Unculturable microorganisms – the source of new antibiotics

Water and soil full of living microorganisms are the major areas of the search for new species and metabolites of antimicrobial action which could be used against human pathogens or in biocontrol of plant infections. Around 80% of currently used antibiotics has been derived from soil microorganisms. For example, actinomycetes are the primary source of compounds such as streptomycin, actinomycin, erythromycin and vancomycin. In the time of growing resistance to known antibiotics it is a high time to come back to the source.

Microbiologic analysis of various soil types revealed, that samples from residential (urbanized) area differs from recreational soil as regards the quantity of species and produced metabolites. Flora in residential soil reveals higher metabolic activity due to constant exposition to substances and wastes from households or farms – such as feaces, food waste and pharmaceuticals (Woappi et al. 2013). In such environment the percentage of resistant strains is 10-20 times higher than in recreational or industrial soil. The horizontal transfer of resistance genes allows for co-existence of strains producing active compounds and resistant strains. This dynamics creates the suitable environment for evolutionary selection of metabolically active strains – due to maintain the balance between species, a selective pressure forces the synthesis of new active compounds and biocontrol of resistant ones. It is therefore the unlimited source of new potent antibiotics.

Unfortunately, standard screening methods for antimicrobial substances which are based mainly on inhibition zones determination, are limited by the need to have particular producing strain isolated. A percentage of species culturable at laboratory conditions is only 1%. The rest 99% is a population of viable but not culturable forms (VBNC). The group of researchers leaded by Prof. Kim Lewis from Northeastern University, Boston, MA (USA) Has find the way to get to this hidden part of iceberg. They employed the iChip (NovoBiotic Pharmaceuticals), a special cultivation device made of a rectangular 7 cm long plate with small wells coved by semi-permeable layer. Flora isolated from soil sample was diluted and loaded in a way that each well contained one microbial cell. Then such prepared iChip has been… buried back in a place of soil sampling, thus on a grass field in Maine, USA.

Ref. Ling et al. 2015

Microorganisms which were grown in such natural environment supplied with soil components, have been tested for the production of antibiotics against human pathogens. This is how teixobactin was discovered – a compound inhibiting bacterial cell wall synthesis by binding to conserved motifs of lipid II (a precursor of peptidoglycan) and lipid III (a precursor of wall teichoic acids). A structure of teixobactin and the predicted biosynthetic gene luster Has been published last year in Nature (Ling et al. 2015). This antibiotic, due to the lack of detectible resistance induction in Staphylococcus aureus and Mycobacterium tuberculosis, is currently a major candidate for clinical application.

The knowledge of active substances and their encoding genes allows for use of other detection methods in situ – such as real time PCR. As a very sensitive method, it allows the amplification of genes of interest in DNA isolated directly from environmental sample, without a cultivation step. Real time PCR has been employed to detect gene phlD encoding a synthesis of DAPG – antimicrobial phenol compound produced by Pseudomonas fluorescens (Mavrodi et al. 2007).

Unculturable species still remain an uncovered part of environmental flora and an unlimited reservoir of antimicrobial metabolites. Luckily, this world is starting to be open for us.

Monika Kossakowska-Zwierucho

Sources:

• Woappi Y, Gabani P, Singh OV. Emergence of Antibiotic-Producing Microorganisms in Residential Versus Recreational Microenvironments. Br Microbiol Res J. 2013 Apr 23;3(3):280-294.
• Mavrodi OV, Mavrodi DV, Thomashow LS, Weller DM. Quantification of 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens in the plant rhizosphere by real-time PCR. Appl Environ Microbiol 2007, 73:5531–5538.
• Ling LL, Schneider T, Peoples AJ, Spoering AL, Engels I, Conlon BP, Mueller A, Schäberle TF, Hughes DE, Epstein S, Jones M, Lazarides L, Steadman VA, Cohen DR, Felix CR, Fetterman KA, Millett WP, Nitti AG, Zullo AM, Chen C, Lewis K. A new antibiotic kills pathogens without detectable resistance. Nature. 2015 Jan 22;517(7535):455-9.