Antimicrobial agents

#The present invention relates to novel polyketide compounds, their preparation, and their use as antimicrobial agents. The invention further relates to antimicrobial agents obtained from a Burkholderia ambifaria strain, or from a variant and/or mutant thereof.

#Bacterial pathogens are prominent in many diseases and the treatment of bacterial infections has become increasingly difficult over recent years with the emergence of a number of antibiotic resistant bacterial strains. Examples include methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE), and multidrug-resistant Gram-negative bacteria such as Acinetobacter baumannii. In addition to the emergence of antibiotic resistant strains, there are many bacterial infections that remain difficult to treat, for example, infections in immuno-compromised patients (e.g. those with AIDS).

#There is therefore an ongoing need to identify new antimicrobial agents that can be used to treat microbial infections effectively, including those caused by drug resistant microbes, for example, infections caused by drug-resistant bacteria.

#The invention will now be further illustrated by the following non-limiting examples and the accompanying figures, in which:

#FIGS. 1 to 126 show 1H NMR, 13C NMR, COSY NMR, 1H-13C HSQC and 1H-13C HMBC spectra of compounds according to the invention.

#In-frame deletions in genes were introduced via double homologous recombination using the suicide plasmid pGPI and the I-SceI expression plasmid pDAI (Flannagan et al., Environ. Microbiol. 10: 1652-1660, 2008). The sequences (500-1000 bp) flanking the gene regions targeted for deletion were amplified from B. ambifaria BCC0203 genomic DNA using Q5 DNA polymerase (NEB). Restriction sites were introduced at the 5′-end of the primers to allow for directional cloning of the PCR products into pGPI. Constructs were mobilized into E. coli SY327 by electroporation and transformants were selected on LB agar plates supplemented with trimethoprim (50 μg/mL). Plasmids were purified from trimethoprim-resistant colonies using the GeneJET Plasmid Miniprep kit (Thermo Scientific) and correct assembly of the mutagenesis constructs was confirmed by Sanger sequencing (GATC Biotech). Validated constructs were transferred into B. ambifaria BCC0203 via triparental mating (Agnoli et al., Mol. Microbiol. 83: 362-378, 2012) and transconjugants were selected using trimethoprim (200 μg/ml) and gentamicin (50 μg/ml). Single B. ambifaria mutants were selected and correct integration of the mutagenesis plasmids into the genome was confirmed by colony PCR. Next, the pDAI plasmid was introduced into the B. ambifaria single crossover mutants by triparental mating using E. coli SY327 (pDAI) and E. coli HB101 (pRK2013) as the donor and helper strain, respectively (Agnoli et al., Mol. Microbiol. 83: 362-378, 2012). Transconjugants were selected on LB agar plates containing tetracycline (200 μg/ml) and gentamicin (50 μg/ml). Single B. ambifaria mutants were selected and correct gene deletion was confirmed by colony PCR and Sanger sequencing. To examine the effect of the gene deletions on enacyloxin biosynthesis, mutant strains were grown at 30° C. on solid minimal medium containing glycerol as a sole carbon source (BSM-G) (O'Sullivan et al., Environ. Microbiol. 9: 1017-1034, 2007). Following incubation for 3 days, the cells were scraped off and ethyl acetate extracts of the agar were analysed by UHPLC-ESI-Q-TOF-MS.

#For production of enacyloxin derivatives, the B. ambifaria BCC0203 deletion mutants were grown in the dark at 30° C. on solid minimal medium containing glycerol as a sole carbon source (BSM-G) (O'Sullivan et al., Environ. Microbiol. 9: 1017-1034, 2007). Following incubation for 3 days, the cells were scraped off and the agar extracted twice using ethyl acetate (1:1). Enacyloxin derivative-containing extracts were concentrated by rotary evaporation in vacuo and the resulting residue was re-dissolved in acetonitrile for purification by preparative HPLC. For the mutasynthetic production of enacyloxin derivatives, B. ambifaria BCC0203 mutant Δ5912-5914 was grown in the dark at 30° C. on BSM-G agar supplemented with 10 mM of a relevant DHCCA analogue (described below). Bromine derivatives of enacyloxin were generated using the same procedure, however the ammonium chloride in the BSM-G was replaced with two equivalents of ammonium bromide.

#Enacyloxin derivative-containing ethyl acetate extracts of the agar were fractionated by preparative HPLC using an Agilent 1260 instrument equipped with a Zorbax SB-C18 column (21.2×100 mm, 5 μm), monitoring absorbance at 360 nm. Mobile phases consisted of water and acetonitrile, each supplemented with 0.1% formic acid. A gradient of 50% B to 100% B over 40 minutes was employed at a flow rate of 10 mL/min. Enacyloxin derivative-containing fractions were pooled, concentrated in vacuo, and subsequently lyophilized. To avoid photo-induced isomerization of the compounds, exposure to light was minimized throughout the entire purification process.