Characterization of mechanisms of quinolone resistance in Pseudomonas aeruginosa strains isolated in vitro and in vivo during experimental endocarditis

Abstract

Mechanisms of resistance to quinolones were characterized in Pseudomonas aeruginosa strains isolated after Tn5 insertional mutagenesis and in resistant strains that emerged during pefloxacin therapy of experimental aortic endocarditis. Quinolone resistance achieved in in vitro-selected mutants Qr_1 and Qr-2 was associated with cross-resistance to several groups of antimicrobial agents, including ,-lactams, tetracycline, and chloramphenicol. A significant reduction of norfloxacin uptake was also observed. After ether permeabilization of the cells, DNA synthesis of these two isolates was as susceptible to norfloxacin as DNA synthesis of the parent strain (PAO1). These results indicate that alteration of outer membrane permeability is the primary determinant of resistance in these isolates. This altered cell permeability was correlated with reduction of outer membrane protein G (25.5 kilodaltons) and loss of a 40-kilodalton outer membrane protein in strain Qr_l. Resistance to quinolones that emerged during experimental endocarditis therapy was associated with both modification of outer membrane permeability (decreased uptake of norfloxacin) and decreased susceptibility of DNA synthesis to norfloxacin. Resistance was limited to quinolones and chloramphenicol. For these strains, norfloxacin inhibitory doses (50%) for DNA synthesis were identical to the drug MICs, suggesting that despite the identification of a permeability change, perhaps due to changes of lipopolysaccharide, the alteration of the quinolone intracellular target(s) susceptibility constitutes the primary determinant of resistance. Also, two distinct levels of norfloxacin resistance of DNA synthesis were found in these isolates, indicating that at least two distinct alterations of the drug target(s) are possible in P. aeruginosa.New fluoroquinolones, such as ciprofloxacin and norfloxacin, have potent antimicrobial activity against gram-positive and gram-negative bacteria, including Pseudomonas aeruginosa (48). Biochemical and genetic evidence has designated the DNA gyrase (subunit A) as the intracellular target of these drugs. Bacterial DNA gyrase is a tetramer of two of each of the subunits, A and B, encoded by the gyrA and gyrB genes, respectively. DNA gyrase catalyzes DNA supercoiling, which serves important functions in DNA replication, recombination, and repair (12). Inhibition of DNA gyrase activities by quinolones leads to inhibition of DNA synthesis, but the mechanisms by which quinolones induce cell death and the exact molecular interactions existing between quinolones, DNA gyrase, and DNA are not clear (24).Mutational resistance to fluoroquinolones in gram-negative bacteria such as Escherichia coli and P. aeruginosa has been associated with modification of the intracellular target of the drug (DNA gyrase) or alteration of outer membrane permeability (21,25,42,48).In E. coli, mutations that affect both gyrase subunits, leading to various levels of resistance to fluoroquinolones and nalidixic acid, have been described (24), while in P. aeruginos...

Norfloxacin Induced Cross-Resistance to Fluoroquinolones and Non fluoroquinolone Groups of Antimicrobial Agents in Pseudomonas aeruginosa

“…This indicates that there was no development of high level of resistance to structurally unrelated antibiotics following the development of norfloxacin resistance, except for one strain, in which minor differences in zone diameters of beta-lactam antibiotics was observed. This finding is not consistent with earlier findings that quinolone resistance developed in in vitro is associated with cross -resistance to several groups of antimicrobial agents (structurally unrelated antibiotics), particularly beta-lactam antibiotics 8,10 . The possibility of development of low level resistance cannot be excluded as the development of low level resistance to these agents may not produce a major change in the zone diameters in vitro study (unless MIC goes very high, no major change occurs in zone diameters).…”

Quinolones: a class of antimicrobial agents

“…Resistance occurs primarily by alterations in bacterial cell wall penetration, with mutant forms of DNA gyrase occurring only rarely (Chamberland et al, 1989). Permeability changes occur either via decreased permeability of the hydrophilic pores (OMP) or through alteration of the active transport (efflux) pump (Kaatz et al, 1991), thereby decreasing the intracellular content of fluoroquinolones.…”

Active efflux of fluoroquinolones in Mycobacterium smegmatis mediated by LfrA, a multidrug efflux pump

“…Unfortunately, fluoroquinolone-resistant clinical isolates of Mycobacterium tuberculosis have already appeared (3,31,33). Mutations conferring resistance to quinolones have been reported for several bacterial species (2), including the gram-negative bacteria Escherichia coli (5,10,22) and Pseudomonas aeruginosa (4,16,28) and the gram-positive bacterium Staphylococcus aureus (11,23,24,36). Two principal mechanisms have been described: (i) alteration of DNA gyrase and (ii) decreased drug accumulation in the cell as a result of either decreased influx or increased efflux.…”