From Soil to New Secondary Metabolites
Soil Bacteria as Successful Producers of New Antibiotics
- Fig. 1: Swarms and fruiting bodies of myxobacteria on agar plates (pictures: Diana Telkemeyer)
- Figure 2: a) Water agar plates with transparent swarming predatory myxobacterium (Myxococcus) on E. coli bait- cross. Magnification on the right: Yellow fruiting bodies b) Soil samples on cellulose filter with cellulose degrading myxobacterium (orange). Magnification on the right: Pure culture of cellulose degrader (Sorangium cellulosum). Top-centre: Fruiting body formation c) Pure liquid cultures of myxobacteria d) Serial dilution test with raw extracts. Highest inhibition in row 4 e) Peak-activity correlation with fractionation plates f) Fermentor for large volume cultivation g) Isolation of natural substances from XAD of a fermented culture in a chemical laboratory h) Sandaracinus amylolyticus on agar plate and i) Structure of Indiacen A produced by S. amylolyticus .
The number of antibiotic-resistant pathogenic bacteria against common classes of antibiotics has increased and this has led to an increasing demand of new active substances. An abundant source for new antibiotics is soil, which harbours an immeasurable number and diversity of bacteria, for example, myxo- and actinobacteria which are well known producers of active substances for a long time. In the past, isolation of soil bacteria and the subsequent screening for bioactivity against a variety of different test organisms led to the discovery of thousands of new metabolites.
Infections with multi-resistant pathogens like vancomycin-resistant enterococci (VRE), methicillin-resistant Staphylococcus aureus strains (MRSA) or extended-spectrum-β-lactamase producing microbes (ESBL) are constantly increasing. Possible causes are uncritical medical prescriptions of antibiotics as well as excessive, often preventive use of antibiotics in animal fattening. In particular nosocomial infections (hospital acquired) are in the focus of the present antibiotic research . Caused by increasing globalization infectious diseases like tuberculosis, thought to be beaten, are powerful motivators to find new active substances.
At the Helmholtz Centre for Infection Research (HZI), myxobacteria have been isolated from environmental samples. Their bioactive potential is being tested against different bacteria, fungi and cell lines. For a couple of years already, in addition fungi and actinobacteria are isolated and tested for antibiotic production, too. However, many substances are already known from these organisms. Nevertheless, by the use of extraordinarily microorganisms, in particular myxobacteria, uncommon actinobacteria and fungi new bioactive secondary metabolites can continually be isolated and published .
Myxobacteria are Gram-negative, aerobic soil and wood inhabitants able to glide on moist surfaces. Most of the more than 50 described species are predators. With exoenzymes they are able to degrade other soil microorganisms and assimilate their nutrients. Two species are also able to degrade cellulose. All known myxobacteria follow a fascinating “social” lifestyle: They grow in swarms and their vegetative cells divide as well as any other bacteria under appropriate nutritional conditions.
However, under nutrient deficiency cells aggregate by swarming and form species-specific “fruiting bodies”. These formations can be of primitive bladder-like form up to complex branched tree like structures, often very colorful (fig. 1). Inside these structures, a part of the vegetative cells convert to dry resistant myxospores.
This means that soil samples can be stored more than 10 years at room temperature, because myxospores can germinate under appropriate conditions even after many years. Due to their large genomes (13 mega base pairs in Sorangium cellulosum  in comparison to 4.6 Mbp in E. coli, for example) myxobacteria are of special interest for natural product researches. Myxobacterial genomes can harbour huge multi-enzyme assembly lines, consisting of polyketide synthases (PKS) and non-ribosomal polypeptide synthetases (NRPS). Most of the myxobacterial secondary metabolites like polyketides, non-ribosomal polypeptides or hybrids of these substance classes are synthesized at these complexes.
Isolation of Myxobacteria and their Metabolites
To isolate myxobacteria from environmental samples two standard cultivation approaches have been established in the past: to trap predatory myxobacteria a bit sample material is placed on water agar plates with living Escherichia coli - bait. After a few days, present myxobacteria swarm out of the sample, lyse the bait and in many cases start to form fruiting bodies, often visible in macroscopic scale. For cellulose degrading genera, the sample is placed on pieces of sterile filter paper, also placed on agar . After approximately two weeks attendant cellulose-degrading myxobacteria start to decompose the filter paper, swarm over the agar and possibly form colorful fruiting bodies, too.
To receive pure cultures, parts of the swarm edge or whole fruiting bodies can be cut/ picked with a sterile needle and transferred to a new agar plate with bait. Subsequently, new isolates are cultivated in different liquid media with various carbon and nitrogen sources. Within the years these media were proven to be appropriate for the production of myxobacterial natural products. A total of 2 % XAD-adsorber resin is added to the cultures to bind the excreted secondary metabolites. On the one hand XAD prevents a possible feedback inhibition of the producer. At the other hand the metabolites are protected against degradation by the myxobacterium.
Identification of Active Metabolites
After extraction of the metabolites from XAD with a solvent, the so called raw-extract can be tested via serial dilution test in 96 well plates for different microorganisms. Substances responsible for inhibitions are detected by high-performance liquid chromatography (HPLC) and fractionation of the raw extract in 96 well plates. HPLC is a chromatographic technique in analytical chemistry used to separate, identify, and quantify components in a mixture. The extract will be diluted in a solvent of decreasing polarity (mobile phase) and passes a column filled with solid adsorbent material (stationary phase).
The substances in the raw extract interact slightly different with the adsorbent material, causing different flow rates for the different components and leading to the separation of these as they flow at varying retention times out the column and pass a detector. After fractionation the N2-dried plate will be inoculated and incubated with the previous inhibited test organism. An inhibition in one or more wells can be assigned to the corresponding retention time as well as to a UV-chromatogram, which is specific for each substance. Measuring the same extract in a HPLC connected with a mass spectrometer results gives information about the molecular masses within the extract, in addition to the existing information namely: activity, retention times and UV-chromatograms. Due to such information both new and known substances can be identified by comparing the data with internal or public data bases.
Despite the high recovery rate for known compounds, new secondary metabolites are steadily being detected by following this procedure. However, in most cases the initial production rate is insufficiently. Now a lot of microbial skills are required: varying biotic and abiotic parameters often leads to an increasing production. When the production in laboratory scale (100 mL) is satisfactory, the transferability of the determined production parameters are scaled up step-wise to higher volumes up to 300 liters. The production in fermentors can be similar, better or worse than in a 100 mL volume.
It is often observed that production of secondary metabolites in oxygen-regulated fermentor scales in comparison to non-regulated lab-flasks decreases dramatically. In this case the production is conducted exclusively in laboratory flask scale. After successful fermentation, whether in flasks or high volume fermentors, chemists isolate the new biological active substance and elucidate the structure by high resolution mass spectrometry (HRMS) and nuclear magnetic resonance spectroscopy (NMR; figure 2).
New Promising Substances
For new isolated substances the minimal inhibition concentration-value is tested against an extended panel of infection relevant organisms. For promising substances information about the mode of action and possible cross resistances to known antibiotics are of high importance. Possible targets of antibiotics are: Inhibition of cell wall-, protein-, or folic acid synthesis or DNA-replication. First, promising substances have to be saved by patents.
However, many substances turned out to be too toxic and had to be excluded from further evaluation processes. Without exceptions all new isolated substances will also be distributed to numerous different test systems of external co-operation partners. It is often observed that a substance inactive in our in-house standard panel attracts attention after testing in an external system.
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Dr. Kathrin I. Mohr
Helmholtz Centre for Infection Research (HZI)
Article on antibiotic resistance:
Webcast on Myxococcus: