Introduction

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Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium commonly found in soil and capable of producing a variety of illness in humans. It is the primary bacterial pathogen found in patients with cystic fibrosis (CF). 13 It is well known that P. aeruginosa is a prolific producer of biofilm and that the biofilm embedded cells are highly recalcitrant to antibacterial drugs, and difficult to eradicate in spite of prolonged antibiotic therapy. 48 Previous investigations have revealed that the initial isolates that colonize the lungs of CF patients are usually susceptible to commonly used antibacterial drugs. However, recurrent and the colonizing strains subsequently becomes less susceptible to antibacterial drugs and frequently develop secondary resistance 9,10

Among the more than 200 Aspergillus species described to date, 1113A. fumigatus is the most common filamentous fungal pathogen in man worldwide. Although typically found as a soil-borne organism, A. fumigatus is commonly spread by airborne asexual spores called conidia. The primary portal of entry in man is the respiratory tract. After an initial local infection, it can subsequently spread causing disseminated infection and subsequent invasive aspergillosis in susceptible individuals. 1416 Among the different Aspergillus species recovered from cultures of CF patients’ respiratory secretions, A. fumigatus is the predominant species, with prevalence as high as 60% in certain geographical regions. Although invasive aspergillosis can occur in CF patients, particularly after lung transplantation, the most common complication is allergic bronchopulmonary aspergillosis, 1720 a condition that frequently causes deterioration of pulmonary function in CF patients.

Given the prevalence of P. aeruginosa and A. fumigatus colonization of the respiratory tracts of CF patients, mixed microbial infections involving these organisms are commonly described. 2123 The mixed infection found in CF patients’ lungs with P. aeruginosa and A. fumigatus could produce a mixed microbial biofilm. 2427 One of our aims is to develop an in vitro model for studying the genesis, physical and chemical characteristics, and the antimicrobial susceptibility of polymicrobial biofilm produced by A. fumigatus and P. aeruginosa. During the development of an in vitro model28 for studying the mixed biofilm produced by P. aeruginosa and A. fumigatus mimicking the mixed microbial infection in CF patients, we found that coculturing A. fumigatus conidia withP. aeruginosa cells resulted in the killing of A. fumigatus. Since mixed infections due to A. fumigatus and P. aeruginosa are commonly found in CF patients, in nosocomial pneumonias, 29,30 and in stem cell transplant recipients, 31,32 we postulated that the hyphae of A. fumigatus will be less susceptible to the fungicidal interaction of P. aeruginosa. We therefore investigated the in vitro interaction of P. aeruginosa with different stages of aspergillus growth including conidia, sporelings, young hyphae and mycelia in cocultures.

Materials and methods

Microorganisms and Culture Conditions:

Fungal isolates:

A. fumigatus clinical isolates 43004 (AF43004), 43135 (AF43135) and53470 (AF53470) obtained from the Microbiology Laboratory of Henry Ford Hospital in Detroit, Michigan, USA were used in this study. The initial cultures on a Sabouraud’s dextrose (SD) agar plate were sub-cultured to verify the viability and purity of the cultures. The colony purified isolates were stored as conidial suspensions in 25% glycerol at -80˚C.

A. fumigatus cultures were grown on SD agar for 4 days at 35˚C to produce conidia. The SD agar containing the fungal growth was sliced into small (5 mm2) pieces, transferred to a 50-ml screw-capped conical culture tube containing 25 ml of sterile distilled water and vortexed for 2 min to disperse the conidia from the conidiophores. The resulting fungal suspension was filtered using several layers of sterile cheese cloth to removemycelia and agar debris. The clarified conidial suspension was standardized by hemocytometer count and stored at 4˚C. A. fumigatus conidia do not germinate at 4˚C and remain viable for several months, thus the same batch of conidial suspension can be used for multiple experiments.

Candida albicans 90028 obtained from American Type Culture Collections (Manassas, VA, USA) was subcultured on SD agar overnight. A pin-head size fresh colony was resuspended in 1 ml sterile distilled water and standardized by hemocytometer count to 1 x 106 cells/ml and then used for SD agar plate inhibition assay.

Bacterial isolates:

Three laboratory (PAO1, PA27853, MJK8) and five clinical (PA56036, PA56314, PA56402, CF39wt, CF39s) isolates of P. aeruginosa as well as one laboratory (SA25923) and three clinical (SA56692, SA56693, SA56694) isolates of S. aureus were used in this study. The laboratory isolates were obtained from the American Type Culture Collections except the small colony variant MJK8 whereas the clinical isolates except CF39wt and CF39s were obtained from the Microbiology Laboratory of Henry Ford Hospital, Detroit, MI, USA. MJK8, CF39wt and CF39s were obtained from Matthew Parsek, Microbiology, University of Washington, Seattle, WA. The cultures were grown overnight on Brain Heart Infusion (BHI) agar to verify purity and viability. The colony purified isolates were stored in 25%glycerol at -80˚C. Since all bacterial isolates grew well on SD agar and SD broth, we used these media to study the interaction of A. fumigatus with P. aeruginosa and S. aureus. One ml aliquots of the overnight cultures were centrifuged for 2 min and the pellets washed three times (1 ml each) with sterile distilled water, resuspended in 1 ml fresh SD broth and subsequently used for various experiments.

Agar Plate Inhibition Assay:

We investigated the interaction of P. aeruginosa and S. aureus with A. fumigatus using two different techniques, a plate inhibition and the colony forming unit (CFU) assays. For the plate inhibition assay, SD agar plates were seeded with A. fumigatus conidia (1 x 106 conidia/plate) and 1 cm diameter wells were cut into the agar using a sterile Cork borer. One-tenth ml aliquots of bacterial cell suspensions in fresh SD broth (1 x 108 cells/ml) or overnight cultures containing stationary phase bacterial cells or culture filtrates were placed in the wells and incubated at 35˚C for 24 h. The diameter of the A. fumigatus inhibition zone was determined for each bacterial species and the culture filtrates. Fresh SD broth (0.1 ml) was used as control. The formation of a clear zone of inhibition around the well indicates inhibition of A. fumigatus growth.

To study the effects of P. aeruginosa and S. aureus on growing A. fumigatus hyphae, a circular colony was grown from a point source of conidial inoculum for two days at 35˚C until it reached a diameter of approximately 3 cm. One cm diameter wells were cut into the agar on the periphery of the growing colony using a sterile Cork borer. One-tenth ml aliquots of various bacterial cell suspensions in fresh SD broth (1 x 108 cells/ml) were placed in the wells and the plates were incubated at 35˚C for 24 h. Fresh SD broth (0.1 ml) was used as control. The absence of fungal growth around the well indicated inhibition of A. fumigatus mycelial growth by the bacterial suspension in the well.

Photomicrography:

A. fumigatus conidia pregrown for various time intervals ranging from 0 h to 24 h at 35˚C and actively growing P. aeruginosa cells (1 x 106 cells/ml) were cocultured on sterile 22 mm plastic microscopic cover slips (Cat. no. 12547, Fisher Scientific Company, Pittsburgh, PA) in 6-well Costar cell culture plates for 24 h at 35˚C in SD broth. The plastic cover slips containing the mixed microbial growth were washed with sterile distilled water and transferred to a clean 6-well Costar cell culture plate and stained with crystal violet (0.04 %) for 30 min at 35˚C. The stained cover slips were washed with sterile distilled water and the excess water was drained off and lightly air-dried. The coverslips were mounted on a standard microscopic slide using nail polish and photographed using a Nikon Microscope Camera System equipped with SPOT image processing computer software (SPOT Imaging Solutions, A Division of Diagnostic Instruments, Inc. 6540 Burroughs, Sterling Heights, MI 48314, USA).

Scanning Electron Microscopy:

A. fumigatus-P. aeruginosa polymicrobial biofilm was developed on Tissue Culture Thermanox 13 mm coverslips in SD broth in 12-well cell culture dishes at 35˚C for 24 h using A. fumigatushyphae pregrown for 18 h. The biofilm was washed 3 times with 2 ml each sterile distilled water, fixed for 60 min in 2% glutaraldehyde in 0.1 M sodium cacodylate (NaCac) buffer (pH 7.4), postfixed in 2% osmium tetroxide in NaCac buffer, dehydrated with a graded ethanol series (25-100%) and critical point dried in CO2. The dried specimens were mounted on aluminum stubs with carbon adhesive tabs and sputter coated with gold-palladium. Biofilm was observed and imaged in a FEI XL30 scanning electron microscope (FEI, Hillsboro, OR) at 10 kV.

CFU Assay:

We used CFU assay to determine the cellular effects of A. fumigatus interaction with P. aeruginosa or S. aureus. Details of specific methods used are included in the legends to Figures 06 and 07. Briefly, cocultures of A. fumigatus and P. aeruginosa or S. aureus were grown in 1 ml SD broth in 24-well Costar cell culture plates at 35˚C for 24 h or 48 h and the mixed microbial adherent cells were washed with sterile distilled water. For the CFU determination, the adherent mixed culture containing fungal and bacterial cells was harvested by scraping the bottom of the cell culture plate wells using sterile swabs into 1ml aliquots of sterile distilled water and vortexed vigorously with 0. 1g sterile glass beads (1mm diameter) to disperse the cells. The resulting cell suspensions were diluted serially from 1 x 101 to 1 x 108 fold and 0.01 ml aliquots were spotted on SD agar plates containing either ciprofloxacin (100 μg/ml) or voriconazole (16 μg/ml) for selective fungal and bacterial growth. The numbers of CFUs of A. fumigatus and the bacteria were determined after 24 h growth at 35˚C.

Heat Inactivation of Culture Filtrate:

P. aeruginosa cultures were grown for 24 h and 48 h in 5 ml SD broth at 35˚C and 1 ml aliquots of the cell suspension were centrifuged in a microcentrifuge at 14,000 rpm for 5 min. The resulting supernatant was transferred to a clean tube and 1 ml aliquots were incubated in a boiling water bath for 10 min and the heat-inactivated culture filtrate was used in the plate inhibition assay to determine the anti-aspergillus activity. Heat untreated culture filtrate was used as positive control.

Statistical Analysis:

Where applicable the data were analyzed statistically by Student’s t-test or one way ANOVA with Dunnett’s multiple comparison posttests using Graphpad Prism Version 5.0 for Windows (GraphPad Software, Inc., La Jolla, CA, USA). A p value ≤0.05 was considered significant. Details of specific statistical test(s) used are given in the corresponding figure legends.ResultsPlate Inhibition Assay:

Figure 01A shows the inhibitory effect of actively growing P. aeruginosa cells in the assay well on the growth of A. fumigatus conidia seeded on SD agar. The fact that the bacterial cells were not in physical contact with A. fumigatus conidia yet was able to elicit an inhibitory effect on the fungal growth indicates that a diffusiblecomponent(s) produced by the growing P. aeruginosa cells was responsible for the inhibitory effect. In contrast, S. aureus failed to inhibit the growth of A. fumigatus indicating that the inhibitory effect is organism dependent.

Anti-aspergillus activity of P. aeruginosa.

Fig. 1: Figure. 01

Anti-aspergillus activity of P. aeruginosa.

A. Plate inhibition assay showing the effects of P. aeruginosa and S. aureus cultures on the germination and growth ofA. fumigatus conidia. Symbols: M, Sabouraud’s dextrose broth (control); A, B and C: P. aeruginosa clinical isolates PA56036, PA56314 and PA56402, or S. aureus clinical isolates SA56692, SA56693 and SA56694, respectively. B. Plate inhibition assay showing the effects of culture filtrates obtained from 24 h cultures of P. aeruginosa and S. aureus clinical isolates on A. fumigatus conidia. Symbols: M, Sabouraud’s dextrose broth (control); A, B, and C: culture filtrates obtained from overnight cultures of P. aeruginosa clinical isolates PA56036, PA56314 and PA56402, or S. aureus clinical isolates SA56692, SA56693 and SA56694, respectively. C. Effects of various growth media on the production of anti-aspergillus factor(s) by P. aeruginosa PAO1. Symbols: BHI, Brain Heart Infusion; MH, Mueller Hinton; LB, Lauria Bertani; SD, Sabouraud Dextrose; RPMI1640, Roswell Park Memorial Institute medium 1640. D. Production of anti-aspergillus factor(s) by various clinical and laboratory strains of P. aeruginosa. MJK8 is a small colony variant derived from P. aeruginosa PAO1. P. aeruginosa CF39wt (normal colony producer) and CF39s (a small colony variant) were isolated from a cystic fibrosis patient. Characteristics and sources of other P. aeruginosa strains used are described in the Materials and Methods section. The data were analyzed by Student’s t-test using Graphpad Prism 5.0 and p≤0.05 was considered significant.

In addition to the actively growing bacterial culture, we examined the effect of stationary phase cultures of P. aeruginosa and S. aureus on the growth of A. fumigatus. Aliquots of P. aeruginosaovernight culture, but not those of S. aureus, inhibited the growth of the fungus to the same extent that obtained when actively growing culture was used (data not shown).The fact that overnight culture of P. aeruginosa containing cells at stationary phase was able to inhibit the growth of A. fumigatus suggests the possible involvement of a diffusible factor(s) released into the growth medium. We therefore examined the effect of culture filtrate obtained from stationary phase cultures of P. aeruginosa and S. aureus in a plate inhibition assay. As shown in Figure 01B, culture filtrates obtained from overnight cultures of P. aeruginosa inhibited the growth of A. fumigatus, whereas that obtained from overnight cultures of S. aureus had no effect. Four commonly used microbial growth media and the mammalian cell culture medium RPMI1640 were used to examine the effect of growth medium on the production and subsequent release of anti-aspergillus factor(s) into the culture filtrate. As shown in Figure 01C all microbiological growth media supported the production of the inhibitory factor(s) more or less similarly and no significant difference in either the production or the activity of the inhibitory factor(s) was obtained. On the other hand the production of the inhibitory factor(s) was significantly poor in RPMI1640 compared to that obtained in other microbiological growth media due to poor growth (p ≤ 0. 0443). P. aeruginosa strains PAO1 and PA27853 used in these experiments provided similar results.

Figure 01D shows a comparison of the relative amount of anti-aspergillus soluble activity produced by various clinical and laboratory isolates of P. aeruginosa. All strains we examined were capable of producing the fungal inhibitory soluble activity. P. aeruginosa PAO1, PA56402 and PA56314 produced more or less similar amounts of the inhibitory activity. The clinical isolates PA56036, CF39wt and CF39s produced the lowest amounts of the inhibitory activity). The small colony variants MJK8 and CF39s produced amounts of anti-aspergillus soluble activity similar to those produced by their isogenic parent strains PAO1 and CF39wt, respectively. A comparison of the relative amounts of the inhibitory activity produced by PAO1 with those of other strains showed that the clinical isolate PA56036, (p ≤0.0527) CF39wt and CF39s (p ≤0.0645) produced significantly less amounts of the inhibitory activity. These results suggest that although the genetic trait to produce the anti-aspergillus soluble activity is inherently present in all strains of P. aeruginosa, the amount of the inhibitory soluble activity they produce may vary significantly from strain to strain.

Since P. aeruginosa is known to produce a variety of small molecular virulence factors that are relatively resistant to heat inactivation, we examined the heat stability of the putative inhibitory factor. Culture filtrate boiled in a water bath for 10 minutes, completely lost its ability to inhibit the growth of A. fumigatus suggesting that the P. aeruginosa inhibitory factor was susceptible to heat inactivation (Data not shown).

Figure 02 shows the dose response and the specificity of 24 h culture filtrate of PAO1 against AF43135.The 24 h culture filtrate produced by P. aeruginosa PAO1 in BHI broth was collected by centrifugation in a microcentrifuge (14,000 rpm, 10 min), diluted to obtain 0.78% to 100% in SD broth, phosphate buffered saline (PBS), sterile distilled water and the inhibitory effect of the diluted culture filtrate was examined by SD agar plate inhibition assay. As shown in panels A and B the bioactivity of the culture filtrate was proportional to the amount present in the assay well at low concentrations and the activity reached near maximum at 6.25% to 12.5% of the culture filtrate (Panel B) and use of more concentrated culture filtrate did not yield increased zone of inhibition. When PBS and sterile distilled water were used as diluent almost identical results were obtained (p≥0.122). On the other hand, when the culture filtrate was diluted in SD broth higher concentrations (25% to 100%) produced significantly larger zone of inhibition (p≤0.001). This is probably due to presence of any odd bacterial cells remained in the supernatant after centrifugation might have grown producing additional inhibitory factor(s). Panel C shows the effects of various concentrations (0% to 100%) of P. aeruginosa PAO1 culture filtrate on the growth of Candida albicans 90028. Even the highest concentration of the culture filtrate showed no inhibitory effect on fungal growth in the plate inhibition assay suggesting that the anti-aspergillus activity elicited by P. aeruginosa culture filtrate is not extended to other fungal species such as pathogenic yeasts and appears to be specific to Aspergillus species.

Since the plate inhibition assay does not necessarily discriminate possible inhibitory effect of P. aeruginosa cells on the germination of conidia only without affecting the mycelial growth, we investigated the effects of P. aeruginosa and S. aureus on actively growing A. fumigatus hyphae. Figure 03 shows that P. aeruginosa cells in the assay well inhibited mycelial growth around the well, whereas S. aureus showed no effect. The growing mycelial front almost completely surrounded the wells containing S. aureus cells. On the other hand, the translucent zone of the fungal colony representing the growing apices of the hyphae failed to reach the well con- taining P. aeruginosa cells due to retarded growth.These results suggest that the putative inhibitory factor(s) produced by P. aeruginosa cells was able to inhibit the apical growth of hyphae.

Dose response and specificity of anti-aspergillus factor(s) produced by P. aeruginosa

Fig. 2: Figure. 02

Dose response and specificity of anti-aspergillus factor(s) produced by P. aeruginosa

A. Plate inhibition assay showing the relative activity of various concentrations (0 % to 100%) of P. aeruginosa PAO1 produced inhibitory factor(s) against A. fumigatus AF43135. B. Plot of culture filtrate concentration (%) against the diameter of inhibition zone. C. Plate inhibition assay showing the effects of various concentrations (0% to 100%) of PAO1 culture filtrate on Candida albicans 90028. Sterile diluent of the culture filtrate was used as 0% concentration (negative control). Experiments in panels A and B were performed two times independently and the data shown in panel B represents the mean of two independent experiments. The data were analyzed by one way ANOVA using GraphPad Prism 5.0 and a p value ≤0.05 was considered significant. PBS vs. sterile DH2O p≥0.122); SD broth vs. PBS or sterile DH2O p ≤0.001.

Plate inhibition assay showing the effects of P. aeruginosa and S. aureus clinical isolates on growing mycelia of A. fumigatus.

Fig. 3: Figure. 03

Plate inhibition assay showing the effects of P. aeruginosa and S. aureus clinical isolates on growing mycelia of A. fumigatus.

Symbols: M, Sabouraud’s dextrose broth (control); A, B and C: P. aeruginosa clinical isolates PA56036, PA56314 and PA56402 or S. aureus SA56692, SA56693 and SA56694, respectively.

Interaction of A. fumigatus Conidia, Sporelings and Young Hyphae with P. aeruginosa Cells in Cocultures:

Figure 04 shows a series of photomicrographic images of cocultures of P. aeruginosa and A. fumigatus conidia pregrown for various time intervals ranging from 0 h to 24 h. As the fungal cells were pregrown for a longer period of time before the addition of P. aeruginosa cells, the formation of a firmly adherent mixed microbial culture of P. aeruginosa and A. fumigatus was substantially increased. Ungerminated conidia cocultured with P. aeruginosa cells for 24 h failed to undergo even the initial stage of conidial germination such as swelling (Panel A). Conidia pregrown for 3 h underwent swelling and cell wall softening, but P. aeruginosa cells added to the culture inhibited subsequent process of germination even after 24 h coculturing (Panel B). Cocultures of P. aeruginosa and A. fumigatus conidia pregrown for 6 h (Panel C) and 9 h (Panel D) showed modest amount of growth but failed to establish a network of hyphae adherent to the polystyrene substrate during coculturing. However, the mixed microbial culture produced a more prominent biofilm growth containing numerous bacterial cells embedded in the extracellular matrix masking the growing hyphae. Panels E and F show photomicrographic images of cocultures of P. aeruginosa and A. fumigatus conidia pregrown for 12 h and 24 h, respectively. The inhibition of the hyphal growth is minimal and the growing hyphae established an extensive network of mycelial growth firmly attached to the surface of the plastic cover slips producing a mixed microbial biofilm.

Photomicrographic images of cocultures of P. aeruginosa 56402 and A. fumigatus 53470 conidia pregrown for various time intervals ranging from 0 h to 24 h at 35˚C

Fig. 4: Figure. 04

Photomicrographic images of cocultures of P. aeruginosa 56402 and A. fumigatus 53470 conidia pregrown for various time intervals ranging from 0 h to 24 h at 35˚C

Symbols: A, coculture of ungerminated conidia with P. aeruginosa; B-F, cocultures of conidia pregrown for 3 h, 6 h, 9 h, 12 h and 24 h with P. aeruginosa. Magnification 400X.

Figure 05 shows low (panels A and B) and high (Panels C and D) magnification scanning electron micrographs of 48 h old A. fumigatus-P. aeruginosa polymicrobial biofilm developed on Thermanox plastic coverslips using A. fumigatus hyphae pregrown for 18 h at 35˚C.The biofilm growth of the mixed microbial culture showed the presence of numerous parallel-packed hyphae (PPH) of A. fumigatus held together as loosely organized ropes by the extracellular matrix (ECM) that enables the P. aeruginosa cells firmly adhere to each other as well as to the fungal hyphae using them as scaffolding forming the polymicrobial biofilm architecture (panel C and D). The ECM is used by the bacterial cells as a cementing agent to bind together the individual cells. A. fumigatus mycelial growth was firmly adhered to plastic coverslips and an increasing number of parallel bundles of hyphae coalesced as mycelial sheaths were present in the more matured (48 h) biofilm. The P. aeruginosa cells were attached to the fungal hyphae as anchoring points and among themselves forming a network for the production of extracellular matrix encasing bacterial cells. The presence of the bacterial cells appeared to have minimal effect on A. fumigatus hyphae as determined by CFU assay.

Low (A and B) and high (C and D) magnification scanning electron micrographs of 48 h old A. fumigatus- P. aeruginosa (AF43135-PAO1) polymicrobial biofilm.

Fig. 5: Figure. 05

Low (A and B) and high (C and D) magnification scanning electron micrographs of 48 h old A. fumigatus- P. aeruginosa (AF43135-PAO1) polymicrobial biofilm.

The bacterial cells are attached to the fungal hyphae using them as scaffolding (Panels C and D) and among themselves using their surface appendages weaving a skeletal network to support the extracellular matrix encasing the bacterial cells. Symbols: PPH, parallel-packed hyphae; ECM, extracellular matrix.

As shown in Figure 06A the fungicidal activity of P. aeruginosa on A. fumigatus conidia was directly proportional to the number of P. aeruginosa and A. fumigatus cells used in cocultures. When 1 x 106 A. fumigatus conidia were cocultured with 1 x 101 to 1 x 106 P. aeruginosa cells, with a P. aeruginosa to A. fumigatus cell ratio of 0.00001 to 1, the bacterial cells killed A. fumigatus conidia significantly at a cell ratio ≥ 0.001, whereas modest to no significant killing was obtained below the cell ratio 0.001. When the cell ratio reached 1, fungal growth was not achieved above the threshold of detection, indicating that A. fumigatus conidia were completely killed by P. aeruginosa cells. Furthermore, both the laboratory and the clinical isolates of P. aeruginosa were able to kill A. fumigatus cells with similar efficiency, suggesting that the fungicidal activity of P. aeruginosa is not strain dependent (Data not shown). In addition to P. aeruginosa, we investigated the interaction of S. aureus cells with A. fumigatus conidia in cocultures. Unlike P. aeruginosa, S. aureus did not demonstrate any fungicidal activity against A. fumigatus conidia. However, fungal growth was significantly inhibited, perhaps due to a population effect and competition for essential nutrients (Data not shown).

Although coculturing of A. fumigatus conidia and P. aeruginosa resulted in cell density dependent killing of conidia as determined by the CFU assay, it was unclear whether the lack of CFU formation by A. fumigatus conidia in the presence of P. aeruginosa was due to the inhibition of conidial germination or due to the inhibition of vegetative growth of sporelings or both. We therefore investigated the interaction of A. fumigates conidia pregrown for various time intervals with P. aeruginosa cells in cocultures. As shown in Figure 06B, in both 24 h and 48 h cocultures the susceptibility of A. fumigatus conidia to the fungicidal effect of P. aeruginosa was steadily declined as the time of germination and growth increased. Conidia grown for 12 h or longer showed no susceptibility to P. aeruginosa when compared to the control for 24 h (p = 0.1421) and 48 h (p = 0.0908) cocultures. However, a comparison of the CFUs produced by germinating conidia in 24 hand 48 h cocultures showed that in the latter the conidia were almost completely killed when pregrown for 0 h to 6 h, whereas only partial killing (p values ranged from 0.0298 to 0.0313) was obtained in 24 h coculture

Figure 06C shows the effect of A. fumigatus conidia and sporelings on P. aeruginosa growth in cocultures. A modest increase (not decrease) in P. aeruginosa cell numbers was seen with increased time of germination and growth of conidia. The highest number of P. aeruginosa cells was obtained when conidia were allowed to germinate and grow for 24 h providing the largest surface area for bacterial adhesion. Thus, the increased P. aeruginosa CFU was obtained mainly because of the increased amounts of mycelia available for P. aeruginosa cells to adhere using the mycelia as scaffolding. The attached bacterial cells together with the fungal hyphae produced a mixed culture in which the bacterial cells were firmly embedded in the mycelial growth that produced a network of extracellular matrix (see Figure 05 for details). This finding suggests that the poor activity of P. aeruginosa cells on A. fumigatus conidia grown for 12 h or longer is not because of decreased growth of bacterial cells in the presence of growing fungal cells.

Interaction of germinated and ungerminated conidia with P. aeruginosa cells.

Fig. 6: Figure. 06

Interaction of germinated and ungerminated conidia with P. aeruginosa cells.

A. Effects of various P. aeruginosa 56402 cell densities on the survival of A. fumigatus 53470 conidia in cocultures.A. fumigatus conidia (1 x 106/ml) were cocultured with varying numbers of P. aeruginosa cells (1 x 101 cells to 1 x 106 cells) in 1 ml SD broth in 24–well Costar cell culture plates for 24 h at 35˚C. The mixed cultures were washed with and resuspended in 1 ml sterile distilled water, diluted 1 x 101 to 1 x 106 fold and 0.01 ml aliquots were spotted on ciprofloxacin (100μg/ml) containing SD agar plates for selective growth of A. fumigatus. The numbers of CFUs for A. fumigatus were determined after 24 h growth at 35˚C and the resulting A. fumigatus CFUs/ml were plotted against P. aeruginosa cell densities used. The experiment was repeated once with PA56402 and AF53470 and twice with PA27853 and AF43135. Similar results were obtained each time. The vertical bar on each data point represents the standard deviation of two independent experiments. B. Survival of A. fumigatus (AF53470) conidia and sporelings as a function of time of germination and growth in 24 h and 48 h cocultures with P. aeruginosa (PA56402) cells. The experiment was repeated once and similar results were obtained each time. The vertical bar on each data point represents the standard deviation of two independent experiments. The data were analyzed by Wilcoxon matched-pairs ranked test and pair-wise comparison of each data set by Student’s t-test for 24 h and 48 h cocultures and a p value ≤0.05 was considered significant. C. Effects of A. fumigatus(AF53470) conidia germination and growth on P. aeruginosa (PA56402) in 48 h cocultures. The experiment was repeated once and similar results were obtained each time. The vertical bar on each data point represents the standard deviation of two independent experiments.

Interaction of A. fumigates Hyphae and P. aeruginosa Cells:

Figure 07 shows the interaction of various clinical isolates of P. aeruginosa or S. aureus cells in 24 h cocultures with A. fumigatus hyphae pregrown for 18 h at 35˚C before the addition of the bacterial cells. As shown in panel A, none of the clinical isolates of P. aeruginosa or S. aureus showed significant fungicidal activity against A. fumigatus hyphae compared to the control where the hyphae were inoculated with an equal volume of SD broth instead of a bacterial cell suspension (p≥0.5672). Similarly, Panel B shows the adherent growth (CFU/ ml) of various clinical isolates ofP. aeruginosa and S. aureus in the presence of A. fumigatus mycelia in cocultures. All six bacterial isolates grew well in the presence of A. fumigatus hyphae (p≥0.6024) precluding the possibility that substantial inhibition of bacterial growth by A. fumigatus hyphae as the reason for the poor fungicidal activity against A. fumigatus hyphae. These results are in sharp contrast to the cell density dependent fungicidal interaction of P. aeruginosa with ungerminated conidia, sporelings and young hyphae (≤9 h old) of A. fumigatus.

Interaction of A. fumigatus 53470 hyphae with various bacterial isolates.

Fig. 7: Figure. 07

Interaction of A. fumigatus 53470 hyphae with various bacterial isolates.

SA. Effects of various clinical isolates ofP. aeruginosa or S. aureus on A. fumigatus hyphae in cocultures. B. Effects ofA. fumigatus hyphae on the growth of variousP. aeruginosa orS. aureus clinical isolates used in cocultures. A. fumigatus conidia (1 x 106/ml) were grown in 1 ml of SD broth in 24-well Costar cell culture plates at 35˚C for 18 h. The adherent hyphae were washed, inoculated with 1 x 106 bacterial cells in 1 ml SD broth and incubated at 35˚C for 24 h. The mixed microbial cultures produced by the cocultured A. fumigatus and the bacterial cells were washed, suspended in 1 ml sterile distilled water, diluted 1 x 101 to 1 x 108 fold and 0.01 ml aliquots of the diluted cell suspensions were spotted on ciprofloxacin (100 μg/ml) or voriconazole (16 μg/ml) containing SD agar plates for selective growth and the CFUs/ml were determined. Each experiment was repeated once, and the vertical bar on each histogram represents the standard deviation of two independent experiments. The data were analyzed by one way ANOVA using Dunnett’s multiple comparison posttest where the CFU values obtained for the control were compared with all the other data sets and p values were ≥0.5672 (Panel A) and ≥0.6024 (Panel B). SA56692, SA56693, SA56694 and PA56036, PA56314, PA56402 denote the clinical isolates of S. aureus and P. aeruginosa, respectively. Sterile SD broth (1 ml each) was used as negative control and treated identically for CFU assay. No fungal or bacterial colonies were obtained from the negative control.

Discussion

Although bacterial-fungal interactions commonly occur in a variety of natural habitats because of the ubiquitous presence of these microorganisms and their propensity to live in communal groups as opposed to free-floating individual cells, we know very little about the consequences of such interactions in these diverse ecological niches. More importantly, bacterial-fungal interactions commonly take place in the human body due to their coexistence as part of the human microbiota. 3335 Such complex interactions may be either beneficial or harmful 3638 to human health. For instance, although the respiratory tracts of CF patients are frequently co-colonized with P. aeruginosa and A. fumigatus, the effects of these organisms on each other or on their host are largely unknown, except that both organisms co-exist in the fibrotic lungs of CF patients as a chronic infection. 22,3941 As a preliminary step for the development of an in vitro model for studyingP. aeruginosa-A. fumigatus mixed microbial biofilm, we examined the interactions of A. fumigatus conidia, sporelings, young hyphae and mycelia with P. aeruginosa.

The fungal growth phase dependent fungicidal interaction of P. aeruginosa with A. fumigatus described in this study is analogous to the fungicidal effect of P. aeruginosa and Acinetobacter baumannii against Candida albicans previously reported by several investigators. 37,38,4244 In the case of C. albicans and P. aeruginosa interaction, the bacterial cells specifically adhered to the fungal filaments and killed them in cocultures. In contrast, the bacterial cells failed to bind to the yeast cells during coincubation and showed no effect on their survival in cocultures. Peleg et al. 37 showed in a tripartite in vivo model using the nematode, Caenorhabditis elegans, that A. baumannii inhibited the filamentation of C. albicans and thus prevented the fungus from infecting the worm effectively. In addition, these investigators showed that the filamentous cells of the fungus were killed by the bacterial cells in coculture, whereas their effect on the yeast cells was minimal. 38,4245 The net result was that in a situation where the worm was coinfected with C. albicans and A. baumannii, the anti-nematode activity of C. albicans was markedly attenuated by the inhibitory effect of A. baumannii on the filamentation of C. albicans, known to be a prerequisite for its infection.

Although physiologically active conidia, sporelings and young hyphae were killed by P. aeruginosa, mycelia were not affected by the inhibitory effect of P. aeruginosa cells. The mechanism of this differential activity of P. aeruginosa against A. fumigatus is not known. It is possible that as the sporelings grow and form an extensive network of hyphae producing a prolificmycelial growth, the cell wall composition of the fungal filament is altered 4650 in such a way that the inhibitory factor(s) is unable to bind to the mature hyphae and elicit a fungicidal effect. Alternatively, it is well known that filamentous fungi, including Aspergillus species, extend their hyphal filaments by tip-growths 5155 and the actively growing region of the hyphae is restricted to the apex of the filaments. So in the case of sporelings and young hyphae, when P. aeruginosa inhibits their growth in cocultures for prolonged periods of time such as 24 h and 48 h, the actively growing hyphal cells are faced with a lethal effect. On the other hand, when mature hyphae interact with P. aeruginosa cells, although the tip growth is inhibited, the bulk of the vegetative mycelia remained recalcitrant to the killing effect and was able to produce fungal colonies during the CFUassay.This finding may provide a possible explanation of why P. aeruginosa and A. fumigatus are able to coexist in the respiratory tracts of the CF patients. The colonizing A. fumigatus may establish a chronic infection producing a collection of hyphae that are recalcitrant to P. aeruginosa fungicidal effect and the two microbes establish a cross-kingdom coinfection in CF airways without any detectable deleterious effect on each other.

P. aeruginosa produces a variety of small molecules that possess the ability to inhibit fungal growth. 43,44 They are generally considered to be virulence factors that enable the survival of the organism against competing species in their natural habitats, such as soil and water. The fact that both P. aeruginosa and A. fumigatus are soil-borne organisms occupying similar ecological niches in nature make them natural competitors for limited resources, such as space and nutrients. In essence, the virulence factors that have evolved in nature are to “out-do” the competition and have emerged as a survival mechanism. However, what is unknown at present is whether the small molecule(s) elicits its effect by directly acting on A. fumigatus or whether it triggers some cascade of events involved with the growth of A. fumigatus cells and thus programs them for their self-destruction by unfavorable physiological events.

Conclusions

Coculturing of P. aeruginosa and A. fumigatus conidia, sporelings and young hyphae (<12 h old) results in the death of A. fumigatus cells initiated by a P. aeruginosa produced anti-aspergillus factor(s) secreted into the culture medium. On the other hand, A. fumigatus hyphae grown for ≥12 h are relatively resistant to the fungicidal effect of P. aeruginosa. All P. aeruginosa strains we examined were capable of producing varying amounts of the anti- aspergillus factor(s). The existence of A. fumigatus with P. aeruginosa in cystic fibrosis patient airways without being eliminated by the bacterial cells has been well documented. The relative immunity of A. fumigatus hyphae from the inhibitory factor(s) may be the key to its survival as a co-inhabitant in the highly specialized airway ecological niche.