Derivatives and intermediates of betulonic and platanic acid as effective antibacterial agents

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Their synthetic derivatives often display improved pharmacological properties and higher levels of efficacy. In this study, we investigated, the antibacterial effects of 13 synthesized oxime derivatives and 4 intermediates of betulonic and platanic acid, comparing them with those of the unmodified forms. Six Gram-positive pathogenic human bacteria, including four resistant strains from the genus Staphylococcus , were selected for testing together with two Gram-negative bacteria. All combinations of the prepared bacterial suspensions and 17 samples (500 − 62.5 µM) were placed on 96-well microtitration plates. The microdilution method was used to measure the absorbance of the samples, from which bacterial growth curves were derived. Resazurin tests were performed to obtain fluorescence values and, thereby, determine the metabolic activity and bactericidal effects. The derivatives and intermediates exhibited higher sensitivity to the Gram-positive bacteria. The most effective samples, for which 90–100% growth inhibition was observed against resistant strains ( Staph. aureus 368, Staph. epidermidis 335), were the oximester derivative of betulonic acid synthesized from 1-adamantylacetic acid ( 17 ; 500 µM) and the C-3 acetate intermediate of platanic acid ( 1 ; 500 µM). Sample 1 also showed 90–100% inhibition against Staph. aureus 670. Thus, our results suggest that the tested triterpenoid derivatives and intermediates have considerable potential for the development of new antibiotic therapies. antimicrobial activity oxime derivatives betulonic acid platanic acid Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The rapid spread of resistant bacterial strains has led to an urgent search for new antibiotics (Karaman, Jubeh, and Breijyeh 2020 ; Davies and Davies 2010 ). WHO has declared antibiotic resistance to be one of the three major threats to public health (Bednarczyk-Cwynar and Zaprutko 2015 ; Giniyatullina et al. 2021 ) and published a list of pathogenic bacteria that are moderately, highly or critically resistant to current treatments (Karaman, Jubeh, and Breijyeh 2020 ; Breijyeh, Jubeh, and Karaman 2020 ). Furthermore, many pathogenic strains have evolved into multidrug-resistant (MDR) forms capable of resisting multiple classes of antibiotics simultaneously (Davies and Davies 2010 ). For example, Staphylococcus aureus is resistant to all β -lactam antibiotics and up to 20 different antimicrobial compounds, which represents most of the available antimicrobial classes (Chung 2020 ). Current approaches to the discovery of new antibacterial agents involve various strategies. The most common of these involve the development of compounds that inhibit resistance mechanisms and the synthesis of derivatives that modify the activity of known antibacterial structures in order to circumvent such mechanisms. More recent strategies comprise phage therapy, the use of nanoparticles, the development of inhibitors of virulence factors or the design of antibacterially effective peptides and proteins (Vila, Moreno-Morales, and Balleste-Delpierre 2020 ). Aside from these approaches, a relatively recent trend is the focus on natural substances with known antibacterial activity (Breijyeh, Jubeh, and Karaman 2020 ). Plant-derived secondary metabolites are a major source of bioactive compounds with potential efficacy in the treatment of bacterial infections. Of these, the class of triterpenoids is of particular interest because they are among the most biologically active compounds and, thus, known for their significant pharmacological effects. For example, they have been used to reduce blood glucose levels and blood pressure. In addition, their anti-inflammatory and antioxidant effects can help protect the liver (Li et al. 2023 ; Khwaza et al. 2021 ). Moreover, they have demonstrated antimicrobial, antiviral and anticancer activities. Triterpenoids occur in nature in diverse structures, including in the form of tetra- or pentacyclic structures (Karaman, Jubeh, and Breijyeh 2020 ; Martinez 2014 ). Pentacyclic triterpenoids are derived from the isoprenoid biosynthetic pathway. This pathway involves the cyclization of oxidosqualene and six isoprene units, meaning that the basic structure of pentacyclic triterpenoids contains 30 carbon atoms (Li et al. 2023 ). They are divided into three classes whose structure is derived from ursane, lupane or oleanane (Chung 2020 ). Currently, research is primarily focused on lupane-type triterpenoids, specifically on lupeol, platanic acid, betulin, and betulinic and betulonic acids. Lupane pentacyclic triterpenoids have four six-membered rings and one five-membered ring (the E ring) in which the isoprenyl group at C-19 is in the α-configuration (Li et al. 2023 ). The antimicrobial activity of betulonic and platanic acids has been demonstrated against Gram-positive bacteria, with greatest sensitivity demonstrated against Staph. aureus and Enterococcus faecalis (Haque et al. 2014 ). Kazakova et al. ( 2010 ) showed that betulonic acid concentrations of up to 80 mg ∙ ml –1 reduced the growth of Staph. aureus , and that concentrations of 90 and 100 mg ∙ ml –1 completely suppressed it. However, for Klebsiella pneumonia growth reduction was manifested, but not complete suppression (Kazakova et al. 2010 ). Because pentacyclic triterpenoids have various limitations - such as low water solubility, low selectivity, poor bioavailability, and short half-life, that currently prevent their clinical application, structural modifications have been proposed to enhance their therapeutic potential. Most such studies have focused on the preparation and evaluation the biological activity of amide or amine derivatives of betulonic and platanic acids (Khwaza et al. 2021 ; Lombrea et al. 2021 ). Bednarczyk-Cwynar et al. (2015) reviewed the synthesis of oxime and oximester derivatives of triterpenoid acids. Subsequently, (Kahnt et al. 2018 ), (Kozubek et al. 2022 ) and (Lombrea et al. 2021 ) all reported promising results with the use of such derivatives. In our previous study, oxime synthesis was employed to successfully prepare completely new derivatives incorporating nitrogen in the structure (Černá et al. 2024 ). This approach appears to be effective, but the antibacterial properties of such derivatives has not yet been fully explored, especially for platanic acid derivatives. In this study, we evaluated the antibacterial effectiveness of a new series of 13 synthesized oxime derivatives, some of which contain a nitrogen heterocycle, and 4 intermediates of betulonic and platanic acids. Using disk diffusion, spectrophotometric assay and the resazurin assay, the bacteriostatic and bactericidal properties of the substances are determined against four model Gram-positive and -negative bacteria: Staph. aureus (CCM 4516) and Enteroc. faecalis (CCM 4224); Escherichia coli (CCM 4517) and Pseudomonas aeruginosa (CCM 1961). All results are compared with those obtained for unmodified forms of betulonic and platanic acids. Because the derivatives and intermediates exhibitited higher sensitivity to the Gram-positive bacteria, the effects are more closely determined for the resistant clinical isolates Staph. epidermidis 335, Staph. hominis 334, Staph. aureus 368 and Staph. aureus 670. Overall, our results suggest that some of the oxime-based derivatives and intermediates have considerable potential for the development of new antibacterial therapies. Material and methods Chemicals and laboratory instruments Chemicals Mueller-Hinton Broth (MHB) and Mueller-Hinton Agar (MHA) were purchased from Oxoid; Resazurin powder was received from HiMedia Laboratories (India); antibiotics were obtained: vancomycin hydrochloride from HiMedia Laboratories and kanamycin sulfate from Sigma-Aldrich. The solvent dimethyl sulfoxide (DMSO) was from Lach-Ner (Czech Republic). Laboratory instruments used included: Class II Biohazard laminar flow cabinet - Clean Air (United Kingdom); Incubation chamber - Memmert GmbH & Co. KG (Germany); Synergy spectrophotometer - BioTek (USA); Vortex mixer - Velp Scientifica (Italy); Densilameter II - Erba Lachema; Digital analytical balance ER 120A - Sartorius; Digital balance PM 400 - Mettler; Autoclave 3870 TUTTNAUER - Tuttnauer. Preparation of solutions and culture media Mueller-Hinton Broth: 22 g MHB powder per 1000 ml distilled H 2 O; Mueller-Hinton Agar: 38 g MHA powder in 1000 ml distilled H 2 O; Resazurin solution: A solution with a concentration of 0.01 mg ∙ ml -1 was prepared; Antibiotic solutions (positive control): A stock solution of 10 mg ∙ ml -1 was used, when working with Gram-positive bacteria a solution of vancomycin with Gram-negative bacteria kanamycin. Overview of microorganisms used Czech Collection of Microorganisms - Staph. aureus CCM 4516; Enteroc. faecalis CCM 4224; E. coli CCM 4517; Pseud. aeruginosa CCM 1961; Clinical isolates obtained in cooperation with the University hospital of Královské Vinohrady (Czech Republic, Prague 10) - Staph. epidermidis 335 M0000335 C0719102; Staph. hominis 334 M0000334 C0719103; Staph. aureus 368 M0000368 G0278785; Staph. aureus 670 M0000371 F0313670. Preparation of the microbial suspension and culture conditions Petri dishes with MHA agar were inoculated with a stock culture of the bacterium and were subsequently left to cultivate for 16 h at 37°C, only in the case of Enteroc. faecalis at 28°C. Preparation of solutions of tested substances The synthesized substances (Černá et al. 2024 ) were first weighed on an analytical balance and dissolved in 1 ml of DMSO to obtain 10 mM solutions. Some samples were not completely dissolved after the addition of the solvent and were therefore placed in a sonication bath. The samples were then diluted 10x with MHB to form 1 mM solutions. Along with these samples, antibiotic solutions were also used in the testing as a positive control and a diluted DMSO solution as a negative control. Antibacterial assay Disc diffusion method The disc diffusion method is mainly a qualitative method used to read the size of the inhibition zones formed on a Petri dish with a bacterial culture. The tested bacterial culture was prepared by 16-hour cultivation on a suitable medium (MHA, 28°C or 37°C). A bacterial suspension solution was then prepared by resuspension in MHB medium and diluted to OD = 0.5 McF. 100 µl of this solution was spread evenly on Petri dishes with MHA agar over the whole surface. The inhibition zones are caused by the gradual diffusion of the sample around the sterile disc, which has been successively spread on the agar with the bacterial suspension. 20 µl of individual solutions of the tested substances in 1 mM were pipetted onto the sterile discs. After a 24-hour incubation, the diameters of the inhibition zones, which represent the area where the growth of the bacteria stopped, were then measured. Information on the bacteriostatic effect of substances was obtained using the diffusion test. Microdilution method The microdilution method is considered an accurate quantitative technique established for determining the bacteriostatic effects of antimicrobial agents. In the dilution plate method, 96-well microtitre plates are used, in which it is possible to measure multiple samples at different concentrations at once. A concentration series of samples (500 − 62.5 mM) is prepared in the plate by successive two-fold dilution, to which is added a 2x diluted bacterial culture solution of 0.5 McF to the corresponding 5∙10 5 CFU ∙ ml -1 . The total volume in each well was set to 200 µl. The determination of each sample of the test substance was performed in triplicate. Subsequently, absorbance values A 625 of bacterial growth in individual wells were recorded for 24 hours using a Synergy spectrophotometer at a wavelength of 625 nm and temperatures of 28°C or 35°C. From these values, growth curves can be constructed and % inhibition and values A 625 of the specific growth rate µ [h -1 ] can be calculated. Equation 1: $$\:\mu\:=\frac{\text{l}\text{o}\text{g}{A}_{2}-\:\text{l}\text{o}\text{g}{A}_{1}}{{t}_{2}-{t}_{1}}$$ A 1 … the average value of the measured absorbance at the point of the beginning of exponential growth, i.e., at time t 1 A 2 … the average value of the measured absorbance at the point of the beginning of exponential growth, i.e., at time t 2 Resazurin assay Cell viability will be measured using the resazurin assay. This assay relies on a reaction in which the color changes from the low-fluorescence reducing blue dye resazurin to the high-fluorescence pink resorufin. After 24 hours of cultivation of the 96-well microtiter plate used for the microdilution test, 100 µl was transferred by pipette to a new 96-well microtiter plate. 100 µl of resazurin blue solution was added to each well immediately afterwards. After approximately 30 min, a color change was observed, which was quantified by measuring fluorescence using a Synergy spectrophotometer. The spectrophotometer was set to two wavelengths λexcitation = 560 nm, λemission = 590 nm. From these measurements, the percentage inhibition and the number of metabolically active cells will be determined, thus a bactericidal effect was detected. Equation 2: $$\:\%\:inhibition=\left(\frac{\left({{\Phi\:}}_{DMSO}-{{\Phi\:}}_{PM}\right)-\left({{\Phi\:}}_{S}-{{\Phi\:}}_{PM}\right)}{\left({{\Phi\:}}_{DMSO}-{{\Phi\:}}_{PM}\right)}\right)\bullet\:100$$ Φ S … average value of the measured fluorescence of the sample Φ DMSO …average value of the measured fluorescence in the presence of DMSO Φ PM …average value of the measured fluorescence of the pure medium MHB Results and Discussion Bacterial infections remain a major global healthcare concerns and the development of novel antibacterial agents continues to be a critical area of research (Haque et al. 2014 ; Lombrea et al. 2021 ). Reports by Kazakova et al. ( 2010 ) have highlighted triterpenoids as promising candidates in the search for new antibacterial agents. Triterpenoids represent a highly effective strategy in treating this type of disease. Based on these findings, this research focuses on the investigation of triterpenoid compounds with potential therapeutic activity against bacterial infections. The triterpenoids selected for this study were platanic acid and betulonic acid. Another reason for their selection was the fact that both of these triterpenoic acids have not yet been fully explored in terms of their biological activity and potential therapeutic applications. On the other hand, they exhibit a range of well-known beneficial properties in the medicinal field. According to the study by Kahnt et al. ( 2018 ), promising results were observed for derivatives of platanic acid after the incorporation of functional groups containing nitrogen atoms (Kahnt et al. 2018 ). Therefore, the research focused on testing derivatives containing an oxime group and evaluating whether the modification of triterpenoic acid leads to improved and enhanced antibacterial activity. The synthesis of oxime derivatives was performed according to the methodology published by (Bednarczyk-Cwynar and Zaprutko 2015 ). This article also describes that these oxime derivatives can undergo further syntheses, such as acylation or alkylation. Additionally, these studies demonstrate that oxime esters in the lupane structure represent a promising pharmacological potential. Information about the synthesis of all tested derivatives is described in our previous research (Černá et al. 2024 ). The antibacterial activity was evaluated using three methods: specifically, the disc diffusion method, the microdilution method, and the resazurin assay. Each compounds was tested in a total of four concentrations (500, 250, 125 and 62.5 µM). In the first step, a screening was performed on four model pathogenic bacteria Staph. aureus , Enteroc. faecalis (Gram-positive) and Pseud. aeruginosa and E. coli (Gram-negative). Preliminary results obtained from the disc diffusion method showed that derivatives 2, 3, 5, 11 and 17 produced inhibition zones of approximately 15 mm in diameter against Staph. aureus . For Enteroc. faecalis , inhibition zones of 15 mm were observed with derivatives 2 and 9 (Table 4 ). In the next step, the derivatives were screened using the microdilution method (Table 5 ). The results showed that most of our samples exhibited activity primarily against Gram-positive bacterial strains. Growth inhibition was observed against Staph. aureus and Enteroc. faecalis for most of the derivatives. These findings are consistent with similar results reported by (Chue K. T., Chang M. S., and N. 2011). In the article by (Haque et al. 2014 ), similar results were reported regarding the lack of activity against Gram-negative bacteria such as E. coli and Pseud. aeruginosa . In the study by Kazakova et al. ( 2010 ), which focused on a similar topic, it was found that among unmodified triterpenoic acids, betulonic acid exhibited the highest antibacterial activity against Staph. aureus . Our results confirm this finding, as unmodified betulonic acid showed better activity compared to unmodified platanic acid. Betulonic acid demonstrated activity against Staph. aureus in both diffusion and microdilution tests, with bacterial growth inhibited to a zero specific growth rate. However, in the resazurin assay, betulonic acid showed no significant inhibition. On the other hand, a high percentage of inhibition was observed for unmodified betulonic acid (93.9% at 500 µM) against Enteroc. faecalis , along with a zero specific growth rate. The higher inhibition of betulonic acid against Enteroc. faecalis compared to Staph. aureus is consistent with the findings of (Haque et al. 2014 ). They measured the antibacterial activity of betulonic acid at a concentration of 50 µM and found 74.3% inhibition against Enteroc. faecalis and 51.1% against Staph. aureus. Kazakova et al. ( 2010 ) measured that the inhibition of Staph. aureus growth caused by betulonic acid was detected at a concentration of 80 mg ∙ ml − 1 (corresponding to 175 mM). In our study, the compounds were tested at a lower concentration, with similar results. In the study by (Poumale et al. 2010), unmodified platanic acid inhibited the growth of Staph. aureus and E. coli in the diffusion method at a concentration of 40 mg ∙ ml − 1 , which corresponds to 87.2 mM. We obtained slightly different results, as unmodified platanic acid did not show significant inhibition against Staph. aureus ATCC strains. However, we observed strong inhibition against a clinical isolate, Staph. aureus 368. Our research revealed enhanced antibacterial activity following the modification of triterpenoic acids. The effects of the acetate intermediate of platanic acid ( 1 ; 500 µM) and the oximester derivative of betulonic acid substituted by the 1-adamantyl acetic acid skeleton ( 17 ; 500 µM) against Staph. aureus was observed ( Fig. 1 , compound 1 ; 500 − 125 µM). The same derivative 17 also showed significant effects against the growth of Enteroc. faecalis . Similarly, also substances 7 , 11 and 12 (500 µM). However, with the reference unmodified betulonic acid, this effect was confirmed especially against Staph, aureus . At the same time, however, 100% inhibition of growth even in lower concentrations, i.e. 125 µM against Staph. aureus occurred with unmodified betulonic acid, 7 , 12 and 1 (figures in suplementary information). With derivative 17 , 100% inhibition of Staph. aureus bacteria occurred even at the lowest tested concentration of 62.5 µM (Fig. 2 ). Derivatives 100% inhibiting the growth of Enteroc. faecalis even at a lower concentration (125 µM) was compound 7 and 17 . These derivatives showed no growth, and their specific growth rate was calculated to be µ = 0.0000 [h⁻¹] for a concentration of 500 µM for derivatives 1 and 17 against Staph. aureus and 7 , 11 and 12 against Enteroc. faecalis . The zero value of the specific growth rate determined against both representatives of Gram-positive bacteria occurred 17 (Table 5 ). The resazurin test proved inhibition of metabolic activity, i.e. bactericidal effects of betulonic acid and tested substances 1 , 4 and 7 with inhibition ranging from 80–100% against Enteroc. faecalis and an intermediate 1 inhibition of 90% against Staph. aureus (Table 6 ). These results indicate that most substances had only bacteriostatic effects on the bacterial cell. Based on the obtained results, a narrower group of samples ( I , II , 1 , 6 , 7 , 12 , 17 ) was selected for further statistical repetition of testing using the same methods. These tests showed and confirmed the most significant effect against Staph. aureus . Inhibition zones measured by the diffusion test up to 11 mm against this bacterium were observed with substances 1 , 17 at a concentration of 500 µM. The zero specific growth rate of Staph. aureus was detected in samples 1 , 7 , 12 and 17 in concentration 250 µM again. Similarly, this was also found in betulonic acid. For derivatives 12 and 17 , a specific growth rate µ = 0.0000 [h⁻¹] was observed at the lowest tested concentration of 62.5 µM. Metabolic activity was inhibited from 80–100% for derivatives 1 , 7 , 12 and 17 , as well as for betulonic acid. Here, derivative 17 was the only one to inhibit metabolism by 100% even at a concentration of 62.5 µM. Due to the significant sensitivity observed against Staph. aureus , these selected derivatives were also tested against resistant clinical isolates obtained from the hospital environment. Specifically, the isolates were Staph. aureus 670, Staph. aureus 368, Staph. hominis 334 and Staph. epidermidis 335. Bacteriostatic properties at a concentration of 500 µM were found mainly against Staph. aureus 670 and 368 and also against Staph. epidermidis 335 in the unmodified betulonic acid and in derivatives 1 , 7 and 17 . However, only samples 1 and 17 achieved significant inhibition of metabolic activity ranging between 90–100% (Table 7 ; Figs. 3 and 4 ). Among the structural modifications of platanic acid, the acetate intermediate ( 1 ) exhibited the most promising biological activity. In the case of betulonic acid, the highest efficacy was observed particularly in its oxime derivatives, including the simple oxime ( 7 ) and two oxime ester derivatives, which were synthesized using pyrazine-2-carboxylic acid ( 12 ) and 1-adamantylacetic acid ( 17 ), respectively. Table 4 The results of bacteriostatic activity, obtained by the diffusion method, against the bacteria Staph. aureus , Enteroc. faecalis , E. coli , and Pseud. aeruginosa . The data correspond to 24-hour exposure of the samples at concentrations of 10 mM after incubation at 37°C or 28°C. Tested substance The average inhibition zone d [mm] Staph. aureus Enteroc. faecalis E. coli Pseud. aeruginosa 1% DMSO - - - - Vancomycin +++ +++ - - Kanamycin - - +++ +++ I - - - - II + - - - 1 + + - - 2 ++ ++ - - 3 ++ - ++ - 4 - - - - 5 ++ - - - 6 - - - - 7 - + - - 8 - - - - 9 - ++ - - 10 - - - - 11 ++ - - - 12 + - - - 13 - - - - 14 - - - - 15 + - - - 16 - - - - 17 ++ - + - +++30 mm ++15 mm + 10 mm -0 mm Table 5 Specific growth rates µ [h⁻¹] of Staph. aureus , Enteroc. faecalis , E. coli , and Pseud. aeruginosa after exposure to the samples at a concentration of 500 µM and incubation at 35°C or 28°C. Tested substance Specific growth rate µ [h⁻¹] Staph. aureus Enteroc. faecalis E. coli Pseud. aeruginosa 1% DMSO 0.0409 0.0335 0.0606 0.0770 Vancomycin 0.0000 0.0000 - - Kanamycin - - 0.0000 0.0000 I 0.0080 0.0167 0.0753 0.0367 II 0.0000 0.0000 0.0559 0.0182 1 0.0000 0.0403 0.1298 0.0314 2 0.0112 0.0107 0,0412 0.0230 3 0.0093 0.0149 0.0603 0.0393 4 0.0088 0.0143 0.0394 0.0319 5 0.0228 0.0101 0.0554 0.0515 6 0.0097 0.0150 0.0543 0.0521 7 0.0238 0.0000 0.0559 0.0254 8 0.0198 0.0175 0.0705 0.0364 9 0.0096 0.0075 0.0278 0.0214 10 0.0233 0.0661 0.0511 0.0346 11 0.0229 0.0000 0.0294 0.0363 12 0.0314 0.0000 0.0297 0.0312 13 0.0717 0.0945 0.0378 0.0400 14 0.0545 0.0282 0.0177 0.0488 15 0.0134 0.0128 0.0223 0.0589 16 0.0279 0.0493 0.0429 0.0261 17 0.0000 0.0000 0.0363 0.0302 The results are subject to a ± 15% error. Table 6. Inhibition of metabolic activity in % obtained from the cell viability test (resazurin assay) in Staph.aureus , Enteroc. faecalis , E. coli , and Pseud. aeruginosa after exposure to the samples at a concentration of 500 µM following 23 hours of incubation at 35°C or 28°C. Tested substance Growth inhibition [%] Staph. aureus Enteroc. faecalis E. coli Pseud. aeruginosa 1 % DMSO 0.0 0.0 0.0 0.0 Vancomycin 100.0 100.0 - - Kanamycin - - 100.0 100.0 I - 5.4 2.6 - II - 93.9 23.2 - 1 96.2 83.0 30.7 24.4 2 - 69.3 - - 3 - 23.3 4.1 - 4 - 86.6 - - 5 - 9.9 - - 6 43.0 74.5 13.0 - 7 - 100.0 6.0 - 8 13.6 55.7 0.9 - 9 10.5 61.0 0.9 - 10 - - - 22.8 11 - 19.0 - - 12 3.2 60.4 - - 13 - - - - 14 12.1 6.5 - - 15 36.9 18.8 16.7 - 16 - 12.6 - 16.6 17 64.6 70.5 - 32.6 The results are subject to a ± 15% error. ­(-Presence of metabolic activity.) Table 7 Inhibition of metabolic activity in % obtained from the cell viability test (resazurin assay) in Staph. aureus 670, Staph. aureus 368, Staph. hominis 334 and Staph. epidermidis 335 after exposure to the samples at a concentration of 500 µM following 23 hours of incubation at 35°C. Tested substance Growth inhibition [%] Staph. aureus 670 Staph. aureus 368 Staph. hominis 334 Staph. epidermidis 335 5% DMSO 0.0 0.0 0.0 0.0 Vancomycin 100.0 100.0 100.0 100.0 I 67.2 100.0 - - II 14.7 68.7 - 100.0 1 97.1 100.0 12.6 100.0 6 73.7 89.8 - 1.2 7 - - 72.1 94.8 12 8.3 53.8 - 5.5 17 56.9 94.4 53.8 99.4 The results are subject to a ± 15% error. (-Presence of metabolic activity.) Conclusion We have demonstrated that the structural modification of betulonic and platanic acids represents a promising strategy for the development of new antibacterial agents with potential applications in the treatment of infectious and cancer-related diseases. In our previous work, we successfully designed and synthesized 13 oxime derivatives and four intermediates of these acids. In the present study, several of these derivatives and intermediates exhibited significantly improved antibacterial properties, particularly against Gram-positive bacteria, including certain resistant clinical isolates. We also hypothesize that such structural modifications may partially contribute to enhanced pharmacological properties, including increased antiviral and anticancer potential. Although the tested derivatives were active against pathogenic Gram-positive bacteria, no activity was observed against Gram-negative strains. Nevertheless, our findings provide valuable insights and open new avenues for further research, particularly toward elucidating the mechanisms underlying these biological effects. Abbreviations A Absorbance CCM Czech Collection of Microorganisms CFU Colony-Forming Units DMSO Dimethyl sulfoxide MDR Multidrug-resistant McF McFarland MHA Mueller-Hinton Agar MHB Mueller-Hinton Broth WHO World Health Organization. Declarations Acknowledgements The research was supported by the Technology Agency of the Czech Republic grant No. TN02000044 and Specific university research of the University of Chemistry and Technology Prague grant No. A2_FPBT_2023_026. Author contribution Petra Lovecká, Uladzimir Bildziukevich and Zdeněk Wimmer designed and supervised the experiments; Lucie Černá and Angelina Massyagutova made antimicrobial testing; Lucie Černá and Petra Lovecká prepared and checked the manuscript. All authors read and approved the final manuscript. Data availability Data will be made available on request. Declarations Ethics approval This article does not contain any studies with animals performed by any of the authors. 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Additional Declarations No competing interests reported. Supplementary Files LucieernSuplementaryinformation.docx Tables.docx Cite Share Download PDF Status: Published Journal Publication published 16 Apr, 2026 Read the published version in Folia Microbiologica → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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1","display":"","copyAsset":false,"role":"figure","size":72097,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth curves of \u003cem\u003eStaph. aureus\u003c/em\u003e after exposure to derivative \u003cstrong\u003e1\u003c/strong\u003e at concentrations of 500 µM, 250 µM, and 125 µM, and incubation at 35°C for 24 hours. The 1% DMSO sample represents the bacterial growth curve \u003cbr\u003e\nas a negative control. The positive control is the bacterial growth curve inhibited by vancomycin at a concentration of 250 µg·ml⁻¹.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8076005/v1/6eb78757ce157d74cd8ecd01.jpg"},{"id":96605531,"identity":"16638518-b9b3-4638-8743-dfa8f5b01489","added_by":"auto","created_at":"2025-11-24 09:23:24","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":74057,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth curves of \u003cem\u003eStaph. aureus\u003c/em\u003eafter exposure to derivative \u003cstrong\u003e17\u003c/strong\u003e at concentrations of 250 µM, 125 µM and 62.5 µM and incubation at 35°C for 24 hours. The 1% DMSO sample represents the bacterial growth curve as a negative control. The positive control is the bacterial growth curve inhibited by vancomycin at a concentration of 250 µg·ml⁻¹.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8076005/v1/a89674b3031e8f87ed29d699.jpg"},{"id":96605841,"identity":"a1d794c9-e4f8-45af-875b-0bd65b8fb60b","added_by":"auto","created_at":"2025-11-24 09:24:11","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":63974,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth curves of resistant \u003cem\u003eStaph. aureus\u003c/em\u003e 368 after exposure to derivative \u003cstrong\u003e1\u003c/strong\u003eat concentrations of 500 µM and incubation at 35°C for 24 hours. The 5% DMSO sample represents the bacterial growth curve as a negative control. The positive control is the bacterial growth curve inhibited by vancomycin at a concentration \u003cbr\u003e\nof 250 µg·ml⁻¹.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8076005/v1/a0fe9475b3a0f09b1235ed49.jpg"},{"id":96590469,"identity":"983433bc-872d-47a9-886e-a7ae78a8c0f3","added_by":"auto","created_at":"2025-11-24 06:21:29","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":63703,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth curves of resistant \u003cem\u003eStaph. aureus\u003c/em\u003e368 after exposure to derivative \u003cstrong\u003e17\u003c/strong\u003e at concentrations of 500 µM and incubation at 35°C for 24 hours. The 5% DMSO sample represents the bacterial growth curve as a negative control. The positive control is the bacterial growth curve inhibited by vancomycin at a concentration \u003cbr\u003e\nof 250 µg·ml⁻¹.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8076005/v1/6ab2c044b4ac2686f7022205.jpg"},{"id":107351089,"identity":"8e51587e-aaee-4e0a-90ce-3141db849247","added_by":"auto","created_at":"2026-04-20 16:09:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1081164,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8076005/v1/117de58e-3f76-4f8d-afac-339d230fa9a4.pdf"},{"id":96590474,"identity":"6f7be701-132f-4c53-9111-50352e28920e","added_by":"auto","created_at":"2025-11-24 06:21:29","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":41099,"visible":true,"origin":"","legend":"","description":"","filename":"LucieernSuplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8076005/v1/30fcddd8fc5a1c394871c8c5.docx"},{"id":96590520,"identity":"2d695a25-485b-40cf-8f9a-bd4166f3319d","added_by":"auto","created_at":"2025-11-24 06:21:31","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":306145,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-8076005/v1/763af0ea106aff645d0493dd.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Derivatives and intermediates of betulonic and platanic acid as effective antibacterial agents","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe rapid spread of resistant bacterial strains has led to an urgent search for new antibiotics (Karaman, Jubeh, and Breijyeh \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Davies and Davies \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). WHO has declared antibiotic resistance to be one of the three major threats to public health (Bednarczyk-Cwynar and Zaprutko \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Giniyatullina et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and published a list of pathogenic bacteria that are moderately, highly or critically resistant to current treatments (Karaman, Jubeh, and Breijyeh \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Breijyeh, Jubeh, and Karaman \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Furthermore, many pathogenic strains have evolved into multidrug-resistant (MDR) forms capable of resisting multiple classes of antibiotics simultaneously (Davies and Davies \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). For example, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e is resistant to all \u003cem\u003eβ\u003c/em\u003e-lactam antibiotics and up to 20 different antimicrobial compounds, which represents most of the available antimicrobial classes (Chung \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Current approaches to the discovery of new antibacterial agents involve various strategies. The most common of these involve the development of compounds that inhibit resistance mechanisms and the synthesis of derivatives that modify the activity of known antibacterial structures in order to circumvent such mechanisms. More recent strategies comprise phage therapy, the use of nanoparticles, the development of inhibitors of virulence factors or the design of antibacterially effective peptides and proteins (Vila, Moreno-Morales, and Balleste-Delpierre \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAside from these approaches, a relatively recent trend is the focus on natural substances with known antibacterial activity (Breijyeh, Jubeh, and Karaman \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Plant-derived secondary metabolites are a major source of bioactive compounds with potential efficacy in the treatment of bacterial infections. Of these, the class of triterpenoids is of particular interest because they are among the most biologically active compounds and, thus, known for their significant pharmacological effects. For example, they have been used to reduce blood glucose levels and blood pressure. In addition, their anti-inflammatory and antioxidant effects can help protect the liver (Li et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Khwaza et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Moreover, they have demonstrated antimicrobial, antiviral and anticancer activities. Triterpenoids occur in nature in diverse structures, including in the form of tetra- or pentacyclic structures (Karaman, Jubeh, and Breijyeh \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Martinez \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePentacyclic triterpenoids are derived from the isoprenoid biosynthetic pathway. This pathway involves the cyclization of oxidosqualene and six isoprene units, meaning that the basic structure of pentacyclic triterpenoids contains 30 carbon atoms (Li et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). They are divided into three classes whose structure is derived from ursane, lupane or oleanane (Chung \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Currently, research is primarily focused on lupane-type triterpenoids, specifically on lupeol, platanic acid, betulin, and betulinic and betulonic acids. Lupane pentacyclic triterpenoids have four six-membered rings and one five-membered ring (the E ring) in which the isoprenyl group at C-19 is in the α-configuration (Li et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The antimicrobial activity of betulonic and platanic acids has been demonstrated against Gram-positive bacteria, with greatest sensitivity demonstrated against \u003cem\u003eStaph. aureus\u003c/em\u003e and \u003cem\u003eEnterococcus faecalis\u003c/em\u003e (Haque et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Kazakova et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) showed that betulonic acid concentrations of up to 80 mg ∙ ml\u003csup\u003e\u0026ndash;1\u003c/sup\u003e reduced the growth of \u003cem\u003eStaph. aureus\u003c/em\u003e, and that concentrations of 90 and 100 mg ∙ ml\u003csup\u003e\u0026ndash;1\u003c/sup\u003e completely suppressed it. However, for \u003cem\u003eKlebsiella pneumonia\u003c/em\u003e growth reduction was manifested, but not complete suppression (Kazakova et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBecause pentacyclic triterpenoids have various limitations - such as low water solubility, low selectivity, poor bioavailability, and short half-life, that currently prevent their clinical application, structural modifications have been proposed to enhance their therapeutic potential. Most such studies have focused on the preparation and evaluation the biological activity of amide or amine derivatives of betulonic and platanic acids (Khwaza et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Lombrea et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Bednarczyk-Cwynar et al. (2015) reviewed the synthesis of oxime and oximester derivatives of triterpenoid acids. Subsequently, (Kahnt et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), (Kozubek et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and (Lombrea et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) all reported promising results with the use of such derivatives. In our previous study, oxime synthesis was employed to successfully prepare completely new derivatives incorporating nitrogen in the structure (Čern\u0026aacute; et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This approach appears to be effective, but the antibacterial properties of such derivatives has not yet been fully explored, especially for platanic acid derivatives.\u003c/p\u003e\u003cp\u003eIn this study, we evaluated the antibacterial effectiveness of a new series of 13 synthesized oxime derivatives, some of which contain a nitrogen heterocycle, and 4 intermediates of betulonic and platanic acids. Using disk diffusion, spectrophotometric assay and the resazurin assay, the bacteriostatic and bactericidal properties of the substances are determined against four model Gram-positive and -negative bacteria: \u003cem\u003eStaph. aureus\u003c/em\u003e (CCM 4516) and \u003cem\u003eEnteroc. faecalis\u003c/em\u003e (CCM 4224); \u003cem\u003eEscherichia coli\u003c/em\u003e (CCM 4517) and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (CCM 1961). All results are compared with those obtained for unmodified forms of betulonic and platanic acids. Because the derivatives and intermediates exhibitited higher sensitivity to the Gram-positive bacteria, the effects are more closely determined for the resistant clinical isolates \u003cem\u003eStaph. epidermidis\u003c/em\u003e 335, \u003cem\u003eStaph. hominis\u003c/em\u003e 334, \u003cem\u003eStaph. aureus\u003c/em\u003e 368 and \u003cem\u003eStaph. aureus\u003c/em\u003e 670. Overall, our results suggest that some of the oxime-based derivatives and intermediates have considerable potential for the development of new antibacterial therapies.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eChemicals and laboratory instruments\u003c/h2\u003e\u003cp\u003eChemicals Mueller-Hinton Broth (MHB) and Mueller-Hinton Agar (MHA) were purchased from Oxoid; Resazurin powder was received from HiMedia Laboratories (India); antibiotics were obtained: vancomycin hydrochloride from HiMedia Laboratories and kanamycin sulfate from Sigma-Aldrich. The solvent dimethyl sulfoxide (DMSO) was from Lach-Ner (Czech Republic). Laboratory instruments used included: Class II Biohazard laminar flow cabinet - Clean Air (United Kingdom); Incubation chamber - Memmert GmbH \u0026amp; Co. KG (Germany); Synergy spectrophotometer - BioTek (USA); Vortex mixer - Velp Scientifica (Italy); Densilameter II - Erba Lachema; Digital analytical balance ER 120A - Sartorius; Digital balance PM 400 - Mettler; Autoclave 3870 TUTTNAUER - Tuttnauer.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePreparation of solutions and culture media\u003c/h3\u003e\n\u003cp\u003eMueller-Hinton Broth: 22 g MHB powder per 1000 ml distilled H\u003csub\u003e2\u003c/sub\u003eO; Mueller-Hinton Agar: 38 g MHA powder in 1000 ml distilled H\u003csub\u003e2\u003c/sub\u003eO; Resazurin solution: A solution with a concentration of 0.01 mg ∙ ml\u003csup\u003e-1\u003c/sup\u003e was prepared; Antibiotic solutions (positive control): A stock solution of 10 mg ∙ ml\u003csup\u003e-1\u003c/sup\u003e was used, when working with Gram-positive bacteria a solution of vancomycin with Gram-negative bacteria kanamycin.\u003c/p\u003e\n\u003ch3\u003eOverview of microorganisms used\u003c/h3\u003e\n\u003cp\u003eCzech Collection of Microorganisms - \u003cem\u003eStaph. aureus\u003c/em\u003e CCM 4516; \u003cem\u003eEnteroc. faecalis\u003c/em\u003e CCM 4224; \u003cem\u003eE. coli\u003c/em\u003e CCM 4517; \u003cem\u003ePseud. aeruginosa\u003c/em\u003e CCM 1961; Clinical isolates obtained in cooperation with the University hospital of Kr\u0026aacute;lovsk\u0026eacute; Vinohrady (Czech Republic, Prague 10) - \u003cem\u003eStaph. epidermidis\u003c/em\u003e 335 M0000335 C0719102; \u003cem\u003eStaph. hominis\u003c/em\u003e 334 M0000334 C0719103; \u003cem\u003eStaph. aureus\u003c/em\u003e 368 M0000368 G0278785; \u003cem\u003eStaph. aureus\u003c/em\u003e 670 M0000371 F0313670.\u003c/p\u003e\n\u003ch3\u003ePreparation of the microbial suspension and culture conditions\u003c/h3\u003e\n\u003cp\u003ePetri dishes with MHA agar were inoculated with a stock culture of the bacterium and were subsequently left to cultivate for 16 h at 37\u0026deg;C, only in the case of \u003cem\u003eEnteroc. faecalis\u003c/em\u003e at 28\u0026deg;C.\u003c/p\u003e\n\u003ch3\u003ePreparation of solutions of tested substances\u003c/h3\u003e\n\u003cp\u003eThe synthesized substances (Čern\u0026aacute; et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) were first weighed on an analytical balance and dissolved in 1 ml of DMSO to obtain 10 mM solutions. Some samples were not completely dissolved after the addition of the solvent and were therefore placed in a sonication bath. The samples were then diluted 10x with MHB to form 1 mM solutions. Along with these samples, antibiotic solutions were also used in the testing as a positive control and a diluted DMSO solution as a negative control.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eAntibacterial assay\u003c/h2\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003eDisc diffusion method\u003c/h2\u003e\u003cp\u003eThe disc diffusion method is mainly a qualitative method used to read the size of the inhibition zones formed on a Petri dish with a bacterial culture. The tested bacterial culture was prepared by 16-hour cultivation on a suitable medium (MHA, 28\u0026deg;C or 37\u0026deg;C). A bacterial suspension solution was then prepared by resuspension in MHB medium and diluted to OD\u0026thinsp;=\u0026thinsp;0.5 McF. 100 \u0026micro;l of this solution was spread evenly on Petri dishes with MHA agar over the whole surface. The inhibition zones are caused by the gradual diffusion of the sample around the sterile disc, which has been successively spread on the agar with the bacterial suspension. 20 \u0026micro;l of individual solutions of the tested substances in 1 mM were pipetted onto the sterile discs. After a 24-hour incubation, the diameters of the inhibition zones, which represent the area where the growth of the bacteria stopped, were then measured. Information on the bacteriostatic effect of substances was obtained using the diffusion test.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\n\u003ch3\u003eMicrodilution method\u003c/h3\u003e\n\u003cp\u003eThe microdilution method is considered an accurate quantitative technique established for determining the bacteriostatic effects of antimicrobial agents. In the dilution plate method, 96-well microtitre plates are used, in which it is possible to measure multiple samples at different concentrations at once. A concentration series of samples (500\u0026thinsp;\u0026minus;\u0026thinsp;62.5 mM) is prepared in the plate by successive two-fold dilution, to which is added a 2x diluted bacterial culture solution of 0.5 McF to the corresponding 5∙10\u003csup\u003e5\u003c/sup\u003e CFU ∙ ml\u003csup\u003e-1\u003c/sup\u003e. The total volume in each well was set to 200 \u0026micro;l. The determination of each sample of the test substance was performed in triplicate. Subsequently, absorbance values A\u003csub\u003e625\u003c/sub\u003e of bacterial growth in individual wells were recorded for 24 hours using a Synergy spectrophotometer at a wavelength of 625 nm and temperatures of 28\u0026deg;C or 35\u0026deg;C. From these values, growth curves can be constructed and % inhibition and values A\u003csub\u003e625\u003c/sub\u003e of the specific growth rate \u0026micro; [h\u003csup\u003e-1\u003c/sup\u003e] can be calculated.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eEquation 1:\u003c/h2\u003e\u003cp\u003e\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\mu\\:=\\frac{\\text{l}\\text{o}\\text{g}{A}_{2}-\\:\\text{l}\\text{o}\\text{g}{A}_{1}}{{t}_{2}-{t}_{1}}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e\u0026hellip; the average value of the measured absorbance at the point of the beginning of exponential growth, i.e., at time \u003cem\u003et\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e\u0026hellip; the average value of the measured absorbance at the point of the beginning of exponential growth, i.e., at time \u003cem\u003et\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eResazurin assay\u003c/h2\u003e\u003cp\u003eCell viability will be measured using the resazurin assay. This assay relies on a reaction in which the color changes from the low-fluorescence reducing blue dye resazurin to the high-fluorescence pink resorufin. After 24 hours of cultivation of the 96-well microtiter plate used for the microdilution test, 100 \u0026micro;l was transferred by pipette to a new 96-well microtiter plate. 100 \u0026micro;l of resazurin blue solution was added to each well immediately afterwards. After approximately 30 min, a color change was observed, which was quantified by measuring fluorescence using a Synergy spectrophotometer. The spectrophotometer was set to two wavelengths λexcitation\u0026thinsp;=\u0026thinsp;560 nm, λemission\u0026thinsp;=\u0026thinsp;590 nm. From these measurements, the percentage inhibition and the number of metabolically active cells will be determined, thus a bactericidal effect was detected.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eEquation 2:\u003c/h2\u003e\u003cp\u003e\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:\\%\\:inhibition=\\left(\\frac{\\left({{\\Phi\\:}}_{DMSO}-{{\\Phi\\:}}_{PM}\\right)-\\left({{\\Phi\\:}}_{S}-{{\\Phi\\:}}_{PM}\\right)}{\\left({{\\Phi\\:}}_{DMSO}-{{\\Phi\\:}}_{PM}\\right)}\\right)\\bullet\\:100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eΦ\u003c/em\u003e\u003csub\u003e\u003cem\u003eS\u003c/em\u003e\u003c/sub\u003e\u0026hellip; average value of the measured fluorescence of the sample\u003c/p\u003e\u003cp\u003e\u003cem\u003eΦ\u003c/em\u003e\u003csub\u003e\u003cem\u003eDMSO\u003c/em\u003e\u003c/sub\u003e\u0026hellip;average value of the measured fluorescence in the presence of DMSO\u003c/p\u003e\u003cp\u003e\u003cem\u003eΦ\u003c/em\u003e\u003csub\u003e\u003cem\u003ePM\u003c/em\u003e\u003c/sub\u003e\u0026hellip;average value of the measured fluorescence of the pure medium MHB\u003c/p\u003e\u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eBacterial infections remain a major global healthcare concerns and the development of novel antibacterial agents continues to be a critical area of research (Haque et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Lombrea et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Reports by Kazakova et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) have highlighted triterpenoids as promising candidates in the search for new antibacterial agents. Triterpenoids represent a highly effective strategy in treating this type of disease. Based on these findings, this research focuses on the investigation of triterpenoid compounds with potential therapeutic activity against bacterial infections. The triterpenoids selected for this study were platanic acid and betulonic acid. Another reason for their selection was the fact that both of these triterpenoic acids have not yet been fully explored in terms of their biological activity and potential therapeutic applications. On the other hand, they exhibit a range of well-known beneficial properties in the medicinal field. According to the study by Kahnt et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), promising results were observed for derivatives of platanic acid after the incorporation of functional groups containing nitrogen atoms (Kahnt et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Therefore, the research focused on testing derivatives containing an oxime group and evaluating whether the modification of triterpenoic acid leads to improved and enhanced antibacterial activity. The synthesis of oxime derivatives was performed according to the methodology published by (Bednarczyk-Cwynar and Zaprutko \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). This article also describes that these oxime derivatives can undergo further syntheses, such as acylation or alkylation. Additionally, these studies demonstrate that oxime esters in the lupane structure represent a promising pharmacological potential. Information about the synthesis of all tested derivatives is described in our previous research (Čern\u0026aacute; et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe antibacterial activity was evaluated using three methods: specifically, the disc diffusion method, the microdilution method, and the resazurin assay. Each compounds was tested in a total of four concentrations (500, 250, 125 and 62.5 \u0026micro;M). In the first step, a screening was performed on four model pathogenic bacteria \u003cem\u003eStaph. aureus\u003c/em\u003e, \u003cem\u003eEnteroc. faecalis\u003c/em\u003e (Gram-positive) and \u003cem\u003ePseud. aeruginosa\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e (Gram-negative). Preliminary results obtained from the disc diffusion method showed that derivatives \u003cb\u003e2, 3, 5, 11\u003c/b\u003e and \u003cb\u003e17\u003c/b\u003e produced inhibition zones of approximately 15 mm in diameter against \u003cem\u003eStaph. aureus\u003c/em\u003e. For \u003cem\u003eEnteroc. faecalis\u003c/em\u003e, inhibition zones of 15 mm were observed with derivatives \u003cb\u003e2\u003c/b\u003e and \u003cb\u003e9\u003c/b\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn the next step, the derivatives were screened using the microdilution method (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The results showed that most of our samples exhibited activity primarily against Gram-positive bacterial strains. Growth inhibition was observed against \u003cem\u003eStaph. aureus\u003c/em\u003e and \u003cem\u003eEnteroc. faecalis\u003c/em\u003e for most of the derivatives. These findings are consistent with similar results reported by (Chue K. T., Chang M. S., and N. 2011). In the article by (Haque et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), similar results were reported regarding the lack of activity against Gram-negative bacteria such as \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003ePseud. aeruginosa\u003c/em\u003e. In the study by Kazakova et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), which focused on a similar topic, it was found that among unmodified triterpenoic acids, betulonic acid exhibited the highest antibacterial activity against \u003cem\u003eStaph. aureus\u003c/em\u003e. Our results confirm this finding, as unmodified betulonic acid showed better activity compared to unmodified platanic acid. Betulonic acid demonstrated activity against \u003cem\u003eStaph. aureus\u003c/em\u003e in both diffusion and microdilution tests, with bacterial growth inhibited to a zero specific growth rate. However, in the resazurin assay, betulonic acid showed no significant inhibition. On the other hand, a high percentage of inhibition was observed for unmodified betulonic acid (93.9% at 500 \u0026micro;M) against \u003cem\u003eEnteroc. faecalis\u003c/em\u003e, along with a zero specific growth rate. The higher inhibition of betulonic acid against \u003cem\u003eEnteroc. faecalis\u003c/em\u003e compared to \u003cem\u003eStaph. aureus\u003c/em\u003e is consistent with the findings of (Haque et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). They measured the antibacterial activity of betulonic acid at a concentration of 50 \u0026micro;M and found 74.3% inhibition against \u003cem\u003eEnteroc. faecalis\u003c/em\u003e and 51.1% against \u003cem\u003eStaph. aureus.\u003c/em\u003e Kazakova et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) measured that the inhibition of \u003cem\u003eStaph. aureus\u003c/em\u003e growth caused by betulonic acid was detected at a concentration of 80 mg ∙ ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (corresponding to 175 mM). In our study, the compounds were tested at a lower concentration, with similar results. In the study by (Poumale et al. 2010), unmodified platanic acid inhibited the growth of \u003cem\u003eStaph. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e in the diffusion method at a concentration of 40 mg ∙ ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which corresponds to 87.2 mM. We obtained slightly different results, as unmodified platanic acid did not show significant inhibition against \u003cem\u003eStaph. aureus\u003c/em\u003e ATCC strains. However, we observed strong inhibition against a clinical isolate, \u003cem\u003eStaph. aureus\u003c/em\u003e 368. Our research revealed enhanced antibacterial activity following the modification of triterpenoic acids.\u003c/p\u003e\u003cp\u003eThe effects of the acetate intermediate of platanic acid (\u003cb\u003e1\u003c/b\u003e; 500 \u0026micro;M) and the oximester derivative of betulonic acid substituted by the 1-adamantyl acetic acid skeleton (\u003cb\u003e17\u003c/b\u003e; 500 \u0026micro;M) against \u003cem\u003eStaph. aureus\u003c/em\u003e was observed (\u003cb\u003eFig.\u0026nbsp;1\u003c/b\u003e, compound \u003cb\u003e1\u003c/b\u003e; 500\u0026thinsp;\u0026minus;\u0026thinsp;125 \u0026micro;M). The same derivative \u003cb\u003e17\u003c/b\u003e also showed significant effects against the growth of \u003cem\u003eEnteroc. faecalis\u003c/em\u003e. Similarly, also substances \u003cb\u003e7\u003c/b\u003e, \u003cb\u003e11\u003c/b\u003e and \u003cb\u003e12\u003c/b\u003e (500 \u0026micro;M). However, with the reference unmodified betulonic acid, this effect was confirmed especially against \u003cem\u003eStaph, aureus\u003c/em\u003e. At the same time, however, 100% inhibition of growth even in lower concentrations, i.e. 125 \u0026micro;M against \u003cem\u003eStaph. aureus\u003c/em\u003e occurred with unmodified betulonic acid, \u003cb\u003e7\u003c/b\u003e, \u003cb\u003e12\u003c/b\u003e and \u003cb\u003e1\u003c/b\u003e (figures in suplementary information). With derivative \u003cb\u003e17\u003c/b\u003e, 100% inhibition of \u003cem\u003eStaph. aureus\u003c/em\u003e bacteria occurred even at the lowest tested concentration of 62.5 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Derivatives 100% inhibiting the growth of \u003cem\u003eEnteroc. faecalis\u003c/em\u003e even at a lower concentration (125 \u0026micro;M) was compound \u003cb\u003e7\u003c/b\u003e and \u003cb\u003e17\u003c/b\u003e. These derivatives showed no growth, and their specific growth rate was calculated to be \u0026micro;\u0026thinsp;=\u0026thinsp;0.0000 [h⁻\u0026sup1;] for a concentration of 500 \u0026micro;M for derivatives \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e17\u003c/b\u003e against \u003cem\u003eStaph. aureus\u003c/em\u003e and \u003cb\u003e7\u003c/b\u003e, \u003cb\u003e11\u003c/b\u003e and \u003cb\u003e12\u003c/b\u003e against \u003cem\u003eEnteroc. faecalis\u003c/em\u003e. The zero value of the specific growth rate determined against both representatives of Gram-positive bacteria occurred \u003cb\u003e17\u003c/b\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe resazurin test proved inhibition of metabolic activity, i.e. bactericidal effects of betulonic acid and tested substances \u003cb\u003e1\u003c/b\u003e, \u003cb\u003e4\u003c/b\u003e and \u003cb\u003e7\u003c/b\u003e with inhibition ranging from 80\u0026ndash;100% against \u003cem\u003eEnteroc. faecalis\u003c/em\u003e and an intermediate \u003cb\u003e1\u003c/b\u003e inhibition of 90% against \u003cem\u003eStaph. aureus\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These results indicate that most substances had only bacteriostatic effects on the bacterial cell.\u003c/p\u003e\u003cp\u003eBased on the obtained results, a narrower group of samples (\u003cb\u003eI\u003c/b\u003e, \u003cb\u003eII\u003c/b\u003e, \u003cb\u003e1\u003c/b\u003e, \u003cb\u003e6\u003c/b\u003e, \u003cb\u003e7\u003c/b\u003e, \u003cb\u003e12\u003c/b\u003e, \u003cb\u003e17\u003c/b\u003e) was selected for further statistical repetition of testing using the same methods. These tests showed and confirmed the most significant effect against \u003cem\u003eStaph. aureus\u003c/em\u003e. Inhibition zones measured by the diffusion test up to 11 mm against this bacterium were observed with substances \u003cb\u003e1\u003c/b\u003e, \u003cb\u003e17\u003c/b\u003e at a concentration of 500 \u0026micro;M. The zero specific growth rate of \u003cem\u003eStaph. aureus\u003c/em\u003e was detected in samples \u003cb\u003e1\u003c/b\u003e, \u003cb\u003e7\u003c/b\u003e, \u003cb\u003e12\u003c/b\u003e and \u003cb\u003e17\u003c/b\u003e in concentration 250 \u0026micro;M again. Similarly, this was also found in betulonic acid. For derivatives \u003cb\u003e12\u003c/b\u003e and \u003cb\u003e17\u003c/b\u003e, a specific growth rate \u0026micro;\u0026thinsp;=\u0026thinsp;0.0000 [h⁻\u0026sup1;] was observed at the lowest tested concentration of 62.5 \u0026micro;M. Metabolic activity was inhibited from 80\u0026ndash;100% for derivatives \u003cb\u003e1\u003c/b\u003e, \u003cb\u003e7\u003c/b\u003e, \u003cb\u003e12\u003c/b\u003e and \u003cb\u003e17\u003c/b\u003e, as well as for betulonic acid. Here, derivative \u003cb\u003e17\u003c/b\u003e was the only one to inhibit metabolism by 100% even at a concentration of 62.5 \u0026micro;M.\u003c/p\u003e\u003cp\u003eDue to the significant sensitivity observed against \u003cem\u003eStaph. aureus\u003c/em\u003e, these selected derivatives were also tested against resistant clinical isolates obtained from the hospital environment. Specifically, the isolates were \u003cem\u003eStaph. aureus\u003c/em\u003e 670, \u003cem\u003eStaph. aureus\u003c/em\u003e 368, \u003cem\u003eStaph. hominis\u003c/em\u003e 334 and \u003cem\u003eStaph. epidermidis\u003c/em\u003e 335. Bacteriostatic properties at a concentration of 500 \u0026micro;M were found mainly against \u003cem\u003eStaph. aureus\u003c/em\u003e 670 and 368 and also against \u003cem\u003eStaph. epidermidis\u003c/em\u003e 335 in the unmodified betulonic acid and in derivatives \u003cb\u003e1\u003c/b\u003e, \u003cb\u003e7\u003c/b\u003e and \u003cb\u003e17\u003c/b\u003e. However, only samples \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e17\u003c/b\u003e achieved significant inhibition of metabolic activity ranging between 90\u0026ndash;100% (Table\u0026nbsp;\u003cspan refid=\"Tab8\" class=\"InternalRef\"\u003e7\u003c/span\u003e; Figs.\u0026nbsp;3 and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAmong the structural modifications of platanic acid, the acetate intermediate (\u003cb\u003e1\u003c/b\u003e) exhibited the most promising biological activity. In the case of betulonic acid, the highest efficacy was observed particularly in its oxime derivatives, including the simple oxime (\u003cb\u003e7\u003c/b\u003e) and two oxime ester derivatives, which were synthesized using pyrazine-2-carboxylic acid (\u003cb\u003e12\u003c/b\u003e) and 1-adamantylacetic acid (\u003cb\u003e17\u003c/b\u003e), respectively.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe results of bacteriostatic activity, obtained by the diffusion method, against the bacteria \u003cem\u003eStaph. aureus\u003c/em\u003e, \u003cem\u003eEnteroc. faecalis\u003c/em\u003e, \u003cem\u003eE. coli\u003c/em\u003e, and \u003cem\u003ePseud. aeruginosa\u003c/em\u003e. The data correspond to 24-hour exposure of the samples at concentrations of 10 mM after incubation at 37\u0026deg;C or 28\u0026deg;C.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eTested substance\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u003cp\u003eThe average inhibition zone \u003cem\u003ed\u003c/em\u003e [mm]\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eStaph. aureus\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eEnteroc. faecalis\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003ePseud. aeruginosa\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1% DMSO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVancomycin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+++\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e+++\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKanamycin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+++\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+++\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eI\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eII\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e++\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e++\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e++\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e++\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e++\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e9\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e++\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e10\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e11\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e++\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e12\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e13\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e14\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e15\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e16\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e17\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e++\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e+++30 mm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e++15 mm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e+\u0026thinsp;10 mm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e-0 mm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSpecific growth rates \u0026micro; [h⁻\u0026sup1;] of \u003cem\u003eStaph. aureus\u003c/em\u003e, \u003cem\u003eEnteroc. faecalis\u003c/em\u003e, \u003cem\u003eE. coli\u003c/em\u003e, and \u003cem\u003ePseud. aeruginosa\u003c/em\u003e after exposure to the samples at a concentration of 500 \u0026micro;M and incubation at 35\u0026deg;C or 28\u0026deg;C.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eTested substance\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u003cp\u003eSpecific growth rate \u0026micro; [h⁻\u0026sup1;]\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eStaph. aureus\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eEnteroc. faecalis\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003ePseud. aeruginosa\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1% DMSO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.0409\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0335\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0606\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0770\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVancomycin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKanamycin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eI\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e0.0080\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0167\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0753\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0367\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eII\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0559\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0182\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0403\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.1298\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0314\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.0112\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0107\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0,0412\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0230\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e0.0093\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0149\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0603\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0393\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e0.0088\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0143\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0394\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0319\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.0228\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0101\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0554\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0515\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e0.0097\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0150\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0543\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0521\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.0238\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0559\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0254\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.0198\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0175\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0705\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0364\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e9\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e0.0096\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.0075\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0278\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0214\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e10\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.0233\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0661\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0511\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0346\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e11\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.0229\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0294\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0363\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e12\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.0314\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0297\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0312\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e13\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.0717\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0945\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0378\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0400\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e14\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.0545\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0282\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0177\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0488\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e15\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.0134\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0128\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0223\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0589\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e16\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.0279\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0493\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0429\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0261\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e17\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0363\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0302\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eThe results are subject to a\u0026thinsp;\u0026plusmn;\u0026thinsp;15% error.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eTable 6. Inhibition of metabolic activity in % obtained from the cell viability test (resazurin assay) in \u003cem\u003eStaph.aureus\u003c/em\u003e, \u003cem\u003eEnteroc. faecalis\u003c/em\u003e, \u003cem\u003eE. coli\u003c/em\u003e, and \u003cem\u003ePseud. aeruginosa\u003c/em\u003e after exposure to the samples at a concentration of 500 \u0026micro;M following 23 hours of incubation at 35\u0026deg;C or 28\u0026deg;C.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 121px;\"\u003e\n \u003cp\u003eTested substance\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" style=\"width: 483px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGrowth inhibition [%]\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cem\u003eStaph. aureus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cem\u003eEnteroc. faecalis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cem\u003ePseud. aeruginosa\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e1 % DMSO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003eVancomycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003eKanamycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e5.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e2.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eII\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e93.9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e23.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e96.2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e83.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e30.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e24.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e69.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e23.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e4.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e86.6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e9.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e43.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e74.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e13.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e6.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e13.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e55.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e10.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e61.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e10\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e22.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e11\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e19.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e3.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e60.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e13\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e14\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e12.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e6.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e15\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e36.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e18.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e16.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e16\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e12.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e16.6\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e17\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e64.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e70.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e32.6\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" style=\"width: 604px;\"\u003e\n \u003cp\u003eThe results are subject to a \u0026plusmn; 15% error.\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026shy;(-Presence of metabolic activity.)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab8\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eInhibition of metabolic activity in % obtained from the cell viability test (resazurin assay) in \u003cem\u003eStaph. aureus\u003c/em\u003e 670, \u003cem\u003eStaph. aureus\u003c/em\u003e 368, \u003cem\u003eStaph. hominis\u003c/em\u003e 334 and \u003cem\u003eStaph. epidermidis\u003c/em\u003e 335 after exposure to the samples at a concentration of 500 \u0026micro;M following 23 hours of incubation at 35\u0026deg;C.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eTested substance\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u003cp\u003eGrowth inhibition [%]\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eStaph. aureus 670\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eStaph. aureus 368\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eStaph. hominis 334\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eStaph. epidermidis 335\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5% DMSO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVancomycin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e100.0\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e100.0\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e100.0\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e100.0\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eI\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e67.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e100.0\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eII\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e14.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e68.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e100.0\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e97.1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e100.0\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e12.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e100.0\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e73.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e89.8\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e72.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e94.8\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e12\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e53.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e17\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e56.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e94.4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e53.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e99.4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e\u003cp\u003eThe results are subject to a\u0026thinsp;\u0026plusmn;\u0026thinsp;15% error.\u003c/p\u003e\u003cp\u003e(-Presence of metabolic activity.)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWe have demonstrated that the structural modification of betulonic and platanic acids represents a promising strategy for the development of new antibacterial agents with potential applications in the treatment of infectious and cancer-related diseases. In our previous work, we successfully designed and synthesized 13 oxime derivatives and four intermediates of these acids. In the present study, several of these derivatives and intermediates exhibited significantly improved antibacterial properties, particularly against Gram-positive bacteria, including certain resistant clinical isolates. We also hypothesize that such structural modifications may partially contribute to enhanced pharmacological properties, including increased antiviral and anticancer potential. Although the tested derivatives were active against pathogenic Gram-positive bacteria, no activity was observed against Gram-negative strains. Nevertheless, our findings provide valuable insights and open new avenues for further research, particularly toward elucidating the mechanisms underlying these biological effects.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAbsorbance\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCCM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCzech Collection of Microorganisms\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCFU\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eColony-Forming Units\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDMSO\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDimethyl sulfoxide\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMDR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMultidrug-resistant\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMcF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMcFarland\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMHA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMueller-Hinton Agar\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMHB\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMueller-Hinton Broth\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eWHO\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eWorld Health Organization.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research was supported by\u0026nbsp;the Technology Agency of the Czech Republic grant No.\u0026nbsp;TN02000044\u0026nbsp;and Specific university research of the University of Chemistry and Technology Prague grant No. A2_FPBT_2023_026.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePetra Loveck\u0026aacute;, Uladzimir Bildziukevich and Zdeněk Wimmer designed and supervised the experiments; Lucie Čern\u0026aacute; and Angelina Massyagutova made antimicrobial testing; Lucie Čern\u0026aacute; and Petra Loveck\u0026aacute; prepared and checked the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations Ethics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with animals performed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBednarczyk-Cwynar, B., and L. Zaprutko. 2015. \u0026apos;Recent advances in synthesis and biological activity of triterpenic acylated oximes\u0026apos;, Phytochem Rev, 14: 203\u0026ndash;31. DOI: 10.1007/s11101-014-9353-5.\u003c/li\u003e\n\u003cli\u003eBreijyeh, Z., B. Jubeh, and R. 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DOI: 10.1016/j.cmi.2019.09.015.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 3 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"antimicrobial activity, oxime derivatives, betulonic acid, platanic acid","lastPublishedDoi":"10.21203/rs.3.rs-8076005/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8076005/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTriterpenoid acids are secondary metabolites known to possess various beneficial biological activities. Their synthetic derivatives often display improved pharmacological properties and higher levels of efficacy. In this study, we investigated, the antibacterial effects of 13 synthesized oxime derivatives and 4 intermediates of betulonic and platanic acid, comparing them with those of the unmodified forms. Six Gram-positive pathogenic human bacteria, including four resistant strains from the genus \u003cem\u003eStaphylococcus\u003c/em\u003e, were selected for testing together with two Gram-negative bacteria. All combinations of the prepared bacterial suspensions and 17 samples (500 − 62.5 µM) were placed on 96-well microtitration plates. The microdilution method was used to measure the absorbance of the samples, from which bacterial growth curves were derived. Resazurin tests were performed to obtain fluorescence values and, thereby, determine the metabolic activity and bactericidal effects. The derivatives and intermediates exhibited higher sensitivity to the Gram-positive bacteria. The most effective samples, for which 90–100% growth inhibition was observed against resistant strains (\u003cem\u003eStaph. aureus\u003c/em\u003e 368, \u003cem\u003eStaph. epidermidis\u003c/em\u003e 335), were the oximester derivative of betulonic acid synthesized from 1-adamantylacetic acid (\u003cstrong\u003e17\u003c/strong\u003e; 500 µM) and the C-3 acetate intermediate of platanic acid (\u003cstrong\u003e1\u003c/strong\u003e; 500 µM). Sample \u003cstrong\u003e1\u003c/strong\u003e also showed 90–100% inhibition against \u003cem\u003eStaph. aureus\u003c/em\u003e 670. Thus, our results suggest that the tested triterpenoid derivatives and intermediates have considerable potential for the development of new antibiotic therapies.\u003c/p\u003e","manuscriptTitle":"Derivatives and intermediates of betulonic and platanic acid as effective antibacterial agents","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-24 06:21:24","doi":"10.21203/rs.3.rs-8076005/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c94932e9-6b30-47c0-8a89-3be06da25981","owner":[],"postedDate":"November 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-20T16:07:12+00:00","versionOfRecord":{"articleIdentity":"rs-8076005","link":"https://doi.org/10.1007/s12223-026-01455-9","journal":{"identity":"folia-microbiologica","isVorOnly":false,"title":"Folia Microbiologica"},"publishedOn":"2026-04-16 15:58:00","publishedOnDateReadable":"April 16th, 2026"},"versionCreatedAt":"2025-11-24 06:21:24","video":"","vorDoi":"10.1007/s12223-026-01455-9","vorDoiUrl":"https://doi.org/10.1007/s12223-026-01455-9","workflowStages":[]},"version":"v1","identity":"rs-8076005","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8076005","identity":"rs-8076005","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}