CC BY
14ORRM
OPEN
ANTIMICROBIAL ACTIVITY OF NOVEL 1-[(2,4-(DI-TERT-BUTYLPHENOXY))-3-DIALKYLAMINO-2-PROPANOL] DERIVATIVES
Nina VRYNCHANU1, Yurii KOROTKIJ2, Nataliia HRYNCHUK1, Irina BOIKO1, Elena SMERTENKO2, Larisa BONDARENKO1
1SI Institute of Pharmacology and Toxicology of NAMS of Ukraine, Kyiv, Ukraine 2 Institute of organic chemistry of NAS of Ukraine, Kyiv, Ukraine
Corresponding author: Nataliia Hrynchuk, e-mail: natali72grynchuk@gmail.com
DOI: 10/38045/ohrm.2021.3.04 CZU: 579.61:547.435+615.281
Keywords: amino-propanol derivatives, antimicrobial activity, bacteria, biofilm.
Cuvinte cheie: de-
rivati de amino-propanol, activi-tate antimicro-bianâ, bacterii, biofilm.
Introduction. The microbial biofilm-forming ability is one of the major aspects of the emerging issue of antibiotic resistance, which makes them tolerant to antibiotics and host defense systems and other external stresses, thus contributing to persistent chronic infections. A series of relevant studies confirmed the high efficiency of aminopropanol derivatives as potential antibacterial and antifungal agents. This present study was aimed to evaluate the antimicrobial activity of new 1-[(2,4-(di-tert-butylphenoxy))-3-dialkylamino-2-propanol] derivatives on the planktonic bacterial/fungal cells and biofilms.
Material and methods. The minimum inhibitory concentrations (MIC) of the new compounds were determined by a standard method, along with their effects on biofilms estimated via the gentian violet adsorption-desorption assay.
Results. The KVM-219 compound showed the most pronounced effect on planktonic bacterial and fungal cells. The MIC values ranged between 0.78 ig/mL to 12.5 |g/mL, depending on the microbial strain. The KVM-316 compound exhibited the strongest inhibitory effect on biofilms, thus preventing their formation by S. aureus (96.1%), E. coli (57.2%), and P. aeruginosa (96.1%).
Conclusions. The 15 newly synthesized 1-[(2,4-(di-tert-butylphenoxy))-3-dialkylamino-2-propanol] derivatives revealed marked antibacterial and antifungal effects on planktonic microorganisms. Most of these compounds showed a strain-specific inhibition of biofilm formation by at least 50% for S. aureus 222, E. coli 311, P. aeruginosa 449 and C. glabrata 404 strains.
ACTIVITATEA ANTIMICROBIANA A NOILOR DERIVATI DE 1 - [(2,4-(DI-TERT-BUTYLPHENOXY)) - 3-DIALKILAMINO-2-PROPANOL]
Introducere. Scopulstudiuluil-a constituit evaluarea efectelor antimicrobieneale derivatilor 1 - [(2,4 (di-tert- butylphenoxy)) - 3- dialkylamino-2-propanol] asupra celulelor planctonice bacteriene/fungice si asupra biofilmelor.
Material si metode. Concentratiile minime inhibitorii (CMI) ale compusilor noi au fost determinate printr-o metoda standard, activitatea antibiofilm a fost testata prin absorbtia violetu-lui degentiana pestructuri formatepeplaci depolistiren, urmata de resolubilizare cu solvent organic si resazurina ca indicator redox.
Rezultate. Efectul cel mai pronuntat asupra celulelor planctonice bacteriene si fungice l-a demonstrat compusul KVM -219, CMI 0,67 ig/ml -12,5 ig/ml, in functie de microorganism, iar asupra biofilmelor - compusul KVM-316. KVM-316 a prevenit formarea biofilmelor de catre S. aureus (96,1%), E. coli (57,2%) si P. aeruginosa (96,1%).
Concluzii. Cei 15 derivati nou sintetizatiai 1 - [(2,4- (di- tert-butylphenoxy)) - 3- dialkylamino -2-propanol] au prezentat efecte antibacterienesi antifungicepronuntate asupra microorga-nismelor planctonice. Majoritatea acestor compusi au inhibat in mod specific formarea biofilmelor de catre tulpinile clinice S. aureus 222, E. coli 311, P. aeruginosa 449 si C. glabrata 404 cu celputin 50%.
27 "J
ORRM
INTRODUCTION
Over the decades, antimicrobial agents have been considered the primary suppressing means for bacterial infections. However, their irrational use led to the emergence and spread of antimicrobial-resistant strains (1, 2). The antimicrobial drug resistance has led to a decrease of infection prevention and control measures and lowered the therapeutic effectiveness thus resulting in a prolonged patient's hospital stay and increased treatment costs. The antibiotic resistance has been regarded nowadays as a major threat to public internal safety across many countries. Therefore, a global action plan to overcome antimicrobial resistance has been developed by World Health Assembly (3). In 2017, WHO presented a list of 12 bacterial species that pose a threat to human health, which are classified into three categories of pathogens, namely critical, high and medium priority, according to the urgency of need for new antibiotics (4). The goal of the global action plan is to ensure, for as long as possible, continuity of successful treatment and prevention of infectious diseases by effective and safe medicines that are quality-assured, used in a responsible way and accessible to all who need them. To achieve this goal, five strategic objectives have been set out: to improve awareness and understanding of antimicrobial resistance; to strengthen knowledge through surveillance and research; to reduce the incidence of infection; to optimize the use of antimicrobial agents; to develop the economic case for sustainable investment that takes account of the needs of all countries; and to increase investments in new medicines, diagnostic tools, vaccines and other interventions (3). Considering all of the above mentioned, the most promising approach is to search for potentially novel antimicrobial agents for combatting antimicrobial resistance.
The microbial biofilm-forming ability is one of the major aspects of the emerging issue of antibiotic resistance, which makes them tolerant to antibiotics and host defence systems and other external stresses, thus contributing to persistent chronic infections (5). Several studies confirmed the high efficiency of aminopropanol derivatives as potential antibacterial and antifungal agents, which actually drew our interest to compounds of this group (6, 7, 8).
The purpose of the present study was to evaluate the antimicrobial activity of new 1-[(2,4-(di-tert-butylphenoxy))-3-dialkylamino-2-propanol] derivatives.
MATERIAL AND METHODS
Bacterial strain and growth conditions
The study was conducted on the gram-positive (Staphylococcus aureus subsp. aureus (ATCC® 25923™), S. aureus 222 (MRSA)) and gram-negative (Escherichia coli (ATCC® 25922™), E. coli 311, Pseudomonas aeruginosa (ATCC® 27853 TM), P. aeruginosa 449) bacterial strains, and yeasts (Candida albicans NTCC 885/653, C. glabrata 404). The bacterial strains were subcultured at 37°G in Mueller-Hinton broth (HiMedia™ Laboratories Pvt Ltd) and Tryptic Soy Broth (TSB) (Merck Millipore) (pH 7.3), yeasts - at 30-32°G in Saburo dextrose broth (HiMedia™ Laboratories Pvt Ltd) (pH 5.6).
Chemicals
The new derivatives of 1-[(2,4-(di-tert-butylphe-noxy))-3-dialkylamino-2-propanol] were first synthesised at the Institute of Organic Chemistry NAS of Ukraine. These compounds were synthesized by using the same procedure (9). Its general structural formula is shown in Figure 1.
R1OCH2CHCH2N-R3 Hal
OH R4
R1 - (2,4-di-tert-butylphenyl); R2,R3 - alkyl, dialkyl, cycloalkyl; R4 - methyl, benzyl, 4-nitrobenzyl, 4-metoxybenzyl, 4-chlorobenzyl, 4- fluorobenzyl, 4-methylbenzyl; Hal - Cl-, (I-).
Figure 1. Structural formula of 1-[(2,4-(di-tert-butylphenoxy))-3-dialkylamino-2-propanol]
new derivatives.
ORRM
Minimum Inhibitory Concentration (MIC) determination
Synthesized compounds antimicrobial activity (I - XV) was tested by the twofold serial dilution method (10, 11) against gram-positive (S. aureus ATCC 25923) and gram-negative (E. coli ATCC 25922, P. aeruginosa ATCC 27853) bacteria, and yeasts (C. albicans NTCC 885/653). Inoculum density was 1-2x105 CFU/mL culture medium (bacteria) and 1-2x104 CFU/mL (yeasts). The 96-well microtiter plates with bacterial cultures were incubated at 35-37°C for 18-24 h, while yeasts - at 30-32oC for 24-48 h. Mueller-Hinton broth and Saburo dextrose broth were used for minimal inhibitory concentration (MIC) determination. The lowest compound concentration inhibiting the microbial growth was considered as the MIC. All assays were performed in triplicate for control of culture growth (as a positive) and cultural media (as a negative).
Quantitative biofilm assay
The anti-biofilm activity of the tested compounds was determined by using the microtiter plate for biofilm formation assay described by O'Toole (12). The overnight cultures were diluted 100fold with fresh TSB medium (bacteria) or Saburo dextrose broth (yeasts). Cell suspensions (100 [iL) were transferred into individual wells of sterile polystyrene 96-well plate. The anti-biofilm effect was estimated by growing strains in media with or without test compounds (2.0xMIC) at
37oC for 24 hours. After incubation, the media were discarded, and plates were rinsed thrice with distillate water to remove nonadherent cells. Adherent cells were stained for 10 -15 min with 0.1% crystal violet. The dye was extracted with ethanol for 15 min to quantify biofilm formation. The optical density was measured at 630 nm via the Absorbance Microplate Reader (model ELx800, BioTek, USA). The measurements were performed in six replications and repeated for at least three times; the values were then averaged.
Statistical Analysis
Newman-Keils (ANOVA) and Kruskal-Wallis criteria were used to assess the results via the STA-TISTICA, version 6.0 (StatSoft. Inc., USA) (13). Data are presented as M±m, where M is the mean value and m is the standard error of the mean.
RESULTS
The studies of antibacterial and antifungal activities confirmed that 1-[(2,4-(di-tert-butylphe-noxy))-3-dialkylamino-2-propanol] new derivatives caused inhibitory effect on S. aureus and C. albicans, with the MIC values ranging from 0.78 to 3.75 Mg/mL and from 1.56 to 20.0 ng/mL, respectively. These compounds revealed no antibacterial effects against gram-negative bacteria, except for the VII and IV compounds, the MIC value for E. coli reaching to 12.5 Mg/mL and 20.0 ng/mL, respectively (tab. 1, 2).
Table 1. Antimicrobial activity (MIC, ^g/mL) of new 1 -(2,4-(di-tert-butylphenoxy)-3- dialkylamino-2-
propanol derivatives.
Compound S. aureus subsp. aureus (ATCC® 25923™) E. coli (ATCC® 25922™) P. aeruginosa (ATCC® 27853™) C. albicans NTCC 885/653
KVM-190 (I) 2.5 >20.0 >20.0 3.75
KVM-266 (II) 5.0 >20.0 >20.0 7.5
KVM-267 (III) 5.0 >20.0 >20.0 7.5
KVM-316 (IV) 5.0 20.0 >20.0 5.0
KVM-251 (V) 3.12 >20.0 >20.0 12.5
KVM-327 (VI) 1.56 >20.0 >20.0 6.25
KVM-219 (VII) 0.78 12.5 >20.0 1.56
KVM-220 (VIII) 2.5 >20.0 >20.0 3.75
KVM-269 (IX) 5.0 >20.0 >20.0 7.5
KVM-268 (X) 5.0 >20.0 >20.0 7.5
KVM-221 (XI) 7.5 >20.0 >20.0 5.0
KVM-222 (XII) 3.75 >20.0 >20.0 2.5
KVM-319 (XIII) 1.56 >20.0 >20.0 20.0
KVM-317 (XIV) 5.0 >20.0 >20.0 5.0
KVM-318 (XV) 1.56 >20.0 >20.0 5.0
I
ORRM
Table 2. Structure of 1-[(2,4-(di-tert-butylphenoxy))-3-dialkylamino-2-propanol] derivatives in correspondence with antibacterial and antifungal activity.
Substituents
MIC, ^g/mL
Compound Ri R2 R3 R4 Hal S. aureus C. albicans
I 2,4-[(CH3)3C]C6H3 CH3 CH3 CH2C6H5 Cl- 2.5 3.75
II 2,4-[(CH3)3C]C6H3 CH3 CH3 CH2(C6H4)-4-NO2 Cl- 5.0 7.5
III 2,4-[(CH3)3C]C6H3 CH3 CH3 CH2(C6H4)-4-OCH3 Cl- 5.0 7.5
IV 2,4-[(CH3)3C]C6H3 CH3 CH3 CH2(C6H4)-4-Cl Cl- 5.0 5.0
V 2,4-[(CH3)3C]C6H3 CH3 C6H11 CH2C6H5 Cl- 3.12 12.5
VI 2,4-[(CH3)3C]C6H3 CH3 C6H11 CH2(C6H4)-4-Cl Cl- 1.56 6.25
VII 2,4-[(CH3)3C]C6H3 (C4H8) CH3 I- 0.78 1.56
VIII 2,4-[(CH3)3C]C6H3 (C4H8) CH2C6H5 Cl- 2.5 3.75
IX 2,4-[(CH3)3C]C6H3 (C4H8) CH2(C6H4)-4-NO2 Cl- 5.0 7.5
X 2,4-[(CH3)3C]C6H3 (C4H8) CH2(C6H4)-4-OCH3 Cl- 5.0 7.5
XI 2,4-[(CH3)3C]C6H3 (C6H12) CH3 I- 7.5 5.0
XII 2,4-[(CH3)3C]C6H3 (C6H12) CH2C6H5 Cl- 3.75 2.5
XIII 2,4-[(CH3)3C]C6H3 [(CH2)2CHCH3(CH2)2] CH2(C6H4)-4-NO2 Cl- 1.56 20.0
XIV 2,4-[(CH3)3C]C6H3 [(CH2)2CHCH3(CH2)2] CH2(C6H4)-4-CH3 Cl- 5.0 5.0
XV 2,4-[(CH3)3C]C6H3 [(CH2)2CHCH3(CH2)2] CH2(C6H4)-4-F Cl- 1.56 5.0
2,4-[(CH3)3C]C6H3 -- 2,4-di-tert-butylphenyl radical.
The most pronounced effect was found in the VII compound. The MIC value against S. aureus was 0.78 |j.g/mL, E. coli - 12.5 |ig/mL, and for C. albicans - 1.56 |j.g/mL
Considering that biofilms are the main mode of microbial existence, the evaluation of their susceptibility to the tested compounds was of great scientific interest. Our clinical trials tested the
following microbial strains: S. aureus 222, E. coli 311, P. aeruginosa 449, and C. glabrata 404.
The obtained results demonstrated that all the tested compounds, except for the XIII compound, prevented S. aureus biofilm formation. The use of 2.0xMIC decreased the mass of MRSA 222 biofilm by 82.5% up to 100 % compared to the untreated culture (tab. 3).
Table 3. Antibiofilm activity
'o ) of new 1-[(2,4-(di-tert-butylphenoxy))-3-dialkylamino-2-propanol]
derivatives.
Compound S. aureus 222 E. coli 311 P. aeruginosa 449 C. glabrata 404
Control 100.0±3.6 Control 100.0±1.2 Control 100.0±9.6 Control 100.0±0.9
KVM-190 (I) 17.5±1.1* 36.1±0.8* 249.1±15.1* 261.3±6.6*
KVM-266 (II) 0.2±0.1* 31.6±5.4* 150.3±6.1* 119.5±6.6
KVM-267 (III) 0.7±0,4* 85.9±0,5* 129.9±3.1* 160.0±43.8*
KVM-316 (IV) 12.7±0.2* 42.8±2.1* 3.9±0.2* 83.2±2.6
KVM-251 (V) 1.2±0.2* 127.8±1.7* 164.1±9.6* 251.3±1.5*
KVM-327 (VI) 0.3±0.2* 77.6±1.7* 61.1±4.3* 3.0±2.4*
KVM-219 (VII) 88.8±1.5 88.0±1.5 37.2±0.2* 111.8±0.0
KVM-220 (VIII) 0.2±0.2* 77.7±0.4* 174.9±3.7* 77.5±2.5
KVM-269 (IX) 0.2±0.1* 91.1±1.7* 69.7±3.3* 9.6±1.9*
KVM-268 (X) 0.1±0,0* 92.8±0.4 145.5±1.6* 3.8±1.9*
KVM-221 (XI) 12.4±4.0* 111.4±0.7* 145.5±3.2* 187.5±7.5*
KVM-222 (XII) 0.0±0.0* 98.1±1.6 131.1±3.2* 161.9±13.0*
KVM-319 (XIII) 51.4±2.9* 93.7±1.1 106.2±3.1 17.3±3.3*
KVM-317 (XIV) 0.6±0.6* 106.9±1.8* 63.0±4.9* 1.3±0.9*
KVM-318 (XV) 0.4±0.3* 96.3±0.1 79.1±1.7* 11.5±5.1*
*p<0.05 in comparison with control.
ORRM
At the same time E. coli biofilms were less susceptible to 1-[(2,4-(di-tert-butylphenoxy))-3-dialkyl-amino-2-propanol] derivatives. Among all tested derivatives, the I, II and IV compounds showed the most pronounced inhibitory effects (63.9%, 68.4% and 57.2%, respectively, p<0.05).
The 1-[(2,4-(di-tert-butylphenoxy))-3-dialkyla-mino-2-propanol] derivatives almost did not inhibit P. aeruginosa biofilm formation. Only two compounds, namely the IV (96.1%) and VII (62.8%) exhibited the most pronounced inhibitory effect, whereas the VI (38.9%), IX (30.3%), XIV (37.0%) and XV (20.9%) derivatives showed a lesser activity. Unlike these ones, the I, II, III, V, VIII, X, XI, XII, and XIII compounds even stimulated the P. aeruginosa biofilm formation.
Thus, according to the study results the most pronounced inhibitory effect on biofilms formation were found for the IV compound, with a 96.1% decrease in S. aureus biofilm mass, 57.2% - E. coli and 96.1% - P. aeruginosa.
As regarding the C. glabrata biofilm formation, a pronounced inhibitory effect was demonstrated by the VI, IX, X, XIII, XIV and XV compounds (82.7%-98.7% biofilm mass decrease).
DISCUSSIONS
The comparative study of the antibacterial and antifungal activity of 1-[(2,4-(di-tert-buty-lphe-noxy)) - 3 -dialkylamino-2 -propanol] derivatives with various substituents in the molecular structure showed that substitutes close to the amino fragment might affect the antimicrobial activity of the tested compounds (tab. 2).
By introducing the N-benzyl dimethylamine (I); N-benzyl pyrrolidine (VIII) and N-benzyl hexa-methyleneamine (XII) groups to the structure of
1-[(2,4- (di-tert-butylphenoxy))-3-dialkylamino-
2-propanol] at the amino fragment, an inhibitory effect towards S. aureus and C. albicans ranging between 2.5-3.75 Mg/mL was registered.
The addition of the substituents on the benzylic radical in the 4-position, namely the nitro group (II, IX), methoxy group (III and X) and chlorine group (IV) exhibited an increased inhibitory activity of 1-[(2,4-(di-tert-butylphenoxy))-3-dial-kylamino-2-propanol] derivatives. Their MIC values for S. aureus and C. albicans ranged between 5.0-7.5 Mg/mL.
Another heterocyclic substituent (4-methylpiper-idine) inserted to the 1-[(2,4-(di-tert-butylphe-noxy)) - 3-dialkylamino-2 -propanol] molecular derivatives, viz. XIV and XV, did not significantly affect their antifungal activities compared with II compound. At the same time, the antibacterial activity of XV compound_(MIC 1.56 Mg/mL) towards S. aureus increased, compared to the II compound (MIC 5.0 Mg/mL).
Upon the insertion of a nitro group at the 4-posi-tion to the benzylic radical, viz. the XIII compound, it showed a reduced antifungal activity (MIC 20.0 Mg/mL) in comparison with XII compound (2.5 Mg/mL).
The replacement of one of the methyl groups for cyclohexyl in the molecule of the V compound led to a decrease of its antifungal activity (MIC 12.5 Mg/mL), while its inhibitory effect towards S. au-reus remained nearly unchanged (MIC 3.12 Mg/mL) compared with the I compound (MIC 2.5 Mg/mL).
The introduction of the chlorine at the 4-position led to completely different consequences: the MIC value of the VI compound showed a two-fold decrease in comparison with the MIC values of the I and V compounds (S. aureus - 1.56-3.12 Mg/mL; C. albicans - 25.0-12.5 Mg/mL). However, this decrease was not statistically significant.
The addition of N-methylpyrrolidine fragment to the amino group allowed to increase the antibacterial and antifungal activity of the VII derivative by 3.2 and 2.4 times, respectively (compared with I and VIII compounds). The MIC values towards S. aureus and C. albicans decreased to 0.78 Mg/mL and 1.56 Mg/mL, respectively.
The replacement of hexamethylenamine pyrroli-dine radical in the XI derivative was accompanied by a decreased inhibitory activity (compared to the VII compound), whereas the MIC values towards S. aureus and C. albicans were 5.0-7.5 Mg/mL.
This present study demonstrated that both tested derivatives 1-[(2,4- (di-tert-butylphenoxy) )-3-di-alkylamino-2-propanol] were able to suppress the gram-positive and gram-negative bacteria in fungal (C. glabrata) planktonic microorganisms and biofilms formation processes. The comparative structure-activity analysis showed that the inhibitory effect depended not only on the molecular structure and position of the substituents of
ORRM
1-[(2,4- (di-tert-butylphenoxy))-3-dialkylamino-
2-propanol] derivatives, but also on the microbial strain used.
Thus, the trials carried out on planktonic and bio
film cultures demonstrated that the newly synthesized derivatives of 1-[(2,4-(di-tert-butylphe-noxy))-3-dialkylamino-2-propanol] exhibited an inhibitory effect against bacteria and fungi.
CONCLUSIONS
1. The studies on planktonic microorganisms demonstrated that the newly synthesized derivatives of 1-[(2,4-(di-tert-butylphenoxy))-3-dialkylamino-2-propanol] have antibacterial and antifungal effects. This research showed that the anti-biofilm effects of the most evaluated compounds could specifically reduce the biofilm formation ability of S. aureus 222, E. coli 311, P. aeruginosa 449 and C. glabrata 404 by at least 50%, depending on the nature of the substituents used in their molecules.
2. The newly synthesized derivatives of 1-[(2,4-(di-tert-butylphenoxy))-3-dialkylamino-2-propa-nol] represent a promising class of chemical compounds, which might lead to the development of novel antimicrobial agents intended for the treatment of many infectious diseases. Further researches are required to study the broad activity spectrum of compounds with the most pronounced antimicrobial action, as well as their antibacterial and antifungal mechanisms, acute toxicity and efficacy in vivo.
CONFLICT OF INTERESTS
Authors have no conflict of interests to declare.
REFERENCES
1. Bowler P, Murphy C, Wolcott R. Biofilm exacerbates antibiotic resistance: Is this a current oversight in antimicrobial stewardship? Antimicrob Resist Infect Control. 2020; 9:162. doi:10.1186/ s13756-020-00830-6
2. Ventola CL. The antibiotic resistance crisis: part 1: causes and threats. P T. 2015; 40(4):277-283.
3. World Health Organization. Antimicrobial resistance. Available from: https://www.who.int/ antimicrobial-resistance/global-action-plan/en/ [Accessed 25.01.2021].
4. World Health Organization. Publishes list of bacteria for which new antibiotics are urgently needed. Available from: https://www.who.int/ news/item/27-02-2017-who-publishes-list-of-bacteria-for-which-new-antibiotics-are-urgently-needed [Accessed 26.02.2021].
5. Sharma D, Misba L, Khan A.U. Antibiotics versus biofilm: an emerging battleground in microbial communities. Antimicrob Resist Infect Control. 2019; 8:76. doi:10.1186/s13756-019-0533-3
6. Babu KS, Reddy PM, Naik VKM, Ramanjaneyulu K. Synthesis and antibacterial screening of five newazomethine derivatives of 2-amino-2-methyl-1-propanol. International Pharmaceutical Sciences and Research. 2019; 10(9):4396-4403. doi:10. 13040/IJPSR.0975-8232.10(9).4396-03
7. Bamou FZ, Le TM, Volford B, Szekeres A, Szakonyi Z. Synthesis and Application of 1,2-Aminoalcohols with Neoisopulegol-Based Octahydrobenzofuran
Core. Molecules. 2019;25(1):21. doi:10.3390/ mol-ecules25010021
8. Dronova M, Vrynchanu N, Varbanets L, Korotkiy Yu, Brovarska O. Arilalophatic aminoalcohol derivative KVM-194 affects E. coli lipo-polysaccharide composition. Farmacia. 2015; 63(4):586-592.
9. Korotkii Yu. V, Vrynchanu N.A, Grinevich S.V, Smertenko O.A. 1-(2,4-di-tert-butyl (phenoxy)-3-(N-benzyl, N-dimethylamino)-2-propanol chloride. Patent UA № 109203. 2015, bull. 14.G.
10. Clinical laboratory testing and in vitro diagnostic test systems - susceptibility testing of infectious agents and evaluation of performance of antimicrobial susceptibility test devices. Part 1: Reference method for testing the in vitro activity of antimicrobial agents against rapidly growing aerobic bacteria involved in infectious diseases (ISO 20776-1:2006). - Geneva: ISO, 2006.
11. Method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for yeasts / Eucast definitive document EDef 7.2 Revision. Available from: http://www.eu-cast.org/ ast_of_fungi/methods_of_antifungal_sus-ceptibility_testing/susceptibility_test-ing_of_yeasts [Accessed 13.07.2020].
12. O'Toole G. A. Microtiter dish biofilm formation assay. J. Vis. Exp. 2011;47(2437).
13. Lapach SN, Chubenko AV, Babich PN. Statistical Methods in Medical and Biological Studies in Excel [Statisticheskie metody v mediko-biologicheskih issledovaniyah s ispolzovaniem Excel]. K.: Morion. 2001.
ORRM
Date of receipt of the manuscript: 31/03/2021 Date of acceptance for publication: 12/06/2021
Nina VRYNCHANU, ORCID ID: 0000-0003-3450-2108 Yurii KOROTKIJ, ORCID ID: 0000-0002-4170-2266 Nataliia HRYNCHUK, ORCID ID: 0000-0002-2069-5917 Irina BOIKO, ORCID ID: 0000-0002-5261-3541 Elena SMERTENKO, ORCID ID: 0000-0002-9241-39 6 Larisa BONDARENKO, ORCID ID: 0000-0001-5107-8148
I
