Differential selection of multidrug efflux mutants by trovafloxacin and ciprofloxacin in an experimental model of Pseudomonas aeruginosa acute pneumonia in rats.

The ability of trovafloxacin and ciprofloxacin to select efflux mutants in vivo was studied in a model of acute Pseudomonas aeruginosa pneumonia in rats. Twelve hours after intratracheal inoculation of 10(6) CFU of P. aeruginosa strain PAO1 enmeshed in agar beads, two groups of 12 rats were treated by three intraperitoneal injections of each antibiotic given every 5 h. Dosing regimens were chosen to obtain a comparable area under the concentration-time curve from 0 to infinity/MIC ratio of 27.9 min for trovafloxacin (75 mg/kg of body weight) and of 32.6 min for ciprofloxacin (12.5 mg/kg). Twelve rats were left untreated and served as controls. Rats were sacrificed 12 h after the last injection (34 h after infection) for lung bacteriological studies. Selection of resistant bacteria was determined by plating lung homogenates on Trypticase soy agar plates containing antibiotic. In untreated animals, the frequency of resistant colonies was 10-fold higher than in agar beads. Compared to controls, both treatment regimens resulted in a 2-log reduction of lung bacterial load. The frequency of resistant colonies was 10-fold less with trovafloxacin than with ciprofloxacin at twice the MIC (7.4 x 10(-5) versus 8.4 x 10(-4), respectively) (P < 0.05) and at four times the MIC (6.2 x 10(-4) versus 5.0 x 10(-5), respectively) (P < 0.05). A multidrug resistance phenotype typical of efflux mutants was observed in all 41 randomly tested colonies obtained from treated and untreated rats. In agreement with in vitro results, trovafloxacin and ciprofloxacin preferentially selected MexCD-OprJ and MexEF-OprN overproducers, respectively. These results demonstrate the differential ability of trovafloxacin and ciprofloxacin to select efflux mutants in vivo and highlight the rapid emergence of those mutants, even without treatment.

Evaluation of the efficacy of prolonged administration of azithromycin in a murine model of chronic toxoplasmosis.

The efficacy of prolonged administration of azithromycin was evaluated in a murine model of lethal chronic toxoplasmosis. Mice were challenged intraperitoneally with cysts of a moderately virulent strain of Toxoplasma gondii, observed for 4 weeks and then allocated to the treatment or control group. All 26 animals given azithromycin 100 mg/kg/day for 100 days were protected compared with 19 of 25 control animals which died (P < 0.001). Nineteen of the 20 mice in the treatment group survived for an additional month while receiving the same azithromycin regimen but viable cysts were identified in the brain tissue of these animals when they were killed. Although there was no significant difference between the groups in terms of the number of cysts in the brain, the administration of azithromycin was associated with a reduction in brain inflammation. The concentrations of azithromycin in the brains of five animals ranged from 0.7 to 2.3 micrograms/g; there was no evidence of accumulation even after 100 doses. Azithromycin merits further evaluation as primary or secondary prophylaxis against toxoplasma encephalitis in individuals at risk of developing this complication.

Quinolones in intracellular infections.

Intracellular parasites are those which spend most of their lives within host cells. The fluoroquinolones demonstrate favourable intracellular pharmacokinetics for the treatment of intracellular infections; these agents diffuse and accumulate in the phagocytes, mainly in the cytosol, and do not associate with cellular organelles. The fluoroquinolones are generally active against Salmonella spp. in vitro, and have been used successfully in the treatment of typhoid fever, Salmonella bacteraemia in patients with AIDS, and chronic enteric carriage. Fluoroquinolone monotherapy has also been found satisfactory in the treatment of tularaemia and Mediterranean spotted fever. Quinolones, alone or in combination with other agents, have also shown promise in animal models of legionellosis and in limited clinical studies. Quinolones, particularly ciprofloxacin and ofloxacin, have notable antimycobacterial activity. Both agents have been used in combination with other antimycobacterial drugs in the treatment of infections caused by Mycobacterium tuberculosis, M. avium-intracellulare complex, rapidly growing mycobacteria and M. leprae, and deserve consideration as part of a multi-drug regimen in otherwise untreatable mycobacterial infections. Clinical data regarding fluoroquinolone monotherapy in brucellosis indicate unacceptable failure rates which preclude the use of these agents in this indication. The quinolones have some efficacy in genital chlamydial infections, but may have limitations in this indication also. In conclusion, as a result of the in vitro activity of the quinolones and their favourable pharmacokinetics, these agents are now an important part of the armamentarium against intracellular infections.

Dietary supplementation with fish oil enhances in vivo synthesis of tumor necrosis factor.

Studies reported here investigate the influence of dietary fat types on cytokine production in response to endotoxin (LPS) challenge. Tumor necrosis factor (TNF) serum levels were markedly higher (by 10-fold) in mice fed chronically a diet rich in fish oil rather than either a diet rich in corn or coconut oil or a low fat diet. This in vivo hyper-responsiveness in LPS-induced TNF production following fish oil consumption concorded with similar exaggerated in vitro TNF release from macrophages exposed to LPS. These data suggest that high consumption of fish oils, by virtue of their high content of omega-3 polyunsaturated fatty acids, can lead to an exaggerated production of mediators of inflammation with potentially adverse consequences on the outcome and severity of infectious diseases.

Development of resistance during ceftazidime and cefepime therapy in a murine peritonitis model.

Resistance emerging after ceftazidime or cefepime therapy was investigated in a peritonitis model. Mice were given a peritoneal challenge (10(8) cfu plus talcum) and treated by either antibiotic (50 mg/kg/dose, which produced similar antibiotic concentrations in peritoneal fluid in both cases). After one or three doses, resistance never developed in Serratia marcescens or Citrobacter freundii infections. After Enterobacter cloacae and Pseudomonas aeruginosa challenge, ceftazidime selected more resistance (21/36 cases) than did cefepime (1/36 cases). In mice challenged with resistant strains selected by ceftazidime therapy, cefepime (six doses) successfully treated 7/18 E. cloacae infections but 0/18 P. aeruginosa infections; ceftazidime was never effective. Neither cefepime nor ceftazidime cured mice infected with the resistant strain selected by cefepime. MICs were poor predictors of further emergence of resistance in mice inoculated with strains classified as susceptible, but antibiotic-containing agar gradients plated with a high inoculum (10(8) cfu) allowed better prediction. In selected clinical situations, cefepime may be preferable because it may be associated with less frequent emergence of resistance.

Activity of minocycline against Toxoplasma gondii infection in mice.

The chemotherapeutic activity of minocycline, a semi-synthetic tetracycline analogue, was evaluated in a murine model of toxoplasmosis. A lethal acute toxoplasmosis was produced by injecting 10(5) tachyzoites of the RH strain of Toxoplasma gondii into the peritoneal cavities of Swiss-Webster mice. When infected mice were treated once daily for 12 days, starting 2 h after challenge, the survival and cure rates were 100% and 40% respectively after minocycline alone (100 mg/kg per day), 0% and 0% after pyrimethamine alone (8.5 mg/kg per day), and 100% and 50% after combination of the two drugs at the same dosages. Absolute survival and cure with minocycline were observed when mice were treated with two daily doses of 100 mg/kg for 12 days. Mice chronically infected with a low virulent strain of T. gondii (Me49) showed a significant reduction in the number of brain cysts after three weeks of treatment with 50 mg/kg per day of minocycline. Minocycline serum levels after a single oral administration of 50 mg/kg or 100 mg/kg to normal mice, peaked at 1.8 mg/l and 10 mg/l after 1 h, respectively, and showed an extended half-life.

Activity of amphotericin B and intraconazole against intraphagocytic Candida albicans.

The activity of amphotericin B and intraconazole against intracellular Candida albicans was determined in vitro using murine resident peritoneal macrophages. Amphotericin B at concentrations of 0.5 and 2 micrograms/ml produced significantly less rapid killing of intracellular than of extracellular Candida albicans as measured in macrophage-free medium. Amphotericin B at concentrations of 0.1 micrograms/ml or itraconazole concentrations of up to 3 micrograms/ml produced only fungistatic or limited fungicidal activity against both intracellular and extracellular organisms. Against intracellular Candida albicans amphotericin B acted by direct antifungal action rather than through stimulation of macrophage function, as demonstrated by the fact that (i) activity persisted when macrophages were successively exposed to amphotericin B, washed and disrupted by sonication, and (ii) no activity was seen when amphotericin B was tested against intracellular amphotericin B-resistant Candida tropicalis or Salmonella typhimurium. Pre-exposure of macrophages to itraconazole (0.4 micrograms/ml) inhibited subsequent killing activity of amphotericin B (2 micrograms/ml) against intracellular susceptible Candida albicans. These experiments validate the conventional methods of susceptibility testing for determining the fungistatic activity of antifungal agents.

How predictable is development of resistance after beta-lactam therapy in Enterobacter cloacae infection?

Certain non-fastidious Gram-negative bacilli, notably Enterobacter cloacae, although classified as susceptible by usual in-vitro susceptibility testing, often become resistant in patients treated with newer beta-lactam antibiotics. Here various in-vitro tests were carried out together with an animal model allowing the quantification of resistance that emerges after short term therapy. Mice were challenged (10(8) cfu plus talcum) intraperitoneally with one each of four strains of Ent. cloacae. Two hours later, a single beta-lactam dose was administered subcutaneously. The following day, the peritoneal bacterial population was analysed by using antibiotic-containing gradient plates. Development of resistance after therapy varied according to the compound considered. Imipenem (50 mg/kg) produced no resistance, and piperacillin (200 mg/kg) only a few, while resistance occurred frequently after therapy with aztreonam (50 mg/kg), ceftazidime (50 mg/kg), cefotaxime (50 mg/kg) and cefpirome (50 mg/kg). MICs increased by at least 16-fold when resistance developed. No simple correlations were found between these in-vivo results and initial MICs, killing kinetics, frequency of resistant variants within the bacterial populations before therapy, initial MIC of these variants or antibiotic concentrations assayed in peritoneal fluid 60 min after dosing. The most reliable predictive in-vitro test appeared to be the determination of resistance emerging in broth containing at least 16 times the MIC of the antibiotic tested. Such a test is unlikely to be used on a routine basis. When a beta-lactam compound seems appropriate for treating an Enterobacter infection, it may be advisable to avoid drugs that are prone to produce resistance in experimental or clinical infections, whatever the results of conventional in-vitro susceptibility tests.

Permeability and penicillin-binding protein alterations in Salmonella muenchen: stepwise resistance acquired during beta-lactam therapy.

A patient with Salmonella muenchen sepsis was unsuccessfully treated with ampicillin. During therapy, four strains that showed stepwise ampicillin resistance and affected other beta-lactams and unrelated antibiotics were isolated sequentially. Resistance was caused by decreased outer membrane permeability associated with diminished expression of porin OmpF. Furthermore, the most resistant isolate overproduced the PBP 3 target molecule.

Resistance emerging after pefloxacin therapy of experimental Enterobacter cloacae peritonitis.

Resistance emerging after pefloxacin therapy was investigated in an experimental Enterobacter cloacae infection. Mice were inoculated intraperitoneally (mean inoculum, 0.9 X 10(8) CFU) with one of four strains initially susceptible to quinolones and treated with a single 25-mg/kg dose of pefloxacin. This therapy produced a net decrease of bacterial counts in the peritoneal fluid, but with the of the isolates, posttherapy (PT1) strains emerged with decreased susceptibilities to quinolones (4- to 1,024-fold), to the structurally unrelated antibiotics (4- to 16-fold) chloramphenicol and trimethoprim, and sometimes to tetracycline and beta-lactam compounds. In a second set of experiments, new mice were similarly infected with PT1 strains and treated with up to five 25-mg/kg doses of pefloxacin. Compared with parent isolates, PT1 strains produced similar disease and peritoneal bacterial count in the control animals. In treated mice posttherapy (PT2) strains emerged that showed 8- to 64-fold increases in quinolone MICs compared with the PT1 strains inoculated. All PT1 and PT2 strains showed altered outer membrane protein patterns, principally marked by a decreased 37,000-molecular-weight band generally accompanied by an increased 42,000-molecular-weight band. Whole cells from all PT1 and PT2 strains, exposed to [14C]pefloxacin for 15 to 60 s, bound significantly less radioactivity than the corresponding parent strains. After partial purification, DNA gyrase extracted from the most resistant isolates (one PT1 and the PT2 strains) showed a 100- to 450-fold 50% inhibitory concentration increase for pefloxacin. Altogether, pefloxacin can select in vivo two types of resistant strain, one with only decreased permeability and another with decreased permeability combined with altered DNA gyrase.

Resistance occurring after fluoroquinolone therapy of experimental Pseudomonas aeruginosa peritonitis.

Resistance emerging after fluoroquinolone therapy was investigated in a murine model of Pseudomonas aeruginosa infection. Mice were infected intraperitoneally by one of six strains and treated with pefloxacin or ciprofloxacin. In mice challenged with a low inoculum (1.6 X 10(5) CFU), no resistance occurred. With a higher inoculum (1.5 X 10(8) CFU) and after a single dose of antibiotic, posttherapy (PT1) strains with decreased susceptibility to quinolones (4- to 32-fold less) were isolated at a variable rate. The presence of talcum (125 mg) in the peritoneal cavity increased the risk of resistance after therapy. Pefloxacin (25 or 200 mg/kg) and ciprofloxacin (25 mg/kg) yielded similar resistance rates (61 to 77%), but ciprofloxacin (10 mg/kg) produced more resistance (83%) than did ciprofloxacin (50 mg/kg) (44%) (P less than 0.02). Combined with a quinolone, ceftazidime (P less than 0.001) or amikacin (P less than 0.01), but not piperacillin, reduced the emergence of resistance. After several doses of ciprofloxacin, it was found that 25-mg/kg doses every 12 h produced more resistance than did 25-mg/kg doses every 8 h or 50-mg/kg doses every 12 h. Compared with the preceding experiments using parent strains, ciprofloxacin and pefloxacin were less efficient in killing bacteria in mice infected with PT1 strains. Moreover, in one of these mice, a highly resistant PT2 strain (64-fold MIC increase for the quinolones) emerged. Besides increased MICs of the quinolones, there was a two- to eightfold increase in imipenem MIC for all PT1 and PT2 strains without alteration of other beta-lactam and aminoglycoside susceptibility. Some PT1 strains also showed a decreased susceptibility to trimethoprim and chloramphenicol. During therapy with a quinolone, resistance can emerge rapidly, especially when there is a large number of bacteria or a foreign body present. This risk may depend on the dosing schedule and may be reduced by combined therapy.

In-vitro activity of newer quinolones against aerobic bacteria.

Nalidixic and five newer 4-quinolones, ciprofloxacin, enoxacin, norfloxacin, ofloxacin and pefloxacin were tested against 576 recent clinical aerobic bacterial isolates. The 4-quinolones were regularly active (MIC90 less than 4 mg/l) against the following bacteria: Staphylococcus aureus, S. epidermidis, S. saprophyticus, different Enterobacteriaceae, Haemophilus influenzae, Campylobacter jejuni, Pseudomonas aeruginosa, Agrobacter spp., Aeromonas spp., Plesiomonas spp., Neisseria meningitidis. Other bacteria were usually intermediately susceptible or resistant: different streptococci, Listeria monocytogenes, Nocardia asteroides, P. maltophilia, Achromobacter xylosoxydans and Alcaligenes denitrificans. Ciprofloxacin was the most potent compound, followed by ofloxacin and pefloxacin, norfloxacin and enoxacin being less active. All the 4-quinolones were much more active than nalidixic acid. The MBC/MIC ratios of the 4-quinolones were between 1 and 2 with a majority of strains, and between 2 and 3 with Streptococcus agalactiae, Str. faecalis and L. monocytogenes. A two- to eight-fold increase of MIC was observed by increasing the inoculum 10,000-fold with most of the strains tested. Susceptible bacterial population of Klebsiella pneumoniae, Enterobacter cloacae, Serratia marcescens and P. aeruginosa contained more clones resistant to nalidixic acid (10(4) to 10(8) at four times the MIC) than to 4-quinolones (10(5) to 10(9) at four times the MIC). Supplementing the media with MgSO4 produced smaller inhibition zone diameters with a disc diffusion method than those obtained with non-supplemented agar, with all quinolone or strains. Less regular effect, or no effect was obtained after supplementation with ZnSO4 or Ca(NO3)2.