In this study, we employed a rapid DSB model previously developed by our group for disinfectant efficacy testing and evaluated the bactericidal efficacy of seven EPA-registered disinfectants against 24 h and 72 h old DSB of S. aureus and P. aeruginosa. Specifically, 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 log10 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 significantly 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 significantly more bactericidal than SH and QT disinfectants. Overall, we demonstrated that prolonged dehydration had varied effects on the bactericidal efficacy of disinfectants against DSB of S. aureus or P. aeruginosa. Specifically, we found that there were no significant differences in the bactericidal efficacies of disinfectants against 24 h and 72 h DSB of S. aureus. There was however, a significantly lower log10 reduction against 72 h DSB of P. aeruginosa compared to 24 h DSB of the same strain.
Bactericidal efficacy varies by strain after prolonged dehydration
Our study found differences in the overall bactericidal efficacy of disinfectants against DSB of S. aureus and P. aeruginosa after prolonged dehydration for 24 h and 72 h. While there was no significant difference in log10 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 efficacy in biofilms compared to planktonic bacteria [29]. Moreover, previous studies [26, 37] have also suggested that unfavorable conditions such as dehydration may trigger bacterial biofilms 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 biofilms [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 efficacy [18]. These factors could account for the reduced bactericidal efficacy against 72 h DSB of P. aeruginosa compared to 72 h DSB of S. aureus.
Product type and class significantly impact disinfectant efficacy against S. aureus DSB
There were significant differences among products, with QA1, QA2, CL, SH and HP1 being more bactericidal than QT. In a related study against S. aureus wet surface biofilms, Lineback et al., demonstrated that one sodium hypochlorite and five hydrogen peroxide disinfectants were significantly 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 biofilms, this may present a significant 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 significantly higher bactericidal efficacies owing to the “rapid” bactericidal mode of action of alcohol [41].
We also found that the mean log10 reductions between HP1 and HP2; QA1 and QA2 were comparable when disinfectants were challenged with S. aureus DSB This finding is consistent with the findings of Lineback et al., 2018 who reported no significant differences among the bactericidal efficacies of five hydrogen peroxide products tested against S. aureus wet surface biofilms [36]. Similarly, in a recent study that evaluated the bactericidal efficacies of six disinfectant wipes against S. aureus ATTC-6538 inoculated on hard-non-porous surfaces, Voorn et al., reported no significant differences in the bactericidal efficacies 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 defined percentage of alcohol added to quaternary ammonium compounds influences bactericidal efficacy; 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 significantly higher log10 reductions against P. aeruginosa DSB than QA1, QT and SH. Our findings 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 antibiofilm activity against one day old P. aeruginosa biofilms as they were biologically active against both viable P. aeruginosa cells and their EPS matrix unlike isopropanol disinfectants [44]. The high efficacy 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 efficacies of 0.5% hydrogen peroxide and 1.312% sodium hypochlorite disinfectants against wet surface biofilms of P. aeruginosa, and found no difference in their efficacies [36]. The same intrinsic factor of a relatively low sodium hypochlorite concentration in SH may also account for the higher bactericidal efficacy of CL compared to SH as in a study by Tiwari et al., 0.60% sodium hypochlorite resulted in superior bactericidal efficacy against clinical isolates of S. aureus biofilms [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 significantly 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 sufficient. Moreover, in a 2018 study by Wesgate et al., the authors reported that quaternary ammonium formulations with side alkyl chains in the C12-16 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 final disinfectant liquid concentration in QA1 than QA2 [46].
P. aeruginosa DSB are harder to inactivate than S. aureus DSB
Our data delineate statistically significant higher average log10 reductions when disinfectants were treated against S. aureus DSB compared to P. aeruginosa DSB. Overall, the low bactericidal efficacy of disinfectants against biofilms is often linked to the EPS matrix [47]. The reduced efficacy 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 biofilm matrix characteristic of P. aeruginosa. Specifically, 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 biofilms [52] and in the biofilm maturation [49]. A spike in alginate and carbohydrate production during biofilm 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 log10 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 log10 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 specifically 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 findings that DSB are harder to kill than planktonic bacteria; all the products tested in this study are EPA registered, indicating high levels of efficacy against planktonic bacteria of S. aureus and P. aeruginosa. To reduce patient safety risks in healthcare facilities, it is critical to conduct baseline disinfectant efficacy testing for product registration using bacteria biofilms representing healthcare environments.
We acknowledge that the scope of our study is limited as we did not investigate the bactericidal efficacy of the tested products against mixed culture bacterial biofilms common on dry contaminated hard-non-porous surfaces in healthcare facilities. We also acknowledge that our study did not specifically investigate disinfectant efficacy against DSB of S. aureus and P. aeruginosa subjected to longer hours of dehydration as this could impact the efficacy 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.