ORIGINAL ARTICLE. M.C. Roberts, O.O. Soge, D. No, S.E. Helgeson and J.S. Meschke. Abstract

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1 Journal of Applied Microbiology ISSN ORIGINAL ARTICLE Characterization of Methicillin-resistant Staphylococcus aureus isolated from public surfaces on a University Campus, Student Homes and Local Community M.C. Roberts, O.O. Soge, D. No, S.E. Helgeson and J.S. Meschke Department of Environmental and Occupational Health Sciences, School of Public Health, University of Washington, Seattle, WA, USA Keywords environmental surfaces, methicillin-resistant Staphylococcus aureus, student home surfaces, university surfaces. Correspondence Marilyn C. Roberts, Department of Environmental and Occupational Health Sciences, , School of Public Health, University of Washington, Seattle, WA , USA. marilynr@u.washington.edu : received 26 January 2011, revised 27 February 2011 and accepted 14 March 2011 doi: /j x Abstract Aim: Isolation and characterization of methicillin-resistant Staphylococcus aureus (MRSA) from frequently touched nonhospital environmental surfaces at a large university, student homes and community sites. Methods and Results: Twenty-four isolates from 21 (4Æ1%, n = 509) surfaces were MRSA positive and included 14 (58%, n = 24) SCCmec type IV, two (8%, n = 24) type I, and eight (33%, n = 24) were not type I-IV (NT). Six different multilocus sequencing types were identified by PCR and sequencing. PCR assays identified one (4Æ2%, n = 24) Panton-Valentine leukocidin (PVL) positive, 22 (92%, n = 24) arginine catabolic mobile element (ACME) positive and 23 (96%, n = 24) multidrug-resistant (kanamycin, macrolide, tetracycline) MRSA isolates. Eleven (46%, n = 24) USA300 isolates were determined by pulsed-field gel electrophoresis. Conclusion: The MRSA-positive environmental surfaces were identified in student homes (11Æ8%, n = 85), the community (2Æ3%, n = 130) and the university (2Æ7%, n = 294). USA300 strains were isolated from the university, student homes and community samples. This is the first report of the animal clone ST97 on urban environmental surfaces. Significance and Impact of the Study: The study highlights the distribution of USA300 on frequently touched surfaces. Whether contact with these MRSA contaminated environmental surfaces are associated with increased risk of transmission of MRSA to people needs further research. Introduction Staphylococcus aureus is carried in the nose of 25 35% of humans, and it is a common cause of serious and lifethreatening infections. Methicillin-resistant S. aureus [MRSA] was first identified fifty years ago, and today, it has become a major nosocomial pathogen. Communityacquired MRSA (CA-MRSA) skin and soft tissue infections, normally associated with USA300 MRSA strains in North America, are on the increase in healthy people with no healthcare exposure or known classical risk factors (Eady and Cove 2003; Klevens et al. 2007). Identified risk factors associated with CA-MRSA outbreaks include sharing of personal care products, frequent skin-to-skin contact, skin abrasions and crowded living conditions. There are less therapeutic options for treatment of MRSA infections, patients often require longer hospital stays, and MRSA infections are more likely to recur which increases the costs as compared to infections because of S. aureus (Rubin et al. 1999). Staphylococcus aureus and MRSA are spread from people-to-people and fomite-to-person, although the potential reservoirs in the community are unknown (Eady and Cove, 2003; Miller and Diep 2008). MRSA-contaminated public transport handrails (Stepanović et al. 2008), beauty salon surfaces (Huijsdens et al. 2008), fire stations, medic trucks and ambulance surfaces have been identified (Brown et al. 2010; Roberts et al. 2011b; Roline et al. 2007). Scott et al. (2009) found MRSA contaminated surfaces in nine (26%) of 35 homes with MRSA isolated Journal of Applied Microbiology 110, ª 2011 The Society for Applied Microbiology 1531

2 from seven of 13 kitchen surfaces and four of 11 bathroom surfaces, and methicillin-susceptible S. aureus [MSSA] was isolated from 12 of the kitchen and ten of the bathroom surfaces. One study found that environmental survival of MRSA improves in the presence of organic material on coins (Tolba et al. 2007). While a second study (Desai et al. 2009) showed MRSA may persist on contaminated surfaces for >5 weeks, suggesting that MRSA may survive for extended time periods. The second study also determined that 3 s of skin contact with a MRSA-contaminated surface or object was all that was required to transfer MRSA to the skin under laboratory conditions. Brook et al. (2009) examined 70 frequently used environmental surfaces in a university of individuals for contamination of S. aureus and MRSA from 420 samples taken from student desktops, computer keyboards, telephone mouthpieces, water fountains, photocopy keypads, vending machines and elevators buttons over a 3-week period in Staphylococcus aureus was isolated from computer keyboards, telephones and elevator buttons; however, no MRSA strains were isolated and S. aureus were not further characterized. More recently we have found 8Æ4% of the 95 environmental surfaces taken from university dental clinics were MRSA positive. In that study, three MRSA were SCCmec type IV, three were Panton-Valentine leukocidin positive (PVL+) and none were USA300 (Roberts et al. 2011a). This study suggested that both dental clinic surfaces and dental students may be reservoirs for MRSA, although the dental clinic services outpatients and thus is more related to a hospital setting than strictly a university environment. In the current study, 24 MRSA isolates were identified and characterized from 21 (4Æ1%, n = 509) environmental surfaces taken from a large university campus, student homes and the local community. Fourteen (58%, n = 24) MRSA were type IV, two (8%, n = 24) type I and eight (33%, n = 24) NT, and 11 (46%, n = 24) isolates from all three sampled areas were USA300. In contrast to earlier studies, 23 of 24 of the MRSA isolates in this study were resistant to 1 other class of antibiotics which would reduce therapeutic options if they caused disease (Eady and Cove, 2003). MRSA-positive surfaces were identified in all three locations (student homes, community and university), suggesting that these contaminated surfaces may be reservoirs for transmission of MRSA to the public. Materials and methods Surfaces sampled A total of 509 frequently touched surfaces including 85 environmental samples from eight undergraduate student housing sites (apartments, houses, parent homes and dorm rooms) including common areas (couch, bathroom surfaces, kitchen, microwave touchpad, TV remote, washing machine) using the Scott et al. study (2009) as a guide, and students cell phones; 294 surfaces on University of Washington (UW) campus (ATM and computer keypads, bathroom handles, elevator buttons, locker handles, vending machine keypads, and water fountain handles, gym equipment); and 130 community sites (parking meters, ATM keypads, public library touch screens) were sampled in (Table 1). The University has > students and employees. Two of the indoor UW ATM keypads were sampled nine separate times over a 6-month time period for the presence of MSSA and MRSA (Table 2). While the other sites were sampled once using the SANICULT TM swab (Starplex Scientific Inc., Etobicoke, ON, Canada) or BBL TM RODAC TM contact plates (Becton Dickinson Co., Franklin Lakes, NJ, USA) for all surfaces and sterile baby wash clothes in the washing machines as previously described (Roberts et al. 2011a). A convenience sampling plan was used. Sample processing BBL TM RODAC TM contact plates (Becton Dickinson Co.) with Bacto Ò Staphylococcus Medium 110 supplemented with 10 lg ml )1 methicillin and 0Æ01% potassium tellurite were incubated in 5% CO 2 at 36Æ5 C. Two millilitres of Bacto Ò m Staphylococcus broth [1Æ5 ] (Difco Laboratories, Sparks, MD, USA) supplemented with a final concentration of 75 lg ml )1 polymyxin B and 0Æ1% potassium tellurite (Sigma Co. St. Louis, MO, USA) was added to the swabs and 50 ml of media for the wash clothes. All were incubated in 5% CO 2 at 36Æ5 C until turbid and black as previously described (Roberts et al. 2011a,b). Positive samples were diluted and plated onto Bacto Ò m Staphylococcus Medium 110 supplemented with 10 lg ml )1 methicillin and 0Æ01% potassium tellurite and Bacto Ò Mannitol Salts Agar (Difco) as previously described (Roberts et al. 2011a,b). All samples were held for 7 days before being labelled as negative. Isolates were biochemically verified as S. aureus, and the meca gene was verified in the MRSA isolates as previously described (Roberts et al. 2011a; Soge et al. 2009). Detection of meca, PVL gene, SCCmec typing, multilocus sequence typing (MLST), and presence of the arginine catabolic mobile element (ACME) The PCR assay conditions for the meca, SCCmec type I-V, and presence of the PVL gene used previously described primers and conditions (Roberts et al. 2011a; 1532 Journal of Applied Microbiology 110, ª 2011 The Society for Applied Microbiology

3 Table 1 Molecular typing characteristics of methicillin-resistant Staphylococcus aureus isolated from 21 public surfaces Isolate Year Location SCCmec ST type PFGE USA 300* ACME Resistance genes Student Homes n = 10 isolates Student #1 J Dorm water fountain NT 8 M No + None Student #2 A Microwave touchpad IV 45 N No + tet(m), msr(a), aadd A Couch armrest, fabric IV 45 N No + tet(m), msr(a), aadd Student #3 S TV remote IV 5 A Yes + tet(m), tet(k), msr(a), aadd S Bathroom floor IV 5 A Yes + tet(m), msr(a), aadd S Bathroom light switch NT 5 A Yes + tet(m), tet(k), aadd Student #4 R Couch IV 8 K Yes + tet(m), msr(a), aadd R Toilet Flush Handle IV 931 K Yes + tet(m), tet(k), msr(a), aadd R Bathroom Door Knob IV 931 K Yes + tet(m), tet(k), msr(a), aadd Student #5 T Washing Machine IV 5 L No + tet(m), tet(k), msr(a), aadd University Campus n = 10 isolates Bathroom 3rd floor NT 5 E No ) tet(m) Bathroom 3rd floor IV 5 A Yes + erm(c), msr(a) 20à 2010 Inside ATM Keypad R IV 5 A Yes + tet(m), tet(k), erm(a), erm(c), msr(a), aadd 36à 2010 Inside ATM Keypad R I 45 D No + tet(m), tet(k), erm(a), erm(c), aadd Inside ATM Keypad T IV 30 C Yes + tet(m), tet(k), erm(a), erm(c), msr(a), aadd Locker handle IV 8 B Yes + tet(m), tet(k), msr(a), aadd Elevator button I 8 F No + tet(m), erm(c), msr(a) Elevator button NT 97 H No + tet(m) Study lounge floor NT 97 H No + tet(m), erm(c) Bathroom 2nd floor NT 97 G No ) tet(m) Community sites near the University campus n = 4 isolates Outside ATM Keypad A NT 5 A Yes + tet(m), msr(a) Outside ATM Keypad B IV 8 I No + tet(m), erm(c), msr(a), aadd 5-3** 2010 Library computer touch screen NT 30 J No + tet(k), aadd 5-4** 2010 Library computer touch screen IV 30 J No + tet(m), tet(k), msr(a), aadd * 80% PFGE identity with USA 300 determined by BioNumerics GelCompar II software;,,** From the same sample; àsame ATM keypad sampled at two different time periods; #20 was isolated and #36 was isolated (see Table 2); PVL positive isolate. Soge et al. 2009). Positive and negative controls were used as previously described in all assays. Those isolates that were not type I-V were labelled nontypeable [NT] for the current study. The PCR assay for the ACME encoded arca gene used primers and conditions described by Diep et al. (2006) with a clinical USA300 as the positive control. The MLST PCR assays were performed as previously described using published primers and conditions (Enright et al. 2000; Roberts et al. 2011a; Soge et al. 2009). Detection of antibiotic resistance genes PCR assays were used to determine the presence of kanamycin resistance gene, aadd; macrolide resistance genes, erm(a), erm(b), erm(c), and msr(a) genes; and tetracycline resistance genes, tet(m) and tet(k), with verification by hybridization using internal 32 P-labelled probe. Positive and negative controls were used for all PCR assays as previously described (Roberts et al. 2011a; Soge et al. 2009). Pulsed-field gel electrophoresis (PFGE) Methicillin-resistant S. aureus isolates were PFGE typed using previously described protocol (McDougal et al. 2003) with SmaI enzyme (Fermentas Inc., Glen Burnie, MD, USA). The different PFGE patterns were labelled A through N (Table 1). The genetic relatedness of the Journal of Applied Microbiology 110, ª 2011 The Society for Applied Microbiology 1533

4 Table 2 Methicillin-susceptible Staphylococcus aureus [MSSA] and methicillin-resistant S. aureus [MRSA] from two indoor university ATM machines Date MSSA MRSA ATM #R No No No Yes No No No Yes No No No No No No Yes No No No ATM #T Yes No No Yes No No No No No No No No No No Yes No Yes No No No isolates to USA300 was determined by Dice coefficient, UPGMA using the GelCompar II software according to the manufacturers instructions (Applied Maths, Inc., Austin, TX USA). Strains that had 80% homology with USA300 were classified as USA300 (Table 1). Results Identification and characterization of MRSA-positive samples Twenty-four MRSA isolates were identified from 21 (4Æ1%, n = 509) different environmental surface samples, and included ten (11Æ8%, n = 85) student house samples, eight (2Æ7%, n = 294) university samples and three (2Æ3%, n = 130) community samples (Table 1). Fourteen (58%, n = 24) of the MRSA isolates were SCCmec type IV of which nine were ( 80% homology) USA300. Two additional isolates, which were SCCmec NT, were also USA300. Two (8%, n = 24) isolates were SCCmec type I, the remaining eight (33%, n = 24) were NT and none of these were USA300. The 24 MRSA represented six different ST types with the most common ST 5 (eight isolates), followed by ST8 (five isolates), ST30, ST40 and ST97 (three isolates each) and ST931 (two isolates). Twentythree (96%, n = 24) of the MRSA isolates carried other antibiotic resistance genes. Twenty-two (92%, n = 24) of the isolates carried tetracycline resistance genes [11 tet(m); 10 tet(k) and tet(m); 1 tet(k)]. Eighteen (75%, n = 24) carried macrolide resistance genes with 12 (50%, n = 24) carrying one antibiotic resistance gene and six (25%, n = 24) carrying 2 macrolide resistance genes. Fifteen isolates carried the msr(a) gene, seven isolates carried the erm(c) gene and three isolates carried the erm(a) gene. Sixteen (67%, n = 24) of the MRSA isolates carried the aminoglycoside resistance gene, aadd (Table 1). Student homes Five (63%, n = 8) undergraduate student homes had MRSA-positive samples (Table 1). The MRSA-positive homes included two students who were living in their family home and three living with other students. The three student homes that were negative for MRSA also lived with other students. The number of MRSA-positive samples ranged from one to three of the 10 surfaces sampled per home. Four different ST-types were identified with two homes having ST5 or ST8 and two homes (Student #3 and #4) were contaminated with USA300 isolates. Student #5 was the only one to have a MRSApositive washing machine. The two MRSA isolated from Student #2 house surfaces were indistinguishable from each other by all typing methods suggesting they represented a single strain. Diversity was found in the SCCmec type, ST type, and number and type of antibiotic resistance genes carried by the three MRSA isolates recovered from each of the Student #3 and Student #4 households. However, the three isolates from each household had indistinguishable PFGE patterns and were related to USA300. The USA300 MRSA isolates from Student #3 home had the same ST type, indistinguishable PFGE pattern, and two of the isolates had the same SCCmec type IV as the university isolates 3-36 from the bathroom floor and 20 from the ATM keypad, although they differed in the number of other antibiotic resistance genes they carried. All 10 MRSA isolates from the student homes were ACME +, nine (90%, n = 10) isolates carried other antibiotic resistance genes and none were PVL + (Table 1). University surfaces Two university ATM keypads were repeatedly sampled nine times over a 6-month time period (Table 2). During that time period, the ATM #R keypad was positive 3 (33%, n = 9) times for S. aureus including twice for MRSA and MSSA, while ATM #T was positive 4 (44%, n = 9) for S. aureus including once with MRSA and three times with MSSA (Table 2). All three of the ATM MRSA differed in ST type (ST5, ST30, ST45), PFGE patterns and 1534 Journal of Applied Microbiology 110, ª 2011 The Society for Applied Microbiology

5 number of different antibiotic resistance genes carried. All three were ACME positive and isolates 20 and 26 were USA300 (Table 1). Five other university samples were MRSA positive and represented three different ST types (ST5, ST8, ST97). Each of these isolates carried 1 other antibiotic resistance gene(s). Isolate was ST8, PVL+, SCCmec type IV, USA300 and carried aminoglycoside, macrolide and tetracycline resistance genes (Table 1). Two university samples contained two different MRSA strains each [3-22, 3-36 and 3-2, 3-8]. The isolates 3-22, 3-36 differed from each other by SCCmec type (IV vs NT), differed by PFGE profile, relatedness to USA300, the presence of the ACME element and the number of antibiotic resistance genes carried and likely represent different strains even though both were ST5 (Table 1). The second two isolates, 3-2, 3-6, differed by SCCmec type (I vs NT), ST type (ST8 vs ST97), PFGE profile and the number of antibiotic resistance genes carried and likely represent different strains. Community surfaces From the 130 community surfaces, 120 of the samples were taken from ATM keypads sampled in 2009 and 2010 and ten were from other surfaces (Table 1). Two of 120 (1Æ7%) ATM keypad samples were MRSA positive and 11 of 120 (9Æ2%) were MSSA positive. The two MRSA isolates differed in SCCmec type (IV vs NT), ST type (ST5 vs ST8), PFGE pattern and antibiotic resistance genes they carried. One strain #40 was USA300, while the second strain #55 was not related to USA300 (Table 1). The two MRSA isolates, 5-3 and 5-4, from the library computer touch screen came from the same sample and were SCCmec type IV, ST30, and had the same PFGE pattern, suggesting that they represented a single strain even if though the isolates differed in the number of antibiotic resistance genes each carried. Discussion This is one of the first studies to isolate and molecularly characterize MRSA from nonhealthcare environmental surfaces. SCCmec typeable isolates [I or IV] were found in 66% of the isolates, with 46% of the isolates USA300 and 92% ACME +. The five of the six MLST types (ST5, ST8, ST30, ST45, ST931) found in the current study are associated with humans and include widely disseminated ST types of CA-MRSA found throughout the world (Tavares et al. 2010; te Witt et al. 2009). In contrast, ST97 is closely associated with cattle and more recently described in both healthy and diseased pigs, although it has not been found contaminating food nor associated with humans as ST398 has (Gomez-Sanz et al. 2010; Lozano et al. 2009). This is the first report of the animal clone ST97 contaminating urban environmental surfaces. Finding three animal associated isolates contaminating an urban University environment was unexpected especially because the ST97 isolates (3-8, 3-10 and 3-6) were taken from three different University Campus Sites. Two of the isolates, 3-8 and 3-10, likely represent a single clone suggesting a common source, while the third isolate was unrelated. The university is a commuter school with students, staff and faculty travelling from surrounding urban and rural communities. Thus, the possibility that the three ST97 MRSA isolates originated in animals and or food and were transferred to the contaminated surfaces in the study is an intriguing hypothesis although it would be difficult to prove. Early studies on community-acquired USA300 found that these isolates were usually susceptible to other classes of antibiotics (Eady and Cove 2003). However, in a more recent study in fire stations, environmental USA300 isolates were multidrug resistant as was found in the current study (Roberts et al. 2011b). Among all the MRSA isolates, 23 (96%, n = 24) carried 1 other antibiotic resistance gene(s) with 92%, n = 24 carrying tetracycline, and 79%, n = 24 carrying macrolide resistance genes and similar to what has previously been found on other hightouched environmental surfaces (Roberts et al. 2011b). USA300 MRSA was isolated from university, student homes and the community environmental samples. Five (63%, n = 8) student homes had 1 3 MRSApositive samples and included both students who lived in their family homes and students living with multiple student roommates. Although this is a very small study, it was >2-fold higher than what was identified by Scott et al. (2009) which identified MRSA in nine (26%) homes samples (Scott et al. 2009). In the current study, three of the homes had multiple samples that were MRSA positive. Of the five homes, two were contaminated with USA300 strains, and four of five homes had MRSA with SCCmec type IV. The MRSA isolates from Student #2 home were indistinguishable from each other, suggesting they represented the same strain from two different samples. The MRSA isolates from Student # 3 and #4 homes did differ, but overall the data suggest that the isolates from each home were clonally related. The MRSA isolates from Student # 3 home were genetically related to two of the university campus MRSA isolates; however, because they were all USA300, it is unclear whether the home and university isolates shared a common source. The Scott et al. s (2009) study did not characterize the MRSA isolates recovered, so it is unknown whether the same strain was found in multiple locations within the MRSApositive homes, or whether any of the isolates were SCCmec type IV or USA300. Journal of Applied Microbiology 110, ª 2011 The Society for Applied Microbiology 1535

6 None of the students that sampled their homes had a MRSA infection although one student (#2) was colonized with a MRSA strain that was genetically distinct in all characteristics from the home isolates (unpublished data). In the current small study, the undergraduate student MRSA carriage rate was 12Æ5% which was higher than a previous study among Texas University students of 7Æ3% carriage rate (Rohde et al. 2009), but lower than the 21% carriage rate found in dental students (Roberts et al. 2011a). A larger study examining more college students and student houses is needed to determine whether college students are at increased risk of MRSA infection. MRSA-positive university samples were found from the bathroom, floors, ATM keypads, elevator buttons, locker handles but not on computer keyboards. One isolate was PVL+ USA300, 3 PVL-USA300 isolates, two type I, and four NT isolates were identified from the university sites. All isolates were multidrug resistant and eight were ACME+. In contrast, Kassem et al. (2007) found 8% of the 24 university computer keyboards were MRSA positive although they did not characterize the isolates. In contrast, in a second university study, no MRSA were identified from 70 frequently used environmental surfaces, although MSSA was isolated in computer keyboards, telephone mouthpieces and an elevator button of a large Midwestern United States urban university (Brook et al. 2009). Multiple sampling of two high use university ATM machines illustrated frequent (38%) contamination with MSSA and MRSA, with USA300 found in two of the three isolates. This was three times higher than the 10Æ9% MSSA and MRSA level found among community ATM machines. Why there were such differences in frequency of MSSA and MRSA between the university and community ATM machines is not clear, and further sampling is needed. The number of other community surfaces sampled was limited in the current study with only computer touch screens from a public library positive. A recent previous study sampled 118 hand-touched surfaces in buses, trains, stations, hotels and public areas of hospital in central London and cultured MSSA from 8% of the sites but did not find MRSA (Otter and French 2009). Whether contact with these MRSA contaminated environmental surfaces are associated with increased risk of transmission of MRSA to people is unknown. However, this study indicates that nonhospital environmental surfaces are potential reservoirs for MRSA. References Brook, J.S., Annand, J.W., Hammer, A., Dembkowski, M.T. and Shulman, S.T. (2009) Investigation of bacterial pathogens on 70 frequently used environmental surfaces in a large urban U.S. university. J Environ Health 6, Brown, R., Minnon, J., Scheider, S. and Vaughn, J. 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