Screening of Fungi for Antimycobacterial Activity Using a Medium-Throughput Bioluminescence-Based Assay

Abstract

There is a real and urgent need for new antibiotics able to kill Mycobacteria, acid-fast bacilli capable of causing multiple deadly diseases. These include members of the Mycobacterium tuberculosis complex, which causes the lung disease tuberculosis (TB) as well as non-tuberculous Mycobacteria (NTM) a growing cause of lung, skin, soft tissue, and other infections. Here we describe a medium-throughput bioluminescence-based pipeline to screen fungi for activity against Mycobacteria using the NTM species Mycobacterium abscessus and Mycobacterium marinum. We used this pipeline to screen 36 diverse fungal isolates from the International Collection of Microorganisms from Plants (ICMP) grown on a wide variety of nutrient-rich and nutrient-poor media and discovered that almost all the tested isolates produced considerable anti-mycobacterial activity. Our data also provides strong statistical evidence for the impact of growth media on antibacterial activity. Chemical extraction and fractionation of a subset of the ICMP isolates revealed that much of the activity we observed may be due to the production of the known anti-mycobacterial compound linoleic acid. However, we have identified several ICMP isolates that retained their anti-mycobacterial activity in non-linoleic acid containing fractions. These include isolates of Lophodermium culmigenum, Pseudaegerita viridis, and Trametes coccinea, as well as an unknown species of Boeremia and an isolate of an unknown genus and species in the family Phanerochaetaceae. Investigations are ongoing to identify the sources of their anti-mycobacterial activity and to determine whether any may be due to the production of novel bioactive compounds.

Antimicrobial Polyketide Metabolites from Penicillium bissettii and P. glabrum

“…In addition to the assays performed by COADD, the compounds citromycetin ( 2 ) and penialdin F ( 4 ) were evaluated for their bioactivity against luminescent derivatives of Mycobacterium abscessus and M. marinum at a maximum concentration of 64 µg/mL using inhouse assays. Luminescence is frequently used as rapid readout of potential antimycobacterial activity [ 28 , 29 , 30 ]. Citromycetin and another compound of the penialdin family, penialdin C, have previously been shown to have activity against the Mycobacterial species M. smegmatis , with minimum inhibitory concentrations (MIC) of 31.2 and 15.6 μg/mL, respectively [ 31 ].…”

“…M. abscessus BSG301 and M. marinum BSG101 [ 28 ] are stable bioluminescent derivatives transformed with the integrating plasmid pMV306G13ABCDE [ 34 ]. This allows light production to be used as a surrogate for bacterial viability [ 28 , 29 , 30 ]. Mycobacterial cultures were grown shaking at 200 rpm in Middlebrook 7H9 broth (Fort Richard, Auckland, New Zealand) supplemented with 10% Middlebrook ADC enrichment media (Fort Richard, Auckland, New Zealand), 0.4% glycerol (Sigma-Aldrich, St. Louis, MO, USA), and 0.05% tyloxapol (Sigma-Aldrich, St. Louis, MO, USA).…”

Antimicrobial Natural Products from Plant Pathogenic Fungi

“…Antimicrobial evaluation against M. abscessus and M. marinum was undertaken using in-house assays with the bioluminescent derivatives M. abscessus BSG301 [ 63 ] and M. marinum BSG101 [ 64 ]. Assays were performed as previously described [ 63 , 68 ]. Specifically, mycobacterial cultures were grown with shaking at 200 rpm in Middlebrook 7H9 broth (Fort Richard, Auckland, New Zealand) supplemented with 10% Middlebrook ADC enrichment media (Fort Richard, Auckland, New Zealand), 0.4% glycerol (Sigma-Aldrich, St. Louis, MO, USA), and 0.05% tyloxapol (Sigma-Aldrich, St. Louis, MO, USA).…”

Avances en la Economía Circular del Ecuador. Una estrategia para la sostenibilidad basada en Métodos BIPLOT

“…Capítulo IV: Biocontrol Ecológico de Moniliasis en Cacao Ecuatoriano: Usando Técnicas Biplot (Grey et al, 2021). Diferentes autores han indicado que las cepas de hongos cultivadas en este medio presentaron las características miceliales más fuertes .…”