Abstract
Quaternary ammonium compound (QAC) disinfectants represent one of our first lines of defense against pathogens. Their inhibitory and bactericidal activities are usually tested through minimum inhibitory concentration (MIC) and time-kill assays, but these assays can become cumbersome when screening many compounds. We investigated how the dynamic surface tension (DST) measurements of QACs correlate with these antimicrobial activities by testing a panel of potent and structurally varied QACs against the gram-positive Staphylococcus aureus and the gram-negative Pseudomonas aeruginosa. We found that DST values correlated well with bactericidal activity in real-world disinfection conditions but not with MIC values. Moreover, no correlation between these two antimicrobial activities of QACs (bactericidal and inhibition) was observed. In addition, we observed that the bactericidal activity of our QAC panel against the gram-negative P. aeruginosa was severely affected in the presence of hard water. Interestingly, we found that the counterion of the QAC affects the killing of bacteria in these conditions, a phenomenon not observed in most MIC assessments. Moreover, some of our best-in-class QACs show enhanced bactericidal activity when combined with a commercially available QAC. In conclusion, we determined that an intrinsic physical property of QACs (DST) can be used as a technique to screen for bactericidal activity of QACs in conditions that mimic real-world disinfection conditions.
Keywords: bacteria, biological activity, MIC assay, time-kill assay, surfactants
Introduction
Biocides are often described as the first line of defense against pathogens and are a critical component for effective infection control and prevention. Quaternary ammonium compounds (QACs) are a common class of cationic biocides widely used in healthcare, agriculture, and household disinfection.[1] Their amphiphilic nature allows them to interact with, and disrupt, membranes resulting in antimicrobial activity against a diverse range of pathogens including bacteria, viruses, fungi, and parasites.[2] However, their effectiveness can be hampered by the effects of hard water. The Centers for Disease Control and Prevention (CDC) lists hard water as one of the physical factors affecting the potency of disinfectants, and it is believed that this is caused by ionic interactions and the formation of insoluble precipitates.[3] This effect has been noted since the late 1940s and represents a key metric for commercialization of disinfectants.[4] In addition, development of resistance to QACs has been increasingly reported, emphasizing the need for new QAC development.[5] The concomitant increase in resistance to biocides and other antimicrobials agents limit the available options for effective control and treatment of pathogens, creating a dire public health scenario.[6–7]
To expand the repertoire of available biocide agents, our group and others have developed additional classes of QACs with diverse structures, as well as their phosphorous analogs.[8–13] In identifying and developing novel antimicrobial compounds efficiently, reliable methods to test important antimicrobial activities are required. The minimum inhibitory concentration (MIC) assay is widely used and defines the lowest concentration of an antimicrobial capable of preventing visible growth of an organism under defined conditions.[14] On the other hand, suspension time-kill assays provide a test that directly measures bactericidal (or killing) activity. Nonetheless, these assays are considered time-consuming, resource-intensive and tedious.[15–16] MIC assays are usually performed with a low inoculum (5×105 CFU/mL) and long incubation time (≥ 18 hours), while suspension time-kill assays are typically performed in a timescale of minutes, oftentimes in hard water, using higher concentrations of compound and a high cell density to mimic the conditions of disinfectant usage. Ideally, a method that predicts effectiveness of QACs based on their chemical properties would facilitate the screening process of compounds before performing biological assays.
There has been a longstanding campaign to correlate antimicrobial activity with physical properties of surfactants, such as critical micelle concentration (CMC).[17–18] Laatiris and coworkers synthetized alkanediyl α,ω-bis(dimethylammonium bromide) gemini surfactants and determined their antimicrobial activity through MIC assays, and their CMC values through conductivity and surface tension measurements.[17] They found that CMC correlated with growth inhibition of S. aureus, but this correlation was absent when tested against E. coli and P. aeruginosa, two gram-negative pathogens. The authors reasoned that this limited correlation was due to the MIC values of their compounds being higher than their CMC values for these bacteria. Similarly, Viscardi and coworkers investigated the correlation of several physical parameters of surfactants to their antimicrobial activity, and found correlation of the hydrophobicity of the compound with antimicrobial activity.[19] To do this, they used the software MacLog P to calculate the C log P values and saw correlation with antimicrobial activity, as determined by suspension bactericidal assay with a 5 minute contact time. However, the lack of correlation to antimicrobial activity against gram-negative pathogens of CMC, the limited temporal resolution of experiments performed in suspension bactericidal assays and the use of specialized software for calculations, reduces the accessibility of generalizable guidelines for surfactant development.
Dynamic surface tension (DST) is another quickly measurable key physical property of amphiphiles that measures the effect of compounds on surface tension over time and can be correlated with the speed of dispersion in a solution.[20] As surface-active compounds, QACs can decrease the surface tension of solutions. In 1926, Frobisher explored how addition of ST reducer or increasing the ST-reducing capacity affected the bactericidal activity of the antiseptic 4-hexylresorcinol.[21] He found that lowering ST could increase bactericidal activity. Building upon this, we decided to explore how DST correlates with both antimicrobial activities: inhibition (measured by MIC assay) and killing (measured by time-kill assay).
The goal of this study was to investigate how the surface activity of a panel of QACs correlates with these inhibitory and bactericidal antimicrobial activities. Specifically, we evaluated how DST values correlate with MIC or log-reduction values (LRV) from time-kill assays performed in conditions mimicking real-world scenarios. In addition, we evaluated how these two antimicrobial activities of QACs correlate between each other. Through our investigations, we found that DST values correlate with LRV but not with MIC values of QACs. In addition, after analyzing the efficacy of commercially available QACs, we found evidence that the counterion of QACs affect their activity in hard water. We leverage this information to increase the effectiveness of our QACs in hard water by combining them with a rapidly diffusible QAC.
Results and Discussion
Selection of QACs and Further Chemical Characterization and Bioactivity
We selected 10 QACs based on structural diversity bioactivity (Figure 1).[22–25] These best-in-class QACs showed promising inhibitory activity against both the gram-positive S. aureus and the gram-negative P. aeruginosa relative to the benchmark and commercially available mono-QACs benzyldimethyldodecylammonium chloride (BAC) and didecyldimethylammonium chloride (DDAC). This QAC panel has a median MIC value of 0.63 μg/mL against S. aureus and 2.5 μg/mL against P. aeruginosa. In contrast, the MIC values of BAC and DDAC were 0.36 μg/mL and 1.4 μg/mL against S. aureus, and 12 μg/mL and 43 μg/mL for P. aeruginosa, respectively. This QAC panel is composed of a selection of bis-cationic QACs with symmetrical alkyl chains of 10 or 12 carbons in length, plus an analogous selection of multi-QACs bearing linear or branched poly-cationic cores.
Figure 1.
Best-in-class quaternary ammonium compounds (QACs) structure and minimal inhibitory concentrations (MIC). Best-in-class QACs were selected based on their biological activity measured using conventional MIC assays for S. aureus (Sa) and P. aeruginosa (Pa) as well as diversity of structures. The commercially available mono-QACs benzalkonium chloride (benzyldimethyldodecylammonium chloride) and didecyldimethylammonium chloride were also included as part of the QAC panel for this study.
QAC Antibacterial Activity Against the Gram-Positive S. Aureus is Not Consistently Affected by Hard Water
Since the QACs display significant inhibitory activity against both gram-positive and gram-negative pathogens by MIC assays, we wanted to assess the bactericidal activity of the QACs through suspension time-kill assays.[8–13] We performed time-kill assays following the American Society for Testing and Materials (ASTM) E2315 standard protocol and report the CFU/mL log reduction values (LRV) at 1, 2, and 5 minutes relative to CFU/mL values at time 0 of untreated controls.[26] This time-assays were performed in both distilled and AOAC 400 μg/mL hard water (400 μg/mL of calcium carbonate), a level of water hardness commonly used when testing disinfectants for commercialization. Our QAC panel was tested using a final concentration of 200 μg/mL. This concentration was selected based on QAC usage during disinfection in real-world scenarios.[5,27] Importantly, we did not observe precipitate formation of our compounds at this level of water hardness at the QAC concentration tested. To accurately measure the killing efficacy of this panel of QACs over short periods of time, their antimicrobial activity must be neutralized after the desired contact time. This neutralizing agent must itself be nontoxic, and it must be able to neutralize the compounds almost instantly. To achieve this, we used Dey-Engley (D/E) neutralization broth and performed neutralization tests following the ASTM E1054 protocol.[28] D/E neutralization broth was developed to improve the reproducibility in testing antimicrobial agents, including QACs.[29]
We were able to effectively neutralize the bactericidal activity of the QACs using a preparation of 2X D/E broth. We did not see significant effects of the neutralizer on the viability of S. aureus SH1000 and P. aeruginosa PAO1 strains after neutralizing the QACs (see Supplemental Figure 1). To increase the throughput of these time-kill assays, the ASTM E2315 standard protocol was adapted to a 96-well plate format with spot plating allowing quick assessment of which compounds were most active by visual inspection (see Supplemental Figures 6 and 7).
Uniform MIC values were observed against the gram-positive S. aureus across the QAC panel, all between 0.34 μg/mL and 0.89 μg/mL (Table 1). However, time-kill assays revealed clear differences in bactericidal effectiveness. While QAC 3 displayed both strong inhibitory and killing activity against S. aureus, some of the QACs that displayed the strongest inhibitory activity by MIC assay (7, 9, 10) were relatively less effective at killing in time-kill assays in early time points. Conversely, compounds 1, 2 and 5 showed average MIC values but good bactericidal activity in deionized water. In addition, we observed that hard water strongly reduced the bactericidal activity of compounds 2, 4 and 7 against S. aureus. This was particularly evident after 5 minutes of contact time for QAC 4 and 7, with a 3-log loss in killing activity compared to that obtained in deionized water. This reduction in killing effectiveness was observed in BAC within 1 minute of contact time but was never observed in DDAC. QACs 1, 3, 5 and 8 behave similarly to BAC and DDAC in hard water against S. aureus, reducing the bacterial populations below the limit of detection after 5 minutes of contact time. Interestingly, QACs 5 and 10 performed better in hard water versus deionized water against S. aureus. Multi-QACs with branched cationic heads (8, 9, and 10) also showed enhanced killing in hard water against S. aureus (Table 1). Compound 4 showed the weakest potency in hard water, displaying almost complete loss of bactericidal activity even after 5 minutes of contact time, but the analogous compound 5 with two additional carbons in the alkyl chains, was markedly different. These results show the differences in inhibitory (MIC) and bactericidal (time-kill) bioactivities of these QACs against S. aureus.
Table 1.
CFU/mL log reduction of S. aureus by QAC panel (n=2).
| Log reduction values (LRV); CFU/mL | |||||||
|---|---|---|---|---|---|---|---|
| 1 minute |
2 minutes |
5 minutes |
|||||
| QACa | MIC (μg/mL) | Deionized Water | Hard Waterb | Deionized Water | Hard Waterb | Deionized Water | Hard Waterb |
| QAC 1 | 0.62 | 1.9 | 1.3 | 2.6 | 3.4 | 5.7 | 6.0 |
| QAC 2 | 0.64 | 3.8 | 2.4 | 6.2 | 3.7 | 6.2 | 5.0 |
| QAC 3 | 0.34 | 4.4 | 3.5 | 5.0 | 5.0 | 5.9 | 6.2 |
| QAC 4 | 0.64 | 0.4 | 0.0 | 0.7 | 0.8 | 3.3 | 0.3 |
| QAC 5 | 0.70 | 1.1 | 2.6 | 2.1 | 6.1 | 2.8 | 6.1 |
| QAC 6 | 0.73 | 1.1 | 2.4 | 3.7 | 2.9 | 2.3 | 3.7 |
| QAC 7 | 0.40 | 0.3 | 0.8 | 0.9 | 2.4 | 6.0 | 3.0 |
| QAC 8 | 0.89 | 0.4 | 1.3 | 1.6 | 3.4 | 3.8 | 6.0 |
| QAC 9 | 0.47 | 0.5 | 0.9 | 0.7 | 2.1 | 2.1 | 5.4 |
| QAC 10 | 0.54 | 0.5 | 1.0 | 0.8 | 2.5 | 1.1 | 4.7 |
| BAC | 0.91 | 4.0 | 2.9 | 4.6 | 5.2 | 5.3 | 6.3 |
| DDAC | 0.36 | 5.5 | 5.1 | 5.8 | 6.1 | 6.1 | 6.1 |
QACs final concentration used was 200 μg/mL.
AOAC 400 μg/mL hard water.
QACs Antibacterial Activity Against the Gram-Negative P. Aeruginosa is Significantly Reduced by Hard Water
P. aeruginosa is a gram-negative opportunistic pathogen of clinical importance due to high antibiotic resistance profiles and its prevalence as a hospital-acquired pathogen, especially in those suffering from cystic fibrosis and other immunocompromised individuals.[30] As a gram-negative bacterium, P. aeruginosa possesses an envelope composed of an asymmetric outer membrane made of an inner phospholipid leaflet and an outer lipopolysaccharide (LPS) leaflet, a phospholipid bilayer inner cytoplasmic membrane, and a thin peptidoglycan cell wall between these membranes.[31] This envelope acts as a barrier to multiple antimicrobials in gram-negative bacteria.[31]
To test how these QACs perform against a gram-negative pathogen in practical applications, we compared the bactericidal effectiveness of our panel against P. aeruginosa in deionized and hard water. In terms of growth inhibition, MIC values showed a wide range of effectiveness in the inhibition of P. aeruginosa, with QAC 4 and 7 showing the weakest inhibition with 41 μg/mL and 6.5 μg/mL, respectively (Table 2). The other QACs in our panel showed comparable MIC values that ranged from 1.34 μg/mL to 2.9 μg/mL. Similarly, bactericidal activity measured by time-kill assays showed a range of bioactivity. Compounds 1 and 2 showed modest inhibitory activities by MIC and good bactericidal activity compared to other QACs. In contrast, compound 7 showed low inhibitory activity relative to other QACs within the panel, but its bactericidal activity was comparable to the other compounds. Similarly, BAC and DDAC showed relatively low inhibitory activity, but potent bactericidal activity as determined in time-kill assays.
Table 2.
CFU/mL log reduction of P. aeruginosa by QAC panel (n=2).
| Log reduction values (LRV); CFU/mL | |||||||
|---|---|---|---|---|---|---|---|
| 1 minute |
2 minutes |
5 minutes |
|||||
| QACa | MIC (μg/mL) | Deionized Water | Hard Waterb | Deionized Water | Hard Waterb | Deionized Water | Hard Waterb |
| QAC 1 | 2.5 | 2.0 | 0.5 | 2.9 | 1.3 | 4.6 | 2.5 |
| QAC 2 | 2.6 | 4.3 | 0.6 | 5.7 | 1.4 | 5.7 | 3.4 |
| QAC 3 | 1.3 | 2.7 | 0.4 | 2.6 | 0.9 | 2.9 | 1.2 |
| QAC 4 | 41 | 2.5 | 0.1 | 2.7 | 0.4 | 3.1 | 0.1 |
| QAC 5 | 2.8 | 2.0 | 0.6 | 1.7 | 0.0 | 3.3 | 0.0 |
| QAC 6 | 2.9 | 2.5 | 0.0 | 3.0 | 0.0 | 3.2 | 0.6 |
| QAC 7 | 6.5 | 2.3 | 0.2 | 1.9 | 0.9 | 3.1 | 0.1 |
| QAC 8 | 1.8 | 2.8 | 0.0 | 3.4 | 0.0 | 4.8 | 0.0 |
| QAC 9 | 1.9 | 2.4 | 0.2 | 2.5 | 0.2 | 3.0 | 0.0 |
| QAC 10 | 2.2 | 1.9 | 0.2 | 1.9 | 0.2 | 3.1 | 0.1 |
| BAC | 43 | 4.5 | 1.8 | 5.3 | 2.2 | 5.9 | 2.4 |
| DDAC | 12 | 3.9 | 4.1 | 5.8 | 5.1 | 5.8 | 5.8 |
QACs final concentration used was 200 μg/mL.
AOAC 400 μg/mL hard water.
The most striking difference we observed in our data was the pronounced and detrimental effects of hard water in the bactericidal activity of all QACs in our panel. We observed that only compounds 1, 2 and 3 retained some of their bactericidal activity against P. aeruginosa in hard water, while the rest of the QACs within the panel were almost completely inactivated in hard water. It is tempting to speculate that the simpler structural characteristics of these QACs (1, 2 and 3) makes them less susceptible to inactivation by hard water compared to the bulkier multi-QACs. It is important to note that DDAC retained its strong bactericidal activity, even in hard water. The observed bactericidal activity patterns in our time-kill data in deionized and hard water show that the inactivating effects of the latter on bactericidal activity against the gram-negative P. aeruginosa is general. Divalent cations are known to stabilize the outer membrane charge of gram-negative bacteria.[32] It is possible that the divalent cations present in hard water have a similar protective effect rendering the QACs ineffective. Our results highlight the need to further explore the nuances of the effects of hard water on cationic antimicrobials, especially against gram-negative pathogens.
Dynamic Surface Tension Reduction Might be a Better Proxy for Bactericidal Activity than MIC Values
With the inhibitory and bactericidal activities of this QAC panel at hand, we explored how the innate ability of these QACs to reduce surface tension correlated with these antimicrobial activities. The dynamic surface tension (DST) of our compounds was measured using a Krüss Bubble Pressure Tensiometer. We found that our QAC panel (and commercial QACs) displayed a range of DST values (Supplemental Information), with the commercially available mono-QACs (chloride salts) displaying the lowest values. Using this information, Pearson correlation coefficients were calculated using the DST values and the log reduction values obtained with the 12 QACs in our panel against S. aureus and P. aeruginosa at 1, 2 and 5 minutes. The statistical significance of the correlation of DST values LRV values against each bacterial strain was determined, as presented in Table 3. Statistically significant positive correlation of DST reduction with bactericidal activity against both S. aureus and P. aeruginosa was observed. This correlation was conserved with bactericidal activity in deionized and hard water at most timepoints with both bacterial strains. Importantly, the DST 500 msec time point showed stronger correlation with bactericidal activity than the 9,800 msec time point, particularly with bactericidal activity within shorter contact times in time-kill assays (1 and 2 minutes). However, is important to note that the correlation of DST values with bactericidal activity is not useful when other co-surfactants are present in the formula since the surface activity of the co-surfactants can mask the contribution of QACs in the DST measurements in addition to the limitations in power of this statistical analysis due to the sample size.
Table 3.
Pearson correlation coefficients calculated using the dynamic surface tension (DST) values and log reduction values (LRV) of the 12 QACs within our panel.
|
S. aureus (LRV) |
P. aeruginosa (LRV) |
|||||
|---|---|---|---|---|---|---|
| DI water | 1 minute |
2 minutes |
5 minutes |
1 minute |
2 minutes |
5 minutes |
| Kruss DST at 500-mseca | 0.81* | 0.79* | 0.78* | 0.64* | 0.72* | 0.65* |
| Kruss DST at 9800-mseca | 0.62* | 0.62* | 0.71* | 0.43 | 0.58* | 0.64* |
| Hard water | ||||||
| Kruss DST at 500-mseca | 0.76* | 0.55 | 0.42 | 0.71* | 0.80* | 0.82* |
| Kruss DST at 9800-mseca | 0.60* | 0.48 | 0.51 | 0.56 | 0.66* | 0.72* |
DST values were measured using a QAC concentration of 1 mg/mL.
Statistically significant correlation P-value<0.05 (critical value of r=0.576). Highlighted values in white: Power (1-β=0.8) by post hoc analysis using G*Power.
To our surprise, we found no significant positive or negative correlation of DST with MIC values (Pearson′s r coefficients < ±0.21 for S. aureus and < ±0.13 for P. aeruginosa) or statistically significant correlation between MIC and log reduction values against S. aureus and P. aeruginosa (Pearson′s r coefficients < ±0.23 for S. aureus and < ±0.41 for P. aeruginosa) in deionized or hard water at any time point tested (Supplemental Information). These data support the idea that the dispersion of QACs measured by DST values can be correlated with bactericidal activity but not MIC values. This could offer a cost-effective method to screen QACs for high bactericidal activity.
Effect of Counterion in Bactericidal Activity in Hard Water
Since we noted that the commercially available BAC and DDAC are the chloride salts, we explored if the counterion (bromide versus chloride salts) had a role in the effectiveness of one of our QAC. To our knowledge, the effects of counterion in the antimicrobial activity of diverse QACs has been explored, but not in relation to the activity in hard water.[33] We decided to investigate whether the inclusion of chloride or bromide counterions had an impact on the activity of QAC 3, as this QAC showed reduced activity in hard water against P. aeruginosa. Interestingly, we found that the chloride salt of QAC 3 was much more active in hard water compared to the bromide salt, both tested at a final concentration of 200 μg/mL (Figure 2). A higher antimicrobial activity has also been associated with the chloride salt of gemini quaternary ammonium salts against fungal pathogens.[34] This counterion-dependent activity increase of QACs has been mainly attributed to effects of the counterion in solubility.[1] It is believed that the counterion does not directly affect the activity of QACs.[19] These data provide additional evidence in support of the importance of the counterion selection during synthesis of QACs, and the potential effect on bactericidal activity.
Figure 2.
Chloride counterion QACs are more tolerant to hard water effectiveness reduction. P-12,12 Br− (3) and P-12,12 Cl− (3 Cl−) were tested by time-kill assay against P. aeruginosa and the reduction in CFU/mL over time was measured in triplicate.
Combination of QACs with DDAC Show Enhanced Bactericidal Activity
Leveraging the information obtained of the effect of the chloride counterion in bactericidal activity, we reasoned that combinations of our QACs with DDAC could overcome the negative effects of hard water on bioactivity because of the effects of the chloride counterions provided by DDAC. We tested these combinations against both S. aureus and P. aeruginosa to ensure that the mixtures of QACs were still as active against both bacterial strains. No major difference was observed in bactericidal activity against S. aureus, with most combinations showing the same potent bactericidal efficacy (Table 4). Importantly, combinations of DDAC with some of the compounds in our QAC panel outperformed the BAC and DDAC combination commonly found in commercial products in shorter timeframes. In contrast, combination of QACs with DDAC against P. aeruginosa showed more pronounced differences in activity. Interestingly, only the combination of QAC 8 with DDAC outperformed the BAC and DDAC combination against P. aeruginosa, with over 1 log reduction higher after 2 minutes of contact time compared to the commercially used QAC combination. Compound 4 also showed the lowest bactericidal activity against P. aeruginosa, which correlates with the results obtained in time-kill assays when testing this QAC alone. Whether the difference in bactericidal activity observed with QAC combinations are due to the interaction of the QACs with the bacterial membranes or interactions between the QACs themselves is not known. These data suggest that combinations of QACs could be used to improve the bactericidal activity against difficult to eradicate gram-negative pathogens, such as P. aeruginosa.
Table 4.
CFU/mL log reduction values (LRVs) of S. aureus and P. aeruginosa by combined QACs (QAC combination concentration=200 μg/mL) in AOAC 400 μg/mL hard water (n=2).
|
S. aureus (LRV) |
P. aeruginosa (LRV) |
|||||
|---|---|---|---|---|---|---|
| Combination |
1 minute |
2 minutes |
5 minutes |
1 minute |
2 minutes |
5 minutes |
| 1 + DDAC | 4.3 | 5.2 | 5.8 | 2.3 | 2.6 | 3.9 |
| 2 + DDAC | 1.9 | 2.2 | 6.1 | 2.0 | 2.3 | 3.9 |
| 3 + DDAC | 5.9 | 5.9 | 5.9 | 1.9 | 2.6 | 2.5 |
| 4 + DDAC | 3.5 | 5.8 | 5.8 | 1.7 | 1.8 | 2.2 |
| 5 + DDAC | 5.8 | 5.8 | 5.8 | 1.9 | 2.7 | 3.3 |
| 6 + DDAC | 5.7 | 5.8 | 5.8 | 2.9 | 3.9 | 4.9 |
| 7 + DDAC | 2.5 | 5.8 | 5.8 | 1.7 | 2.7 | 3.1 |
| 8 + DDAC | 5.7 | 5.7 | 5.7 | 3.0 | 4.6 | 4.1 |
| 9 + DDAC | 5.6 | 4.7 | 6.1 | 2.3 | 2.6 | 3.5 |
| 10 + DDAC | 4.9 | 5.8 | 5.8 | 1.6 | 1.7 | 3.8 |
| BAC + DDAC | 2.8 | 4.2 | 5.7 | 2.8 | 3.1 | 4.7 |
Conclusions
Biocides play an essential role in the control of microbial pathogens in a wide range of industries. Nevertheless, increased usage of biocides such as QACs is leading to higher rates of antimicrobial resistance development.[35] MIC and time-kill assays have been a cornerstone in antimicrobial research, allowing easy determination of inhibitory and bactericidal activity of different antimicrobial agents. However, these assays can become cumbersome when screening numerous antimicrobials. Our data indicates that DST measurements have good correlation with bactericidal activity under real-world disinfection conditions. However, DST values do not correlate with inhibitory activity of QACs determined by MIC assays. Interestingly, we found no statistical correlation between inhibitory capacity and bactericidal activity of in our QAC panel. In addition, we observed that the bactericidal effectiveness of our QAC panel was significantly reduced against gram-negative P. aeruginosa in hard water, but less so against the gram-positive S. aureus (Table 1 and Table 2). To investigate additional factors that affect the potency of QACs in hard water, we evaluated the effect of counterions in QAC activity in hard water and the effectiveness of QAC combinations with DDAC. We found that the chloride salt of a tested QAC outperformed the corresponding bromide salt. Of note, the chloride salts of both BAC and DDAC were used in this study, and they also retained partial and full bactericidal activity in hard water against P. aeruginosa, respectively. Thus, the effect of counterion in QAC bactericidal activity in hard water should be further explored. In addition, we found that rational combinations of QACs could help mitigate the effects of hard water in bactericidal activity. This study provides evidence that DST can be used as a quick and effective technique to broadly screen QACs for bactericidal activity in real-world disinfection conditions. Finally, this study underlines the need to further explore strategies to overcome the detrimental effects of hard water to QAC bactericidal activity as novel and potent biocides are developed.
Supplementary Material
Acknowledgements
Funding was provided by the NIH (GM119426 to W.M.W.; DK126467 to G.G.V.C.; T32 GM008602 to C.A.S.) and the Stepan Company.
Footnotes
Supporting information for this article is available on the WWW under https://doi.org/10.1002/cmdc.202400262
Supporting Information
The authors have cited additional references within the Supporting Information.
Conflict of Interests
The authors have patents covering the compounds disclosed.
Data Availability Statement
The data that support the findings of this study are available in the supplementary material of this article.
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Data Availability Statement
The data that support the findings of this study are available in the supplementary material of this article.