Setting
The study was conducted at Hjørring Hospital, which at the time was part of Vendsyssel Hospital with a catchment population of approximately 200,000 inhabitants of the North Denmark Region. The North Denmark Region covers 18% of the Danish area, includes 10% of the Danish population, and 23% of farms with livestock (20% of all pig farms) numbers available on Statistics Denmark [19] corresponding to an area of high agricultural density. All 1745 employees (including approx. 240 students), were invited to participate in the study that took place over a 3-week period in the autumn of 2015 during normal working hours between 7 am and 8 pm. Information about the study was presented at the hospital’s intranet in text, by a short movie, and through announced meetings. The consecutive inclusion of participants extended for three weeks in September 2015.
Study design
This cross-sectional study was composed of three major parts: i) A questionnaire directed to the employees and a nasal swab analysis; ii) Environmental dust sampling at the hospital; and iii) Dust sampling in the household of the participants (bedrooms) and if relevant related animal sheds.
Questionnaire and nasal swabs
All participants received the questionnaire by e-mail for electronic completion or as handouts. The questionnaire included items of e.g. sex, age, job category, direct or indirect contact (through household members) with animal production (primarily pigs, cattle, poultry and mink) and companion animals, self-evaluated health, earlier disease with a focus on skin disease, and ever having been carrying or infected with MRSA CC398.
Nasal swabs were taken from the anterior nares by the investigators using the ESwab liquid-based collection and transport system (Copan Innovation, Italy) according to the manufacturer’s instructions. Nasal swabs were stored in transport medium (Amies) and processed within 3–4 days at Statens Serum Institut (SSI), Copenhagen, Denmark.
Data were obtained from Statistics Denmark on the proportion of households with members employed in livestock farming in the region of Denmark where staff from the hospital lived.
Environmental dust sampling at the hospital
Short-term sampling of bacteria in bioaerosols was carried out on September 14th and 15th 2015 at the hospital. Four types of active samplers were used (details described later). A total of 20 different areas at the hospital and two reference areas outside the hospital were selected for sampling. Each type of active sampler was placed in at least five and at most 17 of the selected areas so that each area was sampled by at least one and at most five different methods. The presence of employees, patients and visitors in each sampling area during the sampling period was noted, and categorized into none, less than 5, 5 to 10, 10 to 20, and more than 20 persons. Average temperature and relative humidity was measured in the sampling locations during sampling.
Long-term sampling of bioaerosols was performed using passive sampling from September 14th to October 7rd 2015. Both active and passive samplers were placed 1.4 to 2.0 m above ground level. The positions included four locker rooms, two in the bed cleaning areas, nine in different waiting rooms or areas designated for patient reception, two areas in the basement where patients, beds and laundry were transported, one consulting room, one lunchroom, one lunch desk, one main entrance and the outdoor reference.
Dust sampling in homes and related animal sheds
According to the study protocol, 40 participants were to be selected for the sampling of dust in their home and, if relevant in an animal shed: 20 LA-MRSA carriers and 20 non-carriers. Additionally, 10 participants in each group should be in direct or indirect contact with production animals and 10 should be without animal contact. As all the nasal swabs were negative for LA-MRSA (see below), the selection of participants for the home and farm dust sampling was changed, as illustrated in Fig. 1. The final groups were not as intended mutually exclusive, but were selected based on questionnaire information with the aim to include those participants with most contact with pigs, other farm animals or farmers, based on assumed highest LA-MRSA exposure. First, participants with direct or indirect contact with pigs were identified, n = 13. Secondly, participants living on a farm with animals (including hobby farms) were selected, n = 16. The third group of participants were those with direct or indirect contact with mink, n = 3. Finally, participants with direct or indirect contact with cattle, n = 4, and poultry, n = 4 were selected in order to obtain 40 locations for dust sampling. The home dust sample was collected from the bedroom. The animal shed samples were collected from buildings with animals, representing confinement buildings, stables, mink farms as well as small hobby farms. The dust samples from the bedrooms and the animal sheds were collected over 14 days during October–December 2015.
Dust samplers and extraction methods
Four types of active samplers were used in parallel to collect airborne bacteria. Inhalable Gesamtstaubprobenahme samplers (GSP; BGI Inc., Waltham; MA, USA) and IOM (SKC Inc., PA, USA) samplers mounted with 1.0 μm pore size polycarbonate filters (Maine Manufacturing, Sanford, USA) were used at a flow rate of 3.5 l/min and 2.0 l/min, respectively. Sampling was performed in 17 areas with an average sampling period of 118 min. Bacteria from filters were extracted by orbital shaking in a pyrogen-free 0.05% Tween 80 and 0.85% NaCl solution as described previously [20]. The BioSampler (SKC Inc., PA, USA) was used for direct sampling in 20 ml pyrogen-free solution (0.001% Tween 80, 0.85% NaCl). The BioSampler was used for sampling in 5 areas at a flow rate of 7.5 l/min for an average of 50 min during which the sampler was cooled with cooling elements. The BioSampler samples were cultured within 2 h after sampling. All samples from the GSP, IOM, and BioSampler were cultured on Nutrient agar (Oxoid, Basingstoke, UK) supplemented with actidione (50 mg/l cycloheximide) at 25 °C for general cultivation of bacteria (NA plates), on SaSelect agar plates (Bio-Rad, France) at 37 °C for selection of staphylococci (SA plates) and on MRSA–selective agar plates (Oxoid, United Kingdom) at 37 °C for selection of MRSA (MRSA plates). Bacterial colonies were counted after 7 days for NA plates and after 24 h for SA and MRSA plates.
Airborne bacteria were also sampled using a Six-stage Viable Andersen Cascade Impactor (ACI) (N6, Thermo Fisher Scientic Inc. Waltham, MA, USA) in 11 of the areas at a flow rate of 28.3 l/min for 20 min on MRSA plates and for 5 min on NA plates. For calculation of number of colony forming units from the ACI samples, data for the six levels of the sampler were pooled.
EDC samplers (electrostatic dust collectors, ZEEMAN Alphen, the Netherlands) were used for long term sampling as described previously [21] in 8 areas of the hospital and for the dust sampling from bedrooms and animal sheds. EDCs from the hospital were extracted as described by Madsen et al. [22] and cultured on SA and MRSA plates as described above.
EDCs from the bedrooms and animal sheds were extracted as described by Shorter et al. [23]. In brief, the EDCs were extracted twice in 50 mL sterile water with 0.05% Tween20. The extracts were concentrated using centrifugation and subjected to beat milling for mechanical cell disruption before DNA purification. They were then processed similar to nasal swab samples as described in the next section.
MRSA identification and characterization
MRSA was identified according to standard laboratory methods. Nasal swabs and dust samples from EDCs in homes and animal sheds were analysed at SSI. In brief, 200 μl of nasal swab transport medium or EDC extract was enriched in 5 ml of Mueller-Hinton broth (Sigma Aldrich) containing 6.5% NaCl for 18 h at 35 °C. Ten microliters of enriched broth were cultured on Brilliance MRSA 2 agar plates (Oxoid, United Kingdom) and incubated for 20 h at 35 °C. Presumptive MRSA colonies were streaked onto blood agar and grown overnight at 35 °C.
MRSA was identified and characterized using a multiplex PCR assay as described previously [24]. spa typing was performed using the Ridom Staph Type standard protocol [25] and the Ridom SpaServer [26].
Identification of bacteria by MALDI-TOF mass spectrometry
Bacteria colonies from the indoor hospital environment were identified using Matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry (Bruker Daltonics, Bremen, Germany) using Bruker Biotyper 3.1 software with the BDAL standard library. Bacterial colonies from the agar plates were prepared using the extended direct transfer methods as described earlier [27].
Statistics
A two-sample test of proportions was used to test if the distribution of job categories differed between the responders and the entire population of staff and between the individual job categories. The same test was used to validate the proportion with agricultural contact between responders and staff through data from Statistics Denmark. Descriptive statistics were used for the remaining results. The significance level was set at 5%. Statistical analyses were conducted in Stata 12.1 (StataCorp LP, 2011).