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Growth Inhibition of Trichophyton Species by Pseudomonas aeruginosa
James Treat, MD;
William D. James, MD;
Irving Nachamkin, DrPH, MPH;
John T. Seykora, MD, PhD
Arch Dermatol. 2007;143(1):61-64.
ABSTRACT
Objective To assess the ability of Pseudomonas aeruginosa to inhibit the growth of Trichophyton mentagrophytes (TM) and Trichophyton rubrum (TR).
Design Pseudomonas aeruginosa, Escherichia coli, or Staphylococcus epidermidis were grown in co-culture with either TM or TR.
Setting An academic medical center.
Main Outcome Measures The total fungal units and hyphal-spore ratio were measured at days 1, 5, 10, and 15.
Results There was a 73% and 46% reduction of total fungal units and a final hyphal-spore ratio of 0.16 and 0.04, respectively, when TM and TR were co-cultured with P aeruginosa. The number of fungal units increased when TM and TR were cultured with E coli (28% and 42%, respectively), S epidermidis (13% and 18%, respectively), and control media (44% and 62%, respectively), and the hyphal-spore ratio increased to above 30 in the presence of S epidermidis, E coli, and control media.
Conclusion Pseudomonas aeruginosa exhibits growth inhibitory properties against TM and TR.
INTRODUCTION
Interdigital toe web space is a warm, moist, protected environment that predisposes to the proliferation of both dermatophytes and gram-negative organisms.1 Tinea pedis is by far the most common fungal infection.2 Maceration, scaling, and fissures result, allowing for overgrowth of bacteria that normally inhabit this interspace.3 Proliferation of these organisms, which include most prominently Pseudomonas aeruginosa, other gram-negative bacteria (eg, Escherichia coli and Proteus mirabilis), and gram-positive bacteria, leads to an aggressive, painful infection.4 Once these bacterial species propagate, fungi, which initiated the toe web infection, cannot usually be recovered via culture.3 Therefore, growth of bacteria associated with toe web infections may exhibit fungistatic and/or fungicidal properties. In fact, P aeruginosa has been shown to inhibit both Candida albicans and Aspergillus fumigatus in vitro.5-7
To extend these observations to additional fungal species relevant to toe web infections, we conducted a series of co-culture experiments examining how Trichophyton species grow in the presence of various bacteria. The ability of P aeruginosa to affect fungal growth was evaluated, given its association with bacterial toe web infections. Escherichia coli and Staphylococcus epidermidis were chosen as controls to evaluate other relevant bacterial species. The fungal species evaluated were Trichophyton rubrum (TR) and Trichophyton mentagrophytes (TM) because they represent the most common causes of tinea pedis.8-10
METHODS
Both TR and TM were obtained from the American Type Culture Collection (Manassas, Va) and were reconstituted in 1.0 mL of distilled water for 24 hours. Aliquots (30 µL) were inoculated separately onto plates of Sabouraud dextrose agar (Becton Dickinson BBL, Sparks, Md) and grown for 2 weeks at 25°C. Pseudomonas aeruginosa was reconstituted per American Type Culture Collection instructions. Pseudomonas aeruginosa, control bacteria, E coli DH-5 , and S epidermidis (provided by the Clinical Microbiology Laboratory, Hospital of the University of Pennsylvania, Philadelphia), were freshly plated on sheep blood agar 2 days prior to each co-culturing experiment. Colonies of P aeruginosa, E coli, and S epidermidis were each inoculated into Mueller-Hinton (MH) broth, and the bacterial density was adjusted to 4 x 107 colony-forming units (CFU)/mL, as determined by optical density 600 nm.
FUNGAL LAWN ASSAY
Fifty-microliter aliquots of P aeruginosa or E coli grown in MH media or MH control media were inoculated on mature lawns of both TR and TM and observed at 96 hours for morphologic changes in the fungal lawn.
BACTERIA-FUNGAL CO-CULTURE
Approximately 10 mg of TM (8 x 107 CFU) or TR (3 x 106 CFU) was plated on Sabouraud dextrose agar. Then, 30 µL of 4 x 107 CFU/mL of P aeruginosa, E coli, S epidermidis, or MH media alone were separately inoculated into a 1.3-cm demarcated area in the center of plates from each fungus and grown at 25°C. Inhibition of fungal growth was evaluated at 5, 10, and 15 days.
DIRECT VISUALIZATION OF BACTERIAL-FUNGAL INTERACTIONS
One milligram of TM (8 x 106 CFU) and TR (3 x 105 CFU) was smeared evenly onto separate polarized glass slides to cover the area with a thin layer of fungus in a 1.3-cm diameter circle demarcated with a wax pencil. Thirty microliters of MH broth containing 4 x 107 CFU/mL of P aeruginosa, E coli, S epidermidis, or broth alone were inoculated onto the slides containing fungus. Each of the slides marked "15 day" contained a duplicate circle with the same bacteria and fungi or control media. The slides were kept moist by suspending them in a 50-mL tube with 20 mL of deionized water. At 1, 5, 10, and 15 days, slides were removed from the tubes and 2 drops of calcofluor white (Becton, Dickinson, and Company, Franklin Lakes, NJ) were applied and the slides were coverslipped. The number of fungi, including yeast and hyphal forms, in 9 representative high-powered fields (original magnification x40) was evaluated using epifluorescence. A maximum of 100 fungal units was recorded in each field, and if no spores could be found, a 1 was recorded so that a ratio could be calculated. Photomicrographs were taken using light microscopy and epifluorescence. The duplicate 15-day samples were reconstituted with a pipette in 30 µL of gentamicin solution (1 mg/mL) and cultured on Sabouraud agar at 25°C. The growth of these isolates was observed at 96 hours. Separate 15-day samples were reconstituted with 30 µL of MH media and then grown on sheep blood agar and observed at 96 hours. Samples incubated for 15 days were also reinoculated onto sheep blood agar to assess viability.
RESULTS
Co-culture of TM and TR with P aeruginosa resulted in a 73% and 46% reduction of total fungal units (hyphae plus spores), respectively (Figure 1). Numbers of fungal units increased when TM and TR were cultured with E coli (28% and 42%, respectively) and S epidermidis (13% and 18%, respectively), although at a slightly slower rate than when cultured with MH media alone (44% and 62%, respectively). There was one slide of TR grown with P aeruginosa at 15 days that did show a significant number of spores, but this slide had dried out, potentially destroying the P aeruginosa and its inhibitory ability.
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Figure 1. Fungal growth inhibition by Pseudomonas aeruginosa. Bacterial-fungal co-cultures (A, Trichophyton mentagrophytes; B, Trichophyton rubrum) were evaluated to determine the hyphal-spore ratio at days 1, 5, 10, and 15. The ratios are presented graphically for Staphylococcus epidermidis, Escherichia coli, and P aeruginosa. Note the increase in hyphal-spore ratio in each control strain and the dramatic decrease in the P aeruginosa samples. Error bars indicate 95% confidence intervals.
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Co-culture of TM and TR with P aeruginosa decreased the hyphal-spore ratio to 0.16 and 0.04 compared with control TM and TR cultures, respectively. In contrast, the hyphal-spore ratio increased to above 30 in the presence of S epidermidis, E coli, and control media (Figure 1). No fungal organisms could be recovered by culture from a 15-day co-culture of TM or TR with P aeruginosa, while fungi were easily recoverable from co-cultures using E coli or S epidermidis. There was bacterial growth in each control after 15 days of co-culturing.
The fungi, which had been co-cultured for 15 days with control broth, S epidermidis, or E coli, grew successfully at 96 hours when restreaked on Sabouraud agar. The fungi, which had been co-cultured with P aeruginosa for 15 days, showed no growth on Sabouraud agar at 96 hours. A gram stain showed abundant paired cocci in the slides containing S epidermidis and bacilli in the slides containing E coli, growing around the fungal hyphae (Figure 2).
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Figure 2. Gram stains of control bacteria with Trichophyton mentagrophytes (TM) at day 10. Gram stains of Staphylococcus epidermidis (A) and Escherichia coli (B) co-cultured with TM. Note the vigorous growth of TM in the presence of S epidermidis and E coli.
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When freshly plated on Sabouraud agar, TM and TR were both inhibited by P aeruginosa but not the other bacteria or control broth (Table). There was a zone of inhibition of 0.5 to 1.0 cm around the P aeruginosa (Figure 3), but a freeze-thaw–killed sample of P aeruginosa did not inhibit fungal growth. A punch biopsy of the zone of inhibition was taken, and P aeruginosa readily grew when inoculated onto sheep blood agar plates.
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Table. Inhibition (+) or No Inhibition (–) of Fungal Growth by 30 µL of Control Media, Staphylococcus epidermidis, Escherichia coli, or Pseudomonas aeruginosa Inoculated in the Center of Freshly Plated TR or TM
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Figure 3. Zone of fungal inhibition by Pseudomonas aeruginosa. Sabouraud agar inoculated with P aeruginosa (yellow) after freshly streaking Trichophyton mentagrophytes. Note the zone of fungal inhibition by the P aeruginosa.
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Pseudomonas aeruginosa inoculated onto mature lawns of TM and TR degraded the fungus, leading to a hole in the lawn by 96 hours (Figure 4), whereas the drops of both E coli and control media remained as fluid drops on top of the lawns with no apparent effect.
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Figure 4. Pseudomonas aeruginosa eroded through the mature Trichophyton mentagrophytes (TM) fungal lawn at 96 hours. Mature lawn of TM on Sabouraud agar with a 50-µL droplet of control Mueller-Hinton media sitting on top of the fungal lawn, demonstrated by the fact that it has rolled to the side of the lawn (A); a 50-µL aliquot of P aeruginosa shown eroded through the fungal lawn (B).
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COMMENT
Previous experimental observations demonstrated that fungi associated with tinea pedis are difficult to isolate via culture once there is superimposed bacterial infection.3 This observation raises the question of whether the bacteria in toe web infections possess fungistatic or fungicidal properties. Evidence of bacteria possessing antifungal properties has been shown previously for P aeruginosa and can form a glycocalyx biofilm around C albicans that inhibits its growth in the filamentous form. In addition, the pyocyanins produced by P aeruginosa demonstrate antifungal properties against Aspergillus fumigatus and C albicans.5-7
Pseudomonas aeruginosa, the most common pathogen isolated from bacterial toe web infection, specifically inhibited the growth of TM and TR when compared with S epidermidis (a commensal skin colonizer) or E coli (a gram-negative bacterial control). In our experiments, direct co-culture experimental conditions that simulated the moist toe web environment were used. Pseudomonas aeruginosa, but not E coli or S epidermidis, exhibited time-dependent antifungal properties against TM and TR (Figure 1 and Figure 5). No viable fungal elements could be recovered at 15 days via culture after eliminating P aeruginosa with gentamicin. Viable, but not killed, P aeruginosa was able to induce a durable zone of inhibited fungal growth. This observation indicates that live bacteria possess a fungistatic or fungicidal product that may be secreted. However, within the zone of fungal inhibition, viable P aeruginosa were recoverable via culture; therefore, a direct contact–dependent, growth-inhibitory mechanism may also account for these results. Potentially, P aeruginosa may be inhibiting fungal growth via biofilm formation. This co-culture model can be further used to characterize the nature of the fungal growth-inhibitory properties of P aeruginosa. Further experiments are warranted to elucidate the specific antifungal mechanism displayed by P aeruginosa.
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Figure 5. Photographs of fungal growth. Fluorescent photography with calcofluor white highlighting Trichophyton mentagrophytes (TM) hyphae and spores when co-cultured with control Mueller-Hinton media, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa at days 1, 5, 10, and 15. Note the abundant hyphae proliferating in each control compared with only fragmented fluorescent pieces in the sample containing TM co-cultured with P aeruginosa.
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AUTHOR INFORMATION
Correspondence: John T. Seykora, MD, PhD, Department of Dermatology, University of Pennsylvania Medical School, 211a Clinical Research Bldg, 415 Curie Blvd, Philadelphia, PA 19104.
Accepted for Publication: April 29, 2006.
Author Contributions: Study concept and design: Treat, James, Nachamkin, and Seykora. Acquisition of data: Treat and Seykora. Analysis and interpretation of data: Treat, James, and Seykora. Drafting of the manuscript: Treat and Seykora. Critical revision of the manuscript for important intellectual content: Treat, James, Nachamkin, and Seykora. Obtained funding: Seykora. Administrative, technical, and material support: Treat, James, and Nachamkin. Study supervision: James, Nachamkin, and Seykora.
Financial Disclosure: None reported.
Author Affiliations: Departments of Dermatology (Drs Treat, James, and Seykora) and Pathology and Laboratory Medicine (Dr Nachamkin), University of Pennsylvania School of Medicine, Philadelphia.
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