Abstract
The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of nosocomial infections. Its relatively impermeable outer membrane (OM) limits antibiotic entry, and a chromosomally encoded AmpC β-lactamase inactivates β-lactam antibiotics. AmpC expression is linked to peptidoglycan (PG) recycling, and soluble (sLT) or membrane-bound (mLT) lytic transglycosylases are responsible for generating the anhydromuropeptides that induce AmpC expression. Thus, inhibition of LT activity could reduce AmpC-mediated β-lactam resistance in P. aeruginosa. Here, we characterized single and combination LT mutants. Strains lacking SltB1 or MltB had increased β-lactam minimum inhibitory concentrations (MICs) compared to wild type, while only loss of Slt decreased MICs. An sltB1 mltB double mutant had elevated β-lactam MICs compared to either the sltB1 or mltB single mutants (96 vs. 32μg/mL cefotaxime), without changes to AmpC levels. Time-kill assays with β-lactams suggested that increased MIC correlated with a slower rate of autolysis in the sltB1 mltB mutant - an antisuicide phenotype. Strains lacking multiple mLTs were more sensitive to β-lactams and up to 16-fold more sensitive to vancomycin, normally incapable of crossing the OM. Multi-mLT mutants were also sensitive to bile salts and osmotic stress, and were hyperbiofilm formers, all phenotypes consistent with cell envelope compromise. Complementation with genes encoding inactive forms of the enzymes - or alternatively, overexpression of Braun's lipoprotein - reversed the mutants' cell envelope damage phenotypes, suggesting that mLTs help to stabilize the OM. We conclude that P. aeruginosa mLTs contribute physically to cell envelope stability, and that Slt is the preferred target for future development of LT inhibitors that could synergize with β-lactams.
Highlights
Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen and frequent cause of hospital-acquired infections
Among the mechanisms that contribute to antibiotic resistance in P. aeruginosa are the inducible expression of a chromosomally encoded AmpC β-lactamase
To create unmarked gene deletions, appropriate pEX18Gm constructs were transferred to P. aeruginosa strains of interest by conjugation with donor strain E. coli SM10, and mating mixtures were plated on Pseudomonas isolation agar containing 100 μg/mL gentamicin (Gm) to counter-select the donor
Summary
Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen and frequent cause of hospital-acquired infections. It belongs to the ESKAPE group of pathogens (with Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, and Enterobacter spp.). The discovery and development of novel antibiotics and antibiotic adjuvants is urgently required for treatment of these and other pathogens (Rice 2008; Boucher et al 2009; Davies and Davies 2010). Among the mechanisms that contribute to antibiotic resistance in P. aeruginosa are the inducible expression of a chromosomally encoded AmpC β-lactamase. AmpC induction (Jacobs et al 1994; Kraft et al 1999; Moya accumulate sufficient damage to cause death and et al 2009) remain incompletely understood.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
