Unsaturated fatty acids are inhibitors of bacterial conjugation.
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.
Fernandez-Lopez
Raul
R
Departamento de Biología Molecular (Unidad asociada al CIB, CSIC), Universidad de Cantabria, C. Herrera Oria s/n, E-39011 Santander, Spain.
Machón
Cristina
C
Departamento de Biología Molecular (Unidad asociada al CIB, CSIC), Universidad de Cantabria, C. Herrera Oria s/n, E-39011 Santander, Spain.
Longshaw
Christopher M
CM
Cubist Pharmaceuticals (UK) Ltd, 545 Ipswich Road, Slough SL1 4EQ, UK.
Martin
Steve
S
Cubist Pharmaceuticals (UK) Ltd, 545 Ipswich Road, Slough SL1 4EQ, UK.
Molin
Soren
S
Department of Molecular Microbiology, BioCentrum-DTU, DK-2800 Lyngby, Denmark.
Zechner
Ellen L
EL
Institute of Molecular Biosciences, University of Graz, A-8010 Graz, Austria.
Espinosa
Manuel
M
Centro de Investigaciones Biológicas (CIB), CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain.
Lanka
Erich
E
Max-Planck-Institut für Molekulare Genetik, Ihnestrasse 73, Dahlem, D-14195 Berlin, Germany.
de la Cruz
Fernando
F
Departamento de Biología Molecular (Unidad asociada al CIB, CSIC), Universidad de Cantabria, C. Herrera Oria s/n, E-39011 Santander, Spain.
D003227:Conjugation, Genetic / Q000187:drug effects*; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology*; D006090:Gram-Negative Bacteria / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development; D006801:Humans; D049449:Luminescence; D008826:Microbial Sensitivity Tests / Q000379:methods; D011815:R Factors / Q000235:genetics*
D005231:Fatty Acids, Unsaturated