The biocide chlorhexidine (CHX) as well as additional membrane-active agents were shown to induce expression of the mexCD-oprJ multidrug efflux operon, dependent upon the AlgU stress response sigma factor. Hyperexpression of this efflux system in nfxB mutants was also substantially AlgU dependent. CHX resistance correlated with efflux gene expression in various mutants, consistent with MexCD-OprJ being a determinant of CHX resistance.Pseudomonas aeruginosa is an opportunistic human pathogen characterized by an innate resistance to multiple antimicrobials (13), resistance increasingly attributable to the operation of broadly specific, tripartite multidrug efflux systems of the resistance-nodulation-division (RND) family (35,36). One of these, MexCD-OprJ, was originally identified as a determinant of fluoroquinolone resistance (17) but is known to accommodate a variety of clinically relevant antimicrobials (35, 36) as well as biocides (5), dyes, detergents, and organic solvents (27,45,46). MexCD-OprJ is typically quiescent in wild-type cells (20,46), with expression following mutation of the nfxB gene (16,22,23,50) that is divergently transcribed from the mexCDoprJ operon and encodes a repressor of mexCD-oprJ expression (37). Little is known about the signal(s) to which this regulator responds in naturally promoting efflux gene expression, although mexCD-oprJ is inducible by the biocides benzalkonium chloride and chlorhexidine (CHX) (33). These biocides are known to interact with and disrupt bacterial membranes (8), with the possibility that mexCD-oprJ expression is a response to membrane damage/envelope stress. Envelope stress responses (ESRs) are well documented in bacteria (40, 41), with the extracytoplasmic sigma factor RpoE being a key regulator of ESRs in Escherichia coli and other gram-negative bacteria (1,40,41). The RpoE homologue in P. aeruginosa is AlgU, first identified as a regulator of alginate production in mucoid isolates recovered from the lungs of cystic fibrosis patients (15, 28) and shown to be functionally interchangeable with RpoE (51). This study was undertaken to assess the contribution of MexCD-OprJ to biocide resistance in P. aeruginosa and its possible regulation as part of an ESR.Bacterial strains and plasmids used in this study are listed in Table 1. Bacteria were cultivated at 37°C in Luria broth (LB) (34) supplemented with antibiotics to maintain plasmids as needed (for pEX18Tc and derivatives, tetracycline was used [10 g/ml for E. coli and 50 to 100 g/ml for P. aeruginosa]; for pMMB206 and derivatives, chloramphenicol was used [10 g/ml for E. coli and 150 g/ml for P. aeruginosa]; for pK18MobSacB and derivatives, kanamycin was used [50 g/ml for E. coli and 750 to 1,500 g/ml for P. aeruginosa as indicated]; for miniCTX-lacZ and derivatives, tetracycline was used [10 g/ml for E. coli and 25 g/ml for P. aeruginosa]; and for pUC19 and derivatives, ampicillin was used [100 g/ml for E. coli]). AlgU-encoding plasmid pSF02 was constructed by amplifying the algU gene from the chromosome (isol...
Exposure to reactive oxygen species (ROS) (e.g., peroxide) was shown to induce expression of the PA5471 gene, which was previously shown to be required for antimicrobial induction of the MexXY components of the MexXY-OprM multidrug efflux system and aminoglycoside resistance determinant in Pseudomonas aeruginosa. mexXY was also induced by peroxide exposure, and this too was PA5471 dependent. The prospect of ROS promoting mexXY expression and aminoglycoside resistance recalls P. aeruginosa infection of the chronically inflamed lungs of cystic fibrosis (CF) patients, where the organism is exposed to ROS and where MexXY-OprM predominates as the mechanism of aminoglycoside resistance. While ROS did not enhance aminoglycoside resistance in vitro, long-term (8-day) exposure of P. aeruginosa to peroxide (mimicking chronic in vivo ROS exposure) increased aminoglycoside resistance frequency, dependent upon PA5471 and mexXY. This enhanced resistance frequency was also seen in a mutant strain overexpressing PA5471, in the absence of peroxide, suggesting that induction of PA5471 by peroxide was key to peroxide enhancement of aminoglycoside resistance frequency. Resistant mutants selected following peroxide exposure were typically pan-aminoglycoside-resistant, with mexXY generally required for this resistance. Moreover, PA5471 was required for mexXY expression and aminoglycoside resistance in these as well as several CF isolates examined.
Cheese ripening is a complex biochemical process driven by microbial communities composed of both eukaryotes and prokaryotes. Surface-ripened cheeses are widely consumed all over the world and are appreciated for their characteristic flavor. Microbial community composition has been studied for a long time on surface-ripened cheeses, but only limited knowledge has been acquired about its in situ metabolic activities. We applied metagenomic, metatranscriptomic and biochemical analyses to an experimental surface-ripened cheese composed of nine microbial species during four weeks of ripening. By combining all of the data, we were able to obtain an overview of the cheese maturation process and to better understand the metabolic activities of the different community members and their possible interactions. Furthermore, differential expression analysis was used to select a set of biomarker genes, providing a valuable tool that can be used to monitor the cheese-making process.
Expression of the mexXY multidrug efflux operon in wild type Pseudomonas aeruginosa is substantially enhanced by the ribosome-targeting antimicrobial spectinomycin (18-fold) and this is wholly dependent upon the product of the PA5471 gene. In a mutant strain lacking the mexZ gene encoding a repressor of mexXY gene expression, expression of the efflux operon increases modestly (5-fold) and is still responsive (18-fold) to spectinomycin. Spectinomycin induction of mexXY expression in the mexZ mutant is, however, independent of PA5471 suggesting that PA5471 functions as an anti-repressor (dubbed ArmZ for anti-repressor MexZ) that serves only to modulate MexZ's repressor activity, with additional gene(s)/gene product(s) providing for the bulk of the antimicrobial-inducible mexXY expression. Consistent with PA5471/ArmZ functioning as a MexZ anti-repressor, an interaction between MexZ and ArmZ was confirmed using a bacterial 2-hybrid assay. Mutations compromising this interaction (P68S, G76S, R216C, R221W, R221Q, G231D and G252S) were identified and localized to one region of an ArmZ structural model that may represent a MexZ-interacting domain. Introduction of representative mutations into the chromosome of P. aeruginosa reduced (P68S, G76S) or obviated (R216C, R2211W) antimicrobial induction of mexXY gene expression, rendering the mutants pan-aminoglycoside-susceptible. These data confirm the importance of an ArmZ-MexZ interaction for antimicrobial-inducible mexXY expression and intrinsic aminoglycoside resistance in P. aeruginosa.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.