2.1. Hand-Rinse Samples to Recover Skin-Bacteria and Evaluate Their UV-Resistance
Hand-rinse samples were collected from five participants using a “glove method”. Participants inserted each hand, one at a time, into a large powderless latex free nitrile glove filled with 40 mL of municipally sourced, sterile deionized (DI) water. Using their free hand, participants massaged the submerged hand to ensure maximum water to hand contact for bacterial shedding. After approximately 30 s of exposure, participants inserted their other hand and repeated the previous step. Sterile DI water was used instead of tap water to reduce background bacterial input and confounding effects of residual chlorine. Soap was not used so as to reduce potential bacteriostatic effects and to avoid increased turbidity, as the goal was not to simulate greywater production, but rather to isolate skin-borne bacteria for UV-irradiation and enumerate viable cells using two isolation agars. The resulting bacterial suspension was poured into a sterile beaker and thoroughly mixed. A 28.3 mL portion of the sample was poured into a 60 mm sterile Petri dish so as to give a 1 cm greywater depth. A sterile 5 mm × 2 mm stir bar was placed in the dish and the dish was placed onto a magnetic mixer operating at 400 rpm to facilitate mixing without vortexing.
An AquaSense Pearl Beam collimated LED UV reactor (Florence, KY, USA) with a peak wavelength of 256 nm and a half bandwidth of 11.5 nm was used to deliver 256 nm UV-C irradiation to the raw hand-rinse sample using an adapted EPA protocol [
22]. Equation (1) was used to calculate the effective intensity (
Eave) of the collimated beam based on measurable variables [
23]:
The incident intensity (E0) was measured using a NSF certified radiometer (UVP Radiometer, Model UVX-25, Upland, CA, USA). The water height (L) was measured to 1 cm (28.3 mL in a 60 mm cylindrical Petri dish), and a spectrophometer (Thermo Scientific, Genesys 10S UV-VIS, Waltham, MA, USA) was used to measure the absorbance (A) of 256 nm UV in the suspension. The resulting Eave was then multiplied by the exposure time (seconds) in order to calculate the resulting dosage in mJ·cm−2.
To estimate staphylococci counts prior to irradiating the sample, 100 µL of the sample was diluted to 10−1; 100 µL of the dilution was pipetted into 15 mL of sterile 0.85% NaCl buffer then filtered through a 60 mm diameter filter cup apparatus with a 0.22 µm polycarbonate (PC) membrane filter (IsoporeTM GTTP-04700, Cork, IRL) using a vacuum pump. The filtering process was performed a total of six times and the resulting filter papers were placed on tryptic soy agar (TSA) and mannitol salt agar (MSA) plates, both in triplicate.
For UV irradiation of samples, the collimated beam was placed over the Petri dish containing 28.3 mL of a raw hand-rinse sample (as described above), and then irradiated to a dose of 220 mJ·cm−2. In order to achieve a consistent dose for each person’s greywater sample, the absorbance was measured prior to exposure to adjust the exposure time for a resulting dose of 220 mJ·cm−2. After exposing samples to 220 mJ·cm−2, the entire sample was poured into a sterile 50 mL capped test tube and vortexed to ensure adequate mixing. Upon vortexing, 1 mL of the post-irradiated sample was pipetted into 15 mL of sterile 0.85% NaCl buffer and plated using the above described filter-plating technique. TSA and MSA plates were incubated at 37 °C for 18–24 h before assaying colony forming units (CFU).
Given the similarity in growth conditions of the expected staphylococci and micrococci and that no selective medium is known to easily resolve these genera, we confirmed pure isolates using Lysostaphin
TM tablets, known to reliably differentiate between staphylococci (lysis) and micrococci (no lysis) [
24]. Upon assaying, Lysostaphin
TM tablets were used for total staphylococci confirmation for colonies from MSA plates (see [
25] for protocol). Five Lysostaphin
TM confirmations were performed on each of the triplicate MSA plates, for a total of 30 confirmations per sample (15 at 0 mJ·cm
−2 and 15 at 220 mJ·cm
−2). The fraction of lysis positive Lysostaphin
TM confirmation tests was multiplied by the CFU counts from the MSA assays to estimate the fraction of CFU assayed from MSA which were considered totally staphylococci. Equation (2) was used to calculate the log
10 reduction after exposure for each assay:
2.2. Hand-Rinse Isolates
Upon assaying the MSA control plates, two or three colonies from each participant were isolated onto separate MSA plates, which were then incubated for 18–24 h at 37 °C, and then re-streaked at least once more onto MSA plates to ensure purity. Once pure cultures were isolated (total of 14), they were analyzed using a VITEKTM (2 COMPACT) instrument to determine genera and species.
The collimated UV beam procedure was performed on a total of 14 isolates from five different participants as well as a clinical
Staphylococcus aureus strain acquired from the American Type Culture Collection (ATCC),
S.
aureus (ATCC 25923). A presumed
Staphylococcus epidermidis (ATCC 12228) strain was confirmed as
Staphylococcus lentus upon VITEK
TM confirmation and was also used in this study. Overnight cultures grown in Tryptic Soy Broth (TSB) were diluted to 1:100 in a sterile 0.85% NaCl. Once diluted, the above collimated UV beam procedure was performed to irradiate suspended cells (with the following modification). Prior to irradiation, 100 µL of the sample was plated on TSA plates in triplicate at the appropriate dilution to effectively assay control plates. Samples were irradiated to 11 mJ·cm
−2 (slightly less than the approximate dose required for ~4-log
10 reduction of
S. aureus, being a likely targeted reduction level [
7]), and allowing for post exposure detection of at least some samples. Upon irradiation, 100 µL of the sample was plated in triplicate on TSA at the appropriate dilutions to effectively assay the plates, and log
10 reductions were calculated using Equation (2). A portion of the samples were irradiated at 7, 9, and 11 mJ·cm
−2 in order to estimate decay equations.
2.3. Oxybenzone (BP3)
The collimated UV beam procedure described above was performed using
S.
aureus (ATCC 25923) to determine the effects of a sunscreen, oxybenzone, on the performance of UV inactivation. Oxybenzone was used as a test compound due to the relatively high concentrations (reports as high as 0.7 mg·L
−1) when compared to other UV filters/sunscreens [
21]. Since the source of oxybenzone in municipal wastewater is likely from greywater, oxybenzone and other micro-pollutant/PCP concentrations would likely be higher in greywater than in municipal wastewater (which is diluted by blackwater). As shown in a 2005 study by Palmquist and Hanæus [
26], organic compound pollutants were typically in greater concentrations within greywater samples when compared to blackwater and sometimes an order of magnitude higher in concentration. Therefore an oxybenzone concentration range of 1 mg·L
−1 to 10 mg·L
−1 was used due to the expected increase of oxybenzone concentration in greywater. Oxybenzone was dissolved into dimethyl sulfoxide (DMSO) (166.7 µL·L
−1) and pipetted into sterile 0.85% NaCl buffer at two different concentrations: 10 mg oxybenzone·L
−1 and 1 mg oxybenzone·L
−1; DMSO was suspended into two separate buffer solutions (at 166.7 and 16.67 µL·L
−1 for the two solutions respectively) used as controls to adjust for any confounding effects of DMSO on the reduction of
S.
aureus by UV irradiation. Overnight cultures of
S.
aureus (ATCC 25923) suspended in TSB were suspended into the four samples (10 mg oxybenzone·L
−1, 1 mg oxybenzone·L
−1, and respective controls) and irradiated to a UV-C dose of 11.8 mJ·cm
−2 using the previously described collimated beam apparatus protocol. Samples were assayed on TSA plates in at least triplicate prior to and after exposure to UV. Log
10 reductions were quantified using Equation (2). A paired
t-test was performed between oxybenzone and respective controls on the log
10 reduction means.