Disinfectant Ecacy Against Dry Surface Biolms of Staphylococcus Aureus and Pseudomonas Aeruginosa Is Product, Time Point and Strain Dependent

Background: Globally, healthcare associated infections (HAI) are the most frequent adverse outcome in healthcare delivery. Although bacterial biolms contribute signicantly to the incidence of HAI, few studies have investigated the ecacy of common disinfectants against dry surface biolms (DSB). The objective of this study was to evaluate the bactericidal ecacy of seven disinfectants against DSB of Staphylococcus aureus and Pseudomonas aeruginosa. We hypothesized that overall, hydrogen peroxides, sodium dichloro-s-triazinetrione and quaternary ammonium compounds plus alcohol disinfectants will be more bactericidal against DSB than quaternary ammonium. We also hypothesized that regardless of differences in product chemistries, higher bactericidal ecacies against DSB will be exhibited after 24 h of dehydration compared to 72 h. Methods: Wet surface biolms of S. aureus and P. aeruginosa were grown following EPA-MLB-SOP-MB-19 and dehydrated for 24 h and 72 h to establish DSB. Seven EPA-registered disinfectants were tested against dehydrated DSB following EPA-MLB-SOP-MB-20. Results: Overall, quaternary ammonium plus alcohol, sodium dichloro-s-triazinetrione, and hydrogen peroxide products were more ecacious against DSB than quaternary ammoniums for both tested strains. While there was no signicant difference in biolm killing ecacies between 24 h and 72 h S. aureus biolms, signicantly higher log 10 reductions were observed when products were challenged with 24 h P. aeruginosa DSB compared to 72 h P. aeruginosa DSB. Conclusion: Strain type, active ingredient class, and dry time signicantly impact disinfectant ecacy against DSB of S. aureus or P. aeruginosa. statistically compare mean log 10 reductions at 24 and 72 Pair-wise and dry times completed with Tukey adjustments. All statistical procedures completed ecacy of disinfectants against DSB of S. aureus and P. aeruginosa after prolonged dehydration for 24 h and 72 h. While there was no signicant difference in log 10 reductions between 24 h and 72 h DSB of S. aureus, the reverse was true for DSB of P. aeruginosa as 72 h DSB of P. aeruginosa were harder to kill than their 24 h counterparts. In a previous study by our group, we found that 100% of P. aeruginosa DSB established at a dehydration temperature of 21°C were encased in EPS while this was true for only compared to P. aeruginosa DSB exposed to quaternary alcohol and quaternary ammonium products. multiple intrinsic and extrinsic factors. Our study denitively demonstrated that signicant kill levels of the DSB of major healthcare pathogens that cause HAI can be achieved although this is highly dependent on the choice of disinfectant, active ingredient class, DSB “age” and bacteria strain. It is therefore critical for healthcare stakeholders to consider these factors in efforts to reduce HAI rates.


Background
Healthcare associated infections (HAI), a result of diverse interactions among modern healthcare practices, hospital environments, and growing antibiotic resistance, among other factors, pose a crucial threat to human well-being [1]. Globally, the acquisition of HAI is the most frequent adverse outcome in healthcare delivery [2]. In the United States, approximately 633,300 patients are affected by 687,200 HAI [3] with more than 72,000 deaths every year [4]. In Europe, about 4.5 million HAI occur yearly in acute care hospitals [5] with about approximately 135,000 deaths [6]. In low and middle income countries (LMIC), the density of HAI in adult intensive care units is estimated at 47.9 per 1,000 patient days, which is higher than rates in the US and Europe [7]. Comparing HAI incidence rates in developed and LIMC, the incidence rate is seven out of 100 patients in developed economies and ten out of a 100 in LMIC [8].
The prevalence of HAI has been associated with bio lm formation by bacteria [9]. Bacterial bio lms are ubiquitous and represent approximately 99% of the world's known bacterial population [10]. The National Institutes of Health (NIH) of the US estimates that about 80% of all chronic infections are due to bio lm formation [9]. Bio lms are comprised of microbial cells adhered to a surface and to each other, forming a microcolony encased in a polysaccharide dominant matrix [11]. In addition to the cells that inhabit a bio lm, DNA, proteins and biosurfactants are prevalent [12]. Bacterial bio lms are persistent on environmental surfaces due to the ability to adhere to common surfaces and the extracellular polymeric substances (EPS) they produce [13]. The EPS forms a matrix that presents a major barrier to removal from surfaces in healthcare facilities as it is "resistant" to physical stress [14] and shields underlying bacterial cells from direct contact with disinfectants [15]. As a result of the EPS matrix [11Donlan, 2000], the presence of e ux pumps and persister cells [16], bacterial bio lms are about 1,000 times less susceptible to disinfectants than their planktonic counterparts [17]. Additionally, the ability for disinfectants to penetrate the bio lm matrix is affected by the water binding characteristic of the EPS matrix [11], and pH differences among various layers of bio lms [18]. These features may result in the aggregation of organic acids leading to the deactivation of less potent disinfectants that may be non-lethal [18].
Bacterial bio lms have the ability to develop and persist for up to 12 months [19] on wet and dry surfaces in the hospital environment despite repeated cleaning [20]. Dry surface bio lms (DSB) are particularly widespread on surfaces in healthcare facilities [20,21]. In a recent 2018 study by Ledwoch et al., DSB were detected in 95% of 61 samples collected from hospitals in Wales [22]. Such surfaces included commodes, clipboards and sanitizing bottles [22]. DSB have also been detected on indwelling catheters [23]. Although multi-species dry surface bio lms have been detected on a range of surfaces in healthcare facilities, major HAI pathogens as S. aureus [22] and P. aeruginosa are predominant [24,25].
While DSB are widespread on surfaces in healthcare facilities, they are also harder to kill than wet surface bio lms [26]. This is the case as overall, DSB are characterized by a denser EPS matrix than wet surface bio lms [26,27]. Moreover, with prolonged desiccation and starvation of bacteria in DSB, there is an increase in the overall percentage of protein content and slightly decreased carbohydrate content compared to wet surface bio lms [26,28] Being the principal component of bio lms, this increase in the proportion of proteins may further contribute towards the reduced bactericidal e cacy of disinfectants against DSB. [29]. Bio lms may survive longer due to metabolic changes that may result from cell-cell signaling and from the presence of a bio lm matrix that facilitates nutrient recycling and transformation from lysed cells [30].
In the current protocol used by the Environmental Protection Agency (EPA) for bio lm claims on disinfectants, wet surface bio lms of S. aureus and P. aeruginosa are the required test pathogens [31]. Under real world conditions such as in healthcare facilities, disinfectants are relied on to inactivate bacteria on dry surfaces [32], which are usually in the form of DSB [26]. Despite widespread evidence that bacteria in healthcare environments are more likely to be encased in DSB, the standard test for disinfectant e cacy testing and registration with the EPA are conducted using planktonic bacteria or bacteria in wet bio lms. To the best of our ndings, no studies have evaluated the bactericidal e cacy of disinfectants against dry surface bio lms of S. aureus and P. aeruginosa established at different dehydration time points consistent with routine cleaning and disinfection schedules recommended by the CDC [33]. In a previous study, our group developed a rapid model for establishing DSB of S aureus and P. aeruginosa at different time points and at mean log 10 densities su cient for disinfectant e cacy testing [34]. In this study, we evaluated the bactericidal e cacy of seven liquid disinfectants against DSB of S. aureus and P. aeruginosa after 24 h and 72 h of dehydration. We hypothesized that overall, hydrogen peroxide, sodium dichloro-s-triazinetrione, and quaternary ammonium compounds plus alcohol disinfectants will be more bactericidal against DSB than quaternary ammonium disinfectants based on our prior work. We also hypothesized that regardless of differences in product chemistries, higher bactericidal e cacies against DSB will be exhibited after 24 h of bio lm dehydration compared to 72 h of dehydration.
Methods Bacteria strains and disinfectants tested in this study DSB of S. aureus ATTC-6538 and P. aeruginosa ATCC-15442 were established on borosilicate glass coupons (1.27 ± 0.013 cm; Biosurface Tech, Inc.) following Nkemngong et al., 2020 [34]. These strains were selected as they are standard strains of choice for disinfectant e cacy testing [31]. They are also the standard EPA strains for registering disinfectants with claims against wet surface bacterial bio lms [35].  [34]. Wet surface bio lms were established following EPA-MLB-SOP-MB-19 through batch and continuous stir tank reactor (CSTR) phases [35]. The batch medium was 3.0 g/L TSB for S. aureus and 300 mg/L TSB for P. aeruginosa. A 500 ml batch medium held in a CDC bio lm reactor (Biosurfaces Technologies, Inc., Bozeman, MT) was inoculated with one ml of an overnight culture of S. aureus or P. aeruginosa. The batch phase lasted 24 ± 2 h with the CDC bio lm reactor (Biosurfaces Technologies, Inc., Bozeman, MT) mounted on a magnetic hot plate stirrer (Talbays, Thorofare, NJ) set at 60 ± 5 rpm at 36 ± 1°C for S. aureus or 125 ± 5 rpm at 21 ± 2°C for P. aeruginosa. CSTR medium in 20 L of sterile distilled water had a nal concentration of 1.0 g/L TSB for S. aureus and 100 mg/L TSB for P. aeruginosa. CSTR medium was continuously pumped through the CDC bio lm reactor for 24 ± 2 h for both strains.
After wet surface bio lms were established through the batch and CSTR phases, rods from the CDC bio lm reactor; each holding three borosilicate glass coupons were dehydrated for 24 h and 72 h at 25°C or 21°C for S. aureus and P. aeruginosa, respectively. Dry times and dehydration temperatures were informed by Nkemngong et al., 2020 [34].  [34]. Post treatment with disinfectants, DSB of S. aureus or P. aeruginosa were vacuum-ltered onto lter membranes following EPA-MLB-SOP-MB-20 [35]. Negative controls were spread plated following EPA-MLB-SOP-MB-20 [35]. Eight biological replicates were completed for QA and QT products and ve biological replicates for CL, SH and HP products as informed by Lineback et al., 2018 [36].

Statistical analysis
Log 10 reductions resulting from the treatment of coupons with DSB were calculated and used for statistical analyses. Speci cally, mean bacterial log 10 densities per coupon were calculated for disinfectant and PBS-treated coupons. Mean log 10 densities per disinfectant-treated coupon were normalized against the mean log 10 densities of control coupons to determine log 10 reductions. The least squares method of the PROC GLIMMIX procedure was used to analyze and compare mean log 10 reductions (n=70 per strain; N=140; α=0.05) among the seven tested disinfectant products. The same test was used to statistically compare mean log 10 reductions at 24 h and 72 h. Pair-wise comparisons among products, strains, and dry times were completed with Tukey adjustments. All statistical procedures were completed using SAS version 9.4 (SAS Institute, Cary, NC).  Figure 1).

Results
The average log 10 densities of P. aeruginosa DSB per coupon pre-treatment were 7.40 ± 0.75 and 6.77 ± 0.61 after 24 h and 72 h dry times, respectively. There were no signi cant differences between the average log 10 density per coupon after 24 h and 72 h of dehydration (P 0.005).
On average, the mean log 10 reduction per coupon for all tested disinfectants after 24 h and 72 h were 5.50 ± 1.45 and 4.65 ± 1.63, respectively.
Overall and regardless of the product type or active ingredient class, signi cantly higher bactericidal e cacies against DSB of P. aeruginosa were recorded after 24 h compared to 72 h of dehydration (P<0.05; Figure 1).
Mean log 10 reductions for P. aeruginosa DSB were higher for oxidizing agents compared to quaternary ammonium products The mean log 10 density of P. aeruginosa DSB per coupon was 7.08 ± 0.75 after dehydration (24 h and 72 h) and pre-treatment. Overall, product type and active ingredient class were signi cant (P<0.0001; Figure 2 Figure 2). Similarly, CL (5.79 ± 1.40) and QA2 (5.85 ± 0.87) had signi cantly higher log 10 reductions against P. aeruginosa DSB than QA1, QT and SH (P<0.05; Figure 2). However, there were no statistically signi cant differences among QA1, QT and SH (P 0.05; Figure 2). There were also no statistically signi cant differences in the bactericidal e cacies of HP1, HP2, CL and QA2 (P 0.05; Figure 2).
There were statistically signi cant differences among active ingredient classes (CL, HP, SH, QA and QT (P<0.0001; Figure 3). Overall, HP products resulted in a signi cantly higher bactericidal e cacy than QT and SH products (P<0.05; Figure 3). Similarly, CL and QA products had signi cantly higher mean log 10 reductions than QT and SH products (P<0.05; Figure 3). However, there were no differences between QT and SH, CL and HP, HP and QA, and QA and SH products (P 0.05; Figure 3).
Higher bactericidal e cacy against S. aureus DSB than P. aeruginosa DSB Overall, and regardless of the product type, the bacterial strain was statistically signi cant (P P<0.05). The overall mean log 10 reductions for S. aureus and P. aeruginosa were 6.096 ± 1.251 and 4.941 ± 1.505 respectively. Signi cantly higher log 10 reductions were observed when the tested disinfectants were challenged with S. aureus compared to P. aeruginosa (P<0.05).

Discussion
In this study, we employed a rapid DSB model previously developed by our group for disinfectant e cacy testing and evaluated the bactericidal e cacy of seven EPA-registered disinfectants against 24 h and 72 h old DSB of S. aureus and P. aeruginosa. Speci cally, we established DSB of S. aureus and P. aeruginosa at 25°C and 21°C respectively to mimic environmental conditions for the formation of DSB on dry contaminated hard non-porous surfaces in healthcare facilities.
We found that mean log 10 densities per coupon from this study were comparable to the ranges previously reported by Nkemngong et al., 2020 [34]. We found that overall and irrespective of dry time, CL, SH, HP and QA disinfectants were signi cantly more bactericidal against DSB of S. aureus than QT disinfectants. We also found that when DSB of P. aeruginosa were challenged with disinfectants, CL and HP were signi cantly more bactericidal than SH and QT disinfectants. Overall, we demonstrated that prolonged dehydration had varied effects on the bactericidal e cacy of disinfectants against DSB of S. aureus or P. aeruginosa. Speci cally, we found that there were no signi cant differences in the bactericidal e cacies of disinfectants against 24 h and 72 h DSB of S. aureus. There was however, a signi cantly lower log 10 reduction against 72 h DSB of P. aeruginosa compared to 24 h DSB of the same strain.

Bactericidal e cacy varies by strain after prolonged dehydration
Our study found differences in the overall bactericidal e cacy of disinfectants against DSB of S. aureus and P. aeruginosa after prolonged dehydration for 24 h and 72 h. While there was no signi cant difference in log 10 reductions between 24 h and 72 h DSB of S. aureus, the reverse was true for DSB of P. aeruginosa as 72 h DSB of P. aeruginosa were harder to kill than their 24 h counterparts. In a previous study by our group, we found that 100% of P. aeruginosa DSB established at a dehydration temperature of 21°C were encased in EPS while this was true for only 92% of S. aureus DSB established at 25°C [34]. The consistent presence of EPS on DSB of P. aeruginosa at dehydration time points from 24 h to 120 h as previously demonstrated by our group suggested that older DSB of P. aeruginosa developed using our model may be encased in more EPS; making them harder to kill [34]. This is consistent with previous studies that have demonstrated the presence of a thick EPS matrix as a major factor for reduced bactericidal e cacy in bio lms compared to planktonic bacteria [29]. Moreover, previous studies [26,37] have also suggested that unfavorable conditions such as dehydration may trigger bacterial bio lms to produce more EPS. While this may be true for P. aeruginosa DSB as evidenced in our previous study, the same may not be the case for S. aureus DSB as we found that older S. aureus DSB (72 h) were overall encased in less EPS matrix than 24 h bio lms [34]. More EPS production translates into a thicker barrier for disinfectants to bypass before contact with underlying bacteria. Additionally, a thicker EPS matrix may also result in a range of pH, which can impact bactericidal e cacy [18]. These factors could account for the reduced bactericidal e cacy against 72 h DSB of P. aeruginosa compared to 72 h DSB of S. aureus.
Product type and class signi cantly impact disinfectant e cacy against S. aureus DSB There were signi cant differences among products, with QA1, QA2, CL, SH and HP1 being more bactericidal than QT. In a related study against S. aureus wet surface bio lms, Lineback et al., demonstrated that one sodium hypochlorite and ve hydrogen peroxide disinfectants were signi cantly more bactericidal than two quaternary ammonium compounds [36]. This could be explained by the production of reactive oxygen species (ROS) by hydrogen peroxide disinfectants. The production of ROS results in more necrotic death compared to quaternary ammonium compounds as ROS result in DNA damage [38]. Comparatively, quaternary ammonium compounds mainly rely on a positively charged N-atom to bind to cell membranes, creating "pores" for n-alkyl side chains to transverse the cell membrane resulting in lysis and leakage of cytoplasmic contents [39,40]. Considering the denser EPS produced by DSB compared to wet surface bio lms, this may present a signi cant barrier for quaternary ammonium products compared to sodium dichloro-s-triazinetrione, sodium hypochlorite and hydrogen peroxides. Moreover, oxidizing agents such as sodium dichloro-s-triazinetrione, sodium hypochlorite and hydrogen peroxides have low molecular weight active ingredients that when compared to larger molecules such as quaternary ammonium, can more easily bypass the cell membrane to damage internal cellular components [38]. This could further explain the observation that sodium dichloro-s-triazinetrione, sodium hypochlorite and hydrogen peroxide products were overall more bactericidal against DSB of S. aureus than quaternary ammonium. Quaternary alcohol products may have resulted in signi cantly higher bactericidal e cacies owing to the "rapid" bactericidal mode of action of alcohol [41].
We also found that the mean log 10 reductions between HP1 and HP2; QA1 and QA2 were comparable when disinfectants were challenged with S. aureus DSB This nding is consistent with the ndings of Lineback et al., 2018 who reported no signi cant differences among the bactericidal e cacies of ve hydrogen peroxide products tested against S. aureus wet surface bio lms [36]. Similarly, in a recent study that evaluated the bactericidal e cacies of six disinfectant wipes against S. aureus ATTC-6538 inoculated on hard-non-porous surfaces, Voorn et al., reported no signi cant differences in the bactericidal e cacies among three hydrogen peroxide products or three quaternary alcohol products [42]. However, we found that quaternary alcohol products were overall more bactericidal than quaternary ammonium products without alcohol. This suggest that the de ned percentage of alcohol added to quaternary ammonium compounds in uences bactericidal e cacy; alcohol confers a rapid and more potent (tuberculocidal) action against bacteria [41].
HP and CL products are more bactericidal against P. aeruginosa DSB than SH, QT and QA products Overall, CL, QA2, HP1 and HP2 had signi cantly higher log 10 reductions against P. aeruginosa DSB than QA1, QT and SH. Our ndings are similar to those of West et al., who demonstrated that hydrogen peroxide-based disinfectants are overall, more bactericidal against P. aeruginosa allowed to dry on a Formica disc than quaternary ammonium disinfectants [43]. In another study, Tote et al. found that hydrogen peroxides had a stronger antibio lm activity against one day old P. aeruginosa bio lms as they were biologically active against both viable P. aeruginosa cells and their EPS matrix unlike isopropanol disinfectants [44]. The high e cacy of HP1 and HP2 compared to SH against DSB could be explained by the relatively low concentration (0.39%) of sodium hypochlorite in SH as in a 2018 study, Lineback et al. compared the bactericidal e cacies of 0.5% hydrogen peroxide and 1.312% sodium hypochlorite disinfectants against wet surface bio lms of P. aeruginosa, and found no difference in their e cacies [36]. The same intrinsic factor of a relatively low sodium hypochlorite concentration in SH may also account for the higher bactericidal e cacy of CL compared to SH as in a study by Tiwari et al., 0.60% sodium hypochlorite resulted in superior bactericidal e cacy against clinical isolates of S. aureus bio lms [45]. These reports suggest that although sodium hypochlorite is generally more bactericidal than quaternary ammoniums owing to their mode of action, the degree of disinfection is largely concentration dependent.
Although QA2 had a higher quaternary ammonium and lower alcohol content (0.76% quat + 22.5% alcohol) than QA1 (0.5% quat + 55% alcohol) ( Table 1), QA2 demonstrated a signi cantly higher kill against P. aeruginosa DSB than QA1. This suggests that the synergistic effect of quaternary ammonium compounds and alcohol in QA1 may not be su cient. Moreover, in a 2018 study by Wesgate et al., the authors reported that quaternary ammonium formulations with side alkyl chains in the C 12-16 range as is the case for QA1 were more adsorbed to different wipe material types than other formulations [46]. Consequently, and considering that wipes were "wringed" to dispense disinfectant liquid from QA1, the quaternary ammonium compound in QA1 may have been more adsorbed to the wipe material than QA1, resulting in a lower nal disinfectant liquid concentration in QA1 than QA2 [46]. P. aeruginosa DSB are harder to inactivate than S. aureus DSB Our data delineate statistically signi cant higher average log 10 reductions when disinfectants were treated against S. aureus DSB compared to P. aeruginosa DSB. Overall, the low bactericidal e cacy of disinfectants against bio lms is often linked to the EPS matrix [47]. The reduced e cacy of disinfectants, regardless of the product type, observed with Gram-negative P. aeruginosa can be partially explained by the presence of alginate, Psl, Pel [48], and extracellular DNA (eDNA) [49] as important components of the bio lm matrix characteristic of P. aeruginosa. Speci cally, the overproduction of alginates by P. aeruginosa mutants result in the formation of larger microcolonies than wildtype strains [50]. This suggests a role for alginates in decreased susceptibility to antimicrobials [51] compared to non-alginate-producing bacteria such as S. aureus [48]. Pel, on the other hand, plays a vital role in cell-to-cell interactions within these bio lms [52] and in the bio lm maturation [49]. A spike in alginate and carbohydrate production during bio lm formation and maturation confers an overall increase in the net negative charge of the EPS matrix, enhancing the electrostatic attractions between the EPS matrix and positively charged antimicrobials as quaternary ammonium compounds [47]. This limits the diffusion of cationic antimicrobials through the EPS matrix, thus shielding the underlying bacteria from direct antimicrobial contact [47]. However, the cell wall of Gram-positive bacteria such as S. aureus is essentially composed of peptidoglycan and teichoic acid and substances with high molecular weight can traverse the cell wall. [53]. This may explain the higher log 10 reductions observed against S. aureus DSB compared to P. aeruginosa DSB exposed to quaternary alcohol and quaternary ammonium products.
Our results suggest that comparatively higher mean log 10 reductions are achieved when sodium hypochlorite was challenged with S. aureus compared to P. aeruginosa DSB. This could be due to the act that negatively charged disinfectants as sodium hypochlorite destroy the cellular activity of bacterial proteins [54] and are capable of increased penetration of outer cell layers even in unionized state [53]. Similarly, hydroxyl free radicals from HP based products speci cally target sulfhydryl groups, double bonds [55] and destroy bacterial lipids, proteins, and DNA. Our data is in accordance with Lineback et al., 2018 who suggested that sodium hypochlorite products are overall, more effective against P. aeruginosa and S. aureus WSB compared to quaternary ammonium products [36].
Our results support previous ndings that DSB are harder to kill than planktonic bacteria; all the products tested in this study are EPA registered, indicating high levels of e cacy against planktonic bacteria of S. aureus and P. aeruginosa. To reduce patient safety risks in healthcare facilities, it is critical to conduct baseline disinfectant e cacy testing for product registration using bacteria bio lms representing healthcare environments.
We acknowledge that the scope of our study is limited as we did not investigate the bactericidal e cacy of the tested products against mixed culture bacterial bio lms common on dry contaminated hard-non-porous surfaces in healthcare facilities. We also acknowledge that our study did not speci cally investigate disinfectant e cacy against DSB of S. aureus and P. aeruginosa subjected to longer hours of dehydration as this could impact the e cacy levels of commonly used disinfectants. A wider range of disinfectant active ingredients could have also been investigated. However, this study has set the foundation for future investigations of DSB of S. aureus and P. aeruginosa.

Conclusion
Although it is generally agreed that DSB pose a severe challenge for the disinfection of hard non-porous surfaces in healthcare facilities and are a signi cant contributor to the incidence of HAI, the success of any disinfection regime is dependent on multiple intrinsic and extrinsic factors. Our study de nitively demonstrated that signi cant kill levels of the DSB of major healthcare pathogens that cause HAI can be achieved although this is highly dependent on the choice of disinfectant, active ingredient class, DSB "age" and bacteria strain. It is therefore critical for healthcare stakeholders to consider these factors in efforts to reduce HAI rates.