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Pseudomonas aeruginosa strains isolated from patients with persistent lung infections and cystic fibrosis have been found to gradually develop aminoglycoside resistance over time. The aim of this study was to identify potential contributors to low-level aminoglycoside resistance, which may cause such graduated increases in resistance. The Harvard P. aeruginosa PA14 nonredundant library, consisting of approximately 5,800 mutants, was screened for twofold or greater increases in tobramycin resistance. Mutants carrying mutations in a total of 135 unique genes were identified and confirmed to have reduced susceptibility to tobramycin. Many of these genes were involved predominantly in energy metabolism; however, most of these mutants did not exhibit growth defects under the conditions tested, although some exhibited the small-colony phenotype and/or defects in growth under anaerobic conditions. Lipopolysaccharide mutants were also identified, and it was found that tobramycin had a reduced ability to permeabilize the outer membranes of these mutants. The results of this study emphasize the complexity of the interactions that tobramycin may have within the bacterial cell and introduce a large number of novel genes which may play a role in tobramycin resistance.The majority of deaths of cystic fibrosis patients can be attributed to the progressive deterioration of lung function resulting from chronic infection by pathogens such as Pseudomonas aeruginosa (25). Antibiotic treatment of such chronic infections may temporarily suppress symptoms; however, it does not eradicate the pathogen. To overcome the inability of orally and parenterally administered antibiotics to adequately penetrate lung tissue and secretions, the aerosolized aminoglycoside tobramycin was formulated to directly target the site of infection. Clinical trials of aerosolized tobramycin revealed that long-term use of the agent against P. aeruginosa results in small, graduated increases in tobramycin MICs upon repeated isolation of the organism (21, 22), although the mechanisms contributing to this resistance have yet to be described.Tobramycin is a bactericidal agent that targets the 30S ribosome and interferes with protein synthesis. Uptake occurs in three phases: an initial, reversible ionic-binding phase, a phase of slow energized uptake, and finally a phase of very rapid energy-dependent uptake (2, 4, 9). Despite the traditionally held view that antibiotic action can be simplified to interaction with a single target, it is evident that aminoglycosides exert pleiotropic effects on the cell (8, 9), as these effects may be antagonized by a variety of compounds known to affect cellular metabolic processes. Furthermore, the bactericidal nature of aminoglycosides such as tobramycin cannot be accounted for simply by protein synthesis inhibition or misreadings during translation because other protein synthesis inhibitors, such as chloramphenicol, and agents that promote misreading, including modified amino acids, are bacteriostatic. A limited number of cytoch...

Pseudomonas aeruginosa PAO1 lon mutants are supersusceptible to ciprofloxacin, and exhibit a defect in cell division and in virulence-related properties, such as swarming, twitching and biofilm formation, despite the fact that the Lon protease is not a traditional regulator. Here we set out to investigate the influence of a lon mutation in a series of infection models. It was demonstrated that the lon mutant had a defect in cytotoxicity towards epithelial cells, was less virulent in an amoeba model as well as a mouse acute lung infection model, and impacted on in vivo survival in a rat model of chronic infection. Using qRT-PCR it was demonstrated that the lon mutation led to a down-regulation of Type III secretion genes. The Lon protease also influenced motility and biofilm formation in a mucin-rich environment. Thus alterations in several virulence-related processes in vitro in a lon mutant were reflected by defective virulence in vivo.

The increasing number of multidrug resistant bacteria has revitalized interest in seeking alternative sources for controlling bacterial infection. Silver nanoparticles (AgNPs), are amongst the most promising candidates due to their wide microbial spectrum of action. In this work, we report on the safety and efficacy of the incorporation of collagen coated AgNPs into collagen hydrogels for tissue engineering.The resulting hybrid materials at [AgNPs] < 0.4 µM retained the mechanical properties and biocompatibility for primary human skin fibroblasts and keratinocytes of collagen hydrogels; they also displayed remarkable anti-infective properties against S. aureus, S. epidermidis, E. coli and P. aeruginosa at considerably lower concentrations than silver nitrate. Further, subcutaneous implants of materials containing 0.2 µM AgNPs in mice showed a reduction in the levels of IL-6 and other inflammation markers (CCL24, sTNFR-2, and TIMP1). Finally, an analysis of silver contents in implanted mice showed that silver accumulation primarily occurred within the tissue surrounding the implant. IntroductionBiomaterial-associated infections are a significant healthcare problem and have been linked to medical morbidity and death. [2][3][4][5][6] This has motivated the development of materials with anti-infective properties, such as biomaterials loaded with antibiotics. 7 However, with the increasing number of bacteria that are resistant to antibiotics, 8 silver with historically documented anti-microbial activity has re-gained its attractiveness as an alternative to antibiotics. 9 While toxic to bacteria, it unfortunately is also toxic to mammalian cells. 9,10 More recently, therefore, silver nanoparticles (AgNPs) have been evaluated as a safer alternative to ionic silver. 1,[11][12][13][14][15][16][17][18][19][20] The recent work from our team showed that in comparison with silver, biomolecule-coated, photochemically-produced AgNPs can have both bactericidal and bacteriostatic properties with almost negligible cytotoxic effects. 12 We also showed that oxidation of Ag to AgO is most likely the cause of the cytotoxic effects observed with AgNPs. Our overarching goal is to expand the safe use of AgNPs in the development of implantable hybrid-biomaterials with antiinfective properties for future use as scaffolds to enable regeneration of tissue and organs at a risk of bacterial colonization and concomitant biofilm formation like diabetic foot ulcers. Although some collagen-based materials including † Electronic supplementary information (ESI) available: Representative absorption spectra of AgNP@collagen nanoparticles before and after lyophilization. Absorption spectra for the washes obtained from a 1.0 µM AgNP hydrogel over the course of 5 days. Area under the curve (AUC) calculated from the absorption spectra of 500 µm thickness collagen hydrogels prepared using different concentrations of AgNP@collagen. Selected Cryo-SEM images of BDDGE type I collagen-based hydrogels in the absence or presence of 1.0 µM AgNP. An image of a sel...