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

Abstract

Overexpression of multidrug resistance efflux pumps is a relevant mechanism of antibiotic resistance for bacterial pathogens. These systems use to present low levels of basal expression. However, they can be induced by environmental signals or stresses which can lead to situations of phenotypic induced resistance. In contrast to efflux pumps substrates, inducers of these systems have not been thoroughly studied. In this work, we have applied a novel high-throughput methodology in order to identify inducer molecules of the Stenotrophomonas maltophilia SmeVWX and SmeYZ efflux pumps. To that goal, bioreporters in which the expression of the yellow fluorescent protein is linked to the activity of either the smeVWX or the smeYZ promoters were developed and used for the screening of potential inducers of the expression of these efflux pumps using Biolog phenotype microarrays. Confirmation of induction was carried out measuring YFP production along the bacterial growth and by flow cytometry; mRNA levels of smeV and smeY were also determined by real-time RT-PCR after exposure to the selected compounds. Among the 144 tested compounds, iodoacetate, clioquinol (5-chloro-7-iodo-8-hydroxyquinoline) and sodium selenite were found to be smeVWX inducers, while boric acid, erythromycin, chloramphenicol and lincomycin are able to trigger the expression of smeYZ. While the presence of the inducers allowed a decrease in the susceptibility to antibiotics that are known substrates of the efflux pumps, our results indicate that these efflux pumps did not contribute to S. maltophilia resistance to the analyzed inducers.\n\nImportanceMultidrug efflux pumps constitute a category of elements involved in the cellular response to stress that is universally represented; from bacteria to human cells. Besides playing basic roles in cell physiology, these elements are critical elements in the resistance to therapeutic agents, including anti-cancer drugs, antifungals and antibiotics. Stable-inheritable resistance is achieved through mutations in regulatory elements that allow overexpression of these systems. However, much less is known on the effectors, or growing conditions, that might induce their expression, leading to a situation of transient-phenotypic resistance, not detectable by current susceptibility tests, unless the inducer in known. Herein we present a methodology amenable for the high-throughput screening of efflux pumps inducers. The use of phenotype microarrays linked to fluorescence reporters have allowed to identify a set of different inducers for smeVWX and smeYZ. Notably, induction seems to be uncoupled from the detoxification of the inducers by the corresponding efflux pumps. The mechanism of action of each of the inducers for inhibiting bacterial growth allowed us to propose that smeVWX is likely induced as a response to thiol-reactive compounds, while smeYZ is induced by ribosome-targeting antimicrobials. Although applied to a specific bacterium, this method is of application to any type of organism and efflux pump, changing the growing conditions in the case of eukaryotic cells. Since the presence of inducers may change the cell response to therapeutic drugs, the identification of these molecules is of clinical relevance.