Antibacterial and anti-biofilm activities of melittin and colistin, alone and in combination with antibiotics against Gram-negative bacteria.

In vitro antibacterial and anti-biofilm activities of antimicrobial cationic peptides (AMPs) - melittin and colistin - both alone and in combination with antibiotics were evaluated against clinical isolates of Gram-negative bacteria. Minimum inhibitory concentration (MIC) and fractional inhibitory concentration (FIC) index were determined by the microbroth dilution and chequerboard techniques, respectively. The time-kill curve (TKC) method was used for determining the bactericidal activities of AMPs alone and in combination. Measurements of anti-biofilm activities were performed spectrophotometrically for both inhibition of attachment and 24-hour biofilm formation at MIC or subMIC. According to MIC90 values, the most active agents against Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae were colistin, imipenem and ciprofloxacin, respectively. In combination studies, synergistic effects were mostly seen with colistin-imipenem against E. coli and K. pneumoniae (50 and 54%, respectively), colistin-ciprofloxacin against P. aeruginosa (77%). In TKC studies, synergism was observed with almost all expected combinations, even more frequently than chequerboard method. All of the antimicrobial agents were able to inhibit attachment and 24-hour biofilm formation between 0-57% at 1/10 × MIC and 7-73% at 1 × or 1/10 × MIC, respectively. AMPs seem to be a good candidate for antimicrobial chemotherapy with their antibacterial and anti-biofilm activities as a single agent or in combination with antibiotics.

In vitro activities of antimicrobial cationic peptides; melittin and nisin, alone or in combination with antibiotics against Gram-positive bacteria.

The In vitro activities of two antimicrobial cationic peptides, melittin and nisin alone and in combination with frequently used antibiotics (daptomycin, vancomycin, linezolid, ampicillin, and erythromycin), were assessed against clinical isolates of methicillin-susceptible Staphylococcus aureus, methicillin-resistant S. aureus and Enterococcus faecalis. Using the broth microdilution method, minimum inhibitory concentration (MIC) ranges of melittin and nisin against all strains were 2-8 µg/ml and 2-32 µg/ml respectively. In combination studies performed with the microdilution checkerboard method using a fractional inhibitory concentration index of ≤ 0.5 as borderline, synergistic interactions occurred more frequently with nisin-ampicillin combination against MSSA and nisin-daptomycin combination against E. faecalis strains. The results of the time-killing curve analysis demonstrated that the concentration dependent rapid bactericidal activity of nisin, and that synergism or early synergism was detected in most strains when nisin or melittin was used in combination with antibiotics even at concentrations of 0.5 × MIC.

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 nisin alone or in combination with vancomycin and ciprofloxacin against methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains.

BACKGROUND: We investigated the in vitro activities of nisin alone or in combination with vancomycin and ciprofloxacin against methicillin-resistant (MRSA) and -susceptible Staphylococcus aureus (MSSA) strains. METHODS: The minimum inhibitory concentrations were determined by microbroth dilution technique. Antibiotic combinations were assessed using the checkerboard technique. The time-kill curve method was used for determining the bactericidal activity of nisin alone and in combination. RESULTS: For both MSSA and MRSA strains, the minimum inhibitory concentrations of nisin ranged between 4 and 16 mg/l. With a fractional inhibitory concentration of ≥0.5 as borderline, synergistic interactions were seen in three of five isolates with nisin-ciprofloxacin compared to two of five isolates with nisin-vancomycin combinations against both MSSA and MRSA. No antagonism was observed. The results of time-kill curve analysis demonstrated concentration-dependent rapid bactericidal activity of nisin and synergism almost in all strains when nisin was used in combination with ciprofloxacin, and early synergistic interactions in some of the strains when it was used in combination with vancomycin. CONCLUSION: Nisin seems to be a good candidate for further investigations in the treatment of Gram-positive bacteria, alone or in combination with antibiotics.

In vitro activities of various antibiotics, alone and in combination with colistin methanesulfonate, against Pseudomonas aeruginosa strains isolated from cystic fibrosis patients.

BACKGROUND: The in vitro activities of various antibiotics, either alone or in combination with colistin methanesulfonate, were assessed using Pseudomonas aeruginosa strains isolated from cystic fibrosis patients. METHODS: Except for colistin methanesulfonate, minimum inhibitory concentrations were determined by microbroth dilution technique as described by the Clinical and Laboratory Standards Institute (CLSI); for colistin methanesulfonate, a modified method of the CLSI was used. Minimum bactericidal concentrations were determined as described by the CLSI. The in vitro activities of antibiotics in combination were determined by microbroth checkerboard technique, and results were interpreted by fractional inhibitory concentration index. RESULTS: According to MIC values, 100, 98, 96 and 84% of the isolates were found susceptible to amikacin, colistin methanesulfonate, meropenem and ceftazidime, respectively. The minimum bactericidal concentrations were generally equal to or twice as high as those of the minimum inhibitory concentrations. With a fractional inhibitory concentration index of < or =0.5 as borderline, synergistic interactions were more frequent with combinations where amikacin was involved than with those with colistin methanesulfonate. No antagonism was observed. CONCLUSION: The findings of this study may play a useful role in selecting the appropriate combinations when a single agent is inadequate to treat cystic fibrosis patients with P. aeruginosa infections.

CFTR is a pattern recognition molecule that extracts Pseudomonas aeruginosa LPS from the outer membrane into epithelial cells and activates NF-kappa B translocation.

Immune cells are activated during cellular responses to antigen by two described mechanisms: (i) direct uptake of antigen and (ii) extraction and internalization of membrane components from antigen-presenting cells. Although endocytosis of microbial antigens by pattern recognition molecules (PRM) also activates innate immunity, it is not known whether this involves extraction and internalization of microbial surface components. Epithelial cells on mucosal surfaces use a variety of receptors that are distinct from the classical endocytic PRM to bind and internalize intact microorganisms. Nonclassical receptor molecules theoretically could act as a type of endocytic PRM if these molecules could recognize, bind, extract, and internalize a pathogen-associated molecule and initiate cell signaling. We report here that the interaction between the cystic fibrosis transmembrane conductance regulator (CFTR) and the outer core oligosaccharide of the lipopolysaccharide (LPS) in the outer membrane of Pseudomonas aeruginosa satisfies all of these conditions. P. aeruginosa LPS was specifically recognized and bound by CFTR, extracted from the organism's surface, and endocytosed by epithelial cells, leading to a rapid (5- to 15-min) and dynamic translocation of nuclear transcription factor NF-kappa B. Inhibition of epithelial cell internalization of P. aeruginosa LPS prevented NF-kappa B activation. Cellular activation depended on expression of wild-type CFTR, because both cultured Delta F508 CFTR human airway epithelial cells and lung epithelial cells of transgenic-CF mice failed to endocytose LPS and translocate NF-kappa B. CFTR serves as a critical endocytic PRM in the lung epithelium, coordinating the effective innate immune response to P. aeruginosa infection.

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.

In vivo studies on nasal preparations of ciprofloxacin hydrochloride.

Gel formulations of ciprofloxacin hydrochloride (CPH) were prepared with bioadhesive polymers such as hydroxypropyl methylcellulose (HPMC), hydroxyethyl cellulose (HEC) and methylcellulose (MC). They were administered into the nasal cavity of rabbits. A nasal aqueous suspension of CPH with glycerol was also applied. In addition, the effect of Tween 80 as penetration enhancer was examined. The agar plate diffusion technique was applied for the assay of CPH. The results were compared with oral and intravenous administrations. The bioavailability of the CPH gel formulation prepared with HPMC was almost identical to that of the oral route. Other nasal formulations with HEC and MC had bioavailabilities lower than oral preparations. The relative bioavailabilities for the formulation containing HEC and MC were 48.7 and 45.54%, respectively. To increase the bioavailabilities, 1% (w/w) of Tween 80 was added. The bioavailability of these gel formulations increased to 63.54 and 55.72%, respectively. Experiments carried out on rabbits showed that the nasal administration of CPH bioadhesive gel formulation containing HPMC may be an alternative to the oral route.

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.