Vitamin K ₃ Induces the Expression of the Stenotrophomonas maltophilia SmeVWX Multidrug Efflux Pump

Abstract

Stenotrophomonas maltophilia is an opportunistic pathogen with increasing prevalence, which is able to cause infections in immunocompromised patients or in those with a previous pathology. The treatment of the infections caused by this bacterium is often complicated due to the several intrinsic antibiotic resistance mechanisms that it presents. Multidrug efflux pumps are among the beststudied mechanisms of S. maltophilia antibiotic resistance. Some of these efflux pumps have a basal expression level but, in general, their expression is often low and only reaches high levels when the local regulator is mutated or bacteria are in the presence of an effector. In the current work, we have developed a yellow fluorescent protein (YFP)-based sensor with the aim to identify effectors able to trigger the expression of SmeVWX, an efflux pump that confers resistance to quinolones, chloramphenicol, and tetracycline when it is expressed at high levels. With this purpose in mind, we tested a variety of different compounds and analyzed the fluorescence signal given by the expression of YFP under the control of the smeVWX promoter. Among the tested compounds, vitamin K 3 , which is a compound belonging to the 2-methyl-1,4-naphthoquinone family, is produced by plants in defense against infection, and has increasing importance in human therapy, was able to induce the expression of the SmeVWX efflux pump. In addition, a decrease in the susceptibility of S. maltophilia to ofloxacin and chloramphenicol was observed in the presence of vitamin K 3 , in both wild-type and smeW-deficient strains.

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

“…With the aim of measuring the expression of S. maltophilia efflux pumps in response to potential effectors, the use of YFP-based reporters has been employed in this work. In a recent study, we developed the YFP-based reporter strain PBT02 in order to identify potential inducer compounds of the SmeVWX efflux pump in S. maltophilia (30). In the present work, two new reporter strains have been developed, one for measuring smeYZ expression, and another, which contains a constitutive promoter as a control for the normalization of the results.…”

“…In order to test the proper functioning of both reporter strains, bacterial growth and YFP levels were measured for 20 h. Although the validation of PBT02 strain was performed in a previous work (30), it was also included in the analysis. The fluorescence levels given by the PBT10 strain are higher in comparison with those obtained for the already described PBT02 strain (Figure S1), confirming that smeYZ is constitutively expressed in the wild-type S. maltophilia strain (10), while smeVWX expression is very low.…”

“…Since new inducer molecules (all of them with antimicrobial properties) of both SmeVWX and SmeYZ multidrug efflux pumps have been identified, we wanted to elucidate whether these compounds were also substrates of their respective efflux pumps. To test this hypothesis, we used the MBS704 (Δ smeW ) strain (30) and we constructed a D457-derived mutant with a partial deletion in the smeZ gene (PBT100 strain). In order to see whether or not the lack of SmeVWX changed S. maltophilia susceptibility to its potential inducers, D457 and MBS704 (Δ smeW ) strains were grown for 20 h at 37 °C in the presence of clioquinol (78 µM), iodoacetate (0.5 mM) or sodium selenite (0.5 mM) and OD 600 was recorded every 10 min.…”

“…With the aim of identifying conditions or compounds that would induce the expression of the S. maltophilia SmeVWX and SmeYZ efflux pumps, YFP-based reporter strains were developed. In a previous screening using the PBT02 ( P VWX ) strain, we identified that vitamin K3, among 30 tested compounds, was an inducer of the SmeVWX system (30). In the current study, two new reporter strains were constructed.…”

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

“…The RNA extraction from the collected cells was performed as previously described in (40). After a DNase I (Qiagen) treatment, a second treatment using DNase Turbo DNA-free (Ambion) was performed, and the presence of DNA contamination was checked by PCR using primers rpsL_Fw and rpsL_Rv (Table 2).…”