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...
During investigation of susceptibility testing methods for polymyxins, 24 multidrug-resistant clinical isolates of Pseudomonas aeruginosa were observed to have a distinct, reproducible phenotype in which skipped wells were observed during broth microdilution testing for polymyxin B. Possible mechanisms underlying this phenotype were investigated. The effects of various concentrations of polymyxin B on growth, the expression of resistance genes, and outer-membrane permeability were observed. Real-time PCR was performed to compare the expression, in response to selected concentrations of polymyxin B, of genes related to the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems in polymyxin B-susceptible isolate PAO1, polymyxin B-resistant isolate 9BR, and two isolates (19BR and 213BR) exhibiting the skipped-well phenotype. 19BR and 213BR appeared to have similar basal levels of expression compared to that of PAO1 for phoQ, arnB, and PA4773 (from the pmrAB operon), and in contrast, 9BR had 52-and 280-fold higher expression of arnB and PA4773, respectively. The expression of arnB and PA4773 increased in response to polymyxin B in a concentrationdependent manner for 9BR but not for 19BR and 213BR. For these isolates, expression was significantly increased for arnB and PA4773, as well as phoQ, only upon exposure to 2 g/ml polymyxin B but not at a lower concentration of 0.125 g/ml. The sequencing of the pmrAB and phoPQ operons for all three isolates revealed a number of unique mutations compared to that for PAO1. 1-N-phenylnaphthylamine (NPN) was used to study the effect of preincubation with polymyxin B on the self-promoted uptake of polymyxin B across the outer membrane. The preincubation of cells with 2 g/ml polymyxin B affected baseline membrane permeability in 19BR and 213BR and also resulted in a reduced rate of NPN uptake in these isolates and in PAO1 but not in 9BR. The results presented here suggest that the skipped-well isolates have the ability to adapt to specific concentrations of polymyxin B, inducing known polymyxin B resistance genes involved in generating alterations in the outer membrane.The emergence of multidrug-resistant gram-negative organisms and the simultaneous lack of new clinically available antimicrobial agents have led to a resurgence of older compounds such as the polymyxins (6). These agents, including polymyxin B and colistin, have highly potent activity against gramnegative organisms, including Pseudomonas aeruginosa, but were previously abandoned due to a reported high incidence of nephrotoxicity and neurotoxicity (12).Resistance to polymyxin B is predominantly associated with decreased uptake into the bacterial cell resulting from a reduced capacity for initial binding (23). Polymyxin B and other polycationic compounds enter the cell via a process known as self-promoted uptake (10, 11). Polymyxin B binds to outer-membrane lipopolysaccharide (LPS) displacing Mg 2ϩ and disrupting the Mg 2ϩ cross bridges between anionic LPS molecules in the outer membrane, thus leading to membrane des...
Faropenem medoxomil is a new orally administered penem antibiotic. Its chiral tetrahydrofuran substituent at position C2 is responsible for its improved chemical stability and reduced CNS effects, compared with imipenem. Faropenem demonstrates broad-spectrum in vitro antimicrobial activity against many Gram-positive and -negative aerobes and anaerobes, and is resistant to hydrolysis by nearly all beta-lactamases, including extended-spectrum beta-lactamases and AmpC beta-lactamases. However, faropenem is not active against methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium, Pseudomonas aeruginosa or Stenotrophomonas maltophilia. Prospective, multicenter, randomized, double-blind, comparative (not vs placebo) clinical trials of acute bacterial sinusitis (ABS), acute exacerbations of chronic bronchitis (AECB), community-acquired pneumonia (CAP) and uncomplicated skin and skin structure infections (uSSSIs) have demonstrated that faropenem medoxomil has equivalent efficacy and safety compared with cefuroxime, clarithromycin, azithromycin, amoxicillin, cefpodoxime and amoxicillin-clavulanate. The evidence supports faropenem medoxomil as a promising new oral beta-lactam with proven efficacy and safety for the treatment of a variety of community-acquired infections. However, the US FDA recently rejected faropenem for all four indications stating that the clinical trials in ABS and AECB should have been performed versus a placebo. In the CAP studies, the FDA stated that they could not be certain of the validity of the study population actually having the disease and for uSSSI, the FDA stated that only a single trial was not adequate evidence of efficacy for this indication.
Screening of the PA14 genomic transposon mutant library for resistance to ceftazidime, tobramycin, and ciprofloxacin led to the discovery of several mutants that appeared in more than one screen. Testing of the frequency of mutation to ciprofloxacin resistance revealed previously known mutator genes, including mutS and mutL, as well as mutators that have not yet been described for P. aeruginosa, including PA3958 and RadA (PA4609).
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