Antibacterial Activities of Stationary Phase Culture Filtrates of Aspergillus fumigates

Simultaneous static coculturing of A. fumigatus conidia or sporelings with P. aeruginosa cells in vitro results in the initial growth inhibition and eventual death of A. fumigatus cells [1], perhaps elicited by P. aeruginosa produced small diffusible extracellular molecules released into the culture medium [2,3]. In contrast, A. fumigatus hyphae grown for 12h or longer from conidia at 35 °C in a rich fungal growth medium such as Sabouraud’s dextrose (SD) broth are relatively unaffected by the fungicidal effect of P. aeruginosa culture filtrate [1]. The lack of effect of P. aeruginosa culture filtrate on A. fumigatus hyphae could be due to either innate resistance of hyphae to the fungicidal factor(s) and/ or due to the production of an antibacterial factor(s) by the growing A. fumigatus hyphae that either inhibits bacterial growth or blocks the fungicidal activity. Since A. fumigatus is known to produce an array of small molecules [4] possessing antimicrobial properties to safeguard its survival, the latter scenario is a likely event in cocultures of P. aeruginosa and A. fumigatus. We therefore investigated the antibacterial activity of A. fumigatus culture filtrates obtained from 24h and 48h cultures of P. aeruginosa.


Introduction
Simultaneous static coculturing of A. fumigatus conidia or sporelings with P. aeruginosa cells in vitro results in the initial growth inhibition and eventual death of A. fumigatus cells [1], perhaps elicited by P. aeruginosa produced small diffusible extracellular molecules released into the culture medium [2,3]. In contrast, A. fumigatus hyphae grown for 12h or longer from conidia at 35 °C in a rich fungal growth medium such as Sabouraud's dextrose (SD) broth are relatively unaffected by the fungicidal effect of P. aeruginosa culture filtrate [1]. The lack of effect of P. aeruginosa culture filtrate on A. fumigatus hyphae could be due to either innate resistance of hyphae to the fungicidal factor(s) and/ or due to the production of an antibacterial factor(s) by the growing A. fumigatus hyphae that either inhibits bacterial growth or blocks the fungicidal activity. Since A. fumigatus is known to produce an array of small molecules [4] possessing antimicrobial properties to safeguard its survival, the latter scenario is a likely event in cocultures of P. aeruginosa and A. fumigatus. We therefore investigated the antibacterial activity of A. fumigatus culture filtrates obtained from 24h and 48h cultures of P. aeruginosa.

Materials and Methods
A. fumigatus 53470 (a clinical isolate obtained from the Microbiology Laboratory of Henry Ford Hospital, Detroit, MI, USA) was used in this study. Cultures were grown for 24h and 48h in SD broth at 35 °C in 6-well Costar cell culture plates. The culture filtrate (CF) was collected and filter-sterilized (0.22µm Millipore filters). For growth inhibition studies, one ml aliquots of the CF supplemented with fresh SD broth was inoculated with 1x10 7 cells/ ml P. aeruginosa 56402 (a clinical isolate obtained from the Microbiology Laboratory of Henry Ford Hospital in Detroit) in 6-ml polystyrene culture tubes (6 replications) and incubated on a gyratory shaker (170 rpm) at 35 °C for 24h. The growth inhibition of P. aeruginosa was determined spectrophotometrically by determining the optical density at 490nm and by colony forming unit (CFU) assay.
The heat stability of the antibacterial factor(s) of the CF was examined after incubating 1ml aliquots in a boiling water bath for 15min followed by the growth inhibition assays. Heat untreated CF and SD broth (1ml aliquots) were used as controls.
The bactericidal effect of the CF was examined by treating 1x10 7 cells with 1ml CF at 35 °C for 24h. The viability of the treated cells was examined by plating 0.01ml aliquots of the diluted culture on SD agar and determining the CFUs after 24h growth.

Results and Discussion
As shown in Figure 1, both spectrophotometric and CFU assays provided similar results. A. fumigatus CF collected from the 48 h stationary phase cultures almost completely inhibited the growth of P. aeruginosa (control: 3.867x10 11 ± 2.161x10 11 CFU/ ml; CF: 9.333x10 6 ± 1.146x10 7 CFU/ ml; p=0.0071) but that obtained from 24h old culture showed no significant growth inhibition (control: 7.392x10 10 ± 1.312x10 11 CFU/ ml; CF: 5.851x10 10 ± 2.389x10 10 CFU/ ml; p=0.540). A CFU assay of P. aeruginosa cells treated with CF showed that the A. fumigatus produced factor was bacteriostatic and no significant reduction of CFUs was obtained for the CF-treated group compared to the initial inoculum whereas the control group showed a CFU increase

Abstract
Our previous studies performed for the development of polymicrobial biofilm showed that simultaneous coculturing of Aspergillus fumigatus conidia or sporelings with Pseudomonas aeruginosa resulted in the killing of A. fumigatus cells whereas hyphae older than 12h were recalcitrant to the bacterial fungicidal activity. Since A. fumigatus produces an array of antimicrobial molecules, we examined the antibacterial activity of A. fumigatus culture filtrates on bacterial growth to examine the possible role of such small molecules minimizing the fungicidal activity of P. aeruginosa by spectrophotometric and colony forming unit assays. Culture filtrates collected from 48h stationary phase cultures inhibited the growth of P. aeruginosa and S. aureus, but not that of Candida albicans. Colony forming unit assay of culture filtrate-treated P. aeruginosa cells showed that the effect was bacteriostatic. Culture filtrate incubated in a boiling water bath for 15min retained its antibacterial activity compared to that of the unboiled culture filtrate. These results show that stationary phase liquid cultures of A. fumigatus produce a heat stable bacteriostatic compound(s) in liquid culture that is released into the growth medium inhibits the growth of P. aeruginosa. The CF boiled for 15 min in a water bath retained its antibacterial activity (7.450x10 6 ± 3.882x10 6 CFU/ ml) compared to the heat untreated culture filtrate (7.717x10 6 ± 4.490x10 6 CFU/ ml) suggesting that the A. fumigatus produced inhibitory factor is a heat stable molecule(s) (p=0.774). In addition to P. aeruginosa, the stationary phase culture filtrate had similar inhibitory effect on the growth of S. aureus whereas the growth of C. albicans was unaffected.

Keywords
Mycotoxins, including those produced by A. fumigatus, are low molecular weight organic molecules produced by the fungus to protect itself from predators and competitors in its ecological niche. Among the many toxins produced by A. fumigatus, gliotoxin [4] is the most potent and well studied, and it possesses a variety of cytotoxic effects, including triggering apoptosis. We postulated that the CF mediated inhibition of P. aeruginosa is due to gliotoxin or an A. fumigatus produced antibacterial factor(s) perhaps similar to that produced by C. albicans against P. aeruginosa in mixed cultures [5]. Bruns et al. [6] and Sekonyela et al. [7] have recently investigated gene expression profiles of young (24h) and mature (48h) A. fumigatus biofilms by Transcriptomics and Proteomics. Not surprisingly, in matured biofilms and liquid shake cultures overall metabolic activity was significantly reduced but energy is channeled to the enhanced expression of genes coding for the biosynthesis of secondary metabolites. In particular, proteins of the gliotoxin secondary metabolite gene cluster were induced in mature biofilm and shake cultures as determined by real-time PCR and HPLC analysis. Our experiments showed that A. fumigatus stationary phase CF inhibited P. aeruginosa growth due to the production a heat stable bacteriostatic compound(s) secreted into the culture medium, and this bacterial growth inhibitory factor(s) may be at least in part responsible for the poor fungicidal effect of P. aeruginosa CF on A. fumigatus hyphae.
Mature biofilms and shake cultures of A. fumigatus is capable of producing an antibacterial factor perhaps similar to gliotoxin to inhibit P. aeruginosa, and this factor may be at least partly responsible for rending protection to A. fumigatus from the fungicidal activity of P. aeruginosa in polymicrobial biofilm.