Unsaturated fatty acids are inhibitors of bacterial conjugation

Abstract

This report describes a high-throughput assay to identify substances that reduce the frequency of conjugation in Gram-negative bacteria. Bacterial conjugation is largely responsible for the spread of multiple antibiotic resistances in human pathogens. Conjugation inhibitors may provide a means to control the spread of antibiotic resistance. An automated conjugation assay was developed that used plasmid R388 and a laboratory strain of Escherichia coli as a model system, and bioluminescence as a reporter for conjugation activity. Frequencies of conjugation could be measured continuously in real time by the amount of light produced, and thus the effects of inhibitory compounds could be determined quantitatively. A control assay, run in parallel, allowed elimination of compounds affecting cell growth, plasmid stability or gene expression. The automated conjugation assay was used to screen a database of more than 12 000 microbial extracts known to contain a wide variety of bioactive compounds (the NatChem library). The initial hit rate was 1?4 %. From these, 48 extracts containing active compounds and representing a variety of organisms and extraction conditions were subjected to fractionation (24 fractions per extract). The 52 most active fractions were subjected to a secondary analysis to determine the range of plasmid inhibition. Plasmids R388, R1 and RP4 were used as representatives of a variety of plasmid transfer systems. Only one fraction (of complex composition) affected transfer of all three plasmids, while four other fractions were active against two of them. Two separate compounds were identified from these fractions: linoleic acid and dehydrocrepenynic acid. Downstream analysis showed that the chemical class of unsaturated fatty acids act as true inhibitors of conjugation.

Ecology and Evolution as Targets: the Need for Novel Eco-Evo Drugs and Strategies To Fight Antibiotic Resistance

“…There have in fact been important advances in this direction. First, a high-throughput wholecell assay to detect and assay for COINS was developed (50). Using this assay, a conjugation-inhibitory compound called dehydrocrepenynic acid, an unsaturated fatty acid, was discovered, establishing the proof of principle that this type of ecoevo drugs can be found.…”

Cutaneous Wound Healing after Topical Application of <i>Pistacia atlantica</i> Gel Formulation in Rats

“…18 More importantly, it has been found that two FFAs (linoleic acid and dehydrocrepenynic acid) inhibit the bacterial drug resistance by decreasing the transfer frequency of the conjugal DNA. 15,19 Skin is a biological interface with the environment, and is frequently and directly exposed to prooxidative stimuli, which are known to promote the generation of Reactive Oxygen Species (ROSs) and lipid peroxides. 20,21 Wounding is another condition that results in a decrease in antioxidants, 22,23 increase ROSs 24 and elevated lipid peroxide levels.…”

Toxin-Antitoxin Genes of the Gram-Positive Pathogen Streptococcus pneumoniae: So Few and Yet So Many

“…In addition to that, the numbers of clinical isolates of S. pneumoniae that exhibit antibiotic resis-tances have also increased during the past decade (55). Other proposed strategies include the targeting of key virulence factors (101), the inhibition of gene spread by the discovery of conjugation inhibitors (50), bacteriophage therapy, and the use of genomic approaches to identify genes that are essential for microbial survival or virulence. Now that the sequences of a large number of pneumococcal genomes are completed or are under way, it will be possible to search for genes conserved among these bacterial isolates and relate them to other bacteria that can survive in similar sites within the human microbiome: these might represent potential targets for antimicrobials.…”