Biolog phenotype microarray: a tool for the identification of multidrug resistance efflux pumps inducers

Abstract

1Overexpression of multidrug resistance efflux pumps is a relevant mechanism of antibiotic 2 resistance for bacterial pathogens. These systems use to present low levels of basal 3 expression. However, they can be induced by environmental signals or stresses which can 4 lead to situations of phenotypic induced resistance. In contrast to efflux pumps substrates, 5 inducers of these systems have not been thoroughly studied. In this work, we have applied a 6 novel high-throughput methodology in order to identify inducer molecules of the 7Stenotrophomonas maltophilia SmeVWX and SmeYZ efflux pumps. To that goal, 8 bioreporters in which the expression of the yellow fluorescent protein is linked to the activity 9 of either the smeVWX or the smeYZ promoters were developed and used for the screening of 10 potential inducers of the expression of these efflux pumps using Biolog phenotype 11 microarrays. Confirmation of induction was carried out measuring YFP production along the 12 bacterial growth and by flow cytometry; mRNA levels of smeV and smeY were also 13 determined by real-time RT-PCR after exposure to the selected compounds. Among the 144 14 tested compounds, iodoacetate, clioquinol (5-chloro-7-iodo-8-hydroxyquinoline) and sodium 15 selenite were found to be smeVWX inducers, while boric acid, erythromycin, chloramphenicol 16 and lincomycin are able to trigger the expression of smeYZ. While the presence of the 17 inducers allowed a decrease in the susceptibility to antibiotics that are known substrates of 18 the efflux pumps, our results indicate that these efflux pumps did not contribute to S. 19 maltophilia resistance to the analyzed inducers. 20