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American Journal of Infection Control 42 (2014) 1285-90

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Major article

Methicillin-resistant Staphylococcus aureus in public transportation

vehicles (buses): Another piece to the epidemiologic puzzle
Jonathan K. Lutz PhD, CIH a, Joany van Balen DVM b, John Mac Crawford PhD a,
John R. Wilkins III DrPH c, Jiyoung Lee PhD a, d, Rocio C. Nava-Hoet DVM, MSc b,
Armando E. Hoet DVM, PhD b, c, *
Division of Environmental Health Sciences, College of Public Health, The Ohio State University, Columbus, OH
Department of Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State University, Columbus, OH
Division of Epidemiology, College of Public Health, The Ohio State University, Columbus, OH
Department of Food Science and Technology, The Ohio State University, Columbus, OH

Key Words: Background: Little is known about the occurrence and epidemiology of methicillin-resistant Staphylo-
Methicillin-resistant Staphylococcus aureus coccus aureus (MRSA) in public transportation in the United States. This research sought to determine the
Public transportation background prevalence and phenotypic and genotypic characteristics of MRSA strains circulating on
Surface contamination
buses from a large, metropolitan transportation agency.
Methods: Electrostatic wipes were used to collect 237 surface samples from 40 buses randomly selected
from July-October 2010. Six samples were collected from each bus immediately postservice and before
any cleaning and disinfection. Positive isolates were analyzed for antibiotic resistance, staphylococcal
cassette chromosome mec (SCCmec) type, and pulsed-eld gel electrophoresis; and potential epidemi-
ologic factors were examined.
Results: Of the buses, 68% (27/40) were contaminated with S aureus, and 63% (25/40) were contaminated
with MRSA. Seats and seat rails were the surfaces most frequently contaminated, followed by the back
door and stanchions. Most (62.9%) of the MRSA isolates were classied as community-associated MRSA
clones (SCCmec type IV), and 22.9% were health careeassociated MRSA clones (SCCmec type II). Of the
MRSA strains, 65% (5/20) were multidrug resistant.
Conclusion: MRSA was frequently isolated from commonly touched surfaces in buses serving both
hospital and community routes. Phenotypic and genotypic analysis demonstrated that buses may be
effective mixing vessels for MRSA strains of both community and health careeassociated origin.
Copyright 2014 by the Association for Professionals in Infection Control and Epidemiology, Inc.
Published by Elsevier Inc. All rights reserved.

Public transportation is perhaps one of the most important isolated in 2 studies (both in Portugal), with 26% (22/85) and 36%
services in urban life. This critical infrastructure is widely used, (72/199) of sampled buses testing positive for MRSA contamina-
with approximately 10.5 billion trips per year in the United States.1 tion.3-6 MRSA contamination was also found in Japanese trains,
However, little data exist regarding the prevalence of pathogens on with 2.3% of vehicles found as positive.7
public transportation vehicle surfaces in the United States, and no There are several factors that make public transportation vehicles
studies have isolated methicillin-resistant Staphylococcus aureus an ideal setting for the movement and spread of MRSA. First, there
(MRSA) from public transportation vehicles in this country.2 Pre- are undoubtedly colonized and infected individuals using public
vious work has reported the isolation of MRSA on public trans- transportation because the U.S. colonization rate is 0.8%-1.5%.8,9
portation vehicles, but such research has only been conducted in Second, hand-to-fomite contact is expected in public trans-
Europe and Asia.3-7 In prior reports from Europe, MRSA was portation vehicles. This type of contact has been previously impli-
cated in community-associated (CA) MRSA transmission.10 Riders
routinely touch stanchions, seat rails, doors, and seats, especially
* Address correspondence to Armando E. Hoet, DVM, PhD, A100Q Sisson Hall,
during high-volume usage times (as vehicles become crowded, this
1920 Coffey Rd, Columbus, OH 43221.
E-mail address: (A.E. Hoet). hand-to-fomite contact increases). Finally, there is little to no op-
Conicts of interest: None to report. portunity for hand hygiene during and immediately after

0196-6553/$36.00 - Copyright 2014 by the Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.
1286 J.K. Lutz et al. / American Journal of Infection Control 42 (2014) 1285-90

Table 1
Descriptive statistics and comparisons for the presence of Staphylococcus aureus and MRSA on surfaces sampled in buses from a top urban regional transit authority in a large,
Midwestern U.S. city

S aureus, Negative S aureus, MRSA, Negative MRSA,*

Variable n (%) n (%) OR (95% CI) n (%) n (%) OR (95% CI)
Surface samples 41 (17.3) 196 (82.7) NA 35 (14.8) 202 (85.2) NA
Sampling date c2 and Fisher exact tests (P .134) c2 and Fisher exact tests (P .273)
July 12y 14 (24.6) 43 (75.4) 2.5 (0.9-6.7) 13 (22.8) 44 (77.2) 2.2 (0.8-6.1)
August 23 13 (21.7) 47 (78.3) 2.1 (0.8-5.7) 8 (13.3) 52 (86.7) 1.2 (0.4-3.4)
October 11 7 (11.7) 53 (88.3) 1.0 (0.3-3.1) 7 (11.7) 53 (88.3) 1.0 (0.3-3.1)
October 25 7 (11.7) 53 (88.3) Referent 7 (11.7) 53 (88.3) Referent
Facility c2 and Fisher exact tests (P .864) c2 and Fisher exact tests (P .363)
1 21 (17.9) 96 (82.1) 1.1 (0.6-2.1) 20 (17.1) 97 (82.9) 1.4 (0.7-2.9)
2 20 (16.7) 100 (83.3) 15 (12.5) 105 (87.5)
Sample location c2 and Fisher exact tests (P < .001)z c2 and Fisher exact tests (P < .01)z
Seats 13 (32.5) 27 (67.5) 5.9 (1.5-22.9) 13 (32.5) 27 (67.5) 18.8 (2.3-152.2)
Seat rails 13 (35.1) 24 (64.9) 6.7 (1.7-25.9) 11 (29.7) 26 (70.3) 16.5 (2.0-135.6)
Back door 7 (17.5) 33 (82.5) 2.6 (0.6-10.9) 6 (15.0) 34 (85.0) 6.9 (0.8-60.1)
Stanchions 5 (12.5) 35 (87.5) 1.8 (0.4-7.9) 4 (10.0) 36 (90.0) 4.3 (0.5-40.6)
Operators area 3 (7.5) 37 (92.5) Referent 1 (2.5) 39 (97.5) Referent
HVAC return vent 0 (0.0) 40 (100) NA 0 (0.0) 40 (100.0) NA
Multiple routes c2 and Fisher exact tests (P .605) c2 and Fisher exact tests (P .712)
Multiple routes 26 (18.4) 115 (81.6) 1.2 (0.6-2.5) 22 (15.6) 119 (84.4) 1.2 (0.6-2.5)
Same route for day 15 (15.6) 81 (84.4) 13 (13.5) 83 (86.5)
Hospital or nonhospital route c2 and Fisher exact tests (P 1.00) c2 and Fisher exact tests (P 1.00)
Hospital 27 (17.4) 128 (82.6) 1.0 (0.5-2.1) 23 (14.8) 132 (85.2) 1.0 (0.5-2.2)
Nonhospital 14 (17.1) 68 (82.9) 12 (14.6) 70 (85.4)
Ridership c2 and Fisher exact tests (P .173) c2 and Fisher exact tests (P .258)
200/d 24 (20.0) 96 (80.0) 1.5 (0.7-2.9) 20 (16.7) 100 (83.3) 1.4 (0.7-2.8)
0-199/d 17 (14.5) 100 (85.5) 15 (12.8) 102 (87.2)

CI, condence interval; HVAC, heating, ventilating, and air conditioning; MRSA, methicillin-resistant Staphylococcus aureus; NA, not applicable; OR, odds ratio.
*Includes methicillin-susceptible S aureus and all negative results.
On July 12, 57 samples were collected because 3 buses did not have seat rails; 60 samples were collected for each of the remaining sampling dates.
Statistically signicant c2 and Fisher exact tests at P  .05 level.

transportation usage. Previous research has demonstrated that hand Surfaces sampled and methodology
hygiene is the primary method for the prevention of MRSA
transmission.11,12 Sampling was conducted over 4 sampling days, starting in July
Little is known about the background prevalence or variation of 2010 and ending in October 2010 (Table 1). The sampling dates
MRSA strains circulating in the community, especially on fomite were chosen based on project goals and the transportation agencys
surfaces in public transportation vehicles. In this research, the aim availability; each facility was visited twice. In each sampling date,
was to determine the background prevalence and phenotypic and 10 buses were randomly selected from those available to be
genotypic characteristics of MRSA strains contaminating public sampled at the time of visit. Six predetermined sample locations
transportation vehicle surfaces in a large, metropolitan setting. (stanchions; seats; seat rails; back door; drivers area; heating,
Here, the results of a cross-sectional study conducted on 40 buses ventilating, and air conditioning return vent) were collected from
from a Midwestern United States transportation agency are each bus (total of 240 surfaces). Sample locations inside the buses
described. The results of this work provide critical data on the were chosen as those having potentially high levels of skin-to-
epidemiologic characteristics and clonal distribution of MRSA surface contact.
strains contaminating a public environment in this important Sampling was conducted using commercially available elec-
community setting. trostatic wipes (Swiffer, Procter & Gamble, Cincinnati, OH), as
previously described.13 A wipe was unfolded and placed over the
METHODS surface to be sampled in a manner which maximized surface area
contact. Once the sample was collected, the wipe was folded and
Transportation agency placed into a sterile, labeled stomacher bag (Nasco, Fort Atkinson,
WI). To enhance sampling efciency, some samples were
The studied transportation agency is a top 50 (U.S.) urban- collected as a pool. These pooled (composite) samples were
anchored regional transit authority from a large, metropolitan collected from the stanchions, seats, seat rails, and vehicle op-
area. The research team sampled 17% (40/239) of the bus eet (based erators area (which included the steering wheel, arm rests,
on a daily average for each facility), including the selection of an knobs, and headrest). For pooled samples, the same electrostatic
equal number of buses from each depot facility (facilities 1 and 2). cloth was used to wipe all the units of the same surface type (eg,
Specic routes were targeted for sampling using the transportation 9-10 seat rails were sampled with the same cloth), and an
agencys system map. Such routes were chosen based on the attempt was made to sample the same number of seats, seat rails,
following criteria: (1) those that served major hospitals and and similar linear footage of stanchions per bus. Sampling was
nonhospital-related routes (to evaluate the possible variation in always conducted by the same researcher, focusing only on the
hospital-associated [HA] vs CA strains), (2) routes with high rider- surfaces on one side (drivers side) of the bus (to control for any
ship and low ridership (to determine potential effect of crowding potential differences between the 2 sides, which were of different
and human density), and (3) those that served 1 route or multiple conguration). All sampling was conducted while vehicles were
routes (to assess possible differences in the number of strains immediately postservice and before any cleaning and
isolated). disinfection.
J.K. Lutz et al. / American Journal of Infection Control 42 (2014) 1285-90 1287

Strict quality control standards were assured throughout the Conrmation of mecA, staphylococcal cassette chromosome mec
sampling process (such as, but not limited to, technician screening typing, and pulsed-eld gel electrophoresis
for MRSA colonization, use of N-95 masks, and glove changes and
application of alcohol-based hand sanitizer between each sample All isolates phenotypically determined to be MRSA were further
collected). In addition, for each collection date, 2 negative control tested to conrm the presence of the mecA gene. A multiplex
wipes (eld blanks) and a positive control (MRSA ATCC 43300) were polymerase chain reaction technique was used (as described by
held for processing. All collected samples were transported and Oliveira and de Lencastre17 and Milheirio et al,18 with modica-
processed at the Diagnostic and Research Laboratory for Infectious tions described by Van Balen et al14) to conrm and type the
Diseases at The Ohio State University College of Veterinary Medicine. staphylococcal cassette chromosome mec (SCCmec) gene. Results
are reported as types I-VI.
Isolation, identication, and phenotyping Pulsed-eld gel electrophoresis (PFGE) was conducted to further
subtype isolates, which permitted the determination of potential
All samples were transported to the laboratory for processing epidemiologic origin (HA or CA) and clonal relatedness. S aureus
within 1 hour after each sampling event. The details of processing genomic DNA was digested using a SmaI restriction enzyme, ac-
are described elsewhere.13 Briey, all samples were placed in a cording to standardized methods (Centers for Disease Control and
preenrichment media (Trypticase Soy Broth, BD Diagnostic Systems, Prevention-PulseNet). Salmonella serotype Branderup strain H9812
Sparks, MD) at 35 C for 24 hours. After pre-enrichment samples was used as a molecular size marker and digested with XbaI. Elec-
were transferred to BD Mannitol Salt Agar with Oxacillin (2 mG/mL) trophoresis was conducted using a CHEF Mapper XA system (Bio-Rad
plates (BD Diagnostic Systems, Sparks, MD) for 24-48 hours incu- Laboratories, Nazareth, Belgium). All band patterns were analyzed for
bation. Then, 1-3 morphologically unique mannitol-positive colonies clonal relatedness both visually and with BioNumerics version 6.6
were selected per sample for purication, isolation, and further software (Applied Maths, Ghent, Belgium). Clonal relatedness was
analysis. S aureus was identied through morphology, color, size, assigned for all bands using the Dice coefcient and unweighted pair
hemolysis, mannitol fermentation, and the following biochemical group method, using arithmetic averages to achieve dendrograms
tests: catalase reaction (Catalase, BD BBL; Becton, Dickinson and with a 1% band position tolerance.19 Dendrogram analysis, including
Company, Franklin Lakes, NJ); tube coagulase testing (Coagulase cluster and pulsotype interpretation, was performed, as described
plasma rabbit with EDTA, BD BBL Becton, Dickinson and Company); elsewhere,14 with cutoff points of >80% and >98% similarity,
latex agglutination testing (Sure-Vue Color Staph ID, Biokit USA Inc, respectively. Designation of USA types was performed by comparing
Lexington, MA); aniline reaction (ORSAB, Oxoid LTD, Basingstonke, our study isolates to a database obtained from the U.S. Centers for
Hampshire, England); Voges-Proskauer test (Vogues-Proskauer re- Disease Control and Prevention that contains 100 S aureus strains
agents A and B, BD BBL Becton, Dickinson and Company); and (representing the most common band patterns from each USA type in
polymyxin B susceptibility (BD Diagnostic Systems). Coagulase- the United States), using 80% similarity as the cutoff point.
negative isolates were not further processed in this study. All
Statistical analysis of data
S aureus isolates underwent susceptibility testing to determine
methicillin phenotype per the Clinical and Laboratory Standards
Statistical analysis was conducted using PASW Statistics 18.0
Institute procedure and as described by Van Balen et al.14,15 Fresh,
(SPSS Inc, Chicago, IL). Data were analyzed using descriptive
24-hour colonies were inoculated onto oxacillin resistance agar
statistics and c2 and Fisher exact tests (2-sided c2 and Fisher
plates containing 6 mG/mL oxacillin (Oxacillin Screen Agar [OSA], BD
exact analysis was used for all variables except ridership, for which
Diagnostic Systems). All media plates were incubated at 35 C for
a 1-sided analysis was conducted), and simple logistic regression
24 hours. Positive controls were used for all reactions (MRSA: ATCC
was used to determine odds ratios. Total S aureus (MSSA, MRSA)
43300; methicillin-susceptible S aureus [MSSA]: ATCC 29213;
and MRSA prevalence on public transportation vehicles were
methicillin-resistant S pseudintermedius: in-house isolate). Isolates
examined on a per vehicle level and overall (per sample level). For
were classied as MRSA when growth was conrmed on the OSA
all statistical analyses, P  .05 was considered statistically signi-
plates. If no growth was observed on the OSA plates, the isolate was
cant (P  .10 was considered notable). For assessment of ridership,
considered MSSA.
continuous ridership data were obtained and dichotomized into
Resistance testing was conducted using the Kirby-Bauer disk
2 levels (based on a 50% data cut-point) (Table 1): low ridership
diffusion technique (disk concentrations listed accordingly),
(0-199 riders/d) and high ridership (200 riders/d).
following the Clinical and Laboratory Standards Institute standard
methods.15 All MSSA and MRSA isolates underwent phenotyping RESULTS
analysis for antibiotic resistance to 16 antibiotic agents from 9
antibiotic classes: aminoglycosides (gentamicin 1 mG, amikacin A total of 237 surface samples were collected from 40 buses (39
30 mG); b-lactams (ampicillin 10 mG, amoxicillin-clavulanate 30 mG, unique buses with 1 bus randomly repeated), which were equally
oxacillin 1 mG, cephalothin 30 mG, cefpodoxime 10 mG); glycopep- divided between the 2 facilities managed by the transportation
tide (vancomycin); lincosamide (clindamycin 2 mG); tetracyclines agency. All preselected surfaces were collected from each bus
(tetracycline 30 mG, doxycycline 30 mG); macrolide (erythromycin included in the study, with the exception of 3 buses that did not have
15 mG); quinolones (ciprooxacin 2 mG, enrooxacin 5 mG); phe- seat rails. Approximately 16% of the total active eet was sampled
nicol (chloramphenicol 30 mG); and potentiated sulfonamide (sul- over the course of the study, covering 35 of the transportation
famethoxazole-trimethoprim 25 mG). To test for vancomycin systems 68 routes (51%).
resistance, vancomycin screening agar plates (BD BBL Vancomycin At the bus level, S aureus was isolated in 68% (27/40) of buses,
Screen Agar, BD Diagnostic Systems, Sparks, MD) were inoculated and 63% (25/40) of all buses had at least 1 MRSA-positive surface
with 10 mL of the bacterial solution (0.5 McFarland standard). The D sample. At the surface level, S aureus was found in 17% (41/237) of
test was used to detect inducible clindamycin resistance.16 Control all surface samples, and MRSA was found in 15% (35/237) of all
strains were used throughout the phenotyping process, including surface samples. The S aureus and MRSA results (at bus and surface
Enterococcus faecalis (ATCC 29212, ATCC 51299), S aureus (ATCC levels) are summarized in Table 1 and Table 2. The small difference
29253, ATCC 43300, ATCC 29213), Pseudomonas aeruginosa (ATCC observed between S aureus and MRSA contamination rates is highly
27853), and Escherichia coli (ATCC 25922). likely because during the early stages of the isolation process,
1288 J.K. Lutz et al. / American Journal of Infection Control 42 (2014) 1285-90

Table 2
Proportions and ORs of Staphylococcus aureusepositive and MRSA-positive buses from a top urban regional transit authority in a large, Midwestern U.S. city

S aureus, Negative S aureus, MRSA, Negative MRSA,*

Variable n (%) n (%) OR (95% CI) n (%) n (%) OR (95% CI)
Buses 27 (67.5) 13 (32.5) NA 25 (62.5) 15 (37.5) NA
Sampling date c2 and Fisher exact tests (P .134) c2 and Fisher exact tests (P .233)
July 12 9 (90) 1 (10) 9.0 (0.8-100.1) 9 (90) 1 (10) 9.0 (0.8-100.1)
August 23 8 (80) 2 (20) 4.0 (0.6-29.0) 6 (60) 4 (40) 1.5 (0.3-8.8)
October 11 5 (50) 5 (50) 1.0 (0.8-5.8) 5 (50) 5 (50) 1.0 (0.2-5.8)
October 25 5 (50) 5 (50) Referent 5 (50) 5 (50) Referent
Facility c2 and Fisher exact tests (P 1.00) c2 and Fisher exact tests (P .514)
1 14 (70) 6 (30) 1.3 (0.3-4.7) 14 (70) 6 (30) 1.9 (0.5-7.0)
2 13 (65) 7 (35) 11 (55) 9 (45)
Multiple routes c2 and Fisher exact tests (P .733) c2 and Fisher exact tests (P .527)
Multiple routes 17 (70.8) 7 (29.2) 1.5 (0.4-5.6) 16 (66.7) 8 (33.3) 1.6 (0.4-5.7)
Same route for day 10 (62.5) 6 (37.5) 9 (56.3) 7 (43.8)
Hospital or nonhospital route c2 and Fisher exact tests (P .316) c2 and Fisher exact tests (P .502)
Hospital 16 (61.5) 10 (38.5) 0.4 (0.1-2.0) 15 (57.7) 11 (42.3) 05 (0.1-2.2)
Nonhospital 11 (78.6) 3 (21.4) 10 (71.4) 4 (28.6)
Ridership c2 and Fisher exact tests (P .088) c2 and Fisher exact tests (P .257)
200/d 16 (80) 4 (20) 3.3 (0.8-13.3) 14 (70) 6 (30) 1.9 (0.5-7.0)
0-199/d 11 (55) 9 (45) 11 (55) 9 (45)

CI, condence interval; HVAC, heating, ventilating, and air conditioning; MRSA, methicillin-resistant Staphylococcus aureus; NA, not applicable; OR, odds ratio.
*Includes methicillin-susceptible S aureus and all negative results.

a small amount of oxacillin was used. Therefore, a portion of sus- the strains were resistant to erythromycin, 50.0% (10/20) were
ceptible S aureus colonies was probably lost during the isolation resistant to ciprooxacin, and 30.0% (6/20) were resistant to enro-
process. Nonetheless, all results obtained (S aureus, MRSA) are oxacin. Moreover, clindamycin resistance was observed in 45.0%
mentioned and discussed in this article. (9/20) of the strains, where 33.3% (3/9) were considered inducible.
The overall number of positive S aureus and MRSA samples and Of the strains, 65% (13/20) were multidrug resistant (resistant to 3
buses decreased as the study progressed, with the highest contami- classes, as dened in Magiorakos et al).20 Of the strains, 25% (5/20)
nation being found in July (S aureus: 14/57; MRSA: 13/57) and the were resistant to 4 classes, and 1 strain was resistant to 5 antibiotic
lowest in October (S aureus: 7/60; MRSA: 7/60). Also, the degree to classes. All strains were susceptible to amikacin, gentamicin,
which S aureus and MRSA could be isolated varied considerably be- doxycycline, sulfamethoxazole-trimethoprim, and vancomycin.
tween surface sample locations on a bus, with the highest levels of
contamination found on the seat rails (S aureus: 13/40; MRSA: 11/40), DISCUSSION
followed by the seats (S aureus: 13/40; MRSA: 13/40), back door (S
aureus: 7/40; MRSA: 6/40), stanchions (S aureus: 5/40; MRSA: 4/40), Little is known about the overall occurrence and characteristics of
and operators area (S aureus: 3/40; MRSA: 1/40). No S aureus or MRSA MRSA on public transportation vehicle surfaces in the United Sates. In
was found on the heating, ventilating, and air conditioning return this work, the main objective was to determine if buses could harbor
vent (0/40). At the sample and bus levels, multiple variables were MRSA contamination. Further, if MRSA was isolated, the research
considered in the analysis, such as the facility (1, 2), routes covered sought to elucidate the phenotypic and genotypic characteristics of
(single, multiple), if hospitals were served (hospital, nonhospital), this pathogen circulating in the vehicles. S aureus and MRSA strains
and ridership. None of these factors demonstrated a signicant were isolated from 68% and 63% of the buses sampled, respectively.
difference in their association with S aureus or MRSA surface The rate of susceptible S aureus contamination reported in this article
contamination (Table 1 and Table 2). However, high ridership buses could be underestimated because of the isolation process used.
demonstrated a notable difference in S aureus isolation compared Nevertheless, these ndings indicate that public transportation ve-
with low ridership buses (16/20 vs 11/20, respectively). hicles could be a potential source of both S aureus and MRSA to the
people using this service. Using these data, infection control pro-
Antibiotic resistance proles, SCCmec typing, and PFGE fessionals and epidemiologists can better understand the dynamics
and epidemiology of MRSA in the community.
From the 35 MRSA-positive surfaces sampled, a total of 76 MRSA To our knowledge, this is the rst study to report MRSA on public
isolates were obtained and phenotypically characterized. Based on transportation vehicles in the United States. MRSA has been previ-
their morphologic characteristics and antimicrobial susceptibility ously isolated in public transportation systems in Europe and Asia, and
patterns, only 1 unique isolate from each positive surface sampled additional work has resulted in the isolation of susceptible S aureus,
was selected to be genotypically characterized (35 isolates). Detailed but not MRSA.2-5,7 However, considerably higher MRSA prevalence
information of these 35 isolates (locations, surface description, was noted in the current study than in previous work (MRSA-positive
phenotypic prole, SCCmec type, USA type) can be found in Figure 1. vehicles: 63% vs 2.3%-36%).5-7 Low sample number, limited sampling
Most of the isolates (62.9%, 22/35) were classied as SCCmec type IV location, less sensitive sampling technique, inhibitory processing,
and either USA300 or USA400, characteristics present in MRSA clones cleaning practices applied to the buses, and other factors may have
known as CA MRSA. Moreover, 22.9% (8/35) of the isolates were contributed to considerable variation between the studies.3-5,7,21,22
SCCmec type II and USA100, characteristics associated with HA MRSA Further, different regional MRSA colonization rates in the commu-
clones. The results of the dendrogram analysis indicated 15 unique nities using these public transportation services may have also greatly
pulsotypes, with 3 clusters identied. Over 45% (16/35) of the isolates affected the degree to which MRSA could be isolated on buses.23,24
were grouped in cluster 2, and 26% (9/35) were in cluster 1 (Fig 1). Finding MRSA in a nonclinical, community setting (eg, public
Based on the unique combination of their antimicrobial sus- transportation vehicles) has several important implications. First,
ceptibility pattern (SCCmec type and PFGE pulsotype) 20 MRSA these ndings increase understanding about the degree to which
strains were identied. Besides b-lactam resistance, 80.0% (16/20) of MRSA can be found outside of the hospital. Given that 0.8%-1.5% of
J.K. Lutz et al. / American Journal of Infection Control 42 (2014) 1285-90 1289

Fig 1. Clonal dendrogram of methicillin-resistant Staphylococcus aureus isolates from surfaces on buses from a top 50 urban-anchored regional transit authority in a large Mid-
western city. The percentage similarity was calculated with Dice coefcients from the pulsed-eld gel electrophoresis data. Band position tolerance and optimization were set at 1%.
Amc, amoxicillin with clavulanic acid; Amp, ampicillin; Cep, cephalothin; Chl, chloramphenicol; Cip, ciprooxacin; Cli, clindamycin; Cpd, cefpodoxime; Eno, enrooxacin; Ery,
erythromycin; Ind, inducible; Oxa, oxacillin; SCC, staphylococcal cassette chromosome; Tet, tetracycline.

people (in the United States) are colonized with MRSA, it is no either touch the back door surface (at palmed) or grasp 2 vertical
surprise that MRSA has been isolated on a variety of surfaces in the tubes and push on the door. Therefore, throughout a route, there are
community.8,9 However, many community studies that resulted in countless occasions where riders hands contact the back door sur-
MRSA isolation have been conducted in settings that are health care face. In prior research of community surfaces, S aureus contamination
related (eg, re stations and emergency medical services, prison has been found on vehicle rails, stair rails, door and faucet handles,
clinics), after an outbreak (sauna), or limited to household trans- remote controls, and key padsdall surfaces with similar hand-to-
mission.25-27 Not only is the current study one of a few to nd MRSA fomite dynamics as both the bus doors and seat rails.3,4,26,27,30
in public areas of the community, but public transportation also The sampling date was also associated with the degree to which
represents a potentially important setting for transmission. In S aureus and MRSA could be isolated, with 90% and 80% S
addition, despite the fact that previous studies have isolated MRSA aureusepositive buses from the rst 2 dates (90% and 60% MRSA
from the community environment, the bacterium does not appear positive, respectively), but only 50% positive buses from the second
to be ubiquitous. Several published studies have failed to isolate 2 dates (50% both for MRSA). Little is known about any seasonal
MRSA in the community, and one may suspect that many more trends in community MRSA prevalence, but some past research has
unpublished studies have also failed to isolate MRSA.3,4,28,29 noted increases in community MRSA infections during summer
There were multiple factors that were associated with isolating months.31 This may be a reection of increased skin-to-skin and
S aureus and MRSA from a vehicle. Vehicle sample location was an skin-to-fomite contact because of increased temperature and more
important indicator, with seats being the most frequently MRSA- exposed skin (wearing shorts and short sleeves).
positive surface (33%). These results are consistent with prior There remains a paucity of data regarding the underlying
work regarding seat contamination. In a study of re station sur- phenotypic and molecular characteristics of MRSA circulating in the
faces, couches were found to harbor the highest concentration of community. Most published work has either not provided molecular
MRSA.27 Further, Felkner et al30 found MRSA contamination on a data or has come as the direct result of a specic outbreak.3,25-29 Given
prison van seat and clinic chair, and Baggett et al25 isolated MRSA that outbreak strains may have increased tness or occur in scenarios
from a sauna bench. Seat rails, which have a roughened surface that where enhanced transmission may be occurring, they may fail to
may enhance removal of bacteria from hands, were also frequently provide a true reection of the underlying dynamics of MRSA in the
MRSA positive (30%). community.32 Therefore, the current research contributes insight into
Back doors also demonstrated high levels of MRSA contamination which MRSA strains are circulating in a community, which is essential
(15%). These surfaces are a model of hand-to-fomite and fomite-to- to our understanding of pathogen spread and prevention efforts.
hand transmission (although actual transmission has not been Dendrogram analysis showed low diversity among the MRSA
demonstrated in this study). To operate the back door, the rider must isolates, with most isolates (54%, 19/35) classied as SCCmec type IV
1290 J.K. Lutz et al. / American Journal of Infection Control 42 (2014) 1285-90

USA300. These characteristics correspond to the most common CA HICPAC/SHEA/APIC/IDSA Hand Hygiene Task Force. Infect Control Hosp Epi-
demiol 2002;23(Suppl):S3-40.
MRSA strain found in the United States, demonstrating a potential
12. Maltezou HC, Giamarellou H. Community-acquired methicillin-resistant
community-acquired origin.33-36 In addition, 23% (8/35) of the isolates Staphylococcus aureus infections. Int J Antimicrob Agents 2006;27:87-96.
exhibit characteristics present in HA MRSA clones, which may indicate 13. Hoet AE, Johnson A, Nava-Hoet RC, Bateman S, Hillier A, Dyce J, et al. Environmental
a spillover of MRSA strains from health care facilities into the com- methicillin-resistant Staphylococcus aureus in a veterinary teaching hospital
during a nonoutbreak period. Vector Borne Zoonotic Dis 2011;11:609-15.
munity; these results are consistent with previous work.5,7 The most 14. Van Balen J, Kelley C, Nava-Hoet RC, Bateman S, Hillier A, Dyce J, et al. Presence,
predominant strain in U.S. communities (SCCmec type IV USA300) distribution, and molecular epidemiology of methicillin-resistant Staphylococcus
was found in different buses across 3 different sampling dates.34,37 aureus in a small animal teaching hospital: a year-long active surveillance tar-
geting dogs and their environment. Vector Borne Zoonotic Dis 2013;13:299-311.
The results of this environmental study demonstrate that MRSA 15. Clinical and Laboratory Standards Institute. Performance standards for antimi-
contamination is consistently present on multiple surfaces in public crobial susceptibility testing; nineteenth informational supplement. CLSI docu-
transportation vehicles. Some surfaces (ie, seats, seat rails) were ment M100eS19. Wayne (PA): Clinical and Laboratory Standards Institute; 2009.
16. Fiebelkorn KR, Crawford SA, McElmeel ML, Jorgensen JH. Practical disk diffu-
more frequently contaminated with this pathogen; therefore, they sion method for detection of inducible clindamycin resistance in Staphylo-
must be actively targeted for routine cleaning and disinfection. It is coccus aureus and coagulase-negative staphylococci. J Clin Microbiol 2003;41:
also important to highlight that both CA and HA strains were iso- 4740-4.
17. Oliveira DC, de Lencastre H. Multiplex PCR strategy for rapid identication of
lated from these vehicles, in some cases from the same unit structural types and variants of the mec element in methicillin-resistant
(vehicle). This was true regardless if they served hospital routes or Staphylococcus aureus. Antimicrob Agents Chemother 2002;46:2155-61.
not. This nding underscores the conclusion that buses may act as 18. Milheirio C, Oliveira DC, de Lencastre H. Update to the multiplex PCR strategy
for assignment of mec element types in Staphylococcus aureus. Antimicrob
potential mixing vessels for MRSA strains in the community.
Agents Chemother 2007;51:3374-7.
19. Murchan S, Kaufmann ME, Deplano A, de Ryck R, Struelens M, Zinn CE, et al.
Acknowledgments Harmonization of pulsed-eld gel electrophoresis protocols for epidemiolog-
ical typing of strains of methicillin-resistant Staphylococcus aureus: a single
approach developed by consensus in 10 European laboratories and its appli-
The authors wish to thank Shasha Bai, Jade Braman, Melissa cation for tracing the Spre. J Clin Microbiol 2003;41:1574-85.
Mikolaj, Wannasawat Ratphitagsanti, PhD; Chongtao Ge, Sophia 20. Magiorakos A, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al.
Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria:
Dailey, Colleen Shockling-Dent, and Debbie Lutz for their laboratory an international expert proposal for interim standard denitions for acquired
and eld assistance during this project. Also, Duncan MacCannell, resistance. Clin Microbiol Infect 2012;18:268-81.
from the Center for Disease Control and Prevention (CDC), for 21. Kassem II. Concerning public transport as a reservoir of methicillin-resistant
staphylococci. Lett Appl Microbiol 2009;48:268.
facilitating the database containing S. aureus strains with the most 22. Lutz JK, Crawford J, Hoet AE, Wilkins JR, Lee J. Comparative performance of
typical band patterns for each USA type for PFGE characterization. contact plates, electrostatic wipes, swabs and a novel sampling device for the
We also want to thank Herminia de Lencastre, PhD from the Uni- detection of Staphylococcus aureus on environmental surfaces. J Appl Micro-
biol 2013;115:171-8.
versidade Nova de Lisboa in Portugal, for providing MRSA controls
23. Marschall J, Drig P, Mhlemann K. Low prevalence of methicillin-resistant
isolates for the standardization of the SCCmec type multiplex PCR. Staphylococcus aureus (MRSA) carriage in women from former Yugoslavia
Finally, we will like to thank the Network on Antimicrobial Resis- living in Switzerland. Eur J Clin Microbiol Infect Dis 2006;25:535-6.
tance in Staphylococcus aureus (NARSA) program for providing 24. Wall EC, Ghazy A, Bulley S, Ahmad F, Aali A, Lynn W. Medical staff at a London
district hospital are not chronic vectors of MRSA or Clostridium difcile. J Infect
several control strains. NARSA is supported under NIAID, NIH 2009;58:469-71.
contract number HHSN272200700055C. 25. Baggett HC, Hennessy TW, Rudolph K, Bruden D, Reasonover A, Parkinson A,
et al. Community-onset methicillin-resistant Staphylococcus aureus associated
with antibiotic use and the cytotoxin Panton-Valentine leukocidin during a
References furunculosis outbreak in rural Alaska. J Infect Dis 2004;189:1565-73.
26. Scott E, Duty S, McCue K. A critical evaluation of methicillin-resistant Staph-
1. American Public Transportation Association. Public transportation fact book. ylococcus aureus and other bacteria of medical interest on commonly touched
Available from: household surfaces in relation to household demographics. Am J Infect Control
aspx; 2012. Accessed September 26, 2014. 2009;37:447-53.
2. Yeh PJ, Simon DM, Millar JA, Alexander HF, Franklin D. A diversity of antibiotic- 27. Sexton JD, Reynolds KA. Exposure of emergency medical responders to methicillin-
resistant Staphylococcus spp. in a public transportation system. Osong Public resistant Staphylococcus aureus. Am J Infect Control 2010;38:368-73.
Health Res Perspect 2011;2:202-9. 28. Begier EM, Frenette K, Barrett NL, Mshar P, Petit S, Boxrud DJ, et al. A high-
3. Otter JA, French GL. Bacterial contamination on touch surfaces in the public morbidity outbreak of methicillin-resistant Staphylococcus aureus among
transport system and in public areas of a hospital in London. Lett Appl players on a college football team, facilitated by cosmetic body shaving and turf
Microbiol 2009;49:803-5. burns. Clin Infect Dis 2004;39:1446-53.
4. Stepanovi c S, Cirkovi
c I, Djuki
c S, Vukovi
c D, Svabi
c-Vlahovi c M. Public trans- 29. Kazakova SV, Hageman JC, Matava M, Srinivasan A, Phelan L, Garnkel B, et al.
port as a reservoir of methicillin-resistant staphylococci. Lett Appl Microbiol A clone of methicillin-resistant Staphylococcus aureus among professional
2008;47:339-41. football players. N Engl J Med 2005;352:468-75.
5. Simes RR, Aires-de-Sousa M, Conceio T, Antunes F, da Costa PM, de 30. Felkner M, Andrews K, Field LH, Taylor JP, Baldwin T, Valle-Rivera AM, et al.
Lencastre H. High prevalence of EMRSA-15 in Portuguese public buses: a Detection of Staphylococcus aureus including MRSA on environmental surfaces
worrisome nding. PLoS One 2011;6:e17630. in a jail setting. J Correct Health Care 2009;15:310-7.
6. Conceio T, Diamantino F, Coelho C, de Lencastre H, Aires-de-Sousa M. Contami- 31. Kaplan SL, Hulten KG, Gonzalez BE, Hammerman WA, Lamberth L, Versalovic J,
nation of public buses with MRSA in Lisbon, Portugal: a possible transmission route et al. Three-year surveillance of community-acquired Staphylococcus aureus
of major MRSA clones within the community. PLoS One 2013;8:e77812. infections in children. Clin Infect Dis 2005;40:1785-91.
7. Iwao Y, Yabe S, Takano T, Higuchi W, Nishiyama A, Yamamoto T. Isolation and 32. Wagenvoort JH, Sluijsmans W, Penders RJ. Better environmental survival of
molecular characterization of methicillin-resistant Staphylococcus aureus from outbreak vs. sporadic MRSA isolates. J Hosp Infect 2000;45:231-4.
public transport. Microbiol Immunol 2012;56:76-82. 33. Kennedy AD, Otto M, Braughton KR, Whitney AR, Chen L, Mathema B, et al. Epidemic
8. Kuehnert MJ, Kruszon-Moran D, Hill HA, McQuillan G, McAllister SK, community-associated methicillin-resistant Staphylococcus aureus: recent clonal
Fosheim G, et al. Prevalence of Staphylococcus aureus nasal colonization in the expansion and diversication. Proc Natl Acad Sci U S A 2008;105:1327-32.
United States, 2001-2002. J Infect Dis 2006;193:172-9. 34. Deleo FR, Otto M, Kreiswirth BN, Chambers HF. Community-associated
9. Gorwitz RJ, Kruszon-Moran D, McAllister SK, McQuillan G, McDougal LK, methicillin-resistant Staphylococcus aureus. Lancet 2010;375:1557-68.
Fosheim GE, et al. Changes in the prevalence of nasal colonization with Staph- 35. Tenover FC, Tickler IA, Goering RV, Kreiswirth BN, Mediavilla JR, Persing DH.
ylococcus aureus in the United States, 2001-2004. J Infect Dis 2008;197:1226-34. Characterization of nasal and blood culture isolates of methicillin-resistant
10. Miller LG, Diep BA. Clinical practice: colonization, fomites, and virulence: Staphylococcus aureus from patients in United States hospitals. Antimicrob
rethinking the pathogenesis of community-associated methicillin-resistant Agents Chemother 2012;56:1324-30.
Staphylococcus aureus infection. Clin Infect Dis 2008;46:752-60. 36. Talan DA, Krishnadasan A, Gorwitz RJ, Fosheim GE, Limbago B, Albrecht V, et al.
11. Boyce JM, Pittet D, Healthcare Infection Control Practices Advisory Committee. Comparison of Staphylococcus aureus from skin and soft-tissue infections in US
Society for Healthcare Epidemiology of America. Association for Professionals emergency department patients, 2004 and 2008. Clin Infect Dis 2011;53:144-9.
in Infection Control. Infectious Diseases Society of America. Hand Hygiene Task 37. Mediavilla JR, Chen L, Mathema B, Kreiswirth BN. Global epidemiology of
Force. Guideline for Hand Hygiene in Health-Care Settings: recommendations community-associated methicillin resistant Staphylococcus aureus (CA-MRSA).
of the Healthcare Infection Control Practices Advisory Committee and the Curr Opin Microbiol 2012;15:588-95.