bmap-28 and Staphylococcal-Infections

Antimicrobial peptides (AMPs) represent an important part of the innate host immune system. Although they are active against a broad range of pathogens, bacteria have evolved different resistance mechanisms to avoid killing by AMPs. Since not much is known about the impact of efflux pumps on the susceptibility of Gram-positive bacteria to AMPs, especially to the cathelicidins, the aim of this study was to analyze whether Staphylococcus aureus can use efflux pumps to resist the antimicrobial effects of cathelicidins derived from different animal species (human, mouse, rabbit or cattle). For this purpose the minimal inhibitory concentrations (MICs) of S. aureus field isolates for the cathelicidins LL-37, mCRAMP, CAP18, BMAP-27 and BMAP-28 in the presence and absence of different efflux pump inhibitors were determined. Furthermore, the MICs of mutants lacking SecDF, a member of the RND efflux pump family, were determined and compared to the MICs of their respective wildtype and complemented strains. The data demonstrated that after blocking RND-type efflux pumps with 1-(1-naphthylmethyl)-piperazine, the MICs for CAP18, but not those for the other cathelicidins tested, were significantly decreased. In good correlation with these data, significantly decreased MICs for CAP18 and also BMAP-27 have been observed for SecDF knockout mutants as compared to their isogenic wildtype strains. In addition, the MIC values increased again after re-introducing a cloned secDF via plasmid complementation. These results indicated an involvement of SecDF in a reduced efficacy of species-specific cathelicidins against S. aureus.

Topics: Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Bacterial Proteins; Cathelicidins; Cattle; Disease Susceptibility; Enzyme Inhibitors; Humans; Membrane Proteins; Membrane Transport Proteins; Methicillin-Resistant Staphylococcus aureus; Mice; Microbial Sensitivity Tests; Piperazines; Proteins; Rabbits; Staphylococcal Infections

Six different cathelicidin-derived peptides were compared to tobramycin for antibacterial and anti-biofilm effects against S. aureus, P. aeruginosa, and S. maltophilia strains isolated from cystic fibrosis patients. Overall, SMAP-29, BMAP-28, and BMAP-27 showed relevant antibacterial activity (MIC(50) 4-8μg/ml), and in some cases higher than tobramycin. In contrast, indolicidin, LL-37, and Bac7(1-35) showed no significant antimicrobial activity (MIC(50)>32μg/ml). Killing kinetics experiments showed that in contrast to tobramycin the active cathelicidin peptides exert a rapid bactericidal activity regardless of the species tested. All three peptides significantly reduced biofilm formation by S. maltophilia and P. aeruginosa strains at 1/2× MIC, although at a lower extent than tobramycin. In addition, BMAP-28, as well as tobramycin, was also active against S. aureus biofilm formation. Preformed biofilms were significantly affected by bactericidal SMAP-29, BMAP-27 and BMAP-28 concentrations, although at a lesser extent than tobramycin. Overall, our results indicate the potential of some cathelicidin-derived peptides for the development of novel therapeutic agents for cystic fibrosis lung disease.

Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Biofilms; Blood Proteins; Cathelicidins; Cattle; Cystic Fibrosis; Gram-Negative Bacterial Infections; Humans; Microbial Sensitivity Tests; Microscopy, Electron, Scanning; Molecular Sequence Data; Proteins; Pseudomonas aeruginosa; Sheep; Staphylococcal Infections; Staphylococcus aureus; Stenotrophomonas maltophilia; Tobramycin

An experimental study was performed to evaluate the efficacy of BMAP-28 alone and in combination with vancomycin in animal models ureteral stent infection due to Enterococcus faecalis and Staphylococcus aureus. Study included a control group without bacterial challenge to evaluate the sterility of surgical procedure, a challenged control group that did not receive any antibiotic prophylaxis and for each bacterial strain three challenged groups that received (a) 10 mg/kg vancomycin intraperitoneally, immediately after stent implantation, (b) BMAP-28-coated ureteral stents where 0.2-cm(2) sterile ureteral stents were incubated in 1mg/l BMAP-28 solution for 30 min immediately before implantation and (c) intraperitoneal vancomycin plus BMAP-28-coated ureteral stent at the above concentrations. Experiments were performed in duplicate. Ureteral stents were explanted at day 5 following implantation and biofilm bacteria enumerated. Our data showed that rats that received intraperitoneal vancomycin showed the lowest bacterial numbers. BMAP-28 combined with vancomycin showed efficacies higher than that of each single compound. These results highlight the potential usefulness of this combination in preventing ureteral stent-associated in gram-positive infections.

Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Biofilms; Disease Models, Animal; Drug Synergism; Drug Therapy, Combination; Female; Microbial Sensitivity Tests; Molecular Sequence Data; Molecular Weight; Proteins; Rats; Rats, Wistar; Staphylococcal Infections; Staphylococcus aureus; Stents; Ureter; Vancomycin

An in vitro antibiotic susceptibility assay for Staphylococcus aureus biofilms developed on 96-well polystyrene tissue culture plates was performed to elucidate the activity of the 27 residues cathelicidin peptide BMAP-28, quinupristin/dalfopristin (Q/D), linezolid, and vancomycin. Efficacy studies were performed in a rat model of staphylococcal CVC infection. Silastic catheters were implanted into the superior cava. Twenty-four hours after implantation the catheters were filled with BMAP-28. Thirty minutes later rats were challenged via the CVC with 1.0x10(6) CFU of S. aureus strain Smith diffuse. Administration of antibiotics into the CVC at a concentration equal to the MBC observed using adherent cells, or at a much higher concentration (1024 microg/mL) began 24 h later. The inhibition activities of all antibiotics against adherent bacteria were at least two-four-fold lower that against freely growing cells. When antibiotics were used in BMAP-28 pre-treated wells, they showed higher activities. The in vivo studies showed that when CVCs were pre-treated with BMAP-28 or with a high dose of antibiotics, biofilm bacterial load was reduced from 10(7) to 10(3) CFU/mL and bacteremia reduced from 10(3) to 10(1) CFU/mL. When CVCs were treated with both BMAP-28 and antibiotics, biofilm bacterial load was further decreased to 10(1) CFU/mL and bacteremia was not detected. These results suggest that CVC pre-treated with BMAP-28 represents an attractive choice for the treatment of device-related infections caused by staphylococci.

Topics: Animals; Anti-Bacterial Agents; Anti-Infective Agents; Antimicrobial Cationic Peptides; Cathelicidins; Catheterization, Central Venous; Disease Models, Animal; In Vitro Techniques; Male; Proteins; Rats; Rats, Wistar; Staphylococcal Infections; Staphylococcus aureus; Treatment Outcome

A mouse model of staphylococcal sepsis was used to evaluate the efficacy of RNAIII-inhibiting peptide (RIP) combined with the cathelicidin BMAP-28. Preliminary in vitro studies showed that both peptides, alone or combined, were able to inhibit the lipoteichoic acid-induced production of tumor necrosis factor alpha and nitric oxide by RAW 264.7 cells. For in vivo experiments, the main outcome measures were lethality, quantitative blood cultures, and detection of tumor necrosis factor alpha and interleukin 6 plasma levels. BALB/c mice were injected i.v. with 2.0 x 10(6) colony-forming units of live Staphylococcus aureus ATCC 25923 or with 5.0 x 10(8) heat-killed cells of the same strain. All animals were randomized to receive i.v. isotonic sodium chloride solution, 10-mg/kg RIP, alone or in combination with 2-mg/kg BMAP-28, 7-mg/kg imipenem, or 7-mg/kg vancomycin, immediately and at 6 hours after bacterial challenge. In in vivo experiments performed with live bacteria, all compounds reduced lethality rates and bacteremia when compared with controls. In general, combined-treated groups had significantly lower bacteremia when compared with single-treated groups. Lowest lethality rates and bacteremia were obtained when RIP was administered in combination with BMAP-28 or vancomycin. In the experiments performed using heat-killed organisms, only BMAP-28 demonstrated significant efficacy on lethality rates and cytokines plasma levels when compared with controls. RIP combined with BMAP-28 exhibited the highest efficacy on all main outcome measurements. These data were observed on both immediate and delayed treatments. These results highlight the capacity of RIP and BMAP-28 to reduce the septic effects of bacterial cell components and exotoxins, and suggest their potential use in the treatment of severe staphylococcus-associated sepsis.

Topics: Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Cell Line, Tumor; Disease Models, Animal; Drug Therapy, Combination; Interleukin-6; Lipopolysaccharides; Macrophages; Male; Mice; Mice, Inbred BALB C; Nitric Oxide; Oligopeptides; Proteins; Staphylococcal Infections; Staphylococcus aureus; Survival Analysis; Teichoic Acids; Tumor Necrosis Factor-alpha

A mouse model of staphylococcal sepsis was used to compare the efficacy of the bovine antimicrobial peptide BMAP-28, a compound of the cathelicidin family, with that of conventional antibiotics.. Prospective, randomized, controlled animal study.. Research laboratory in a university hospital.. BALB/c male mice.. BALB/c mice were injected intravenously with 2.0 x 10(6) colony-forming units of live Staphylococcus aureus ATCC 25923 or with 5.0 x 10(8) heat-killed cells of the same strain. All animals were randomized to receive intravenously isotonic sodium chloride solution, 2 mg/kg BMAP-28, 7 mg/kg imipenem, 7 mg/kg vancomycin, 7 mg/kg clindamycin, and 7 mg/kg clarithromycin immediately and at 6 hrs after bacterial challenge.. Lethality, quantitative blood cultures, and detection of tumor necrosis factor-alpha and interleukin-6 plasma levels. In the experiments performed with live bacteria, all compounds reduced lethality rates and bacterial growth compared with controls. Imipenem and vancomycin exhibited the highest efficacy on these main outcome measures. In the experiments performed using heat-killed organisms, only BMAP-28 demonstrated significant efficacy on lethality rates, tumor necrosis factor-alpha, and interleukin-6 plasma levels compared with controls.. These results highlight the capacity of BMAP-28 to reduce the effects of components of the bacterial cells and suggest that it may be beneficial in the treatment of severe staphylococcal infections in concert with other antimicrobial agents.

Topics: Analysis of Variance; Animals; Anti-Bacterial Agents; Disease Models, Animal; Drug Interactions; Enzyme-Linked Immunosorbent Assay; Interleukin-6; Male; Mice; Mice, Inbred BALB C; Probability; Proteins; Random Allocation; Sensitivity and Specificity; Shock, Septic; Staphylococcal Infections; Survival Rate; Treatment Outcome; Tumor Necrosis Factor-alpha

Topics: Animals; Bacteremia; Cells, Cultured; Cytokines; Disease Models, Animal; Macrophages; Mice; Nitric Oxide; Proteins; Risk Factors; Sensitivity and Specificity; Staphylococcal Infections