2.59) Season of training start Winter 1.00 Spring 1.52 (0.34, 6.68) Summer 1.29 (0.25, 6.72) Fall 3.84 (0.17, 87.3) Week of training Week n+1 vs. n (n=1, 2,15) 0.97 (0.83, 1.13) Whether there was any SSTI case in platoon prior to enrollment No 1.00 Yes 1.12 (0.31, 4.06) Frequency of hand sanitizer use, per day 3 times 1.00 Four or more times 0.64 (0.16, 2.5) 33

506 507 508 509 510 Frequency of washing towels 1 time/week or never 1.00 Daily/Several times per week 1.81 (0.51, 6.37) Frequency of washing uniform 1 time/week or never 1.00 Daily/Several times per week 0.65 (0.15, 2.88) 34