Journal of Applied Microbiology , 2024, 135 , lxae147 https://doi.org/10.1093/jambio/lxae147 Advance access publication date: 2 July 2024 Research Article The int er activ e effects of medicinal dy es with con v entional antimicrobials against skin pathogens Rhea Ramfol and Sandy van Vuuren * Department of Pharmacy and Pharmacology, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg 2193, South Africa ∗Corresponding author. Department of Pharmacy and Pharmacology, Faculty of Health Sciences, University of Witwatersrand, Johannesburg 2193, South Africa. E-mail: Sandy.vanvuuren@wits.ac.za Abstract Aims: This study aimed to explore potential synergistic effects of medicinal dyes with antimicrobials against pathogens responsible for skin infections. Methods and results: Antimicrobial testing was conducted using minimum inhibitory concentrations and minimum bactericidal/fungicidal con- centration assa y s. T he fractional inhibitory inde x ( �FIC) of combinations w as calculated, and isobolograms w ere constructed on selected combi- nations. Toxicity studies were conducted using the brine-shrimp lethality assay. Combination (1:1 ratio) studies noted that 26% of dye-antibiotic combinations were synergistic against the Gram-positive strains, 15% against the Gram-negative strains, and 14% against the yeasts. The Mer- curochrome: Betadine ® combination noted synergy at ratios against all the Staphylococcus aureus strains with �FIC values ranging from 0.05 to 0.48. The combination of Gentian violet with Gentamycin noted a 15-fold decrease in toxicity, and a selectivity index of 977.50 against the Esc heric hia coli (DSM 22314) strain. Time-kill studies were conducted on the combinations with the highest safe selectivity index (SI) value and lo w est safe SI v alue i.e. Gentian violet with Gentam y cin and Malachite green with Neom y cin. B oth combinations demonstrated better antimicrobial activity in comparison to the independent values and the controls. Conclusion: This study highlights the potential for medicinal dye combinations as a treatment for skin infections. Impact Statement Many antibiotics have become ineffective in treating the simplest of infections thereby sparking new interest in combination therapy. Research into medicinal dyes has all been forgotten in the shadow of antibiotic efficacy. The current study provides a reasonable option for combatting antimicrobial resistance by examining the potential of combination therapy of medicinal dyes with antimicrobials. Not only do these combinations provide feasible synergistic options, but also target clinically resistant pathogens. Ke yw or ds: medicinal dyes; commercial antimicrobials; skin infections; antimicrobial resistance; combinations; synergy; toxicity a i a m 1 2 o s b m c c e w t a v a s l w s D ow nloaded from https://academ ic.oup.com /jam bio/article/135/7/lxae147/7704455 by jam estem lett user on 04 July 2024 Introduction The skin is one of the first line defences against microbial inva- sion (Williamson et al. 2017 ). Infections caused by antibiotic- resistant bacteria, especially skin and soft tissue infections, have become prevalent in hospital and community settings. This has led to a change in the approach to empiric antibiotic use (Lushniak 2014 ). Antibiotic resistance is now a worldwide crisis, as difficulty in treating microbial pathogens has led to a high rate of mortality (Frieri et al. 2017 ). Many antibiotics have become ineffective in treating the simplest of infections, and this has led to a high number of hospitalizations, fail- ures in treatment, and the development of more drug-resistant pathogens (Martens and Demain 2017 ). Therefore, treatment plans have changed from monother- apy to combination therapy (Poustchi et al. 2021 ). Combina- tion therapy can reduce resistance, lower treatment costs, and even reduce the therapeutic dose needed, and this has been found to be an effective strategy (Sullivan et al. 2020 , Poustchi et al. 2021 ). One such strategy is the possible combination with alterative antimicrobials such as dyes. Dyes were introduced into medicine in the 19th century by Koch and Ehrlich and have been used for centuries to treat various medical conditions (Wainwright 2014 ). The popular- ity decreased due to the discovery of antibiotics as well as their Received 3 January 2024; revised 21 May 2024; accepted 1 July 2024 © The Author(s) 2024. Published by Oxford University Press on behalf of Applie under the terms of the Creative Commons Attribution License ( https:// creativecom and reproduction in any medium, provided the original work is properly cited. dverse side effects, ranging from tissue colouration to toxic- ty and mutagenicity (Wainwright 2014 ). Research into dyes s an antiseptic has been largely lacking in recent years and ost clinical work involving dyes has been carried out before 945, after which, antibiotics took the spotlight (Wainwright 014 ). Commonly used over-the-counter dyes such as Gentian vi- let, Iodine tincture, and Mercurochrome are commercially old in various grocery stores and pharmacies. As described y Wainwright ( 2008 ), there are two major factors as to why edicinal dyes are regularly used; firstly, is the ability to dis- olour the skin, and secondly is that dyes have been shown to ause mutation in vivo , which can be a cause for concern. As ffective as dyes are, they are not without risk as it is the case ith most medicines and, indeed, antibiotics. Dyes are known o have toxicity (Wainwright 2008 ). It is therefore critical to ssess the toxicity of the individual dyes and combinations at aried concentrations to determine the safety. The purpose of this study was to determine the interactive ntimicrobial and toxicity effects of seven medical dyes (Fuch- ine, Gentian violet, Iodine tincture, Malachite green, Methy- ene blue, Mercurochrome, and Potassium permanganate) ith seven conventional antimicrobials (Chlorohexidine, Fu- idic acid, Nystatin, Ketoconazole, Miconazole, Povidone io- d Microbiology International. This is an Open Access article distributed mons.org/ licenses/ by/ 4.0/ ), which permits unrestricted reuse, distribution, https://doi.org/10.1093/jambio/lxae147 https://orcid.org/0000-0002-4859-1845 mailto:Sandy.vanvuuren@wits.ac.za https://creativecommons.org/licenses/by/4.0/ 2 Ramfol and van Vuuren d T M D A t ( b a s c t P t l c p M ( c a s a 1 c p r t m g a A T m ( t c L l g c c c ( c d c 5 i 4 a i c m ( t M b T d d a T b t c o t ( t c b p b u p v f e 1 w G t μ l c c t 2 r w i t w M ( t a s w s F v I b c r a t t D ow nloaded from https://academ ic.oup.com /jam bio/article/135/7/lxae147/7704455 by jam estem lett user on 04 July 2024 ine, and Mupirocin) associated with skin infection treatment. he impact of toxicity was also considered. aterials and methods ye and antibiotic preparation selection of dyes for consideration was based on a review of he literature where antimicrobial activity has been observed Owen 1913 , Smith 1915 , Gupta et al. 2008 , Berrios and Ar- iser 2011 , Wainwright 2014 , Rosa et al. 2015 , Korkmaz et l. 2019 ). The dyes (indicated for topical use only) were Fuch- ine (Sigma-Aldrich), Methylene blue (Sigma-Aldrich), Mala- hite green (Saarchem), Potassium permanganate (ACE), Gen- ian violet, Iodine tincture, and Mercurochrome (Dis-Chem harmacies). The dyes were initially prepared to a concen- ration of 32 mg/ml −1 in sterile water, except Gentian vio- et (5.00 μg/ml −1 ), Iodine tincture (25.00 μg/ml −1 ), and Mer- urochrome (20.00 μg/ml −1 ) as they were procured at the pre- ared concentrations. The antibiotics Fusidic acid ( ±100%), Mupirocin ( ≥92%), iconazole (99.50%), Gentamycin ( ±100%), Neomycin 90%–100%), Nystatin ( ≥4400 USP units per mg), and Keto- onazole ( ±100%) (all obtained from Sigma-Aldrich), as well s Betadine ® and Steriscrub (Dis-Chem, South Africa) were elected based on use to treat skin infections. The antibiotics nd antifungals were prepared at a starting concentration of 0.00 μg/ml −1 , while Betadine ® (100.00 μg/ml −1 ) was pro- ured at a prepared concentration, and Steriscrub was pre- ared to a concentration of 32 000.00 μg/ml −1 diluted in 1:1 atio of water to acetone. A preliminary test was conducted o determine the viable concentrations (32 000.00 μg/ml −1 for edicinal dyes and 10.00 μg/ml −1 for antibiotics and antifun- als) required for further dilution that still note antimicrobial ctivity. ntimicrobial analysis he pathogens were selected based on their role in causing der- atological infections. The strains included reference strains American Type Culture Collection ATCC; Davies Diagnos- ics, Johannesburg, South Africa). Clinical strains were re- eived from the NHLS Infection Control and Microbiology aboratory (National Health Laboratory Services), and se- ected resistant strains (Deutsche Sammlung von Mikroor- anismen; DSM) were obtained from the German culture ollection (The Leibniz Institute). The bacterial strains in- luded: Staphylococcus aureus (ATCC 46644), S. aureus clini- al strain, methicillin resistant Staphylococcus aureus (MRSA) ATCC 43300), Gentamycin–methicillin resistant Staphylo- occus aureus (GMR S A) (ATCC 33592), Staph ylococcus epi- ermidis (ATCC 12228), Staphylococcus epidermidis clini- al strain, Staphylococcus epidermidis resistant strain (ATCC 1625), Pseudomonas aeruginosa (ATCC 27858), P. aerug- nosa clinical strain, P. aeruginosa resistant strain (ATCC 6316), Esc heric hia coli (ATCC 8379), E. coli clinical strain, nd E. coli resistant strain (DSM 22314). The yeast strains ncluded Candida albicans (ATCC 10231), C. albicans clini- al strain and C. albicans resistant strain (ATCC 90028). All icroorganisms were cultured in Tryptone Soya broth (TSB) Oxoid). Bacterial strains were incubated at 37 ◦C for 24 h and he yeasts at 37 ◦C for 48 h. inimum inhibitory concentration and minimum actericidal/fungicidal assay o evaluate the antimicrobial activity of samples indepen- ently and in combination, the Clinical and Laboratory Stan- ards Institute (CLSI) guidelines ( 2020 ) was followed. In 96-well micro-titre plate, 100.00 μl of TSB was added. he medicinal dyes, commercial antimicrobials and/or com- inations were added into the first row of wells according o the following ratios: 100.00 μl of selected dye and/or ommercial antimicrobial and for combinations, 50.00 μl f dye and 50.00 μl antimicrobial (1:1 ratio). A posi- ive control of a broad-spectrum commercial antimicrobial Ciprofloxacin at 0.01 mg/ml −1 for bacteria and Ampho- ericin B at 0.10 mg/ml −1 for the yeasts), a negative solvent ontrol of 32.00 mg/ml −1 water to acetone and a negative roth control of sterile TSB was added to each micro-titre late. The positive control was added to ensure antimicro- ial susceptibility of pathogens. The TSB culture control was sed to ensure that the media will support the growth of the athogen. The negative control was added to ensure the sol- ent does not exhibit antimicrobial activity. Thereafter, a two- old serial dilution was carried out. Pathogens were added into ach well (100.00 μl) with the approximate inoculum size of × 10 8 colony forming units (CFU)/ml −1 . Microtitre plates ere then sealed with a sterile adhesive seal (Macherey-Nagel mbH & Co KG) and incubated as per respective incuba- ion conditions. After the appropriate incubation period, 40 l of the indicator, 0.4 mg/ml −1 p -iodonitrotetrazolium vio- et (Sigma-Aldrich), was added to each well. The lowest con- entration that inhibits growth was the minimum inhibitory oncentration (MIC) end-point value and was shown with he absence of purple-pink colour in the well (Hübsch et al. 014 ). The MIC assays are largely dependent on a colorimet- ic change; therefore, interpretation of results can be difficult hen dyes or dye combinations are used, due to the stain- ng properties. To overcome this problem with staining due to he dyes minimum bactericidal (MBC) assays were conducted, hereby a loop of culture was taken from each well from the IC assays and streaked onto subdivided Tryptone Soya agar TSA) plates. The plates were then incubated under the op- imal incubation conditions. The absence of growth on the gar plates indicated the bactericidal or fungicidal activity, re- pectively. The study was done in triplicate and comparisons ere made between how the clinical and reference strains re- ponded to the test inhibitors. ractional inhibitory concentration index and aried ratio concentrations nteractions between the combinations of dyes and antimicro- ials were studied using the sum of the fractional inhibitory oncentration ( �FIC). The �FIC was calculated where (a) epresents the dye sample and (b) represents the commercial ntimicrobial sample (van Vuuren and Viljoen 2011 ) (Equa- ion 1 ): FIC (i) = MIC (a) in combination with (b) MIC (a) independently FIC (ii) = MIC (b) in combination with (a) MIC (b) independently (1) The FIC index is then calculated using the following equa- ion: �FIC = FIC (i) + FIC (ii). The interactions were Dyes with conventional antimicrobials 3 t t w v 2 1 ( b 2 T T ( t t e s o r a r s s 1 t u a c u w t μ T n p t t a R A a T e f S u c s t G b t m a t D ow nloaded from https://academ ic.oup.com /jam bio/article/135/7/lxae147/7704455 by jam estem lett user on 04 July 2024 classified as being synergistic ( �FIC is ≤ 0.5), additive ( > 0.5– 1.0), indifferent ( > 1.0–≤ 4.0) or antagonistic ( > 4.0) (van Vu- uren and Viljoen 2011 ). If a pattern was observed i.e. majority of the combinations noted synergy or antagonism when tested against a specific pathogen group (Gram-positive, Gram-negative strains, and yeasts) then that combination was chosen for further varied ratio testing. The dye samples and commercial antimicrobials in combination were prepared in nine different ratios (9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and 1:9) and the MIC values de- termined as per the method described in the ‘Minimum in- hibitory concentration and minimum bactericidal/fungicidal assay’ section. Data points for each ratio were plotted first on an excel spreadsheet and then transferred to GraphPad Prism ® (Ver- sion 5) software where an isobologram was constructed. The �FIC and varied ratio concentrations were conducted in trip- licate. Toxicity studies To determine the toxicity of the medicinal dyes and commer- cial antimicrobials the brine shrimp lethality assay (BLSA) was carried out (Hübsch et al. 2014 ). The medicinal dyes were tested at concentration of 500.00 μg/ml −1 except Gen- tian violet, Iodine tincture, and Mercurochrome, which were tested at the predetermined procured concentration as a start- ing point. The antibiotics and antifungals were also tested at 500.00 μg/ml −1 except Betadine ®, which was already at a pre- determined concentration. Combinations tested in the toxicity studies were based on their antimicrobial activity (synergistic and antagonistic interactions), varied ratio testing and the in- dividual toxicity studies of the medicinal dyes and commercial antimicrobials (non-toxic values). Both synergistic and antag- onistic interactions were considered equally important. For the BSLA, Tropical sea salt weighing 32.00 g was dissolved in 1.00 l of distilled water. Dried, brine-shrimp ( Artemia franciscana ) eggs (Ocean Nutrition™) weighing 0.50 g was added to the artificial sea water. To achieve a high hatch rate, a rotary pump (Kiho) was used to aerate the water and disperse the eggs. The eggs were incubated for 18–24 h at ambient temperature. A 220–240 V lamp was used as a source of light and warmth to help the hatching process. Once hatched, 400.00 μl of saltwater containing live brine-shrimp (40–60 brine-shrimp) was added to each micro-titre plate well. In each well, 400.00 μl of the sample (antimicrobials, dyes, or combinations) was added. The negative control used was salt water with a concentration of 32.00 g/l. The positive control that was used is 1.60 mg/ml −1 potassium dichromate (Sigma- Aldrich). After 24 and 48 h, the plates were observed un- der a light microscope (Olympus) and the dead shrimp were counted. Lastly, a lethal dose of 50 μl acetic acid (100% v/v) (SAAR Chem-Trade Pvt. Ltd) was added in each well and a to- tal count of dead shrimp were taken; thereafter, the percentage of mortality calculated (Equation ) A percentage mortality of 50% or greater is considered toxic (Bussmann et al. 2011 ). The tests were conducted in triplicate. Percentage mortality (%) = Total number of dead shrimp at 24 / 48 h Total number of shrimp × 100 (2) If the sample (dye, commercial antimicrobial, or combi- nation) was determined to be toxic, the concentration of he sample was lowered whereby a dose-response was de- ermined from a series of concentrations. An LC 50 value as then determined and classified as highly toxic (LC 50 alue < 249 μg/ml −1 ), moderate toxicity (LC 50 value of 50–499 μg/ml −1 ) and low toxicity (LC 50 value of 500– 000 μg/ml −1 ) (Bussmann et al. 2011 ). The selectivity index SI), which is defined as the ratio of toxicity to antimicro- ial activity (MIC) was calculated (Equation 3 ) (Elisha et al. 017 ). SI = L C 50 MIC (3) ime-kill studies ime-kill studies were conducted on selected combinations including individual samples) that demonstrated the best an- imicrobial activity with the least toxicity when combined, i.e. he highest SI index. In this study, the highest SI index and low- st SI index where a non-toxic relationship was observed was elected to determine the bactericidal or bacteriostatic effect ver time. The time-kill assay method was adapted from Kur- imboccus et al. ( 2021 ). A quantity of 100.00 μl of TSB was dded into the first row of a 48-well micro-titre plate. The emaining rows were filled with 900.00 μl of sterile 0.90% odium chloride (NaCl) solution. A 0.5 McFarland’s turbidity tandard of culture (approximate inoculum concentration of × 10 6 CFU/ml −1 ) was prepared, and 100.00 μl thereof was ransferred into the first row of the microtitre plate. A vol- me of 100.00 μl of the individual medicinal dye, commercial ntimicrobial, and each combination (medicinal dye: commer- ial antimicrobial) was added into the first well of each col- mn. Thereafter 100.00 μl was transferred to the subsequent ell containing 0.90% NaCl. Five successive dilutions were hen performed and then incubated at 37 ◦C for 24 h. A 50.00 l sample was withdrawn at 0, 3, 6, and 24 h and spread onto SA plates and incubated at 37 ◦C for 24 h, after which the umber of colonies were counted. The logarithm of CFU/ml −1 resent in the original well with time were calculated (Equa- ion 4 ). Log ( CFU / m l −1 ) = log 10 Colony count × 1 × 10 9 (4) A positive control of ciprofloxacin (0.01 mg/ml −1 ) was used o ensure the pathogen was susceptible. A culture control was lso included, and the assay was conducted in triplicate. esults ntimicrobial activity of the commercial ntimicrobials o study the susceptibilities for the combinations—it was nec- ssary to initially determine the MIC/MBC breakpoint ranges or the commercial antimicrobials ( Supplementary Tables S1 – 3 ). The EUCAST and CLSI breakpoint ranges have been sed as a susceptibility reference with most of the commer- ial antimicrobials falling within the determined ranges. Fu- idic acid noted low MIC/MBC values within the range of he EUCAST criteria against all S. epidermidis strains. The ram-negative pathogens demonstrated the most suscepti- ility to Betadine ® with MBC values ranging from 1.56 o 2.60 μg/ml −1 . Overall, the pathogens demonstrated the ost resilience against Steriscrub. Ketoconazole, Miconazole, nd Nystatin. When tested against C. albicans A100, Nys- atin demonstrated the most potent antimicrobial activity https://academic.oup.com/jambio/article-lookup/doi/10.1093/jambio/lxae147#supplementary-data https://academic.oup.com/jambio/article-lookup/doi/10.1093/jambio/lxae147#supplementary-data 4 Ramfol and van Vuuren w A a M d c e s A T i c l b ( d s d d 8 G G t s S d r T t l e t r w a s n r s t a d d r g c 1 A p M t l a a i t Ta b le 1 . Th e an tim ic ro bi al ac tiv ity (M B C va lu es in μ g/ m l − 1 ) o f th e m ed ic in al dy es St ap hy lo co cc us au re us St ap hy lo co cc us ep id er m id is Ps eu do m on as ae ru gi no sa E sc he ri c h ia co li C an di da al bi ca ns D ye s R ef er en ce st ra in (A T C C 64 46 6) C lin ic al st ra in (c lin ic al 64 37 93 8) R es is ta nt st ra in R ef er en ce st ra in (A T C C 12 22 8) C lin ic al st ra in (c lin ic al B A 16 ) R es is ta nt st ra in (A T C C 51 62 5) R ef er en ce st ra in (A T C C 27 85 3) C lin ic al st ra in (D SM 46 31 6) R es is ta nt st ra in (A T C C 46 31 6) R ef er en ce st ra in (A T C C 83 79 ) C lin ic al st ra in (c lin ic al 66 43 7 87 3) R es is ta nt st ra in (D SM 22 31 4) R ef er en ce st ra in (A T C C 10 23 1) C lin ic al st ra in (A 10 0) R es is ta nt st ra in (A T C C 90 02 8) M R SA (A T C C 43 30 0) G M R SA (A T C C 33 59 2) Fu ch si ne 31 2 . 50 50 0 . 00 50 0 . 00 25 .0 0 6 . 25 1 . 56 10 0 . 00 80 00 .0 0 20 0 . 00 80 00 .0 0 16 6 . 67 20 00 .0 0 26 66 .6 7 25 0 . 00 20 0 . 00 20 8 . 33 G en ti an vi ol et 0 . 03 0 . 03 0 . 03 0. 00 02 5 0 . 00 2 0. 00 00 3 0 . 01 0 . 01 0 . 01 0 . 52 0 . 01 0 . 03 0 . 02 0. 00 02 5 0. 00 02 5 0 . 00 1 Io di ne ti nc tu re 1 . 88 1 . 56 1 . 56 0 . 78 0 . 16 0 . 00 2 0 . 39 0 . 78 0 . 78 0 . 78 0 . 78 0 . 78 0 . 78 0 . 78 1 . 56 0 . 78 M al ac hi te gr ee n 1 . 00 2 . 50 1 . 00 0 . 03 0 . 00 09 0 . 00 2 1 . 00 40 0 . 00 0 . 63 80 0 . 00 1 . 67 10 .0 0 10 .0 0 0 . 32 0 . 63 5 . 00 M er cu ro ch ro m e 0 . 44 0 . 27 0 . 13 0 . 42 0 . 14 0 . 01 6 0 . 06 > 5 . 00 0 . 08 > 5 . 00 0 . 31 0 . 08 2 . 50 0 . 42 0 . 08 0 . 08 M et hy le ne B lu e 20 .0 0 50 .0 0 20 8 . 33 50 .0 0 12 .5 0 20 .0 0 40 .0 0 > 80 00 .0 0 13 3 . 33 > 80 00 .0 0 40 .0 0 80 0 . 00 80 0 . 00 12 .5 0 26 .6 7 26 .6 7 Po ta ss iu m Pe rm an ga na te 70 00 .0 0 > 80 00 .0 0 > 80 00 .0 0 > 80 00 .0 0 > 80 00 .0 0 80 00 .0 0 80 00 .0 0 > 80 00 .0 0 > 80 00 .0 0 > 80 00 .0 0 > 80 00 .0 0 > 80 00 .0 0 > 80 00 .0 0 80 00 .0 0 40 00 .0 0 > 80 00 .0 0 M R S A : m et hi ci lli n- re si st an t St ap hy lo co cc us au re us , G M R SA : g en ta m yc in -m et hi ci lli n re si st an t St ap hy lo co cc us au re us . D ow nloaded from https://academ ic.oup.com /jam bio/article/135/7/lxae147/7704455 by jam estem lett user on 04 July 2024 ith an MIC/MBC value of 0.20 μg/ml −1 . Candida albicans TCC 90028 demonstrated the most resistance to all three ntifungals—Ketoconazole, Miconazole, and Nystatin with BC values of > 25.00 μg/ml −1 . The Gram-positive strains emonstrated better susceptibility to the commercial antimi- robials; however, no patten was observed among the refer- nce, clinical and resistant strains where one strain was con- istently susceptible in comparison to the other. ntimicrobial activity of the medicinal dyes he MBC values of the selected medicinal dyes are given n Table 1 . Most of the pathogens demonstrated high sus- eptibility to the medicinal dyes. Values ranged from the owest concentration of 0.00003 to > 8000.00 μg/ml −1 . The est antimicrobial activity was attributed to Gentian violet 0.00003 μg/ml −1 against S. epidermidis clinical strain), Io- ine tincture (0.002 μg/ml −1 against S. epidermidis clinical train and Malachite green (0.0009 μg/ml −1 against S. epi- ermidis ATCC 12228) in comparison to the other medicinal yes tested. Relatively high MBC values (ranging from 1.56 to 000.00 μg/ml −1 ) were observed against Fuchsine for the ram-positive strains, Gram-negative strains, and the yeasts. entian violet demonstrated the lowest MBC values among he dyes (0.00003–0.52 μg/ml −1 ). Iodine tincture demon- trated the lowest MBC value of 0.002 μg/ml −1 against the . epidermidis clinical strain. Furthermore, the Iodine tincture emonstrated the same MBC value among all the strains— eference, clinical and resistant of P. aeruginosa and E. coli . he S. epidermidis strains demonstrated higher susceptibility o Malachite green in comparison to the other pathogens. The owest MBC value of 0.0009 μg/ml −1 was observed against S. pidermidis ATCC 12228. Mercurochrome had the second lowest MBC values among he S. aureus strains after Gentian violet. The MBC values anged from 0.13 to 0.44 μg/ml −1 . The highest susceptibility as noted by S. epidermidis (BA 16), the clinical strain with n MBC value of 0.016 μg/ml −1 . Methylene blue noted the econd highest MBC values among the dyes. The P. aerugi- osa strains (ATCC 27853 and ATCC 46316) demonstrated esilience against Methylene blue and Mercurochrome. All pathogens demonstrated a high resilience to Potas- ium permanganate with MBC values ranging from 4000.00 o > 8000.00 μg/ml −1 . All strains behaved as expected against ll controls. The Gram-positive and Gram-negative bacteria emonstrated susceptibility to Ciprofloxacin and the yeasts emonstrated susceptibility to Amphotericin B with values anging from 1.25 to 2.50 μg/ml −1 . Growth of all microor- anisms was noted against the culture control and the negative ontrol. :1 Combinations total of 567 combinations were tested against the Gram- ositive strains, Gram-negative strains, and yeasts, and the BC/ �FIC values were determined (Ramfol 2023 ). When ested, predominantly indifference (38.81%) was noted, fol- owed by additive interactions (27.87%), synergy (20.46%), nd lastly antagonism with 12.87%. A summary of all inter- ctions is presented in Fig. 1 . The synergistic and antagonistic nteractions were the primary focus of this study, and hence hese are elaborated on in Tables 2 and 3 . Dyes with conventional antimicrobials 5 Figure 1. Summary of the 1:1 dye: commercial antimicrobial interactions. The most synergistic and antagonistic interactions were observed against the Gram-positive strains. T T T b d n M d t 4 n c s a 2 a d 1 2 p T T w t t s t s a t d t D ow nloaded from https://academ ic.oup.com /jam bio/article/135/7/lxae147/7704455 by jam estem lett user on 04 July 2024 Mercurochrome with Betadine ® was the combination to yield the most (eight interactions) synergy with �FIC values ranging from 0.05 to 0.48 (Table 2 ). This was followed by the combination of Fuchsine with Gentamycin (seven interac- tions) with �FIC values ranging from 0.001 to 0.50. The most synergistic interaction ( �FIC value of 0.001) was observed for the combination of Fuchsine with Gentamycin when tested against P. aeruginosa DSM 46316. The highest number (six interactions) of antagonistic inter- actions was observed with Fuchsine with Steriscrub, followed by Malachite green in combination with Betadine (five inter- actions). The most antagonistic combination (Malachite green with Betadine) was observed against MR S A ATCC 43300 with an �FIC value of 1000.50 (Table 3 ). Malachite green in combination with Fusidic acid also noted a high �FIC value of 1000.20 against MR S A ATCC 43300. Further attention was paid to a selection of combina- tions that noted synergy and antagonism (bold in Tables 2 and 3 ). Toxicity as well as varied ratio studies were conducted on these combinations. Furthermore, the two combinations demonstrating potent antimicrobial activity (Table 2 , bold and italics) were further investigated in different ratios to deter- mine if synergy was apparent at varied concentrations. Toxicity analysis To xicity anal ysis of commercial antimicrobials The commercial antimicrobials predominantly demonstrated low percentage mortalities and were deemed non-toxic (Ta- ble 4 ). Betadine ®, however, demonstrated the highest percent- age mortality of 100% at 24 and 48 h with an LC 50 value of 0.20 μg/ml −1 at both 24 and 48 h. Gentamycin demonstrated the lowest percentage mortality of 0% at 24 h 1.11% at 48 h. The antifungals tested also noted low percentage mortalities. o xicity anal ysis of medicinal d yes he various toxicity profiles of the medicinal dyes are noted in able 5 . At 24 h, Fuchsine, Malachite green, and Methylene lue were observed to be non-toxic. At 48 h, all medicinal yes demonstrated high percentage mortalities. Four medici- al dyes (Gentian violet, Iodine tincture, Malachite green, and ercurochrome) noted a percentage mortality of 100%. Io- ine tincture demonstrated the greatest toxicity of all dyes ested with the percentage mortality rate of 100% at 24 and 8 h even when the dye concentrations were decreased. It also oted the lowest LC 50 value of 0.0001 μg/ml −1 at 24 and 48 h onfirming the toxicity of the dye. Malachite green demon- trated a percentage mortality of 38.20% at 24 h and 100% t 48 h with a low LC 50 value of 3.91 μg/ml −1 noted at both 4 and 48 h. Methylene blue demonstrated the lowest percent- ge mortality of 33.33% at 24 h and 84.95% at 48 h. The dye emonstrated an LC50 value of 250.00 μg/ml −1 at 24 h and 25.00 μg/ml −1 at 48 h. The LC 50 values of 125 μg/ml −1 at 4 h and 15.63 μg/ml −1 at 48 h were observed for Potassium ermanganate. o xicity anal ysis of combinations he medicinal dyes exhibited very high toxicity and therefore ere combined with various commercial antimicrobials to de- ermine if the toxicity could be reduced. Selected combina- ions (synergy and antagonism) (Tables 2 and 3 , bold) were elected for the brine shrimp lethality assay, and the toxici- ies are shown in Table 6 . The dyes independent toxicities are hown in the table for comparison. The percentage mortalities t the LC 50 values (the non-toxic values) are also shown in the able as a reference only. The combination of Gentian violet with Gentamycin emonstrated the greatest decrease in dye toxicity. Hence, his combination was the most synergistic combination. An art/lxae147_f1.eps 6 Ramfol and van Vuuren Table 2. Summary of synergistic combinations grouped by dye combinations. Combinations �FIC value Pathogen/strain Fuchsine + Betadine ® 0 .46 S. aureus ATCC 64466 Fuchsine + Fusidic acid 0 .16 S. aureus Clinical 6437938 Fuchsine + Gentamycin 0 .10 S. aureus ATCC 64466 0 .50 S. aureus Clinical 6437938 0 .50 MR S A ATCC 43300 0 .28 S. epidermidis ATCC 12228 0 .12 S. epidermidis ATCC 51625 0 .001 P. aeruginosa DSM 46316 0 .50 P. aeruginosa ATCC 46316 Fuchsine + Ketoconazole 0 .04 C. albicans Clinical A100 Fuchsine + Neomycin 0 .10 S. aureus ATCC 64466 0 .50 S. aureus Clinical 6437938 0 .21 MR S A ATCC 43300 0 .31 S. epidermidis ATCC 12228 0 .002 P. aeruginosa DSM 46316 Fuchsine + Steriscrub 0 .38 S. epidermidis ATCC 51625 0 .44 P. aeruginosa DSM 46316 0 .10 E. coli Clinical 66437873 0 .42 E. coli DSM 22314 Gentian violet + Betadine ® 0 .25 S. aureus ATCC 64466 0 .09 S. epidermidis ATCC 12228 0 .30 P. aeruginosa ATCC 46316 0 .44 E. coli Clinical 66437873 Gentian violet + Fusidic acid 0 .33 S. aureus ATCC 64466 Gentian violet + Gentamycin 0 .26 S. aureus ATCC 64466 0 .15 MR S A ATCC 43300 0 .36 P. aeruginosa ATCC 46316 0 .50 E. coli DSM 22314 Gentian violet + Mupirocin 0 .27 S. aureus ATCC 64466 0 .15 MR S A ATCC 43300 0 .02 S. epidermidis ATCC 12228 Gentian violet + Neomycin 0 .27 S. aureus ATCC 64466 0 .16 MR S A ATCC 43300 0 .04 S. epidermidis ATCC 12228 0 .42 E. coli DSM 22314 Gentian violet + Steriscrub 0 .25 S. aureus ATCC 64466 0 .06 S. epidermidis ATCC 12228 0 .31 S. epidermidis ATCC 12228 0 .33 P. aeruginosa ATCC 46316 Iodine tincture + Betadine ® 0 .46 S. aureus ATCC 64466 0 .37 MR S A ATCC 43300 0 .15 S. epidermidis ATCC 12228 Iodine tincture + Gentamycin 0 .42 S. aureus ATCC 64466 Iodine tincture + Mupirocin 0 .43 S. aureus ATCC 64466 0 .16 S. epidermidis ATCC 12228 Iodine tincture + Neomycin 0 .44 S. aureus ATCC 64466 0 .34 S. aureus Clinical 6437938 0 .26 MR S A ATCC 43300 Iodine tincture + Steriscrub 0 .40 S. aureus ATCC 64466 0 .37 P. aeruginosa ATCC 27853 0 .25 P. aeruginosa ATCC 46316 0 .22 E. coli Clinical 66437873 0 .14 C. albicans ATCC 10231 0 .28 C. albicans ATCC 90028 Malachite green + Miconazole 0 .46 C. albicans Clinical A100 Malachite green + Mupirocin 0 .10 S. aureus Clinical 6437938 0 .13 S. epidermidis Clinical BA16 Malachite green + Neomycin 0 .02 S. aureus Clinical 6437938 0 .02 S. epidermidis Clinical BA16 0 .38 E. coli ATCC 8379 0 .50 E. coli DSM 22 314 Malachite green + Steriscrub 0 .38 S. epidermidis ATCC 51625 Mercurochrome + Betadine ® 0 .27 S. aureus ATCC 64466 0 .40 S. aureus Clinical 6437938 0 .45 MRSA ATCC 43300 0 .05 GMR S A ATCC 33592 Table 2. Continued Combinations �FIC value Pathogen/strain 0 .48 S. epidermidis ATCC 12228 0 .40 S. epidermidis Clinical BA16 0 .38 E. coli ATCC 8379 0 .07 C. albicans ATCC 90028 Mercurochrome + Fusidic acid 0 .39 S. aureus ATCC 64466 Mercurochrome + Ketoconazole 0 .09 C. albicans ATCC 10231 Mercurochrome + Mupirocin 0 .36 S. aureus ATCC 64466 0 .44 GMR S A ATCC 33592 Mercurochrome + Neomycin 0 .30 MR S A ATCC 43300 0 .50 GMR S A ATCC 33592 Mercurochrome + Steriscrub 0 .41 S. epidermidis ATCC 12228 Methylene blue + Betadine ® 0 .50 S. aureus ATCC 64466 0 .50 S. epidermidis ATCC 12228 Methylene blue + Gentamycin 0 .30 MR S A ATCC 43300 0 .50 GMR S A ATCC 33592 0 .15 P. aeruginosa DSM 46316 U E. coli Clinical 66437873 Methylene blue + Ketoconazole 0 .01 C. albicans Clinical A100 Methylene blue + Neomycin 0 .30 MR S A ATCC 43300 0 .41 GMR S A ATCC 33592 0 .18 P. aerugi nosa DSM 46316 0 .06 E. coli Clinical 66 437873 Methylene blue + Nystatin 0 .06 C. albicans Clinical A100 Methylene blue + Steriscrub 0 .50 S. epidermidis ATCC 12228 0 .07 S. epidermidis Clinical BA16 0 .38 S. epidermidis ATCC 51625 0 .10 E. coli Clinical 66 437873 Potassium Permanganate + Betadine ® 0 .40 S. aureus ATCC 64466 0 .28 S. epidermidis Clinical BA16 0 .08 S. epidermidis ATCC 51625 Potassium Permanganate + Gentamycin 0 .33 S. epidermidis Clinical BA16 Potassium Permanganate + Steriscrub 0 .09 S. epidermidis Clinical BA16 Bold: combinations chosen for further testing. U: the mean MBC values were not absolute values (either > 8000.00 μg/ml or < 0.01 μg/ml), and hence tentative FIC values were established. o c c a N c f c p i t d i f e t t t v i o D ow nloaded from https://academ ic.oup.com /jam bio/article/135/7/lxae147/7704455 by jam estem lett user on 04 July 2024 ver 15-fold decrease is observed with Gentian violet when ombined with the commercial antimicrobial. Three other ombinations demonstrated a decrease in toxicity and were s follow; Fuchsine with Gentamycin, Malachite green with eomycin, and Malachite green with Miconazole. Mer- urochrome combined with Betadine ® demonstrated a five- old increase in toxicity when combined. Interestingly, this ombination noted the best activity against the Gram-positive athogens. Fuchsine with Steriscrub also noted a decrease n toxicity when combined. Out of the synergistic combina- ions tested, 66.67% of these combinations demonstrated a ecrease in toxicity when tested against the brine shrimp. As expected, most of the antagonistic combinations noted ncreased toxicity (i.e. antagonism); however, there were a ew combinations that demonstrated decreased toxicity (syn- rgism) while not having potent antimicrobial activity. Out of he antagonistic combinations tested, 50% of these combina- ions demonstrated a decrease in toxicity when tested against he brine shrimp. While Gentian violet demonstrated decreased toxicity pre- iously (when combined in a synergistic 1:1 combination), t did not demonstrate a decrease in toxicity in the antag- nistic (1:1) combinations, which may be attributed to the Dyes with conventional antimicrobials 7 Table 3. Summary of antagonistic combinations grouped by dye combinations. Combinations �FIC value Pathogen/strain Fuchsine + Betadine ® 4 .04 S. epidermidis Clinical BA16 29 .87 S. epidermidis ATCC 51625 4 .08 E. coli DSM 22 314 10 .67 C. albica ns ATCC 10231 Fuchsine + Fusidic acid 68 .01 S. epidermidis Clinical BA16 5 .81 S. epidermidis ATCC 51625 Fuchsine + Gentamycin 68 .01 S. epidermidis Clinical BA16 Fuchsine + Ketoconazole U C. albicans ATCC 90028 Fuchsine + Miconazole U C. albicans ATCC 90028 Fuchsine + Mupirocin 72 .01 S. epidermidis Clinical BA16 5 .01 S. epidermidis ATCC 51625 Fuchsine + Neomycin 4 .01 GMR S A ATCC 33592 65 .00 S. epidermidis Clinical BA16 Fuchsine + Nystatin 4 .08 C. albicans ATCC 10231 U C. albicans ATCC 90028 Fuchsine + Steriscrub 5 .94 GMR S A ATCC 33592 106 .89 S. epidermidis Clinical BA16 U P. aeruginosa ATCC 27853 U P. aeruginosa ATCC 46316 U E. coli Clinical 66 437873 5 .13 C. albicans Clinical A100 Gentian violet + Betadine ® 6 .79 S. aureus Clinical 6437938 5 .99 P. aeruginosa ATCC 27853 Gentian violet + Fusidic acid 5 .04 S. aureus Clinical 6437938 Gentian violet + Gentamycin 8 .02 S. epidermidis ATCC 12228 4 .01 P. aeruginosa DSM 46316 Gentian violet + Mupirocin 107 .78 S. aureus Clinical 6437938 Gentian violet + Neomycin 4 .02 P. aeruginosa DSM 46316 Gentian violet + Steriscrub 5 .09 GMRSA ATCC 33592 Iodine tincture + Betadine ® 62 .63 S. epidermidis Clinical BA16 Iodine tincture + Fusidic acid 4 .91 GMRSA ATCC 33592 8 .81 S. epidermidis ATCC 12228 4 .50 S. epidermidis Clinical BA16 Iodine tincture + Gentamycin 126 .00 S. epidermidis Clinical BA16 Iodine tincture + Mupirocin 31 .75 S. epidermidis Clinical BA16 8 .01 S. epidermidis ATCC 51625 Iodine tincture + Neomycin 125 .25 S. epidermidis Clinical BA16 Iodine tincture + Steriscrub 41 .72 S. epidermidis Clinical BA16 Malachite green + Betadine ® 1000 .50 MR S A ATCC 43300 4 .43 S. epidermidis ATCC 12228 5 .33 S. epidermidis Clinical BA16 5 .60 P. aeruginosa clinical strain 4 .27 E. coli DSM 22314 Malachite green + Fusidic acid 1000 .20 MR S A ATCC 43300 7 .88 S. epidermidis Clinical BA16 5 .81 S. epidermidis ATCC 51625 Malachite green + Mupirocin 13 .02 MR S A ATCC 43300 5 .01 S. epidermidis ATCC 51625 Malachite green + Neomycin 5 .31 P. aeruginosa ATCC 46316 Malachite green + Steriscrub 17 .42 S. epidermidis ATCC 12228 10 .42 S. epidermidis Clinical BA16 Mercurochrome + Gentamycin 5 .50 S. epidermidis ATCC 51 625 Mercurochrome + Mupirocin 7 .00 S. epidermidis ATCC 51625 Mercurochrome + Neomycin 10 .48 S. epidermidis ATCC 51625 Mercurochrome + Steriscrub 5 .15 S. epidermidis Clinical BA16 4 .37 S. epidermidis ATCC 51625 10 .60 E. coli DSM 22 314 Methylene blue + Betadine ® 4 .03 E. coli DSM 22314 6 .41 C. albicans ATCC 10231 Methylene blue + Fusidic acid 20 .23 S. epidermidis ATCC 51625 Methylene blue + Gentamycin 4 .09 P. aeruginosa ATCC 46316 Methylene blue + Miconazole U C. albicans ATCC 90028 Methylene blue + Mupirocin 4 .59 MR S A ATCC 43300 17 .03 S. epidermidis ATCC 51625 Potassium Permanganate + Fusidic acid U S. aureus ATCC 64466 Potassium Permanganate + Gentamycin U S. aureus ATCC 64466 D ow nloaded from https://academ ic.oup.com /jam bio/article/135/7/lxae147/7704455 by jam estem lett user on 04 July 2024 8 Ramfol and van Vuuren Table 3. Continued Combinations �FIC value Pathogen/strain U P. aeruginosa ATCC 27853 Potassium Permanganate + Mupirocin U S. aureus ATCC 64466 16 .28 S. epidermidis Clinical BA16 16 .53 S. epidermidis ATCC 51625 Potassium Permanganate + Neomycin U S. aureus ATCC 64466 U P. aeruginosa ATCC 27853 U E. coli ATCC 8379 Potassium Permanganate + Nystatin 7 .25 C. albicans Clinical A100 Bold: combinations chosen for further testing. U: the mean MBC values were not absolute values (either > 8000.00 μg/ml or < 0.01 μg/ml), and hence tentative FIC values were established. Table 4. The toxicity analysis of the commercial antimicrobials. Conventional antimicrobial Percentage mortality (%) LC 50 ( μg/ml) 24 h 48 h 24 h 48 h Betadine ® 100 .00 100 .00 0 .20 0 .20 Fusidic acid 9 .78 46 .79 500 .00 500 .00 Gentamycin 0 .00 1 .11 500 .00 500 .00 Ketoconazole 8 .31 29 .58 500 .00 500 .00 Miconazole 0 .72 6 .62 500 .00 500 .00 Mupirocin 35 .34 72 .22 500 .00 125 .00 Neomycin 4 .10 5 .84 500 .00 500 .00 Nystatin 0 .00 40 .17 500 .00 500 .00 Steriscrub 46 .61 92 .84 500 .00 250 .00 Controls Negative control (Salt water 32 mg/ml) 1 .86 6 .38 N/A N/A Positive control (Potassium dichromate) 100 .00 100 .00 N/A N/A Bold: toxic values. Table 5. The toxicity analysis of the medicinal dyes. Medicinal dye Percentage mortality (%) LC 50 ( μg/ml) 24 h 48 h 24 h 48 h Fuchsine 39 .38 90 .36 125 .00 31 .25 Gentian violet 100 .00 100 .00 0 .01 0 .001 Iodine Tincture 100 .00 100 .00 0 .0001 0 .0001 Malachite green 38 .20 100 .00 3 .91 3 .91 Mercurochrome 100 .00 100 .00 0 .02 0 .01 Methylene blue 33 .33 84 .95 250 .00 125 .00 Potassium permanganate 71 .42 94 .30 125 .00 15 .63 Controls Negative control (Salt water 32 mg/ml) 1 .86 6 .38 N/A N/A Positive control (Potassium dichromate) 100 .00 100 .00 N/A N/A Bold: toxic values. w d s T i M d t l d i o M c f T M o c c d a a t t T T G s D ow nloaded from https://academ ic.oup.com /jam bio/article/135/7/lxae147/7704455 by jam estem lett user on 04 July 2024 eak antimicrobial activity. Iodine tincture with Fusidic acid emonstrated the greatest decrease in toxicity, while Fuch- ine with Nystatin demonstrated a much smaller decrease. wo other combinations demonstrated a decrease in toxic- ty of the medicinal dyes; Mercurochrome with Steriscrub and ethylene blue with Gentamycin. Interestingly, two medicinal yes, Fuchsine and Methylene blue when in combination with he commercial antimicrobials, respectively, demonstrated the owest decrease in toxicity which could be due to the fact the yes were not highly toxic initially. While understanding which combinations are antagonistic s important, the synergistic interactions were further focused n. Two combinations: Gentian violet with Gentamycin and alachite green with Neomycin, demonstrated potent antimi- robial activity and reduced toxicity, hence, were chosen for urther in-depth testing. t he selectivity inde x es ost of the medicinal dyes were considered toxic, while the pposite is true for the commercial antimicrobials. The 1:1 ombinations have shown potential in decreased toxicity once ombined. It is therefore important to determine the SI of the yes, commercial antimicrobials, and combinations to ensure non-toxic relationship to the host while maintaining potent ntimicrobial activity. The SI also helps ensure the dye’s an- imicrobial activity is not due to a toxic metabolite and rather he dye itself. he SIs of the Gram-positive pathogens able 7 demonstrates the SI of the individual dyes against the ram-positive pathogens. Most of the medicinal dyes demon- trated low SI values. The LC 50 values that are added in the able are mainly just a reference of the toxicity of each sample. Dyes with conventional antimicrobials 9 Table 6. The toxicity of the 1:1 combinations. Combinations Concentration ( μg/ml) Percentage mortality (%) at LC 50 value LC 50 value of combination ( μg/ml) Change in toxicity of dye when combined with commercial antimicrobials Dye tested independently Dye in combination with commercial antimicrobial Synergistic combinations Fuchsine + Gentamycin 31 .25 62 .50 41 .04 125 .00 Decrease Fuchsine + Steriscrub 31 .25 3 .91 40 .21 7 .81 Increase Gentian violet + Gentamycin 0 .001 0 .01 30 .21 15 .64 Decrease Malachite green + Neomycin 3 .91 7 .81 34 .59 15 .62 Decrease Malachite green + Miconazole 3 .91 7 .81 46 .39 15 .62 Decrease Mercurochrome + Betadine ® 0 .01 0 .004 45 .33 0 .20 Increase Antagonistic combinations Fuchsine + Nystatin 31 .25 15 .63 42 .50 31 .26 Decrease Gentian violet + Betadine ® 0 .001 0 .001 35 .36 0 .20 No change Gentian violet + Neomycin 0 .001 0 .0001 2 .91 0 .24 Increase Gentian violet + Steriscrub 0 .001 0 .001 43 .33 1 .95 No change Iodine tincture + Fusidic acid 0 .0001 0 .05 40 .05 2 .00 Decrease Malachite green + Betadine ® 3 .91 1 .95 12 .67 2 .15 Increase Mercurochrome + Steriscrub 0 .01 0 .02 23 .61 7 .83 Decrease Methylene blue + Gentamycin 125 250 .00 34 .41 500 .00 Decrease Table 7. The SI values of the dyes and commercial antimicrobials against the Gram-positive pathogens. LC 50 SI Staphylococcus aureus Staphylococcus epidermidis Reference strain (ATCC 64466) Clinical strain (clinical 6437938) MRSA (ATCC 43300) GMRSA (ATCC 33592) Reference strain (ATCC 12228) Clinical strain (clinical BA16) Resistant strain (ATCC 51625) Dyes Fuchsine 31 .25 0 .10 0 .06 0 .06 1 .25 5 .00 20 .03 0 .31 Gentian violet 0 .001 0 .03 0 .03 0 .03 4 .00 0 .64 32 .26 0 .10 Iodine tincture 0 .0001 0 .0001 0 .0001 0 .0001 0 .0001 0 .001 0 .06 0 .0003 Malachite green 3 .91 3 .91 1 .56 3 .91 130 .33 4344 .44 U 3 .91 Mercurochrome 0 .01 0 .02 0 .04 0 .08 0 .02 0 .07 0 .64 0 .17 Methylene blue 125 .00 6 .25 2 .50 0 .60 2 .50 10 .00 6 .25 3 .13 Potassium permanganate 15 .63 0 .002 U U U U 0 .002 0 .002 Antimicrobials Betadine ® (Povidone-Iodine) 0 .20 0 .03 0 .11 0 .03 0 .02 0 .20 0 .08 0 .03 Fusidic acid 500 .00 200 .00 200 .00 200 .00 6250 .00 12 500 .00 6250 .00 7142 .86 Gentamycin 500 .00 4 .00 20 .00 20 .00 80 .00 3125 .00 6250 .00 1612 .90 Mupirocin 125 .00 5 .00 5 .00 3 .99 781 .25 1562 .50 3125 .00 1562 .50 Neomycin 500 .00 40 .00 20 .00 40 .00 12 .00 793 .65 1612 .90 793 .65 Steriscrub (Chlorohexidine) 250 .00 1 .18 1 .00 2 .00 1 .88 10 .00 1 .50 1 .25 U: the mean MBC values were not absolute values (either > 8000.00 μg/ml or < 0.01 μg/ml), and hence the SI was unable to be calculated. Bold: non-toxic values; LC 50 values: added as a reference. n h c 0 s e S v a S D ow nloaded from https://academ ic.oup.com /jam bio/article/135/7/lxae147/7704455 by jam estem lett user on 04 July 2024 Malachite green demonstrated the highest SI values of 130.33 against GMR S A ATCC 33952 and 4344.44 against S. epider- midis ATCC 12228. Fuchsine and Gentian violet noted a high SI value of 20.03 and 32.26 against S. epidermidis Clinical BA16, respectively. Iodine tincture demonstrated the lowest SI value of 0.0003 against the S. epidermidis ATCC 51625. Io- dine tincture also noted the lowest SI values of 0.0001–0.06 against the other Gram-positive strains. Unlike the medicinal dyes, most of the commercial antimi- crobials demonstrated high SI values (a good SI-which is a on-toxic relationship with the sample and the host while aving potent antimicrobial activity). Betadine ® was the only ommercial antimicrobial that noted low SI values of 0.03– .20 for all Gram-positive pathogens. While it did demon- trate good antimicrobial efficacy among the S. aureus and S. pidermidis strains, it noted highly toxic values, hence the low I values. Fusidic acid and Neomycin demonstrated high SI alues against all S. aureus and S. epidermidis strains. Fusidic cid demonstrated the highest SI value of 12500.00 against . epidermidis ATCC 12228. This means that Fusidic acid 10 Ramfol and van Vuuren Table 8. The SI values of the dyes and commercial antimicrobials against the Gram-negative pathogens. Sample LC 50 SI Pseudomonas aeruginosa Esc heric hia coli Reference strain (ATCC 27853) Clinical strain (DSM 46316) Resistant strain (ATCC 46316) Reference strain (ATCC 8379) Clinical strain (clinical 66437873) Resistant strain (DSM 22314) Dyes Fuchsine 31 .25 0 .004 0 .16 0 .004 0 .19 0 .02 0 .01 Gentian violet 0 .001 0 .10 0 .10 0 .002 0 .10 0 .03 0 .05 Iodine tincture 0 .0001 0 .0001 0 .0001 0 .0001 0 .0001 0 .0001 0 .0001 Malachite green 3 .91 0 .01 6 .21 0 .005 2 .34 0 .39 0 .39 Mercurochrome 0 .01 U 0 .13 U 0 .03 0 .13 0 .004 Methylene blue 125 .00 U 0 .94 U 3 .13 0 .16 0 .16 Potassium permanganate 15 .63 U U U U U U Antimicrobials Betadine ® (Povidone-Iodine) 0 .20 0 .10 0 .08 0 .09 0 .13 0 .08 0 .13 Gentamycin 500 .00 200 .00 2 .00 200 .00 20 .00 U 47 .98 Neomycin 500 .00 U 20 .00 125 .00 2 .00 2 .00 3 .00 Steriscrub (Chlorohexidine) 250 .00 1 .25 0 .63 1 .25 3 .75 0 .13 6 .00 U: the mean MBC values were not absolute values (either > 8000.00 μg/ml or < 0.01 μg/ml), and hence the SI was unable to be calculated. Bold: non-toxic values; LC 50 values: added as a reference. p a T A t S s m a G 0 n S A T M b g v S g s o g 1 2 T T T f u p c c b 0 S o c ( t b g E h t F V S l s t s m w t 5 o s i t c s r o n m f t T T n D ow nloaded from https://academ ic.oup.com /jam bio/article/135/7/lxae147/7704455 by jam estem lett user on 04 July 2024 roved to have good antimicrobial activity against the strains nd is also safe to use based on the BSLA studies. he SIs for the Gram-negative pathogens ll medicinal dyes noted low SI values ranging from 0.0001 o 6.21 (Table 8 ). Iodine tincture demonstrated the lowest I values of 0.0001 among all the P. aeruginosa and E. coli trains. Unlike the Gram-positive strains, most of the com- ercial antimicrobials noted low SI values (Table 8 ). Over- ll, Betadine ® demonstrated the lowest SI values (as with the ram-positive strains) with SI values ranging from 0.08 to .13. Steriscrub also noted low SI values against the P. aerugi- osa and E. coli strains. Gentamycin demonstrated the highest I value of 200.00 against the P. aeruginosa ATCC 27853 and TCC 46316. he SIs for the yeasts ost of the medicinal dyes demonstrated low SI values (Ta- le 9 ) and, hence, were toxic even though they demonstrated ood antimicrobial activity. Iodine tincture noted the lowest SI alue of 0.0001. Only two medicinal dyes demonstrated high I values-both against C. albicans ATCC 10231. Malachite reen noted an SI value of 12.22, and Methylene blue demon- trated an SI value of 10.00. Betadine ® noted low SI values f 0.05–0.15 against the C. albicans strains. All three antifun- als demonstrated high SI values against the C. albicans ATCC 0231 and A100 strain. Nystatin noted the highest SI value of 500.00 against C. albicans A100. he SIs of the combinations able 10 provides the SI values of the combinations tested. he synergistic and antagonistic combinations were selected or further study based on the MIC/MBC and toxicity val- es. Synergistic combinations were chosen as they have the otential to become a treatment option, while the antagonistic ombinations show which combinations have the potential to ause adverse effects and should not be used. Fuchsine in com- ination with Steriscrub demonstrated the lowest SI value of .17 against the E. coli strain. Four combinations noted high I value; Fuchsine and Gentamycin (SI = 100.03), Gentian vi- let and Gentamycin (SI = 977.50), Malachite green with Mi- onazole (SI = 128.87), and Malachite green with Neomycin SI = 50.37). As expected, 90% of the antagonistic combina- ions demonstrated a toxic relationship; therefore, the com- inations cannot be used due to the high toxicity. Malachite reen and Betadine ® noted the lowest SI value of 0.12 against . coli DSM 22314. Only one combination demonstrated a igh SI value of 320.61, which was Methylene blue with Gen- amycin against P. aeruginosa ATCC 46316. urther in-depth studies aried ratio studies ynergy was observed only in the 1:1 ratio of Gentian vio- et with Gentamycin when tested against the E. coli resistant train (Fig. 2 ). Ratios with Gentian violet in a higher concen- ration mainly demonstrated synergy with additive effects in elected ratios as detailed in methods. The varied ratios were determined using the MIC/MBC ethod mentioned in Antimicrobial analysis. The �FIC value as calculated and interactions were determined, which were hen plotted on a graph using the GraphPad Prism ® (Version ) software, and an isobologram was created. The majority f the points plotted are on the line or below, which demon- trates synergy. The red point in the isobologram is the 1:1 nteraction. The combination noted synergy and some addi- ive effects therefore it was chosen for further analysis. The ombination noted potent antimicrobial activity in compari- on to other combinations tested (Ramfol 2023 ). Malachite green with Neomycin tested against the E. coli esistant strain also demonstrated a synergistic interaction nly in a 1:1 ratio, and additive interactions were predomi- antly noted for the other ratios (Fig. 3 ). The varied ratios were determined using the MIC/MBC ethod previously mentioned. The combination was chosen or further analysis as it noted one of the best antimicrobial ac- ivities in comparison to other combinations (Ramfol 2023 ). ime-kill studies ime-kill studies were then conducted on these two combi- ations based on the non-toxic SI. While both combinations Dyes with conventional antimicrobials 11 Table 9. The SI values of the dyes and commercial antimicrobials against the yeasts. Sample LC 50 SI Candida albicans Reference strain (ATCC 10231) Clinical strain (A100) Resistant strain (ATCC 90028) Dyes Fuchsine 31 .25 0 .13 0 .16 0 .15 Gentian violet 0 .001 4 .00 4 .00 1 .00 Iodine tincture 0 .0001 0 .0001 0 .0001 0 .0001 Malachite green 3 .91 12 .22 6 .21 0 .78 Mercurochrome 0 .01 0 .02 0 .13 0 .13 Methylene blue 125 .00 10 .00 4 .69 4 .69 Potassium permanganate 15 .63 0 .002 0 .004 U Antimicrobials Betadine ® (Povidone-Iodine) 0 .20 0 .15 0 .08 0 .05 Ketoconazole 500 .00 26 .67 159 .74 U Miconazole 500 .00 80 .00 400 .00 U Nystatin 500 .00 80 .00 2500 .00 U Steriscrub (Chlorohexidine) 250 .00 0 .31 0 .31 0 .31 U: the mean MBC values were not absolute values (either > 8000.00 μg/ml or < 0.01 μg/ml), and hence the SI was unable to be calculated. Bold: non-toxic values; LC 50 values: added as a reference. Table 10. SI of synergistic and antagonistic combinations. Combinations Corresponding pathogens LC 50 SI Synergistic combinations Fuchsine + Gentamycin P. aeruginosa ATCC 46316 125 .00 100 .03 Fuchsine + Steriscrub E. coli DSM 22314 7 .81 0 .17 Fuchsine + Steriscrub P. aeruginosa DSM 46316 7 .81 0 .52 Gentian violet + Gentamycin E. coli DSM 22314 15 .64 977 .50 Malachite green + Miconazole C. albicans A100 15 .62 128 .87 Malachite green + Neomycin E. coli DSM 22314 15 .62 50 .37 Mercurochrome + Betadine ® S. aureus Clinical 6437938 0 .20 5 .11 Mercurochrome + Betadine ® MR S A ATCC 43300 0 .20 2 .25 Mercurochrome + Betadine ® S. epidermidis ATCC 12228 0 .20 3 .00 Mercurochrome + Betadine ® S. epidermidis Clinical BA16 0 .20 5 .11 Mercurochrome + Betadine ® S. aureus ATCC 64466 0 .20 1 .55 Antagonistic combinations Fuchsine + Nystatin C. albicans ATCC 10231 31 .26 1 .78 Gentian violet + Betadine ® P. aeruginosa ATCC 27853 0 .20 1 .68 Gentian violet + Neomycin P. aeruginosa DSM 46316 0 .24 4 .35 Iodine tincture + Fusidic acid GMR S A ATCC 33592 2 .00 4 .51 Iodine tincture + Fusidic acid S. epidermidis Clinical BA16 2 .00 2 .23 Malachite green + Betadine ® S. epidermidis ATCC 12228 2 .15 0 .90 Malachite green + Betadine ® S. epidermidis Clinical BA16 2 .15 0 .23 Malachite green + Betadine ® E. coli DSM 22314 2 .15 0 .12 Mercurochrome + Steriscrub E. coli DSM 22314 7 .83 1 .58 Methylene blue + Gentamycin P. aeruginosa ATCC 46316 500 .00 320 .61 LC 50 values: added as a reference; bold: non-toxic values. T W w r D ow nloaded from https://academ ic.oup.com /jam bio/article/135/7/lxae147/7704455 by jam estem lett user on 04 July 2024 have noted potent antimicrobial activity and a non-toxic re- lationship, it is also important to determine the bacterici- dal/bacteriostatic ability over time. Both combinations were tested against the E. coli resistant strain. he combination of Gentian violet and Gentamycin hen a time-kill analysis was undertaken on Gentian violet ith Gentamycin, a slower killing action against the E. coli esistant strain was observed in comparison to the positive 12 Ramfol and van Vuuren Figure 2. Isobologram of combinations of Gentian violet with Gentam y cin against E. coli DSM 22314. Six synergistic interactions were observed. No antagonism was evident. Figure 3. Isobologram of combinations of Malachite green with Neom y cin against E. coli DSM 22314. Only one synergistic (1:1 ratio) was observed. c G a s r r s l d t t s c T A N t o i n d n c T 2 N c o i t p a b o D W f p d p F t o a r m c 0 a b i S o a a i h e l n l p e t o b a w D ow nloaded from https://academ ic.oup.com /jam bio/article/135/7/lxae147/7704455 by jam estem lett user on 04 July 2024 ontrol (Fig. 4 ). Gentamycin demonstrated a similar pattern to entian violet, however, at 6–24 h, Gentamycin demonstrates more bactericidal effect, which is indicated by the change in teepness of the curve. Gentian violet demonstrated a bacte- iostatic effect on the E. coli resistant strain as not all bacte- ia were killed after 24 h. The current study noted bacterio- tatic activity when Gentamycin was tested. (interrupted black ine). The combination of Gentian violet and Gentamycin emonstrated better activity (the green line) in comparison to he samples when tested individually. This shows that Gen- amycin with Gentian violet acts synergistically and demon- trates bacteriostatic activity better than the Ciprofloxacin ontrol (Fig. 4 ). he combination of Malachite green and Neomycin s with the previous combination, Malachite green and eomycin demonstrated a slow killing action (Fig. 5 ). From 0 o 24 h, Malachite green demonstrates a bacteriostatic effect n the pathogen. Neomycin demonstrates bacteriostatic activ- ty similar to Malachite green; however, at 6–24 h, Neomycin oted better activity in comparison to Malachite green as in- icated by the slightly steeper curve. The combination of both Malachite green and Neomycin oted synergy as seen by the green line. From 0 to 6 h, the ombination notes a bacteriostatic effect with a steady decline. hereafter, a steep decline of pathogen viability was noted. At 4 h, the pathogen is completely killed. Interestingly, the combination of Malachite green and eomycin demonstrated better activity in comparison to the ombination of Gentian violet and Gentamycin. Gentian vi- let with Gentamycin demonstrated a higher SI value, hence t was the most non-toxic combination and noted a high an- imicrobial activity, however, Malachite green with Neomycin roved to be a better combination with bactericidal activity nd even with a lower SI value. It is also important to note that oth combinations demonstrated better activity to the antibi- tic controls in this study. iscussion hile there are many other notable uses of medicinal dyes, the ocus of this study is on skin infections. Fuchsine is known to ossess anaesthetic, bactericidal (Gram-positive), and fungici- al properties. It has been used to treat burns, eczema, and yodermas (Gupta et al. 2008 , Berrios and Arbiser 2011 ). uchsine dye in a 2%–5% solution has also been used for he treatment of burns (Balabanova et al. 2003 ). Gentian vi- let (also known as crystal violet) has been known to exhibit ntitrypanosomal, antifungal, anthelminthic, and antibacte- ial properties (Berrios and Arbiser 2011 ). Other than der- atological treatments, Gentian violet has been used in a ream to treat vaginal infections and has been effective in a .5% aqueous solution used to treat oropharyngeal candidi- sis (Balabanova et al. 2003 ). Iodine tincture has historically een used as a first line treatment for cuts and abrasions as t exhibits antifungal and antiseptic properties (Owen 1913 , mith 1915 ). In the early 1900s, Iodine was used as a pre- perative preparation as it was found to have antimicrobial ctivity against Gram-positive, Gram-negative bacteria, fungi, nd viruses (Morgan et al. 1996 ). The popularity decreased as t tends to burn the skin after prolonged use. Povidone-iodine as since replaced Iodine tincture as an antimicrobial (Morgan t al. 1996 ). Malachite green was previously known as Bril- iant green; however, current preparations of Brilliant green do ot contain malachite, a term used for the green colour of the eaves of the Malvaceae family of plants. Malachite green has rimarily been used against mycobacterial infections; how- ver, it has also been used as an antiseptic and antifungal to reat superficial pyodermas. The dye is also used as a sec- ndary treatment of topical skin conditions (Berrios and Ar- iser 2011 , Rosa et al. 2015 ). Mercurochrome has been used s a long-acting antibacterial and antifungal agent to prevent ound sepsis while also aiding in wound healing (Wainwright art/lxae147_f2.eps art/lxae147_f3.eps Dyes with conventional antimicrobials 13 Figure 4. Time kill of Gentian violet and Gentam y cin. T he combination noted better activity when compared to individual samples and the controls tested. Figure 5. Time-kill studies of Malachite green and Neom y cin. T he combination of Malachite green and Neom y cin demonstrated potent bactericidal activity in comparison to individual samples and controls. a ( f w t ( f a D ow nloaded from https://academ ic.oup.com /jam bio/article/135/7/lxae147/7704455 by jam estem lett user on 04 July 2024 2008 , Korkmaz et al. 2019 ). Mercurochrome was previously used intravenously and was known as a ‘true bloodstream an- tiseptic’ (Cleary 1925 ). Ehrlich had used Methylene blue to treat malaria. Later, Methylene blue was found to have ac- tivity against onychomycosis (Wainwright 2014 , Souza et al. 2017 ). Potassium permanganate solution was previously used in the treatment of impetigo. It was also used to treat tinea pedis and ringworm (Ive 1973 ), and is known to have disin- fectant, deodorizing, and astringent properties. It may be used s a cleansing agent for wounds when in a weaker solution SAMF 2020 ). While these aspects provide a good grounding or dyes to be used as antimicrobials, the use in combination ith conventional antibiotics has not been given adequate at- ention. This study focuses on this in depth. When examining the antibiotic breakpoints Supplementary Tables S1 –S3 ) the EUCAST ( 2020 ) guidelines or breakpoint MIC values were used for comparison. Fusidic cid noted low MIC/MBC values within the range of the art/lxae147_f4.eps art/lxae147_f5.eps https://academic.oup.com/jambio/article-lookup/doi/10.1093/jambio/lxae147#supplementary-data https://academic.oup.com/jambio/article-lookup/doi/10.1093/jambio/lxae147#supplementary-data 14 Ramfol and van Vuuren E t n ( v c t a S b i m s F i s G o t G t p a s t t 2 a f p c b a t m t w e M [ t e M a e b c b t c r p d d m a a c i a C n f t B c B e h t f t k d w t f a s fi s u i f b ( o N s t a m c d e h v i A i r a l ( m m fi t r o m c t w M d h a l A o e t D ow nloaded from https://academ ic.oup.com /jam bio/article/135/7/lxae147/7704455 by jam estem lett user on 04 July 2024 UCAST criteria against all S. epidermidis strains. A study esting the efficacy of Betadine ® against various organisms oted an MIC range of 4.00–1024.00 μg/ml −1 against E. coli Gmur and Karpi ́nski 2020 ). The current study notes MIC alues lower than 4.00 μg/ml −1 , demonstrating better sus- eptibility. In 2016, a study noted the MIC value of Steriscrub o be 16.00 μg/ml −1 and the MBC value of 32.00 μg/ml −1 , much lower value to the current study (Akca et al. 2016 ). teriscrub has been used repeatedly in a clinical settings and acteria repeatedly exposed to Steriscrub may have resulted n resistance (Hashemi et al. 2019 ). There have been various older studies noting the uses of edicinal dyes for the treatment of skin infections. An older tudy on the antibacterial activity of aniline dyes noted that uchsine can inhibit the growth of Gram-positive bacteria, ncluding S. aureus (Krumwiede and Pratt 1914 ). In a later tudy, it was noted that Fuchsine had a bacteriostatic effect on ram-positive bacteria (Rogosa 1934 ). While these are much lder studies, the results of the current study are in line with he literature. Dumisa et al. ( 2020 ), noted the MBC values of MR S A ATCC 33592 to be 1000.00 μg/ml −1 , vastly higher han the current study. The expiry date and storage of the sam- le in the previous study are unknown, and this could have ttributed to the vast difference in the MBC values. Potas- ium permanganate has been on the World Health Organiza- ion’s (WHO) essential drug list due to the antiseptic proper- ies and has been broadly used in wound care (Agnihotri et al. 019 ). When tested against the pathogens in the current study, high resistance rate was noted. With the dye being used so requently used for skin infections, resistant mechanisms may ossibly have developed. A study of general antiseptics was onducted against C. albicans , which demonstrated suscepti- ility to Potassium permanganate at 10 000.00 μg/ml −1 (Abed nd Hussein 2016 ). In this study, the highest concentrations ested were 8000.00 μg/ml −1 . When the 1:1 combinations of medicinal dyes with com- ercial antimicrobials were tested comparisons with litera- ure were virtually non-existent and only other combinations ere noted. Other combination studies demonstrating syn- rgy included a study on photodynamic therapy (PDT) with ethylene blue in combination with four different surfactants SDS (anionic), CTAC (cationic), HPS (zwitterionic), and Tri- on X-100 (non-ionic)] were tested against C. albicans (ref- rence strain) (Lyon et al. 2012 ). The combination of both ethylene blue PDT and surfactants proved to have better ctivity than when tested individually (Lyon et al. 2012 ). This arlier study affirms that the use of Methylene blue in com- ination has the potential to yield synergistic effects, which is onfirmed in the current study, but with alternate antimicro- ials. Dumisa et al. ( 2020 ), noted a few antagonistic combina- ions when Methylene blue and Malachite green were tested in ombination with various medicinal plants against the S. au- eus and C. albicans strains. While this study cannot be com- ared to the current study as medicinal plants were used, it oes demonstrate that antagonism may occur with medicinal yes. Except for Betadine, Mupirocin, and Steriscrub, all com- ercial antimicrobials were found to be non-toxic. Fusidic cid topically is well tolerated and is currently used in a cream nd ointment called Fucidin (SAMF 2020 ). When used topi- ally, Ketoconazole may cause itchiness, dryness, and sting- ng at the site of application. Hypersensitivity reactions such s anaphylaxis and urticaria may also occur (Sinawe and asadesus 2024 ). While the antifungal application has been oted to have some serious toxic effects (Mourad and Per- ect 2018 , Sinawe and Casadesus 2020), demonstrated a non- oxic concentration of 500 μg/ml −1 . At high concentrations, etadine ® causes necrosis, while at low concentrations, it auses apoptosis (Punjataewakupt et al. 2019 ). The use of etadine ® may delay wound healing, which may be caused by pithelial cell death (Punjataewakupt et al. 2019 ). Betadine ® as also been noted to cause acute kidney injury when used opically (Punjataewakupt et al. 2019 ). Betadine ® has been ound to have the most toxicity in the current study, substan- iating the previous literature found. Gentamycin has been nown to cause otoxicity, hearing loss, and vestibular disor- ers (Noack et al. 2017 ). Nephrotoxicity has also been noted ith the use of Gentamycin, in patients who have had ex- ended treatment (Pasmooij 2018 ). These effects have been requently reported when Gentamycin was used parentally nd with multiple dose treatment regimens. When used as a ingle dose treatment regimen, however, a lower toxicity pro- le was noted (Hayward et al. 2018 ). In this study, the brine hrimp lethality assay was conducted, and a single dose was sed, and effects were tested at 24 and 48 h, possibly lead- ng to the low toxicity value observed. Not many adverse ef- ects of Miconazole are noted. However, it can cause stinging, urning, pruritus, and contact dermatitis when used topically McKeny et al . 2021 ). Hübsch et al. ( 2014 ) tested the toxicity f Nystatin in the brine shrimp lethality assay and noted that ystatin demonstrated no toxicity at 24 and 48 h. The current tudy supports these results. Intravenously, Nystatin is highly oxic; hence, topicay use is only recommended (Scheibler et l . 2017 ). Dumisa et al. ( 2020 ) determined the toxicity of the edicinal dyes; however, not many other studies have been onducted over the years. Brine-shrimp toxicity assays con- ucted on dyes and combinations with plant extracts (Dumisa t al. 2020 ), showed that the dyes tested were toxic. Debate as existed on the safety and oncogenic properties of Gentian iolet, as previous studies have shown that Gentian violet can nteract with deoxyribonucleic acid (DNA) cells (Maley and rbiser 2013 ). Earlier studies on mice have noted an increase n hepatocellular carcinomas as well as an increase in thy- oid cancerswhen fed large quantities of Gentian violet (Maley nd Arbiser 2013 ). Other studies believed that Gentian vio- et was safe to use and did not have major contra-indications Maley and Arbiser 2013 ). Gentian violet is currently sold in ajor grocery stores and pharmacy retail stores with mini- al side effects noted (Prabha et al. 2020 ), making the earlier ndings by Maley and Arbiser ( 2013 ) substantiated. Iodine incture was noted as being the most toxic dye in the cur- ent study with the lowest LC 50 values. Iodine can cause seri- us corrosive damage to the gastrointestinal tract and mucous embranes (Owen 2018 ). Exposure to the skin and eyes may ause severe burns, and lethal doses have varied in concen- ration from 200 mg to 20 g (Owen 2018 ). Iodine tincture as ith Gentian violet is sold in retail stores without reservation. alachite green was also noted to be one of the most toxic yes in the study by Dumisa et al. ( 2020 ). Malachite green as been reported to cause reproductive abnormalities as well s being a carcinogen and has teratogenic properties. This has ed the dye to being banned in the USA, Canada, and Europe. s it is a potent antimicrobial, it is still steadily used in various ther countries such as South Korea and China (Gopinathan t al. 2015 ). Previous studies have noted a dose-dependent oxicity with Methylene blue (Clifton and Leikin 2003 ). Dyes with conventional antimicrobials 15 t ( a a G N c e ( g A w ( d C T a d s w t f s T b a t t o A T s e t I s c S S C p A R M ( t D T i D ow nloaded from https://academ ic.oup.com /jam bio/article/135/7/lxae147/7704455 by jam estem lett user on 04 July 2024 Relatively lower toxicity was observed in comparison to the other medicinal dyes. An old study reported that the inges- tion of Potassium permanganate crystals resulted in corrosive burns to the mouth, esophagus, and trachea (Southwood et al. 1987 ). Once the toxicity of the individual dyes was tested, the medicinal dyes were then tested in combination. Little to no research on the toxicity of dye combinations was found. Line- zolid is used to treat tuberculosis; however, it is a toxic drug to the host. A combination of bedaquiline, pretomanid, and linezolid has been used to treat drug resistant tuberculosis; preserving the drug’s efficacy as well as reducing the toxicity of linezolid (Bigelow et al. 2020 ). While this study does not relate to the current medicinal dye study, it does demonstrate that combinations of antimicrobials are able to have better efficacy as well as reduced toxicity to the host. The SIs also help ensure the dye’s antimicrobial activity is not due to a toxic metabolite and rather the dye itself. Dzoyem et al. ( 2016 ) suggested that the good selectivity index (high SI values) is due to the sample (the medicinal dye or commer- cial antimicrobial) antimicrobial activity rather than a toxin associated with a metabolite of the sample. The SI values of the Gram-positive pathogens overall were much lower in com- parison to the commercial antimicrobials. The high SI values ( > 10.00) mean that Malachite green, Fuchsine, and Gentian violet are safe to use with low toxic effects. Iodine tincture noted the lowest SI values among all dyes, which means that while the dye has a potent antimicrobial activity, it is still highly toxic against the host as well. While the medicinal dyes have proved to have potent effi- cacy against the Gram-negative pathogens, their efficacy could possibly be due to a toxic metabolite and is unsafe to use on the host. The low SI values means that it does demonstrate po- tent antimicrobial activity and low toxic effects; hence, it may be considered safe. While this Iodine tincture did demonstrate potent antimicrobial activity against the yeasts, it also proved to be highly toxic hence, is not an ideal choice or safe to use. Malachite green and Methylene blue noted an SI value > 10.00 demonstrating good antimicrobial efficacy while having min- imal adverse effects to the host. The high SI value noted for Nystatin indicates that a good antimicrobial activity is shown, and the antimicrobial noted fewer toxic effects. The four com- binations noted potent antimicrobial activity against each of the pathogens, which is due to the samples and not a toxic metabolite as well as a non-toxic relationship with the host making them ideal combinations for use. Further in-depth (various ratio studies) were presented on only the two best overall combinations. Gentamycin and Neomycin both come from the same class of antibiotics (aminoglycosides), yet, in combination, Neomycin demon- strates more additive effects. Dumisa et al. ( 2020 ) conducted varied ratio studies on crystal violet in combination with var- ious plant extracts and noted synergy among all ratios, which demonstrated the potential of varied ratio testing among medicinal dye combinations. Time-kill studies were conducted on two combinations (Gentian violet in combination with Gentamycin and Mala- chite green in combination with Neomycin) based on the pos- itive SI observed. Gentian violet has been observed to have a bacteriostatic effect on various bacteria (Ingraham 1933 , Ro- gosa 1934 ). This has been demonstrated in the current study as well. While Gentamycin is known for its bactericidal ac- tivity, a previous study conducting time-kill studies on Gen- amycin observed bactericidal activity only after 6 and 48 h Bortolin et al. 2017 ). Gentamycin has also been used in combination with other ntimicrobials for an enhanced synergistic effect (Krause et l. 2016 ). One example to note would be the combination of entamycin with β-lactams (Krause et al. 2016 ). The second combination chosen was Malachite green and eomycin, which noted bactericidal activity better than the ontrols tested. The medical dye used noted a bacteriostatic ffect in the current study which has been observed previously Chaudhary et al. 2017 ). Neomycin comes from the same roup of aminoglycosides as Gentamycin (Krause et al. 2016 ). s with Gentamycin, Neomycin is also most often combined ith β-lactams due to the synergy the combination provides Krause et al. 2016 ). As proven in the current study, Neomycin oes provide a synergistic effect when combined with the dye. onclusion he medicinal dyes have demonstrated potent antimicrobial ctivity, much more than the commercial antimicrobials but etermining the toxicity of medicinal dyes is important con- idering reports of adverse effects. This study has shown that hen dyes are combined with a selection of commercial an- ibiotics there is the potential to decrease not only the toxic ef- ects but enhance antimicrobial efficacy. This was particularly een for the combination of Malachite green with Neomycin. his study has successfully shown the potential to use com- inations of medicinal dyes and commercial antimicrobials gainst pathogens that have been known to cause skin infec- ions. Future studies should examine a more clinical approach o dye: antibiotic therapy with the aim of lessening the burden f currently prescribed antibiotics. c kno wledg ements he financial assistance of the University of the Witwater- rand (Faculty Research Council—FRC) is hereby acknowl- dged. Mrs. Phumzile Madondo and Londiwe Mathobela are hanked for laboratory assistance. Dr Teena Thomas (NHLS nfection Control and Microbiology Laboratory) at Univer- ity of Witwatersrand is thanked for the contribution of the linical strains. upplementary data upplementary data is available at JAMBIO Journal online. onflict of interest : The authors declare that there are no com- eting interests. uthor contributions hea Ramfol (Data curation, Formal analysis, Investigation, ethodology, Writing – original draft), and Sandy van Vuuren Conceptualization, Funding acquisition, Project administra- ion, Resources, Supervision, Writing – review & editing) ata availability he data underlying this article are available in the article and n the online supplementary material. https://academic.oup.com/jambio/article-lookup/doi/10.1093/jambio/lxae147#supplementary-data 16 Ramfol and van Vuuren R A A A B B B B B C C C C D D E F G G G H H H I I K K K K L L M M M M M N O O P D ow nloaded from https://academ ic.oup.com /jam bio/article/135/7/lxae147/7704455 by jam estem lett user on 04 July 2024 eferences bed AR , Hussein IM. In vitro study of antibacterial and antifungal activity of some common antiseptics and disinfectants agents. K uf a J Vet Med Sci 2016; 7 :148–59. https:// doi.org/ 10.36326/kjvs/ 2016/v 7i1B4255 gnihotri G , Gandhi S, Lio PA. Colorful dyes and other vibrant topi- cal creams as treatments for dermatological conditions. Drugs Ther Perspect 2019; 35 :491–9. kca AE , Akca G, Topçu FT et al . The comparative evaluation of the antimicrobial effect of propolis with chlorhexidine against oral pathogens: an in vitro study. Biomed Res Int 2016; 2016 :3627463. https:// doi.org/ 10.1155/ 2016/3627463 alabanova M , Popova L, Tchipeva R. Dyes in dermatology. Clin Dermatol 2003; 21 :2–6. https:// doi.org/ 10.1016/ S0738-081X(02)0 0330-9 errios RL , Arbiser JL. Effectiveness of gentian violet and simi- lar products commonly used to treat pyodermas. Dermatol Clin 2011; 29 :69–73. https:// doi.org/ 10.1016/ j.det.2010.08.009 igelow KM , Tasneen R, Chang YS et al . Preserved efficacy and reduced toxicity with intermittent linezolid dosing in combination with be- daquiline and pretomanid in a murine tuberculosis model. Antimi- crob Agents Chemother 2020; 64 :e01178–20. https:// doi.org/ 10.112 8/AAC.01178-20 ortolin M , Bidossi A, de Vecchi E et al . In vitro antimicrobial activity of chlorquinaldol against microorganisms responsible for skin and soft tissue infections: comparative evaluation with gentamicin and fusidic acid. Front Microbiol 2017; 8 :1–10. https:// doi.org/ 10.3389/ fmicb.2017.01039 ussmann RW , Malca G, Glenn A et al . Toxicity of medicinal plants used in traditional medicine in Northern Peru. J Ethnopharmacol 2011; 137 :121–40. https:// doi.org/ 10.1016/ j.jep.2011.04.071 haudhary H , Gupta D, Gupta C. Multifunctional dyeing and finishing of polyester with sericin and basic dyes. J Text Inst 2017; 108 :314– 24. https:// doi.org/ 10.1080/ 00405000.2016.1165401 leary I . The use of mercurochrome in typhoid fever. Am J Nurs 1925; 25 :97–99. lifton J , Leikin JB. Methylene blue. Am J Ther 2003; 10 :289–91. https: // doi.org/ 10.1097/ 00045391- 200307000- 00009 LSI . Performance Standards for Antimicrobial Susceptibility Testing , 30th edn, Pennsylvania, USA: Clinical and Laboratory Standards In- stitute, 2020. umisa M , Aiyegorob OA, van Vuuren S. Medicinal plant: dye combinations—impact on antimicrobial potency and toxicity. S Afr J Bot 2020; 135 :188–200. https:// doi.org/ 10.1016/ j.sajb.2020.09.002 zoyem JP , Aro AO, McGaw LJ et al . Antimycobacterial activity against different pathogens and selectivity index of fourteen medic- inal plants used in Southern Africa to treat tuberculosis and respira- tory ailments. S Afr J Bot 2016; 102 :70–74. https:// doi.org/ 10.1016/ j.sajb.2015.08.002 lisha IL , Botha FS, Madikizela B et al . Acetone leaf extracts of some South African trees with high activity against Esc heric hia coli also have good antimycobacterial activity and selectivity index. BMC Complement Altern Med 2017; 17 :1–5. https:// doi.org/ 10.1186/ s1 2906- 017- 1831- z rieri M , Kumar K, Boutin A. Antibiotic resistance. J Infect Pub- lic Health 2017; 10 :369–78. https:// doi.org/ 10.1016/ j.jiph.2016.08 .007 mur MK , Karpi ́nski TM. Povidone-iodine in wound healing and pre- vention of wound infections. Eur J Biol Res 2020; 10 :232–9. opinathan R , Kanhere J, Banerjee J. Effect of malachite green toxicity on non-target soil organisms. Chemosphere 2015; 120 :637–44. http s:// doi.org/ 10.1016/ j.chemosphere.2014.09.043 upta VK , Mittal A, Gajbe V et al . Adsorption of basic fuchsin us- ing waste materials-bottom ash and deoiled soya-as adsorbents. J Colloid Interface Sci 2008; 319 :30–39. https:// doi.org/ 10.1016/ j.jcis .2007.09.091 ashemi MM , Holden BS, Coburn J et al . Proteomic analysis of re- sistance of Gram-negative bacteria to chlorhexidine and impacts on susceptibility to colistin, antimicrobial peptides, and ceragenins. Front Microbiol 2019; 10 :210. https:// doi.org/ 10.3389/ fmicb.2019 .00210 ayward RS , Harding J, Molloy R et al . Adverse effects of a single dose of gentamicin in adults: a systematic review. Br J Clin Pharmacol 2018; 84 :223–38. https:// doi.org/ 10.1111/ bcp.13439 übsch Z , Van Zyl RL, Cock IE et al . Interactive antimicrobial and tox- icity profiles of conventional antimicrobials with Southern African medicinal plants. S Afr J Bot 2014; 93 :185–97. https:// doi.org/ 10.1 016/j.sajb.2014.04.005 ngraham MA . The bacteriostatic action of gentian violet and dependence on the oxidation-reduction potential. J Bacteriol 1933; 26 :573–98. https:// doi.org/ 10.1128/ jb.26.6.573-598.1933 ve FA . Diseases of the skin. Treatment of skin infections and infesta- tions. Br Med J (Clin Res Ed) 1973; 4 :475–8. https:// doi.org/ 10.113 6/bmj.4.5890.475 orkmaz S , Ceylan ME, Ceylan G et al . Auditory and histopathological effects of topical mercurochrome treatment in rats with tympanic membrane perforation. J Int Adv Otol 2019; 15 :22–27. https://doi. org/ 10.5152/ iao.2018.5489 rause KM , Serio AW, Kane TR et al . Aminoglycosides: an overview. Cold Spring Harb Perspect Med 2016; 6 :a027029. https:// doi.org/ 10 .1101/cshperspect.a027029 rumwiede C , Jr, Pratt JS. Observations on the growth of bacteria on media containing various aniline dyes. J Exp Med 1914; 19 :20–27. https:// doi.org/ 10.1084/ jem.19.1.20 urrimboccus F , Orchard A, Danckwerts MP et al . Antimicrobial for- mulation of Chrysopogon zizanioides essential oil in an emulsified lotion for acne. Planta Med 2021; 88 :1256–62. ushniak BD . Antibiotic resistance: a public health crisis. Public Health Rep 2014; 129 :314–6. https:// doi.org/ 10.1177/ 0033354914129004 02 yon JP , Rezende RR, Rabelo MP et al . Synergic effect of photody- namic therapy with methylene blue and surfactants in the inhibi- tion of Candida albicans . Mycopathologia 2012; 175 :159–64. https: // doi.org/ 10.1007/ s11046- 012- 9601- 4 aley AM , Arbiser JL. Gentian violet: a 19th century drug re-emerges in the 21st century. Exp Dermatol 2013; 22 :775–80 https://doi.org/ 10.1111/exd.12257 artens E , Demain AL. The antibiotic resistance crisis, with a focus on the United States. J Antibiot (Tokyo) 2017; 70 :520–6. https://doi.or g/ 10.1038/ ja.2017.30 cKeny PT , Nessel TA, Zito PM. Antifungal antibiotics. StatPearls [online article] . StatPearls Publishing, 2021. Available from: https: // www.ncbi.nlm.nih.gov/books/NBK538168/ organ JP , Haug RH, Kosman JW. Antimicrobial skin preparations for the maxillofacial region. J Oral Maxillofac Surg 1996; 54 :89–94. https:// doi.org/ 10.1016/ S0278- 2391(96)90312- 2 ourad A , Perfect JR. Tolerability profile of the current antifungal ar- moury. J Antimicrob Chemother 2018; 73 :i26–32. oack V , Pak K, Jalota R et al . An antioxidant screen identifies candi- dates for protection of cochlear hair cells from gentamicin toxicity. Front Cell Neurosci 2017; 11 :242. https:// doi.org/ 10.3389/ fncel.20 17.00242 wen E . Tincture of iodine as a household remedy. Lancet North Am Ed 1913; 182 :1063–4. https:// doi.org/ 10.1016/ S0140-6736(01)775 20-4 wen K . Iodine. In: Olson KR, Anderson IB, Benowitz NL, Blanc PD, Clark RF, Kearney TE, Kim-Katz SY, Wu AHB (eds), Poisoning and Drug Overdose , United States of America: The McGraw-Hill Companies, 2018, 1000. asmooij AM . Topical gentamicin for the treatment of genetic skin dis- eases. J Invest Dermatol 2018; 138 :731–4. https:// doi.org/ 10.1016/ j.jid.2017.12.008 https://doi.org/10.36326/kjvs/2016/v7i1B4255 https://doi.org/10.1155/2016/3627463 https://doi.org/10.1016/S0738-081X(02)00330-9 https://doi.org/10.1016/j.det.2010.08.009 https://doi.org/10.1128/AAC.01178-20 https://doi.org/10.3389/fmicb.2017.01039 https://doi.org/10.1016/j.jep.2011.04.071 https://doi.org/10.1080/00405000.2016.1165401 https://doi.org/10.1097/00045391-200307000-00009 https://doi.org/10.1016/j.sajb.2020.09.002 https://doi.org/10.1016/j.sajb.2015.08.002 https://doi.org/10.1186/s12906-017-1831-z https://doi.org/10.1016/j.jiph.2016.08.007 https://doi.org/10.1016/j.chemosphere.2014.09.043 https://doi.org/10.1016/j.jcis.2007.09.091 https://doi.org/10.3389/fmicb.2019.00210 https://doi.org/10.1111/bcp.13439 https://doi.org/10.1016/j.sajb.2014.04.005 https://doi.org/10.1128/jb.26.6.573-598.1933 https://doi.org/10.1136/bmj.4.5890.475 https://doi.org/10.5152/iao.2018.5489 https://doi.org/10.1101/cshperspect.a027029 https://doi.org/10.1084/jem.19.1.20 https://doi.org/10.1177/003335491412900402 https://doi.org/10.1007/s11046-012-9601-4 https://doi.org/10.1111/exd.12257 https://doi.org/10.1038/ja.2017.30 https://www.ncbi.nlm.nih.gov/books/NBK538168/ https://doi.org/10.1016/S0278-2391(96)90312-2 https://doi.org/10.3389/fncel.2017.00242 https://doi.org/10.1016/S0140-6736(01)77520-4 https://doi.org/10.1016/j.jid.2017.12.008 Dyes with conventional antimicrobials 17 S S S S T V W W W D ow nloaded from https://academ ic.oup.com /jam bio/article/135/7/lxae147/7704455 by jam estem lett user on 04 July 2024 Poustchi F , Amani H, Ahmadian Z et al . Combination therapy of killing diseases by injectable hydrogels: from concept to medical applica- tions. Adv Healthcare Mater 2021; 10 :1–56. https:// doi.org/ 10.100 2/adhm.202001571 Prabha N , Arora RD, Ganguly S et al . Gentian violet: revisited. Indian J Dermatol Venereol Leprol 2020; 86 :600–3. https:// doi.org/ 10.410 3/ijdvl.IJDVL _ 579 _ 19 Punjataewakupt A , Napavichayanun S, Aramwit P. The downside of antimicrobial agents for wound healing. Eur J Clin Microbiol Infect Dis 2019; 38 :39–54. Ramfol R . The Combination of Medicinal Dyes with Conven- tional Antimicrobials: Potential for Synergy in Topical Skin Infec- tions . Thesis for M. Pharmacy, The University of Witwatersrand, 2023. Rogosa M . The Bacteriostatic Action of Gentian Violet, Crystal Vio- let, Basic Fuchsin, and Acid Fuchsin on Certain Gram-positive Bac- teria . Thesis for M. Science, University of Massachusetts Amherst, 1934. Rosa L , da Silva F, Nader S et al . Antimicrobial photody- namic inactivation of Staphylococcus aureus biofilms in bone specimens using methylene blue, toluidine blue ortho and malachite green: an in vitro study. Arch Oral Biol 2015; 60 : 675–680. Scheibler E , Garcia MCR, Medina da Silva R et al . Use of nystatin and chlorhexidine in oral medicine:p, indications and pitfalls with focus on geriatric patients. Gerodontology 2017; 34 :291–98. Sinawe H , Casadesus D. Ketoconazole . [Updated 2023 Jun 26]. In: Stat- Pearls [Internet]. : ; . Available from: https://www.ncbi.nlm.nih.gov /books/NBK559221/ Smith HL . The value of tincture of iodine as a bactericide. Lancet North Am Ed 1915; 185 :345–6. https:// doi.org/ 10.1016/ S0140-6736(00)5 2944-4 Received 3 January 2024; revised 21 May 2024; accepted 1 July 2024 © The Author(s) 2024. Published by Oxford University Press on behalf of Applied Mic terms of the Creative Commons Attribution License ( https:// creativecommons.org/ licen any medium, provided the original work is properly cited. outh African Medicines Formulary (SAMF) . Rossiter D, Blackman M, Barnes K , (eds.) 13th edn. Erasmuskloof, Pretoria: Health and Med- ical Publishing Group of the South African Medical Association, 2020. outhwood T , Lamb CM, Freeman J. Ingestion of potassium perman- ganate crystals by a three-year-old boy. Med J Aust 1987; 146 :639– 40. https:// doi.org/ 10.5694/ j.1326-5377.1987.tb120444.x ouza LWF , Souza SWT, Botelho ACC. Randomized controlled trial comparing photodynamic therapy based on methylene blue dye and fluconazole for toenail onychomycosis. Dermatological Ther- apy 2017; 27 :43–47. https:// doi.org/ 10.1111/ dth.12042 ullivan GJ , Delgado NN, Maharjan R et al . How antibiotics work to- gether: molecular mechanisms behind combination therapy. Curr Opin Microbiol 2020; 57 :31–40. https:// doi.org/ 10.1016/ j.mib.20 20.05.012 he European Committee on Antimicrobial Susceptibility Testing . Breakpoint tables for interpretation of MICs and zone diameters, version 10. 2020. https:// www.eucast.org/ (23 December 2023, date last accessed). an Vuuren S , Viljoen A. Plant-based antimicrobial studies–methods and approaches to study the interaction between natural products. Planta Med 2011; 77 :1168–82. https:// doi.org/ 10.1055/ s- 0030- 125 0736 ainwright M . Dyes in the development of drugs and pharmaceuticals. Dyes Pigm 2008; 76 :582–9. https:// doi.org/ 10.1016/ j.dyepig.2007. 01.015 ainwright M . In defence of ‘dye therapy’. Int J Antimicrob Agents 2014; 44 :26–29. https:// doi.org/ 10.1016/ j.ijantimicag.2014.02.013 illiamson DA , Carter GP, Howden BP. Current and emerging topical antibacterials and antiseptics: agents, action, and resistance patterns. Am Soc Clin Microbiol 2017; 30 :827–60. https:// doi.org/ 10.1128/ CMR.00112-16 robiology International. This is an Open Access article distributed under the ses/by/ 4.0/ ), which permits unrestricted reuse, distribution, and reproduction in https://doi.org/10.1002/adhm.202001571 https://doi.org/10.4103/ijdvl.IJDVL_579_19 https://www.ncbi.nlm.nih.gov/books/NBK559221/ https://doi.org/10.1016/S0140-6736(00)52944-4 https://doi.org/10.5694/j.1326-5377.1987.tb120444.x https://doi.org/10.1111/dth.12042 https://doi.org/10.1016/j.mib.2020.05.012 https://www.eucast.org/ https://doi.org/10.1055/s-0030-1250736 https://doi.org/10.1016/j.dyepig.2007.01.015 https://doi.org/10.1016/j.ijantimicag.2014.02.013 https://doi.org/10.1128/CMR.00112-16 https://creativecommons.org/licenses/by/4.0/ Introduction Materials and methods Results Discussion Conclusion Acknowledgements Supplementary data Author contributions Data availability References