Study design and ethic
A cross-sectional study was performed at the VTH-CU, the Small Animal Teaching Hospital of the Faculty of Veterinary Science. The samples were collected during May 2014-2016. Study was approved by Institutional Animal Care and Use Committee (113/56), Faculty of veterinary science, Chulalongkorn university and the Ethical Review Committee for Research Involving Human Research Subjects, Health Science Group, CU (137/57), Research and innovation for society, Chulalongkorn University. All methods were performed in accordance with relevant guidelines and regulations.
The hospital setting
The VTH-CU is the primary veterinary hospital in Bangkok, Thailand, with approximately 140,000 patient visits each year. The hospital comprises five divisions: Emergency and Intensive Care, General Medicine, Surgical, Gynecological, and Special Rooms (Cardiology, Kidney & Urine care, Diabetes, Dermatological, Cancer, Feline disease, and Neurological).
In 2014, approximately 389 pet patients per day visited the VTH-CU, divided as 96, 30, 27, and eight at the general medicine, gynaecological, dermatological, and surgical rooms, respectively. The routine of cleaning management in the VTH-CU was comprised as follows. The floors were washed with 2.5% (w/v) quaternary ammonium compound (UMONIUM38®; Laboratoire Huckert's International, Thailand) between 3.30–4.00 PM. The examination tables, stethoscopes, syringe plates, waiting branch, drug cabinet, keyboard, and knot door were cleaned with 0.5% (w/v) UMONIUM38® when the items were not in use. The cotton for wound dressing did not change rooms when empty. The disinfectant water in forceps jars, which is 1% (w/v) povidone-iodine (Betadine® solution; Pathumthani, Thailand), was changed every day in all rooms.
Populations and sample collections
Environmental samples
A total of 216 samples were retrieved from the environmental surfaces in nine parts of the VTH-CU; the division of general medicine (M), vaccination room (V), gynaecology (G), dermatology (SW), surgery (S), post-surgery care (P), intensive care unit (ICU), and hallway (lower and upper; H). On the sample collection day, the medicine, vaccination, and dermatological rooms were located in the new building of the VTH-CU. The gynaecology, surgery, post-surgery care, and hallways were in parts of the old building. The criteria of sample collections are described in Table 1. The requirements for room selection were (i) having at least eight pet patients per day and (ii) the room was cleaned at least once per day. One environmental sample was collected two times within the same day; before clinics opened at 7.30–8.00 AM and after the clinics had been cleaned at 3.30–4.00 PM. In each room, samples were collected from five parts of the floor [31] at the central examination room. In the case of medical instruments, if there was more than one item in that room, then the most used item was chosen for sampling [32]. All surfaces were sampled using sterile cotton swabs. The cotton was dipped into 2 mL of peptone saline dilution (PSD; 100 mg/mL peptone and 850 mg/mL sodium chloride in distilled water). The moistened swab was rolled on the surface, the cotton part cut off into the same tube and kept on ice until cultured.
Veterinary staff samples
Before this experiment started, the written informed consent was obtained from veterinary staff. Study in human was approved by the Ethical Review Committee for Research Involving Human Research Subjects, Health Science Group, CU (137/57), Research and innovation for society, Chulalongkorn University. Samples from veterinary staff were taken from one veterinary nurse per unit and 16 veterinarians who had worked more than 40 h/week at the VTH-CU for two years. Note that routinely, veterinarians and other staff wear a protective mask during working hours. They were asked to provide a sterile cotton swab wipe from their nasal cavities [18]. The sterile cotton swabs were dipped into 2 mL of peptone saline diluent (PSD) in a sterile test tube (No. 9820, Cole-Parmer®, Thailand) before nasal sampling [18]. After sampling, the cotton part was cut into the same tube, contained on ice and cultured within 2 h.
Dog samples
Before this experiment started, the written informed consent was obtained from dog owners. Study in animal was approved by Institutional Animal Care and Use Committee (113/56), Faculty of veterinary science, Chulalongkorn university. A total of 14 samples were collected from the wound abscess of 14 dogs in surgery, post-surgery care, and the dermatological room on the same day as the staff and environmental samplings. Outpatient dogs with wound infection or dermatitis were chosen from each clinical room with the authorisation of the owner's veterinarian and permission. The moist, sterile cotton swabs were dipped in 2 mL of PSD into a sterile test tube [18], then rolled on the wound sites of dogs, and treated as above.
Identification of CoPS and MRCoPS
Samples were cultured within 2 h of the collection in an enrichment culture. A total of 0.1 mL of the suspension was spread on Baird-Parker agar (DifcoTM, France) without and with 0.5 µg/mL oxacillin (screening plate) Staphylococci selection. After incubation at 35ºC for 48 h, black Staphylococci-like colonies were selected. Three presumptive Staphylococci colonies were purified on tryptic soy agar (TSA; DifcoTM, France) and confirmed with the coagulase test. All CoPS were identified by primary and secondary biochemical tests, as previously reported [33]. Multiplex-PCR (M-PCR) and matrix-assisted laser desorption/ionisation–time of flight mass spectrometry (MALDI-TOF) were used for characterisation as previously reported [34, 35], where S. aureus ATCC 25923, S. pseudintermedius CVMC 0108, S. schleiferi subsp. coagulans CVMC 0208 (canine origin), S. intermedius CVMP 0309, and S. delphini CVMP 0109 were used as control strains. All CoPS were screened using the oxacillin disk diffusion method. The oxacillin (1 µg) and cefoxitin (30 µg) breakpoints were used to confirm MRCoPS, as defined by the CLSI recommendation [36]. All oxacillin-resistant isolates were characterised as MRCoPS and approved by the presence of the mecA gene by M-PCR as reported [37], and using S. aureus ATCC 25923 and MRSA N315 as the internal control strains.
Antibiogram, SCCmec typing, and clone typing of the CoPS and MRCoPS isolates
All MRCoPS strains were further determined for their antibiograms and SCCmec types by the disk diffusion method [36] with clindamycin (DA; 2 µg), doxycycline (DO; 30 µg), gentamicin (CN; 10 µg), erythromycin (E; 15 µg), mupirocin (MUP; 200 µg), and sulfamethoxazole/trimethoprim (SXT; 25 µg), and using S. aureus ATCC 25923 as the control strain. The susceptibility level was interpreted following the Clinical and Laboratory Standards Institute (CLSI) recommendation [2013], while the determination of the DO breakpoint followed that of Maaland [38]. In addition, the breakpoint of amoxicillin/clavulanic acid is not presented in this edition of CLSI [2013].
The conserved fragments of the mec gene complex and ccr gene complex were detected by M-PCR to identify the SCCmec type [39]. To determine the clonal relationship between CoPS and MRCoPS, pulsed-field gel electrophoresis (PFGE) was performed as recommended [18]. Briefly, bacterial DNA was plugged into Seakem® agarose (Bio-Rad, USA) and cut with the Cfr9I enzyme. In the case of uncut samples, ApaI was used as the restriction enzyme [8]. The DNA fragments were then separated on a CHEFIII at 6 V/cm, 14 ºC, 120º in a 1% (w/v) pulsed-field grade agarose gel with switching at 5–15 s for 18 h and 15–60 s for 5 h. Gels were stained with red safeTM Nucleic Acid Staining Solution (Scientific, NSW, AUS), destained in water, and then digitally captured under UV light [40]. The Lambda Ladder PFG Marker (New England BioLabs, Beverly, Mass) was used as the size ladder. The Bionumeric program associated with the dice coefficient [1.5] was used for dendrogram construction (UPGMA with 1.0% position tolerance). Individual patterns were discriminated based on 80% similarity.
Data analysis
Statistic 22 for Microsoft Windows (SPSS Inc.; Chicago, IL, USA) was used for statistical analysis. The prevalence of CoPS, MRCoPS, and MSCoPS positive surfaces are described as a percentile, while the odds ratio (OR) explained the risk factor in each room compared to the reference room, which was chosen as the room with the lowest level of MRCoPS-positive surfaces.