With increasing efforts to reuse wastewater treatment plant (WWTP) products in agriculture, assessing their impact on soil-plant systems is crucial, while the effects of accompanying antibiotic residues on soil microbial communities have not yet been adequately studied. This study focuses on clarithromycin (CLR), highly present in wastewater, and investigates the CLR-degradation potential of plant-associated microorganisms. Phaseolus vulgaris plants were grown in raised beds filled with Haplic Cambisol and amended with or without WWTP products (treated wastewater, biosolid, or composted biosolid), as a source of CLR residues. The rhizosphere microbiomes after biosolid amendments was significantly enriched by Pseudomonadaceae as assessed by 16S rRNA metagenomics and cultures enriched by CLR revealed dominance of Proteobacteria. However, no degradation of CLR by microbial consortia or enrichment cultures was observed, suggesting the multiplication of CLR-resistant bacteria with other resistance mechanisms. Cultivation-based approach combined with antibiotic modulation assays and subsequent LC-MS analysis confirmed the complete CLR removal by seven phylogenetic groups of actinomycetes in vitro. The proportion of isolates indicated that the rhizosphere is a natural reservoir for CLR-inactivating microorganisms; however, the amendment of soils with WWTP products can significantly increase their abundance and diversity.

• Keywords
• Streptomyces, Antimicrobial resistance, Biodegradation, Macrolides, Micropollutants, Soil microbiome,
• MeSH
• Anti-Bacterial Agents * pharmacology   MeSH
• Phaseolus * microbiology   drug effects   growth & development   MeSH
• Phylogeny MeSH
• Clarithromycin * pharmacology   metabolism   MeSH
• Microbiota drug effects   MeSH
• Wastewater * chemistry   MeSH
• Soil Microbiology MeSH
• Rhizosphere * MeSH
• RNA, Ribosomal, 16S genetics   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Anti-Bacterial Agents * MeSH
• Clarithromycin * MeSH
• Wastewater * MeSH
• RNA, Ribosomal, 16S MeSH

The taxonomic position of three actinobacterial strains, BCCO 10_0061T, BCCO 10_0798T, and BCCO 10_0856T, recovered from bare soil in the Sokolov Coal Basin, Czech Republic, was established using a polyphasic approach. The multilocus sequence analysis based on 100 single-copy genes positioned BCCO 10_0061T in the same cluster as Lentzea waywayandensis, strain BCCO 10_0798T in the same cluster as Lentzea flaviverrucosa, Lentzea californiensis, Lentzea violacea, and Lentzea albidocapillata, and strain BCCO 10_0856T clustered together with Lentzea kentuckyensis and Lentzea alba. Morphological and chemotaxonomic characteristics of these strains support their assignment to the genus Lentzea. In all three strains, MK-9(H4) accounted for more than 80 % of the isoprenoid quinone. The diagnostic diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. The whole-cell sugars were rhamnose, ribose, mannose, glucose, and galactose. The major fatty acids (>10 %) were iso-C15 : 0, anteiso-C15 : 0, iso-C16 : 0, and C16 : 0. The polar lipids were diphosphatidylglycerol, methyl-phosphatidylethanolamine, phosphatidylethanolamine, hydroxy-phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylinositol. The genomic DNA G+C content of strains (mol%) was 68.8 for BCCO 10_0061T, 69.2 for BCCO 10_0798T, and 68.5 for BCCO 10_0856T. The combination of digital DNA-DNA hybridization results, average nucleotide identity values and phenotypic characteristics of BCCO 10_0061T, BCCO 10_0798T, and BCCO 10_0856T distinguishes them from their closely related strains. Bioinformatic analysis of the genome sequences of the strains revealed several biosynthetic gene clusters (BGCs) with identities >50 % to already known clusters, including BGCs for geosmin, coelichelin, ε-poly-l-lysine, and erythromycin-like BGCs. Most of the identified BGCs showed low similarity to known BGCs (<50 %) suggesting their genetic potential for the biosynthesis of novel secondary metabolites. Based on the above results, each strain represents a novel species of the genus Lentzea, for which we propose the name Lentzea sokolovensis sp. nov. for BCCO 10_0061T (=DSM 116175T), Lentzea kristufekii sp. nov. for BCCO 10_0798T (=DSM 116176T), and Lentzea miocenica sp. nov. for BCCO 10_0856T (=DSM 116177T).

• Keywords
• Actinomycetes, Lentzea, polyphasic taxonomy, post-mining sites,
• MeSH
• Actinobacteria * MeSH
• Actinomycetales * MeSH
• Bacteria MeSH
• DNA, Bacterial genetics   MeSH
• Phosphatidylethanolamines MeSH
• Phylogeny MeSH
• Fatty Acids chemistry   MeSH
• RNA, Ribosomal, 16S genetics   MeSH
• Sequence Analysis, DNA MeSH
• Bacterial Typing Techniques MeSH
• Coal MeSH
• Base Composition MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Czech Republic MeSH
• Names of Substances
• DNA, Bacterial MeSH
• Phosphatidylethanolamines MeSH
• Fatty Acids MeSH
• RNA, Ribosomal, 16S MeSH
• Coal MeSH

Notwithstanding the fact that streptomycetes are overlooked in clinical laboratories, studies describing their occurrence in disease and potential pathogenicity are emerging. Information on their species diversity in clinical specimens, aetiology and appropriate therapeutic treatment is scarce. We identified and evaluated the antibiotic susceptibility profile of 84 Streptomyces clinical isolates from the Czech Republic. In the absence of appropriate disk diffusion (DD) breakpoints for antibiotic susceptibility testing (AST) of Streptomyces spp., we determined DD breakpoints by correlation with the broth microdilution method and by the distribution of zone diameters among isolates. Correlation accuracy was high for 9 antibiotics, leading to the establishment of the most valid DD breakpoints for Streptomyces antibiotic susceptibility evaluation so far. Clinical strains belonged to 17 different phylotypes dominated by a cluster of strains sharing the same percentage of 16S rRNA gene sequence identity with more than one species (S. albidoflavus group, S. hydrogenans, S. resistomycificus, S. griseochromogenes; 70% of isolates). AST results showed that Streptomyces exhibited intrinsic resistance to penicillin, general susceptibility to amikacin, gentamycin, vancomycin and linezolid, and high percentage of susceptibility to tetracyclines and clarithromycin. For the remaining antibiotics, AST showed inter- and intra-species variations when compared to available literature (erythromycin, trimethoprim-sulfamethoxazole), indicating a region-dependent rather than species-specific patterns.

• MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Linezolid MeSH
• Microbial Sensitivity Tests MeSH
• RNA, Ribosomal, 16S genetics   MeSH
• Streptomyces * genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• Linezolid MeSH
• RNA, Ribosomal, 16S MeSH

(1) Background: Manumycins are small actinomycete polyketides with prominent cancerostatic and immunosuppressive activities via inhibition of various eukaryotic enzymes. Their overall activity towards human cells depends on the structural variability of both their polyketide chains, mainly the upper one. In our genetic screening project to find novel producers of anti-inflammatory manumycins, the strain Saccharothrix espanaensis DSM44229 was identified as containing a novel manumycin-type biosynthetic gene cluster (BGC). (2) Methods: The biosynthetic genes appeared to be silent under all assayed laboratory conditions. Several techniques were used to activate the BGC, including: (i) heterologous expression in various hosts, (ii) overexpression of putative pathway-specific regulatory genes, and (iii) overexpression of a bottleneck cyclizing aminolevulinate synthase gene in both natural and heterologous producers. (3) Results: Multiple novel manumycin-type compounds were produced at various levels by genetically-modified strains, sharing a tetraene lower chain structure with a colabomycin subgroup of manumycins, but possessing much shorter and saturated upper chains. (4) Conclusions: A cryptic manumycin-type BGC was successfully activated by genetic means to gain production of novel manumycin-type compounds for future comparative activity assays. Heterologously produced compounds were identical to those found after final activation of the BGC in the original strain, proving the intactness of the cloned BGC.

• Keywords
• Saccharothrix, actinomycetes, cancerostatics, colabomycin, cryptic BGC activation, immunomodulators, manumycin, secondary metabolites,
• Publication type
• Journal Article MeSH