Hyperoxia and prolongation of aminoglycoside-induced postantibiotic effect in Pseudomonas aeruginosa: role of reactive oxygen species.

Hyperoxia prolongs the postantibiotic effect (PAE) of the aminoglycoside tobramycin in Pseudomonas aeruginosa. We tested the hypothesis that the PAE is prolonged because hyperoxia increases free radical flux while tobramycin inhibits the induction of antioxidant defenses. Exposure of P. aeruginosa to hyperoxia (100% O2) for 1 h increased superoxide dismutase, catalase, and glutathione levels. In the presence of tobramycin (1x the MIC), the induction of antioxidant defenses by hyperoxia was nearly abrogated. Neither preexposure of P. aeruginosa to hyperoxia nor supplementation with the antioxidants copper(II) (diisopropylsalicylate)2 (superoxide dismutase-like), catalase, or dimethyl sulfoxide abolished prolongation of the PAE of tobramycin induced by hyperoxia.     

Hyperoxia and the antimicrobial susceptibility of Escherichia coli and Pseudomonas aeruginosa.

We have tested the ability of hyperoxia (98% O2-2% CO2 at 2.8 atmospheres absolute [ca. 284.6 kPa]) to enhance killing of Escherichia coli (serotype O18 or ATCC 25922) by nitrofurantoin, sulfamethoxazole, trimethoprim, gentamicin, and tobramycin. We have also looked for interactions between hyperoxia and the aminoglycosides against Pseudomonas aeruginosa ATCC 27853. Hyperoxia significantly enhanced bacteriostatic activity of nitrofurantoin and trimethoprim as measured by MIC testing. The possibility exists that these effects might be due to the method required to tests MICs under hyperoxic conditions rather than to the effect of hyperoxia itself. In addition, hyperoxia enhanced killing of bacteria by trimethoprim as measured by MBC testing. Hyperoxia decreased numbers of E. coli by 1.3 log10 and P. aeruginosa by 2.7 log10 in cation-supplemented Mueller-Hinton broth medium. The bacteriostatic effects of hyperoxia did not affect MICs of gentamicin or tobramycin. The lack of interaction between hyperoxia and gentamicin or tobramycin was confirmed by determining the number of viable bacteria remaining after 24 h of exposure to hyperoxia by using a pour plate method. We conclude that hyperoxia potentiates the antimicrobial activity of the reduction-oxidation-cycling antibiotic tested (nitrofurantoin) and of one of the antimetabolites tested (trimethoprim). Hyperoxia does not enhance the bactericidal effects of gentamicin and tobramycin, which require oxidative metabolism for transport into bacterial cells.     

Deletion of the multiple-drug efflux pump AcrAB in Escherichia coli prolongs the postantibiotic effect

The mechanism of the postantibiotic effect (PAE) was examined in Escherichia coli. Drugs exhibited longer-lasting PAEs in an acrAB mutant, suggesting that intracellular drug concentrations influence the duration of the PAE. With specific assays for tetracycline and erythromycin, a direct link between intracellular persistence of antibiotics and maintenance of the PAE was established.     

Antimicrobial activity of subinhibitory concentrations of ciprofloxacin against Pseudomonas aeruginosa as determined by the killing curve method and the postantibiotic effect.

This investigation used the postantibiotic effect (PAE) and killing curves to examine the antimicrobial activity of subinhibitory (1/8x, 1/4x and 1/2x MIC) and inhibitory (1x MIC) concentrations of ciprofloxacin against mucoid (M) and nonmucoid (NM) urinary isolates of Pseudomonas aeruginosa. Subinhibitory concentrations (1/8x, 1/4x and 1/2x MIC) of ciprofloxacin produced PAEs with no difference between M and NM strains. For NM strains, those with low MICs (< or = 1.0 mg/l) to ciprofloxacin produced significantly longer PAEs than isolates with high MICs (> 1 mg/l). Killing curve studies demonstrated that subinhibitory concentrations of ciprofloxacin produce little effect (1/8x MIC) or stasis (1/4x and 1/2x MIC) of growth for several hours. Only 1x MIC was bactericidal for several strains. At 1/2x and 1x MIC, bacterial inhibition was greater against NM versus M isolates. The M phenotype of P. aeruginosa reduces killing by ciprofloxacin but not the PAE.     

Influence of pH on adaptive resistance of Pseudomonas aeruginosa to aminoglycosides and their postantibiotic effects.

Adaptive resistance to aminoglycosides in Pseudomonas aeruginosa and other gram-negative bacilli is usually induced by the initial exposure to the drug. We investigated the influence of pH on the adaptive resistance of a clinical P. aeruginosa strain to aminoglycosides in vitro and on their postantibiotic effects. For adaptive resistance, the first-exposure concentrations of both amikacin and netilmicin were one, two, four, and eight times the MIC of each drug and the second-exposure concentrations were two times the MIC of each drug. Adaptive resistance was greater and more prolonged with higher initial aminoglycoside concentrations, and the bactericidal effects of the aminoglycosides were concentration dependent at pH 7.4. At pH 6.5, the killing rates of amikacin and netilmicin were far lower than those observed at pH 7.4. At pH 5.5, amikacin and netilmicin exerted practically no bactericidal effect on the P. aeruginosa strain used. However, with media at pH 5.5 and 6.5, adaptive resistance of P. aeruginosa preexposed to amikacin and netilmicin was also clearly exhibited, with the degree of adaptive resistance depending on the bactericidal effects of both drugs on nonpreexposed controls. Maximal adaptive resistance occurred between 0 and 4 h after preexposure. The postantibiotic effects of amikacin and netilmicin against the P. aeruginosa strain were shown to be concentration dependent and were reduced at acidic pHs. No changes in outer and inner membrane proteins occurred during the adaptive-resistance interval.     

Antimicrobial activity of subinhibitory concentrations of aminoglycosides against Pseudomonas aeruginosa as determined by the killing-curve method and the postantibiotic effect

This investigation used the postantibiotic effect (PAE) and killing curves to provide data on the antimicrobial activity of subinhibitory (1/8x, 1/4x and 1/2x minimum inhibitory concentration; MIC) and inhibitory (1x MIC) concentrations of amikacin, gentamicin and tobramycin against Pseudomonas aeruginosa. Subinhibitory concentrations (1/4x and 1/2x MIC) of aminoglycosides demonstrated a reproducible PAE. At 1/4x MIC, the order of duration of the PAE was approximately 15 min for all aminoglycosides, while at 1/2x MIC all three aminoglycosides displayed a similar PAE of approximately 40 min. Killing-curve studies demonstrated that subinhibitory concentrations of aminoglycosides either decrease bacterial growth for several hours (1/4x and 1/2x MIC) or produce stasis of growth for several hours (1/8x MIC). Only inhibitory aminoglycoside concentrations (1x MIC) proved to be bactericidal. Subinhibitory concentrations of aminoglycosides decrease bacterial growth and produce a PAE against P. aeruginosa.     

Antibiotic exposure and its relationship to postantibiotic effect and bactericidal activity: constant versus exponentially decreasing tobramycin concentrations against Pseudomonas aeruginosa.

In vitro postantibiotic effects (PAEs) exhibited by a standard strain of Pseudomonas aeruginosa following exposure to tobramycin at constant concentrations were compared to those at exponentially decreasing concentrations. Exposure to a constant concentration showed more extensive bacterial killing and resulted in longer PAEs at comparable areas under the concentration-time curves above the MIC. This phenomenon suggests a significant contribution of pharmacokinetics to antimicrobial pharmacodynamics.     

Postantibiotic effect of imipenem on Pseudomonas aeruginosa.

Imipenem (formerly N-formimidoyl thienamycin) and ceftazidime were investigated for their postantibiotic effect on Pseudomonas aeruginosa. Four strains of P. aeruginosa in the logarithmic phase of growth were exposed for 1 and 2 h to concentrations of antibiotics achievable in human serum. Recovery periods of test cultures were evaluated after dilution or addition of beta-lactamase. A consistent postantibiotic effect against all strains was obtained with imipenem but not with ceftazidime. Although ceftazidime did not have a postantibiotic effect, it did suppress the growth of the organisms at concentrations equivalent to one-third of the MIC. The clinical implications of these effects need further evaluation.     

Inoculum effect of new beta-lactam antibiotics on Pseudomonas aeruginosa.

The degree of the inoculum effect shown by the new beta-lactam antibiotics with Pseudomonas aeruginosa was investigated, and the antibiotics were divided into three groups based upon the observations. The group 1 antibiotics (cefotaxime, moxalactam, cefoperazone, azlocillin, piperacillin, and aztreonam) demonstrated a large inoculum effect, were poorly bactericidal, produced aberrant, elongated bacilli, and did not inhibit the increase in turbidity of high inocula during an 18-h incubation. The group 2 antibiotics (ceftazidime and ticarcillin) were slowly bactericidal, caused minimal formation of aberrant, elongated bacilli, and slowly decreased the turbidity of high inocula. The group 3 antibiotics (imipenem and gentamicin) were bactericidal, did not cause the formation of elongated bacilli, and decreased the turbidity of high inocula rapidly. Data are presented which suggest that the inoculum effect seen with the group 1 beta-lactam antibiotics is related to (i) the poor intrinsic antibactericidal activity of these antibiotics for P. aeruginosa at the inocula tested and (ii) failure of these antibiotics to inhibit the formation of aberrant and filamentous bacilli, which can result in increased bacterial mass and turbidity.     

Resistance to pefloxacin in Pseudomonas aeruginosa.

Mechanisms of resistance to pefloxacin were investigated in four isogenic Pseudomonas aeruginosa strains: S (parent isolate; MIC, 2 micrograms/ml), PT1 and PT2 (posttherapy isolates obtained in animals; MICs, 32 and 128 micrograms/ml, respectively), and PT2-r (posttherapy isolate obtained after six in vitro subpassages of PT2; MIC, 32 micrograms/ml). [2-3H]adenine incorporation (indirect evidence of DNA gyrase activity) in EDTA-permeabilized cells was less affected by pefloxacin in PT2 and PT2-r (50% inhibitory concentration, 0.27 and 0.26 microgram/ml, respectively) than it was in S and PT1 (50% inhibitory concentration, 0.04 and 0.05 microgram/ml, respectively). Reduced [14C]pefloxacin labeling of intact cells in strains PT1 and PT2 correlated with more susceptibility to EDTA and the presence of more calcium (P less than 0.05) and phosphorus in the outer membrane fractions. Outer membrane protein analysis showed reduced expression of protein D2 (47 kDa) in strains PT1 and PT2. Other proteins were apparently similar in all strains. The addition of calcium chloride (2 mM) to the sodium dodecyl sulfate-solubilized samples of outer membrane proteins, before heating and Western blotting, probed with monoclonal antibody anti-OmpF showed electrophoretic mobility changes of OmpF in strains PT1 and PT2 which were not seen in strain S. Calcium-induced changes were reversed with ethyleneglycoltetraacetate. Decreased [14C]pefloxacin labeling was further correlated with an altered lipopolysaccharide pattern and increased 3-deoxy-D-mannooctulosonic acid concentration (P less than 0.01). These findings suggested that resistance to pefloxacin is associated with altered DNA gyrase in strain PT2-r, with altered permeability in PT1, and with both mechanisms in PT2. The decreased expression of protein D2 and the higher calcium and lipopolysaccharide contents of the outer membrane could be responsible for the permeability deficiency in P. aeruginosa.     

Pseudomonas aeruginosa antibodies in human plasma.

Plasma samples from healthy adults were examined for antibodies to the common protective antigen (OEP) [1] by passive hemagglutination (HA) reaction [2] with the finding that the majority of them had an antibody titer of 60 or lower. 2-Mercaptoethanol (2-ME) treatment, however, caused a decrease in HA titer down to 16 or lower in most of the cases, suggesting that the OEP antibody resides mostly in IgM and slightly in IgG. The finding was corroborated by gel filtration on Sephadex G-200. It appears likely that IgA is not implicated in the HA titer since IgA was HA negative. However, the detection of enzymatic activity by enzyme-linked immunosorbent assay (ELISA) [9] still suggests a possibility that OEP antibody resides in IgA. On the other hand, antibodies to proteolytic enzymes, especially protease and elastase [3], elaborated by Pseudomonas aeruginosa, were found to be present in most cases at a titer of 16 or lower by HA reaction. It was also found that they reside mostly in IgM.     

Transferable imipenem resistance in Pseudomonas aeruginosa.

We isolated an imipenem-resistant strain, GN17203, of Pseudomonas aeruginosa. The strain produced a beta-lactamase that hydrolyzed imipenem. The beta-lactamase was encoded by a 31-MDa plasmid, pMS350, which belongs to incompatibility group P-9. The plasmic conferred resistance to beta-lactams, gentamicin, and sulfonamide and was transferable by conjugation to P. aeruginosa but not to Escherichia coli. The molecular weight of the purified enzyme was estimated to be 28,000, and the isoelectric point was 9.0. The enzyme showed a broad substrate profile, hydrolyzing imipenem, oxyiminocephalosporins, 7-methoxycephalosporins, and penicillins. The enzyme activity was inhibited by EDTA, iodine, p-chloromercuribenzoate, CuSO4, and HgCl2 but not by clavulanic acid or sulbactam.     

Carbenicillin resistance of Pseudomonas aeruginosa.

Four strains of Pseudomonas aeruginosa obtained from clinical isolates which are carbenicillin resistant were studied to find the cause(s) of resistance to this beta-lactam antibiotic. The electrophoresis patterns of the four strains (PH20610, PH20815, PH4011, and PH4301) were found to be different from those of a wild-type strain, P. aeruginosa NCTC 10662, and appeared to lack penicillin-binding protein 2. Affinity of other penicillin-binding proteins from strains PH20610 and PH20815 for carbenicillin seemed to be normal or slightly diminished. Electrophoretic patterns of penicillin-binding proteins from strains PH4011 and PH4301 had more profound differences, since the affinities of their penicillin-binding proteins 1a, 1b, and 4 for carbenicillin were decreased by nearly two orders of magnitude relative to the preparations from the wild-type strain. Kinetic studies on binding of carbenicillin to penicillin-binding proteins both in isolated membrane preparations and in intact cells revealed that carbenicillin penetration into resistant cells was a much slower process than in susceptible cells, suggesting that the outer envelope structures serve as an efficient barrier against carbenicillin entry into our P. aeruginosa strains from clinical isolates.     

Complement-mediated phagocytosis of Pseudomonas aeruginosa.

The nature of the opsonic factors in nonimmune human serum for six blood culture isolates of Pseudomonas aeruginosa was investigated by measuring uptake of [3H] adenine-labeled bacteria by human PMNs. Normal human serum, C2- and C4-deficient sera, zymosan-treated serum, and immunoglobulin-deficient sera were used as opsonic sources. Heat inactivation of each of these serum sources markedly reduced its opsonic capacity for all Pseudomonas strains, suggesting that the serum C system was essential for opsonization. Five strains were opsonized in the absence of the classical C pathway; however, kinetic studies revealed that opsonization proceeded at a faster rate when the classical pathway was present. In spite of markedly reduced factor B and C3 levels, zymosan-treated serum retained significant opsonic activity for one of the strains tested. Four strains were poorly opsonized by immunoglobulin-deficient serum, and C activation by these strains appeared to depend upon the presence of antibodies. Two strains, however, were effectively opsonized in a relative absence of antibodies. Thus, in the nonimmune state, phagocytosis of P. aeruginosa is mediated primarily via the C system, and antibodies appear to play a role in the opsonization of some but perhaps not all Pseudomonas strains.     

The bactericidal activity and postantibiotic effect of trospectomycin.

Trospectomycin sulfate (trospectomycin, TRS) is a novel, broad-spectrum, aminocyclitol antibiotic that is being developed clinically for the treatment of upper respiratory tract infections, bacterial vaginosis, pelvic inflammatory disease, and gonorrhea. This study investigated the bactericidal activity (by time-kill kinetics) and the postantibiotic effect (PAE) of TRS. Species-dependent bacteriostatic/bactericidal activity was observed for TRS; the antibiotic was bacteriostatic for Staphylococcus epidermidis, Enterococcus faecalis, and Escherichia coli, and bactericidal for Haemophilus influenzae, Neisseria gonorrhoeae, Moraxella catarrhalis, and Bacteroides fragilis (one of two test strains). When TRS was tested at four times its minimum inhibitory concentration or at a maximum test concentration of 32 micrograms/ml, with a 1-hr exposure period, the following PAE values were recorded: S. epidermidis 30032, 1.8 hr, En. faecalis ATCC 29212, 1.6 hr, E. coli UC 311, 1.5 hr, E. coli UC 9451, 1.5 hr, H. influenzae 30063, greater than 4.0 hr, B. fragilis ATCC 25285, 5.2 hr, and B. fragilis UC 12199, 6.7 hr. The broad-spectrum PAE that was observed for TRS is somewhat unique compared with other antibiotics.