Antifungal efficacy of a citrus fruit extract against Antifungal efficacy of citrus fruit extracts against Candida albicans cells Daniel Joel Kobric Masters of Science Discipline of
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Antifungal efficacy of a citrus fruit extract against
Candida albicans cells
by
Daniel Joel Kobric
A thesis submitted in conformity with the requirements for the degree of Masters of Science
2.3.4 Immune response to the components of the cell wall ..............................................................7 2.4 Biofilms of Candida .......................................................................................................................7
2.5 Commensalism vs. virulence ......................................................................................................11 2.5.1 Host-fungal interactions .........................................................................................................12
2.5.1.1 Host ligands....................................................................................................................................12 2.5.2 Candidal infections- oral and esophageal ..............................................................................12
v
2.5.2.1 Candida in periodontal disease, caries, and endodontic infections................................................13 2.6 Treatment protocols....................................................................................................................14
2.6.1 Current treatments ..................................................................................................................14 2.6.2 Antimycotic drugs and mechanisms of action .......................................................................15
2.6.3 C. albicans methods of drug resistance to antimycotics ........................................................17 2.6.4 Resistance to polyenes ...........................................................................................................18 2.6.5 Resistance to azoles................................................................................................................18 2.6.6 Resistance to pyrimidine analogues .......................................................................................19 2.6.7 Resistance to echinocandins...................................................................................................19 2.6.8 Resistance to allylamines .......................................................................................................19 2.6.9 Combination therapy ..............................................................................................................19
4. Materials and Methods........................................................................................................ 25
4.1 Fungal cultures and growth conditions.....................................................................................25 4.2 Test compounds ...........................................................................................................................25 4.3 Biofilm growth .............................................................................................................................25
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4.4 Determination of the minimum inhibitory concentration .......................................................26 4.5 Treatment of biofilm with test compounds ...............................................................................26 4.6 Scanning electron microscopy....................................................................................................26 4.7 Resistance assay...........................................................................................................................27 4.8 pH assay .......................................................................................................................................27 4.9 Drug synergy................................................................................................................................28 4.10 Statistical analysis .....................................................................................................................28
5.1 Determination of CFU from stationary phase cultures ...........................................................29 5.2 Antimicrobial susceptibility to B320 and nystatin ...................................................................29 5.3 Antimycotic activity of B320 ......................................................................................................29 5.4 Combined application of B320 and nystatin.............................................................................30 5.5 Determination of synergistic antimycotic activity....................................................................30 5.6 Resistance assay...........................................................................................................................31 5.7 SEM analysis................................................................................................................................31
6.1 B320 as an antifungal..................................................................................................................40 6.2 Effects of pH on Activity of B320...............................................................................................41 6.3 MIC of B320.................................................................................................................................41 6.4 Effects of combinations of B320 and nystatin on C. albicans ..................................................42 6.5 Development of resistance to B320 and nystatin ......................................................................43 6.6 Cell morphology of C. albicans with and without treatment ..................................................44 6.7 Possible sources of error .............................................................................................................44 6.8 Summary ......................................................................................................................................46
7. Conclusions and future studies........................................................................................... 47
Table 4. Fractional inhibitory concentrations (FIC) and FIC indices (FICI).
WT strain MICalone MICcombination FICa FICIb
Nystatin 10 IU/ml 3 IU/ml 0.3
B320
0.005% 0.005% 1.0
1.3
NYSR strain MICalone MICcombination FICa FICIb
Nystatin 100 IU/ml 30 IU/ml 0.3
B320 0.005% 0.001% 0.2
0.5
aFIC = MIC of B320 or nystatin in combination/MIC of B320 or nystatin alone. bFICI = FIC of
B320 + FIC of nystatin. The interaction is identified as synergistic (FICI < 0.5), indifferent (FICI
0.5 – 4.0) or antagonistic (FICI > 4.0).
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Table 5. Development of drug resistance.
Passagea # B320 MIC (%) Nystatin MIC (IU/ml)
2 0.01 30
4 0.01 30
6 0.01 50
8 0.01 50
10 0.01 50
12 0.01 >100
14 0.01 >100
a C. albicans WT strain was cultivated in 0.5X MIC of tested compound. At every second
passage, a passage was used for MIC determination.
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Figure 4. Percentage of cell survival of 24-h-old C. albicans WT biofilms following treatment
with B320 (0.5, 1, 10%), nystatin (100,000 IU/ml), or glycerin (control) for 60 s. Results are the
mean ± SD of three independent experiments. *statistically significance difference (p<0.0001)
between 10% B320 and all other test compounds.
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Figure 5. Percentage of cell survival of 24-h-old C. albicans WT biofilms following treatment
with 10% B320 (pH 2.8; non-pH-adjusted), 10% B320pH (pH 6.8; pH adjusted with NaOH), and
carrierpH solution (pH 2.8; adjusted with HCl) for 60 s. Results are the mean ± SD of three
independent experiments. *Statistical significance: B320 at pH 2.8 or pH 6.8 vs. carrier at pH 2.8
or saline control (not shown) (p<0.0001); no statistically significant difference between B320 at
pH 6.8 and pH 2.8 (p= 0.3468).
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Figure 6. Percentage of cell survival of 6-h, 24-h, and 72-h-old C. albicans WT biofilms
following treatment with 10% B320, nystatin 100,000 IU/ml, or 10% B320 with 90,000 IU/ml
NYS for 60 s. Results are the mean ± SD of three independent experiments. Statistically
significant difference between B320 (#) and nystatin or control for 24-h and 72-h biofilms
(p<0.0001). Statistically significant difference between B320+NYS (*) and nystatin or control
treatments at 6-h (p<0.03), 24-h (p<0.0001), and 72-h (p<0.0001); no statistically significant
difference between B320 and B320+NYS (combination) at any time point.
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Figure 7. Percentage of cell survival of 6-h-, 24-h-, and 72-h-old C. albicans NYSR biofilms
following treatment with 10% B320, NYS 100,000 IU/ml, or 10% B320 with 90,000 IU/ml NYS
for 60 s. Results are the mean ± SD of three independent experiments. Statistically significant
difference (p<0.0001) between B320+NYS and B320 at 24-h (*). Statistically significant
difference between B320 +/-NYS and control or nystatin treatments at 6-h, 24-h, and 72-h. No
statistically significant difference between NYS and control treatment at any time point.
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Figure 8. Scanning electron micrograph of 24-h-old C. albicans WT biofilms exposed to B320
(10%), nystatin (100,000 IU/ml), or B320 (10%) combined with nystatin (90,000 IU/ml) for 60 s.
There is formation of an amorphous mass of cell debris following exposure the combination
(arrow). Magnification of 1,000 X.
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Figure 9. Scanning electron micrograph of 24-h-old C. albicans NYSR biofilms exposed to B320
(10%), nystatin (100,000 IU/ml), or B320 (10%) combined with nystatin (90,000 IU/ml) for 60 s.
Formation of an amorphous mass of cell debris following exposure to the combination (arrows).
Magnification of 1,000 X. (Note: no hyphae observed).
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6. Discussion The components of B320 include extracts from lemon, bergamot, orange, and lime. These
ingredients make up the citrus extracts, which include flavonoids and essential oils. Determining
the flavonoids present in the fruits used to manufacture B320 is relatively easy (see review
(110)), however, the composition of the mixture of citrus biomass would require further analysis
as the composition of B320 is proprietary and therefore was unavailable to the investigator.
However, the antifungal properties of the flavonoids and essential oils have been previously
investigated. Citrus essential oils have been shown to possess antimicrobial properties against
C. albicans (113). The antifungal properties of the essential oils may be related to activation of
quorum-sensing pathways, which direct the cessation of Candida hyphal growth and induces
yeast cell dispersion. The polyphenol curcumin acts to kill Candida by disrupting cell membrane
sterols, making it an effective antifungal agent (114). Since we do not know the concentration of
the components of B320, it cannot be speculated as to what particular component(s) is
responsible for the biological activity of this antimycotic agent. However, the polyphenols
(hesperindin, rutin, and naringenin) are likely to be present in B320, we can hypothesize as to
how the agent may work and which components could be responsible for the antifungal
properties observed in this study. Moreover, the findings reported here are consistent with the
literature in that polyphenols from citrus fruits are effective as antimycotic agents (115). The
hypothesis is that the polyphenols present in B320 act to disrupt the plasma membrane. It is also
possible that the polyphenols trigger a cascade leading to cell death as >90% cell death is
observed after a 60 s application. However, the checkerboard assay for the combined application
of a B320 and nystatin with planktonic C. albicans biofilm cells demonstrated an indifferent
result unlike those in the literature showing synergistic effects between nystatin and a polyphenol
(curcumin) (45, 90).
6.1 B320 as an antifungal
In this investigation it has been demonstrated that concentrations of 0.5%, 1%, and 10% of B320
possess significantly greater (p<0.05) fungicidal properties relative to nystatin at 100,000 IU/ml
(Figure 4). A 10% solution of B320 applied to a 24-h old biofilm for 60 s causes ~95% of the
cells to die relative to a saline control (Figure 6), which is substantially greater than what would
be obtained by the use of nystatin. We can also glean from Figure 6 that when B320 is applied
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for 60 s to a 6-h and 72-h old WT biofilms, 99% and 90% of the cells die, respectively. Taken
together, these data demonstrate that B320 is an efficient fungicidal agent, and only needs to be
applied for a period of 60 s, a finding that was reproduced across several of the different
experiments shown here. B320 mechanism of action is still to be determined but based on other
studies we can speculate on what they may include. It may act to disperse yeast cells in the
biofilm or act to induce cell death via a quorum-sensing regulating pathway. As well, B320
components may act in a manner that increases the fluidity of the membrane by itself or by
disrupting ergosterol synthesis. It may inhibit formation of hyphae by reducing the availability of
oxidizing agents required for host invasion thereby decreasing the virulence of C. albicans.
Recent work with curcumin has suggested that the antifungal properties of polyphenol may be
related to yeast cell dispersion or cell death via a quorum-sensing regulating pathway (114).
Others have also shown that the polyphenol curcumin (a polyphenol) acts synergistically with
antifungals known as azoles.
6.2 Effects of pH on Activity of B320
The pH of B320 solution is about 2.8. This was of some concern since it was possible that the
low pH, and not the so-called active ingredients in B320 might have been responsible for the
antimycotic activity of B320. Moreover, if this agent is to be used in the mouth, then a pH of 2.8
could theoretically cause irritation to the patients’ mucosa and could lead to dental
demineralization lesions. Therefore it was critically important to determine whether the acidic
pH of B320 was responsible for its antimycotic effects. As shown in Figure 5, it was not the
effect of low pH itself that killed C. albicans cells. Moreover, when the pH was adjusted
upwards to a more physiological level, the antimycotic activity of B320 was retained. This
basically suggests that if B320 were approved for use in humans, the pH of the solution could be
raised without losing the antimycotic activity. It is also important to point out that since altering
the pH of B320 can be a rather time-consuming effort, and since the activity of B320 is identical
at the different pH’s tested, it was decided that it would be appropriate to continue our
experimentation using the original formula for B320 without adjusting the pH.
6.3 MIC of B320
The MIC assay allows for speculation as to the putative mechanism that might explain how B320
is able to kill C. albicans cells. We postulate that the antimycotic effects of B320 are possibly
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related to the ergosterol content of the cell membrane. In this regard, we observed that the MIC
for NYSR strain, required 100x more B320 at HCD compared to LCD (Table 3). This is in
contrast to what was observed with WT planktonic cells where those at HCD required only 10x
greater concentration of B320 relative to the LCD cells. The decreased ergosterol content of the
NYSR cells may yield fewer targets for B320 to affect the membrane. However, the NYSR cells
require twice the amount of B320 at HCD vs. WT cells. The difference observed between the
WT and NYSR strains of this species in the MIC assay may be related to the ergosterol content of
the cells. However, further studies would be required to verify this hypothesis.
Another research group recently tested two bioflavonoid containing product, derived from citrus
fruits (Citrox®) on planktonic and biofilm cells (115). Their results are similar to those reported
here however their agent was applied for a 24-h duration to C. albicans biofilms and planktonic
cells, whereas our results were obtained after a 60 s application. Other notable issues concerning
the apparent differences between the findings reported here and those reported with Citrox®
were that the other investigators used the ‘McFarland standard’, which uses E. coli as the culture
from which the OD is determined to make a standard curve. In our study, standard curves were
generated using the test species of interest, C. albicans. It would seem to be more scientifically
valid to generate standard curves for calculation of cell counts by using the microbial species of
interest as opposed to another microbe. However, it also has to be recognized that the others’
use of E. coli for generation of their standard curves might not be completely invalid. In this
regard, while this approach might not actually produce true and accurate CFU counts for
C. albicans, but would constitute a systematic error in all tests meaning at least that intra-
experimental comparisons would be somewhat reliable. In addition it was unclear as to whether
the findings reported by others (115) were obtained using biofilms or planktonic presentations of
C. albicans. At any rate, our findings confirmed that the MIC of B320 against planktonic cells
of C. albicans at low and high cell density was 0.005% and 0.05% B320, respectively.
6.4 Effects of combinations of B320 and nystatin on C. albicans
This combination approach of antifungal and citrus extracts is not new. Terpenoids, including
essential oils, are known to impair yeast cell membrane integrity. Together with an azole, the
synergistic effect exhibited by these two agents affects the yeast plasma membrane and cell
signaling (112). Using biofilms of C. albicans (WT and NYSR) it was possible to observe
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synergistic-like effects with regard to the killing of C. albicans by combining B320 with nystatin
(Figures 6 and 7). Based on the data it is probable that that biofilms at different stages of
maturity might respond differently to these antimycotic agents. As shown in this study, it appears
that early and intermediate biofilms are most susceptible to the synergistic-like effect of B320
and nystatin. However, no statistically significant difference between B320 alone or in
combination with nystatin was found after a multivariate analysis.
When it comes to considering more mature biofilms, the effects of B320 alone or in combination
with nystatin do not have synergistic effects while the cell survival rates are similar (~10%).
Using a checkerboard assay with planktonic cells of the WT and NYSR strains at LCD, synergy
was not observed using the FICI calculation. However, the NYSR cells with a FICI value of 0.5 is
quite close to the being indicative of synergy between B320 and nystatin (FICI < 0.5). As
reported above, the effects of B320 and nystatin were also tested against a nystatin-resistant
strain of C. albicans. This strain, as reported via the ATCC database, does not express ERG2, a
gene responsible for the conversion of fecosterol to episterol along the ergosterol pathway
altering membrane fluidity. It is hypothesized that the polyphenol acts to further disrupt the
already altered membrane lacking ergosterol, thus imparting antifungal effects to this nystatin-
resistant strains of C. albicans. Additionally, it can also be hypothesized that the polyphenol
somehow makes the nystatin-resistant strain susceptible to nystatin.
There is likely an alteration in the mature biofilm (cells or ECM) that prevents full synergy
between B320 and nystatin, as this is not observed in younger biofilms. Although synergy
between B320 and nystatin could not be shown in the planktonic yeast, statistical analysis
showed a statistically significant difference between B320 and nystatin with the intermediate
biofilms formed by the NYSR strain. Furthermore, even if pure synergy could not always be
demonstrated, there is at the very least an important additive effect when both compounds are
used.
6.5 Development of resistance to B320 and nystatin
Given the problems relating to the continual development of microbial resistance to
anti-infective medications, it was decided that it was necessary to demonstrate whether or not,
C. albicans could develop resistance to B320. Planktonic cells were incubated with levels of the
antimycotic agents at doses below the previously determined MIC. It was found that when cells
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were incubated with sub-MIC levels of the antimycotic agents, they were able to form biofilms.
If the cells developed a biofilm at or above the MIC then it was concluded that the organism had
developed resistance to the agent used. Resistance to B320 was never observed in WT cells over
a two-week period of treatment. When WT cells were exposed to nystatin they were able to
overcome the MIC and develop biofilms at the MIC levels. A 10-fold increase in the MIC level
was observed as the MIC of nystatin increased from 10 to 100 IU/ml. These findings are
consistent with the development of resistance to nystatin for WT strain. This has been reported
by others, where 5-fold increases in resistance were noted by using this approach over a period
of 20 days (116). However, we can only conclude that resistance does not occur after 14 days
and further testing would be required to determine if resistance develops after more time.
6.6 Cell morphology of C. albicans with and without treatment
The SEM results of both C. albicans strains examined (WT and NYSR) demonstrated visible
changes following treatment with the combination of B320 and nystatin. These results are
congruent with a study by Sohn et al. on C. albicans (ATCC 10231) (117). In their investigation
it was surmised that the flavonoid alters the cell wall/membrane resulting in clefting, surface
collapse and wrinkled appearance, like that of a ‘shredded fabric’, of candidal cells, which is
observed in Figure 8 and Figure 9. Of interest is that their result is after incubation of C. albicans
with the flavonoid for 2 h at 30°C. Our results were based on a 60 s exposure at room
temperature. However, we cannot conclude that the morphological change is demonstrative of
cell death.
6.7 Possible sources of error
Any errors that may have occurred during the carefully planned experiments are likely due to the
following.
1) Difficulty in preparing the required concentrations of reagents is related to the high
viscosity of B320. This may affect the accuracy of the prepared solution, specifically the
10% B320 which was used in all the experiments. Additionally, exceptionally small
volumes were used in the microtiter plates; this may have led to some errors as well.
However, completing experiments with three technical replicates and in duplicate or
triplicate minimized this possibility.
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2) Washing of the biofilms to remove the test agent (B320, nystatin). This action likely
disrupted some of the biofilm. The test agent may have also been left on the biofilm (not
washed off completely) resulting in treatment times greater than 60 s. As experiments
were completed multiple times and the results across different experiments had
reproducible results, it is probable that the potential sources of error discussed here had a
minimal impact or at most constituted a systematic error that would have affected all
cultures treated with one or the other agent. Furthermore, although intraoral use of an
antimycotic rinse involves expectoration after about a 30 s rinse, it is highly likely that
the antimycotic agent is still present (substantivity). In fact, if that was not the case, the
effectiveness of oral rinses might be quite poor. At any rate, given the potential for a
systematic error, intra-experimental comparison of data was deemed reliable while inter-
experimental comparison of data (apart from relative changes) could not be done.
3) There were some concerns associated with the use of microtiter plates and the
measurement of turbidity (OD) to determine viability. The reagents: B320, YPG broth
and nystatin are yellow in colour and can absorb incident light in a spectrophotometer at
various wavelengths. As a result of this, other investigators have used XTT (a tetrazolium
dye) to define cell viability. Candida metabolizes XTT to an orange colour.
Spectrophotometry is then used to compare the XTT reaction at specific wavelengths.
The protocol used for this investigation provided a step to wash away the yellow colour
with saline. The wavelengths used (490 nm or 530 nm) were kept constant within each
experimental protocol to minimize error further.
4) We observed what appeared to be synergy-like between B320 and nystatin with biofilm
but not planktonic C. albicans cells. Several studies have examined the effects of
combining natural products with antifungal agents. In some cases there was synergy
(118) and in others the effect was additive or indifferent (119). Given the inconclusive
results obtained for synergy, another method to test for synergy may be required. The
rationale for stating this is that the biofilm data suggest that there might well be
synergistic antimycotic effects when B320 and nystatin are combined, at least in early
biofilms. However, it must be recognized that both types of tests (biofilm and planktonic
cultures) are important, and could very well yield different types of interactions between
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B320 and nystatin, since C. albicans exists in the mouth and elsewhere in both the
planktonic and biofilm configurations.
6.8 Summary
Overall this study demonstrated the antimycotic potency of a citrus fruit extract solution, B320
on C. albicans. A kill rate of over 90% after a 60 second application on a biofilm was observed
and pH played no role in the antimycotic activity of B320. No synergy was demonstrated
between B320 and nystatin (a polyene) on a planktonic population. C. albicans could not be
shown to develop resistance to B320 unlike what was observed for cells treated with nystatin.
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7. Conclusions and future studies The citrus derived agent known as B320 is effective as an antifungal agent alone or in
combination with the polyene antifungal nystatin. It mediates killing of C. albicans in biofilm
and planktonic forms. The mechanism of action of B320 may be multifaceted. Further study of
the mechanism of action of this agent and the possible synergistic effects of B320, a citrus fruit
extract, with various antimycotic pharmaceuticals is required. Future studies using the flavonoids
present in lemons, bergamot, limes, and oranges are available from Sigma-Aldrich and can be
tested alone or in combination based on their relative concentration in the specified citrus fruits
(110). The determination of the minimum biofilm eradication concentration (MBEC) may be
more useful than the MIC. The MIC value pertains to planktonic cells whereas the MBEC relates
to biofilms. The techniques described in (120) can be used in future experiments. Further, animal
models should be used to test the efficacy in vivo before proceeding to human studies. This is
required to determine the most appropriate concentration of agent that is required to act
effectively, and whether it should be used alone or in combination with other antimycotic agents.
We could also further investigate the resistance to B320 by testing over a longer period of time
(>14 days). And since B320 is not specific to C. albicans, the effect of B320 on the normal oral
flora would also need to be evaluated.
This agent could represent an exciting and novel treatment alternative for superficial candidiasis
and possibly other biofilm related human illnesses given that it also has antibacterial effects. It is
as if there has been a re-awakening of ‘traditional medicine’ in the western world, which could
lead to safer and more effective treatments for various disorders (121).
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