The reclaimed water spray irrigation site included in this study is located in the Mid-Atlantic region of the USA. The site was chosen based on the willingness of the site operator to participate in the study. This site receives treated wastewater from a tertiary wastewater treatment plant (Mid-Atlantic WWTP1) [7,12] that serves an urban area, processing 681,390 m³/day of wastewater, with a peak capacity of 1.51 million m³/day. The incoming wastewater (influent) at Mid-Atlantic WWTP1 includes domestic and hospital wastewater, and the following treatment steps are employed at the plant: screens, primary clarifier, activated sludge reactors, secondary clarifier, sand filters, chlorination, dechlorination and discharge. At the time of the study, the chlorination dose was 2–3 mg/L, followed by dechlorination with sodium bisulfite such that the chlorine residual in the effluent was <0.1 mg/L.

The effluent (discharge) from Mid-Atlantic WWTP1 is sent to our spray irrigation study site through an enclosed pipe. Once the treated wastewater reaches the spray irrigation site, it passes through an aluminum screen and is then treated with 254 nanometer wavelength ultraviolet (UV) radiation bulbs that produce a minimum of 30,000 microwatt seconds per square centimeter. After UV treatment, the water is pumped into an open-air storage pond at a rate of 230,000 gallons per day with a peak capacity of 4 million gallons. Based on turf irrigation needs, water from the storage pond is pumped to spray heads throughout the site. Spray irrigation workers also carry backpack spray systems to irrigate additional areas. The spray irrigation site employs eight full-time employees and approximately 22 seasonal employees each year.

This study was approved by the University of Maryland College Park, Institutional Review Board, IRB Protocol 09-0211. A total of 43 subjects were enrolled in the study; 19 spray irrigation workers from the study site who were occupationally exposed to reclaimed water, and 24 office worker controls from an academic work setting who were not exposed to reclaimed water or healthcare settings on the job. Study subjects were selected through a convenience sample based on employment status. Office worker controls were matched by age (±2 years), sex, and race to the spray irrigation workers and recruited into the study in person and over email. Individuals were excluded from participation if they reported a nosebleed three days prior to sample collection to avoid dislodging blood clots.

Participants were asked to complete a short survey containing questions related to sociodemographics, as well as questions related to risk factors associated with MRSA colonization and previous MRSA diagnosis. The survey also asked participants about previous work in healthcare facilities and household members who work in healthcare facilities, because S. aureus nasal colonization rates among healthcare workers is greater than the 20%–30% colonization rate found in the general population [24,25]. The survey was filled out by participants on site at each sampling event.

A total of 94 nasal (48 from spray irrigation workers and 46 from office worker controls) and 94 dermal swab samples (48 from spray irrigation workers and 46 from office worker controls) were collected between August 2009 and February 2011 when the irrigation spray heads were in use. Participants were sampled at multiple time points when possible. On average, participants were each sampled 2.19 times. Nasal swabs were collected using Liquid Stuart Medium Transport swabs (Copan, Brescia, Italy). The swab was inserted approximately 1.25 cm into the participant’s right nostril and gently rotated five times on the inside wall of the nostril [23,26]. Dermal swabs were also collected using the same type of swabs. An approximately five-by-five cm area of the participant’s right forearm was swabbed by rolling the swab back and forth 15 times [27]. All samples were transported to the laboratory at 4 °C and processed within 24 h.

All media was obtained from Becton, Dickinson and Company (Franklin Lakes, NJ, USA). Nasal and dermal swabs were streaked onto Baird Parker agar for isolation of total S. aureus and Enterococcosel agar for isolation of total Enterococcus spp. Baird Parker agar plates were incubated at 37 °C for 24 h, while Enterococcosel agar plates were incubated at 41 °C for 24 h. Resulting black colonies with halos on Baird Parker, and colonies with a black precipitate on Enterococcosel agar were considered presumptive S. aureus and enterococci, respectively. These colonies were purified on Brain Heart Infusion agar and archived in Brucella broth with 15% glycerol at −80 °C. S. aureus ATCC 43300 and Enterococcus faecalis ATCC 29212 were used as positive controls and phosphate buffered saline was used as a negative control throughout the isolation process.

S. aureus were identified using the Gram stain, the coagulase test (Becton, Dickinson and Company), and the catalase test. For confirmation of S. aureus and MRSA differentiation, the S. aureus-specific nuc gene, the MRSA-specific mecA gene, and a 16S rDNA internal control were PCR amplified in a multiplex reaction as described previously [7,28]. DNA extraction was performed using the MoBio UltraClean® Microbial DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA, USA) per the manufacturer’s recommendations. Briefly, PCR amplification consisted of an initial denaturing step of 95 °C for 3 min, followed by 34 cycles of denaturing at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s, with a final extension at 72 °C for 5 min. S. aureus ATCC 43300 was used as a positive control for PCR amplification of nuc and mecA genes.

Enterococci were presumptively identified using the Gram stain, the catalase test, and by detection of pyrrolidonyl peptidase (pyr) activity (Remel, Lenexa, KS, USA). Confirmation was accomplished using a modified multiplex PCR assay previously described by Micallef et al. [29]. Genomic DNA from enterococci was extracted by heat lysis as described previously [29]. Briefly, the PCR reaction targeted the ddl genes of Enterococcus faecalis and E. faecium, the vanC1 and vanC2/3 genes of E. gallinarum and E. casseliflavus, and a 16S rDNA internal control [29]. PCR amplification consisted of an initial denaturing step of 95 °C for 3 min, followed by 35 cycles of denaturing at 94 °C for 30 s, annealing at 54 °C for 30 s, and extension at 72 °C for 30 s, with a final extension at 72 °C for 5 min. Positive controls used for PCR amplification were E. faecalis ATCC 51299, E. faecium ATCC 51559, E. casseliflavus ATCC 25788, and E. gallinarum ATCC 49573. Colonization among study participants was defined as MRSA, MSSA, VRE, or VSE isolated and confirmed from any swab sample (dermal or nasal) during the study period.

Antimicrobial susceptibility testing was performed using the Sensititre® microbroth dilution system with GPN3F minimal inhibitory concentration (MIC) plates (Trek Diagnostic Systems Inc., Cleveland, OH, USA) in accordance with the manufacturer’s instructions on all PCR-confirmed S. aureus (n = 97) and Enterococcus spp. (n = 20) isolates. Briefly, overnight cultures were transferred to sterile demineralized water (Trek Diagnostic Systems) to achieve a 0.5 McFarland standard. Then, 30 μL of the suspension for S. aureus and 50 μL of the suspension for enterococci was transferred to sterile cation-adjusted Mueller Hinton broth (Trek Diagnostic Systems), and 50 μL of the broth solution was then dispensed into the GPN3F MIC plates (Trek Diagnostic Systems Inc.) with the following antibiotics: erythromycin (ERY; 0.25–4 μg/mL), clindamycin (CLI; 0.12–2 μg/mL), quinupristin/ dalfopristin (SYN; 0.12–4 μg/mL), daptomycin (DAP; 0.25–8 μg/mL), vancomycin (VAN; 1–128 μg/mL), tetracycline (TET; 2–16 μg/mL), ampicillin (AMP; 0.12–16 μg/mL), gentamicin (GEN; 2–16, 500 μg/mL), levofloxacin (LEVO; 0.25–8 μg/mL), linezolid (LZD; 0.5–8 μg/mL), ceftriaxone (AXO; 8–64 μg/mL), streptomycin (STR; 1,000 μg/mL), penicillin (PEN; 0.06–8 μg/mL), rifampin (RIF; 0.5–4 μg/mL), gatifloxacin (GAT;1–8 μg/mL), ciprofloxacin (CIP; 0.5–2 μg/mL), trimethoprim/sulfamethoxazole (SXT; 1/19–4/76 μg/mL), and oxacillin + 2%NaCl (OXA+; 0.25–8 μg/mL). Plates were incubated at 37 °C and read after 18–24 h. Enterococcus faecalis ATCC 29212 and S. aureus ATCC 29213 were used as quality control strains. MICs were recorded as the lowest concentration of an antimicrobial that completely inhibited bacterial growth [30]. Resistance breakpoints published by the Clinical and Laboratory Standards Institute were used [30]. Multidrug resistance (MDR) was defined as resistance to two or more classes of antibiotics.

Descriptive statistics were reported including the percentages of study participants that were positive for MSSA and VSE by worker classification. Differences in sociodemographic variables between spray irrigation workers and controls were compared using the chi-square or Fisher’s exact test. Statistical analyses of antibiotic resistance data were limited to MSSA (n = 32) and VSE (n = 3) isolates expressing unique antimicrobial resistance profiles; this allowed us to reduce bias that could be introduced by including possible clones. A two-sample t-test was used to compare the number of antibiotics that isolates expressed resistance against. Logistic regression models were used (1) to determine if the odds of ever being colonized with the bacteria of interest were different for spray irrigation workers compared to controls, while controlling for education, duration of job, yearly income, and current smoking status; and (2) to conduct analyses of spray irrigation worker odds of colonization. A generalized linear mixed effects model (GLMM) was used to evaluate the odds of being colonized with S. aureus over time by occupational status. In all cases, p-values of ≤0.05 were defined as statistically significant. All statistical analyses were performed using Stata/IC 10 (StatCorp LP, College Station, TX, USA) or SAS 9.2 (SAS Inc., Cary, NC, USA).

The participation rate for the study was 88% (43/49). Participants ranged in age from 17 to 66 years and both spray irrigation workers and office worker controls were composed largely of Caucasian males (Table 1). The mean age of the spray irrigation workers and controls was 34 and 33 years, respectively. None of the participants in either group reported previous diagnoses of MRSA. Of the sociodemographic variables collected from participants, there were a few significant differences between spray irrigation workers and controls. Education levels and yearly income differed by exposure group (p<0.001; p = 0.01). Spray irrigation workers reported “currently smoking” (p = 0.002) and “smoking more than 100 cigarettes in the past six months” (p < 0.001) more than the controls (Table 1). Slightly more controls reported either personally having worked in a healthcare setting or having a household member who had worked in a healthcare setting, but these differences were not statistically significant.