Anti-fungal activity of sulfamethoxazole toward Aspergillus species

Article (PDF Available)inBiological & Pharmaceutical Bulletin 28(5):773-8 · June 2005with81 Reads
Source: PubMed
Abstract
Invasive mycosis has significantly increased in frequency among immunocompromised hosts leading to excessive morbidity and mortality. The combination of sulfamethoxazole (SMX) and trimethoprim (TMP) has been used extensively for the treatment and prophylaxis of infections by various microbes. The purpose of this study is to estimate the anti-fungal activity of SMX-TMP and examine the mechanism of activity. To investigate the antimicrobial activity of SMX-TMP in vitro, a mixture of SMX and TMP at 5:1 was serially diluted and added to potato dextrose agar medium or C-limiting agar medium. Aspergillus species were inoculated on the medium plate with SMX-TMP. The growth of A. fumigatus and A. oryzae was inhibited by addition of SMX-TMP. The anti-Aspergillus effect depended on not TMP but SMX and that was inhibited by p-aminobenzoic acid (PABA). A. niger was not sensitive against SMX-TMP in PDA medium, but sensitive in C-limiting medium. Those results showed that the activity depends on culture medium. Furthermore, addition of human serum did not influence the activity of SMX. The finding in this study suggested that SMX might be effective against Aspergillus species in clinical practice and prophylaxis treatment.
Opportunistic infections are becoming increasingly com-
mon due to the growing number of individuals immunocom-
promised by chemotherapy or immunosuppressants, such as
cyclosporine and steroids.
1)
For example, immunosuppres-
sants are necessary for the treatment of autoimmune diseases
such as rapidly progressive glomerulonephritis (RPGN),
nephrotic syndrome.
2)
Recently, fugal infections have espe-
cially become a problem.
3—5)
Invasive Aspergillosis induced
by Aspergillus species has significantly increased in fre-
quency among immunocompromised hosts leading to exces-
sive morbidity and mortality.
6,7)
The high mortality of As-
pergillosis is due in part to difficulties of diagnosis in that
signs and symptoms are often nonspecific and usually appear
at the late stages of disease.
8)
Therefore, prophylaxis of As-
pergillus infection is important in therapy for autoimmune
disease.
9)
Sulfamethoxazole (SMX) is a sulfa drug that inhibits the
metabolism of folate in microorganisms. It acts as a competi-
tive antagonist of p-aminobenzoic acid (PABA), which is a
component of the biosynthesis of folic acid. Then it inhibits
the enzyme dihydropteroate synthase (DHPS), which cat-
alyzes the condensation of PABA with pteridine, forming di-
hydropteroic acid. Clinically, SMX has been used in combi-
nation with trimethoprim (TMP) which inhibits another en-
zyme, dihydrofolate reductase (DHFR), involved in the me-
tabolism of folate. Because SMX and TMP inhibit different
enzymes in the same pathway, their combination (SMX-
TMP) shows antimicrobial activity synergistically and
against a broad spectrum of gram-positive and gram-negative
bacteria.
SMX-TMP has been used extensively for treatment and
prophylaxis to prevent pneumocystis carinii pneumonia in
AIDS patients and in other immunocompromised indivi-
duals.
10—12)
Moreover, it was reported that SMX is active in
vitro against Aspergillus species and therefore might help to
prevent invasive Aspergillosis in AIDS patients receiving
SMX-TMP.
13,14)
Such reports suggest that sulfa drugs have
anti-fungal activity. Although there have been many papers
about the anti P. carinii activity of sulfonamides, there have
been few about anti-Aspergillus species and Candida
species. Therefore, we investigated the anti-Aspergillus activ-
ity of SMX-TMP in detail.
The purpose of this study is to evaluate the anti-fungal ac-
tivity of SMX-TMP and examine the mechanism of this ac-
tivity. Furthermore, we estimated in vitro whether SMX-
TMP actually acts in the human body by adding human
serum to medium with drugs and fungi.
MATERIALS AND METHODS
Materials SMX, TMP, folic acid (FA) and PABA were
purchased from Sigma Chemical Company (U.S.A). Filter
paper was purchased from ADVANTEC Toyo Roshi kaisha,
Ltd, Japan. Aspergillus fumigatus IFO 30870, Aspergillus
niger IFO 6342, Aspergillus oryzae IFO 30103, Candida al-
bicans IFO 1385, and Candida parapsilosis IFO 1068 were
purchased from the Institute for Fermentation, Osaka, Japan
(IFO). The fungi were maintained on plate of Potato dextrose
agar (PDA) purchased from Difco, U.S.A. at 27 °C and trans-
ferred to a new medium once every three months. Human
sera were collected from healthy donors.
Media PDA and C-limiting medium, originally de-
scribed by Shepherd and Sullivan,
15)
were used in experi-
ments on antimicrobial activity. The C-limiting agar medium
that we used contained (per liter) sucrose 10 g, (NH
4
)
2
SO
4
2 g, KH
2
PO
4
2g, CaCl
2
·2H
2
O 0.05 g, MgSO
4
·7H
2
O 0.05 g,
ZnSO
4
·7H
2
O 1 mg, CuSO
4
·5H
2
O 1mg, FeSO
4
·7H
2
O 0.01 g,
biotin 25
m
g, and agar 10 g, with a final pH of 5.2.
Measurement of Anti-fungal Activity by Plate Culture
Added with Antimicrobial Agent SMX and TMP were
dissolved in methanol at an initial concentration of 50 mg/ml.
SMX is now used mainly as a mixture with TMP
32)
and a
May 2005 Biol. Pharm. Bull. 28(5) 773—778 (2005) 773
To whom correspondence should be addressed. e-mail: ohnonao@ps.toyaku.ac.jp © 2005 Pharmaceutical Society of Japan
Anti-fungal Activity of Sulfamethoxazole toward Aspergillus Species
Shunsuke HIDA,
a
Masaharu YOSHIDA,
b
Iwao NAKABAYASHI,
b
Noriko N. MIURA,
a
Yo shiyuki ADACHI,
a
and Naohito OHNO
*
,a
a
Laboratory for Immunopharmacology of Microbial Products, School of Pharmacy, Tokyo University of Pharmacy and
Life Science; 1432–1 Horinouchi, Hachioji, Tokyo 192–0392, Japan: and
b
Division of Nephrology, Hachioji Medical
Center of Tokyo Medical University; 1163 Tate-machi, Hachioji, Tokyo 193–0944, Japan.
Received December 22, 2004; accepted February 26, 2005; published online March 2, 2005
Invasive mycosis has significantly increased in frequency among immunocompromised hosts leading to ex-
cessive morbidity and mortality. The combination of sulfamethoxazole (SMX) and trimethoprim (TMP) has been
used extensively for the treatment and prophylaxis of infections by various microbes. The purpose of this study is
to estimate the anti-fungal activity of SMX-TMP and examine the mechanism of activity. To investigate the an-
timicrobial activity of SMX-TMP in vitro, a mixture of SMX and TMP at 5 : 1 was serially diluted and added to
potato dextrose agar medium or C-limiting agar medium. Aspergillus species were inoculated on the medium
plate with SMX-TMP. The growth of A. fumigatus and A. oryzae was inhibited by addition of SMX-TMP. The
anti-Aspergillus effect depended on not TMP but SMX and that was inhibited by p-aminobenzoic acid (PABA).
A. niger was not sensitive against SMX-TMP in PDA medium, but sensitive in C-limiting medium. Those results
showed that the activity depends on culture medium. Furthermore, addition of human serum did not influence
the activity of SMX. The finding in this study suggested that SMX might be effective against Aspergillus species
in clinical practice and prophylaxis treatment.
Key words sulfamethoxazole; trimethoprim; Aspergillus species; Aspergillus fumigatus; chemotherapy; prophylaxis
mixture SMX and TMP at a ratio of 5 : 1 has marketed in
Japan. Therefore, SMX-TMP as an antimicrobial agent was
prepared by mixing SMX and TMP at a ratio of 5 : 1, and
dissolved similarly. Then, the agents were diluted 2 times se-
rially in methanol at a concentration of 25, 12.5, 6.25 or
3.125 mg/ml. The diluted forms were added to PDA or C-
limiting agar medium in order to obtain final concentrations
of 500, 250, 125, 62.5 and 31.25
m
g/ml while the medium
melted.
A. fumigatus, A. niger, A. oryzae, C. albicans, and C.
parapsilosis were passaged at an interval of 7 d at 27 °C by
subculturing on PDA plates to obtain adequate sporulation.
Conidia of Aspergillus species were collected and suspended
in saline. In the case of Candida species, a single colony was
removed and suspended in saline. This homogeneous suspen-
sion (10
m
l) was inoculated onto the center of a plate of PDA
or C-limiting agar medium. The inoculation was performed
in four plates. Each strain was incubated stationary for 7 d at
27 °C.
After incubation, the diameter of the giant colony was
measured.
Anti-fungal Activity of SMX Alone, TMP Alone or the
Combination SMX and TMP were dissolved in methanol
at an initial concentration of 10 mg/ml. SMX-TMP was pre-
pared at 10 mg/ml. The agents were added to C-limiting agar
medium in order to obtain final concentrations of 100
m
g/ml
while the medium melted. As described in protocol, suspen-
sion of A. fumigatus, A. niger or A. oryzae were prepared and
placed onto the center of a plate. After incubation for 7 d, the
colony size was measured.
Reversal of the Inhibition of the Antimicrobial Agent
by Exogenous Folic Acid and p-Aminobenzoic Acid FA
and PABA were dissolved in saline at 1 mg/ml. Then they
were diluted 2 times serially in saline at a concentration of
500, 250, 125, or 0
m
g/ml. The diluted FA and PABA were
added to PDA plates with SMX-TMP at 100
m
g/ml in order
to obtain final concentrations of 10, 5, 2.5, 1.25 and 0
m
g/ml
while the medium melted. A PDA plate with methanol in-
stead of SMT-TMP was prepared as a control.
As described above, A. fumigatus was inoculated, the di-
ameter of the giant colony was measured.
Reversal of Anti-A. niger Activity by Exogenous PABA
in C-Limiting Agar Medium with SMX-TMP PABA w a s
dissolved in saline at 1mg/ml and was diluted 2 times serially
in saline at a concentration of 500, 250, 125 or 0
m
g/ml. The
diluted PABA was added to PDA plate with SMX-TMP at
100
m
g/ml in order to obtain final concentrations of 10, 5,
2.5, 1.25 and 0
m
g/ml while the medium melted. A C-limit-
ing agar plate with methanol instead of SMT-TMP was pre-
pared as a control.
As described above, conidia of A. niger were collected and
inoculated into the center of a plate of C-limiting agar. The
diameter of the giant colony was measured.
Detection of Inhibitors for the Anti-A. niger Activity of
SMX-TMP in PDA Water soluble fraction of PDA (sPDA)
was prepared from Potato dextrose agar (Difco, U.S.A.) by
centrifugation. After centrifugation, the supernatant was fil-
trated for sterilization. A part of sPDA was dialyzed to re-
move low molecular weight materials including PABA. After
the dialysis, supernatant fluid (dPDA) was centrifuged and
filtrated for sterilization. These fractions were prepared be-
fore use.
Pieces of filter paper cut in a circle of diameter 6 mm were
sterilized by autoclave. After adequately drying, the pieces
were soaked with saline as a control, PABA in saline at
100
m
g/ml, the fraction of sPDA and the fraction of dPDA.
Conidia of A. niger were collected and suspended in saline.
The suspension (1 ml) of A. niger was inoculated homoge-
neously onto a plate of C-limiting agar with SMX-TMP at
100
m
g/ml or 0
m
g/ml. Then, pieces of filter paper soaked
with saline (upper left), PABA (upper right), sPDA (lower
left) or dPDA (lower right), were placed on PDA plates with
or without SMX-TMP. A. niger was incubated stationary for
2d at 27 °C.
After incubation, it was observed whether A. niger grew
around the filter paper with saline, PABA, sPDA or dPDA.
Detection of Inhibitors for the Activity of SMX-TMP in
Human Serum Pieces of filter paper cut in a circle 6 mm
in diameter were sterilized by autoclave. After adequately
drying, those pieces were soaked with saline as a control,
PA BA in saline at 100
m
g/ml, Folic acid in saline at
100
m
g/ml or human serum.
Conidia of A. fumigatus were collected and suspended in
saline as described above. The suspension (1 ml) was inocu-
lated homogeneously onto a plate of PDA medium with
SMX-TMP at 100
m
g/ml or 0
m
g/ml. The inoculation was
performed in duplicate.
Then, the circular pieces of filter paper soaked with saline
(upper left), FA (upper right), PABA (lower left) or human
serum (lower right), were placed on PDA plates with or with-
out SMX-TMP. A. fumigatus on the plate was incubated sta-
tionary for 2 d at 27 °C.
After incubation, it was observed whether A. fumigatus
grew around the filter paper with saline, FA, PABA or human
sera.
RESULTS
SMX Shows Antimicrobial Activity in Vitro against As-
pergillus Species and C. albicans, but the Activity De-
pended on Culture Medium A. fumigatus, A. niger and A.
oryzae as Aspergillus species were incubated on PDA
medium. Colony sizes of A. fumigatus and A. oryzae de-
creased with the increase of SMX-TMP in medium. But the
colony of A. niger was unchanged despite of a larger dose of
drug (Fig. 1A). Notably, the colony of A. fumigatus de-
creased in size by 50% on addition of SMX-TMP at about
100
m
g/ml.
With a similar protocol, A. fumigatus, A. niger and A.
oryzae were incubated on C-limiting agar medium that con-
tained serially diluted SMX-TMP. In the absence of drugs,
the colony of Aspergillus species in C-limiting agar medium
was smaller than that in PDA medium (Fig. 1B). Moreover,
the 50% inhibition dose of SMX-TMP against Aspergillus
species was lower than that in PDA medium. It is of note that
Aspergillus niger which was resistant to SMX-TMP in PDA
medium, was sensitive to SMX-TMP in C-limiting agar
medium.
Figure 1 showed that the anti fungal activity of SMX-TMP
differed among Aspergillus species. Therefore, the activity
against Candida albicans and Candida parapsilosis were ex-
amined using a similar protocol. Neither C. albicans nor C.
774 Vol. 28, No. 5
parapsilosis was sensitive to SMX-TMP in PDA medium
(Fig. 2A). In C-limiting medium, C. albicans was sensitive to
SMX-TMP (Fig. 2B), but C. parapsilosis was not sensitive
despite of high medicine dose at 500
m
g/ml. This result
shows that effect of SMX-TMP varies in fungal strain.
Clinically, SMX and TMP are used as a mixture of 1 : 5.
To examine whether either SMX or TMP has an anti-As-
pergillus effect, SMX or TMP was added to PDA medium
and A. fumigatus was cultured on these medium. Figure 3A
shows that SMX exhibited anti A. fumigatus activity but
TMP did not. Furthermore, The activity of SMX or TMP
alone was investigated against other Aspergillus species. All
other Aspergillus species were sensitive against SMX alone
and SMX-TMP as well as A. fumigatus (Fig. 3B).
Next, to examine whether A. niger produces inhibitors of
antibiotics, we performed a coculture of A. niger with A. fu-
migatus in PDA medium with SMX-TMP. If A. niger pro-
duces anti antibiotic components, they may affect the growth
of A. fumigatus. But the colony size of A. fumigatus was the
same as in the case of the single culture (Fig. 4). The result
ruled out that A. niger produces components against anti
chemotherapeutic agent.
p-Aminobenoate Reversed the Effects of the Active
Sulfa Drugs, But Folate Did Not Sulfonamides such as
SMX act as a competitor antagonist of PABA and demon-
strate anti microbial activity by inhibiting folate synthesis. To
evaluate whether the anti fungal activity depends on inhibi-
tion of folate synthesis, folic acid or PABA were added to
PDA medium with SMX-TMP at 100
m
g/ml and A. fumiga-
tus was cultured in this medium. However, despite a high
concentration (10
m
g/ml), addition of folate could not reverse
the inhibition by SMX-TMP (Fig. 5A). It was suggested that
A. fumigatus could not use folic acid for the metabolism of
folate. Therefore, PABA that is competed by SMX was added
to PDA medium with drugs. The addition of PABA reversed
the effects of SMX-TMP (Fig. 5A). The quantity that had an
effect on reversion was smaller than 1
m
g/ml. The result sug-
gested that Aspergillus species needs PABA for folate usage.
From the results in Fig. 1, it was clear that the sensitivity
of A. niger to SMX-TMP differed with the culture media.
Therefore, to examine whether the sensitivity of A. niger de-
pends on PABA in the medium, A. niger was cultured in the
C-limiting medium plate with PABA and SMX-TMP at
100
m
g/ml. Colony size of A. niger increased dependent on
the dose of PABA added to the plate (Fig. 5B). Furthermore,
because PDA may contain inhibitors of SMX-TMP like
May 2005 775
Fig. 1. Anti-Aspergillus Activity of the Combination of SMX and TMP
Conidia from Aspergillus species were suspended in saline, and the suspension was
placed onto the center of PDA (A) or C-limiting agar (B), a synthetic medium with
SMX-TMP at serial concentrations. After incubation for 7 d at 27 °C, giant colonies
were observed. Symbols in the figure represent ; A. fumigatus, ; A. oryzae, ; A.
niger. The error bars here represent standard deviations.
Fig. 2. Anti-Candida Activity of the Combination of the SMX and TMP
A single colony from Candida albicans and Candida parapsilosis was suspended in
saline, and the suspension was placed at the center of PDA (A) or C-limiting agar (B)
medium with SMX-TMP at serial concentrations. A. fumigatus was incubated as a con-
trol. After incubation for 7 d at 27 °C, the diameter of the giant colony was. Symbols in
the figure represent ; A. fumigatus, ; Candida albicans, ; Candida parapsilosis.
The error bars here represent standard deviations.
Fig. 3. Anti-fungal Activity of SMX Alone, TMP Alone, or the Combina-
tion
Conidia from A. fumigatus, A. niger, A. oryzae and the yeast of C. albicans was sus-
pended in saline, (A) the suspension of A. fumigatus was placed at the center of PDA
medium with SMX-TMP (), SMX alone () or TMP alone () at serial concentra-
tions. After incubation, the colony size was measured. (B) The other fungal suspension
was placed at the center of C-limiting medium with SMX-TMP, SMX alone, or TMP
alone at 100
m
g/ml. According to similar protocol, the colony size was measured. The
error bars here represent standard deviations.
Fig. 4. Mixed Culture of A. fumigatus and A. niger in PDA Medium with
SMX-TMP
Conidia from A. fumigtus and A. niger were suspended in saline, and the suspension
was placed onto PDA medium with SMX-TMP at 0 (A) or 100
m
g/ml (B). The culture
was performed with A. fumigatus alone (Left), A. niger alone (middle) or both A. fumi-
gatus and A. nigar (Right). No interaction of A. fumigatus and A. niger in plates with or
without SMX-TMP was observed.
PA BA as a cause of why SMX-TMP did not affect A. niger in
PDA medium, we examined whether the addition of the
water-soluble fraction of PDA inhibits the effect of SMX-
TMP on A. niger. A part of the water-soluble fraction of
PDA was dialyzed to remove low weight molecules such as
PA BA (Mw 137). Round pieces of filter paper were soaked
with saline, PABA, the sPDA and dPDA, and were placed on
A. niger culture in the presence of SMX-TMP at 100
m
g/ml
in C-limiting agar medium. The culture was performed for
48 h at 27 °C. Colonies of A. niger germinated around the
paper treated with PABA and sPDA, but not dPDA (Fig. 6B).
The results suggested that sPDA contains inhibitors of SMX-
TMP whose molecular weights are low such as PABA and A.
niger is insensitive to SMX-TMP because of these inhibitors.
No Factors that Inhibit the Activity of SMX-TMP Exist
in Human Serum As shown in Fig. 6, SMX-TMP does not
have anti-fugal activity in the presence of PABA. Similar in-
hibitors may be present in patient’s blood. Therefore, it was
examined whether the addition of human serum inhibits the
effects of SMX-TMP. Pieces of filter paper with a diameter
of 6 mm were soaked with saline, PABA, Folic acid or human
serum, and placed on A. fumigatus culture in the presence of
SMX-TMP at 100
m
g/ml. Cultures were performed for 48 h
at 27 °C. After 48 h, colonies of A. fumigatus germinated
around the paper treated with PABA but not human serum
(Fig. 6A). In the case of folic acid, growth was not as good as
the result in Fig. 5A. This result suggested that factors which
inhibit the activity of SMX-TMP do not exist in human
serum and SMX-TMP shows anti-fungal activity in vivo.
DISCUSSION
In the present study, we confirmed the anti-fungal effect of
SMX and TMP used against a broad spectrum of gram-posi-
tive and gram-negative bacteria. The addition of approxi-
mately 100
m
g/ml of SMX-TMP into media decreased of the
colony size by 50%. Cmax of SMX and TMP is respectively
50—60
m
g/ml and 1.5—2.5
m
g/ml. Therefore, the concentra-
tion of 100
m
g/ml may be a little high level, but actually it
can be this concentration.
33,34)
Furthermore, we have shown
that the anti-fungal effect depends on not TMP but SMX and
that it is inhibited by PABA which antagonizes SMX. Those
results suggested that SMX-TMP used as a bactericidal agent
has not only an anti-bacterial effect but also an anti-fungal
one. In other words, treatment with SMX-TMP may protect
immunocompromised patients from fungal infections. There
may, therefore be other medicines that have unknown benefi-
cial effects.
SMX inhibited the growth of A. fumigatus and A. oryzae
but not A. niger, Candida albicans or Candida parapsilosis
in PDA (Figs. 1A, 2A). However, in C-limiting agar medium
a synthetic medium, the growth of A. niger and C. albicans
were suppressed by adding SMX (Fig. 1B). Moreover, addi-
tion of PABA, not folic acid, restored the growth of As-
pergillus species (Fig. 5, 6). These results suggested that the
effect of SMX depends on the components of the culture
medium. Verweij et al. reported anti-Aspergillus activity of
SMX.
13)
They used two media, RPMI 1640 and yeast nitro-
gen base (YNB). RPMI medium contains four times more
PA BA than YNB. The result showed that the MICs obtained
with RPMI 1640 medium were significantly higher than
those obtained with YNB medium. In the present study, we
also used two media, C-limiting medium and PDA medium.
C-limiting medium is a synthetic medium and does not con-
tain PABA, whereas PDA medium is a natural medium that
may contain many nutrients, such as PABA and folic acid, for
the growth of microorganism. Figure 6B shows that PDA
medium contains inhibitors of SMX-TMP activity. There-
fore, the presence of PABA explains the difference in the
anti-A. niger activity of SMX differs between the two culture
media. But, A. fumigatus was inhibited in both C-limiting
and PDA medium. Furthermore, in C-limiting medium, the
colony of A. fumigatus was smaller than that in PDA
medium, whereas there was little change with the two media
in the colony size of A. niger (Fig. 1). The difference in
colony growth of A. fumigatus and A. niger may depend on
the folate synthetic pathway. A. fumigatus and A. oryzae
which are highly sensitive to sulfa drugs such as SMX may
need many folate components, whereas A. niger may require
little folate. Therefore, Fig. 5 suggested that Aspergillus
species have folate synthesis pathway and need for growth.
In study for utilization of exogenous folaes in yeast, one mu-
tant lacked DHPS and DHFS grow up by addition of exoge-
nous folinic acid but not folic acid.
35)
Aspergillus species
776 Vol. 28, No. 5
Fig. 5. Reversal of Anti-chemotherapeutic Agent Inhibition by Exogenous
Folic Acid and p-Aminobenzoic Acid
(A) Serially diluted FA () and PABA () were added to PDA with SMX-TMP at
100
m
g/ml. Conidia from A. fumigatus were suspended in saline, and the suspension
was placed onto the center of prepared plates. A PDA plate without SMX-TMP was
used as a control. After incubation for 7 d at 27 °C, the diameter of the giant colony was
measured the colony size. (B) Serially diluted PABA () was added to C-limiting agar
medium with SMX-TMP at 100
m
g/ml. Conidia from A. niger were suspended in
saline, and the suspension was placed onto the center of the prepared plates. C-limiting
agar medium without SMX-TMP was used as a control. After incubation for 7 d at
27 °C, the diameter of the giant colony was measured. The error bars here represent
standard deviations.
Fig. 6. Detection of Inhibitors for the Activity of SMX-TMP in Human
Serum and PDA Medium
(A) Conidia from A. fumigatus were suspended in saline, and the suspension was
placed in PDA medium with SMX-TMP at 0 (left plate in Fig. 6A) or 100
m
g/ml
(right). Filter paper treated with saline (No. 1), Folic acid (No. 2), PABA (No. 3) or
human serum (No.4), was placed on the PDA plate with agents and A. fumigatus.
(B) The suspension of A. niger was placed in C-limiting agar medium with SMX-
TMP at 0 (left plate in Fig. 6B) or 100
m
g/ml (right). Filter paper treated with saline
(No. 1), PABA (No. 2), water-soluble fraction of PDA (No. 3) or dialyze water-soluble
fraction of PDA (No. 4), was placed on the plate with the agents and A. niger. A. niger
grew around the circular pieces treated with PABA and sPDA.
may not intake folic acid despite of eukaryotic cells. It may
help with the development of new antimicrobial agents and
application of conventional antimicrobial agents to research
the metabolic biochemistry of the fungus.
The combination SMX-TMP has been used extensively for
the treatment and prevention of Pneumocystis carinii pneu-
monitis.
10—12)
Although Pneumocystis carinii lacks the major
fungal sterol, ergosterol, P. carinii is closely related to
fungi.
16)
Moreover, it has been reported that the combination
of SMX and TMP acts synergistically against the dimorphic
fungus Paracoccidioides brasiliensis.
17,18)
Those reports sug-
gest that SMX-TMP acts toward fungus. In the present study
(Fig. 1) and other reports,
13,14)
SMX acted against Aspergillus
species. The results suggest that SMX-TMP has a general
anti-fungal effect. However, the effect of SMX-TMP toward
Candida species was weaker than toward Aspergillus species.
(Fig. 2). Some research suggests that sulfonamides, such as
SMX, alone have anti-P. carinii activity without Trimetho-
prim.
19—21)
Figure 3 shows also that the anti-Aspergillus ef-
fect depended on SMX, not TMP. Thus, the dihydropteroate
synthase (DHPS) that is a target enzyme of sulfonamides
may differ between sulfonamide-sensitive fungi, such as P.
carinii, Aspergillus and non-sensitive fungi, such as C. albi-
cans. The gene sequence of DHPS from Saccharomyces
cerevisiae and P. carinii was already revealed.
11,22,23)
Phylo-
genetic analysis of the DHPS sequence revealed that it’s
closely related to ascomycete fungi.
23)
Furthermore, a gene
similar to the DHPS gene from C. albicans was also re-
vealed.
24)
However, no gene sequence of DHPS from As-
pergillus species has been reported yet. The sequence of
DHPS from Aspergillus species may resemble the sequence
from P. carinii more than that from Candida species, espe-
cially, Candida parapsilosis, given the effect of the sulfon-
amides. Research on the Aspergillus DHPS gene will be
needed for the understanding and usage of sulfonamides
such as SMX-TMP.
In this study, the anti-Aspergillus activity of SMX was re-
stored by the addition of a small amount of PABA, less than
1
m
g/ml (Fig. 5). If inhibitors of sulfonamides, such as
PA BA, do exist in human serum and tissue, anti-microbial
susceptibility testing in vitro may be meaningless clinically.
However, Figure 6A shows that factors inhibiting the activity
of SMX-TMP do not exist in human serum. This result sug-
gests that SMX-TMP would act against Aspergillus species
in a clinical setting. Although it is important to use animal
models of infection for the assessment of medicines,
21,25—27)
such experiments are complex and not easy for non-profes-
sionals of microbiology.
Tang et al. demonstrated that PABA requiring mutant of A.
nidulans was nonphathogenic in murine models of invasive
pulmonary Aspergillosis.
36)
In research for pathogenicity of
A. fumigatus, Sandhu et al. reported that the anxotrophic mu-
tants which have absolute growth requirement for PABA
were completely avirulent to mice.
37)
Those results supports
that SMX could be used as therapeutic agent of Aspergillo-
sis. Further studies of folate metabolism in Aspergillus
species are needed for clinical application of sulfamides.
Adverse reactions to SMX-TMP have frequently been re-
ported.
28—31)
The most frequent reactions include rash,
nuetropenia, and fever. However, some evidence suggests
that at least some of the adverse reactions to SMX-TMP
might be due to the TMP component
19,28,31)
and that sulfon-
amides such as SMX alone have anti-P. carinii activity.
20,21)
In the present study, SMX alone had anti-Aspergillus activity.
Thus, sulfonamides may be useful for the prophylaxis of fun-
gal infections by Aspergillus species and P. carinii etc.
Acknowledgements The authors thank Mr. Kazuaki
Hirao for technical assistance. The authors are grateful to
Shionogi & Co., Ltd, for supplying the SMX and TMP which
used preliminary experiment. This work was partly sup-
ported by a grant for private universities provided by the
Ministry of Education, Culture, Sports, Science and Technol-
ogy and by the Japan Private School Promotion Foundation.
REFERENCES
1) Myskowski P. L., White M. H., Ahkami R., Dermatol. Clin., 15, 295—
305 (1997).
2) Klein M., Radhakrishnan J., Appel G., Annu. Rev. Med., 50, 1—15
(1999).
3) van Burik J. A., Magee P. T., Annu. Rev. Microbiol., 55, 743—772
(2001).
4) Anaissie E., Clin. Infect. Dis., 14 (Suppl. 1), S43—S53 (1992).
5) Latge J. P., Calderone R., Curr. Opin. Microbiol., 5, 355—358 (2002).
6) Denning D. W., Clin. Infect. Dis., 26, 781—803, quiz 804—805
(1998).
7) Hope W. W., Denning D. W., Clin. Microbiol. Infect., 10, 2—4 (2004).
8) Perfect J. R., Marr K. A., Walsh T. J., Greenberg R. N., DuPont B., de
la Torre-Cisneros J., Just-Nubling G., Schlamm H. T., Lutsar I., Es-
pinel-Ingroff A., Johnson E., Clin. Infect. Dis., 36, 1122—1131
(2003).
9) Mahfouz T., Anaissie E., Curr. Opin. Investig. Drugs, 4, 974—990
(2003).
10) Carr A., Tindall B., Penny R., Cooper D. A., AIDS, 6, 165—171
(1992).
11) Lane B. R., Ast J. C., Hossler P. A., Mindell D. P., Bartlett M. S.,
Smith J. W., Meshnick S. R., J. Infect. Dis., 175, 482—485 (1997).
12) Hughes W. T., Annu. Rev. Med., 42, 287—295 (1991).
13) Afeltra J., Meis J. F., Vitale R. G., Mouton J. W., Verweij P. E., Antimi-
crob. Agents Chemother., 46, 2029—2031 (2002).
14) Afeltra J., Meis J. F., Mouton J. W., Verweij P. E., AIDS, 15, 1067—
1068 (2001).
15) Shepherd M. G., Sullivan P. A., J. Gen. Microbiol., 93, 361—370
(1976).
16) Kaneshiro E. S., J. Eukaryot. Microbiol., 49, 367—373 (2002).
17) Restrepo A., Arango M. D., Antimicrob. Agents Chemother., 18, 190—
194 (1980).
18) Stevens D. A., Vo P. T., Antimicrob. Agents Chemother., 21, 852—854
(1982).
19) Hughes W. T., Killmar J., Antimicrob. Agents Chemother., 40, 962—
965 (1996).
20) Kunz S., Junker U., Blaser J., Joos B., Meyer B., Zak O., O’Reilly T., J.
Antimicrob. Chemother., 36, 137—155 (1995).
21) Walzer P. D., Kim C. K., Foy J. M., Linke M. J., Cushion M. T., An-
timicrob. Agents Chemother., 32, 96—103 (1988).
22) Sen-Gupta M., Guldener U., Beinhauer J., Fiedler T., Hegemann J. H.,
Yeast, 13, 849—860 (1997).
23) Ma L., Imamichi H., Sukura A., Kovacs J. A., J. Infect. Dis., 184,
1358—1362 (2001).
24) Jones T., Federspiel N. A., Chibana H., Dungan J., Kalman S., Magee
B. B., Newport G., Thorstenson Y. R., Agabian N., Magee P. T., Davis
R. W., Scherer S., Proc. Natl. Acad. Sci. U.S.A., 101, 7329—7334
(2004).
25) Shibuya K., Naoe S., Yamaguchi H., Contrib. Microbiol., 2, 130—138
(1999).
26) Hughes W. T., Gray V. L., Gutteridge W. E., Latter V. S., Pudney M.,
Antimicrob. Agents. Chemother., 34, 225—228 (1990).
27) Bartlett M. S., Shaw M. M., Smith J. W., Meshnick S. R.., Antimicrob.
Agents Chemother., 42, 934—935 (1998).
28) Hughes W. T., LaFon S. W., Scott J. D., Masur H., J. Infect. Dis., 171,
May 2005 777
1295—1301 (1995).
29) Gordin F. M., Simon G. L., Wofsy C. B., Mills J., Ann. Intern. Med.,
100, 495—499 (1984).
30) Medina I., Mills J., Leoung G., Hopewell P. C., Lee B., Modin G.,
Benowitz N., Wofsy C. B., N. Engl. J. Med., 323, 776—782 (1990).
31) Floris-Moore M. A., Amodio-Groton M. I., Catalano M. T., Ann.
Pharmacother., 37, 1810—1813 (2003).
32) Wormser G. P., Keusch G. T., Heel R. C., Drugs, 24, 459—518 (1982).
33) Bruun J. N., Ostby N., Bredesen J. E., Kierulf P., Lunde P. K., Antimi-
crob. Agents Chemother., 19, 82—85 (1981).
34) Joos B., Blaser J., Opravil M., Chave J. P., Luthy R., Antimicrob.
Agents Chemother., 39, 2661—2666 (1995).
35) Bayly A. M., Berglez J. M., Patel O., Castelli L. A., Hankins E. G.,
Coloe P., Hopkins Sibley C., Macreadie I. G., FEMS Microbiol. Lett.,
204, 387—390 (2001).
36) Tang C. M., Smith J. M., Arst H. N., Jr., Holden D. W., Infect. Immun.,
62, 5255—5260 (1994).
37) Sandhu D. K., Sandhu R. S., Khan Z. U., Damodaran V. N., Infect.
Immun., 13, 527—532 (1976).
778 Vol. 28, No. 5
    • "Antibody phage display is one of the technologies that can lead to the identification of novel target molecules (Akahori et al., 2009). A number of published reports describe the in vitro antifungal activity against A. fumigatus species with the existing drugs (Moore et al., 2001; Hida et al., 2005; Kahn et al., 2006). Schutte et al. (Schutte et al., 2009) identified the putative Crf splice variant and generation of recombinant antibodies for the specific detection of A. fumigatus for histopathological detection system for A. fumigatus by immunofluorescence and a serum diagnostic assay by ELISA. "
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    Full-text · Article · Mar 2011
    • "Sulphonamides are synthetic antimicrobial agents derived from sulfonic acid. These molecules act as competitive inhibitors of para-aminobenzoic acid, which is a necessary intermediate in these organisms for the synthesis of folic acid during DNA synthesis [5]. Sulfadiazine (SD,Fig. "
    [Show abstract] [Hide abstract] ABSTRACT: The effect of the frequently used antibiotic sulfadiazine (SD) was studied on a bacteria membrane mimetic model system by using differential scanning calorimetric (DSC), small- and wide-angle X-ray scattering (SWAXS) and freeze-fracture methods. The membrane model system consisted of dipalmitoylphosphatidylethanolamine (DPPE, 0.8 molar ratio) and dipalmitoylphosphatidylglycerol (DPPG, 0.2 molar ratio). The SD molar ratio (relative to the lipids) was varied between 10(-3) and 1. In the presence of SD, two transitions between the gel and liquid crystalline phases appear at 60.5 degrees C and about at 65 degrees C. In the temperature domain of the gel phase, the subcell of the chain packing is strongly temperature dependent indicating the increased dominance of the hydration forces during the first transition and the location of SD molecules in the neighbourhood of the polar lipid head groups. The second transition is accompanied by the changes in the nanometer-scale layer arrangements observed by SAXS and in the mum-scale morphology observed by freeze-fracture. Above the temperature of the second transition, the SD-induced metastable structures undergo further formations to produce a more homogeneous state favoured by the geometrical packing of the cylindrical-shaped lipid molecules.
    Full-text · Article · Mar 2007
  • [Show abstract] [Hide abstract] ABSTRACT: In vitro susceptibilities of ten clinical isolates, including five strains of Cryptococcus neoformans var. grubii and five strains of Aspergillus fumigatus, were determined against nine sulfa drugs using a microdilution method. Among the five tested media, minimum inhibitory concentration (MIC) values were observed only in YNB medium: no detectable level MIC value of less than 125 microg/ml was observed in the four remaining media against Cryptococcus species. Of the nine sulfa drugs, of which sulfaphenazole showed the highest antifungal activity, the MIC values for A. fumigatus and C. neoformans var. grubii were, respectively, 64 microg/ml and 4-8 microg/ml, suggesting high susceptibility of C. neoformans to sulfa drugs.
    Article · Feb 2007
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