Screening and Characterization of Staphylococcus aureusfrom Ophthalmology Clinic Surfaces: A Proposed Surveillance Tool


To screen environmental surfaces of an outpatient ophthalmic clinic for methicillin-susceptible and methicillin-resistant Staphylococcus aureus (MSSA and MRSA); to identify the most commonly contaminated surfaces and to phenotype and genotype all collected isolates.


A single institution, 1-year prospective environmental study.


Commonly touched surfaces in examination rooms and common areas were targeted and sampled on a quarterly basis for 1 year. Samples were collected using electrostatic cloths and swabs. S. aureus was isolated using nonselective and selective media. Morphologic characteristics and standard biologic testing were used to confirm staphylococcal species. S. aureus isolates were phenotypically (Kirby-Bauer method) and genotypically characterized (mecA confirmation, SCCmec typing and pulsed-field gel electrophoresis). Dendrogram analysis was used to establish genetic relatedness between the isolates.


Of 112 total samples, 27 (24%) and 5 (4%) were MSSA- and MRSA-positive, respectively. Both community-associated (SCCmec IV, USA300) and hospital-associated (SCCmec II, USA100) MRSA isolates were found. No single surface remained consistently positive with the same isolate over time, and molecular analysis demonstrated high levels of diversity among isolates. Doorknobs, slit-lamp headrests and chinrests, and computer keyboards were commonly found to be contaminated.


The proposed surveillance protocol successfully allowed the detection of both MSSA and MRSA contaminating important high-touch surfaces in a representative ophthalmology clinic. Frequently contaminated surfaces must be targeted for routine cleaning and disinfection because there is a constant introduction of clones over time. Hence, other clinics may consider implementing and adapting surveillance tools, like the one described here, to help them control these important nosocomial pathogens.

Methicillin-susceptible and methicillin-resistant Staphylococcus aureus (MSSA and MRSA) are pathogens of growing concern to clinicians. In the past 2 decades, an increase in MRSA colonization has been observed in the United States. This is especially true of community-associated MRSA, which is increasing in the general population and is known to cause severe skin and pulmonary infections in individuals without traditional risk factors.

In ophthalmology, manifestations of MSSA and MRSA infection include preseptal cellulitis, dacryocystitis, blepharoconjunctivitis, orbital cellulitis, blebitis, keratitis/corneal ulcer, and endophthalmitis. Among these infections, community-associated MRSA has been reported as 1 of the emerging causes involved. Thus, as the prevalence of community-associated MRSA increases in the general population, infection-control measures, which are critical to the inpatient environment, could be adapted for outpatient settings to evaluate for S. aureus surface contamination and to evaluate and improve the effectiveness of clinic cleaning and disinfection practices.

Studies in hospital settings have shown that commonly-touched surfaces, such as computer keyboards and blood-pressure cuffs, among others, could be potential sources of S. aureus colonization and infection in patients. MSSA and MRSA can survive for days to months on inanimate surfaces. In light of this, the widespread presence of hospital-associated MRSA and the increasing prevalence of community-associated MRSA, one must question whether surfaces in the outpatient setting also pose transmission risks for patients and employees.

To identify effectively the surfaces most commonly contaminated with pathogens like MSSA and MRSA and allow for molecular typing, an economic, standardized sampling protocol has been developed. This tool is valuable to evaluate the effectiveness of cleaning and disinfection practices and could contribute to a surveillance program with the goal of minimizing potential patient and personnel exposure to MSSA and MRSA strains as well as other nosocomial pathogens.

The purpose of this study was to use a standardized sampling protocol to screen ophthalmic clinic equipment and commonly touched surfaces for MSSA and MRSA; to identify the most commonly contaminated clinic surfaces; and to further phenotype and genotype such isolates. This study could provide valuable information for the development of surveillance programs that evaluate and improve future cleaning and disinfection protocols for outpatient clinical settings.


Experimental Design

This study was carried out in a large academic ophthalmology setting. A standardized method (described below) was used to sample 12 randomly selected examination rooms and shared imaging rooms in 2 ophthalmology clinic buildings belonging to the same system (1 freestanding outpatient clinic, clinic 1; and 1 hospital-adjoining clinic, clinic 2). To be able to cover as many locations as possible from the clinic, the same surface in 3 different examination rooms was sampled as a pool. For example, all tonometer tips from each set of 3 rooms were pooled into 1 sample. Each group of rooms was denoted group A, group B, etc. ( Table 1 ). Items sampled in each examination room included patient-contact surfaces, such as slit-lamp, headrests and chinrests and tonometer tips; employee-contact surfaces such as computer keyboards and hand-sanitizer dispensers; and general contact surfaces such as doorknobs. Excluding shared imaging areas, 12 examination rooms of 43 (28%) were sampled ( Table 1 ).

Table 1

Staphylococcus surveillance results: detection of methicillin-susceptible and methicillin-resistant Staphylococcus aureus on ophthalmology clinic surfaces over time

Location Source (Number in Pool) a Collection Method Type of Contact Result 11/2010 Result 02/2011 Result 05/2011 Result 08/2011
Clinic 1
Group A Tonometer tips (3) Swab P
Group A Headrests/chinrests (3) Cloth P MSSA MSSA
Group A Computer keyboards (3) Cloth S MRSA MRSP
Group A Hand sanitizer dispensers (3) Cloth S
Group A Doorknobs (3) Cloth G MSSA MSSA
Group B Tonometer tips (3) Swab P
Group B Headrests/chinrests (3) Cloth P MSSA MSSA
Group B Computer keyboards (3) Cloth S MSSA MSSA
Group B Hand sanitizer dispensers (3) Cloth S MSSP MSSA
Group B Doorknobs (3) Cloth G MSSA MSSA MSSA
Group C Tonometer tips (3) Swab P
Group C Headrests/chinrests (3) Cloth P MRSA MRSA MSSA
Group C Computer keyboards (3) Cloth S
Group C Hand sanitizer dispensers (3) Cloth S
Group C Doorknobs (3) Cloth G MSSA
Imaging A Headrests/chinrests (2) Cloth P
Imaging A B-scan & A-scan probes (2) Swab P
Imaging A Computer keyboards (2) Cloth S MSSA
Imaging A Doorknobs (3) Cloth G MSSA MSSA
Clinic 2
Group D Tonometer tips (3) Swab P
Group D Headrests/chinrests (3) Cloth P MSSA MRSA
Group D Computer keyboards (3) Cloth S MSSA
Group D Hand sanitizer dispensers (3) Cloth S MSSA MSSA
Group D Doorknobs (3) Cloth G MSSA MSSA
Imaging B Headrests/chinrests (2) Cloth P MRSA
Imaging B B-scan & A-scan probes (2) Swab P
Imaging B Computer keyboards (2) Cloth S MSSA
Imaging B Doorknobs (3) Cloth G MSSA MSSP

G = general contact; Group = group of pooled exam rooms; Imaging = pooled surfaces from shared imaging equipment; MRSA = methicillin-resistant Staphylococcus aureus ; MRSP = methicillin-resistant Staphylococcus pseudintermidius ; MSSA = methicillin-susceptible Staphylococcus aureus ; MSSP = methicillin-susceptible Staphylococcus pseudintermidius ; P = patient contact; S = staff contact.

a The same surfaces in each room were sampled in pools of 3 with either a premoistened cotton-tipped swab (Swab) or an electrostatic cloth (Cloth). Some imaging-area samples had fewer surfaces from which to sample; number of surfaces included in the pool is denoted in column 2.

Shared imaging equipment and imaging room contact surfaces (doorknobs, keyboards, head/chinrests, A- and B-scan ultrasound probes) in both clinic buildings were also tested. In these areas, a total of 3 surfaces were not always available, so the number of samples included in each pool can be found in Table 1 . The sampled surfaces were identical in all subsequent samplings. Overall, a total of 28 pooled samples from both examination and imaging rooms were collected on each date, representing a total of 76 individual surfaces.

Sampling was performed quarterly for 1 year. For the initial survey, rooms were selected randomly, and the same cadre of rooms and surfaces was screened for each subsequent survey. In each case, sampling was conducted at the end of the workday, after the clinics had closed and before daily cleaning and disinfection procedures were performed by the housekeeping staff.

Sample Collection and Processing

Samples were systematically collected from commonly touched surfaces by either cotton-tipped swabs (for smaller surface areas) that were premoistened to enhance sensitivity or by 10.4 × 8.0 inch (26.5 × 20.3 cm) dry electrostatic cloths (for larger surface areas). The electrostatic nature of the cloth enhances sensitivity of bacterial collection. For doorknob samples, the area immediately surrounding the doorknob was also sampled, up to about 1 foot from the knob and on both sides of the door. Slit-lamp headrests and chinrests were sampled at both forehead points and chin points of contact. Sampling was conducted by trained personnel wearing clean clothing covers and gloves, which were changed between each sample collected. After each sample was collected, swabs were immediately placed in tubes containing sterile trypticase soy broth (TSB, BD BBL; Becton, Dickinson) with 2.5% NaCl, while cloths were placed in sterile empty transportation bags (Nasco Whirl-Pak; Fort Atkinson, WI, USA). All samples were taken immediately to the Diagnostic and Research Laboratory on Infectious Diseases and processed as previously described, with a few modifications. Briefly, a pre-enriched medium (TSB with 2.5% NaCl) was added to each bag containing the dry cloths. Tubes with swabs as well as bags with cloths were incubated for 24 hours at 35°C, then grown on mannitol salt agar plates (BD BBL mannitol salt agar; Becton, Dickinson). Finally, 3 colonies with typical mannitol reactions were plated on 5% sheep blood plates (Remel; blood agar [trypticase soy agar, TSA, with 5% sheep blood]; Thermo Fisher Scientific, Lenexa, KS) to be further characterized. Typical morphologic colonies of S. aureus were confirmed by standard biological testing. The MRSA phenotype was confirmed by growth on oxacillin screen agar plates (OSA, BD BBL Becton-Dickinson) containing 6 μg/mL of oxacillin supplemented with NaCl following the Clinical Laboratory Standards Institute protocols.

Phenotyping and Genotyping

Antimicrobial resistance of all S. aureus isolates was established by the Kirby Bauer method using an array of 15 antibiotics (ampicillin 10 μg, amoxicillin/clavulanate 30 μg, oxacillin 1 μg, cephalothin 30 μg, cefpodoxime 10 μg, ciprofloxacin 2 μg, erythromycin 15 μg, gentamicin 1 μg, amikacin 30 μg, clindamycin 2 μg, tetracycline 30 μg, doxycycline 30 μg, trimethoprim/sulfamethoxazole 25 μg, moxifloxacin 5 μg, and chloramphenicol 30 μg). Resistance to vancomycin was tested using vancomycin screen agar plates (BD BBL Vancomycin Screen Agar; Becton-Dickinson). For quality control purposes, the following strains were included: S. aureus (ATCC 43300); S. aureus (ATCC 29213); S. aureus (ATCC 25923); Enterococcus faecalis (ATCC 23212); Escherichia coli (ATCC 25922); and Pseudomonas aeruginosa (ATCC 27853).

Molecular characterization of S. aureus isolates was performed as previously described. Briefly, pulsed-field gel electrophoresis was performed in a CHEF mapper system (Bio-Rad Laboratories, Nazareth, Belgium) to separated restriction fragments obtained from the digestion of chromosomal DNA with SmaI. Salmonella serotype Branderup strain H9812 was digested with XbaI and used as a molecular size marker. Relatedness between isolates was evaluated by dendrogram analysis of the resulting pulsed-field gel electrophoresis band patterns using commercial software (BioNumerics, v 6.6, Applied Maths, Ghent, Belgium). The dendrogram was generated by using the Dice coefficient and the unweighted pair group method with arithmetic averages, with a 1% band position tolerance. Based on percentage of similarity, band patterns were classified as clones or the same pulsotype (≥98%) and grouped in clusters (≥80%). To establish US types, all S. aureus isolates obtained in this study were compared against a Centers for Disease Control and Prevention (CDC) database containing 100 S. aureus strains with the most typical band patterns for each US type, using ≥80% similarity as the cutoff point. For MRSA isolates, mecA gene confirmation and SCCmec typing was performed as well.


During the year, S. aureus was detected on 30 of 112 (28% overall; prevalence 14%–39% over time) total samples. Of these, 25 were MSSA, and 5 were MRSA ( Table 2 ). MSSA prevalence ranged from 14% to 29%, and MRSA ranged from 0 to 11%. One surface tested positive for both MRSA and MSSA simultaneously.

Table 2

Prevalence of positive Staphylococcus aureus on surfaces in the ophthalmology clinic over time

Date Total surfaces sampled All S. aureus MSSA MRSA
11/2010 28 6/28 (21%) 5 (18%) 1 (4%)
02/2011 28 11/28 (39%) 8 a (29%) 3 (11%)
05/2011 28 9/28 (32%) 8 a (29%) 1 (4%)
08/2011 28 4/28 (14%) 4 (14%) 0 (0%)
TOTAL 112 30/112 (27%) 25 (22%) 5 (4%)

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Jan 8, 2017 | Posted by in OPHTHALMOLOGY | Comments Off on Screening and Characterization of Staphylococcus aureusfrom Ophthalmology Clinic Surfaces: A Proposed Surveillance Tool

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