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Applied and Environmental Microbiology, June 2000, p. 2678-2681, Vol. 66, No. 6
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Quantification of Siderophore and Hemolysin from
Stachybotrys chartarum Strains, Including a Strain Isolated
from the Lung of a Child with Pulmonary Hemorrhage and
Hemosiderosis
Stephen J.
Vesper,1,*
Dorr G.
Dearborn,2
Okan
Elidemir,3 and
Richard
A.
Haugland1
National Environmental Research Laboratory,
U.S. Environmental Protection Agency, Cincinnati, Ohio
452681; Case Western Reserve
University, Department of Pediatrics, Rainbow Babies and Children
Hospital, Cleveland, Ohio 441062; and
Pediatric Pulmonary Section, Baylor College of Medicine,
Houston, Texas 770303
Received 3 January 2000/Accepted 8 March 2000
 |
ABSTRACT |
A strain of Stachybotrys chartarum was recently
isolated from the lung of a pulmonary hemorrhage and hemosiderosis (PH)
patient in Texas (designated the Houston strain). This is the first
time that S. chartarum has been isolated from the lung of a
PH patient. In this study, the Houston strain and 10 strains of
S. chartarum isolated from case (n = 5) or
control (n = 5) homes in Cleveland were analyzed for
hemolytic activity, siderophore production, and relatedness as measured
by random amplified polymorphic DNA analysis.
| |
TEXT |
The fungus Stachybotrys
chartarum (Ehrenb. ex Link) Hughes(= S. atra Corda) has
been associated with a number of human health problems, including
potentially fatal pulmonary hemorrhage and hemosiderosis (PH) in
infants (3, 4, 5, 9, 10, 16). Recently, Elidemir et al.
(6) isolated a strain of S. chartarum (designated
here as the Houston strain) from the lungs of a child with progressive
respiratory symptoms and PH. S. chartarum was also found in
the child's water-damaged home. The child recovered after removal from
the house and subsequent remediation of the home.
In this study, hydroxamate-type siderophore production and hemolytic
activity by case and control strains from Cleveland were quantified and
compared to those of the Houston strain. Random amplified polymorphic
DNA (RAPD) analysis of the Houston strain was also compared with those
of case and control strains from Cleveland (22).
Quantification of siderophore production and hemolytic activity by
S. chartarum when incubated with human blood.
Five
strains of S. chartarum isolated from PH control houses in
Cleveland (58-07, 58-16, 58-17, 58-18, and 63-01) and five from case
houses (51-08, 51-11, 58-02, 58-06, and 63-07) (15) and the
Houston strain were used. The strains of S. chartarum were
grown on wet wallboard pieces as previously described (22). Human blood used in this study was taken from one of the authors by his
physician using 7-ml vacuum tubes containing sodium heparin (Becton
Dickinson, Franklin Lakes, N.J.), and the tubes were then refrigerated
overnight. The next day the plasma was separated from the packed red
blood cells (RBC) and the buffy coat containing the white blood cells
was removed and discarded. To 250 ml of Trypticase soy broth (Becton
Dickinson, Sparks, Md.) was added 1.5 ml of the packed RBC and 2 ml of
plasma, and then the sample was mixed continuously while 10-ml aliquots
were dispensed into 50-ml polypropylene tubes (Corning Inc., Corning,
N.Y.). Approximately 4 × 104 conidia of each strain
were added to each of three replicate tubes. The tubes were placed on
an incubator-shaker (LabLine Inc, Melrose Park, Ill.) set at 36 ± 1°C and mixed at 200 rpm.
After 72 h of incubation, quantification of hydroxamate-type
siderophore was performed by the method of Emery and Neilands (7, 8). The readings of absorbance at 264 nm were converted to hydroxamic acid concentrations based on a standard curve (data not
shown). The data were analyzed by using the Mann-Whitney rank sum test
in the SigmaStat 2.0 computer program (SPSS Inc., Chicago, Ill.). The
five case strains and the Houston strain produced significantly more
(P = <0.001) of the hydroxamate-type siderophore than
the five strains from control houses (Fig.
1). The Houston strain was found to be
similar to the case strains in siderophore production but significantly
different from the control house strains (P = 0.009).
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FIG. 1.
Quantification of the hydroxamate-type siderophore from
S. chartarum. Triplicate human blood broth tubes were
inoculated with conidia of each strain after 4, 5, or 6 weeks of growth
on wet wallboard. Five strains of S. chartarum isolated from
PH control houses in Cleveland (no. 1 through 5: 58-07, 58-16, 58-17, 58-18, and 63-01), five from case houses (no. 6 through 10: 51-08, 51-11, 58-02, 58-06, and 63-07) (15), and the Houston strain
were used.
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|
After 120 h of incubation, the hemolytic activity of the cultures
was determined by measuring the absorbance of the supernatant
at 540 nm, as described by Nomura et al. (
17). The data were
analyzed by using the Mann-Whitney rank sum test in the SigmaStat
2.0 computer program (SPSS Inc.). The five strains from PH case
houses and
the Houston strain, as a group, produced significantly
more
(
P = <0.001) hemolytic activity than the five control
strains
(Fig.
2). However, the hemolytic
activity of one control strain
(58-18) was comparable to that of the
case strains.
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FIG. 2.
Quantification of hemolytic activity from S. chartarum. Triplicate human blood broth tubes were inoculated with
conidia of each strain after 4, 5, or 6 weeks of growth on wet
wallboard. Five strains of S. chartarum isolated from PH
control houses in Cleveland (no. 1 through 5: 58-07, 58-16, 58-17, 58-18, and 63-01), five from case houses (no. 6 through 10: 51-08, 51-11, 58-02, 58-06, and 63-07) (15), and the Houston strain
were used.
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|
DNA extraction and RAPD analysis of S. chartarum
strains.
The DNA of each strain was extracted using a bead-beating
method (13). The 936-bp segments of the nuclear rRNA gene
operon from all of the strains, analyzed by methods described
previously (12), were identical to the sequences found
earlier for 15 other S. chartarum strains isolated from many
parts of the world (data not shown) (12). Since the nuclear
rRNAs from all strains are essentially identical, a RAPD analysis of
the strains was carried out in an attempt to find differences among the
strains based on the entire genomic DNA.
After the DNA was extracted from each strain, it was randomly amplified
using the R28 primer (5'-ATGGATCCGC) and PCR protocol
described by Fujimori and Okuda (
11) as modified by Vesper
et
al. (
22). Table
1 shows the
RAPD analysis for the Houston strain.
The analysis of the Cleveland
strains was previously published
(
22). Phylogenetic
relationships of the strains and strain distances
were inferred from
the RAPD data using the branch-and-bound option
of the Phylogenetic
Analysis Using Parsimony (PAUP) program, version
3.1 (Sinauer
Associates, Sunderland, Mass.) as described by Vesper
et al.
(
22).
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TABLE 1.
Matrix showing the presence or absence of the 26 bands
produced by RAPD analysis of the Houston strain of
S. chartarum
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|
The RAPD analysis was used to compare the Houston strain to the case
and control groups of strains from Cleveland. The Houston
strain showed
a 91% relatedness to three of the case strains from
Cleveland (Fig.
3) but no significant relationship to any
of the
control strains (Fig.
4).
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FIG. 3.
Phylogram of the five case house strains of S. chartarum from Cleveland and the Houston strain. The phylogram
presented is the most parsimonious tree inferred from the binary data
using the branch-and-bond option in PAUP, version 3.1. The scale bar
represents the distance resulting from one character change. The value
above the branches is the percentage of 1,000 bootstrap analysis
replicates in which the branches were found.
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FIG. 4.
Phylogram of the five control house strains of S. chartarum from Cleveland and the Houston strain. The phylogram
presented is the most parsimonious tree inferred from the binary data
using the branch-and-bond option in PAUP, version 3.1. The scale bar
represents the distance resulting from one character change. Values
above the branches are the percentages of 1,000 bootstrap analysis
replicates in which the branches were found.
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Hemolysis of sheep RBC.
The Houston strain was grown on wet
wallboard, and the presence or absence of hemolytic activity of the
conidia was measured weekly for 8 weeks as described by Vesper et al.
(22). The conidia from the Houston strain were found to
produce the hemolysin consistently from weeks 1 to 8 when tested on
sheep blood agar (data not given).
Concluding remarks.
Although a definite cause-and-effect
relationship between S. chartarum and PH has not been
established, the American Academy of Pediatrics' Committee on
Environmental Health recently recommended that for infants under the
age of 1 year, chronically moldy, water-damaged environments should be
avoided (1). The isolation of S. chartarum from
the lung of a PH-afflicted child is the best evidence yet of the link
with PH. RAPD analysis of the Houston strain suggests that it is
related to some of the PH case strains from Cleveland but not to the
control house strains tested here.
In the 1930s in Eastern Europe and Russia, many thousands of horses
died after consuming
Stachybotrys-contaminated fodder
(
10). Sarkisov and Orshanskaiya (
21) reported
that they were
able to isolate
Stachybotrys alternans (=
chartarum) from several
internal organs of some of these
horses, suggesting in vivo growth
of the
fungus.
For a fungus or other microorganism to colonize a mammalian host, it
must have mechanisms to obtain iron (
2). Iron can
frequently
be the limiting factor for the growth of pathogens,
and the production
of siderophores and hemolysins by pathogens
is a typical mechanism for
obtaining iron (
18,
19,
20,
23).
Recently, Vesper et al.
(
22) demonstrated that strains of
S. chartarum
can produce a hemolysin. Most of the case strains (
22)
and
the Houston strain produced the hemolysin during the entire
8-week
test, but none of the strains from control houses in Cleveland
were
consistently able to express the hemolysin (
22).
The fact that the hemolysin is produced more consistently and in larger
amounts in the case strains and Houston strain than
in most control
strains suggests that hemolysis may have a role
in PH pathology. One
control strain (58-18) was similar to the
case and Houston strains in
hemolysin production. It may be that
the presence of a case house type
strain of
S. chartarum in a
home is only one factor required
for PH expression in an infant.
Based upon epidemiological analysis
(
4,
5,
9), other
factors that seem to be involved include
environmental tobacco
smoke and lack of breast
feeding.
Holzberg and Artis (
14) demonstrated that hydroxamate-type
siderophores were produced by nine different fungal pathogens.
We
report here for the first time that
S. chartarum can also
produce
a hydroxamate-type siderophore (additional siderophores cannot
be ruled out). Under in vitro cultural conditions, the case strains
and
Houston strain produced significantly more of this siderophore,
suggesting that they may be better prepared to obtain iron in
a host.
However, what happens in vivo is not
known.
S. chartarum has, at least in vitro, a number of mechanisms
for obtaining iron which could help it survive in a mammalian
host.
With these mechanisms and its arsenal of toxins (
15),
it
seems reasonable that some strains might be pathogenic under
some
conditions or at least able to colonize a host. It will be
essential to
develop an animal model that conforms to the observed
human PH
pathology in order to understand potential pathophysiological
roles of
S. chartarum.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: U.S. EPA, 26 W. M. L. King Dr., M.L. 314, Cincinnati, OH 45268. Phone:
(513) 569-7367. Fax: (513) 569-7117. E-mail:
Vesper.Stephen{at}EPA.gov.
| |
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Applied and Environmental Microbiology, June 2000, p. 2678-2681, Vol. 66, No. 6
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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