In vitro pharmacodynamic properties of colistin methanesulfonate and amikacin against Pseudomonas aeruginosa.

PURPOSE: In vitro pharmacodynamic properties of colistin methanesulfonate and amikacin were investigated by studying time-kill kinetics and post-antibiotic effect (PAE) against strains of Pseudomonas aeruginosa isolated from patients with cystic fibrosis. METHOD: Synergy was investigated at 0.5 ×, 1 × and 5 × MIC of antibiotics using time-kill curve method. PAEs were determined by the standard viable counting method where bacteria in the logarithmic phase of growth were exposed for 1 h to the antibiotics at 1 × or 20 × MIC, alone and in combinations. Synergy and additive effects were detected at 1 × MIC, at 24 h. RESULTS: Some of the strains produced an earlier synergistic effect at 12 h. No antagonism was observed. Colistin methanesulfonate and amikacin produced PAEs 1.16 ± 0.10 to 2.25 ± 0.16 h and 0.96 ± 0.15 to 2.69 ± 0.32 h, respectively. When the antibiotics were used in combination the PAEs were prolonged to a value of 3.88 ± 0.25 h. Consequently, the CONCLUSIONS: Findings of this study may play useful role in selecting the appropriate combinations when a single agent is inadequate, and may have important information for optimizing the dose intervals.

In-vitro activities of various antibiotics, alone and in combination with amikacin against Pseudomonas aeruginosa.

The in-vitro activities of various antibiotics, either alone or in combination with amikacin were assessed using clinical isolates of Pseudomonas aeruginosa. The minimum inhibitory concentrations (MIC) of these antibiotics were determined by microbroth dilution method against 50 clinical strains. The MIC values showed that 96, 94, and 74% of the isolates were susceptible or moderately susceptible to amikacin, meropenem and ceftazidime, respectively. The in vitro activities of ceftazidime and meropenem in combination with amikacin were determined by microbroth chequerboard technique and results were interpreted using the fractional inhibitory concentration (FIC) index. With a FIC index of < or =0.5 as borderline, synergistic interactions were more frequent with ceftazidime (70.8%) than with meropenem (40%). No antagonism was observed.

Impact of heterogeneity within cultured cells on bacterial invasion: analysis of Pseudomonas aeruginosa and Salmonella enterica serovar typhi entry into MDCK cells by using a green fluorescent protein-labelled cystic fibrosis transmembrane conductance regulator receptor.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel that also serves as a receptor for entry of Pseudomonas aeruginosa and Salmonella enterica serovar Typhi into epithelial cells. To evaluate heterogeneity in CFTR protein expression in cultured cells and the effect of heterogeneity on internalization of different P. aeruginosa and serovar Typhi strains, we used two-color flow cytometry and confocal laser microscopy to study bacterial uptake by Madin-Darby canine kidney (MDCK) type I epithelial cells stably expressing a green fluorescent protein (GFP)-CFTR fusion construct (MDCK-GFP-CFTR cells). We found a strong correlation between cell size and GFP-CFTR protein expression, with 60 to 70% of cells expressing low levels of GFP-CFTR protein, 20 to 30% expressing intermediate levels, and <10% expressing high levels. The cells were sorted into low-, intermediate-, or high-level producers of CFTR protein; in vitro growth of each sorted population yielded the same distribution of CFTR protein expression as that in the original population. Cells expressing either low or high levels of CFTR protein internalized bacteria poorly; maximal bacterial uptake occurred in the cells expressing intermediate levels of CFTR protein. Treatment of MDCK cells with sodium butyrate markedly enhanced the production of CFTR protein without increasing cell size; butyrate treatment also increased the proportion of cells with internalized bacteria. However, there were fewer bacteria per butyrate-treated cell and, for P. aeruginosa, there was an overall decrease in the total level of bacterial uptake. The most highly ingested bacterial strains were internalized by fewer total MDCK-GFP-CFTR cells, indicating preferential bacterial uptake by a minority of epithelial cells within a given culture. Confocal fluorescence microscopy showed that P. aeruginosa and serovar Typhi induced cytoplasmic accumulation of CFTR protein close to the plasma membrane where the bacteria were adherent. These results show that within a population of MDCK-GFP-CFTR cells, there are cells with markedly different abilities to ingest bacteria via CFTR, the majority of the P. aeruginosa and serovar Typhi cells are ingested by the one-fourth to one-third of the cells that exhibit an intermediate size and level of CFTR protein expression, and overexpression of the CFTR receptor does not increase total bacterial uptake but rather allows more epithelial cells to ingest fewer total bacteria.

Production and characterization of a set of mouse-human chimeric immunoglobulin G (IgG) subclass and IgA monoclonal antibodies with identical variable regions specific for Pseudomonas aeruginosa serogroup O6 lipopolysaccharide.

The heavy- and light-chain variable regions from a murine monoclonal antibody that recognize Pseudomonas aeruginosa serogroup O6 lipopolysaccharide (LPS) were used to generate a series of chimeric mouse-human monoclonal antibodies with identical variable regions. The murine variable-region gene segments were cloned into an immunoglobulin (Ig) cDNA expression vector that contained the human kappa light-chain and IgG1 constant regions. The IgG1 heavy-chain constant region was then replaced with the human IgG2, IgG3, IgG4, or IgA1 heavy-chain constant region. The five different expression vectors were transfected into Chinese hamster ovary cells for antibody production. The chimeric antibodies exhibited immunoreactivity and affinity similar to that of the parental murine IgG antibody toward whole cells of a serogroup O6 strain. In vitro complement deposition assays demonstrated that the chimeric IgG4 and IgA antibodies did not mediate the deposition of complement component C3 onto the surface of either purified LPS or whole bacteria. The chimeric IgG1 and IgG3 antibodies were similar in their ability to deposit C3 onto the surface of both bacteria and LPS, while IgG2 antibody was more effective at depositing C3 onto the surface of bacteria than onto purified LPS. The pattern of opsonophagocytic activity of the chimeric monoclonal antibodies was similar to that of complement deposition onto bacterial cells in that the chimeric IgG1 and IgG3 had the highest opsonic activity. Although IgG2 deposited more C3 onto the bacterial surface than did IgG4 or IgA, all three of these isotypes had low opsonic activity against the serogroup O6 target strain. This series of related antibodies will help reveal functional differences in efficacy among protective antibodies to P. aeruginosa and will be critical for defining the optimal formulation of either a vaccine for active immunization or a polyclonal intravenous IgG or monoclonal antibody cocktail for passive immunotherapy.

Prophylactic and therapeutic efficacy of immunoglobulin G antibodies to Pseudomonas aeruginosa lipopolysaccharide against murine experimental corneal infection.

PURPOSE: To evaluate the efficacy of lipopolysaccharide (LPS)-specific antibodies administered prophylactically or therapeutically to protect against corneal challenge with Pseudomonas aeruginosa. METHODS: The prophylactic efficacy of active immunization with purified P. aeruginosa LPS was evaluated in a murine corneal-scratch model of P. aeruginosa keratitis. The same model was used to evaluate both the prophylactic and the therapeutic efficacy of systemic passive transfer of variable region-identical, isotype-switched, LPS-specific, murine immunoglobulin M (IgM) and immunoglobulin G (IgG) monoclonal antibodies (mAbs). The mAbs were injected intraperitoneally at various times either before or after corneal challenge and the corneal response was monitored macroscopically. In addition, immune rabbit sera were used to evaluate the efficacy of treatment. RESULTS: Active immunization with homologous, but not heterologous, LPS before challenge reduced the severity of corneal disease and protected challenged mice against permanent corneal damage. Passive transfer of the LPS-specific IgM mAb 1F6 before challenge did not prevent corneal damage at any dose tested and had no effect on the course of disease. However, results of dose-response studies of the passive transfer of a variable region-identical IgG2b mAb, 2H3, before challenge indicated a 50% protective dose of 11.8 micrograms. When mAb 2H3 was administered at a dose of 50 micrograms before challenge and the challenge inoculum was increased, all mice were protected from corneal damage up to a challenge inoculum of 2.2 x 10(8) CFU/eye. When given 2 or 4 hours after corneal challenge with P. aeruginosa strain 6294 (which invades corneal epithelial cells during infection) but not when given at 8 or 24 hours, 50 micrograms of mAb 2H3 conferred significant protection (P < 0.05). The maximal interval after challenge during which this antibody could be administered and still protect 50% of mice was calculated by probit analysis to be 9.4 hours. Administration of homologous LPS-specific rabbit antiserum to mice at various times after challenge with P. aeruginosa strain 6206 (which is cytotoxic to corneal epithelial cells and does not remain in these cells during infection) resulted in significant protection when administered 4 or 8 hours after infection. Although probit analysis could not be performed with the available data, 50% of mice were completely protected when the antiserum was given up to 24 hours after challenge. CONCLUSIONS: In an experimental model of P. aeruginosa keratitis, systematically delivered IgG antibodies directed against the O-side-chain antigens of P. aeruginosa, LPS conferred protection against severe corneal damage when administered both prophylactically and therapeutically.

Postantibiotic effect of imipenem, alone and in combination with amikacin, on Pseudomonas aeruginosa.

Imipenem and amikacin, alone and in combination, were investigated for their postantibiotic effect (PAE) on Pseudomonas aeruginosa. Four clinical strains of P. aeruginosa in the logarithmic phase of growth were exposed for 1 h to antibiotics, alone and in combination. Recovery periods of test cultures were evaluated after dilution using viable counting. Imipenem produced a PAE ranging from 0.7 to 1.55 h. Similar PAEs were induced by amikacin (ranging from 0.65 to 2 h). In combination, imipenem and amikacin produced as a final PAE (ranging from 1.6 to 2.65 h), a rough mathematical sum of the individual effects. The finding of this study may have important implications for the timing of doses during therapy with antimicrobial combinations.

In-vitro activities of various antibiotics, alone and in combination with amikacin against Pseudomonas aeruginosa.

The in-vitro activities of various antibiotics, either alone or in combination with amikacin were assessed in clinical isolates of Pseudomonas aeruginosa. Clinically relevant synergic interactions were most frequent with piperacillin (92%), ceftazidime (87%) and aztreonam (84%), and less frequent with imipenem (35%). Ciprofloxacin-amikacin showed only an additive effect.

Comparison of imipenem and five other antipseudomonal agents against gentamicin-susceptible and -resistant Pseudomonas aeruginosa.

The in vitro activities of imipenem, aztreonam, piperacillin, ciprofloxacin and amikacin were tested by the microbroth dilution technique against 86 clinical isolates of Pseudomonas aeruginosa. Imipenem and ciprofloxacin were the most active agents against gentamicin-susceptible P. aeruginosa. Only imipenem inhibited gentamicin-resistant P. aeruginosa at < or = 8 micrograms/ml. The finding that none of the gentamicin-resistant strains were resistant to imipenem and amikacin indicated the superiority of these antibiotics to the other agents in hospital-associated gentamicin-resistant P. aeruginosa infections.