Bacillus subtilis induces morphological changes in Fonsecaea pedrosoi in vitro resulting in more resistant fungal forms in vivo

Interactions among microorganisms may be the cause of morphological modifications, particularly in fungal cells. The aim of this work was to examine the changes that occur in cells of the fungus Fonsecaea pedrosoi after in vitro co-culturing with Bacillus subtilis and to explore the results of this interaction in vivo in an experimental murine infection. B. subtilis strain was inoculated into a 15-day pure culture of F. pedrosoi. In vitro, after 48 hours of co-culturing, the fungal cells were roundish. The secretion of fungal dark pigments and production of terminal chlamydoconidia were observed in hyphae after one week. In the in vivo study, two animal groups of 30 BALB/c mice each were employed. One group was inoculated intraperitoneally with hyphal fragments from the co-culture of bacteria and fungi; the other group was infected only with F. pedrosoi hyphae. After seven days of infection, both animal groups developed neutrophilic abscesses. Phagocytosis of bacilli by macrophages occurred at three days. At later periods, generally after 25 days, only roundish cells similar to sclerotic bodies remained in the tissues while hyphae were eliminated by 15 to 20 days. These fungal forms originated mainly from terminal chlamydoconidia. The co-culturing between bacteria and fungi may constitute a mechanism to rapidly obtain resistant fungal forms for host defenses, especially for chromoblastomycosis (CBM) experimental infections.


INTRODUCTION
Gram-positive bacilli are widespread in the environment, particularly in soils.These organisms have been used for several biotechnological applications, for example, as biocontrollers, probiotics, bioregulators, and as producers of antibiotics, insecticides and enzymes (1)(2)(3)(4)(5)(6).Gram-positive bacteria such as the ones from Bacillus genera can secrete a variety of substances that are inhibitory to the growth of environmental and phytopathogenic fungi (7)(8)(9)(10).This organism has also been reported to inhibit a fungal chromoblastomycosis (CBM) agent in vitro (11).It has also been reported that Bacillus spp.can induce morphological changes in dematiaceous fungi (12)(13)(14).
Dematiaceous fungi are saprobes found mainly in soils and organic matter functioning as decomposition agents (15).A natural characteristic of these microorganisms is their dark cellular pigment, which is the result of a melanization process.Melanin has been linked to high virulence, resistance in adverse environmental conditions and low susceptibility to antifungal drugs (16,17).Many dematiaceous fungi are harmful to their hosts, in particular the Herpotrichiellaceae family, which includes a large number of CBM agents (18).Worldwide, Fonsecaea pedrosoi is the species most frequently isolated in CBM disease, especially in humid areas (19).Generally, infection occurs after a traumatic inoculation with vegetable fragments that are contaminated with the fungus (20)(21)(22).A typical sign of the disease is the presence of parasitic forms that are commonly known as sclerotic cells (bodies) in the tissue (20).
In a preliminary study, we isolated an environmental gram-positive bacterium from Brazilian soil that antagonizes several fungi, including some dematiaceous species (23).Herein, we studied the interactions between this bacterium and a clinical strain of F. pedrosoi in two manners: firstly, we examined the in vitro changes in fungal cells after co-culturing with bacillus, and secondly, we explored the in vivo significance of this interaction.

Animals
Two groups of 30 male BALB/c mice each weighing about 23 g (6 to 8-week-old), were used throughout this study.The specific-pathogenfree animals were purchased from CEDEME/ UNIFESP (Brazil).The protocol used was approved by the UNIFESP Ethics Committee under project number 0808/05.

Microorganisms
F. pedrosoi strain (EPM -380/03) was isolated from a patient with CBM examined in the Dermatology Outpatient Department, UNIFESP, in 2003.It was cultivated on Sabouraud dextrose agar (SDA -Difco Laboratories, USA) supplemented with 80 mg/L gentamycin at a temperature of 28°C, with periodic transfers at 15-day intervals into a new SDA medium containing antibiotics.An environmental bacillus isolated from random soil samples collected from the campus of the Federal University of Mato Grosso (Cuiabá, Mato Grosso state, Brazil) was used in this study; as in previous co-culture assays, it was able to inhibit the growth of some phytopathogenic fungi and to induce the release of dark pigment by dematiaceous molds.This bacterium was identified as B. subtilis by classical and molecular methods (23).

F. pedrosoi Hyphae
Cultures of F. pedrosoi were incubated for 15 days on Sabouraud dextrose broth (SDB) with and without gentamycin, pH 5.7, while shaking at 150 rpm at 28°C.

Co-culture of F. pedrosoi and B. subtilis
The B. subtilis strain was inoculated with a sterilized loop into 15-day F. pedrosoi cultures on SDA or SDB without antibiotic and maintained at a temperature of 28°C.SDB cultures were agitated at a rate of 150 rpm for one week.These co-cultures of cells were analyzed daily by optical microscopy.

Inocula
Broth cultures were vortexed with 4 mm glass beads for 20 minutes and filtered on sterilized gauze to retain macroscopic globoid mycelia.With a 50-mL needle, the filtrates were drained and dispensed several times in a 100 mL glassbeaker to break up small mycelia-clusters and obtain solitary hyphal fragments.Viability of inocula was assessed with LIVE/DEAD® Cell Vitality Assay Kit (L34951, Invitrogen, USA) by fluorescence microscopy.The viability of inoculum suspensions generally was defined by the presence of more than 97% of cells being alive.The suspensions containing fungal forms were adjusted to a final concentration of 1 x 10 6 cells in a Neubauer chamber.The separation between septa was used to distinguish individual hyphal cells, which were then counted.

Infection
Ten minutes prior to infection, animals were anesthetized by intraperitoneal route with 0.4 μL of Anasedan® and Dopalen® 0.35 mL/kg (Vetbrands, Brazil).Approximately 100 µL of the suspension containing 1 x 10 6 hyphal cells (about 1 x 10 5 short hyphal fragments) was inoculated intraperitoneally (IP) with a 25x8/21G1 needle.One group of mice was infected IP with hyphal fragments from the co-culture of fungi and bacteria, while the other group was inoculated only with F. pedrosoi hyphae.The kinetics of the infection was followed for one month.Three animals were sacrificed every three days, and the abscesses were immediately removed.Part of the infected tissue specimens was cultivated on SDA.This experimental infection was performed in duplicate.

Histopathology
Fragments of fresh tissue samples from abscesses were left in 10% formalin for 12 hours.The specimens were embedded in paraffin, and serial sections of 3 to 5-µm from the blocks were stained with hematoxylin-eosin (HE) and by the Brown-Brenn Method (tissue gram stain).

Dark-Pigment Secretion on Media Culture
Viable F. pedrosoi cells from the SDB cocultures with B. subtilis were observed daily over the course of the week.The secretion of a chestnut-brown pigment, probably melanin, was observed in co-cultures on SDA (Figure 1 -A) and SDB (Figure 1 -B) media.Cultures with antibiotic did not show dark pigments (Figure 1 -C).

Modifications of Fungal Morphogenesis
Usually, the morphology of F. pedrosoi hyphal cells on broth media with antibiotic is linear (Figure 2 -A), with lower conidium production by conidiophores.After 48 hours of co-culturing with B. subtilis, however, the F. pedrosoi hyphae were roundish (Figure 2 -B).The hyphal conidiogenesis and conidiophore (phialid-type) production were also enhanced (Figure 2 -C).Terminal globoid cells (chlamydoconidia-like) of 5 to 10 µm in diameter, usually with a thick wall, were found in hyphae after a week of co-culturing (Figure 3).

Murine Infection with F. pedrosoi Hyphae and Co-culture of Fungal Cells and B. subtilis
In both animal groups, we observed an intense inflammatory response and palpable lesions of approximately 8 mm in diameter a week postinfection.A capsulated neutrophilic abscess with a necrotic center was found at the inoculation site.Cellular layers were composed of distinct sets of specific cells, such as neutrophils in the central region, foamy macrophages in the periphery, and fibroblasts surrounding the abscess.A scheme showing abscess layers is presented in Figure 4. Lymphocytes were rarely detected.Dematiaceous hyphae were found in a necrotic center.Grampositive bacilli had been phagocytized by macrophages by the third day post-infection with the co-culture.After one week, however, only F. pedrosoi cells remained and were recovered from the infected tissue.Fungal forms similar to sclerotic bodies, in particular those proceeding from the terminal chlamydoconidia, were observed in histological sections of the abscess produced by the infection with the co-cultured cells (Figure 5).These brown-rounded cells were in vivo less susceptible than hyphae to the microbicidal action of phagocytes.In general, most of the animals that were infected with the co-cultured cells healed after 25 to 30 days, while the clinical and mycological healing of mice infected with only F. pedrosoi hyphae were frequently achieved by 15 to 20 days of infection.

DISCUSSION
Gram-positive bacilli and fungi are important bioregulators widespread in soil (2,24,25).The coexistence of microorganisms in the environment during evolution has promoted several interactions including predation, symbiosis, mutualism, competition and antibiosis (3,26).Microbial antagonisms have been studied due to their numerous biotechnological applications, particularly those related to development of new biocontrol forms (1,3,25).Many Bacillus species present natural biocidal activities against fungi and other bacteria, and are frequently described as biocontrollers of phytopathogens (7,13,25,27,28).In the present study, an environmental bacterium (B.subtilis strain) with a good antagonic activity against environmental fungi and phytopathogens (23) was co-cultured with F. pedrosoi (clinical isolate).
The melanin secretion by the dematiaceous fungus increased after co-culturing with Bacillus and was continued later in broth and agar media.The ability of microbes to synthesize melanins is related to virulence and pathogenicity (17).Fungal melanogenesis was found to be stimulated after co-culturing Cryptococcus neoformans with Klebsiella aerogenes (29).Homogentisic acid, derived from the bacterial metabolism, seems to be capable of inducing fungal melanization (30).Several pathogenic species can be transformed into melanized forms or secrete melanin in order to increase antifungal resistance or evade host defense mechanisms (16,17).It is possible that the melanin secretion by F. pedrosoi is associated with bacterial antibiosis or that metabolic substances are capable of inducing the melanogenesis pathway; however, more studies are necessary to confirm these hypotheses.In the present work, even though we observed the microbial growth and survival of two microorganisms in laboratory conditions, the results observed are likely to occur in nature.
Chlamydoconidia are roundish cellular forms, with a thick wall, found in filamentous fungi at intercalary or terminal positions in the hyphae.Generally, these cells are formed under environmental stress and improve fungus survival under adverse conditions.The formation of chlamydoconidia-shaped cells was observed after the interaction of dematiaceous fungi with B. subtilis or with bacterial metabolic products (13,14).The introduction of bacillus into 15day cultures of F. pedrosoi caused morphological changes in the fungal hyphae, including the presence of terminal chlamydoconidiashaped cells.Previously, it has been noted that Pseudomonas cepacia mutants that are defective in the production of antifungal compounds fail to induce cellular abnormalities in phytopathogenic fungi, suggesting that antibiosis may be responsible for fungal morphological changes (31).Hence, our experiments raise concerns about the possibility that resistant fungal forms may be spread in nature as a result of several factors, including microbial interactions.
Curiously, we observed in histological sections that the host inflammatory response was not fully capable of eliminating all of the F. pedrosoi forms in the tissue.Some chlamydoconidia-shaped cells persisted a few days longer than the hyphae in host tissue.For this, morphological changes may be an important factor in fungal resistance to host defenses.Although these cells were more resistant to host immunity, all mice had healed by 25 days post-infection.
In the history of CBM research, several works have focused on establishing an experimental infection that is similar to the human form of the disease (32)(33)(34)(35)(36). Currently, however, there is no suitable animal model for this purpose.Nonetheless, chlamydoconidia-shaped cells have been shown to be worthy of future studies on the development of experimental infections, in which B. subtilis or other antagonistic strains can be used in co-cultures with CBM agents, especially because these forms may be precursors of sclerotic bodies.Recently, we developed a murine chronic infection after inoculation of round cells and chlamydoconidia from F. pedrosoi cells cultivated for long periods (23).
In conclusion, our results show that the interaction in vitro between F. pedrosoi and B. subtilis stimulated melanin secretion and induced morphological changes in a clinical isolate of F. pedrosoi.Chlamydoconidia-shaped cells were more resistant to the host immune response than hyphae.Thus, co-culturing antagonistic bacteria and CBM pathogens may be an interesting method for obtaining fungal forms that are more resistant to host defense.

Figure 1 .
Figure 1.Chestnut-brown pigment secretion after one week of co-culturing F. pedrosoi with B. subtilis.(A) Plate co-culture on SDA medium, (+) positive reaction of pigment secretion proximal to bacterial colonies and (-) negative region.(B) Flask co-culture on SDB medium without antibiotic.(C) Pure culture of F. pedrosoi cultivated for 15 days on SDB medium with antibiotic.Bs: Bacillus subtilis colony.

Figure 3 .
Figure 3. Fonsecaea pedrosoi terminal roundish-cell observed in vitro after a week of co-culturing with B. subtilis strain, 1000x.