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Methicillin-resistant Staphylococcus aureus (MRSA) is a public health threat within the general community, thereby warranting identification of MRSA reservoirs within the community. Computer terminals in schools were sampled for S. aureus and methicillin-resistant staphylococci. The overall prevalence of MRSA on computer keyboards was low: 0.68% for a postsecondary institution and 2% and 0% for two secondary institutes. The MRSA isolate from the postsecondary institution did not correspond to the Canadian epidemic clusters, but is related to the USA 700 cluster, which contains strains implicated in outbreaks within the U.S. The isolate from the secondary institute's keyboard was typed as CMRSA7 (USA 400), a strain that has been implicated in both Canadian and U.S. epidemics. Methicillin-resistant S. haemolyticus and S. epidermidis were also isolated from keyboards, indicating that a mixed community of methicillin-resistant staphylococci can be present on keyboards. Although the prevalence was low, the presence of MRSA combined with the high volume of traffic on these student computer terminals demonstrates the potential for public-access computer terminals and computer rooms at educational institutes to act as reservoirs. [PUBLICATION ABSTRACT]
INTERNATIONAL PERSPECTIVES
Abstract
Methicillin-resistant Staphylococcus aureus (MRSA) is a public health threat within the general community, thereby warranting identification of MRSA reservoirs within the community. Computer terminals in schools were sampled for S. aureus and methicillin-resistant staphylococci. The overall prevalence of MRSA on computer keyboards was low: 0.68% for a postsecondary institution and 2% and 0% for two secondary institutes. The MRSA isolate from the postsecondary institution did not correspond to the Canadian epidemic clusters, but is related to the USA 700 cluster, which contains strains implicated in outbreaks within the U.S.
The isolate from the secondary institute's keyboard was typed as CMRSA7 (USA 400), a strain that has been implicated in both Canadian and U.S. epidemics. Methicillin-resistant S. haemolyticus and S. epidermidis were also isolated from keyboards, indicating that a mixed community of methicillin-resistant staphylococci can be present on keyboards. Although the prevalence was low, the presence of MRSA combined with the high volume of traffic on these student computer terminals demonstrates the potential for public-access computer terminals and computer rooms at educational institutes to act as reservoirs.
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Introduction
Staphylococcus species are commonly divided into two groups: pathogenic S. aureus and coagulase-negative staphylococci (CoNS). CoNS include multiple species and are generally regarded as only opportunistically pathogenic. The frequency of methicillin resistance in CoNS is notably high and it has been suggested that this may provide a reservoir to propagate methicillin resistance into other Staphylococcus species (Lindsay & Holden, 2004).
The occurrence of methicillin-resistant Staphylococcus aureus (MRSA) in hospitals was first reported in 1961 (Jevons, 1961). Numerous nosocomial MRSA outbreaks occur annually due to the widespread prevalence of MRSA within hospitals (Klein, Smith, & Laxminarayan, 2007). Recently, highly virulent strains of MRSA have been identified in individuals with no history of recent hospitalizations and no evidence of having predisposing risk factors. These strains have been referred to subsequently as community-associated MRSA (CAMRSA) and have become a global infectious threat (reviewed in Diep & Otto, 2008). In the U.S., 33% of current MRSA infections are due to infections of community origin (Klevens et al., 2007).
Compared to the U.S., Australia, and other nations, MRSA rates in Canada have been relatively low; however, they have increased 16-fold from 1995 to 2005 from 0.46 per 1,000 hospital admissions to 7.6 per 1,000 hospital admissions (Webster, Rennie, Brosnikoff, Chui, & Brown, 2007). Two main strains have been implicated in the majority of CA-MRSA infections in Canada: CMRSA 7 (also known as USA 400/MW2) and CMRSA 10 (also known as USA 300) (Christianson, Golding, Campbell, & Mulvey, 2007).
Identifying reservoirs for pathogenic organisms is an important step in implementing intervention methods to prevent the spread of disease. Studies examining routes of transmission of hospital-associated MRSA (HAMRSA) have shown that hospital keyboards can represent a significant reservoir; the incidence of keyboard contamination by MRSA in these studies ranged from 8% to 42% (Bures, Fishbain, Uyehara, Parker, & Berg, 2000; Devine, Cooke, & Wright, 2001; Fellowes, Kerstein, & Azadian, 2006; Neely et al, 2005). The high number of users on computer terminals in public settings such as libraries and computer labs at schools creates an opportunity for the transmission of bacteria (Anderson & Palombo, 2009), suggesting they may be a possible reservoir for CA-MRSA.
MRSA prevalence on keyboards within a community setting was recently investigated by researchers at the University of Toledo (Kassem, Siglar, & Esseili, 2007). Twenty-four publicaccess computer keyboards were sampled and two of the keyboards were found to be contaminated with MRSA. The presence of MRSA combined with the high volume of traffic on public computer terminals is a concern and may contribute to the spread of this pathogen in the community. Using selective and differential media we investigated the prevalence of S. aureus and methicillin-resistant staphylococci contamination on public-access computer terminals at the University of Regina and two secondary schools (grades 10-12) within the Regina area.
Methods
Specimen Collection
Keyboards in the Archer Library at the University of Regina were sampled repeatedly over several nonconsecutive days with a minimum of seven days between sampling dates during the months of October-November 2007 and January-February 2008. Both high traffic (n = 7) and low traffic keyboards (n = 13) were included in the sampling. High-traffic computers are standing terminals located at the main entrance of the library and are used by many individuals for short periods of time, whereas low-traffic computers are sit-down terminals used for longer periods of time resulting in fewer users on any given day.
Computer keyboards were also sampled by high school students at two high schools in the Regina area on March 5 (HS #1) and March 27 (HS #2), 2009. A total of 50 individual keyboards from two computer labs were sampled one time from HS #1 while 71 individual keyboards were sampled one time from three computer labs at HS #2. These computer labs are accessed by the majority of the student population and are in use throughout the day.
Sterile cotton swabs dipped in sterile phosphate buffered saline (PBS, Fluka) were passed over the entire surface of all letter keys, space bar, and enter key. Swabs were cut so that only the cotton swab was placed directly into tryptic soy broth (TSB) and incubated overnight at 370C with agitation. A control swab dipped in phosphate buffered saline and briefly exposed to the air was also incubated in TSB along with the keyboard samples.
Isolation and Identification of Staphylococcus Colonies
After incubation, turbid TSB tubes were subcultured onto mannitol salt agar (MSA) medium, a selective medium used to isolate putative Staphylococcus species and differentiate S. aureus (Chapman, 1943), and incubated for 48 hours at 370C. One hundred pL of the turbid TSB culture was also inoculated into TSB supplemented with oxacillin (2 mg L-I) and incubated overnight at 370C with agitation (Jonas, Speck, Daschner, & Grundmann, 2002) prior to plating onto MSA and BairdParker agar (Baird-Parker, 1962; Oxoid). Oxacillin, which is in the same class of drugs as methicillin, is used since methicillin is no longer commercially available. Additionally, oxacillin maintains its activity during storage better than methicillin. Colonies arising on MSA and Baird-Parker agar exhibiting morphology appropriate to S. aureus were further characterized using gram-staining, testing for catalase, and coagulase testing (Pastorex Staph-Plus kit, Bio-Rad). Catalase and coagulase positive isolates were subcultured onto MRSASelect medium (Bio-Rad) and oxacillin screen agar (OSA) medium.
Isolates that grew on OSA and MRSASelect were inoculated onto lysogeny broth plates and sent to the Saskatchewan Disease Control Laboratory (Regina, Saskatchewan) for automated identification and antibiotic susceptibility testing. Antimicrobial susceptibility testing was performed using automated instrumentation (MicroScan WalkAway plus System). Interpretive criteria for MIC values were applied as recommended by the Clinical and Laboratory Standards Institute (Clinical and Laboratory Standards Institute, 2011).
Genomic Profiling of MRSA and Other MRS Strains
Profiling of the MRSA strains involved S. aureus protein A gene (spa) typing (Shopsin et al., 1999), detection of Panton-Valentine leukocidin (PVL) toxin gene, and methicillin-resistance mecA gene detection by multiplex polymerase chain reaction (PCR) as described by McDonald and coauthors (2005). Pulsed-field gel electrophoresis (PFGE) as described by Mulvey and co-authors (2001) was used when necessary. Spa types and PFGE profiles of MRSA isolates were compared to local and national databases (Saskatchewan Disease Control Laboratory and Canadian Nosocomial Infections Surveillance Program) to determine if they were members of known clusters or match any previously observed clinical strains. Classification based on PFGE profile followed the recommendation of Tenover and co-authors (1997) whereby if the typical number of fragment differences compared to the outbreak pattern is greater or equal to seven, then they are not related. Indistinguishable, closely related, and possibly related strains have 0, 2-3, and 4-6 fragment differences from the outbreak pattern, respectively (Tenover et al., 1997).
Determining Survival of Staphylococcus spp. on Keyboards
Individual computer keyboard keys were removed from standard keyboards, cleaned, and autoclaved prior to inoculation with individual Staphylococcus strains. Staphylococcus species were provided by the Saskatchewan Disease Control Laboratory. The HA-MRSA was a CMRSA-2 (PVL-) strain while the CAMRSA strain was a CMRSA-7 (PVL+) strain. Isolates were enriched overnight at 350C on TSB (with oxacillin for MRSA isolates). Cells were adjusted to optical density of 0.9 at 620 nm (approximately 5 ? 10° cells). Twenty µ? of the cell suspension were inoculated onto individual keyboard keys. For each strain a total of 36 keys were inoculated, allowing each sampling day to be conducted in triplicate. A negative control (20 mL sterile PBS) was also inoculated onto 12 keys. The keys were kept in the laboratory at ambient temperature and humidity. On a daily basis for a period of 12 days bacteria were recovered from the keys, in triplicate, by swabbing the entire surface of each key with a sterile swab moistened in PBS. The swab was cut to ensure no cross contamination, the keyboard keys were both placed in a sterile 50 mL tube containing 5 mL TSB, and the tube was vortexed for one minute in order to recover all the cells. The bacteria were subsequently enumerated by spread plating serial dilutions onto TSA medium.
Statistical Analysis
The bacterial counts obtained for each strain were compiled and the Weibull type model (Marfan, Couvert, Gaillard, & Leguerine, 2002) was used to fit them:
...
where N represents the bacterial density (CFUs per keyboard key) observed at time t (in days), N0 is the initial bacterial density (in CFUs per keyboard key), and d is the time (in days) for the first decimal reduction in bacterial cell number. The model was fitted using the nls function of the R software version 2.0.1 (Ihaka & Gentleman, 1996). A one-way ANOVA test was carried out in order to examine the influence of the different strains on the d parameter values. Multiple comparisons of the d values were then made using pairwise i-tests (Bonferroni correction).
Results
The computer keyboards from the three schools experienced different levels of S. aureus contamination (Table 1). Higher incidences of S. aureus were observed on the high school keyboards. Two MRSA strains were isolated during this survey, one originating from a single high-traffic keyboard (Arch 7) at the University of Regina library and the other from a HS #1 keyboard. The University of Regina keyboards, including Arch 7, were sampled several times during OctoberNovember 2007 and January-February 2008; however, the MRSA strain was only detected once on the Arch 7 keyboard during the October-November sampling period.
The two MRSA isolates were further characterized using spa typing. The MRSA isolate from the University of Regina library (UR-I) has a spa type 664 and has a repeat succession 07-23-12-12-17-20-17-12-17. This spa type is not present in the Saskatchewan Disease Control Laboratory (SDCL) or the Canadian Nosocomial Infection Surveillance Program (CNISP) spa typing databases. It is found, however, within the Ridom SpaServer (Harmsen et al., 2003). Six isolates with this spa type are present in the database and all were originally isolated in Sweden. Because of the relatively uncharacterized nature of the isolate, PFGE was performed for further identification. The UR-I isolate's PFGE pattern clustered with the CMRSA7 profile; however, it has greater then seven fragment differences compared to its closest related strain. Therefore, UR-I is a distant relative to CMRSA7 (Figure 1).
Furthermore, the PFGE fingerprint of UR-I did not correspond to any patterns from MRSA isolates obtained from Saskatchewan patients that were stored in the SDCL database. The PFGE profile was subsequently compared to the PFGE national database of CNISR The PFGE pattern of isolate UR-I did match to three clinical isolates in this database, 02S1336 (isolated in 2002), 06S1154 (isolated in 1995), and N08-00209 (isolated in 2008), indicating that this strain can be associated with human disease. The strains found in this cluster are related to the USA700 cluster, which has been found in both community and nosocomial settings (Tenover et al., 2008). The MRSA isolate from HS #1 (Lumi) has the spa type tl28, which is the spa type found in the CA-MRSA strain lineage CMRSA7/ USA400, one of the two prominent community acquired MRSA strains in the U.S. and Canada (Baba et al., 2002; Christianson et al., 2007). This lineage and USA300/CMRSA10 are considered highly clinically significant and together with the hospital associated MRSA strains, CMRSA 1 to 6 and 9, they represent over 80% of all reported MRSA infections in Canada (Simmonds, Dover, Louie, & Keays, 2008). CA-MRSA strains often carry the genes coding for the PVL toxin (Tenover et al., 2008). Both the MRSA strains isolated in this study tested negative for the presence of the PVL genes (data not shown).
The incidence of oxacillin-resistant bacteria contaminating the keyboards was particularly high in the high schools (Table 1), although the prevalence observed in the university library was also relatively high. The high frequency of growth in oxacillin-supplemented TSB was investigated further and several coagulase-negative Staphylococcus isolates obtained in the University of Regina sampling were screened for growth on OSA. Four coagulase-negative Staphylococcus isolates grew on OSA and were confirmed to be oxacillin resistant (Table 2) and a PCR assay verified the presence of the mecA gene (not shown). Biochemical typing identified the strains as S. epidermidis and S. haemolyticus.
The two distinct MRSA isolates shared similar antibiotic resistance profiles (Table 2). The methicillin-resistant S. epidermidis and S. haemolyticus (MRSE and MRSH) isolates had distinctive profiles and were generally resistant to more antibiotics than the MRSA isolates.
To determine the length of time a keyboard may remain contaminated with MRSA, the survival of Staphylococcus strains on keyboards was also investigated. Figure 2a shows the survival curves for the Staphylococcus spp. used in the study. A large percentage of cells were inactivated rapidly during the first day of incubation. The rate of die off decreased, however, and persistent recovery of cells was possible following 12 days of incubation. Considering the 95% confidence interval overlap, no significant differences occurred between the mean d values for the HA-MRSA (S2), S. aureus (S3), and S. epidermidis (S4) strains (Figure 2b). This statement was also confirmed by using the Bonferroni correction test (p > .05). The CA-MRSA (Sl) strain had a significantly higher survival rate, however, when compared to the S. aureus and S. epidermidis strains (Bonferroni, ? < .01).
Discussion
The primary mode of transmission of S. aureus is thought to be direct skin-to-skin contact (Miller & Diep, 2008). Computer keyboards have been recognized, however, as an alternative important reservoir for pathogenic bacteria, such as MRSA, within hospital and clinical settings (Fellowes et al., 2006; Shultz, Gill, Zubairi, Huber, & Gordin, 2003; Wilson et al., 2006). Moreover, recent attention has also focused on the potential role of public computer keyboard terminals as reservoirs for pathogens like MRSA (Anderson & Palombo, 2009; Kassem et al., 2007).
In our study, computer keyboards at educational institutions were selected since these keyboards receive relatively high volumes of users. The degree of contamination on the keyboards by S. aureus varied widely between institutions, with absolute prevalences ranging from 18% to 60%. These ranges are similar to other studies on public keyboard terminals at universities; for instance, Anderson and Palombo (2009) reported prevalences of S. aureus on multiple-user keyboards ranging from 40% to 60%. Kassem and co-authors (2007) reported a prevalence of 21% on multiple-user university keyboards.
The keyboard sampling at the University of Regina allowed for a comparison between high-traffic and low-traffic keyboards. Of the samples taken from low- traffic keyboard terminals, the prevalence of S. aureus was 13% (9/70) while the high traffic terminals the prevalence was 22% (17/77). Perhaps not surprisingly, the single MRSA strain from the University of Regina was isolated from one of the high-traffic computer keyboards. The keyboards at high schools are considered high traffic given the large numbers of students who access the computer labs on a daily basis, and this frequency likely contributes to the high incidence of S. aureus on these terminals. Intuitively it seems reasonable to expect higher contamination on multiple-user keyboards. The results of our study and that of Anderson and Palombo (2009) and Kassem and co-authors (2007) reinforce the emphasis that should be placed on disinfection of high-traffic computer keyboards, especially, as well as placing hand sanitizers near high-traffic public computer keyboards.
MRSA was identified at two of the three institutions with an absolute prevalence of 0.68% and 2.0%. This result is in agreement with the limited data on MRSA prevalence on public computer terminals, where the incidence of MRSA on computer keyboards from another university setting was 8.3% (2/24) (Kassem et al., 2007). Brooke and co-authors (2009) did not detect any MRSA isolates from university keyboards (30 samples total).
Spa typing and PFGE profiling were used to characterize the MRSA isolates from this study. UR-I is an uncommon CA-MRSA isolate while Lum-1 is an isolate within the prevalent CMRSA 7 group that is commonly implicated in CA-MRSA infections in both Canada and the U.S. Only sporadic cases of UR-1-like strains have been observed (Figure lb) (Mulvey et al., 2005). The isolates were further characterized for the presence of the PVL genes. CA-MRSA strains isolated in clinical situations often carry the genes coding for the PVL toxin. PVL causes tissue necrosis and leukocyte destruction by forming pores in cellular membranes (Lina et al., 1999), and the PVL genes are commonly associated with CA-MRSA virulence (Diederen & Kluytmans, 2006; Diep & Otto, 2008; Etienne, 2005).
Interestingly, both the CA-MRSA strains isolated in our study did not possess the genes for PVL. Recent research comparing clinical isolates from the CA-MRSA USA400 (CMRSA 7) group indicated that only 22.3% of the isolates were PVL positive and the PVL-negative isolates shared similar clinical characteristics and virulence to the PVLpositive isolates, suggesting PVL may not be absolutely necessary for CA-MRSA virulence (Zhang, McClure, Elsayed, Tan, & Conly 2008). UR-I and Lum-1 were also characterized for additional antibiotic resistance phenotypes. Both isolates had a resistance profile typical of CA-MRSA strains (Chambers & Deleo, 2009).
The survival of MRSA on keyboards is an important consideration, as the duration of persistence will directly impact the potential risk for transmission of the pathogen to keyboard users. Our study found that MRSA and methicillin-susceptible Staphylococcus aureus (MSSA) can persist for at least 12 days on keyboards, thereby allowing for possible transmission to multiple users who access a contaminated keyboard. This is similar to reports of MRSA persisting on laminated tabletops for more than 12 days (Huang, Mehta, Weed, & Price, 2006). The slight but significantly higher survival rate in the CAMRSA strain is noteworthy and merits further investigation.
The high prevalence of oxacillin-resistant bacteria on the keyboards and the subsequent isolation of MRSE and MRSH on the computer keyboards is worth noting. It is tempting to speculate that MSSA may gain resistance genes when colonizing environments that contain methicillin-resistant coagulase-negative staphylococci. In fact, it has been suggested that the SCCmec elements, which confer methicillin resistance to Staphylococcus species, are derived from coagulase-negative staphylococci (Lindsay & Holden, 2004). The mechanisms for the transfer of SCCmec elements are not well understood, however, and require further study. Notably MSSA and methicillinresistant coagulase-negative staphylococci (S. epidermidis and S. haemolyticus) were isolated from the same keyboard on separate sample dates during the University of Regina sampling, and since S. aureus can survive on keyboards for extended periods of time (Duckworth & Jordens, 1990 and Figure 2) it is possible for co-contamination to occur. Furthermore, the fact that MRSE and MRSH isolates were also resistant to selected macrolides and other antibiotics is notable. A mixed staphylococcal community of antibiotic-resistant genotypes occurring on the keyboards may contribute to development of newly acquired resistances in CA-MRSA isolates. Therefore, further studies on the transfer of antibiotic resistance from MR-CoNS to MSSA and MRSA in the environment is warranted.
Conclusion
In conclusion, computer terminals at the University of Regina and high schools within the Regina area were found to be contaminated with various staphylococci species, including normal flora, methicillin-resistant coagulase-negative staphylococci, and potentially pathogenic MRSA. Although the incidences of MRSA were low, the keyboards still presented a possible reservoir. Survival of Staphylococcus spp. were detected up to 12 days postinoculation of computer keyboards. Children have been noted as a population at risk for infection by CA-MRSA (Adcock, Pastor, Medley, Patterson, & Murphy, 1998), suggesting that further sampling of computer labs in elementary schools and promoting awareness to personal hygiene following use of multiuse computer keyboards across all educational institutes may have merit in helping to control the spread of CA-MRSA.
Reducing the risk of transmission from keyboards may benefit from the routine disinfection of keyboards, particularly on high-traffic computers in university and public libraries. Recent technologies have been developed that have been mainly deployed in hospital settings. For example, the use of keyboard designs that facilitate effective disinfection with chemical disinfectants have been considered for hospital settings (Rutala, White, Gergen, & Weber, 2006; Wilson, Ostro, Magnussen, & Cooper, 2008). Using ultraviolet light to sanitize keyboards has also been tested for eliminating bacterial contamination of keyboards in hospital settings, although the efficiency of disinfection remains unclear (Martin, Qin, Braden, Migita, & Zerr, 2011; Sweeney & Dancer, 2009). In general, increasing public awareness about the risk of using public facilities and providing antimicrobial hand sanitizing stations in areas with open-access keyboards may help lessen the risk of transmittance and potential for infections.
Acknowledgements: We thank the teachers and students at HS #1 and HS #2 for help with computer keyboard sampling. We thank staff at the Saskatchewan Centre for Disease Control for technical assistance and providing access to resources. TOR is funded through a scholarship provided by the Botswana Ministry of Education and Skills Development (MoESD). The lab of CKY is supported through an NSERC Discovery Grant and the Canada Research Chairs program and infrastructure from the Canadian Foundation for Innovation.
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Tyler T. Boa
Teddie O. Rahube
Bastien Fremaux, PhD
University of Regina
Paul N. Levett, PhD
Saskatchewan Health
Christopher K. Yost, PhD
University of Regina
Corresponding Author. Christopher Yost, Associate Professor, Biology Department, University of Regina, Regina, Saskatchewan, Canada S4S 0A2. E-mail: [email protected].
Copyright National Environmental Health Association Jan/Feb 2013