Pharmacokinetic and pharmacodynamic evaluation of the atypical tetracyclines chelocardin and amidochelocardin in murine infection models.

IMPORTANCE: There is a strong need to find novel treatment options against urinary tract infections associated with antimicrobial resistance. This study evaluates two atypical tetracyclines, namely chelocardin (CHD) and amidochelocardin (CDCHD), with respect to their pharmacokinetics and pharmacodynamics. We show CHD and CDCHD are cleared at high concentrations in mouse urine. Especially, CDCHD is highly effective in an ascending urinary tract infection model, suggesting further preclinical evaluation.

Amidochelocardin Overcomes Resistance Mechanisms Exerted on Tetracyclines and Natural Chelocardin.

The reassessment of known but neglected natural compounds is a vital strategy for providing novel lead structures urgently needed to overcome antimicrobial resistance. Scaffolds with resistance-breaking properties represent the most promising candidates for a successful translation into future therapeutics. Our study focuses on chelocardin, a member of the atypical tetracyclines, and its bioengineered derivative amidochelocardin, both showing broad-spectrum antibacterial activity within the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) panel. Further lead development of chelocardins requires extensive biological and chemical profiling to achieve favorable pharmaceutical properties and efficacy. This study shows that both molecules possess resistance-breaking properties enabling the escape from most common tetracycline resistance mechanisms. Further, we show that these compounds are potent candidates for treatment of urinary tract infections due to their in vitro activity against a large panel of multidrug-resistant uropathogenic clinical isolates. In addition, the mechanism of resistance to natural chelocardin was identified as relying on efflux processes, both in the chelocardin producer Amycolatopsis sulphurea and in the pathogen Klebsiella pneumoniae. Resistance development in Klebsiella led primarily to mutations in ramR, causing increased expression of the acrAB-tolC efflux pump. Most importantly, amidochelocardin overcomes this resistance mechanism, revealing not only the improved activity profile but also superior resistance-breaking properties of this novel antibacterial compound.

Semisynthesis and biological evaluation of amidochelocardin derivatives as broad-spectrum antibiotics.

To address the global challenge of emerging antimicrobial resistance, the hitherto most successful strategy to new antibiotics has been the optimization of validated natural products; most of these efforts rely on semisynthesis. Herein, we report the semisynthetic modification of amidochelocardin, an atypical tetracycline obtained via genetic engineering of the chelocardin producer strain. We report modifications at C4, C7, C10 and C11 by the application of methylation, acylation, electrophilic substitution, and oxidative C-C coupling reactions. The antibacterial activity of the reaction products was tested against a panel of Gram-positive and Gram-negative pathogens. The emerging structure-activity relationships (SARs) revealed that positions C7 and C10 are favorable anchor points for the semisynthesis of optimized derivatives. The observed SAR was different from that known for tetracyclines, which underlines the pronounced differences between the two compound classes.

Engineering Atypical Tetracycline Formation in Amycolatopsis sulphurea for the Production of Modified Chelocardin Antibiotics.

To combat the increasing spread of antimicrobial resistance and the shortage of novel anti-infectives, one strategy for the development of new antibiotics is to optimize known chemical scaffolds. Here, we focus on the biosynthetic engineering of Amycolatopsis sulphurea for derivatization of the atypical tetracycline chelocardin and its potent broad-spectrum derivative 2-carboxamido-2-deacetyl-chelocardin. Heterologous biosynthetic genes were introduced into this chelocardin producer to modify functional groups and generate new derivatives. We demonstrate cooperation of chelocardin polyketide synthase with tailoring enzymes involved in biosynthesis of oxytetracycline from Streptomyces rimosus. An interesting feature of chelocardin, compared with oxytetracycline, is the opposite stereochemistry of the C4 amino group. Genes involved in C4 transamination and N,N-dimethylation of oxytetracycline were heterologously expressed in an A. sulphurea mutant lacking C4-aminotransferase. Chelocardin derivatives with opposite stereochemistry of the C4 amino group, as N,N-dimethyl- epi-chelocardin and N,N-dimethyl-2-carboxamido-2-deacetyl- epi-chelocardin, were produced only when the aminotransferase from oxytetracycline was coexpressed with the N-methyltransferase OxyT. Surprisingly, OxyT exclusively accepted intermediates carrying an S-configured amino group at C4 in chelocardin. Applying medicinal chemistry approaches, several 2-carboxamido-2-deacetyl- epi-chelocardin derivatives modified at C4 were produced. Analysis of the antimicrobial activities of the modified compounds demonstrated that the primary amine in the R configuration is a crucial structural feature for activity of chelocardin. Unexpectedly, C10 glycosylated chelocardin analogues were identified, thus revealing the glycosylation potential of A. sulphurea. However, efficient glycosylation of the chelocardin backbone occurred only after engineering of a dimethylated amino group at the C4 position in the opposite S configuration, which suggests some evolutionary remains of chelocardin glycosylation.

A polyphasic approach leads to seven new species of the cellulose-decomposing genus Sorangium, Sorangium ambruticinum sp. nov., Sorangium arenae sp. nov., Sorangium bulgaricum sp. nov., Sorangium dawidii sp. nov., Sorangium kenyense sp. nov., Sorangium orientale sp. nov. and Sorangium reichenbachii sp. nov.

Seventy-three strains of Sorangium have been isolated from soil samples collected from all over the world. The strains were characterized using a polyphasic approach and phenotypic, genotypic and chemotype analyses clarified their taxonomic relationships. 16S rRNA, xynB1, groEL1, matrix-assisted laser desorption/ioniziation time-of-flight mass spectrometry and API-ZYM analyses were conducted. In addition, from selected representative strains, fatty acids, quinones and phospholipids were analysed. In silico DNA-DNA hybridization and DNA-DNA hybridization against the current type species of Sorangiumcellulosum strain Soce 1871T (DSM 14627T) completed the analyses. Finally, our study revealed seven new species of Sorangium: Sorangium ambruticinum (Soce 176T; DSM 53252T, NCCB 100639T, sequence accession number ERS2488998), Sorangium arenae (Soce 1078T; DSM 105768T, NCCB 100643T, ERS2489002), Sorangium bulgaricum (Soce 321T; DSM 53339T, NCCB 100640T, ERS2488999), Sorangium dawidii (Soce 362T; DSM 105767T, NCCB 100641T, ERS2489000), Sorangium kenyense (Soce 375T; DSM 105741T, NCCB 100642T, ERS2489001), Sorangium orientale (Soce GT47T; DSM 105742T, NCCB 100638T, ERS2501484) and Sorangium reichenbachii (Soce 1828T; DSM 105769T, NCCB 100644T, ERS2489003).

Genome-wide mutant profiling predicts the mechanism of a Lipid II binding antibiotic.

Identifying targets of antibacterial compounds remains a challenging step in the development of antibiotics. We have developed a two-pronged functional genomics approach to predict mechanism of action that uses mutant fitness data from antibiotic-treated transposon libraries containing both upregulation and inactivation mutants. We treated a Staphylococcus aureus transposon library containing 690,000 unique insertions with 32 antibiotics. Upregulation signatures identified from directional biases in insertions revealed known molecular targets and resistance mechanisms for the majority of these. Because single-gene upregulation does not always confer resistance, we used a complementary machine-learning approach to predict the mechanism from inactivation mutant fitness profiles. This approach suggested the cell wall precursor Lipid II as the molecular target of the lysocins, a mechanism we have confirmed. We conclude that docking to membrane-anchored Lipid II precedes the selective bacteriolysis that distinguishes these lytic natural products, showing the utility of our approach for nominating the antibiotic mechanism of action.

Racemicystis persica sp. nov., a myxobacterium from soil.

A novel myxobacterium, strain MSr11462T, was isolated in 2015 from a soil sample collected form Kish Island beach, Persian Gulf, Iran. It displayed general myxobacterial features like Gram-negative staining, rod-shaped vegetative cells, gliding on solid surfaces, microbial lytic activity, fruiting-body-like aggregates and myxospore-like structures. The strain was mesophilic, aerobic and showed a chemoheterotrophic mode of nutrition. It was resistant to many antibiotics like gentamycin, polymyxin, fusidic acid and trimethoprim, and the key fatty acids of whole-cell hydrolysates were iso-C15 : 0, C16 : 0, iso-C17 : 0, C18 : 1, iso-C17 : 1 2-OH, C18 : 1 2-OH, iso-C15 : 0 OAG (O-alkylglycerol) and C16 : 1 OAG. The 16S rRNA gene sequence showed highest similarity (98.6 %) to Racemicystis crocea strain MSr9521T (GenBank accession no. KT591707). The phylogenetic analysis based on 16S rRNA gene sequences and matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) spectroscopy data supports a novel species of the family Polyangiaceae and the genus Racemicystis. DNA-DNA hybridization showed only about 50 % similarity between the novel strain and the phylogenetically closest species, Racemicystis. crocea MSr9521T. On the basis of a comprehensive taxonomic study, we propose a novel species, Racemicystis persica sp. nov., for strain MSr11462T (=DSM 103165T=NCCB 100606T).