Biolog Phenotype Microarray Is a Tool for the Identification of Multidrug Resistance Efflux Pump Inducers

Abstract

Multidrug resistance efflux pumps frequently present low levels of basal expression. However, antibiotic-resistant mutants that overexpress these resistance determinants are selected during infection. In addition, increased expression of efflux pumps can be induced by environmental signals/cues, which can lead to situations of transient antibiotic resistance. In this study, we have applied a novel high-throughput methodology in order to identify inducers able to trigger the expression of the SmeVWX and SmeYZ efflux pumps. To that end, bioreporters in which the expression of the yellow fluorescent protein (YFP) is linked to the activity of either or promoters were developed and used for the screening of potential inducers of the expression of these efflux pumps using Biolog phenotype microarrays. YFP production was also measured by flow cytometry, and the levels of expression of and in the presence of a set of selected compounds were also determined by real-time reverse transcription-PCR (RT-PCR). The expression of was induced by iodoacetate, clioquinol, and selenite, while boric acid, erythromycin, chloramphenicol, and lincomycin triggered expression. The susceptibility to antibiotics that are known substrates of the efflux pumps decreased in the presence of the inducers. However, the analyzed multidrug efflux systems did not contribute to resistance to the studied inducers. To sum up, the use of fluorescent bioreporters in combination with Biolog plates is a valuable tool for identifying inducers of the expression of bacterial multidrug resistance efflux pumps, and likely of other bacterial systems whose expression is regulated in response to signals/cues.

Co-Occurrence of Colistin and Meropenem Resistance Determinants in a Stenotrophomonas Strain Isolated from Sewage Water

“…1, strain G4 was more closely related to S. rhizophila rather than S. maltophilia and Xanthomonas species, based on whole genomic level phylogeny with S. rhizophila DSM14405, four S. maltophilia and a variety of closely related Xanthomonas fully sequenced genomes in GenBank. However, the more accurate species assignment of strain G4 needs further clarified with the Biolog examination 23 and more fully sequenced Stenotrophomonas strains.…”

Novel inducers of the expression of multidrug efflux pumps that triggerPseudomonas aeruginosatransient antibiotic resistance

“…One example of this situation is the induction of MexXY-OprM in P. aeruginosa by aminoglycosides, which are substrates of this efflux pump (59). However, in other cases, as it happens in the case of S. maltophilia SmeVWX and SmeYZ efflux pumps (37), some of their inducers are not substrates of these efflux pumps.…”

“…The distribution of every normalized luminescence value obtained from each biosensor strain was represented in a box plot in order to determine the threshold from which one value will be consider as indicator of significant induction or repression. This threshold was determined as previously described (37), using the formula Q 3 + 1.5 X IQR for the induction or Q 1 – 1.5 X IQR for the repression, being Q 3 the upper quartile, Q 1 the lower quartile and IQR the interquartile range for each data set.…”

“…The flasks were incubated at 37°C and 250 rpm until exponential phase of growth (OD 600nm = 0.6) was reached. Then, the optimal concentrations of each tested compound (10 μg/ml of dequalinium chloride, 2 mg/ml of procaine and 2 mg/ml of atropine) were added to each flask and cultures were incubated for 90 minutes with shaking, as previously described in (37), to perform the induction assay; bacterial cultures without any compound or with ethanol in the case of dequalinium chloride, the solvent of this compound, were used as negative controls, and a mexCD-oprJ overexpressing strain ( nfxB *) (Table 1), grown in the absence of inducer, was used as a control of mexCD-oprJ overexpression. Afterwards, 10 ml of each culture were pelleted by centrifugation at 7000 rpm and 4°C for 20 minutes.…”

“…Previous studies have shown that mexAB-oprM expression is induced by oxidative stress (30), triclosan and pentachlorophenol (31), while envelope stress, benzalkonium chloride, chlorhexidine, tetraphenylphosphonium chloride, ethidium bromide, rhodamine 6G or antimicrobial human peptides IL-37 (32–35) are inducers of mexCD-oprJ expression. By the screening of a set of compounds present in Biolog Phenotype Microarrays, an approach that has been previously validated as a useful strategy to discover novel inducers of the expression of the genes that encode efflux pumps (36, 37), we have detected different molecules that induce the expression of mexAB-oprM and mexCD-oprJ in P. aeruginosa. For further analysis, we focused on molecules that this bacterial pathogen can potentially encounter when producing an infection, as procaine, atropine or dequalinium chloride.…”