antibiotics Article Repurposing Disulfiram as an Antimicrobial Agent in Topical Infections Maria Lajarin-Reinares 1,2, * , Eloy Pena-Rodríguez 1 , Mariona Cañellas-Santos 3 , Elisabet Rosell-Vives 3 , Pilar Cortés 2 , Montserrat Llagostera Casas 2 , Maria Àngels Calvo 4 and Francisco Fernandez-Campos 1, * 1 Topical & Oral Development, Research and Development Reig Jofre Laboratories, 08970 Barcelona, Spain 2 Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain 3 Biotechnology Department, Research and Development Reig Jofre Laboratories, 08970 Barcelona, Spain 4 Department of Animal Health and Anatomy, Faculty of Veterinary, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain * Correspondence: mlajarin@reigjofre.com (M.L.-R.); mran82@gmail.com (F.F.-C.); Tel.: +34-935-507-718 (M.L.-R.) Abstract: Antimicrobial drugs applied topically offer several advantages. However, the widespread use of antibiotics has led to increasing antimicrobial resistance. One interesting approach in the drug discovery process is drug repurposing. Disulfiram, which was originally approved as an anti- alcoholism drug, offers an attractive alternative to treat topical multidrug resistance bacteria in skin human infections. This study aimed to evaluate the biopharmaceutical characteristics of the drug and the effects arising from its topical application in detail. Microdilution susceptibility testing showed antibacterial activity against Gram-positive bacteria Staphylococcus aureus and Streptococcus pyogenes. Dermal absorption revealed no permeation in pig skin. The quantification of the drug retained in pig skin demonstrated concentrations in the stratum corneum and epidermis, enough to treat Citation: Lajarin-Reinares, M.; skin infections. Moreover, in vitro cytotoxicity and micro-array analyses were performed to better Pena-Rodríguez, E.; Cañellas-Santos, understand the mechanism of action and revealed the importance of the drug as a metal ion chelator. M.; Rosell-Vives, E.; Cortés, P.; Casas, Together, our findings suggest that disulfiram has the potential to be repurposed as an effective M.L.; Calvo, M.À.; antibiotic to treat superficial human skin infections. Fernandez-Campos, F. Repurposing Disulfiram as an Antimicrobial Agent Keywords: disulfiram; skin; Staphylococcus aureus; repurposing; DNA arrays; antibiotics; in Topical Infections. Antibiotics 2022, antimicrobial resistance; Streptococcus pyogenes 11, 1752. https://doi.org/10.3390/ antibiotics11121752 Academic Editor: Filip Boyen 1. Introduction Received: 12 October 2022 Accepted: 2 December 2022 Human skin is one of the first lines of defense against microbial invasion. Healthy Published: 4 December 2022 skin harbors a diverse range of bacteria, collectively known as the skin microbiome, and depending on the host, bacterial, and environmental factors, this bacterial population Publisher’s Note: MDPI stays neutral may be protective or harmful [1]. Staphylococcus aureus and group A streptococci are the with regard to jurisdictional claims in two most frequently encountered pathogens causing primary and secondary infections of published maps and institutional affil- the skin and skin wounds. Gram-negative organisms such as Pseudomonas aeruginosa are iations. sometimes involved in cutaneous infections [2]. Antimicrobial drugs applied topically offer several advantages over systemic adminis- tration, including the avoidance of systemic toxicity and side effects, decreased induction of Copyright: © 2022 by the authors. bacterial resistance, high local concentration of antibacterial agents in the infected site, low Licensee MDPI, Basel, Switzerland. costs and they also allow the administration of molecules that cannot be given systemati- This article is an open access article cally [1,3,4]. However, the widespread use of topical antibiotics (particularly mupirocin and distributed under the terms and fusidic acid) has led to increasing antimicrobial resistance (AMR) in some settings, making conditions of the Creative Commons their use extremely difficult and limiting the potential efficacy of such agents [3,4]. Thus, Attribution (CC BY) license (https:// leading to the selection of more resistant Staphylococcus aureus strains. Studies conducted creativecommons.org/licenses/by/ in order to better understand the impact of topical antimicrobial use on the development 4.0/). Antibiotics 2022, 11, 1752. https://doi.org/10.3390/antibiotics11121752 https://www.mdpi.com/journal/antibiotics Antibiotics 2022, 11, 1752 2 of 14 of resistant S aureus strains have demonstrated that exposing both MSSA (Methicillin- sensitive S aureus) and MRSA (Methicillin-resistant S aureus) to fusidic acid and mupirocin resulted not only in the rapid selection of MRSA over MSSA but also the emergence of MDR (Multi-drug resistant) isolates from the original culture [5]. One of the most important human streptococci pathogens of skin is Streptococcus pyogenes, which can produce superficial impetigo or more deep-seated cellulitis but also more se- vere invasive infections such as sepsis, necrotizing fasciitis, and streptococcal toxic shock syndrome [6]. In streptococcal infections, penicillin remains the antibiotic of choice. Im- portantly, clindamycin-resistant isolates have also been reported in Europe, including France and Finland, and Asian countries, with alarming rates of 94% in China [7]. Other species, such as Pseudomonas aeruginosa, are less common but they are a major problem leading to high mortality rates due to the appearance of drug-resistant strains, especially in critically ill and immunocompromised patients [8]. The spectrum of P. aeruginosa cutaneous diseases includes localized and often self-limited infections, such as hot-tub folliculitis, chloronychia, hot-foot syndrome and interdigital intertrigo. Ecthyma gangrenosum and sometimes subcutaneous nodules are manifestations in immunocompromised hosts and as such present a medical emergency [7]. Given global concerns regarding antibiotic resistance and relatively limited therapeu- tic options, especially for some species such as S. aureus, the appropriate use of topical agents and the prevention of further resistance are critical [1]. The development of a new antimicrobial is a very slow process and is frequently beset with numerous pitfalls. Repur- posing approved drugs is a promising alternative strategy that reduces the time and cost of antibiotic development, as it takes advantage of existing toxicology and pharmacokinetic data from preclinical and clinical trials [9]. Disulfiram (D), first commercialized as Antabuse® , is a dithiocarbamate that was approved by the US Food and Drug Administration (FDA) in 1951 as a drug to treat alcoholism. It has been widely used in clinics for over 70 years without severe side effects [10]. D is an inhibitor of aldehyde dehydrogenase (ALDH) and it inhibits all the currently identified cytosolic and mitochondrial ALDH isoforms. This fact results in the specific accumulation of acetaldehyde, causing discomfort in patients who drink alcohol as a strategy for their dishabituation [10,11]. A cutaneous emulsion consisting of 2% D has been registered in Sweden since 1982 and in Iceland since 1997 for the treatment of scabies and pediculosis in adults and children [12]. Different in vitro and in silico studies have shown that D may be an active substance against the hepatitis C virus, fungi, parasites, bacteria, and cancer [9,10,13–15]. A different mechanism of action was proposed to explain these antimicrobial and antiproliferative activities. D has been reported to induce apoptosis, show metal ion-dependent antineo- plastic activity and arrest angiogenesis. It has also been shown to inhibit the activating transcription factor/cyclic-AMP-responsive element-binding protein. Moreover, recent studies have shown that D inhibits the activity of the ATPS-binding cassette (ABC) drug transport complex, which is responsible for the development of multiple drug resistance in cancer and fungal infection treatments [16]. D is an electrophile (Figure 1) that readily forms disulfides with thiol-bearing substances. Bacteria possess a diverse range of intracellular cofactors (e.g., coenzyme A), metabolites (e.g., glutathione, mycothiol, and bacillithiol), and enzymes (e.g., thioredoxin) containing thiophilic residues, which D can potentially modify via thiol–disulfide exchange to evoke antimicrobial effects [17]. The use of D as an antimicrobial agent with a different mechanism of action, in comparison with other classical antibiotics, offers an attractive alternative to treating topical, multiresistant bacteria. In the context of limited-availability antibiotics and the increase in AMR to them, we focused on repurposing D as an alternative to conventional antimicrobial compounds for superficial human skin infections. Thus, in this study, we present a detailed evaluation of the biopharmaceutical characteristics of the disulfiram drug and the effects derived from topical application. Antibiotics 2022, 11, 1752 3 of 15 Antibiotics 2022, 11, 1752 3 of 14 Figure 1. Chemical structure of disulfiram molecule (D). Figure 1. Chemical structure of disulfiram molecule (D). 2. Results and Discussion InAntibiotic 2.1. the context of limited-availability Susceptibility Testing antibiotics and the increase in AMR to them, we focused on repurposing D as an alternative to conventional antimicrobial compounds for S. aureus, P. aeruginosa, and S. pyogenes were selected for susceptibility testing as repre- superficial sentativehuman strainsskin infections. related to skin Thus, in this infection andstudy, we present frequently involveda detailed evaluation with AMR of in humans. theThe biopharmaceutical minimum inhibitory characteristics of the disulfiram concentrations drug and (MICs) against thesethe effectsare species derived from summarized topical application. in Table 1. As can be seen, D was effective against the Gram-positive bacteria tested, but it did not effectively inhibit Pseudomonas spp. growth, a Gram-negative bacterium. 2. Results S. aureusand Discussion exhibited the greatest susceptibility, with a MIC of 8 µg/mL. This result agreed 2.1.with Antibiotic those Susceptibility of published Testing reports, where it was found to exhibit a potent activity with the same MIC value S. aureus, [9,17]. Inand P. aeruginosa, theS.case of S. pyogenes, pyogenes were selectedD had fora susceptibility MIC of 32 µg/mL. testingThis value as rep- differs from resentative the previously strains published related to skin value, infection and16 µg/mL, which frequently involved could beAMR with due tointhe different humans. Thestrain used, MGAS1882 minimum [18]. Moreover,(MICs) inhibitory concentrations the MIC against in P. aeruginosa resultthese species arecorrelates summarizedwell in with published data, with a MIC value of more than 64 µg/mL [17]. Conventional Table 1. As can be seen, D was effective against the Gram-positive bacteria tested, but it antibiotics did(clindamycin not effectively and gentamicin) inhibit were used Pseudomonas spp. as controls growth, for the bacteriabacterium. a Gram-negative susceptibility method. S. aureus Clinical Laboratory Standards Institute (CLSI) guidelines suggest that exhibited the greatest susceptibility, with a MIC of 8 µg/mL. This result agreed with those comparing the MICs of different antibiotics should not be based solely on the numerical of published reports, where it was found to exhibit a potent activity with the same MIC value but rather on how far the MIC is from the breakpoint [19]. The CLSI groups MIC values value [9,17]. In the case of S. pyogenes, D had a MIC of 32 µg/mL. This value differs from into three categories: thesusceptible, intermediate, previously published and16resistant, value, based on µg/mL, which clinical could be duedatato and research.strain the different Regarding used, D, there are no defined clinical breakpoints because there are no published MGAS1882 [18]. Moreover, the MIC result in P. aeruginosa correlates well with published data on natural mutants resistant to it, which could be due to the low solubility of data, with a MIC value of more than 64 µg/mL [17]. Conventional antibiotics (clindamycin disulfiram in aqueous andmedia, preventing gentamicin) wereexperiments used as controlsfrom for being theperformed at higher concentrations. bacteria susceptibility method. ClinicalTherefore, La- it was difficult to use the classical approach to compare D activity with other antibiotics. boratory Standards Institute (CLSI) guidelines suggest that comparing the MICs of differ- entTable antibiotics should activity 1. Antibacterial not be based solelycompared of disulfiram on the numerical value and with gentamicin but clindamycin. rather on how far the MIC is from the breakpoint [19]. The CLSI groups MIC values into three categories: susceptible, intermediate, and resistant, based on clinical data MICand research. Regarding D, Species clinical breakpoints because there are no published data on natural there are no defined Disulfiram Clindamycin Gentamicin mutants resistant to it, which could be due (µM) (µg/mL) to the low solubility (µg/mL) of disulfiram (µM) (µg/mL)in aqueous (µM) media, Streptococcus preventing pyogenes experiments from 32 being performed 108 at 0.125 higher 1 concentrations. – There- – fore, it was difficult to Staphylococcus use the classical aureus 8 approach 27 to compare– D activity – with 0.125 other antibi- 0.262 otics. Pseudomonas aeruginosa >64 >216 – – 0.250 0.523 Table 1.The Antibacterial activity differential of of effect disulfiram compared D against with gentamicin Gram-positive and clindamycin. and Gram-negative bacteria tested could be due to two possible mechanisms of action: Cu 2+ MICchelation and compound affinity for the thiol group of cysteine. Proper protein targeting and maturation are fundamental Species Disulfiram Clindamycin Gentamicin to the homeostasis of all organisms. In Gram-negative bacteria, secreted proteins mature (µg/mL) (µM) (µg/mL) (µM) (µg/mL) (µM) in an oxidative periplasm. Gram-positive bacteria lack this protected compartment due Streptococcus pyogenes 32 108 0.125 1 to the absence of an outer membrane. Thus, their secreted proteins must mature -- -- in an Staphylococcus unregulated aureus environment. 8 27 cysteine-- residues -- Furthermore, perform a0.125 variety of0.262 essential Pseudomonas aeruginosa >64 >216 -- -- functions in protein, such as metal binding and enzyme catalysis. However, the 0.250 0.523 reactive thiol groups can also negatively impact the function of a protein by forming incorrect The differential disulfide effect bonds [20]. Theofgreater D against Gram-positive exposure and Gram-negative of Gram-positive bacteria to bacteria D could tested increase could the be due to two reactivity possiblethiols of cysteine mechanisms of action: Cu and, consequently, 2+ chelation and compound affinity not allow the correct maturation of the forprotein, the thiolleading group to of the cysteine. deathProper protein targeting of the bacteria. and maturation Additionally, are fundamental the non-effectiveness detected to the homeostasis ofbacteria in Gram-negative all organisms. can be In Gram-negative explained since P.bacteria, aeruginosa secreted proteins is endowed mature with several mechanisms in the periplasmic space to adapt to copper fluctuations [21]. Antibiotics 2022, 11, 1752 4 of 14 Moreover, P. aeruginosa is a pathogen characterized by the secretion of virulent factors such as the zinc metalloprotease pseudolysisin, also known as LasB or pseudomonas elas- tase. This virulence factor is highly toxic, causing tissue damage and invasion, processing components of the immune system to cause immunomodulation, and acting intracellularly to initiate bacterial biofilm growth. The inhibition of bacterial virulence factors has gained attention recently as an antimicrobial strategy that is non-destructive to the bacteria. It has been proposed as a second-generation class of antibiotics. By attenuating virulence mechanisms without challenging bacterial cell viability directly, these antibiotic agents would potentially place little or no pressure on the bacterial cell for the emergence of resis- tant strains [22]. Although disulfiram does not directly reduce the bacterial cell viability, it may reduce virulence factors. Additional studies should be carried out to check this possible effect. 2.2. Ex Vivo Skin Absorption Experiment To evaluate the permeation profile of D, Franz diffusion cells were used. The use of human skin is usually preferred in these kinds of experiments; however, due to ethical and economic reasons, this tissue is frequently unavailable. In this case, porcine skin is a good surrogate due to its structural similarity to human skin in terms of hair growth density (~20 hairs/cm2 ) and the presence of structures such as Langerhans cells and rete ridges; stratum corneum thickness and contents such as glycosphingolipids and ceramides; stratified, multilayered, keratinizing epithelium; thickness of the viable epidermis (~70 µm); and collagen fiber arrangement in the dermis [23]. Therefore, an ex vivo permeated study with pig skin was used to characterize the absorption of D. In this case, D concentrations were not detected in the chromatogram in any replicate (n = 11) at any time in the tested concentration (3.65 mg/cm2 ). This lack of permeability of D in pig skin has also been described in human skin [24]. It is an advantageous property because D can cause adverse effects when it is systemically available due to the inhibition of aldehyde dehydrogenase and dopamine beta-hydroxylase [25]. 2.3. Quantification of Disulfiram in Skin Disulfiram, as shown by permeation studies in Section 2.2, was not able to permeate the dermis (D was not detected in the receptor medium in any replicate). However, D could penetrate and be retained in the first layers of the skin (stratum corneum and epidermis). The concentration of the active ingredient in these layers is important to determine whether there are effective concentrations to combat superficial infections. Therefore, the amount of disulfiram retained in pig skin (SC and total skin) was determined. Before the experiment, the density of the skin was calculated. The mean densities of the non-dermatomized and dermatomized pig skins were very similar: 1.05 g/mL and 1.04 g/mL, respectively. 2.3.1. Quantification of D in SC Using the Tape-Stripping Technique Before HPLC analysis, we checked the selectivity of the method and the adhesive of the tape used did not produce interferences during the test. The mean amount of D obtained on each strip of the five replicates is shown in Figure 2. A concentration of 48 µg in the SC was obtained (corresponding to 38% of the dose applied in the permeation experiment: 110 µg). Before calculating the concentration of D in SC, the amount of extracted skin was cal- culated in each strip group (Table 2) by weighing the strips before and after each extraction. The mean skin retained was 2.20 mg, and the final concentration of D was 20 mg/mL. The concentration obtained was superior to the MIC values found for S. aureus and S. pyogenes. These results are interesting because superficial skin infections take place in the stratum corneum and epidermis, and this concentration is sufficient to ensure effectiveness. Antibiotics 2022, 11, 1752 5 of 15 obtained on each strip of the five replicates is shown in Figure 2. A concentration of 48 µg Antibiotics 2022, 11, 1752 in the SC was obtained (corresponding to 38% of the dose applied in the permeation ex- 5 of 14 periment: 110 µg). Figure 2. Figure Amount Amount 2. of of D per D per strip (n = strip (n = 5). The 5). The results results show the show meanthe andmean and standard standard deviation.deviation. Table Before 2. Amount the calculating of skin retired (mg) for concentration of each D ingroup stripamount SC, the (n = 3) with its standard of extracted deviation. skin was calculated in each strip group Strip (Table 2) by weighing the Mean (mg) strips before and after each SD ex- traction. The mean skin retained was 2.20 mg, and the final concentration of D was 20 1 0.43 0.26 mg/mL. The concentration obtained was superior to the MIC values found for S. aureus 2 0.39 0.20 and S. pyogenes. These3–7 results are interesting because superficial skin infections take 0.68 0.18place in the stratum corneum8–12 and epidermis, and this concentration 0.39 is sufficient to ensure 0.16 ef- fectiveness. 13–17 0.23 0.06 18–20 0.07 0.00 Table 2. Amount of skin retired (mg) for each group strip (n = 3) with its standard deviation. 2.3.2. Disulfiram Concentration Retained in the Dermatomized Skin Strip Mean (mg) SD 1 Over the years, the literature has documented 0.43 different methodologies 0.26for quantifying drug2 amounts within the skin. The techniques 0.39 include skin extraction 0.20measurements, horizontal stripping and sectioning, the removal of hair follicles, quantitative autoradio- 3–7 0.68 0.18 graphy, and spectroscopic methods [26]. Given that our target is the SC and epidermis, 8–12 0.39 0.16 the drug content in the epidermis layer was obtained. After tape-stripping the skin, as 13–17 0.23 0.06 described in Section 2.3.1, the rest of the skin tissue was cut into pieces of 10 mg, and 18–20were homogenized with mobile these 0.07 phase using the MagNa Lyser 0.00instrument. After HPLC analysis, the mean amount detected was 4.32 ± 4.83 µg. Considering the previously 2.3.2. Disulfiram obtainedConcentration Retained skin density (1.04 in the g/mL), the Dermatomized Skin obtained was 450 µg/mL. mean D concentration Over Thistheconcentration years, the literature has documented is still higher compareddifferent with the methodologies MIC value of both for quantify- microorganisms, ing drugconfirming amounts within the skin. of the suitability TheD techniques for treatinginclude skin extraction skin infections, even if measurements, the infection applies to deeper horizontal skin layers. stripping and sectioning, the removal of hair follicles, quantitative autoradiog- raphy, and spectroscopic methods [26]. Given that our target is the SC and epidermis, the 2.4. IninVitro drug content the Cytotoxicity Evaluation epidermis layer was obtained. After tape-stripping the skin, as de- Cytotoxicity scribed in Section was 2.3.1, the resttested of theinskin human-transformed tissue was cut into keratinocyte pieces of 10(HEK001) cells with the mg, and these cell proliferation were homogenized withmeasured mobile phaseby anusing adenosine triphosphate the MagNa Lyser (ATP) luminescent instrument. assay, which is After HPLC analysis,directly the mean related to thedetected amount presencewas of metabolically active 4.32 ± 4.83 µg. cells [27]. the Considering Thispreviously method was ob-preferred to other standard methods to determine cell viability, such as tained skin density (1.04 g/mL), the mean D concentration obtained was 450 µg/mL. This the MTT (tetrazolium salt) assay. isInstill concentration this case, compared higher MTT was with not used because the MIC valuethe of colorimetric assay is based both microorganisms, con- on the firming reduction of tetrazolium the suitability dye mediated of D for treating by nicotinamide skin infections, even if adenine dinucleotide the infection applies phosphate to (NADPH). deeper skin layers. D is an aldehyde dehydrogenase inhibitor and it affects NADPH levels. The results with MTT could be inconclusive; therefore, this method was discarded. The topical application of active ingredients may cause skin irritation and it is neces- sary to develop rapid assays to assess potentially damaging effects. An evaluation of the potential for an ingredient or product to cause skin irritation is one of the various studies undertaken in the overall safety assessment process. However, due to increasing concern over animal experimentation, in vitro alternatives must now be developed. Cell cytotoxicity (NADPH). D is an aldehyde dehydrogenase inhibitor and it affects NADPH levels. The results with MTT could be inconclusive; therefore, this method was discarded. The topical application of active ingredients may cause skin irritation and it is neces- sary to develop rapid assays to assess potentially damaging effects. An evaluation of the potential for an ingredient or product to cause skin irritation is one of the various studies Antibiotics 2022, 11, 1752 undertaken in the overall safety assessment process. However, due to increasing concern 6 of 14 over animal experimentation, in vitro alternatives must now be developed. Cell cytotoxi- city assays are among the most common in vitro bioassay methods used to predict the toxicityare assays ofamong substances in various the most common tissues [28,29]. in vitro In this bioassay case, weused methods havetoinvestigated the cy- predict the toxicity of substances totoxic inDvarious effect of tissues on human [28,29]. In for keratinocytes thisitscase, we have evaluation investigated potential theskin to cause cytotoxic irrita- effect tion. of D on human keratinocytes for its evaluation potential to cause skin irritation. After After 7272 hh of of treatment, treatment, in in aa testing testing range range from from 0.20 0.20 to to 33.36 µM, which 33.36 µM, which corresponds corresponds from from 0.06 to 9.89 µg/mL, the D resulted in a cytotoxic compound 9.89 µg/mL, the D resulted in a cytotoxic compound with an IC50 value with an IC 50 value of of 6.97 6.97 µM (2.31 µg/mL) (95% confidence interval (CI), 6.76 µM (2.31 µg/mL) (95% confidence interval (CI), 6.76 to 7.18) (Figure 3).to 7.18) (Figure 3). 110 100 90 80 % Cell viability 70 60 50 40 30 20 10 0 0.1 1 10 100 D concentration (µM) Figure 3. Figure 3. In In vitro vitro cell cell culture culture studies. studies. HEK001 HEK001 cell cell viability viability with with luminometric luminometric assay assay after after 72 72 hh post post treatment with D. Data are represented as the mean (n = 3) of the cell viability percentage, treatment with D. Data are represented as the mean (n = 3) of the cell viability percentage, referringrefer- ring to the untreated cell (negative control) and positive control with 1% of sodium dodecyl sul- to the untreated cell (negative control) and positive control with 1% of sodium dodecyl sulfate (SDS). fate (SDS). We found differences in the cell’s viability compared with other authors. R. Thakare et al. [9] We found reported an IC50differences of 250 µg/mLin thefor cell’s viability D using Verocompared cells andwith MTTother assay.authors. OthersR.such Thakare as J. et al.et[9]al.reported Zha [30] who an used IC50 ofRaji 250and µg/mL for cell Molt4 D using linesVero andcells MTTand MTT assay assay. Others reported an IC50such of as J. Zha 0.793 ± 0.08 et al. µM[30] andwho used 1.314 ±Raji 0.229andµM,Molt4 cell lines and respectively. The MTT assay of sensitivity reported an IC50 of the cytotoxicity 0.793 used assay ± 0.08differs µM and 1.314 ± on depending 0.229 the µM, respectively. different mechanisms,The which sensitivity leadsoftothe cellcytotoxicity death [31]. assay used Further, differs depending as commented before,onthethe differentdehydrogenase aldehyde mechanisms, which leadscaused inhibition to cell death [31]. by D may Further, with interfere as commented the results before, the aldehyde of the MTT dehydrogenase assay, which could explaininhibition causeddifferences. these observed by D may interfere E. Pena etwith the who al. [32], results of the tested theMTT sameassay, which(dexamethasone) compound could explain these observed in two differentdiffer- cell lines ences.HEK001 E. Penaand et al.HaCaT, reported [32], who lower tested the cytotoxicity same compound concentrations (dexamethasone) for the firstdifferent in two cell line but cell not linesfor the second HEK001 one. They and HaCaT, relatedlower reported thesecytotoxicity results to the immortalization concentrations procedure for the first cell to obtain the HaCaT, which can modify their cell sensibility to line but not for the second one. They related these results to the immortalization proce-xenobiotics. HEK001 cells dureare metabolically to obtain the HaCaT,less which active can thanmodify HaCaTtheir cellscell and this could sensibility tomean that they xenobiotics. have HEK001 fewer defense mechanisms against D. Therefore, these considerations could explain the differences detected with R. Thakare. Moreover, Raji and Molt4 are tumor lines and so they have the signaling system altered and their sensibility to xenobiotics is different from other cells’ sensibility. For this reason, it is important to consider what effect is expected in relation to the predicted cell death mechanism. To better understand the cytotoxicity effect in HEK001 cells, a microarray analysis was performed. Even though D was cytotoxic in low concentrations in HEK001 cells, it is important to consider that cytotoxicity tests are useful in screening chemicals for their intrinsic and relative toxicities, but it is impossible to tell whether predictions based on cytotoxicity data alone are sufficiently accurate for labeling and classifying a new chemical according to its likely acute toxicity in vivo [33]. There are difficulties in carrying out a direct extrapolation from the in vitro cell studies to the in vivo topical administration, for example, because cell culture lacks some of the properties of intact skin, such as the presence of stratum corneum and lipids, which limits the permeation of xenobiotics. In a cytotoxicity assay, cells are exposed directly to the active substances. The solubility of D is 4.09 mg/L and Antibiotics 2022, 11, 1752 7 of 14 it has a log P of 3.88 [34], which gives it poor water solubility; it tends to remain retained between the lipids of the skin, so the D exposed to keratinocytes would be considerably lower than the in vitro concentrations. Further, D has been used for years in humans after oral and topical administration and it is considered a safe product [24]. Thus, although a cytotoxic assay was used as a prescreening tool for skin irritation, due to D characteristics, the cytotoxicity results may be overestimated in comparison with the in vivo possible effect. Further studies are needed to evaluate the consistency and magnitude of possible irritant effects on the skin. 2.5. Functional Analysis of Expressed Genes An expression profile of HEK001 cells after a 48 h treatment with 6.59 µM of D was performed. This concentration was a little bit lower than the D’s IC50 and the contact time was a little bit lower than the one used in the cytotoxicity studies so as not to induce signifi- cant cell death, which could hide the effect on gene expression. With this concentration, an effect on the cell gene expression was ensured and total cellular mortality was prevented. Forty-nine genes presented with a differential expression after treatment with D versus cells without treatment. The differentially expressed genes were further classified into two groups: upregulated genes (Table 3) and downregulated genes (Table 4). The main association between the altered genes is represented in Figure 4. Table 3. Upregulated genes. Target prediction was performed using Affymetrix software. A list of differentially expressed (more than 1.5-fold, t-test p-value < 0.05) putative targets was obtained. The ratio column corresponds to the fold change in expression for each gene relative to the control, calculated from the normalized values. Gen Symbol Gen Description (D) Raw (Control) Raw Ratio CLU|MIR6843 clusterin|microRNA6843 312.08 106.16 3.08 CCDC138 Coiled-coil domain-containing protein 138 82.23 48.90 1.75 MT1F Metallothionein-1F 3379.27 2011.00 1.75 DNAH14 Dynein axonemal heavy chain 14 60.23 35.91 1.69 ZNF483 Zinc finger protein 483 77.93 46.19 1.69 WDR78 Dynein axonemal intermediate chain 4 80.69 53.16 1.71 BVES-AS1 BVESantisenseRNA1 59.32 35.94 1.66 Dolichyl pyrophosphate Man9GlcNAc2 ALG6 85.31 49.07 1.65 alpha-1,3-glucosyltransferase RASSF6 Ras association domain-containing protein 6 52.74 33.33 1.64 DOCK11 Dedicator of cytokinesis protein 11 161.39 107.55 1.64 MNS1 Meiosis-specific nuclear structural protein 1 129.81 88.17 1.61 PAIP2|CTB-43P18.1 Poly(A)bindingproteininteractingprotein2 128.98 84.64 1.57 CNTRL Centriolin 78.00 48.35 1.59 SGOL2 Shugoshin 2 186.99 117.74 1.59 NUP133 Nuclear pore complex protein Nup133 161.03 99.01 1.57 ZNF780A Zinc finger protein 780A 105.86 67.45 1.57 CCDC66 Coiled-coil domain-containing protein 66 91.98 57.60 1.59 GLMN Glomulin 36.51 22.98 1.58 KDM4D Lysine-specific demethylase 4D 49.39 32.68 1.53 SNX25 Sorting nexin-25 127.76 89.91 1.57 ARHGEF26 Rho guanine nucleotide exchange factor 26 47.67 35.02 1.57 COX assembly mitochondrial CMC1 97.74 62.22 1.53 protein homolog FASTKD1 FAST kinase domain-containing protein 1 259.25 172.60 1.52 DNAJC21 DnaJ homolog subfamily C member 21 497.80 327.50 1.52 DDX5 ATP-dependent RNA helicase DDX5 142.99 101.83 1.56 CEP83 Coiled-coil domain-containing protein 41 86.58 56.97 1.52 BUD31 Protein BUD31 homolog 29.50 19.38 1.51 KIN DNA/RNA-binding protein KIN17 131.16 84.77 1.50 Antibiotics 2022, 11, 1752 8 of 14 Table 4. Downregulated genes. Target prediction was performed using Affymetrix software. A list of differentially expressed (more than 1.5-fold, t-test p-value < 0.05) putative targets was obtained. The ratio column corresponds to the fold change in expression for each gene relative to the control, calculated from the normalized values. Gen Symbol Gen Description (D) Raw (Control) Raw Ratio Ankyrin repeat domain-containing ANKRD36 38.41 67.57 2.28 protein 36A YWHAG 14-3-3 protein gam 25.03 46.32 1.87 EXT1|hunera Jeck2013ALT 88.64 147.16 1.98 QRICH2 Glutamine-rich protein 2 33.80 65.17 1.82 YBX1 Nuclease-sensitive element-binding protein 1 47.70 69.96 1.68 DGCR8 Microprocessor complex subunit DGCR8 74.69 118.81 1.60 C1orf198 Chromosome1openreadingframe198 43.49 66.29 1.64 Forkhead-associated domain-containing FHAD1 46.94 71.21 1.60 protein 1 PLXNB3 Plexin-B3 56.35 83.87 1.56 CYGB Cytoglobin 62.20 98.36 1.58 CCDC84 Coiled-coil domain-containing protein 84 167.37 242.60 1.60 F10 Coagulation factor X 70.55 101.19 1.56 PRR21 Proline rich 21 19.56 28.10 1.57 Polypeptide GALNT14 29.99 44.42 1.55 N-acetylgalactosaminyltransferase 14 ADCY2 Adenylate cyclase type 2 31.16 46.58 1.55 KIRREL2 Kin of IRRE-like protein 2 27.16 39.19 1.54 RPS6KA1 Ribosomal protein S6 kinase alpha-1 34.27 48.01 1.52 AP1M1 AP-1 complex subunit mu-1 142.99 204.18 1.53 Antibiotics 2022, 11, 1752 PRR26 Proline-rich protein 26 40.47 60.82 9 of 15 1.50 CRYM Thiomorpholine-carboxylate dehydrogenase 34.46 52.06 1.51 WNT8B Wingless-type mmtv integration site family 20.00 26.53 1.50 Figure 4. Interaction network of the main altered genes (upregulated and downregulated) for their Figure 4. Interaction network of the main altered genes (upregulated and downregulated) for their response response to to disulfiram. disulfiram. Each Each gene gene is represented is represented as aasnode a node andand each each lineline represents represents thethe number number of of interactions between genes. Image was created using the STRING interactions between genes. Image was created using the STRING Database.Database. As seen in Table 3, there was an upregulated expression of several genes in the samples As seen in Table 3, there was an upregulated expression of several genes in the sam- (28 genes) from the treated cells (HEK001 6.59 µM D after 48 h) in comparison with the ples (28 genes) from the treated cells (HEK001 6.59 µM D after 48 h) in comparison with non-treated cells (control). Considering that the expression came from cells that have the non-treated cells (control). Considering that the expression came from cells that have survived the drug exposure, multiple genes related to cell division were overexpressed; they are involved in cell cycle arrest after the detection of DNA damage to ensure its cor- rect repair and they activate signaling pathways as a mechanism of resistance to D. These genes were SGOL2, KDM4D, DNAJC21, MNS1, DOCK11, and DDX5. Additionally, again Antibiotics 2022, 11, 1752 9 of 14 survived the drug exposure, multiple genes related to cell division were overexpressed; they are involved in cell cycle arrest after the detection of DNA damage to ensure its correct repair and they activate signaling pathways as a mechanism of resistance to D. These genes were SGOL2, KDM4D, DNAJC21, MNS1, DOCK11, and DDX5. Additionally, again related to cell division, there was an overexpression of genes such as KIN, which is involved in DNA replication and the cellular response to DNA damage. The gene whose expression was most affected was the microRNA (miRNA) CLU|MIR6843. miRNAs were recently discovered to be regulatory expression molecules. Consisting of ≈22 non-coding nucleotides that regulate gene expression via hybridization with messenger RNAs (mRNAs), mRNA degradation and the translational inhibition of targeted transcripts are consequences of this process [35]. In this case, it affected the clusterin (CLU) gene, which has been independently identified as being involved with various fields without clear relationships between them. It is associated with many biological functions, including tissue differentiation and remodeling, membrane recycling, lipid transportation, cell–cell or cell–substratum interac- tion, cell motility, cell proliferation, and cell death [36]. The affectation in this multifunctional gene could play an important role in the cytotoxicity of D. It is important to mention the alteration of the MT1F gene. Metallothioneins (MTs) have a high cysteine residue content, which binds various heavy metals. They are one of the most important biological mechanisms used to protect against toxic metal exposure. Increasing levels of cellular zinc ions activate transcription factor 1 (MTF-1), the major transcriptional regulator [37]. D is a chelator of ions, especially divalent ions such as zinc and copper. D acts as a Cu/Zn ionophore, facilitating cellular accumulation [38]. Therefore, it can alter the cellular levels of these ions and, consequently, produce cell death. Possible mechanisms of MT1F activation of D include induction of zinc influx, generation of reactive oxygen species that induce the release of zinc from MTs, or direct binding to MTF-1 or another protein necessary for MTF-1 transcriptional activation [37]. Moreover, two additional genes related to zinc were altered: ZNF483 and ZNF780A. Both are zinc finger proteins, implicated in transcriptional regulation. Another important upregulated gene is RASSF6. It is involved in the induction of apoptosis; it may act as a Ras effector protein or suppress the serum-induced basal levels of NF-kB. This result is consistent with the results of other authors, who reported the inhibition of NF-kB and the apoptosis by disulfiram and other carbamates [39,40]. Out of the 49 genes affected, 21 were downregulated (Table 4). An alteration of the expression of genes related to the expression of transmembrane protein, adhesion, and cell migration, such as PLXNB3, KIRREL2, and PLXNB3, was observed. These alterations could decrease the cell’s protection mechanisms and make it more vulnerable to death. Moreover, signaling genes such as RPS6KA1, YWHAG, and ADCY2 also decreased. The chelating effect of D could explain the interruption of signal pathways where divalent ions participate. These results are also in agreement with other authors, who reported the cytotoxicity effect induced by the sequestration and alteration of intracellular and extracellular levels of zinc [41]. D has been shown to affect cell ion levels, producing alterations in signaling pathways and cell division. Other authors reported additional pathway alterations such as the inhibition of proteasome signaling [42,43]. Therefore, cytotoxicity was not caused by a single mechanism but by different mechanisms that induce the final apoptosis of the cell. Concerning the mechanism of resistance, metallothionines may have an important role. These results suggest that the chelating character of D could be related to its antibac- terial effect. For example, Nabil M. Abraham et al. reported that a metal ion chelator can inhibit S. aureus biofilm formation and viability and that metallic cations, such as Ca2+ and Mg2+ , play a role in bacterial growth [44]. Nevertheless, additional studies are needed to determine the mechanism of action of D as an antimicrobial. Antibiotics 2022, 11, 1752 10 of 14 3. Materials and Methods 3.1. Materials Disulfiram, cocoa butter, stearic acid, and cetostearyl alcohol were kindly gifted by Bioglan AB (Malmö, Sweden). Methanol and KH2 PO4 (Scharlab, S.L., Sentmenat, Spain) were used as mobile phases. Phosphate-buffered saline (PBS) (Sigma-Aldrich, Saint Louis, MO, USA) and hydroxypropyl-beta-cyclodextrin (HPCD) (Pracofar, S.L., Martorell, Spain) were used as receptor media in skin permeation experiments. For cytotoxic evaluation, dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Burlington, MO, USA), keratinocyte serum- free medium, L-glutamine, and epidermal growth factor (hEGF) (ThermoFisher, Waltham, MC, USA) were used. Mueller–Hinton broth (MHB) and chocolate agar were purchased from Condalab (Madrid, Spain). Gentamicin (Sigma-Aldrich, Saint Louis, MO, USA), clindamycin (Sigma-Aldrich, Saint Louis, MO, USA), and lysed horse blood (Themo Fisher, Landsmeer, The Netherlands) were used for antibacterial studies. 3.2. Antibiotic Susceptibility Testing The strains used in the study were from American Type Culture Collection and were purchased through LGC Standards, S.L.U (Barcelona, Spain). Disulfiram was studied against S. aureus (ATCC® 25923), S. pyogenes (ATCC® 12344), and P. aeruginosa (ATCC® 27853). The MIC of D was determined using a broth microdilution assay in 96-well microplates in accordance with method described inCLSI guidelines [19]. MIC was defined as the lowest drug concentration that inhibited visual growth. The overnight culture was adjusted to a 0.5 McFarland and diluted to 1:100 in media; MHB, in the case of S. aureus and P. aeruginosa, and MHB with 5% LHB for S. pyogenes, were treated with serial dilutions of each antibiotic. Gentamicin was used as a positive antibiotic effect control for S. aureus and P. aeruginosa, and clindamycin was used for S. pyogenes. Stock solutions of D (10 mg/mL) were prepared in ethanol. Gentamicin and clindamycin were prepared in the same concentration in each growth medium. D was tested in amounts ranging from 64 to 0.125 µg/mL due to solubility limitations. The final ethanol concentration of these dilutions was below 0.5% to not alter the bacterial growth. Negative controls were used with the respective media and the same ethanol concentration as the samples with D was used. Positive controls were performed with bacteria with the respective media and without D. Gentamicin and clindamycin were tested in ranges from 512 to 0.125 µg/mL. The plates were sealed with adhesive film and incubated at 37 ◦ C for 18–24 h and then the MIC was determined. For each test compound, MIC determination was carried out independently three times. 3.3. Production of Disulfiram Emulsion The oil phase was a mixture of cocoa butter (7% w/w), stearic acid (6% w/w), cetostearyl acid (1% w/w), and D (2% w/w). Fats were melted at 75 ◦ C and the water phase (water q.s 100% with preservative) was heated at the same temperature in a water bath. Then, both phases were mixed and homogenized at 11.000 rpm with an Ultra-Turrax (IKA T-25, Staufen im Breisgau, Germany) for 10 min. 3.4. Ex Vivo Skin Absorption Experiment Abdominal pig skin (n = 11) was obtained from a local slaughterhouse (Barcelona, Spain). Permeation studies were accomplished in vertical Franz diffusion cells (VidraFoc, Barcelona, Spain) with a permeation area of 1.54 cm2 . The skin was cut with a thickness of approximately 0.5 mm using a dermatome CG1371 (Nouvag AG, Goldach, Switzerland). Then, 15% HPCD on PBS at pH 5.5 was used as a receptor medium to maintain sink conditions throughout the experiment, and it was kept at 32 ◦ C and stirred at 500 rpm. A transepidermal water loss (TEWL) measurement device (Delfin technologies, Kuopio, Finland) was used to evaluate skin integrity. Samples from a receptor compartment (300 µL) were taken at fixed times and replaced by an equivalent volume of fresh receptor medium at the same temperature, and they were analyzed using the HPLC method (Waters 2695, Milford, MA, USA) [24]. Sampling times were 1, 2, 3, 4, 5, 20, 21, 22, 23, and 24 h. Antibiotics 2022, 11, 1752 11 of 14 The disulfiram emulsion was dosed in a compartment in the equivalent human pre- scription dose (3.65 mg/cm2 ) [24] to study skin absorption. Placebo formulation was tested as a negative control of permeation and to check the lack of analytical interferences due to the presence of excipients. 3.5. Quantification of Disulfiram in Skin after Permeation Experiments 3.5.1. Determination of Pig Skin Density Three replicates of each skin were used. The density (ρ) of a solid is the ratio between the mass and the volume it occupies. The volume occupied by the skin was determined using Equation (1): M + M2 − M3 Volume = 1 (1) ρ where M1 is the weight of the skin, M2 is the weight of the flask filled with water, M3 is the weight of the flask filled with water and the skin, and ρ is the density of the water (1 g/cm3 ). 3.5.2. Tape-Stripping Study After the permeation experiment, five of the eleven Franz cells with the pig skin were used to study the disulfiram retained in the stratum corneum. Prior to tape-stripping, the emulsion residue was removed with a swab soaked in PBS. After 24 h, the strips (Tesa® 4101 PV2, Shanghai, China) were carefully adhered to the diffusional skin area and a constant weight (345 g) was used to press the tape onto the skin surface for 10 s. The following tape strip groups were carried out: strip 1, strip 2, strips 3–7, strips 8–12, strips 13–17, and strips 18–20. The tape strips were placed in 50 mL Falcon tubes and we added 4 mL of mobile phase. Then, the samples were sonicated for 15 min in an ultrasonic bath (JP Selecta™, Abrera, Spain) and analyzed using the same HPLC method in Section 3.4. 3.5.3. Determination of the Concentration Retained in the Dermatomized Skin After tape-stripping, the skin sections were used to extract the retained D. In total, 10 mg of the area of each replicate per duplicate was cut and introduced into the MagNa Lyser instrument (Roche, Sant Cugat del Valles, Spain) with 600 µL of mobile phase. The homogenization of the tissue was carried out with 5 cycles of 90 s at 6500 rpm. Finally, the samples were analyzed using the HPLC method. 3.6. In Vitro Cytotoxicity Evaluation HEK001 (CRL2404, ATCC, Manassas, VA, USA) cells were seeded in 96-well plates (n = 6) at 10,000 cells/well in 50 µL of medium (keratinocyte serum-free medium, supple- mented with 2 mM L-glutamine and 5 ng/mL of hEGF). The cells were incubated at 37 ◦ C in 5% CO2 and a 95% air-humidified atmosphere for 24 h. After 24 h of incubation, the cells were treated with 100 µL of D solution in cell media at different concentrations (33.36 to 0.20 µM). A stock solution was prepared in DMSO. The DMSO concentration in the well was below 1% and a DMSO control was carried out. Negative control of cytotoxicity was performed with cells in the same media and positive control of mortality with 1% of SDS (ThermoFisher, Waltham, MA, USA). The exposure period was 72 h. At the end of the incubation time, a CellTiter-Glo® Luminescent Cell Viability Assay (Dojindo Molecular Technologies, Rockville, MD, USA) was used to evaluate cytotoxicity. CellTiter-Glo® reagent was added (100 µL) directly to every well plate cultured in serum- free medium and incubated for 10 min. Luminescence was measured with a Luminometer Victor X3 (Perkin Elmer, Waltham, MA, USA). Cytotoxicity plots and IC50 values were obtained. Cell survival was calculated considering the 100% viability of the untreated Antibiotics 2022, 11, 1752 12 of 14 control cells (negative control) and 100% of mortality positive control. The percentage of cell viability was calculated using the following equation: (LNegative control − LExperimental value) /(LNegative control − LPositive control ) × 100 (2) where LExperimental value is the luminescence of the sample, LNegative control is the mean lumi- nescence of cells in media control, and LPositive control is the absorbance with 1% SDS control. 3.7. Microarrays and Data Analysis HEK001 cells were seeded in 6-well plates at 250,000 cells/well in 2 mL of medium (keratinocyte serum-free medium and L-glutamine (99:1)). The cells were incubated at 37 ◦ C in 5% CO2 and a 95% air-humidified atmosphere for 24 hr. After 24 h of incubation, the cells were treated with 6.59 µM of D. The compound was tested in duplicate and cells with medium without D were used as a control (n = 3). The exposure period was 24 h. After the incubation time, the total RNA from each sample was extracted and purified from HEK001 cells using a RNeasy® Plus Mini insolation Kit (Qiagen, Hilden, Germany) according to the instructions provided by the manufacturer. Total RNA concentration and quality were spectrophotometrically measured using the absorbance ratio 260:280 nm with NanoDropTM Lite (ThermoFisher Scientific, Waltham, MA, USA). Once the RNA was obtained, cDNA was synthesized with PxE Thermal Cycler (ThermoFisher, Waltham, MA, USA) according to the manufacturer’s recommendations. For microarray analysis, GeneChip® Clariom S Human Array (ThermoFisher Scientific, Waltham, MA, USA) was used. Data were generated and processed with Affymetrix software (Santa Clara, CA, USA). Gene expression data from the samples were compared using a one-way t-test using stringent transcript cut-off criteria with fold change (FC) > 1.5 and p-value ≤ 0.05. 4. Conclusions Disulfiram is effective against S. aureus and S. pyogenes and is a potential alternative to classic antibiotics in the treatment of skin infections. Skin permeation studies showed no permeation in pig skin confirming the suitable topical use in superficial human infections. The drug concentration in pig skin tissue was higher than the MIC value of both microor- ganisms, being potentially effective in the treatment of cutaneous infections. Disulfiram showed cytotoxicity in transformed human keratinocyte cells and several upregulated and downregulated genes after its incubation. The main affected genes were related to cell proliferation signaling and the metabolism of cell ions, which is in agreement with the potential mechanism of action of the drug as a metal ion chelator, essential for cell home- ostasis. However, the implementation of D in clinical use has encountered some challenges and further research should be carried out, for example, to evaluate the local tolerance in vivo after D topical administration. Further exploration of the molecular mechanism of disulfiram as an antibacterial agent is needed. Taken together with these results, disulfiram could be a good candidate as an alternative treatment for skin infections, considering the global concern of limited therapeutic options. Author Contributions: Conceptualization, F.F.-C., M.L.C., M.À.C., P.C. and E.R.-V.; methodology, M.L-R., M.C.-S., E.P.-R., F.F-C, P.C., M.L.C. and E.R.-V.; validation, M.L.-R., M.C.-S. and E.P.-R.; formal analysis, M.L.-R. and M.C.-S.; investigation, M.L.-R. and M.C.-S.; resources, F.F.-C., M.L.C. and E.R.-V.; data curation, M.L.-R., M.C.-S., E.P.-R., F.F.-C., P.C., M.L.C. and E.R.-V.; writing—original draft preparation, M.L.-R.; writing—review and editing, M.L.-R., M.C.-S., E.P.-R., F.F.-C., P.C., M.L.C., M.À.C. and E.R.-V.; visualization, M.L.-R. and M.C.-S.; supervision, F.F.-C., M.L.C. and M.À.C.; project administration, F.F.-C., M.L.C. and M.À.C.; funding acquisition, E.R.-V. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Generalitat of Catalonia, Industrial Doctorate Program, grant number 2020 DI 97. Institutional Review Board Statement: Not applicable. Antibiotics 2022, 11, 1752 13 of 14 Informed Consent Statement: Not applicable. Data Availability Statement: Data are available upon request due to intellectual property. Acknowledgments: We thank the valuable contribution of our technical assistants: Mari Carmen Moreno (Laboratory Reig Jofre, Sant Joan Despí, Barcelona, Spain) and Carles Ciutat (Molecular Biology department, Faculty of Pharmacy, University of Barcelona, Spain). Conflicts of Interest: M.L.-R., E.P.-R., M.C.-S., E.R.-V., and F.F.-C. are employees of Laboratory Reig Jofre. They belong to the R&D department and do not participate in marketing or commercial activities. The data presented were conducted under general good laboratory practices. The other authors do not declare any conflicts of interest. References 1. Williamson, D.A.; Carter, G.P.; Howden, B.P. Current and emerging topical antibacterials and antiseptics: Agents, action, and resistance patterns. Clin. Microbiol. Rev. 2017, 30, 827–860. [CrossRef] [PubMed] 2. 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