Five disinfectant towelette products (Table 1) were tested against S. aureus (ATCC 6538) and P. aeruginosa (ATCC 15442) at five contact times (30 s, one min, two min, three min, and 10 min). Three biological replicates were conducted for each disinfectant, contact time, and bacteria permutation. Experimental procedures were adapted from EPA MB-33-00 [24].
Table 1
Description of disinfectant towelettes
Disinfectant Productsa | Active Ingredientsb | Dilution at Use | Label Contact Timed |
HP1 | 0.5% hydrogen peroxide | RTUc | 1 min |
QAC1 | 0.25% n-Alkyl (68% C12, 32% C14) dimethyl ethylbenzyl ammonium chlorides, 0.25% n-Alkyl (60% C14, 30% C16, 5% C12, 5% C18) dimethyl benzyl ammonium chlorides, 55% isopropyl alcohol) | RTU | 2 min |
QAC2 | 0.125% n-Alkyl (68% C12, 32% C14) dimethyl ethylbenzyl ammonium chlorides, 0.125% n-Alkyl (60% C14, 30% C16, 5% C12, 5% C18) dimethyl benzyl ammonium chlorides | RTU | 3 min |
QAC3 | 0.14% n-Alkyl (68% C12, 32% C14) dimethyl ethylbenzyl ammonium chlorides), 0.14% n-Alkyl (60% C14, 30% C16, 5% C12, 5% C18) dimethyl benzyl ammonium chlorides | RTU | 3 min |
QAC4 | Octyl decyl dimethyl ammonium chloride, dioctyl dimethyl ammonium chloride, didecyl dimethyl ammonium chloride, n-Alkyl (50% C14, 40% C12, 10% C16) dimethyl benzyl ammonium chloride (848 ppm active at 1:256 use-dilution) | 1:256 | 10 min |
a Abbreviated naming scheme for commercially available EPA-registered disinfectants used in this study; |
b Concentrations of active ingredients at use-dilution; |
c Ready-to-use; |
d Recommended label contact time for standard use.
Preparation Of The Test Suspension
Test suspensions with soil load were prepared according to EPA MB-33-00 [24]. Briefly, 10 mL of tryptic soy broth (TSB; BD, New Jersey, USA) were inoculated with 100 µL of thawed bacterial stock and incubated at 36 ± 1°C for 16–24 h with 200 rpm shaking. Soil components were added to the bacterial cultures to produce a test suspension containing 0.25% (w/v) bovine serum albumin (Thermo Fisher, Waltham, MA), 0.35% (w/v) yeast extract (Thermo Fisher, Waltham, MA), and 0.08% mucin (MilliporeSigma, Burlington, MA).
Preparation Of The Wiping Templates
Wiping templates were defined as 2 ft x 2 ft squares on Formica® laminate sheets (Midwest Manufacturing, Eau Claire, WI) (Fig. 1). Templates were disinfected prior to inoculation by applying 10% bleach, followed by a neutralization step with 0.3% hydrogen peroxide. The board was then rinsed three times with sterile ultrapure water followed by a final application of 70% ethanol. Each template was inoculated with five 10 µL droplets of test suspension in the designated inoculation area (Fig. 1). This volume yielded an average recovery of 1.2 x 107 CFU for S. aureus and 2.8 x 107 CFU for P. aeruginosa upon drying. The templates were left undisturbed under ambient conditions until the inoculum was fully dried (approximately 1–2 hours).
Preparation Of The Disinfectant Wipes
Four of the five test disinfectants (HP1, QAC1, QAC2, and QAC3; Table 1) were purchased as ready-to-use pre-saturated wipes. Prior to use, canisters were inverted several times to distribute the disinfectant throughout the canister and lids were wiped with 70% ethanol to minimize contamination during handling. Prior to testing, the first three wipes were removed from the canister to ensure that the wipes used for testing contained an even distribution of liquid disinfectant. QAC4 was purchased as a concentrate and diluted 1:256 in synthetic hard water and applied to EasyWipes (Diversey Holdings LTD, Fort Mill, SC). Synthetic hard water was prepared using the guidance for AOAC Hard Water as described in EPA MB-30-02 [25]. EasyWipes were cut to approximately 6” x 6.8”, a size comparable to that of the ready-to-use wipes used in the study. The dry wipes were impregnated on the day of use using a liquid-to-towelette ratio of 4.9 mL diluted disinfectant per wipe based on the suggested ratio provided on the EasyWipes canister.
Disinfectant Testing
A disinfectant wipe was applied to the upper left-hand corner of the wiping template and moved across the template manually using four horizontal passes (Fig. 2a). This design allowed for partial depletion of the disinfectant towelette prior to wiping over the inoculation zone. Contact time was initiated at the end of the final pass. After the defined contact time had elapsed for each product, a neutralizing surface swab (World Bioproducts LLC, Woodinville, WA) was passed over the sampling area three times to collect the sample (Fig. 2b). The swab was vortexed for 30 s [26] prior to serial dilution of the neutralizing buffer in phosphate buffered saline. Dilutions were filtered over 0.2 um polyethersulfone filters (Pall Corporation, Port Washington, NY) using an EZ-Fit™ Manifold filtration system (MilliporeSigma, Burlington, MA). Filters were plated onto tryptic soy agar (TSA; BD, Franklin Lakes, NJ) and incubated at 36 ± 1°C for 24–48 h prior to counting. A non-inoculated template served as a negative control and two inoculated, untreated templates served as positive controls. Negative controls were swabbed with macrofoam swabs (World Bioproducts LLC, Woodinville, WA) saturated with sterile phosphate buffered saline and processed as described above.
Terminal Cleaning
After all templates were sampled, a terminal cleaning protocol was performed to remove residual soils prior to further testing. A 10% bleach solution was applied for a minimum of 10 min followed by a neutralization step with 0.3% hydrogen peroxide. A multi-purpose cleaning spray (Babyganics, Westbury, NY) was applied and wiped dry with paper towels. The board was then rinsed once with ultrapure water followed by a final application of 70% ethanol.
Data analysis
Dilutions yielding colony counts of 20–200 CFU were used for calculating bacterial recoveries. If two or more dilutions for a given test yielded a count within this range, the dilution with the higher total CFU was used. When no colonies were present across any dilutions filtered, a count of 1 CFU was used for calculations at the lowest dilution filtered. All colony counts from untreated and treated templates were log-transformed. Bacterial log reduction was calculated by subtracting the log density of a treated template from the average log density of the two control templates for a given replicate. Three biological replicates were performed for each combination of disinfectant and organism.
Statistical analyses were conducted in SAS software, Version 9 of the SAS system for Linux. Copyright© 2012–2018 SAS Institute Inc. SAS and all other SAS Institute Inc. product or service names are registered trademarks or trademarks of SAS Institute Inc., Cary, NC, USA. A general linear model was performed to determine the overall effects of contact time and product type on disinfectant efficacy for each organism studied. A Tukey’s t-test post-hoc analysis was conducted to determine differences among individual contact times and products for a given organism. Figures were prepared using Microsoft Office Suite (Microsoft Office 365) and GraphPad Prism version 9.4.1 for Mac OS X, GraphPad Software, San Diego, CA, USA.