Infect Chemother. 2024 Dec;56(4):522-533. English.
Published online Nov 26, 2024.
© 2024 by The Korean Society of Infectious Diseases, Korean Society for Antimicrobial Therapy, The Korean Society for AIDS, and Korean Society of Pediatric Infectious Diseases
Original Article

Effect of Antimicrobial Wipes on Hospital-Associated Bacterial and Fungal Strains

Hye-Sun Chun,1 Chulmin Park,1 Dukhee Nho,1,2 Raeseok Lee,1,2 Sung-Yeon Cho,1,2 Chang-Joo Kim,3 view all
    • 1Vaccine Bio Research Institute, College of Medicine, The Catholic University of Korea Seoul, Korea.
    • 2Division of Infectious Diseases, Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea.
    • 3R2ELab, Seoul, Korea.
Received August 21, 2024; Accepted November 05, 2024.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Healthcare-associated infections (HAI) caused by multidrug-resistant organisms have emerged as a significant global issue, posing substantial challenges to healthcare systems. Low- and intermediate-level disinfectants are extensively utilized for cleaning and disinfecting surfaces in hospitals to mitigate environmental transmission of HAI. Therefore, the need for more effective and environmentally safe disinfectants is increasing. This study aimed to assess the effect of antimicrobial wipes used for surface cleaning and disinfection in healthcare environments.

Materials and Methods

A microbe library comprising 188 bacterial and fungal isolates, including multidrug-resistant strains, was established and used to evaluate the antimicrobial effect of three types of antimicrobial wipes: A (didecyldimethylammonium chloride [DDAC] 0.31% and 3-(trimethoxysilyl)-propyldimethyloctadecyl ammonium chloride [Si-QAC] 0.45%); B (benzalkonium chloride [BAK] 0.63%); and C (DDAC 0.5% and BAK 0.9%). The antimicrobial effect of the wipes was assessed and compared in three assays: rapid bactericidal effect assay of the three wipes, minimum inhibitory concentration (MIC) assay of DDAC and BAK, and a time-kill assay of the DDAC and Si-QAC combination.

Results

The rapid antimicrobial effect evaluation showed that both wipes A and C, which contain a combination of two quaternary ammonium compounds (QACs), exhibited similar antimicrobial effect (P=0.8234). Antimicrobial wipe A demonstrated better effect against Gram-positive bacteria and fungi than wipe C (P <0.05). The antimicrobial efficacy of the A wipe against Mycobacterium strains was superior to that of both the B and C wipes. Moreover, DDAC exhibited MIC50 values that were 2 to 3-fold lower than those of BAK for Gram-negative bacteria and fungi. The time-kill assay results for the DDAC and Si-QAC combination exhibited a growth reduction of >3 logs for Staphylococcus aureus and Enterococcus faecium, whereas approximately 2 logs of reduction was observed for Escherichia coli and Pseudomonas aeruginosa at 3 hour.

Conclusion

The results suggest that antimicrobial wipes containing relatively lower concentrations of QAC (wipe A) achieve similar rapid bactericidal effect as that of those with higher concentrations (wipe C). For Gram-negative bacteria, including multidrug-resistant strains and fungal isolates, DDAC presented lower MICs compared with BAK. Furthermore, the combination therapy with DDAC and Si-QAC demonstrated enhanced efficacy compared to treatment with either agent alone, except in the case of Klebsiella strains. Further research is needed to develop antimicrobial wipes that minimize the environmental impact while ensuring effective disinfection.

Keywords
Health care associated infections; Quaternary ammonium compounds; Multiple drug resistance

Environmental contamination in healthcare settings is strongly associated with the incidence of hospital-associated infections (HAI) caused by key pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE), Clostridioides difficile, carbapenemase-producing carbapenem-resistant Enterobacteriaceae (CP-CRE), and Acinetobacter baumannii. Among these pathogens, multidrug-resistant organisms are associated with treatment failure in patients with underlying diseases. Hand hygiene and environmental cleaning are essential measures for preventing transmission of these infections [1, 2, 3]. Environmental cleaning using commercial surface disinfectant wipes is widely used to mitigate cross-infection and transmission of HAI associated with hospital environments or shared medical equipment.

Hospital surface disinfection predominantly employs low- and intermediate-level disinfectants including quaternary ammonium compounds (QAC), ethyl or isopropyl alcohol, chlorine-releasing agents, and enhanced hydrogen peroxide (3% concentration) [4]. Unlike flammable substances (alcohol), corrosive agents (chlorine-releasing agents), or those with material compatibility concerns (hydrogen peroxide, chlorine, and alcohol), high cost (hydrogen peroxide), and high toxicity (chlorine), QAC disinfectants are extensively utilized in hospital settings. Despite their relatively narrow spectrum, their wide material compatibility, low cost, and non-corrosive properties make them popular [5].

QACs include benzalkonium chloride (BAK), didecyldimethylammonium chloride (DDAC), and 3-propyldimethyloctadecyl ammonium chloride (Si-QAC) [1]. BAK is commonly used as a preservative in ophthalmic formulations or detergents, where it disrupts cell membranes by interacting with lipid components, exhibiting antimicrobial activity in vitro [6, 7]. DDAC, known for its strong initial adsorption onto cell walls, offers rapid bactericidal effects and is widely used for disinfection in various settings, such as restaurants, hospitals, and factories [8, 9]. Si-QAC, a novel organosilane compound, adheres to surfaces such as steel, glass, and plastic, thereby providing effective disinfection [10, 11, 12]. However, due to environmental concerns, BAK is now subject to stringent regulatory standards (Ministry of Environment Notification No. 2024-89) [13]. Increased environmental exposure to BAK has led to the emergence of resistant bacteria [14], posing substantial risks to human health and ecosystems [15]. This has driven the development of disinfectants with enhanced antimicrobial effect and safety for human health [12].

This study aimed to evaluate and compare the antimicrobial effect of three widely available antimicrobial wipes for hospital surface disinfection, focusing on their effect against multidrug-resistant bacteria from hospitals and environmental sources. Additionally, this study sought to assess the effect of different QACs to guide the selection of the most appropriate formulation and evaluate combinations of QACs using time-kill assays to improve antimicrobial effect.

1. A library of multidrug-resistant strains and pathogenic strains

A total of 188 multidrug-resistant bacterial and fungal strains were assembled into a multidrug-resistant strain library, using strains isolated from the clinical and environmental settings of a tertiary university hospital, the Korean Collection for Type Cultures, and the National Culture Collection for Pathogens (Table 1). The strains included 61 Gram-positive and 86 Gram-negative bacteria, including resistant pathogens; 4 non-tuberculous Mycobacterium species; and 37 fungal isolates.

Table 1
The list of bacteria and fungi in the microbe library used in this study, including multi-drug resistant and susceptible strains

Most bacterial strains were cultivated on tryptic soy agar (TSA, BD Korea, Seoul, Korea) or in tryptic soy broth (TSB, BD Korea) at 35°C. Streptococcus and Listeria strains were inoculated onto blood agar plates (BAP, Duksan, Korea) and incubated for 24 h in a 5% CO2 incubator. Legionella was cultured on BCYEagar (MBcell, Seoul, Korea), while fungi including Candida were incubated on Sabouraud dextrose agar (BD Korea) for a period exceeding 48 h. Clostridium was cultivated anaerobically under limited-air conditions at 35°C in a CO2 incubator using reinforced Clostridial Medium (BD Korea) for over one week. Following confirmation of endospore formation, cultures were resuspended in distilled water for further experimentation. Bacillus species were incubated on nutrient agar (BD Korea) at 35°C for over one week. Upon confirmation of endospore formation, the cultures were resuspended in distilled water for further experimental procedures [16]. Mycobacterium species were cultured on 7H10 agar (BD Korea) at 35°C in an incubator for more than 14 days before being used for experimentation.

2. Ethics statement

This study was approved by the Institutional Review Board of Seoul St. Mary’s Hospital (No. KC24EIDI0125), which waived the requirement for informed consent because of the anonymous nature of the bacterial and fungal isolates.

3. Rapid antibacterial surface test of anti-microbial wipes against HAI-associated strains.

Three commercially available antimicrobial wipes were selected: A (Bio Spike Guard, Chong Kun Dang Pharm, Seoul, Korea: DDAC, 0.31% and Si-QAC, 0.45%), B (BAK, 0.63%), and C (DDAC, 0.5% and BAK, 0.9%) [17].

After a 24-h incubation of bacterial strains in 3 ml of TSB or on BAP or BCYE agar, the suspensions were adjusted to a McFarland 0.5 standard (1×108 CFU/mL). The adjusted suspensions were diluted 100-fold with 0.85% NaCl to a final concentration of 1×106 CFU/mL.

For experimental purposes, each antimicrobial wipe was divided into one-eighth sections to ensure that a uniform quantity of the disinfectant solution was applied to the surface, reflecting the actual use of these wipes. As a positive control, we prepared a 20 mg/ml stock solution of DDAC (Merck Korea, Seoul, Korea) and used 10 µL (0.2 mg) from this stock. We then prepared a 48-well plate with the following wells: a negative control well, wells cleaned with each of the three antimicrobial wipes, and positive control wells with 10 µL of DDAC. Each diluted bacterial suspension was inoculated (120 µL) into the wells. After inoculation, 3 µL samples were collected at each surface contact time point (5 min, 15 min, 30 min, and 1 h) and plated onto square TSA plates. The plates were then incubated for 24 h, and bacterial growth was assessed visually. Growth indices were defined according to the growth rate compared to that of untreated disinfectant wipes (1, no growth; 2, 0–10% growth; 3, 10–60% growth; 4, 60–90% growth; and 5, 90–100% growth).

4. Minimum inhibitory concentration (MIC) of QACs (DDAC, BAK)

MICs were determined using the broth microdilution method, following the guidelines established by the Clinical and Laboratory Standards Institute [1820]. Disinfectant susceptibility testing was performed on 175 strains selected from our library, as previously described. The cultivation protocols for these strains were the same as those used for the rapid bactericidal testing.

Stock solutions of the QACs were prepared with DDAC at 20,000 µg/mL and BAK (Merck Korea) at 53,000 µg/mL. Dilution series ranging from 1 to 128 µg/mLwere prepared. Each disinfectant (100 µL) was added to a 96-well plate. The bacterial suspension was diluted 100-fold to a concentration of 1×106, and 100 µL of this dilution was added to each well. The plates were then incubated at 37°C for 24 h. The disinfectants and bacterial suspensions were diluted in Mueller–Hinton broth (BD Korea). After incubation, MIC was visually assessed. The MIC50 values for each bacterial strain were determined, and the differences in susceptibility were compared between the two disinfectants. MIC50 was defined as the concentration at which 50% of the strains for a species exhibited inhibition.

5. Time-kill test of SI-QAC and DDAC combination

All the relevant information and technology for the combined synthesis of DDAC and Si-QAC to increase bactericidal and antimicrobial performance were provided solely by R2ELab Inc. Based on the rapid antimicrobial results against multidrug resistant strains, two representative strains each of Gram-negative (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa) and Gram-positive bacteria (S. aureus and Enterococcus faecium) were selected. The strains were selected based on their specific characteristics as determined in prior experiments. The selected E. coli strains were CRE (CV_BS_EC1) and non-CRE (CV_BS_EC7). For K. pneumoniae, the selected strains were CRE (CV_BS_K14) and CRE (CV_BS_K15). The P. aeruginosa strains included carbapenem-resistant Pseudomonas (CRP) (CV_BS_P29) and non-CRP (CV_BS_P34). For S. aureus, the strains selected were MRSA (CV_BS_S48) and vancomycin-intermediate S. aureus (VISA) (CV_BS_S60). The E. faecium strains were VSE (CV_BS_EF63) and VRE (CV_BS_EF65). The cultures were incubated in 3 ml of TSB for 24 h and then diluted in tryptone solution chloride solution (TSCS) medium to achieve McFarland 0.5 (1×106 CFU/ml). When using QAC alone, a stock solution of 20,000 µg/mL DDAC was prepared and inoculated to achieve a final concentration of 1/2 MIC. SI-QAC was coated onto a 6-well plate and dried before use. For the combination therapy, wells were first coated with Si-QAC and allowed to dry. Then, 10 µL of DDAC at a final concentration of 1/2 MIC was added to achieve the combination. Each well of a 6-well plate, prepared according to each treatment, was inoculated with 1 mL of the diluted bacterial suspension. The plate was incubated at 37°C, and 100 µL samples were taken at 15 min, 30 min, 1 h, 2 h, and 3 h. The remaining disinfectant was neutralized before use. Neutralization was performed using membrane filtration with an Amicon Ultra 100 k limit 0.5 mL filter (Millipore, Merck Korea), and TSCS was used as the diluent [21]. The neutralized samples were serially diluted in phosphate buffer solution (PBS, Merck Korea, Seoul, Korea), and 10 µL of each dilution was spread onto square TSA agar plates. After incubation for 24 h, the viable cell counts were measured. Experimental results were obtained from triplicate measurements, with final values reported as the mean.

6. Statistical analysis

All statistical analyses were performed using Prism (version 10, GraphPad Software LLC.; https://www.graphpad.com). The rapid bactericidal effect results of the antimicrobial wipes were compared using a paired t-test with two-tailed P-values, and statistical significance was established at P <0.05. The time-kill curve was analyzed using simple linear regression.

1. Rapid antibacterial evaluation of anti-microbial wipes against HAI-associated strains.

At 15 min, the comparison between Wipes A and C did not reveal a significant difference in antimicrobial activity (P=0.0823). In contrast, significant differences were observed between Wipes A and B (P <0.0001) and between Wipes B and C (P <0.0001). At 30 min, no significant difference was observed between Wipes A and C (P=0.3160). However, significant differences were noted between Wipes A and B (P <0.0001) and between Wipes B and C (P <0.0001). After 1 h, Wipes A and C showed no significant difference (P=0.8234), whereas significant differences were observed between Wipes A and B (P <0.0001) and between Wipes B and C (P <0.0001). Overall, Wipes A and C exhibited comparable antimicrobial effect (Fig. 1 A, B, and C).

Figure 1
Analysis of rapid antimicrobial effects of disinfectant wipes on pathogens in the microbe library. Wipes A includes DDAC 0.31% & Si-QAC 0.45%, Wipes B include BAK 0.63%, and Wipes C includes DDAC 0.5% and BAK 0.9%. Relative microbial growth was measured in duplicates. The growth indices on Y-axis were defined according to relative microbial growth ratio to that of untreated disinfectant wipes (1, no growth; 2, 0–10% growth; 3, 10–60% growth, 4, 60–90% growth; 5, 90–100% growth).
aindicates P <0.0001, bP <0.01, cP <0.05, and not significant>0.05. (A) Time-point evaluation of microbial growth of pathogens from the microbe library after disinfectant wipes treatment (n=156) at 15 min, (B) 30 min, and (C) 60 min. (D) Time-point evaluation of relative growth of molds from the microbe library after disinfectant wipes treatment (n=30) at 15 min, (E) 30 min, and (F) 60 min.

DDAC, didecyldimethylammonium chloride; Si-QAC, 3-(trimethoxysilyl)-propyldimethyloctadecyl ammonium chloride; BAK, benzalkonium chloride.

When evaluating the effect on E. coli strains, Wipe A demonstrated 99% antimicrobial effect against 46% (6/13) of the strains at 15 min, which increased to 61% (8/13) at 1 h. For Klebsiella strains, Wipe A exhibited 99% antimicrobial effect against 69% (9/13) of the strains at 15 min and 100% (13/13) at 1 h. After 1 h of exposure to Wipe B, only 15% (2/13) of E. coli strains and 54% (7/13) of Klebsiella strains exhibited 99% effect. Wipe C demonstrated 99% bactericidal effect against 92% (12/13) of both E. coli and Klebsiella strains within 5 min. For Pseudomonas strains, Wipe A showed 99% bactericidal effect at 50% (5/10) after 15 min, which increased to 70% (7/10) at 1 h. Wipe B achieved 99% effect against 50% (5/10) of both strains at 1 h. Wipe C reached 99% effect in 60% (6/10) of the cases at 15 min, rising to 70% (7/10) at 1 h. For Acinetobacter strains, Wipe A showed 99% effect against 90% (9/10) of the strains at 15 min and 100% (10/10) at 1 h. Wipe B achieved 99% effect against 50% (5/10) of the strains at 1 h. Wipe C demonstrated 99% antimicrobial effect at 100% (10/10) within 5 min. (Supplementary Table 1).

For Streptococcus strains, Wipes A and C exhibited 99% antimicrobial effect within 5 min for 100% (11/11) of the strains, whereas Wipe B demonstrated 99% antimicrobial effect for 82% (9/11) of the strains. For Staphylococcus strains, Wipe A showed 99% antimicrobial effect for 93% (14/15) of the strains within 5 min. Wipe B exhibited 99% antimicrobial effect against 47% (7/15) of the strains at 15 min, which increased to 67% (10/15) at 1 h. Wipe C achieved 99% antimicrobial effect against 100% (11/11) of the strains from 5 min onward. For Enterococcus strains, Wipe A demonstrated 99% effect against 94% (15/16) of the strains at 15 min and 100% (16/16) at 1 h. Wipe B achieved 99% antimicrobial effect against 31% (5/16) of the strains at 1 h. Wipe C exhibited 99% antimicrobial effect against 100% (16/16) of the strains from 5 min onward.

Regarding the antimicrobial effect of Wipes A and C against mold over time, Wipe A demonstrated significantly greater effect than Wipe C at 15 min (P=0.0043), 30 min (P=0.0029), and 1 h (P=0.0388). Wipe A achieved 99% antimicrobial effect against 37% (10/27) of the strains after 15 min, which increased to 82% (22/27) after 1 h. In comparison, Wipe C showed 99% antimicrobial effect against 30% (8/27) of the strains at 15 min and against 67% (18/27) of the strains at 1 h (Fig. 1 D, E, and F). The analysis for the B wipe was conducted, but it was excluded from further consideration due to its lack of antimicrobial effect (data not shown).

Wipe A also exhibited superior antimicrobial effect against spore-forming Clostridium and Bacillus species, as well as Mycobacterium species (Table 2). In contrast, Wipes B and C showed no effect against Bacillus cereus within 30 min and against M. avium for up to 1 h. Furthermore, none of the wipes, including Wipe A, were effective against M. fortuitum (Table 2).

Table 2
Rapid antimicrobial effects of disinfectant wipes on endospore-forming bacteria and Mycobacteriuma

2. MIC of QACs (DDAC, BAK)

For E. coli, the MIC50 values were 4 µg/mL for DDAC and 32 µg/mL for BAK. Klebsiella strains showed MIC50 values of 8 µg/mL for DDAC and 32 µg/mL for BAK, P. aeruginosa had 8 µg/mL for DDAC and 64 µg/mL for BAK, and A. baumannii demonstrated 4 µg/mL for DDAC and 16 µg/mL for BAK. Overall, the MIC values for DDAC were two to three times lower than those for BAK. Notably, DDAC exhibited consistently lower MIC values than BAK for Gram-negative bacteria, with differences reaching up to 3-fold. Regarding S. aureus, the MIC50 values were 2 µg/mL for both DDAC and BAK; for Enterococcus sp. strains, the MIC50 were 2 µg/mL for DDAC and 4 µg/mL for BAK; for S. epidermidis, the MIC50 were 2 µg/mL for both DDAC and BAK; for Streptococcus strains, the MIC50 were 2 µg/mL for both DDAC and BAK; and for L. monocytogenes, the MIC50 were 2 µg/mL for DDAC and 4 µg/mL for BAK. For Gram-positive bacteria, the MIC50 values for DDAC were comparable to or slightly lower than those for BAK (Table 3).

Table 3
Analysis of the-MIC values of didecyldimethylammonium and benzalkonium chloride in the microbe librarya

Analysis of MIC50 values showed that Aspergillus strains had MIC50 of 2 µg/mL for DDAC and 8 µg/mL for BAK; Fusarium and Mucorales strains demonstrated MIC50 of 4 µg/mL for DDAC and 16 µg/mL for BAK; and Candida sp. strains had MIC50 of 2 µg/mL for DDAC and 8 µg/mL for BAK. For fungi, DDAC demonstrated lower MIC values than BAK, similar to the findings for Gram-negative bacteria, with a typical difference of approximately 2-fold (Table 3).

3. Time-kill test of DDAC and Si-QAC

In the time-kill analysis against E. coli, DDAC+Si-QAC treatment in the CRE strain (CV_BS_EC1) showed a 2-log reduction in the log10 value, decreasing from 6.24 at 0 h to 4.23 at 3 h. For strain non-CRE (CV_BS_EC7), the treatment of DDAC+Si-QAC showed a 3-log reduction in the log10 value, decreasing from 6.23 at 0 h to 3.18 at 3 h. In contrast, the time-kill results for K. pneumoniae indicated that for strain CRE (CV_BS_K14), there was no change in log10 values in DDAC+Si-QAC from 6.19 at 0 h to 6.23 at 3 h, indicating that there was indifference effect. Similarly, for strain CRE (CV_BS_K15), an indifference effect was observed with a log10 value of 6.81 at 0 h to 7.01 after 3 h in DDAC+Si-QAC. The time-kill results for carbapenem resistant P. aeruginosa CV_BS_P29 strain showed that the log10 value decreased by 1 log from 6.27 at 0 h to 5.19 after 3 h upon DDAC+Si-QAC treatment. For strain carbapenem susceptible P. aeruginosa (CV_BS_P34), the log10 value upon treatment with DDAC+Si-QA appeared from 6.63 at 0 h to 4.40 after 3 h, indicating a 2 log reduction (Fig. 2A–F).

Figure 2
The time-kill analysis of multidrug-resistant and susceptible strains treated with disinfectants (comprising both DDAC and Si-QAC or alone). The live bacterial counts were performed at 0 min, 15 min, 30 min, 1 h, 2 h, and 3 h. (A) The time-kill curves of carbapenem-susceptible Escherichia coli strain CV_BS_E7 and (B) carbapenem-resistant Escherichia coli strains CV_BS_E1 (C) The time-kill curves of carbapenem-resistant Klebsiella pneumoniae strains CV_BS_K14 and (D) CV_BS_K15 (E) The time-kill curves of carbapenem-susceptible Pseudomonas aeruginosa strain CV_BS_P34 and (F) carbapenem-resistant P. aeruginosa strains CV_BS_P29 (G) The time-kill curves of methicillin-resistant Staphylococcus aureus strain CV_BS_S48 and (H) vancomycin-intermediate S. aureus strains CV_BS_S60 (I) The time-kill curves of vancomycin-susceptible Enterococcus faecium strain CV_BS_EF63 and (J) vancomycin-resistant E. faecium strain CV_BS_EF65.
DDAC, didecyldimethylammonium chloride; Si-QAC, 3-(trimethoxysilyl)-propyldimethyloctadecyl ammonium chloride.

The time-kill results for MRSA CV_BS_S48 strain indicated that the log10 value upon DDAC + Si-QAC treatment was 6.94 at 0 h and 1.00 after 3 h, indicating a decrease of more than 5 log. For strain VISA (CV_BS_S60), the log10 value decreased from 6.27 at 0 h to 3.15 after 3 h for DDAC + Si-QAC, indicating a 3 log reduction. Results of the time-killing curve test for strain VSE (CV_BS_EF63) showed a 4 log reduction, with log10 values decreasing from 6.76 at 0 h to 2.00 after 3 h for in the treatment of DDAC + Si-QAC. Similarly, for strain VRE (CV_BS_EF65), we found that the log10 value decreased by 4 log from 6.99 at 0 h to 2.00 after 3 h upon DDAC + Si-QAC treatment (Fig. 2G–J).

The surge in disinfectant usage has been accompanied by a significant increase in cases of toxic event, with the severity of disinfectant-related toxicity exacerbated during the COVID-19 pandemic [22]. Hrubec et al. reported that 80% of the study participants had detectable levels of QACs in their blood, which was likely due to increased exposure to these compounds [23]. Among the widely used QACs, BAK has been classified by the United States Environmental Protection Agency into the following toxicity categories: category I (highly irritating to eyes and skin), category II (oral and inhalation routes), and category III (dermal route). Additionally, BAK is recognized as a hazardous substance in aquatic environments, leading to strict regulatory standards under both European and Korean legal regulations. Excessive use of disinfectants and consequent environmental exposure have also resulted in bacteria developing tolerance or resistance to BAK, which has contributed to cross-resistance to antimicrobials. This creates a vicious cycle that poses a significant threat to HAI control [14].

In the present study, we evaluated the effect of antimicrobial wipes containing varying concentrations of QACs, including formulations of one or two types of QACs, against HAI-associated strains and multidrug-resistant populations. The antimicrobial effect was evaluated using surface tests, simulating the real-world application of ready-to-use disinfecting wipes. By assessing the inhibitory concentrations of commonly used QACs, such as DDAC and BAK, we aimed to identify relatively effective QACs at concentrations that pose lower environmental risks to HAI-associated strains.

A wipe containing a formulation of DDAC and Si-QAC demonstrated antimicrobial efficacy comparable to, or in some cases superior to, wipes containing higher quantities of BAK, both with and without DDAC, in the surface test. Notably, Wipe A exhibited significantly enhanced efficacy against Gram-positive strains (Supplementary Table 1), Mycobacterium species (Table 2), and molds (P <0.05) (Fig. 1). This suggests that Si-QAC may enhance the antimicrobial effect of DDAC despite its lower concentration. We also observed some degree of antimicrobial effect against endospore-forming strains in the rapid antimicrobial test (Table 2). Endospore-forming pathogens, such as Clostridium and Bacillus, present significant challenges in disinfection due to the inherent resistance of endospores to antimicrobial agents, leading to persistent infections in healthcare settings [9]. However, inducing endospore formation and executing the associated experimental procedures are technically challenging [9]. It is well established that endospores are resistant to QACs, which are known to possess sporiostatic rather than sporicidal properties [9, 12]. We did not include a neutralization step in the rapid screening tests of this study. Consequently, the observed antimicrobial effect against endospores may reflect a sporiostatic rather than sporicidal action due to the absence of neutralization.

Subsequently, we analyzed the cumulative MIC of DDAC and BAK across populations of HAI-related microbial species. The MIC50 values for DDAC were found to be 4- and 8-fold lower than those of BAK in Gram-negative species, 2-fold lower in Gram-positive species, and 4-fold lower in fungal species (Table 3). These results indicate that BAK exhibits a higher MIC compared to DDAC in populations of HAI-associated strains, suggesting an increased potential for tolerance or resistance to BAK. This observation may reflect the possibility that hospital-associated microorganisms have developed increased tolerance due to frequent environmental exposure to BAK disinfectants, which are widely used in hospital settings.

In this study, the incorporation of Si-QAC, a surface-coating agent, in combination with DDAC, demonstrated enhanced effect not only against bacteria but also against significant pathogens such as carbapenem-resistant Enterobacteriaceae (CRE) and azole-resistant Aspergillus species (Supplementary Table 1). Si-QAC has been shown to damage bacteria upon contact with coated surfaces, thereby augmenting the effect of disinfectants [8, 10]. This approach could provide a viable alternative for safely managing hospital environments using effective QACs. Additionally, time-kill analysis revealed that the combination of DDAC with Si-QAC exhibited enhanced activity effect compared to DDAC alone, particularly in both resistant and susceptible strains of S. aureus (CV_BS_S48, CV_BS_S60) and E. faecium (CV_BS_EF, CV_BS_EF65) (Fig. 2). These findings suggest that the combinational use of these agents may reduce overall QAC usage and decrease the likelihood of resistance development.

The disinfectant products we evaluated are known to have been tested for their disinfectant effectiveness using test strains (standard strains) as part of the reporting/approval process by regulatory authorities (e.g., Ministry of Environment). The results of these tests are also specified on each product's information on the regulatory online (https://ecolife.me.go.kr/ecolife) [17]. While these test strains are widely accepted, they have limitations when it comes to evaluating effectiveness against resistant strains that pose significant clinical challenges. Therefore, in this study, we believed it would be more meaningful to assess disinfectant products that have already proven effective against test strains by evaluating them against a variety of strains that are more commonly detected in surface environments. Hence, for this study, we built a library that included a wide range of resistant strains from clinical and environmental settings for evaluation.

This study has several limitations. When conducting surface tests, various factors such as contact time, wipe material, and cross-contamination must be considered to reflect practical applicability. In this study, we primarily focused on evaluating contact time, as well as the type and concentration of disinfectants, while making efforts to minimize cross-contamination during the experimental process. However, we acknowledge a limitation in that we did not analyze other variables, such as different solvents or wipe materials. Future studies should address these factors, as well as other variables discussed in previous research [24], such as contact duration, material composition, and cross-contamination. Due to the considerable time and effort required to utilize and quantify a large number of test strains, the rapid evaluation of disinfectants was conducted without a neutralization step, which is a notable limitation. Additionally, while this study aimed to include as many HAI strains, including multidrug-resistant microorganisms, as possible, there remain limitations in the diversity and number of strains tested. Efficacy also varies slightly by surface type and wipe material. Recent findings suggest that biofilm formation on hospital surfaces reduces disinfection effectiveness; further research is needed to assess whether mixed quaternary ammonium compounds can mitigate this issue [25, 26].

In summary, the rapid antimicrobial screening results suggest that wipes containing complex QACs (Wipes A & C) exhibit greater antimicrobial activity in surface tests compared to those containing single QACs (Wipe B). Additionally, wipes with relatively lower concentrations of QACs (Wipe A) achieve a bactericidal effect comparable to those with higher concentrations (Wipe C). For HAI-associated strains, including multidrug-resistant and fungal isolates, DDAC exhibited relatively lower inhibitory concentrations compared to BAK. Furthermore, the time-kill assay results revealed that the combined use of DDAC and Si-QAC exhibited either significantly enhanced or slightly improved antimicrobial activity against E. coli, P. aeruginosa, S. aureus, and E. faecium strains used in this experiment, compared to their individual use. Additional research is warranted to develop antimicrobial wipes that minimize environmental impact while ensuring effective disinfection. Our findings suggest that the judicious use of lower concentrations of DDAC and Si-QAC would be effective against HAIs caused by multidrug-resistant bacteria and fungi. This approach could provide a novel and safer alternative for managing hospital environments and controlling HAIs.

Supplementary Table 1

Analysis of the rapid antimicrobial effect of disinfectant wipes on pathogens obtained from the microbe library.

Click here to view.(74K, xls)

Notes

Funding:This work was supported by The Catholic University of Korea Research and Development grant (No. 52024D018900001) from ChongKun Dang Pharm.

Conflict of Interest:DGL received grants through his institution’s research program funded by ChongKun Dang Pharm. Also, DGL is the editor-in-chief of the Infect Chemother; however, he was not involved in the peer reviewer selection, evaluation, or decision process for this article. CJK is the CEO of R2ELab, Inc. and declares a potential conflict of interest, as they may receive benefits related to the development of the products mentioned in this study. None of the other authors declare any conflicts of interest.

Author Contributions:

  • Conceptualization: HSC, CMP, DGL.

  • Data curation: HSC, DHN, RSL.

  • Formal analysis: HSC, CMP.

  • Methodology: HSC, CMP, CJK.

  • Software: CMP, DHN, RSL.

  • Validation: HSC, SYC.

  • Investigation: HSC, CMP, DGL.

  • Writing-original draft: HSC, CMP.

  • Writing-review & editing: HSC, CMP, DHN, RSL, SYC, CJK, DGL.

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