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Antibiotics 2022, 11, 1743. https://doi.org/10.3390/antibiotics11121743 www.mdpi.com/journal/antibiotics
Article
Increasing the Efficacy of Treatment of Staphylococcus
aureus–Candida albicans Mixed Infections with Myrtenol
Ruba Y. Mahmoud 1, Elena Y. Trizna 1, Rand K. Sulaiman 1, Roman S. Pavelyev 1, Ilmir R. Gilfanov 1,2,
Svetlana A. Lisovskaya 3,4, Olga V. Ostolopovskaya 1,3, Larisa L. Frolova 5, Alexander V. Kutchin 5,
Galina B. Guseva 6, Elena V. Antina 6, Mikhail B. Berezin 6, Liliya E. Nikitina 1,3,†,* and Airat R. Kayumov 1,†,*
1 Institute of Fundamental Medicine and Biology, Kazan Federal University, 420008 Kazan, Russia
2 Varnishes and Paints Department, Kazan National Research Technological University, 420015 Kazan, Russia
3 Faculty of Medicine and Biology, Kazan State Medical University, 420012 Kazan, Russia
4 Scientific Research Institute of Epidemiology and Microbiology, 420015 Kazan, Russia
5 Institute of Chemistry, Federal Research Center “Komi Scientific Centre”, Ural Branch, Russian Academy
of Sciences, 167000 Syktyvkar, Russia
6 G.A. Krestov Institute of Solution Chemistry of Russian Academy of Sciences, 153045 Ivanovo, Russia
* Correspondence: nikitl@mail.ru (L.E.N.); kairatr@yandex.ru (A.R.K.); Tel.: +7 903 307-50-70 (L.E.N.);
Tel.: +7 904 665-19-08 (A.R.K.)
† These authors contributed equally to this work.
Abstract: Infectious diseases caused by various nosocomial microorganisms affect worldwide both
immunocompromised and relatively healthy persons. Bacteria and fungi have different tools to
evade antimicrobials, such as hydrolysis damaging the drug, efflux systems, and the formation of
biofilm that significantly complicates the treatment of the infection. Here, we show that myrtenol
potentiates the antimicrobial and biofilm-preventing activity of conventional drugs against S. aureus
and C. albicans mono- and dual-species cultures. In our study, the two optical isomers, (-)-myrtenol
and (+)-myrtenol, have been tested as either antibacterials, antifungals, or enhancers of conventional
drugs. (+)-Myrtenol demonstrated a synergistic effect with amikacin, fluconazole, and ben-
zalkonium chloride on 64–81% of the clinical isolates of S. aureus and C. albicans, including MRSA
and fluconazole-resistant fungi, while (-)-myrtenol increased the properties of amikacin and flucon-
azole to repress biofilm formation in half of the S. aureus and C. albicans isolates. Furthermore, myr-
tenol was able to potentiate benzalkonium chloride up to sixteen-fold against planktonic cells in an
S. aureus–C. albicans mixed culture and repressed the adhesion of S. aureus. The mechanism of both
(-)-myrtenol and (+)-myrtenol synergy with conventional drugs was apparently driven by mem-
brane damage since the treatment with both terpenes led to a significant drop in membrane poten-
tial similar to the action of benzalkonium chloride. Thus, due to the low toxicity of myrtenol, it
seems to be a promising agent to increase the efficiency of the treatment of infections caused by
bacteria and be fungi of the genus Candida as well as mixed fungal–bacterial infections, including
resistant strains.
Keywords: mixed infections; myrtenol; benzalkonium chloride; drug synergism; Staphylcoccus au-
reus; Candida albicans
1. Introduction
Infectious diseases caused by various nosocomial bacteria and fungi like Enterobac-
teriaceae (Klebsiella sp and Escherichia coli), Staphylococcus aureus, Candida albicans, Crypto-
coccus neoformans, and many others affect worldwide both immunocompromised and rel-
atively healthy persons [1]. In addition to the most vulnerable populations of patients,
such as neonatal, old, and AIDS-infected patients and persons with an intravenous cath-
eter, in the last three years, SARS-CoV2 led to an increased risk of mortality and a longer
Citation: Mahmoud, R.Y.; Trizna,
E.Y.; Sulaiman, R.K.; Pavelyev, R.S.;
Gilfanov, I.R.; Lisovskaya, S.A.;
Ostolopovskaya, O.V.; Frolova, L.L.;
Kutchin, A.V.; Guseva, G.B.; et al.
Increasing the Efficacy of Treatment
of Staphylococcus aureus–Candida
albicans Mixed Infections with
Myrtenol. Antibiotics 2022, 11, 1743.
https://doi.org/10.3390/
antibiotics11121743
Academic Editors: Jorge H. Leitão,
Nitin Amdare and Joana R. Feliciano
Received: 17 November 2022
Accepted: 28 November 2022
Published: 2 December 2022
Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional
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Copyright: © 2022 by the authors. Li-
censee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and con-
ditions of the Creative Commons At-
tribution (CC BY) license (https://cre-
ativecommons.org/licenses/by/4.0/).
Antibiotics 2022, 11, 1743 2 of 17
course of ICU stays [2–4]. Antimicrobial therapy remains the only way to target patho-
genic microorganisms and save lives. Although conventional antimicrobial agents use
various strategies to repress the growth of pathogens, bacteria and fungi have different
tools to evade them, making the development and spread of antimicrobial resistance
(AMR) one of the factors that complicates the treatment of infectious diseases [5]. It has
been shown in the last decades that, in many cases, several pathogens rather than only
one are associated with disease [6]. These polymicrobial infections are often characterized
by more intense symptoms than any of the effects noticed by one microbe alone and in-
creased resistance to treatment [7,8]. S. aureus and C. albicans, an important, dangerous
twosome, have been shown to form a bacterial–fungal environment and were coisolated
from different infections, including periodontitis, cystic fibrosis, denture stomatitis, uri-
nary tract infections, burn wound infections, and infections of medical devices, such as
central venous catheters [9]. In bacterial–fungal coinfection, each counterpart has been re-
ported to contribute to resistance [10–12]. Moreover, 94% of S. aureus isolates are tolerant
to penicillin and its derivatives [13], and even cephalosporins and carbapenems often be-
come ineffective against this bacterium, leading to increased mortality of S. aureus-associ-
ated infections [14]. Some of the resistance mechanisms of S. aureus are limiting the drug
uptake, modifying the drug target, inactivating the drug, and active drug efflux [15]. C.
albicans also busts resistance to antifungals along the course of treatment [16] via trans-
forming between several morphological forms (blastospores, pseudohyphae, and hyphae)
[17], decreasing the permeability of drugs, and expressing efflux pumps or compromised
drug import [18].
In addition, biofilm formation plays an important role in S. aureus and C. albicans
protection. Biofilms are microbial communities (either mono- or polymicrobial) where the
cells are embedded into a matrix consisting of polysaccharides, proteins, and nucleotides
produced by the cells themselves [9,19,20]. The biofilm is formed in several stages, includ-
ing attachment to biotic or abiotic surfaces, maturation, and detachment (dispersal of ma-
ture biofilm) [21]. While in biofilm, microorganisms are characterized by a decreased sus-
ceptibility to antimicrobials due to the diffusional barrier for the latter as well as being
more virulent and capable to adhere to surfaces and form new biofilms [22]. Therefore,
the development of new approaches to increase the susceptibility of pathogenic microor-
ganisms to conventional antimicrobials could be promising in overcoming the AMR prob-
lem.
Various classes of compounds were reported to be able to potentiate the efficiency of
antimicrobials against planktonic- and biofilm-embedded bacteria and fungi: derivatives
of 5(H)furanone [23,24], various hydrolytic enzymes [25,26], and essential oils [27,28]. Ter-
penes, the active fraction of essential oils from plant extracts, make up the largest group
of secondary metabolites of plants (over 50,000 known substances) [29]. Monoterpenes
consist of two isoprene units and naturally occur in plants and essential oils [30] and are
introduced as key ingredients in the design and production of novel biologically active
compounds because of anti-inflammatory, antimicrobial, anticonvulsant, analgesic, anti-
viral, anticancer, antituberculosis, and antioxidant biological activities [31–33]. Addition-
ally, some researchers have described the ability of terpenes to inhibit the formation of S.
aureus biofilms as well as their antimicrobial and antifungal activity [34,35]. Myrtenol is a
monoterpene bicyclic derivative that has been well known for its antimicrobial activity
[36]. Myrtenol exhibited antibacterial activity against S. aureus and Acinetobacter baumannii
[37,38] and has repressed the growth of C. albicans, R. nigricans, A. fumigates, and F. solani
fungi species [36]. Several chemically synthesized myrtenol derivatives demonstrated sig-
nificant in vitro antifungal activity against Physalospora piricola with better or comparable
antifungal activity than those of positive controls (the commercial fungicides azoxystrobin
and chlorothalonil) [32]. In addition, the combination of myrtenol and antifungal agents
reduced the effective concentrations of the latter with synergistic and additive effects
[39,40]. The mechanism of myrtenol action is discussible. It has been suggested that myr-
tenol possibly damages the fungal membrane, affecting the change in the functional state
Antibiotics 2022, 11, 1743 3 of 17
of integrin-like proteins, which can lead to the disruption of morphogenesis of the fungal
cell [36].
Here, we show that myrtenol potentiates the antimicrobial and biofilm-preventing
activity of conventional drugs against S. aureus and C. albicans mono- and dual-species
cultures.
2. Results
2.1. Antibacterial and Antifungal Activity of Myrtenol
The antimicrobial activity of myrtenol was evaluated on S. aureus ATCC 29213 as
well as four methicillin-sensitive clinical isolates of S. aureus (MSSA), seven methicillin-
resistant isolates of S. aureus (MRSA), and 10 clinical isolates of C. albicans. (−)-myrtenol
and (+)-myrtenol exhibited low both antibacterial and antifungal activities (Tables 1 and
2). Worth noting, the minimum bactericidal concentration (MBC) either fit or exceeded
the minimum inhibiting concentration (MIC) two-fold, suggesting the bactericidal/fungi-
cidal property of terpenes. Furthermore, MRSA and MSSA were of similar susceptibility
to myrtenol, and the resistance to fluconazole did not affect the susceptibility of C. albicans
isolates to terpene.
Table 1. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
values (expressed in µg/mL) of (−)-myrtenol, (+)-myrtenol, amikacin, and benzalkonium chloride
(BAC) against S. aureus isolates.
Strains (−)-Myrtenol (+)-Myrtenol Amikacin BAC
MIC MBC MIC MBC MIC MBC MIC MBC
S. aureus
ATCC 29213 (MSSA) 1024 1024 512 512 4 8 0.5 1
S. aureus
18 (MSSA) 1024 1024 1024 1024 16 32 0.25 1
S. aureus
25 (MSSA) 1024 1024 1024 1024 8 16 0.25 0.5
S. aureus
26 (MSSA) 1024 1024 512 512 16 16 0.5 1
S. aureus
27 (MSSA) 1024 1024 512 512 4 16 0.5 1
S. aureus
1053 (MRSA) 1024 1024 2048 2048 128 512 0.5 2
S. aureus
1065 (MRSA) 1024 1024 512 512 128 256 0.5 2
S. aureus
1130 (MRSA) 512 1024 512 512 256 512 0.5 2
S. aureus
1145 (MRSA) 1024 512 512 512 64 1024 1 1
S. aureus
1167 (MRSA) 2048 2048 512 512 256 512 0.5 2
S. aureus
1168 (MRSA) 2048 2048 512 512 8 8 0.5 1
S. aureus
1173 (MRSA) 512 512 256 256 256 256 0.5 1
Table 2. Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC)
(expressed in µg/mL) of (−)-myrtenol, (+)-myrtenol, fluconazole and benzalkonium chloride (BAC)
against C. albicans isolates.
Strains (−)-Myrtenol (+)-Myrtenol Fluconazole BAC
MIC MFC MIC MFC MIC MFC MIC MFC
C. albicans
722 2048 2048 1024 1024 8 8 0.5 2
C. albicans
761 1024 1024 2048 2048 8 8 0.5 1
C. albicans
661FR 1024 1024 2048 2048 512 512 0.5 1
C. albicans
672 FR 1024 1024 2048 2048 512 512 0.5 0.5
C. albicans
688 FR 1024 1024 2048 2048 512 512 0.5 1
C. albicans
701 FR 2048 2048 2048 2048 512 512 1 1
C. albicans
703 FR 1024 1024 1024 1024 512 512 0.5 2
C. albicans
748 FR 1024 1024 2048 2048 512 512 1 2
C. albicans
762 FR 1024 1024 2048 2048 512 512 1 2
C. albicans
763 FR 2048 2048 2048 2048 512 512 0.5 1
2.2. Myrtenol Potentiates Both Antibacterial and Antifungal Agents
Antibiotics 2022, 11, 1743 4 of 17
The synergism of myrtenol with antimicrobials was assessed using the chequerboard
approach. For S. aureus, the concentrations of amikacin or benzalkonium chloride were in
the range of 0.06–4 × MIC, and myrtenol was added to concentrations of 0.125–1 × MIC.
After 24 h of incubation, the fractional inhibitory concentration index (FICI) was calcu-
lated for both the growth and biofilm repression assessed by crystal violet staining (Sup-
plementary Tables S1-S4). (−)-Myrtenol exhibited a synergistic effect with amikacin with
an FIC index in the range of 0.3–0.5 on 42% of clinical isolates of S. aureus; (+)-myrtenol
led to a four-fold decrease of the MIC of antibiotics against 75% of the studied isolates
regardless of their susceptibility to methicillin (MRSA or MSSA). On the other isolates, the
combined use of amikacin and either (−)- myrtenol or (+)-myrtenol led to an additive effect
(Table 3). On the biofilm-preventing activity, the synergistic effect of (−)-myrtenol and (+)-
myrtenol with amikacin was observed for 42% and 33% of isolates, respectively (Table 3).
In a combination of myrtenol with benzalkonium chloride, in most cases, the additive
effect was observed against the planktonic cells of S. aureus isolates. (−)-Myrtenol potenti-
ated the antiseptic only against one strain, and (+)-myrtenol demonstrated synergy with
benzalkonium chloride against four isolates (33%). Regarding biofilm prevention, (−)-
myrtenol significantly increased the efficiency of benzalkonium chloride against 50% of
isolates with an FICI ranging from 0.16–0.38 while (+)-myrtenol significantly increased the
effectiveness of the antiseptic in only 17% of isolates.
Table 3. FICI values of amikacin and benzalkonium chloride in combination with either (−)-myr-
tenol or (+)-myrtenol against various isolates of S. aureus.
Strains
Amikacin Benzalkonium Chloride
Growth Repression Biofilm Prevention Growth Repression Biofilm Prevention
(−)-Myr-
tenol
(+)-Myr-
tenol
(−)-Myr-
tenol
(+)-Myr-
tenol
(−)-Myr-
tenol
(+)-Myr-
tenol
(−)-Myr-
tenol
(+)-Myr-
tenol
S. aureus
ATCC (MSSA) 0.30 0.50 0.38 1.00 1.25 0.75 0.75 1.50
S. aureus
18 (MSSA) 0.50 0.31 1.12 0.75 0.75 0.5 0.63 0.75
S. aureus
25 (MSSA) 0.75 0.31 1.00 0.50 2.25 0.5 1.50 0.75
S. aureus
26 (MSSA) 0.75 0.75 0.75 1.25 0.75 0.75 0.75 0.28
S. aureus
27 (MSSA) 0.38 0.50 0.75 1.00 0.75 0.75 0.50 1.50
S. aureus
1053 (MRSA) 0.75 0.38 0.38 0.38 0.75 0.5 0.63 0.38
S. aureus
1065 (MRSA) 0.75 0.50 1.12 1.25 0.75 1.25 0.19 2.25
S. aureus
1130 (MRSA) 0.75 0.38 1.00 1.00 0.75 0.75 1.00 1.00
S. aureus
1145 (MRSA) 0.75 0.50 0.25 0.63 1.25 0.5 0.16 0.63
S. aureus
1167 (MRSA) 0.31 0.75 0.31 0.31 0.5 1.25 0.38 0.75
S. aureus
1168 (MRSA) 0.38 0.31 0.28 0.31 0.75 1.25 0.38 1.25
S. aureus
1173 (MRSA) 0.75 0.75 0.625 1.50 1.25 1.25 0.16 0.53
Fraction of strains with shown
synergy 42% 75% 42% 33% 8% 33% 50% 17%
Next, the synergistic effect of myrtenol with fluconazole and benzalkonium chloride
against C. albicans was evaluated. The fractional inhibitory concentration index (FICI) was
calculated for planktonic cell growth repression and biofilm formation prevention (Sup-
plementary Tables S5-S8). (+)-Myrtenol exhibited synergy with fluconazole in 64% of the
C. albicans isolates while (−)-myrtenol mainly led to an additive effect, and synergy was
only observed in 36% of isolates (Table 4). By contrast, when assessing the biofilm repres-
sion, (−)-myrtenol had an FICI less or equal to 0.5 for six out of 11 isolates while (+)-myr-
tenol was only for four out of 11 (Table 4). (-)-Myrtenol also demonstrated synergism with
benzalkonium chloride in relation to planktonic cells for five isolates, and the combined
use of (+)-myrtenol with antiseptic showed a clear synergistic effect in relation to nine
isolates. A similar result was obtained for C. albicans biofilm repression. Most of the iso-
lates (seven out of 11) were more sensitive to the combination of antiseptic with (+)-
Antibiotics 2022, 11, 1743 5 of 17
myrtenol while the use of (−)-myrtenol with benzalkonium chloride showed an FICI less
or equal to 0.5 for only four isolates (Table 4).
Table 4. FICI values of fluconazole and benzalkonium chloride in combination with either (−)-myr-
tenol or (+)-myrtenol against various isolates of C. albicans.
Strains
Fluconazole Benzalkonium Chloride
Growth Repression Biofilm Prevention Growth Repression Biofilm Prevention
(−)-Myr-
tenol
(+)-Myr-
tenol
(−)-Myr-
tenol
(+)-Myr-
tenol
(−)-Myr-
tenol
(+)-Myr-
tenol
(−)-Myr-
tenol
(+)-Myr-
tenol
C. albicans
722 1.25 1.25 0.37 0.40 0.50 0.50 1.25 0.31
C. albicans
761 1.25 1.25 0.37 0.50 0.75 0.50 0.38 0.50
C. albicans
661 FR 1.25 0.75 1.25 1.25 0.75 0.38 1.25 0.50
C. albicans
672 FR 0.27 0.50 0.28 0.50 0.75 0.50 0.38 0.75
C. albicans
688 FR 1.25 0.75 0.26 1.25 0.50 0.50 0.75 0.75
C. albicans
701 FR 0.28 0.27 0.75 0.75 0.75 0.75 0.50 0.38
C. albicans
703 FR 0.38 0.27 0.31 4.25 0.75 0.50 0.75 0.75
C. albicans
748 FR 1.25 0.27 0.26 1.25 0.75 0.50 0.75 0.50
C. albicans
762 FR 1.25 0.27 1.25 1.25 0.38 0.50 0.50 0.38
C. albicans
763 FR 0.27 0.50 0.75 0.30 0.50 1.25 0.75 0.50
Fraction of strains with shown
synergy 36% 64% 54% 36% 45% 81% 36% 72%
Thus, these data indicate that myrtenol reduces the effective concentrations of anti-
microbial and antifungal drugs, which, in turn, reduces both the general toxic effect on
the host organism and the risk of resistance development by bacterial and fungal cells.
2.3. Myrtenol Increases the Antimicrobial and Antifungal Activity of Benzalkonium Chloride
against an S. aureus and C. albicans Mixed Culture
Since the benzalkonium chloride demonstrated synergy with myrtenol against both
S. aureus and C. albicans, the effect of their combined use against the fungal–bacterial
mixed culture community was assessed. For this purpose, S. aureus and C. albicans were
cocultivated in a BM broth in a 24-well plate in the presence of benzalkonium chloride in
the concentration range of 0, 0.25, 0.5, 1, 4, or 8 µg/mL solely or in combination with (−)-
myrtenol and (+)-myrtenol at a concentration of 256 µg/mL. After a 24 h incubation, the
viability of planktonic bacterial and fungal cells was assessed by counting CFUs in a series
of ten-fold dilutions followed by plating on selective media for the differentiation of S.
aureus and C. albicans. The sole benzalkonium chloride led to a three-log decrease of viable
S. aureus and the death of C. albicans planktonic cells only at 8 µg/mL (Figure 1). In the
presence of either myrtenol, (−) or (+), a significant increase in the efficiency of the anti-
septic was observed, and the complete death of both C. albicans and S. aureus planktonic
cells was observed at a concentration of 0.5–1 µg/mL, suggesting an eight- to sixteen-fold
increase in the antiseptic’s efficiency by terpene. Worth noting, while the combination of
both (−)-myrtenol and (+)-myrtenol with benzalkonium chloride led to the prevention of
the adherence of S. aureus, although at 4 µg/mL of antiseptic, no significant effect on C.
albicans adherence was observed.
Antibiotics 2022, 11, 1743 6 of 17
Figure 1. Viability of S. aureus and C. albicans in mixed culture in presence of benzalkonium chloride
with concentrations 0, 0.25, 0.5, 1, 4, and 8 µg/mL separately and in combination with (−)-myrtenol
and (+)-myrtenol at a concentration of 256 µg/mL. The viability of bacterial and fungal cells was
assessed after 24 h growth in culture liquid and after 48 for adherent cells. The viable cells were
counted after a series of ten-fold dilutions followed by plating on selective media for differentiation
of S. aureus and C. albicans.
2.4. Myrtenol Damages the Cell Membrane of Bacterial and Fungal Cells
Since damage to the cell membrane has been proposed for various terpenes as the
mechanism of antimicrobial action [41], the effect of myrtenol on the membrane potential
of bacterial and fungal cells was investigated. Cells were preincubated with the fluores-
cent dye DioC2(3) which can be reduced on the membrane of intact cells; then the myr-
tenol was added until 0.5–2×MIC, and the fluorescence was recorded during 30 min of
incubation. As can be seen from Figure 2, in the presence of (−)-myrtenol and (+)-myrtenol,
the fluorescence intensity of S. aureus cells decreased compared to untreated cells in a
dose-dependent manner, confirming a drop in membrane potential, apparently, because
of its damage. A similar drop in fluorescence was observed in cells treated with ben-
zalkonium chloride, which also permeates the cell membrane, while no changes were de-
tected in ampicillin-treated cells. These data clearly suggest that both (−)-myrtenol and
(+)-myrtenol apparently damage the bacterial membrane, thus facilitating the penetration
of antimicrobials into the cell. Treatment of C. albicans cells with low concentrations of (−)-
myrtenol did not affect the fluorescence, similar to fluconazole, although the latter also
affects the integrity of the membrane via repression of the conversion of lanosterol to er-
gosterol (Figure 3). By contrast, (−)-myrtenol led to a significant decrease in fluorescence
comparable with the effect of benzalkonium chloride.
Antibiotics 2022, 11, 1743 7 of 17
Figure 2. Relative membrane potential of S. aureus cells expressed in fluorescence units of DioC2(3)
reduced on an intact cell membrane resulting in green fluorescence. Cells were grown for 18 h in LB
broth, washed with PBS, and resuspended until a final density of 106 CFU/mL in PBS supplemented
with DioC2(3) to a final concentration of 10 µM/mL. After a 30 min incubation at 25°C, compounds
of interest were added to the samples. Fluorescence detection was performed for 30 min with 5 min
intervals with excitation and emission wavelengths of 497 and 520 nm, respectively.
Figure 3. Relative membrane potential of C. albicans cells expressed in fluorescence units of DioC2(3)
reduced on an intact cell membrane resulting in green fluorescence. Cells were grown for 18 h in
Sabouraud broth, washed with PBS, and resuspended until a final density of 105 CFU/mL in PBS
supplemented with DioC2(3) to a final concentration of 10 µM/mL. After a 30 min incubation at
25°C, compounds of interest were added to the samples. Fluorescence detection was performed for
Antibiotics 2022, 11, 1743 8 of 17
30 min with 5 min intervals with excitation and emission wavelengths of 497 and 520 nm, respec-
tively.
To evaluate whether myrtenol binds to the membrane or diffuses into the cell, con-
focal laser scanning microscopy was performed to check the localization of terpenes in
bacterial cells. For this, S. aureus and C. albicans cells were incubated for 15 min in the
presence of myrtenol fused with a fluorophore (myrtenol-lum). Synthesis, physicochemi-
cal properties, and spectral data of BF2-ms-(4-((1″R)-6″,6″-dimethylbicyclo[3.1.1]hept-2″-
ene-2″)ylmethoxycarbonylpropyl)-3,3′,5,5′-tetramethyl-2,2′-dipyrromethene (mentioned
as “lum”) were described in detail in our previous paper [42]. To visualize the membranes
of bacteria and fungi, cells were additionally stained with CalcoFluor-White (CFW). Fig-
ures 4 and 5 show that myrtenol was evenly distributed in S. aureus cells (green fluoresce)
while the fluorophore was observed only over the cell surface, suggesting that myrtenol
diffuses through the membrane. A similar result was shown for C. albicans cells (Figures
4 and 5).
(a) (b)
Figure 4. Myrtenol-lum penetration into S. aureus cells. (a) The localization of either (−)-myrtenol or
(+)-myrtenol carrying the fluorophore BODIPY (Myrtenol-lum) assessed by confocal laser scanning
microscopy. The solely fluorophore (lum) and Calcofluor-White (CFW) membrane dyes served as
references. (b) Penetration of myrtenol-lum into S. aureus cells. Myrtenol-lum was added to S. aureus
cells, and the sole fluorophore (lum) was used as a control. After 4, 8, 16, 32, and 64 min of incuba-
tion, cells were harvested, washed with PBS, and residual fluorescence was measured. The half-time
of penetration (t½) was 24±1.3 min and 26±1.5 min for (−)-myrtenol and (+)-myrtenol, respectively,
while for the fluorophore solely (lum) was t½> 5000 min.
Antibiotics 2022, 11, 1743 9 of 17
(a) (b)
Figure 5. Myrtenol-lum penetration into C. albicans cells. (a) The localization of either (−)-myrtenol
or (+)-myrtenol carrying the fluorophore BODIPY (myrtenol-lum) assessed by confocal laser scan-
ning microscopy. The solely fluorophore (lum) and Calcofluor-White (CFW) membrane dyes served
as references. (b) Penetration of myrtenol-lum into C. albicans cells. Myrtenol-lum was added to C.
albicans cells, and the sole fluorophore (lum) was used as a control. After 4, 8, 16, 32, and 64 min of
incubation, cells were harvested, washed with PBS, and residual fluorescence was measured. The
half-time of penetration (t½) was 24±1.3 min and 18±1.2 min for (−)-myrtenol and (+)-myrtenol, re-
spectively, while for the fluorophore solely (lum) was t½> 5000 min.
In the next step, the rate of penetration of myrtenol into bacterial and fungal cells was
assessed. Myrtenol containing a fluorophore in its structure (Myrtenol-lum) was added to
S. aureus and C. albicans cells. The pure fluorophore (lum) itself was used as a control.
After 4, 8, 16, 32, and 64 min of incubation, cells were harvested, washed with PBS, and
the fluorescence in suspension was measured using a Tecan Infinite 200 Pro microplate
reader (Switzerland). Cells without any added compounds were considered point zero,
and the cell suspension with the fluorescent compound was considered 100%. As can be
seen from Figures 4 and 5, the half-time of maximal penetration (t½) of (+)-myrtenol-lum
was 26±1.5 min and 18±1.2 min for S. aureus and C. albicans, respectively. For (−)-myrtenol-
lum, the calculated t½ was 24 ± 1.3 min while the t½ of the sole fluorophore was t½> 5000
min in both bacterial and fungal cells, suggesting the interaction of myrtenol with the
membrane.
3. Discussion
The worldwide spread of pathogenic bacteria and micromycetes resistant or tolerant
to conventional antimicrobials drastically decreases the number of available options for
the treatment of infectious diseases and thus becomes a global challenge for healthcare
[43–46]. Furthermore, the coexistence of different microorganisms in mixed communities
leads to additional difficulties in treatment compared to monospecific infections [8,11,47].
Due to interbacterial and bacterial–fungal interactions in consortia, their counterparts
Antibiotics 2022, 11, 1743 10 of 17
change metabolism and morphology that consequently leads to altered susceptibility to
antimicrobials [7,48,49]. Therefore, the development of either novel universal antimicro-
bials or approaches to potentiate conventional ones could be tools to overcome the toler-
ance of microorganisms to antimicrobials.
Essential oils have been shown as both potential antimicrobials and enhancers of con-
ventional antimicrobials [27,28]. In particular, the bicyclic monoterpene myrtenol, a ter-
pene from the myrtenol tree, is able to repress the growth of bacteria [37,38] and fungi [36]
as well as reduce the effective concentrations of some antifungals [39,40]. In our study, the
two optical isomers of myrtenol, (−)-myrtenol and (+)-myrtenol, were tested as either an-
tibacterial, antifungal, or enhancers of conventional drugs. As can be seen from Tables 3
and 4, (+)-myrtenol demonstrated the synergistic effect with amikacin, fluconazole, and
benzalkonium chloride on most of the clinical isolates of S. aureus and C. albicans while
(−)-myrtenol exhibited synergy with conventional drugs only on a third of the isolates.
Thus, in the presence of myrtenol, the MICs of amikacin, fluconazole, and benzalkonium
chloride were reduced up to sixteen-fold (see Supplementary file), reaching medically rel-
evant concentrations. On the contrary, (−)-myrtenol more readily increased the property
of amikacin and fluconazole to repress biofilm formation by the S. aureus and C. albicans
isolates, respectively. The reason for such selectivity remains questionable since the half-
time penetration of both (−)-myrtenol and (+)-myrtenol into either S. aureus or C. albicans
was similar at 18–24 min (Figures 4 and 5). Additionally, the confocal microscopy of
treated cells revealed similar intracellular localization of (−)-myrtenol and (+)-myrtenol
fused to the fluorophore. However, in the membrane integrity assay, (+)-myrtenol led to
a faster drop in the membrane potential of treated C. albicans cells (Figure 3), which allows
for speculation about either the specificity of (+)-myrtenol to any molecular target or a
higher tropism to the membrane at least in fungal cells. The last assumption may be less
probable since no difference in the effect of either (−)-myrtenol or (+)-myrtenol on the S.
aureus membrane could be observed (Figure 2). Nevertheless, the mechanism of both (−)-
myrtenol and (+)-myrtenol synergy with conventional drugs is apparently driven by
membrane damage since the treatment with both terpenes led to a drop in membrane
potential similar to the action of benzalkonium chloride (Figures 2 and 3), the membrane-
permeating agent [50–52].
As has been reported in many works, S. aureus and C. albicans are opportunistic path-
ogens that live in the same niche and are capable of forming mixed-species consortia.
These consortia appear widely on various mucosa, including the mouth, vaginal tract, etc.
[10]. In this form, their resistance to antimicrobial and antifungal drugs increases signifi-
cantly [53,54]. Hence, we tested whether either (−)-myrtenol or (+)-myrtenol could poten-
tiate the antiseptic benzalkonium chloride against a mixed culture of S. aureus and C. albi-
cans. As can be seen from Figure 1, in this case, both isomers of myrtenol were able to
potentiate benzalkonium chloride up to sixteen-fold against planktonic cells, which al-
lows for reduction of the concentration of this toxic antiseptic for the treatment of various
mucosa with the same efficiency. On the other hand, the increase in antiseptic efficiency
decreases the risk of resistance development by pathogens [55]. Unfortunately, while the
combination of myrtenol with antiseptic could completely repress the adhesion of S. au-
reus, no effect of terpene on C. albicans adhesion repression by benzalkonium chloride
could be observed. This effect is probably due to the highly adaptive capabilities of the
fungal cells that make it possible to neutralize the negative effect of antimycotics at their
low concentrations.
Taken together, our data allow for the suggestion of myrtenol as a tool to increase the
susceptibility of pathogens to antimicrobials. While the terpene will apparently not be
effective against resistant strains, its combined use with antimicrobials could be helpful
when treating tolerant isolates. The lack of toxicity of terpenes [56,57] makes them a harm-
less and potential therapeutic agent to increase the efficiency of the treatment of bacterial
and fungal infections mediated by resistant strains. Thus, in much previous research, nei-
ther cytotoxicity nor acute toxicity on animals has been found for relatively high
Antibiotics 2022, 11, 1743 11 of 17
concentrations of myrtenol, up to 600 mg/L in vitro and 1.3 g per kg in vivo [58–60] It is
worth mentioning that a crucial benefit from using the described compounds is that their
resource is almost inexhaustible [61,62]. Thus, the knowledge of the clinical and economic
burden of antibiotic-resistant mixed infections, coupled with the benefits of the availabil-
ity of such compounds, will allow for optimal control and improved patient safety [63].
4. Materials and Methods
4.1. Chemistry
The (+)- or (-)-myrtenol were synthesized by the oxidation of (+)- or (-)-α-pinene with
tert-butyl hydroperoxide in the presence of catalytic amounts of SeO2 according to the
reported procedure [64]. The myrtenal formed during the reaction (content 70−75% by
Gas liquid chromatography) was isolated through a water-soluble sulfite derivative (al-
dehyde purity is 97−98%) with subsequent NaBH4 reduction of the aldehyde into myr-
tenol. A yield of 40–42% was observed. The spectral data and physical constants associ-
ated with the compounds obtained fit with the literature data. Synthesis, physicochemical
properties, and spectral data of BF2-ms-(4-((1″R)-6″,6″-dimethylbicyclo[3.1.1]hept-2″-ene-
2″)ylmethoxycarbonylpropyl)-3,3′,5,5′-tetramethyl-2,2′-dipyrromethene (mentioned as
“lum”) were described in detail in our previous paper [42]. A solution of ester 1 (0.128
mmol, 1 equiv) in isopropanol (5 mL) was stirred with 0.1 N KOH (2 mL) under argon
atmosphere at room temperature using thin layer chromatography (TLC) in a 1:10 methyl
tert-butyl ether (MTBE)−CCl4 system to monitor the reaction progress. After almost com-
plete transformation (1−2 h), the mixture was evaporated. Then, 20 mL of toluene and
diluted aqueous HCl were added to the mixture with intensive stirring. The organic layer
was separated and evaporated in vacuo. Then, 0.154 mmol (1.2 equiv) of (−)- or (+)-myr-
tenol and 0.128 mmol of DMAP in 20 mL of dichloromethane (DCM) were added. After
complete dissolution, 0.384 mmol of HATU was added to the mixture. The progress of the
reaction was monitored by TLC with a 1:10 MTBE−CCl4 system. After completion of the
reaction (about 5 h), the solvent was removed in vacuum and the product was purified by
silica gel column chromatography. A 1:19 MTBE−CCl4 mixture served as an eluent. A
yield of 59% was observed. The stock solutions of (−)-myrtenol and (+)-myrtenol were
prepared in pure DMSO at a concentration of 20 g/L. Working solutions were prepared in
a bacterial growth medium with a final concentration of DMSO of no more than 5%, which
is nontoxic for both bacterial and fungal strains. Amikacin (Sigma), benzalkonium chlo-
ride (Sigma), and fluconazole (Sigma) were used as reference antimicrobials.
4.2. Strains and Cultivation Conditions
A methicillin-sensitive Staphylococcus aureus ATCC 29213 as well as 10 clinical
MRSA isolates obtained from the Republican Clinical Hospital, Laboratory of Clinical Bac-
teriology in Kazan were used in this study (see Table 1). The bacterial strains were stored
in 50 % (V/V) glycerol stocks at -80 °C and freshly streaked on LB plates followed by their
overnight growth at 37 °C before use. Ten clinical isolates of Candida albicans (see Table
2 for resistance details) from the patients of Kazan Scientific Research Institute of Epide-
miology and Microbiology (Kazan, Russia) obtained during the year 2019 were used. Iso-
lates were identified as C. albicans by using AuxaColor 2 Colorimetric sugar-assimilation
yeast-identification kit (Bio-Rad) and confirmed on MALDI-TOF mass spectrometry
(Bruker Biotyper system, Bruker Daltonics, Germany). Fungal strains were stored as a 50%
glycerol stock at −80 °C and grown in the RPMI broth. The overnight cultures were used
to adjust an optical density to 0.5 McFarland (equivalent to 108 cells/mL) in growth me-
dium and used as a working suspension. To obtain a mature biofilm, fungal and bacterial
cells were seeded in TC-treated culture plates (at 106 cells/mL) and grown under static
conditions for 48 h at 37°C in BM-broth [65] supplemented with 1% glucose. Mannitol salt
agar and Sabouraud agar supplemented with ciprofloxacin (20 µg/mL) were used for the
Antibiotics 2022, 11, 1743 12 of 17
differential count of CFUs of S. aureus and C. albicans, respectively, in S. aureus–C. albicans
mixed cultures.
4.3. Determination of the Minimum Inhibitory (MIC) and the Minimum Bactericidal/Fungicidal
Concentrations (MBC/MFC)
The minimum inhibitory concentration (MIC) was determined by serial microdilu-
tion in 96-well plates according to the EUCAST rules for antimicrobial susceptibility test-
ing [66]. The highest final concentration of each compound was 512 µg/mL. The next wells
contained two-fold decreasing concentrations of compound in the range of 0.5–1024
µg/mL. The wells were seeded with microbial culture to obtain density of 106 CFU/mL in
a volume of 200 µL per well. The plates were incubated under static conditions at 37°C
for 24 in case of bacterial culture and 48 h for yeast. The growth was assessed by measur-
ing the optical density at wavelength of 600 nm. The minimum inhibitory concentration
of the compound was defined as the concentration providing complete suppression of the
visible growth of cells. The minimum bactericidal/fungicidal concentration (MBC/MFC)
was determined by seeding 5 µL of culture fluid from wells with no visible growth in 3
mL of fresh nutrient broth. The MBC/MFC was considered the minimum concentration of
the studied compound, which ensures the complete absence of growth [67].
4.4. Determination of the Biofilm Prevention Concentration (BPC)
To determine the biofilm prevention concentration (BPC), bacterial and fungal cells
were grown in 96-well adhesive plates for 48 h under static conditions at 37 °C in BM
broth in wells of 200 µL with an initial density of 106 CFUs / ml in the presence of the test
substances. Next, staining with crystal violet was carried out as described in [68]. The
minimum biofilm inhibitory concentration was defined as the lowest concentration
providing no visible staining of the residual biofilm.
4.5. Analysis of the Antimicrobial Effect in the Combined Use of Antimicrobial Agents
(Chequerboard Approach)
The chequerboard approach was used to assess the possibility of increasing the ef-
fectiveness of other antimicrobial agents with myrtenol. The experimental methodology
was similar to the determination of the MIC in 96-well plates. Each plate contained serial
dilutions of a myrtenol derivative and various compounds in a chequerboard pattern, as
described previously [69]. One of the antimicrobial substances [A] was twice diluted hor-
izontally, and the other [B] vertically on a 96-well plate. The result was a combination of
77 concentrations of antimicrobial compounds [A] and [B]. The extreme lines and columns
contained only one of the considered substances to determine their MICs directly in the
experiment. The initial concentration of each studied antimicrobial agent was 4×MIC. The
final concentration of bacterial and fungal cells in the wells was 0.5×105 CFU/mL. The
plates were incubated at 37°C for 20 h. Then, the optical density OD600 was measured on
an Infinite 200 PRO plate spectrophotometer (Tecan, Switzerland). Each test was run in
triplicate and included a growth control without the addition of any antimicrobial agent.
The fractional inhibitory concentration index (FICI) for each double combination was cal-
culated as follows:
FICI =
[]
[] +[]
[]
(1)
Interpretation of the obtained FICI values was carried out according to [70,71]; FICI
≤ 0.5 corresponded to synergy, 0.5 < IFIC ≤ 4 to an additive effect while IFIC > 4 corre-
sponded to antagonism.
4.6. Evaluation of Viability of Bacterial and Fungal Cells
To assess the viability of planktonic cells, samples from the upper layer of the culture
liquid were taken. Then the culture liquid was removed from the wells; wells were
Antibiotics 2022, 11, 1743 13 of 17
washed several times with a sterile NaCl solution (0.9%) to remove planktonic and de-
tached cells. The biofilms were mechanically destroyed, and cells were resuspended in a
sterile NaCl solution (0.9%). The viability of cells was evaluated by the drop plate assay
with minor modifications [72]. Serial ten-fold dilutions from each well were prepared, and
5 µL of suspension was dropped on Mannitol salt agar and Sabouraud agar with ciprof-
loxacin (20 µg/mL) to differentiate S. aureus and C. albicans cells, respectively. After 24 h
of incubation at 37°C, the number of colonies on the plates was counted; the values were
averaged and expressed as CFU/mL.
4.7. Membrane Potential Evaluation
Membrane potential was evaluated by the detection of 3,3’-diethyloxacarbocyanine
iodide (DioC2(3)) fluorescence as an indicator of the membrane potential level. Bacterial
or fungal cells were grown for 18 h in LB broth with stirring, then harvested and washed
with PBS. Cells were resuspended until a final density of 106 CFU/mL was reached in PBS
supplemented with DioC2(3) to a final concentration of 10 µM/mL. C. albicans cells were
resuspended until a final density of 105 CFU/mL. After a 30 min incubation at 25°C, com-
pounds were added to the samples. Fluorescence detection was performed for 30 min with
5 min intervals using carboxyfluorescein (FAM) wavelength detection (the excitation and
emission wavelengths were 497 and 520 nm, respectively).
4.8. Estimation of the Penetration Rate of Myrtenol into Bacterial and Fungal Cells
To assess the penetration rate of terpenoids into bacterial cells, (-)-myrtenol-lum and
(+)-myrtenol-lum, which contain a fluorophore (lum) in their structures, were used. Bac-
terial and fungal cells were grown overnight at 37 °C in LB culture medium with agitation,
then washed with BPS (pH = 7.4), and resuspended in a buffer to an optical density of 0.5
by McFarland. Either (-)-myrtenol-lum or (+)-myrtenol-lum was added to a final concen-
tration of 10 µg/mL and incubated at 25 °C in the dark. Pure fluorophore was used as a
control. After 4, 8, 16, 32, and 64 min of incubation, 150 µL of the suspension was taken;
cells were harvested by centrifugation, washed with PBS, and then resuspended in 150 µL
of the buffer. The fluorescence was measured using a Tecan Infinite 200 Pro microplate
reader (at an excitation and emission wavelength of 485 and 520 nm, respectively). The
time required to obtain half of the maximum fluorescence of stained cells (t 1/2) was cal-
culated by plotting log10 (time) as a function of percent fluorescence (taking into account
the fluorescence of unstained cells as 0% and the fluorescence of cell suspension in buffer
with test compound (10 µg/mL) as 100%) in GraphPad Prism 6. Additionally, the pene-
tration of either (−)-myrtenol-lum or (+)-myrtenol-lum in bacterial and fungal cells and
their localization there were assessed by confocal laser scanning microscopy on micro-
scope. (−)-Myrtenol-lum, (+)-myrtenol-lum, or pure fluorophore were added to the cells
at a concentration of 10 µg/mL. Cell membranes were additionally stained with calcofluor
dye (1 mg/mL). As a result, the membranes that were stained in blue (excitation emission)
and green fluorescence (excitation emission) indicated the localization of terpenes in the
cells.
4.9. Data Analysis
All experiments were performed in three biological replicates with three technical
replicates in each experiment. The data were analyzed and visualized using GraphPad
Prism version 6.00 for Windows (GraphPad Software, USA, www.graphpad.com). In each
experiment, a comparison with a negative control was performed using the nonparamet-
ric Kruskal–Wallis test of variance. Significant differences from control were considered
at p<0.05.
5. Conclusions
Antibiotics 2022, 11, 1743 14 of 17
Both (−)-myrtenol and (+)-myrtenol have weak antibacterial and antifungal activity
while demonstrating nonstrain-specific bactericidal and fungicidal effects and exhibiting
synergism with amikacin and benzalkonium chloride in relation to planktonic cells and
biofilms. The mechanism of these effects appears as a consequence of the membranotropic
property of the compound against bacterial and fungal cells. This may be considered as
further validation that these compounds contribute to an increase in the effectiveness of
various antimicrobial, antifungal, and antiseptic drugs, manifesting synergy with these
compounds. Moreover, our findings confirm that terpene derivatives increase the effec-
tiveness of benzalkonium chloride against microorganisms in the mixed community of S.
aureus and C. albicans. Thus, due to the low toxicity of terpenes, these compounds could
become promising agents in the treatment of infections caused by bacteria and by fungi
of the genus Candida as well as mixed fungal–bacterial infections, including resistant
strains.
Supplementary Materials: Table S1. MIC, FIC, and FICI values of amikacin in combination with
either (−)-myrtenol or (+)-myrtenol (expressed in µg/mL) against various isolates of S. aureus. Table
S2. Biofilm preventing concentrations (BPC), fractional biofilm preventing concentrations, and FICI
values of amikacin in combination with either (−)-myrtenol or (+)-myrtenol (expressed in µg/mL)
against various isolates of S. aureus. Table S3. MIC, FIC, and FICI values of benzalkonium chloride
(BAC) in combination with either (−)-myrtenol or (+)-myrtenol (expressed in µg/mL) against various
isolates of S. aureus. Table S4. Biofilm preventing concentrations (BPC), fractional biofilm preventing
concentrations, and FICI values of benzalkonium chloride (BAC) in combination with either (−)-
myrtenol or (+)myrtenol (expressed in µg/mL) against various isolates of S. aureus. Table S5. MIC,
FIC, and FICI values of fluconazole in combination with either (−)-myrtenol or (+)-myrtenol (ex-
pressed in µg/mL) against various isolates of C. albicans. Table S6. Biofilm preventing concentrations
(BPC), fractional biofilm preventing concentrations, and FICI values of fluconazole in combination
with either (−)-myrtenol or (+)-myrtenol (expressed in µg/mL) against various isolates of C. albicans.
Table S7. MIC, FIC, and FICI values of benzalkonium chloride (BAC) in combination with either (−)-
myrtenol or (+)-myrtenol (expressed in µg/mL) against various isolates of C. albicans. Table S8. Bio-
film preventing concentrations (BPC), fractional biofilm preventing concentrations, and FICI values
of benzalkonium chloride (BAC) in combination with either (−)-myrtenol or (+)-myrtenol (expressed
in µg/mL) against various isolates of C. albicans.
Author Contributions: Conceptualization, L.L.F., A.V.K., G.B.G., E.V.A., M.B.B., L.E.N. and A.R.K;
Data curation, A.R.K; Formal analysis, L.E.N.; Funding acquisition, A.R.K and E.V.A.; Investigation,
R.Y.M., E.Y.T., R.K.S., R.S.P., I.R.G., S.A.L., O.V.O., L.L.F., A.V.K., G.B.G., E.V.A. and M.B.B.; Meth-
odology, R.Y.M., E.Y.T., R.K.S., R.S.P., I.R.G., O.V.O., L.L.F., A.V.K., G.B.G., E.V.A. and M.B.B.; Pro-
ject administration, E.Y.T., L.E.N. and A.R.K; Resources, L.E.N. and A.R.K; Supervision, L.E.N. and
A.R.K; Validation, L.E.N. and A.R.K; Visualization, R.Y.M. and E.Y.T.; Writing—original draft,
R.Y.M., E.Y.T., R.K.S. and A.R.K; Writing—review and editing, R.Y.M., E.Y.T., R.K.S. and A.R.K. All
authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Russian Science Foundation (grant N 20-64-47014 to
A.R.K.). The synthesis of BODIPY (BF2-ms-methoxycarbonylpropyl-3,3′,5,5′-tetramethyl-2,2′-dipyr-
romethene) was carried out in the ISC RAS under the Russian Science Foundation Project (grant N
20-63-47026 to E.V.A.).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: All data are included in the manuscript
Acknowledgments: This research was performed in frames of the Kazan Federal University Strate-
gic Academic Leadership Program (PRIORITY-2030).
Conflicts of Interest: The authors declare no conflict of interest.
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