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Applied and Environmental Microbiology, July 1999, p. 3175-3181, Vol. 65, No. 7
0099-2240/99/$04.00+0
Hemolysis, Toxicity, and Randomly Amplified Polymorphic DNA
Analysis of Stachybotrys chartarum Strains
Stephen J.
Vesper,1,*
Dorr G.
Dearborn,2
Iwona
Yike,2
W. G.
Sorenson,3 and
Richard
A.
Haugland1
National Exposure Research Laboratory, U.S.
Environmental Protection Agency, Cincinnati, Ohio
452681; Department of Pediatrics,
Case Western Reserve University, Rainbow Babies and Childrens
Hospital, Cleveland, Ohio 441062; and
Division of Respiratory Disease Studies, National
Institutes of Occupational Safety and Health, Morgantown, West
Virginia 265053
Received 19 January 1999/Accepted 6 April 1999
 |
ABSTRACT |
Stachybotrys chartarum is an indoor air, toxigenic
fungus that has been associated with a number of human and veterinary
health problems. Most notable among these has been a cluster of
idiopathic pulmonary hemorrhage cases that were observed in the
Cleveland, Ohio, area. In this study, 16 strains of S. chartarum isolated from case (n = 8) or control
(n = 8) homes in Cleveland and 12 non-Cleveland
strains from diverse geographic locations were analyzed for hemolytic
activity, conidial toxicity, and randomly amplified polymorphic DNA
banding patterns. In tests for hemolytic activity, strains were grown
at 23°C on wet wallboard pieces for an 8-week test period. Conidia
from these wallboard pieces were subcultured on sheep's blood agar
once a week over this period and examined for growth and clearing of
the medium at 37 or 23°C. Five of the Cleveland strains (all from
case homes) showed hemolytic activity at 37°C throughout the 8-week
test compared to 3 of the non-Cleveland strains. Five of the Cleveland
strains, compared to two of the non-Cleveland strains, produced highly
toxic conidia (>90 µg of T2 toxin equivalents per g [wet weight]
of conidia) after 10 and 30 days of growth on wet wallboard. Only 3 of
the 28 strains examined both were consistently hemolytic and produced
highly toxic conidia. Each of these strains was isolated from a house
in Cleveland where an infant had idiopathic pulmonary hemorrhage.
| |
INTRODUCTION |
Beginning in 1993, some infants,
living in water-damaged homes in Cleveland, Ohio, developed cases of
idiopathic pulmonary hemorrhage (IPH) (2). An investigation
by the Centers for Disease Control and Prevention concluded that
Stachybotrys chartarum was likely connected to this disease
(10, 11, 23). Stachybotrys chartarum (Ehrenb. ex
Link) Hughes (= S. atra Corda) is a toxigenic fungus that
grows on wet, cellulose-based products such as paper, cardboard, and
wallboard (12) and has been associated with human health
problems (4, 6, 18, 19).
Since 1993, there have been more than 40 IPH cases in the Cleveland
metropolitan area, with the majority of cases clustered in a small
geographic area. These cases have resulted in a 30% mortality rate
(7). For a 3-year period (1993 to 1995), 12% of all sudden
infant death syndrome deaths in the cluster area were actually IPH
deaths (7). The IPH problem is not limited to Cleveland. In
an informal survey, more than 140 cases of IPH have been reported in
infants across the United States during the past 5 years
(8).
The first cluster of IPH cases was reported from Greece in the early
1980s (5). Other reported clusters of the illness have
occurred in Chicago, Ill., between 1992 and 1994 (3); in
Detroit, Mich., between 1992 and 1995 (24); in Ann Arbor, Mich., between 1988 and 1993 (27); and in Milwaukee, Wis.,
between 1993 and 1996 (15). No apparent etiology was found
for these other clusters of IPH. But clusters of
Stachybotrys-related illness and death are well documented
in the veterinary literature.
In the 1930s, an epizootic of stachybotryotoxicosis (ST) in horses
began in Eastern Europe and Russia and ended about 1944 (13). No similar major outbreak seems to have occurred since then, but incidents of ST still occur in domestic animals in Eastern Europe and Russia and some other parts of the world (28).
Toxins produced by this fungus are considered the cause of the disease (9).
When viewed in relation to the widespread occurrence of S. chartarum around the world (21), the outbreak of
disease suspected to be caused by this organism can be considered to be
sporadic. The goal of this study was to determine if the strains of
S. chartarum isolated from homes in Cleveland where infants
developed IPH are unique compared to strains from control homes or
strains from other diverse geographic locations.
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MATERIALS AND METHODS |
Hemolytic test of S. chartarum strains on SBA.
Conidia of each strain were inoculated onto and grown on 5- by 5-cm
pieces of sterile, 9.4-mm (3/8 in.)-thick wallboard placed in 100- by
15-mm petri dishes containing 20 ml of sterile water and incubated at
23°C. More sterile water was added to the petri dishes after 4 weeks
of incubation, so that there was always free water around the wallboard
pieces during the 8-week incubation.
Every 7 days for 8 weeks, conidia from each strain were recovered with
a sterile cotton swab from each wallboard piece. The conidia were
transferred to plates of 5% sheep blood agar (SBA; Becton Dickinson,
Sparks, Md.) and incubated at 37 and 23°C. After 10 days of
incubation, the plates were checked for hemolytic activity. Clearing of
the medium beyond the edge of the fungal growth was considered to be an
indication of hemolytic activity (hemolytic positive).
Hemolytic activity after heat treatment.
After 5 weeks of
growth on wallboard, conidia from each of the 28 strains were directly
blotted onto four sets of duplicate sterile, borosilicate filters
(Millipore, Bedford, Mass.). One set of filters was heat treated at
86°C for 20 min, and another set was held at 23°C. Finally, each
filter was placed on SBA and incubated at 37°C for 10 days, and
hemolysis was noted for each strain.
Toxicity of S. chartarum strains.
The strains of
S. chartarum were evaluated for their toxicity by the method
of Yike et al. (30), which uses luciferase translation in a
mammalian cell-free system to assess protein synthesis inhibition, an
activity characteristic of trichothecene mycotoxins. Strains were grown
on wallboard as described above. After 10 and 30 days of growth on
wallboard, conidia from each of the strains were blotted directly onto
9-mm-diameter sterile, borosilicate filters (Millipore). The wet weight
of the conidia on the filters was determined, and the filters and
conidia were then freeze-dried and held at 4°C prior to performing
the toxicity assays.
The filters with adhering conidia were extracted with 10 ml of 95%
ethanol at room temperature overnight and then sonicated
at room
temperature for 30 min. The sonication was repeated with
5 ml of fresh
ethanol, and the combined extracts were filtered
through
0.22-µm-pore-size Millex-GV filters (Millipore) and evaporated
in a
Speed Vac Plus centrifuge (model SC210A; Savant Instrument,
Farmingdale, N.Y.). Extracts were reconstituted in 200 µl of 95%
ethanol, diluted, and passed through Ultrafree-MC 5000 NMWL
microcentrifuge
filters
(Millipore).
In vitro translation of firefly luciferase mRNA by using Flexi rabbit
reticulocyte lysate (Promega, Milwaukee, Wis.) was carried
out in the
presence or absence of the standard toxin, T-2 (Sigma,
St. Louis, Mo.),
and conidial extracts. Inhibition of protein
translation caused by the
extracts and purified T-2 toxin was
evaluated by measuring the
luminescence of newly synthesized luciferase.
Dose-response curves for
the standard toxin and conidial extracts
were generated, and the
concentrations causing 50% inhibition
were determined by using the
TableCurve curve-fitting program
(Jandel Scientific, Chicago, Ill.).
T-2 toxin equivalents in the
conidial extracts were derived from
comparison of the 50% inhibitory
concentrations for the standard
toxin.
DNA extraction and randomly amplified polymorphic DNA (RAPD)
analysis of S. chartarum strains.
The 12 non-Cleveland
strains used in this study are listed in Table
1. Each strain was grown on potato
dextrose agar (Becton Dickinson, Sparks, Md.) for 7 days at 23°C.
Conidia were collected from each strain by using a sterile cotton swab
and resuspended in sterile water to give a final concentration of
107 conidia per ml (as determined by hemocytometer counts).
The DNA of each strain was extracted by the bead-beating method
(20) as previously modified (16). Briefly, this
involves adding 0.3 g of glass beads (G-1277; Sigma, St. Louis,
Mo.) to 2 ml of a conical-bottom, screw-cap tube (PGC Scientifics,
Gaithersburg, Md.) and autoclaving. Then, 100 µl of lysis buffer and
300 µl of binding buffer from the Elu-Quik Kit (Schleicher & Schuell, Keene, N.H.), followed by 10 µl (i.e., 105 conidia) of
each conidial suspension was added to each tube. The tubes were shaken
rapidly in a Mini-Beadbeater (Biospec Products, Bartlesville, Okla.)
for 1 min at the maximum rate. The released DNA was processed as
described in the directions for the Elu-Quik Kit.
The recovered DNA was randomly amplified by using the R28 primer
(5'-ATGGATCCGC) and the PCR protocol described by Fujimori
and Okuda (
14), modified as follows. The R28 primer was
synthesized
by PE Applied Biosystems (Foster City, Calif.). The RAPD
reaction
was done in a total volume of 50 µl containing 0.2 µl of a
50
nM solution of the R28 primer, 1 µl of PCR nucleotide mix
(1581295;
Boehringer, Indianapolis, Ind.), 0.3 µl of Expand High
Fidelity
PCR System DNA polymerase (84367521; Boehringer), 5 µl of
10×
PCR buffer with MgCl (83073623; Boehringer), 5 µl of a sterile
bovine serum albumin (Fraction V; Sigma) solution containing 2
mg of
water per ml, 7 µl of a 50% solution of glycerol in water,
26.5 µl
of sterile deionized water, and 5 µl of the purified DNA
solution.
The PCR was conducted at 92°C for 1 min, followed by
30 cycles of
denaturation at 92°C for 45 s, annealing at 34°C
for 60 s, and extension at 72°C for 90 s; then, the final extension
was
done at 72°C for 10 min in a thermal cycler (PTC-200; MJ Research,
Watertown, Mass.).
The PCR products were separated on a 0.7% agarose gel. The gels were
run at 100 V for 6 h and stained for 40 min with SYBR
Green (FMC,
Rockland, Maine) at a concentration of 10 µl per 100
ml of water. The
gels were imaged with the FluorImager 595 (Molecular
Dynamics,
Sunnyvale, Calif.), and the molecular weight of each
band was
determined by using the associated image analysis program,
Fragment NT.
Molecular size standards consisting of 2,000-, 1,200-,
800-, 400-, 200-, and 100-bp fragments (Life Technologies, Grand
Island, N.Y.) were
run in triplicate on each
gel.
RAPD analysis of each strain's DNA was replicated three to seven times
in separate gels. Only bands that appeared in at least
80% of the
analyses were considered positive for that strain.
The results were
assembled in a 1-0 matrix depending upon whether
a band was present (1 matrix) or absent (0 matrix), as described
by Fujimori and Okuda
(
14). Phylogenetic relationships of the
strains and strain
distances were inferred from these data by
using the branch and bond
option of the Phylogenetic Analysis
Using Parsimony (PAUP) program,
version 3.1 (Sinauer Associates,
Sunderland, Mass.). A distance
analysis was performed on each
strain to determine which was the least
similar. This strain was
used as the outgroup in the phylogenetic
evaluation. The PAUP
bootstrap with a branch-and-bound search (1,000 times replication)
was used to estimate the distance between strains,
and the levels
of support for the branches of the most parsimonious
trees with
at least 90% occurrence were used as the cutoff for
significance.
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RESULTS |
Hemolysis of SBA by S. chartarum strains.
Tables
2 and 3
show the results of the weekly hemolytic assays on SBA at 37 and
23°C, respectively. When incubated at 37°C, five of the Cleveland
strains (all from case homes) and three of the non-Cleveland strains
were consistently capable of demonstrating hemolytic activity
throughout the 8-week test (Table 2). By week 5 of the incubation, all
28 strains were demonstrating hemolytic activity. None of the strains
were consistently hemolytic when incubated at 23°C (Table 3).
Hemolytic activity after heat treatment.
None of the strains
on the filters heat treated at 86°C for 20 min produced a hemolytic
response on SBA at 37°C, but the conidia of all strains on SBA that
were not heat treated grew and produced a hemolytic response (Fig.
1).
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FIG. 1.
Appearance of strain 58-06 conidia on SBA after conidia
on filters were heat treated (left) or not heat treated (right). After
5 weeks of growth on wallboard, conidia from strain 58-06 were directly
blotted onto borosilicate filters. The filter on the left was heat
treated at 86°C for 20 min, and the one on the right was held at
23°C. Filters were placed on SBA and incubated at 37°C for 10 days.
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Toxicity of S. chartarum strains.
The conidia of
all 28 strains of S. chartarum tested in this study showed
some level of toxicity, as measured by protein synthesis inhibition,
when grown on wet wallboard (Table 4).
However, only seven strains (five from Cleveland and two non-Cleveland
strains) were consistently highly toxic, i.e., toxicities were greater than 90 µg of T-2 toxin equivalents per g (wet weight) of conidia. One Cleveland strain (58-08) and three non-Cleveland strains (NC-1, NC-2, and NC-4) were intermediate in toxicity. The other 17 strains had
toxicities consistently less than 20 µg of T-2 toxin equivalents per
g (wet weight) of conidia.
RAPD analysis of S. chartarum strains.
The RAPD
analysis of S. chartarum strains produced 26 bands. The
matrices resulting from this analysis are shown in Tables 5 and 6.
Two bands (15 and 21) were found in all strains of S. chartarum analyzed in this study. When the highly toxic and/or consistently hemolytic strains are analyzed as a group for phylogenetic relationships, strains 51-06 and 58-02 are most distant from the other
nine strains in this group (Fig. 2). If
the consistently hemolytic strains are analyzed as a group (Fig.
3), none of the strains show significant
relationships. If the highly toxic strains are analyzed as a group,
then 51-06 and 58-02 are significantly different from the other highly
toxic strains (Fig. 4).
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TABLE 5.
Matrix showing the presence ("1") or absence
("0") of the 26 bands produced by RAPD analysis of the strains
of S. chartaruma
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TABLE 6.
Frequency of occurrence of bands in the strains of
S. chartarum that were both highly toxic and consistently
hemolytic (A) compared to the other 25 strains in the
study (B)a
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FIG. 2.
Phylogram of the 11 strains of S. chartarum
which were highly toxic and/or consistently hemolytic. The phylogram
presented is the most parsimonious tree inferred from the binary data
(Table 5) by using the branch-and-bond option in PAUP 3.1. The scale
bar represents the distance resulting from one character change.
Distance analysis of the data set was performed in PAUP to determine
which strain was the least similar. This strain was used as the
outgroup in the phylogenetic evaluation. Values appearing above the
branches are percentages of 1,000 bootstrap analysis replicates in
which the branches were found. Only percentages greater than 50% are
shown. A strain number followed by an asterisk indicates a strain that
came from an IPH case house in Cleveland.
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FIG. 3.
Phylogram of the seven strains of S. chartarum which were consistently hemolytic. The phylogram
presented is the most parsimonious tree inferred from the binary data
(Table 5) by using the branch-and-bond option in PAUP 3.1. The scale
bar represents the distance resulting from one character change.
Distance analysis of the data set was performed in PAUP to determine
which strain was the least similar. This strain was used as the
outgroup in the phylogenetic evaluation. Values appearing above the
branches are percentages of 1,000 bootstrap analysis replicates in
which the branches were found. Only percentages greater than 50% are
shown. A strain number followed by an asterisk indicates a strain that
came from an IPH case house in Cleveland.
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FIG. 4.
Phylogram of the seven highly toxic strains of S. chartarum. The phylogram presented is the most parsimonious tree
inferred from the binary data (Table 5) by using the branch-and-bond
option in PAUP 3.1. The scale bar represents the distance resulting
from one character change. Distance analysis of the data set was
performed in PAUP to determine which strain was the least similar. This
strain was used as the outgroup in the phylogenetic evaluation. Values
appearing above the branches are percentages of 1,000 bootstrap
analysis replicates in which the branches were found. Only percentages
greater than 50% are shown. A strain number followed by an asterisk
indicates a strain that came from an IPH case house in Cleveland.
Strains 51-06 and 58-02 were significantly different from the other
highly toxic strains, as indicated by the 90 on the line connecting to
the rest of the strains.
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| |
DISCUSSION |
S. chartarum is the etiologic agent of ST, a
potentially fatal disease of horses and other large domestic animals
caused by consuming moldy feed (13). Even though S. chartarum has a worldwide distribution, the incidence of ST is
limited to certain geographic areas, especially Eastern Europe and
Russia (28). Toxins produced by S. chartarum are
generally considered responsible for the ST deaths (9), but
additional factors might also have been involved. Sarkisov and
Orshanskaiya (26) described infections of the horse's liver, kidney, lymphatic system, and spleen, from which they were able
to isolate Stachybotrys. This suggests that, under some
circumstances, this fungus was not only toxigenic but also infectious
in horses.
Currently available information on the levels of exposure to airborne
S. chartarum conidia for children with diagnosed cases of
IPH (11) suggests that toxic effects alone may not be
sufficient to explain these illnesses. This possibility was further
suggested by demonstrations that the amounts of toxins produced by
isolates from the IPH case homes were no greater than those produced by isolates from control homes (17). These observations suggest that some additional factor(s) may contribute to IPH.
Virulence factors, such as hemolysins, are often associated with
pathogenic microorganisms (22). In some cases, the ability of pathogenic microorganisms to acquire iron from a mammalian host by
producing a hemolysin has been shown to be of critical importance in
establishing infections (1, 25, 29). The four infants who
had lived in the homes where the eight case strains were isolated had
hemoglobinuria, a finding consistent with a hemolysin playing a role in
the pathophysiology of IPH.
All 16 strains of S. chartarum studied from case and control
houses produced a hemolytic agent at 37°C (Table 2). However, the
strains isolated from case houses showed hemolytic activity 89% of the
time (57 of 64 observations) at 37°C compared to strains isolated
from control houses, which were hemolytic only 58% of the time (37 of
64 observations). Chi-square analysis of these results indicates that
case strains and control strains were significantly different
(P < 0.001) in the frequency of hemolytic-activity
expression. When SBA plates were incubated at 23°C, the case strains
were hemolytic only 53% of the time (33 of 64 observations) compared to only 30% of the time (19 of 64 observations) for control strains (Table 3). Chi-square analysis of these results indicates that case
strains and control strains were significantly different (P < 0.001) in their frequency in expressing hemolysis at 23°C. Comparing all strains incubated at 37°C to all strains incubated at
23°C, the frequency of hemolytic expression was also statistically different (chi-square result of P < 0.001), indicating
that 37°C is the preferred temperature for S. chartarum
hemolysis expression.
The identity of the hemolysin has not been determined. Forgacs
(13) indicated that S. chartarum conidia were
killed by heat treatment but that toxins in the spores were unaffected
by this treatment. Because heat-killed conidia in this study are not
hemolytic, it seems unlikely that the hemolysin is any of the known
S. chartarum toxins.
This study has demonstrated that there are highly toxic and
consistently hemolytic strains of S. chartarum. The
frequency of occurrence of highly toxic strains from Cleveland was
about the same as for the non-Cleveland sources; i.e., 31% of the
Cleveland strains were highly toxic compared to 17% of the
non-Cleveland strains (Table 4). The frequency of consistently
hemolytic strains at 37°C was only slightly higher among the
Cleveland strains (31%) than among the non-Cleveland strains (25%)
(Table 2). Despite these indications that S. chartarum
strains from the Cleveland area are not much more likely to be either
highly toxic or highly hemolytic than strains from other locations, as
a whole, results from this study suggest that the combination of these
characteristics may be unique to certain Cleveland case strains.
Only three strains of the 28 tested (51-06, 51-11, and 58-02) were both
highly toxic and consistently hemolytic. All three of these strains
came from homes in Cleveland where infants became sick. This
observation raises the interesting possibility that a combination of
hemolysins and toxins may be required to induce the IPH disorder. Two
of these three strains (51-06 and 58-02) were statistically different
from the other highly toxic strains based on RAPD analysis (Fig. 4).
The RAPD analysis of strains 51-06, 51-11, and 58-02 compared to that
of the other 25 strains in this study (Table 6) indicates a potentially
diagnostic pattern for combined highly toxic and consistently hemolytic
strains. There are five bands (bands 4, 10, 18, 25, and 26) that occur
in this group of three strains whose frequency of occurrence differs by
more than 50% from the other set of strains. The binary pattern, bands
4(0), 10(1), 18(1), 25(0), and 26(0), is unique to this group. Knowing
this pattern may help screen other strains for these combined virulence factors.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: U.S. EPA, 26 W. M. L. King Drive, 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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Abstract
Introduction
