# Tetracycline Effects onCandida Albicans Virulence Factors

Authors: Logan McCool; Hanh Mai; Michael Essmann; Bryan Larsen
Journal: Infectious Diseases in Obstetrics and Gynecology
 (2008)

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## Abstract

Object. To determine if tetracycline, previously reported to increase the probability of developing symptomatic vaginal yeast infections, has a direct effect on Candida albicans growth or induction of virulent phenotypes.
Method. In vitro, clinical isolates of yeast were cultivated with sublethal concentrations of tetracycline and yeast cell counts, hyphal formation, drug efflux pump activity, biofilm production, and hemolysin production were determined by previously reported methods.
Results. Tetracycline concentrations above 150 μg/mL inhibited Candida albicans, but at submicrogram/mL, a modest growth increase during the early hours of the growth curve was observed. Tetracycline did not inhibit hyphal formation at sublethal concentrations. Hypha formation appeared augmented by exposure to tetracycline in the presence of chemically defined medium and especially in the presence of human serum. Efflux pump CDR1 was upregulated and a nonsignificant trend toward increased biofilm formation was noted.
Conclusion. Tetracycline appears to have a small growth enhancing effect and may influence virulence through augmentation of hypha formation, and a modest effect on drug efflux and biofilm formation, although tetracycline did not affect hemolysin.  It is not clear if the magnitude of the effect is sufficient to attribute vaginitis following tetracycline treatment to direct action of tetracycline on yeast.


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## Body

## 1. Introduction

Epidemiological  studies purport
that the use of certain antibiotics during pregnancy  increases susceptibility
to vulvo-vaginal candidiasis [1, 2]. This condition is generally believed to
result from the suppression of a bacterial flora in the face of antibacterial
drugs that fail to inhibit Candida, resulting
in its overgrowth and subsequent emergence of symptoms. Although this concept is often repeated in
textbooks, surprisingly little direct evidence exists to support the concept that
vaginal candidiasis is caused by prior antibiotic treatment. A report by Burns etal. [3] provided correlation
between antibiotic use and vaginal colonization and candidiasis.  Since the role of antibiotics in eliciting
yeast vaginitis is medical dogma, there has been little reason to expend effort
on developing actual data on the relationship between antibiotics and candidiasis.In 1998, Glover and Larsen [4],
curious about the magnitude of the impact of antibiotics on yeast vaginitis, unexpectedly
found that nonantibiotic-treated pregnant patients actually became symptomatic
more frequently than did the patients treated with antibiotics. This prompted a
search in the literature for evidence of a direct effect of antibiotic
treatment on yeast colonization.  The
clearest evidence for antibiotic effects on yeast colonization came from the
paper by Caruso [1] who implicated tetracycline as increasing yeast
vaginal  colonization. Tetracycline was also implicated by Oriel and Waterworth
[5] who indicated that 14 days of tetracycline or minocycline treatment
increased vaginal Candida culture
positive rate from 13% to 29%.Although tetracycline was not employed
in Glover and Larsen’s analysis [4] because their study focused on pregnant
women, they did speculate that tetracycline may have had some direct effect on Candida independent of its effect on
competing vaginal bacterial flora. Previous studies showed that estradiol and
other drugs were able to affect the growth of Candida and the expression of certain virulence-related
factors [6, 7], implying that a variety of chemical compounds including tetracycline, may
exert a direct effect on Candida apart
from or in addition to its antimicrobial activity. It is against this
background that we investigated whether tetracycline may have direct effects on
the growth or expression of virulence attributes of Candida albicans. Such a discovery could illuminate the potential
relationship of antibiotics, particularly tetracycline, and candidiasis that is
distinct from the antimicrobial effect on the normal flora.


## 2. Materials and Methods

Human isolates ofCandida albicans were available in our
laboratory culture collection. These were originally obtained from gynecologic
specimens and identified as C. albicans by observing microscopic morphology, germ-tube formation in human serum, and
production of brown colonies on BIGGY agar. Candida were maintained at −80°C, and when needed, revived on Sabouraud’s
Dextrose Agar and held at 4°C until use in a specific
experiment. Prior to any experiment, the
organisms were removed from the refrigerated plate culture and subcultured in
an appropriate liquid medium to create an actively-growing starter culture.Six human isolates ofC. albicans were used. Three of these
had previously been shown to have growth stimulation in the presence of estrogen
(GT 387, GT 142, GT 397) and the other three showed restricted growth in the
presence of estrogen (GT 188, GT 132, 986). In addition, a special indicator organism, CaSA1 [8] which has a green
fluorescent protein gene inserted in place of one allele of the CDR1 structural gene, was used to
investigate regulation of CDR1. This gene encodes an ATP binding cassette
molecular pump that confers multidrug resistance on the organism and is
upregulated both by azole antifungal drugs as well as estradiol [9].Growth yield studies were performed
by diluting an overnight starter culture prepared in Yeast Nitrogen Broth with
0.5% glucose (YNB), and the culture was diluted104-fold in fresh YNB with and without additives
at final concentrations as described in the individual experiments. Experiments employed tetracycline or estrogen
(1, 3, 5[10]-estra-triene-3, 17β-diol, referred to hereafter as 17β-estradiol)
as additives in the growth experiments. 17β-estradiol was employed as a
positive control in some experiments since we knew from prior studies what
effect this compound had on the test strains used. When experimental cultures
were prepared, they were incubated overnight at 30°C or 37°C before
counts were made by hemacytometer.  For
consistency, hyphal forms were counted on the basis of the presumed number of
nuclei visualized. Budding cells were
counted as 2 when a constriction at the bud neck had formed between the mother
and daughter cells. While overnight
growth in YNB did produce some hyphal forms, they were not predominant and
contributed little to the overall counts performed by hemacytometer. For growth-rate determination, overnight
starter cultures were diluted ten folds into fresh medium and grown for approximately 2 hours to
ensure the culture we note
in stationary phase of growth.Evaluation of germination inCandida was performed by making a heavy
suspension of the test organism in serum or YNB, with or without tetracycline
and incubating at 37°C for 30 minutes and 3 hours. Cells are examined at each time point and presence
or absence of germ tubes determined by microscopic examination at 100x and 400x. Video microscopy was available to compare
images obtained from different culture conditions.Induction of theCDR-1 gene under various growth
conditions was analyzed by flow cytometry in the green fluorescence channel
(FL1) as described in our previous studies [8]. Data were normalized in relation to control values, and fluorescence of
treated cells was reported as fold-increase-over control (FIOC). Previous work
from our laboratory sought to determine if germinated or hyphal cells provided
a stronger fluorescent signal than yeast cells. If the instrument is gated on higher forward scatter values, we found
that mean channel fluorescence for high forward scatter particles did not
contribute disproportionately to the mean channel fluorescence of the
population.Induction of biofilm formation was
measured by creating biofilm on the bottoms of flat-well microtiter plates.
Briefly, overnight cultures of the selected test strains ofCandida were grown in unsupplemented
growth media or in media supplemented with 17β-estradiol or tetracycline at 30°C
or 37°C. Starter cultures of selected
yeast strains were diluted 10−4-fold and allowed to adhere to well
bottoms for 1 hour after which all wells were vigorously washed with a plate
washer to remove unbound yeast. Media
with or without test additives was replaced in the wells, and biofilm was
allowed to develop through overnight incubation at 37°C. At the conclusion of biofilm development, the
wells again were exhaustively washed and overlaid with 100 μL of 0.5% glucose
and 5 μL of Alimar Blue metabolic indicator. Plates were read
spectrophometrically at 30 minute intervals for 2 hours, and the area under the
time—absorbance600 curve (AUC) was taken as an indication of the amount of biomass adherent to the
wells.Hemolysin production was measured by
exposing supernatants of spentCandida cultures
to a 1% human erythrocyte suspension in phosphate buffered saline, removing
unlysed erythrocytes by centrifugation and reading the hemoglobin from lysed
erythrocytes in the supernatant fluid by spectrophotometry at 540 nm.


## 3. Results

### 3.1. Effects on Fungal Growth

The first question addressed by
this study was whether tetracycline could increase fungal growth, since direct growth
enhancement could explain why prior studies reported increased yeast
colonization after tetracycline therapy. To establish whether any concentration of tetracycline altered growth ofCandida albicans strains, various concentrations
were tested for their effects on overnight growth; concentrations of tetracycline
between 5 and 0.15 mg/mL were inhibitory toward Candida albicans with 5 mg/mL reducing hemacytometer counts to near
zero and 150 μg/mL reducing counts by half compared to the control containing
no tetracycline. While hemacytometer counts were useful in distinguishing the
obvious difference between tetracycline-treated and control cultures at high
tetracycline level, we turned to viable plate counts to elucidate more subtle
differences in growth of yeast exposed to sublethal concentrations of the drug.
In at least three separate trials with duplicate plate counts (not hemacytometer)
determinations, the effect of 0.1 and 0.3 μg/mL proved variable with
tetracycline-containing cultures having from 54–126% of control counts. Those
organisms previously shown to be stimulated by 17β-estradiol showed an
insignificant growth increment in the presence of tetracycline, whereas those
not stimulated by estrogen did not show increased growth. The magnitude of this
tendency was not sufficient to draw firm conclusions about possible growth
stimulation by tetracycline (Figure 1).Figure 1
The effect of adding a very low concentration of tetracycline
to chemically defined growth medium (YNB) on overnight viable counts of 6
strains of yeast is shown. Three strains of yeast (GT188, 986, and GT132) were
previously known to be nonsensitive to 17β-estradiol (did not show increased
growth yield in the presence of1×10−5 M/L 17β-estradiol), and 3 strains (GT387, 397,
and GT142) were previously shown to be sensitive to growth yield stimulation in
the presence of 17β-estradiol. Standard deviations from
triplicate determinations are shown by error bars. White bars represent growth in YNB, and black
bars represent growth in YNB with 0.1 μg/mL tetracycline.Because antibiotics may cause fitness-enhancing
generalized stress responses at sublethal concentrations and stress responses
may be induced rapidly, we also examined viable plate counts at 6 hours in lieu
of overnight growth for strain 142. Tetracycline concentrations from 0.3–5μg/mL increased counts by as much as four
folds (Table 1) indicating
an effect on growth may be transient but apparently not sustained long enough
to produce significantly more growth after overnight culture.Table 1
Effect of tetracycline in YNB on growth of strain 142 at 6 hours (37°C).
Tetracycline concentration (μg/mL)Viable plate count (average of duplicate platings)04.2× 1040.34.7× 1040.68.4× 1041.37.6× 1042.57.1× 10451.8× 105106.0× 104


### 3.2. Yeast Germination

The ability ofCandida albicans to form
germ tubes, pseudohyphae, or hyphae is considered an expression of the more
virulent forms of the organism. We tested several strains of yeast initially with 0.3 μg/mL of tetracycline in
liquid media and human serum to determine if tetracycline altered hyphal
production. YNB, a chemically-defined medium, showed the presence of many germ tubes after 3 hours
incubation at 37°C, and in human serum, even more extensive germ-tube
formation was seen. We were unable to discern an effect of tetracycline on
germination in either YNB or serum. These cultures were held at 37°C overnight and viewed
again. In YNB, there were mostly yeast
forms and a substantial number of pseudohyphae, and a similar morphologic
appearance was found among tetracycline-treated YNB cultures. In contrast,
culture in serum caused Candida transformation
to mostly hyphal growth. It was clear that tetracycline did not interfere with hyphal
formation at 0.3 μg/mL and may have enhanced hyphal formation. To investigate
this further, we tested additional concentrations of tetracycline (1, 3, and 5 μg/mL) and found that at 5 μg/mL it was possible to obtain evidence that in
both YNB (Figure 2) and in serum (Figure 3) tetracycline seemed to enhance
hyphal formation. The effect was particularly profound when serum was present,
a condition possibly more representative of conditions that prevail in vivo.Photomicrographs ofCandida
albicans (Strain GT142) grown overnight at 37°C in YNB or YNB
plus tetracycline (5 μg/mL). The yeast tended toward germination and
filamentation in YNB, and tetracycline did not inhibit this tendency. Images
are representative of many fields that indicate filamentous growth in the
presence of tetracycline that
is augmented and fungal masses appear larger than in control (YNB) cultures.
(a)(b)Photomicrographs ofCandida
albicans (Strain GT142) grown overnight at 37°C in serum or
serum plus tetracycline (5 μg/mL). The tendency of tetracycline to enhance
filamentous growth is particularly evident in the right-hand panel.
(a)(b)


### 3.3. Regulation of CDR1

In addition to effects on growth, tetracycline might alter the expression of virulence factors related to
colonization and symptoms in antibiotic-treated patients. The drug efflux pumpCDR1 has been shown to be promiscuously
upregulated by 17β-estradiol and coumarin and may be part
of a generalized stress response that enhances fitness of Candida [8]. With the availability of the CaSA1
organism [8], we
were able to examine the effect of tetracycline on CDR1 expression. The effect
on CDR1 expression was tested by incubation of CaSA 1 with or without  tetracycline
at 0.1, 0.3, 5, and 10 μg/mL after overnight incubation at 37°C. Flow cytometry was used to determine the
relative amount of GFP produced and the known inducer of CDR1 ; 17β-estradiol (at 1×10−5 M) was
used as a positive control. The maximum increase caused by tetracycline was
only a 30% increase relative to negative controls (Table 2). This level of
induction was significant (p=0.05, unpaired 2-tailed t-test) when compared to
the unstimulated CaSA 1 organism but was unremarkable compared to the known
inducer, estradiol (p=0.0004, 2-tailed unpaired t-test versus CaSA 1),
especially in view of the fact that the highest molar concentration
tetracycline was greater than that of estradiol.Table 2
Flow cytometry evaluation of CDR1 expression in response tetracycline exposure.
ConditionFl-1hour (mean channel fluorescence)FIOC (fold increase over control fluorescence)CaSA1 (organism background)5.07±0.881.0Estradiol (positive control)34.56±4.276.86±0.3910μg/mL tetracycline6.52±0.261.31±0.235μg/mL tetracycline5.54±0.791.10±0.130.3μg/mL tetracycline4.12±0.250.84±0.190.1μg/mL tetracycline3.77±0.110.76±0.13


### 3.4. Biofilm

Biofilm is increasingly being recognized as an important contributor to pathogenesis by mucosal organisms. The
potential for tetracycline to alter the biofilm production under in vitro
culture conditions was examined to gain support for a role of tetracycline in
enhancing symptomatic vulvovaginal candidiasis. As illustrated in Figure4,
overnight culture in the presence of tetracycline was not unequivocally associated
with increased production of biofilm at 37°C, although a slight, though not
statistically different, increase in biofilm metabolic activity was noted for 5
of 6 Candida strains tested.  It
was clear that this biological assay was characterized by substantial
variability as seen by the wide standard deviations and perhaps with a refined
method, the tendency toward biofilm formation in the presence of tetracycline
could be confirmed. In parallel with this study, the effect of nanomolar
estradiol induced a nonsignificant trend toward increased level of biofilm activity
(data not shown) at 37°C and as such provided data that appeared very similar
to tetracycline exposure.Figure 4
Biofilm was produced in the YNB growth media with or without the addition of tetracycline (0.1μg/mL). Biofilm was measured by allowing the metabolic indicator Alimar Blue to
be reduced by the biomass remaining after vigorous washing of the wells. The
relative biofilm measure represents the dye reduction over a 2-hour period
measured at 600 nm spectrophotometrically. Of note, 5 of the 6 strains tested
showed a tendency toward increased biofilm metabolic activity when exposed to
tetracycline, though the variability of the biological assay limited definite
conclusions regarding excess biofilm production.


### 3.5. Hemolysin

The final potential virulence
property evaluated in this study wasCandida albicans was hemolysin
which may aid the organism in acquiring iron in vivo. Hemolytic activity
in spent cultures  of Candida is typically consistently detected, but the
magnitude  of
effect is relatively low amounting to less than  5% hemolysis of a
1% erythrocyte suspension in 30 minutes. Hemolytic activity is summarized
in Figure 5 and shows that baseline hemolytic activity (in the absence of
tetracycline or estradiol) varied with strain, and minimal effect was observed
when tetracycline (0.1 μg/mL), or nanomolar estradiol was added
to the growth medium. Higher concentrations of tetracycline did not
appear to affect the hemolytic activity of spent cultures.Figure 5
Very little hemolytic activity was detected in cultures
ofCandida albicans regardless of the
presence or absence of added tetracycline (0.1 μg/mL). The greatest amount f
hemolysis detected was 4% lysis of a 1% erythrocyte suspension. As shown by the
figure, there was minimal difference between strains employed in this
evaluation. It may be concluded that tetracycline has a negligible effect on
hemolytic activity of Candida.


## 3.1. Effects on Fungal Growth

The first question addressed by
this study was whether tetracycline could increase fungal growth, since direct growth
enhancement could explain why prior studies reported increased yeast
colonization after tetracycline therapy. To establish whether any concentration of tetracycline altered growth ofCandida albicans strains, various concentrations
were tested for their effects on overnight growth; concentrations of tetracycline
between 5 and 0.15 mg/mL were inhibitory toward Candida albicans with 5 mg/mL reducing hemacytometer counts to near
zero and 150 μg/mL reducing counts by half compared to the control containing
no tetracycline. While hemacytometer counts were useful in distinguishing the
obvious difference between tetracycline-treated and control cultures at high
tetracycline level, we turned to viable plate counts to elucidate more subtle
differences in growth of yeast exposed to sublethal concentrations of the drug.
In at least three separate trials with duplicate plate counts (not hemacytometer)
determinations, the effect of 0.1 and 0.3 μg/mL proved variable with
tetracycline-containing cultures having from 54–126% of control counts. Those
organisms previously shown to be stimulated by 17β-estradiol showed an
insignificant growth increment in the presence of tetracycline, whereas those
not stimulated by estrogen did not show increased growth. The magnitude of this
tendency was not sufficient to draw firm conclusions about possible growth
stimulation by tetracycline (Figure 1).Figure 1
The effect of adding a very low concentration of tetracycline
to chemically defined growth medium (YNB) on overnight viable counts of 6
strains of yeast is shown. Three strains of yeast (GT188, 986, and GT132) were
previously known to be nonsensitive to 17β-estradiol (did not show increased
growth yield in the presence of1×10−5 M/L 17β-estradiol), and 3 strains (GT387, 397,
and GT142) were previously shown to be sensitive to growth yield stimulation in
the presence of 17β-estradiol. Standard deviations from
triplicate determinations are shown by error bars. White bars represent growth in YNB, and black
bars represent growth in YNB with 0.1 μg/mL tetracycline.Because antibiotics may cause fitness-enhancing
generalized stress responses at sublethal concentrations and stress responses
may be induced rapidly, we also examined viable plate counts at 6 hours in lieu
of overnight growth for strain 142. Tetracycline concentrations from 0.3–5μg/mL increased counts by as much as four
folds (Table 1) indicating
an effect on growth may be transient but apparently not sustained long enough
to produce significantly more growth after overnight culture.Table 1
Effect of tetracycline in YNB on growth of strain 142 at 6 hours (37°C).
Tetracycline concentration (μg/mL)Viable plate count (average of duplicate platings)04.2× 1040.34.7× 1040.68.4× 1041.37.6× 1042.57.1× 10451.8× 105106.0× 104


## 3.2. Yeast Germination

The ability ofCandida albicans to form
germ tubes, pseudohyphae, or hyphae is considered an expression of the more
virulent forms of the organism. We tested several strains of yeast initially with 0.3 μg/mL of tetracycline in
liquid media and human serum to determine if tetracycline altered hyphal
production. YNB, a chemically-defined medium, showed the presence of many germ tubes after 3 hours
incubation at 37°C, and in human serum, even more extensive germ-tube
formation was seen. We were unable to discern an effect of tetracycline on
germination in either YNB or serum. These cultures were held at 37°C overnight and viewed
again. In YNB, there were mostly yeast
forms and a substantial number of pseudohyphae, and a similar morphologic
appearance was found among tetracycline-treated YNB cultures. In contrast,
culture in serum caused Candida transformation
to mostly hyphal growth. It was clear that tetracycline did not interfere with hyphal
formation at 0.3 μg/mL and may have enhanced hyphal formation. To investigate
this further, we tested additional concentrations of tetracycline (1, 3, and 5 μg/mL) and found that at 5 μg/mL it was possible to obtain evidence that in
both YNB (Figure 2) and in serum (Figure 3) tetracycline seemed to enhance
hyphal formation. The effect was particularly profound when serum was present,
a condition possibly more representative of conditions that prevail in vivo.Photomicrographs ofCandida
albicans (Strain GT142) grown overnight at 37°C in YNB or YNB
plus tetracycline (5 μg/mL). The yeast tended toward germination and
filamentation in YNB, and tetracycline did not inhibit this tendency. Images
are representative of many fields that indicate filamentous growth in the
presence of tetracycline that
is augmented and fungal masses appear larger than in control (YNB) cultures.
(a)(b)Photomicrographs ofCandida
albicans (Strain GT142) grown overnight at 37°C in serum or
serum plus tetracycline (5 μg/mL). The tendency of tetracycline to enhance
filamentous growth is particularly evident in the right-hand panel.
(a)(b)


## 3.3. Regulation of CDR1

In addition to effects on growth, tetracycline might alter the expression of virulence factors related to
colonization and symptoms in antibiotic-treated patients. The drug efflux pumpCDR1 has been shown to be promiscuously
upregulated by 17β-estradiol and coumarin and may be part
of a generalized stress response that enhances fitness of Candida [8]. With the availability of the CaSA1
organism [8], we
were able to examine the effect of tetracycline on CDR1 expression. The effect
on CDR1 expression was tested by incubation of CaSA 1 with or without  tetracycline
at 0.1, 0.3, 5, and 10 μg/mL after overnight incubation at 37°C. Flow cytometry was used to determine the
relative amount of GFP produced and the known inducer of CDR1 ; 17β-estradiol (at 1×10−5 M) was
used as a positive control. The maximum increase caused by tetracycline was
only a 30% increase relative to negative controls (Table 2). This level of
induction was significant (p=0.05, unpaired 2-tailed t-test) when compared to
the unstimulated CaSA 1 organism but was unremarkable compared to the known
inducer, estradiol (p=0.0004, 2-tailed unpaired t-test versus CaSA 1),
especially in view of the fact that the highest molar concentration
tetracycline was greater than that of estradiol.Table 2
Flow cytometry evaluation of CDR1 expression in response tetracycline exposure.
ConditionFl-1hour (mean channel fluorescence)FIOC (fold increase over control fluorescence)CaSA1 (organism background)5.07±0.881.0Estradiol (positive control)34.56±4.276.86±0.3910μg/mL tetracycline6.52±0.261.31±0.235μg/mL tetracycline5.54±0.791.10±0.130.3μg/mL tetracycline4.12±0.250.84±0.190.1μg/mL tetracycline3.77±0.110.76±0.13


## 3.4. Biofilm

Biofilm is increasingly being recognized as an important contributor to pathogenesis by mucosal organisms. The
potential for tetracycline to alter the biofilm production under in vitro
culture conditions was examined to gain support for a role of tetracycline in
enhancing symptomatic vulvovaginal candidiasis. As illustrated in Figure4,
overnight culture in the presence of tetracycline was not unequivocally associated
with increased production of biofilm at 37°C, although a slight, though not
statistically different, increase in biofilm metabolic activity was noted for 5
of 6 Candida strains tested.  It
was clear that this biological assay was characterized by substantial
variability as seen by the wide standard deviations and perhaps with a refined
method, the tendency toward biofilm formation in the presence of tetracycline
could be confirmed. In parallel with this study, the effect of nanomolar
estradiol induced a nonsignificant trend toward increased level of biofilm activity
(data not shown) at 37°C and as such provided data that appeared very similar
to tetracycline exposure.Figure 4
Biofilm was produced in the YNB growth media with or without the addition of tetracycline (0.1μg/mL). Biofilm was measured by allowing the metabolic indicator Alimar Blue to
be reduced by the biomass remaining after vigorous washing of the wells. The
relative biofilm measure represents the dye reduction over a 2-hour period
measured at 600 nm spectrophotometrically. Of note, 5 of the 6 strains tested
showed a tendency toward increased biofilm metabolic activity when exposed to
tetracycline, though the variability of the biological assay limited definite
conclusions regarding excess biofilm production.


## 3.5. Hemolysin

The final potential virulence
property evaluated in this study wasCandida albicans was hemolysin
which may aid the organism in acquiring iron in vivo. Hemolytic activity
in spent cultures  of Candida is typically consistently detected, but the
magnitude  of
effect is relatively low amounting to less than  5% hemolysis of a
1% erythrocyte suspension in 30 minutes. Hemolytic activity is summarized
in Figure 5 and shows that baseline hemolytic activity (in the absence of
tetracycline or estradiol) varied with strain, and minimal effect was observed
when tetracycline (0.1 μg/mL), or nanomolar estradiol was added
to the growth medium. Higher concentrations of tetracycline did not
appear to affect the hemolytic activity of spent cultures.Figure 5
Very little hemolytic activity was detected in cultures
ofCandida albicans regardless of the
presence or absence of added tetracycline (0.1 μg/mL). The greatest amount f
hemolysis detected was 4% lysis of a 1% erythrocyte suspension. As shown by the
figure, there was minimal difference between strains employed in this
evaluation. It may be concluded that tetracycline has a negligible effect on
hemolytic activity of Candida.


## 4. Discussion

This study focused on the possibility that growth ofCandida
albicans and the
expression of virulence attributes may be altered by the exposure of this
fungal pathogen to tetracycline. A long-standing medical dogma states that
antibiotic therapy is often accompanied by development of symptomatic yeast
vaginitis, though in a study we conducted during pregnancy we found that antibiotics
other than tetracycline did not promote yeast vaginitis. Because an older study
of tetracycline [1] linked prolonged treatment with increasing rates of vaginal
yeast colonization, we intended to look for direct effects of tetracycline on
yeast growth and virulence attribute expression.Tetracycline,
like 17 ß-estradiol, has a phenolic A ring, although the hydroxyl group in
tetracycline is in the 2 instead of 3 position as it is in estradiol.
Nevertheless, our previous finding that 17 ß-estradiol at concentrations
ranging from1×10−5 to 1×10−9 M/L increases Candida growth in some strains, and
certain virulence attributes are elevated by 17 ß-estradiol, encouraged the
current study since it was not clear precisely what chemical signature
accounted for some of the previously observed direct effects of 17 ß-estradiol
on yeast including growth stimulation and upregulation of drug efflux
mechanisms.Because
many biological effects of 17β-estradiol
have been observed inCandida albicans [6–11], we reasoned
that tetracycline may have chemical similarities that could elicit similar
direct effects on the organism such as increasing growth or upregulating
virulence factors. These biological
effects could help explain the role of tetracycline in symptomatic yeast
vaginitis in a manner that goes beyond the medical dogma that attributes
vaginal infection to bacterial killing and simultaneous fungal sparing.
However, tetracycline does appear to have antifungal activity, at least when
present in high concentration as noted in the present study, raising new
questions about old beliefs about pathogenesis of vaginal candidiasis.After
submitting the present manuscript we became aware that White’s group published
an important study [12] that indicated that tetracycline  was
inhibitory toward Candida, Cryptococcus, and Aspergillus. However, their research focused on in vitro
tetracycline concentrations that were 1-2 orders of magnitude larger than the
levels investigated in this paper. Still, it is important to realize that tetracycline, while not used as
an antifungal drug, does have inhibitory properties that are dose related.In the
current study, we focused on tetracycline concentrations in the range of 0.1–10μg/mL (2.1×10−7 M to 2.1×10−5 M) which is in the range of typical serum
concentrations among individuals being treated [13]. At higher concentrations (3–5 mg/mL),
tetracycline proved to be antibiotic toward Candida
albicans, particularly when the yeast strains were cultivated
overnight with the drug. A slight increase in growth was obtained with
incubation times shorter than overnight with submicrogram/mL concentrations
among some, but not all, yeast strains. Moreover,
the magnitude of growth increases was quite small and not sufficient to support
the hypothesis that growth stimulation by tetracycline was the likely mechanism
predisposing to yeast vaginitis. We noted that the tendency of growth effects
of tetracycline seemed to mirror those we had previously seen for estradiol,
underscoring our belief that as a chemical entity, tetracycline might have some
biological effects similar to estradiol. These growth studies may deserve
additional evaluation in the future.Although preliminary
growth studies suggested thatCandida
albicans, like bacteria, may be adversely affected by high levels of tetracycline,
and the small magnitude of growth stimulation at low concentrations makes
direct growth stimulation an unlikely sole explanation for increased vaginitis
among tetracycline treated women. However, a growth increment coupled with
elevated virulence of yeast offers a potential explanation for tetracycline
promotion of symptomatic yeast infection.Four-virulence
attributes were studied mainly at the phenotypic level and appeared to be
affected variously by exposure of the yeast to tetracycline. Most striking
among these was the finding that germination and related transition from yeast
to hyphal growth were  augmented in the presence of tetracycline, and
this was especially true when serum was present. This finding has practical
relevance to the potential effects of tetracycline in vivo because the hyphal
form of the organism is reputed to be its most virulent state. Had this study
indicated that tetracycline prevents hyphal transformation instead of
augmenting it, it would be difficult to suggest how tetracycline treatment
might contribute to yeast vaginitis.For
sometime, we have hypothesized that the drug efflux pumpCDR1 may serve the organism to enhance its fitness in hostile environments [11]. To examine
the effect of tetracycline on CDR1 expression, a special recombinant indicator
strain was employed which explains why this experiment did not involve the
panel of test organisms used in the remainder of the study. The yeast CaSA1
produces green fluorescent protein in response to azole drugs and exposure to
estradiol [8]; and when sublethal concentrations of tetracycline were
introduced to cultures of CaSA1, it was found that a slight (30%) increase in
fluorescence occurred at 37°C at 10 μg/mL, but not
at lower levels. In the recent study from White’s group [12], they failed to
find upregulation of CDR1 at the
levels of tetracycline they employed. Higher levels of tetracycline are likely
to suppress protein synthesis as suggested by our growth studies CDR1 expression that was not studied at concentrations of
tetracycline that inhibited fungal growth. Because CDR1 expression
appears to be adaptive under adverse conditions, this factor could
incrementally help the yeast to survive under hostile conditions; although as
demonstrated by our positive controls, the upregulation of CDR1 in the presence of tetracycline was far below levels attained
with equimolar levels of estradiol (5 μg/mL tetracycline was approximately equal
to the molarity of the estradiol positive control).The
virulence attributes of biofilm and hemolysin production were also examined,
and there was a tendency toward increased biofilm formation which, though not
significant in magnitude, was at least fairly consistent across test strains
evaluated. There was clearly a notable degree of variability (see Figure4)
among replicate tests for biofilm production, but because it was a biological
rather than strictly analytical method, the variability was not surprising, but
did undermine our ability to unequivocally demonstrate tetracycline as a
stimulant of biofilm.Hemolysin, as shown by Figure5 which in Candida,
is poorly characterized, tended to be slightly increased in the presence of
tetracycline, but the effect was modest and consequently unlikely to have
biological significance. It is difficult to claim with certainty from this
study that any tendency of women to develop vaginitis symptoms after
tetracycline therapy is due to direct effects of the drug on biofilm or
hemolysin, but the slight effects on early growth and more profound effects on hyphal formation could
contribute to yeast vaginitis.It is
worth noting that a plethora of genes inCandida have been identified as having an association with virulence. This list is
large and continues to grow, but importantly available molecular methods for
analysis of expression of virulence genes could be applied to Candida exposed to tetracycline. We have
not yet delved into studies of expression of these specific genes because we
preferred in this preliminary work, to establish a sufficient biological cause
on a phenotypic level to warrant additional studies of putative virulence
genes.We
conclude from this investigation that tetracycline, though displaying an
antifungal effect at high concentrations, has a small be detectable stimulating
effect on growth rate at sublethal concentrations. This effect in conjunction
with the more potent is the effect of tetracycline on hyphal morphogenesis, and
possible enhancement of biofilm formation may contribute to the tetracycline's
relationship to symptomatic yeast vaginitis. While not unequivocally
demonstrating that tetracycline affectsCandida and thereby predisposing to vaginitis, it does suggest that future research
should pursue this hypothesis.Finally,
we return to the medical dogma that prompted this report. The concept that
vaginal symptoms developing after tetracycline therapy is merely the result of
reduction in bacterial populations with a simple overgrowth of yeast is
commonly repeated. This explanation, in light of the current study, appears too
simplistic as high levels of tetracycline inhibitCandida which should reduce the risk of vaginitis, but antifungal
concentrations may not be attainable with typical doses. In contrast, sublethal
concentrations appear to affect the physiology of the yeast in a way consistent
with predisposition to symptomatic yeast vaginitis which may modify the view of
pathogenesis of vaginitis following tetracycline treatment.



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*Source: 493508-2008-05-29.xml*