Antimicrobial activity of filamentous fungi isolated from highly antibiotic-contaminated river sediment

Background: Filamentous fungi are well known for their production of substances with antimicrobial activities, several of which have formed the basis for the development of new clinically important antimicrobial agents. Recently, environments polluted with extraordinarily high levels of antibiotics have been documented, leading to strong selection pressure on local sentinel bacterial communities. In such microbial ecosystems, where multidrug-resistant bacteria are likely to thrive, it is possible that certain fungal antibiotics have become less efficient, thus encouraging alternative strategies for fungi to compete with bacteria. Methods: In this study, sediment of a highly antibiotic-contaminated Indian river was sampled in order to investigate the presence of cultivable filamentous fungi and their ability to produce substances with antimicrobial activity. Results: Sixty one strains of filamentous fungi, predominantly various Aspergillus spp. were identified. The majority of the Aspergillus strains displayed antimicrobial activity against methicillin-resistant Staphylococcus aureus, extended-spectrum beta-lactamase-producing Escherichia coli, vancomycin-resistant Enterococcus faecalis and Candida albicans. Bioassay-guided isolation of the secondary metabolites of A. fumigatus led to the identification of gliotoxin. Conclusion: This study demonstrated proof of principle of using bioassay-guided isolation for finding bioactive molecules

Effects of PAMPs on gliotoxin production by <i>A. fumigatus</i> during a 13-day period of incubation at 37°C.

<p>The graphs depict gliotoxin concentrations as a function of time for triplicate liquid cultures incubated with LPS (<b>A</b>), LTA (<b>B</b>) and PG (C) at concentrations of 0.3, 1 or 5 μg/ml. In all experiments, the maximum gliotoxin concentration rose as the PAMP concentration was increased. The highest gliotoxin concentrations were measured between days five and eleven. Error bars show the standard error of the mean for the triplicate experiments.</p

The gliotoxin concentration in the growth media of the four subgroups with 40 cultures each and their standard errors of mean.

<p>The Mann-Whitney test indicated that the reference group had a significantly (P<0.001) lower gliotoxin concentration than the other three, indicated by (*), which were grown in the presence of 5 μg of LPS, LTS or PG per ml.</p

The influence of the PAMPs at concentrations of 0.1–100 μg/ml on the production of gliotoxin by <i>A. fumigatus,</i> based on triplicate <i>A. fumigatus</i> cultures incubated at 37°C for seven days.

<p>The gliotoxin concentrations are normalized against those for the control group (38 μM), and the error bars show the standard error of the reported mean. The gliotoxin level increased in all cultures exposed to PAMPs at concentrations above 0.1 μg/ml.</p

HPLC chromatograms at 254 nm and MS spectra of liquid cultures of <i>A. fumigatus</i> after seven days of incubation.

<p><b>A:</b> Overlaid chromatograms for the control culture (below) and cultures exposed to 5 μg PG/ml (above). The grey box indicates the gliotoxin peaks. The PG-culture chromatogram is almost identical to that of the control and those for cultures containing LTS and LPS, with the exception of the intensity of the gliotoxin peak at 19.5 minutes. This indicates that the presence of PAMPs did not induce the production of any new metabolites or in any other way radically altered the metabolic profile. <b>B:</b> A representative MS spectrum showing the ion pattern of gliotoxin, which features prominent ions at 263.1, 245.1 and 227.1, m/z together with various background ions. The mother ion at 327.4 m/z is also indicated.</p

Induction of Gliotoxin Secretion in Aspergillus fumigatus by Bacteria-Associated Molecules

Aspergillus fumigatus is the most common causative agent of mold diseases in humans, giving rise to life-threatening infections in immunocompromised individuals. One of its secreted metabolites is gliotoxin, a toxic antimicrobial agent. The aim of this study was to determine whether the presence of pathogen-associated molecular patterns in broth cultures of A. fumigatus could induce gliotoxin production. Gliotoxin levels were analyzed by ultra-performance liquid chromatography and mass spectrometry. The presence of a bacteria-derived lipopolysaccharide, peptidoglycan, or lipoteichoic acid in the growth media at a concentration of 5 mu g/ml increased the gliotoxin concentration in the media by 37%, 65%, and 35%, respectively. The findings reveal a correlation between the concentrations of pathogen-associated molecular patterns and gliotoxin secretion. This shows that there is a yet uncharacterized detection system for such compounds within fungi. Inducing secondary metabolite production by such means in fungi is potentially relevant for drug discovery research. Our results also give a possible explanation for the increased virulence of A. fumigatus during bacterial co-infection, one that is important for the transition from colonization to invasiveness in this pulmonary disease