APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2000, p. 2678–2681 0099-2240/00/$04.00⫹0 Copyright © 2000, American Society for Microbiology. All Rights Reserved.
Vol. 66, No. 6
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
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 ⴝ 5) or control (n ⴝ 5) homes in Cleveland were analyzed for hemolytic activity, siderophore production, and relatedness as measured by random amplified polymorphic DNA analysis. 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). 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
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-
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.
* 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:
[email protected]. 2678
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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.
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. 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 TABLE 1. Matrix showing the presence or absence of the 26 bands produced by RAPD analysis of the Houston strain of S. chartarum Band no. and bandwidth (bp)
No. of bands in strain Houston
1 (2026–1970) ................................................................................. 0 2 (1923–1889) ................................................................................. 0 3 (1818–1750) ................................................................................. 1 4 (1708–1657) ................................................................................. 0 5 (1601–1592) ................................................................................. 0 6 (1553–1517) ................................................................................. 0 7 (1509–1451) ................................................................................. 0 8 (1438–1416) ................................................................................. 0 9 (1399–1367) ................................................................................. 0 10 (1357–1347) ............................................................................... 1 11 (1300–1251) ............................................................................... 1 12 (1211–1153) ............................................................................... 1 13 (1094–1004) ............................................................................... 0 14 (995–956) ................................................................................... 1 15 (931–884) ................................................................................... 0 16 (872–822) ................................................................................... 1 17 (754–702) ................................................................................... 1 18 (700–664) ................................................................................... 1 19 (656–610) ................................................................................... 1 20 (599–566) ................................................................................... 1 21 (534–481) ................................................................................... 1 22 (469–421) ................................................................................... 0 23 (402–381) ................................................................................... 1 24 (367–332) ................................................................................... 1 25 (310–282) ................................................................................... 0 26 (223–206) ................................................................................... 1
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.
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). 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). 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
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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 hydroxamatetype 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.
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.
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.
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