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The antimicrobial peptide distinctin consists of two peptide chains linked by a disulfide bridge; it presents a peculiar fold in water resulting from noncovalent dimerization of two heterodimeric molecules. To investigate the contribution of each peptide chain and the S-S bond to distinctin biochemical properties, different monomeric and homodimeric peptide analogues were synthesized and comparatively evaluated with respect to the native molecule. Our experiments demonstrate that the simultaneous occurrence of both peptide chains and the disulfide bond is essential for the formation of the quaternary structure of distinctin in aqueous media, able to resist protease action. In contrast, distinctin and monomeric and homodimeric analogues exhibited comparable antimicrobial activities, suggesting only a partial contribution of the S-S bond to peptide killing effectiveness. Relative bactericidal properties paralleled liposome permeabilization results, definitively demonstrating that microbial membranes are the main target of distinctin activity. Various biophysical experiments performed in membrane-mimicking media, before and after peptide addition, provided information about peptide secondary structure, lipid bilayer organization, and lipid-peptide orientation with respect to membrane surface. These data were instrumental in the generation of putative models of peptide-lipid supramolecular pore complexes.

To investigate the efficacy of piperacillin/tazobactam combined with indolicidin in the prevention of lethality in two rat models of polymicrobial peritonitis.Prospective, randomized, controlled animal study.Research laboratory in a university hospital.Adult male Wistar rats.Adult male Wistar rats were given an intraperitoneal injection of 1 mg of Escherichia coli 0111:B4 lipopolysaccharide or had intraabdominal sepsis induced by cecal ligation and puncture. For each model, all animals were randomized to receive isotonic sodium chloride solution intraperitoneally, 1 mg/kg indolicidin, 120 mg/kg piperacillin/tazobactam, and 1 mg/kg indolicidin combined with 120 mg/kg piperacillin/tazobactam. Each group included 20 animals.Main outcome measures were: bacterial growth in blood, peritoneum, spleen, liver, and mesenteric lymph nodes; endotoxin, interleukin-6, and tumor necrosis factor-alpha concentrations in plasma; and lethality. All compounds reduced significantly bacterial growth and lethality compared with saline treatment. Treatment with indolicidin resulted in significant decrease in plasma endotoxin and cytokine levels, whereas piperacillin/tazobactam exerted the opposite effect. The combination between indolicidin and piperacillin/tazobactam proved to be the most effective treatment in reducing all variables measured.Indolicidin may have potential therapeutic usefulness alone and when associated with piperacillin/tazobactam in polymicrobial peritonitis.

Multiple animal models exist for the study of biofilm infections and their inhibitors in vivo. The infection models described in this chapter range from the simple nematode-killing and amoeba-plate-killing assays, to models with more relevance to human disease like the pulmonary and cellulitis infection models in mice, the graft prosthesis, and the central venous catheter infection models in rats, and the endocarditis and osteomyelitis infection models in rabbits.

We investigated the effect of topical temporin A in the management of methicillin-resistant strain of Staphylococcus aureus (MRSA)-infected experimental surgical wounds in mice. The wound, cut through the panniculus carnosus of BALB/c mice, was inoculated with 5 × 10 7 colony-forming units of MRSA. Mice were treated with Allevyn, temporin A-soaked Allevyn, Allevyn and daily intraperitoneal teicoplanin (7 mg/kg), temporin A-soaked Allevyn and daily intraperitoneal teicoplanin. Main outcome measurements were: quantitative bacterial culture, histological examination with assessment of micro-vessel density and of vascular endothelial growth factor (VEGF) expression in tissue sections, and VEGF plasma levels alike. Treatment with temporin-A associated with teicoplanin injection significantly reduced bacterial load to 0.85 × 10 1 ± 0.1 × 10 1 CFU/ml. Histological examination showed that infected mice receiving temporin A-soaked Allevyn (with or without teicoplanin) had a higher degree of granulation tissue formation and collagen deposition compared to the other treated groups. A significant increase in serum VEGF expression was observed in mice receiving temporin A topically and temporin A topically associated with intraperitoneal teicoplanin. In conclusion our results demonstrated that temporin A is effective in the management of infected wounds, by a significant bacterial growth inhibition and acceleration of wound repair process.

Ureteral stents coated with the quorum-sensing inhibitor RNAIII-inhibiting peptide (RIP) were implanted in rat bladders and shown to suppress Staphylococcus aureus formation on the stent and in urine and was especially effective when combined with teicoplanin. Coating ureteral stents with RIP thus increases the efficacy of teicoplanin in preventing ureteral stent-associated staphylococcal infections.

We investigated the efficacy of Tachyplesin III alone or combined with piperacillin-tazobactam (TZP) to prevent biofilm formation in vitro and in a rat model of Pseudomonas aeruginosa ureteral stent infection. We have observed that in vitro TZP, in presence of Tachyplesin III, showed minimal inhibitory concentrations (MIC)s twofold and minimal bactericidal concentrations (MBC)s eightfold lower. The in vivo study showed that rats that received intraperitoneal TZP showed the lowest bacterial numbers. Tachyplesin III combined with TZP showed efficacies higher than that of each single compound. Coating ureteral stents with Tachyplesin III is able to inhibit bacterial growth up to 1,000 times.

Objective: To investigate the efficacy of rifampin and colistin in three experimental rat models of Pseudomonas aeruginosa sepsis. Design: Prospective, randomized, controlled animal study. Setting: Research laboratory in a university hospital. Subjects: Adult male Wistar rats. Interventions: Adult male Wistar rats were given a) an intraperitoneal injection of 1 mg of P. aeruginosa 10 lipopolysaccharide; b) 2 × 1010 colony-forming units of P. aeruginosa ATCC 27853; and c) 2 × 1010 colony-forming units of one clinically multiresistant strain of P. aeruginosa. For each model, all animals were randomized to receive intravenously isotonic sodium chloride solution, 10 mg/kg rifampin, 1 mg/kg colistin, and 10 mg/kg rifampin plus 1 mg/kg colistin. Measurements and Main Results: Lethality, bacterial growth in blood and peritoneum, and endotoxin and tumor necrosis factor-α concentrations in plasma were measured. Colistin exerted a strong antimicrobial activity and achieved a significant reduction of plasma endotoxin and tumor necrosis factor-α concentration compared with control and rifampin-treated groups. Rifampin exhibited no antimicrobial activity with no substantial impact on endotoxin and tumor necrosis factor-α plasma concentrations. The combination of colistin and rifampin resulted in a significant reduction in bacterial count compared with colistin monotherapy, whereas no significant difference was found in positive hem cultures and mortality rates between the two groups. Conclusions: Colistin and rifampin might have a role in the therapy of multiresistant P. aeruginosa infection.

Yeast-like fungi are the most common cause of fungal infections in humans. Actually, in the age of opportunistic infections and increasing resistance, development of modern antifungal agents becomes a very important challenge. This paper describes synthesis and antimicrobial assay of four naturally occurring peptide antibiotics (aurein 1.2, citropin 1.1, temporin A, uperin 3.6) and three chemically engineered analogues actually passing clinical trials (iseganan, pexiganan, omiganan) against Candida strains isolated from patients with infections of the oral cavity or respiratory tract. The peptides were synthesized using solid-phase method and purified by high-performance liquid chromatography. Biological tests were performed using the broth microdilution method. The antifungal activity of the peptide antibiotics was compared to that of nystatin and amphotericin B. We found synthetic peptides to be generally less potent than amphotericin B or nystatin. However, some of the naturally occurring peptides still retained reasonable antifungal activities which were higher than these of iseganan, pexiganan or omiganan. We think that the naturally occurring peptide antibiotics included in our study can be a good matrix for development of novel antifungal compounds.

An experimental study has been performed to compare the in vitro activity and the in vivo efficacy of tachyplesin III, colistin, and imipenem against a multiresistant Pseudomonas aeruginosa strain. In vitro experiments included MIC determination, time-kill, and synergy studies. For in vivo studies, a mouse model of sepsis has been used. The main outcome measures were bacterial lethality, quantitative blood cultures, and plasma levels of lipopolysaccharide, tumor necrosis factor alpha, and interleukin-6. The combination of tachyplesin III or colistin with imipenem showed in vitro synergistic interaction. A significant increase in efficacy was also observed in vivo: combination-treated groups had significantly lower levels of bacteremia than did groups treated with a single agent. Tachyplesin III combined with imipenem exhibited the highest efficacy on all main outcome measurements. These results highlight the potential usefulness of these combinations and provide therapeutic alternatives for serious infections caused by gram-negative bacteria in the coming years.

The quorum-sensing inhibitor RIP inhibits staphylococcal TRAP/ agr systems and both TRAP- and agr -negative strains are deficient in biofilm formation in vivo, indicating the importance of quorum sensing to biofilms in the host. RIP injected systemically into rats has been found to have strong activity in preventing methicillin-resistant Staphylococcus aureus graft infections, suggesting that RIP can be used as a therapeutic agent.

Cyclospora is a coccidian, previously referred to as a cyanobacterium-like body, with an epidemiology similar to that of Cryptosporidium parvum. In recent years, several studies have shown that Cyclospora is not a rare opportunistic pathogen but rather is the cause of common, worldwide intestinal infections in healthy adults and children. Previous reports of diarrhea resulting from Cyclospora sp. have been linked to travelers and immunocompromised patients. The species has a worldwide distribution. Although Cyclospora infection has been reported from Southeast Asia, Papua New Guinea, Indonesia, India, Pakistan, Nepal, the Middle East, North Africa, the United Kingdom, the Caribbean, the United States, Central America, and South America, the true prevalence of this parasite in any population is unknown. The parasite is associated with prolonged self-limiting and relapsing watery diarrhea, anorexia, fatigue, and sometimes myalgia. Fever is infrequent.

The in vitro activities of the lipopeptides palmitoyl (Pal)-Lys-Lys-NH 2 and Pal-Lys-Lys against gram-positive cocci were investigated. Enterococci and streptococci demonstrated higher susceptibilities than staphylococci and Rhodococcus equi . A positive interaction was shown when the lipopeptides were combined with beta-lactams and vancomycin. These results suggest that lipopeptides are promising candidates for antimicrobial therapy for infections caused by gram-positive organisms.

The in vitro activity of the cathelicidin tritrpticin was investigated against multidrug-resistant Pseudomonas aeruginosa . The isolates were susceptible to the peptide at concentrations of 0.50 to 8 mg/liter. Tritrpticin completely inhibits lipopolysaccharide procoagulant activity at a 10 μM concentration. Fractionary inhibitory concentration indexes (0.385, 0.312, and 0.458) demonstrated synergy between the peptide and β-lactams.

Sepsis remains a major cause of morbidity and mortality in hospitalized patients, despite intense efforts to improve survival. The primary lead for septic shock results from activation of host effector cells by endotoxin, the lipopolysaccharide (LPS) associated with cell membranes of gram-negative bacteria. For these reasons, the quest for compounds with antiendotoxin properties is actively pursued. We investigated the efficacy of the amphibian skin antimicrobial peptide temporin L in bindingEscherichia coliLPS in vitro and counteracting its effects in vivo. Temporin L strongly bound to purifiedE. coliLPS and lipid A in vitro, as proven by fluorescent displacement assay, and readily penetrated intoE. coliLPS monolayers. Furthermore, the killing activity of temporin L againstE. coliwas progressively inhibited by increasing concentrations of LPS added to the medium, further confirming the peptide's affinity for endotoxin. Antimicrobial assays showed that temporin L interacted synergistically with the clinically used β-lactam antibiotics piperacillin and imipenem. Therefore, we characterized the activity of temporin L when combined with imipenem and piperacillin in the prevention of lethality in two rat models of septic shock, measuring bacterial growth in blood and intra-abdominal fluid, endotoxin and tumor necrosis factor alpha (TNF-α) concentrations in plasma, and lethality. With respect to controls and single-drug treatments, the simultaneous administration of temporin L and β-lactams produced the highest antimicrobial activities and the strongest reduction in plasma endotoxin and TNF-α levels, resulting in the highest survival rates.

An experimental study was designed to investigate the efficacy of BMAP-27, a compound of the cathelicidin family, in neutralizing Escherichia coli 0111:B4 lipopolysaccharide (LPS) in bile duct-ligated mice. Main outcome measures were: endotoxin and TNF-alpha concentrations in plasma, evidence of bacterial translocation in blood and peritoneum, and lethality. Adult male BALB/c mice were injected intraperitoneally with 2 mg/kg E. coli 0111:B4 LPS 1 week after sham operation or bile duct ligation (BDL). Six groups were studied: sham with placebo, sham with 120 mg/kg tazobactam-piperacillin (TZP), sham with 1 mg/kg BMAP-27, BDL with placebo, BDL with 120 mg/kg TZP, and BDL with 1mg/kg BMAP-27. After LPS, TNF-alpha plasma levels were significantly higher in BDL mice compared to sham-operated animals. BMAP-27 achieved a significant reduction of plasma endotoxin and TNF-alpha concentration when compared with placebo- and TZP-treated groups. On the other hand, both TZP and BMAP-27 significantly reduced the bacterial growth compared with saline treatment. Finally, LPS induced 60% and 55% lethality in BDL placebo- and TZP-treated treated mice and no lethality in sham-operated mice, while only BMAP-27 significantly reduced the lethality to 10%. In light of its dual antimicrobial and anti-endotoxin properties, BMAP-27 could be an interesting compound to inhibit bacterial translocation and endotoxin release in obstructive jaundice.

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.

We investigated the efficacy of LL-37, the C-terminal part of the only cathelicidin in humans identified to date (termed human cationic antimicrobial protein), in three experimental rat models of gram-negative sepsis. Adult male Wistar rats (i) were given an intraperitoneal injection of 1 mg Escherichia coli 0111:B4 LPS, (ii) were given 2 × 10 10 CFU of Escherichia coli ATCC 25922, or (iii) had intra-abdominal sepsis induced via cecal ligation and puncture. For each model, all animals were randomized to receive intravenously isotonic sodium chloride solution, 1-mg/kg LL-37, 1-mg/kg polymyxin B, 20-mg/kg imipenem, or 60-mg/kg piperacillin. Lethality; growth of bacteria in blood, peritoneum, spleen, liver, and mesenteric lymph nodes; and endotoxin and tumor necrosis factor alpha (TNF-α) concentrations in plasma were evaluated. All compounds reduced lethality compared to levels in controls. Endotoxin and TNF-α plasma levels were significantly higher in conventional antibiotic-treated rats than in LL-37- and polymyxin B-treated animals. All drugs tested significantly reduced bacterial growth compared to saline treatment. No statistically significant differences between LL-37 and polymyxin B were noted for antimicrobial and antiendotoxin activities. LL-37 and imipenem proved to be the most effective treatments in reducing all variables measured. Due to its multifunctional properties, LL-37 may become an important future consideration for the treatment of sepsis.

We performed in vitro studies to elucidate the bactericidal activity of the antibiotics in an adherent-cell biofilm model. Efficacy studies were performed in a staphylococcal central venous catheter (CVC) infection rat model. Silastic catheters were implanted into the superior cava. Via the CVC the rats were challenged with 1.0 × 10 6 CFU of a live Staphylococcus aureus strain. Twenty-four hours later, the antibiotic-lock technique was started. All animals were randomized to receive daily isotonic sodium chloride solution, quinupristin-dalfopristin (Q/D), linezolid, vancomycin, or ciprofloxacin at the minimal bactericidal concentration (MBC) and at 1,024 μg/ml in a volume of 0.1 ml that filled the CVC. The main outcome measures were MICs and MBCs for both planktonic and adherent cells, quantitative culture of the catheters and surrounding venous tissues, and quantitative peripheral blood cultures. The killing activities of all antibiotics against the adherent bacteria were at least fourfold lower than those against freely growing cells, with the exception of Q/D, which showed comparable activities against both adherent and planktonic organisms. Overall, Q/D at 1,024 μg/ml produced the greatest reduction in the number of cells recovered from the catheters, while at the same concentration, Q/D and vancomycin demonstrated higher activities than ciprofloxacin or linezolid in reducing the number of organisms recovered from the blood cultures. This study points out that treatment outcome of device-related infections cannot be predicted by the results of a standard susceptibility test such as the MIC. Our findings suggest that the clinically used antibiotics cannot eradicate the CVC infection through the antibiotic-lock technique, even at a concentration of 1,024 μg/ml.

The in vitro activity of uperin 3.6, alone or combined with six antibiotics, against gram-positive cocci, including Rhodococcus equi , methicillin-resistant staphylococci, and vancomycin-resistant enterococci, was investigated. All isolates were inhibited at concentrations of 1 to 16 mg/liter. Synergy was demonstrated when uperin 3.6 was combined with clarithromycin and doxycycline.

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 citropin 1.1, rifampin and minocycline. 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 citropin 1.1 (10 microg/mL). Thirty minutes later the rats were challenged via the CVC with 1.0 x 10(6) CFU of S. aureus strain Smith diffuse. Administration of antibiotics into the CVC (the antibiotic lock technique) began 24 h later. The study included: one control group (no CVC infection), one contaminated group that did not receive any antibiotic prophylaxis, one contaminated group that received citropin 1.1-treated CVC, two contaminated groups that received citropin 1.1-treated CVC plus rifampin and minocycline at concentrations equal to MBCs for adherent cells and 1024 microg/mL in a volume of 0.1 mL that filled the CVC and two contaminated groups that received rifampin or minocycline at the same concentrations. All catheters were explanted 7 days after implantation. Main outcome measures were: minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), synergy studies, quantitative culture of the biofilm formed on the catheters and surrounding venous tissues, and quantitative peripheral blood cultures. MICs of conventional antibiotics against the bacteria in a biofilm were at least four-fold higher than against the freely growing planktonic cells. In contrast, when antibiotics were used on citropin 1.1 pre-treated cells they showed comparable activity against both biofilm and planktonic organisms. The in vivo studies show that when CVCs were pre-treated with citropin 1.1 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 both with citropin 1.1 and antibiotics, biofilm bacterial load was further reduced to 10(1) CFU/mL and bacteremia was not detected, suggesting 100% elimination of bacteremia and a log 6 reduction in biofilm load. Citropin 1.1 significantly reduces bacterial load and enhances the effect of hydrophobic antibiotics in the treatment of CVC-associated S. aureus infections.

Background Medical devices used in clinical practice are often associated with biofilm-associated staphylococcal infections. Methods An in vitro antibiotic susceptibility assay of Staphylococcus aureus biofilms using 96-well polystyrene tissue-culture plates was performed to test the effects of RNAIII-inhibiting peptide (RIP), ciprofloxacin, imipenem, and vancomycin. Efficacy studies were performed using a rat model of central venous catheter (CVC)-associated infection. Twenty-four hours after implantation, the catheters were filled with RIP (1 mg/mL). Thirty minutes later, rats were challenged, via the CVC, with 1.0 x 10(6) cfu of S. aureus strain Smith diffuse. The antibiotic-lock technique was begun 24 h later. Results Minimum inhibitory concentrations of antibiotics in biofilms were at least 4-fold higher than those against the freely growing planktonic cells. When they were first treated with RIP, the cells in biofilms became as susceptible to antibiotics as did planktonic cells. These data were confirmed by the in vivo studies. In particular, when CVCs were treated with both RIP and antibiotics, the biofilm bacterial load was further reduced to 1 x 10(1) cfu/mL, and bacteremia was not detected, suggesting that there was 100% elimination of bacteremia and a 6 log10 reduction in biofilm bacterial load. Conclusion RIP significantly reduces bacterial load and enhances the effect of antibiotics in the treatment of CVC-associated S. aureus infections.

Objective: To study the in vitro activity of temporin A, a basic, highly hydrophobic, antimicrobial peptide amide derived from the skin of the European red frog Rana temporaria, alone and in combination with co-amoxiclav, imipenem, ciprofloxacin, linezolid and vancomycin, against 42 nosocomial isolates of Enterococcus faecalis. Fourteen of these were resistant to vancomycin. Methods: Antimicrobial activity of temporin A was measured by MIC, MBC and time-kill studies and by the chequerboard titration method. Results: All isolates were inhibited at concentrations of 1 to 16 mg/L. Combination studies carried out with E. faecalis ATCC 29212 and ATCC 51299 demonstrated synergy only when the peptide was combined with co-amoxiclav and imipenem. Conclusions: Our findings show that temporin A is active against E. faecalis and that its activity could be enhanced when it is combined with other antimicrobial agents.

The in vitro activities of three amphibian peptides magainin II amide, citropin 1.1 and temporin A alone and in combination with eight clinically used antimicrobial agents (imipenem, ceftazidime, clarithromycin, vancomycin, amikacin, polymyxin E, ciprofloxacin and linezolid) were investigated against several multidrug-resistant Pseudomonas aeruginosa and Staphylococcus aureus strains isolated from surgical wound infections. Antimicrobial activities were measured by MIC, MBC and time-kill studies. P. aeruginosa strains were more susceptible to magainin II amide and less susceptible to temporin A. S. aureus isolates were highly susceptible to temporin A and citropin 1.1. The combination studies showed synergy between citropin 1.1 and clarithromycin. Magainin II amide and temporin A showed synergism with imipenem and ceftazidime. Finally, all peptides showed synergistic effects with polymyxin E. These results provide evidence for the potential use of these antimicrobial peptides in the topical or systemic treatment of surgical wound infections.

Objective 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. Design Prospective, randomized, controlled animal study. Setting Research laboratory in a university hospital. Subjects BALB/c male mice. Interventions 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. Measurements and main results 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. Conclusion 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.

Background Morbidity and mortality from staphylococcal toxic shock remain high, despite the availability of antibiotics to which the microorganism is sensitive. Methods In in vitro experiments, the ability of temporin A to inhibit lipoteichoic acid-induced production of tumor necrosis factor (TNF)- alpha and nitric oxide (NO) was determined. Also, mouse models of staphylococcal sepsis were used to evaluate the efficacy of temporin A alone and in combination with imipenem. BALB/c mice were injected intravenously with live Staphylococcus aureus or heat-killed cells and then received either isotonic sodium chloride solution, 2 mg/kg temporin A, 7 mg/kg imipenem, or 2 mg/kg temporin A in combination with 7 mg/kg imipenem immediately and 6 h after challenge. The main outcome measures were lethality rates, plasma bacterial counts, and plasma TNF- alpha and interleukin (IL)-6 levels. Results The in vitro experiments showed that temporin A did not cause TNF- alpha or NO release. In the in vivo experiments with live bacteria, both compounds reduced lethality rates and bacterial growth. Imipenem exhibited the highest efficacy. The combination-treated group had significantly lower bacterial counts than did the singly-treated groups and the lowest lethality rates. In the experiments with heat-killed cells, only temporin A demonstrated significant efficacy with respect to lethality and reduction of plasma TNF- alpha and IL-6 levels. Discussion This study shows that temporin A can reduce the stimulatory effects of bacterial cell components and suggests that it may be beneficial in the treatment of severe staphylococcal sepsis.

OBJECTIVES This study was designed to investigate the antimicrobial and anti-endotoxin activity of MSI-78, a synthetic cationic peptide analogue of magainin 2. METHODS The in vitro antimicrobial activity of MSI-78 was investigated against the commercially available quality control strain Escherichia coli ATCC 25922. In addition, three rat models of septic shock were investigated: (i) rats were injected intraperitoneally with 1 mg Escherichia coli 0111:B4 LPS; (ii) rats were given an intraperitoneal injection of 2 x 10(10) cfu of Escherichia coli ATCC 25922; (iii) intra-abdominal sepsis was induced via caecal ligation and puncture. All animals were randomized to receive after 360 min intravenously isotonic sodium chloride solution, 1 mg/kg MSI-78, or 60 mg/kg piperacillin. Main outcome measures were: abdominal exudate and plasma bacterial growth, plasma endotoxin and tumour necrosis factor alpha (TNF-alpha) concentrations, and lethality. RESULTS Our in vitro data showed that MSI-78 possesses a strong activity against Escherichia coli. The in vivo studies showed that all compounds reduced the lethality when compared to controls. MSI-78 showed a slightly higher antimicrobial activity than piperacillin and achieved a substantial decrease in endotoxin and TNF-alpha plasma concentrations than the beta-lactam. CONCLUSIONS Because of its strong double anti-endotoxin and antimicrobial activities MSI-78 could be an interesting compound for Gram-negative septic shock treatment.

Staphylococcal bacteria are a prevalent cause of infections associated with foreign bodies and indwelling medical devices. Bacteria are capable of escaping antibiotic treatment through encapsulation into biofilms. RNA III-inhibiting peptide (RIP) is a heptapeptide that inhibits staphylococcal biofilm formation by obstructing quorum-sensing mechanisms. K 4 -S4(1-13) a is a 13-residue dermaseptin derivative (DD 13 ) believed to kill bacteria via membrane disruption. We tested each of these peptides as well as a hybrid construct, DD 13 -RIP, for their ability to inhibit bacterial proliferation and suppress quorum sensing in vitro and for their efficacy in preventing staphylococcal infection in a rat graft infection model with methicillin-resistant Staphylococcus aureus (MRSA) or S. epidermidis (MRSE). In vitro, proliferation assays demonstrated that RIP had no inhibitory effect, while DD 13 -RIP and DD 13 were equally effective, and that the chimeric peptide but not DD 13 was slightly more effective than RIP in inhibiting RNA III synthesis, a regulatory RNA molecule important for staphylococcal pathogenesis. In vivo, the three peptides reduced graft-associated bacterial load in a dose-dependent manner, but the hybrid peptide was most potent in totally preventing staphylococcal infections at the lowest dose. In addition, each of the peptides acted synergistically with antibiotics. The data indicate that RIP and DD 13 act in synergy by attacking bacteria simultaneously by two different mechanisms. Such a chimeric peptide may be useful for coating medical devices to prevent drug-resistant staphylococcal infections.

Sepsis remains a serious clinical problem despite intense efforts to improve survival. It is a major cause of morbidity and mortality in hospitalized patients and all immunocompromised subjects.1–4 The lipopolysaccharide (LPS), composed of an O-polysaccharide chain, a core sugar, and a lipophilic fatty acid (lipid A), is one of the most important bacterial components involved in the pathogenesis of Gram-negative infection activating the host effector cells through stimulation of receptors on their surface.1,5 These target cells secrete large quantities of inflammatory cytokines, such as tumor necrosis factor (TNF), interleukin (IL)-1, IL-6, and IL-8, platelet-activating factor, arachidonic acid metabolites, erythropoietin, and endothelin.5–7 The underlying principles of therapy have remained unchanged for decades. They are based on prompt institution of antimicrobial agents aimed at the inciting pathogen, source control directed at removal of the infection nidus whenever possible, and support of organ dysfunction.8 Nevertheless, several studies have indicated that exposure of Gram-negative organisms to antibacterial agents can result in endotoxin release. However, even though some observations have suggested that this phenomenon could have deleterious effects, its clinical relevance has not been well defined.9–11 To date, the list of potential therapeutic targets has been growing, and many compounds have been used to treat septic shock: monoclonal antibodies to endotoxin, IL-1 receptor antagonists, antioxidants, and various antiinflammatory therapies; despite these advances, the mortality from sepsis-related diseases have remained substantially unchanged.1–4,12 Antimicrobial peptides are positively charged molecules isolated from a wide variety of animals and plants. Most of these molecules, natural or synthetic, are amphipathic and have a broad spectrum of activity against bacteria, fungi, and protozoa; in addition, in recent years they received increasing attention because they possess anti-endotoxin activity.13–19 In fact, polycationic peptides bind to the negatively charged residues of LPS of the outer membrane by electrostatic interactions involving the negatively charged phosphoryl-groups and by hydrophobic interactions involving the acyl chains of lipid A. This binding is the key mechanistic step in the killing of Gram-negative organisms and endotoxin inactivation.19 Among these compounds, cecropin B is a positively charged peptide that was originally isolated from the hemolymph of the giant silk moth. Similarly to other polycationic peptides, it possesses 2 important activities: broad antimicrobial spectrum and anti-endotoxin activity.17–19 The present experimental study was performed to assess the potential therapeutic role of cecropin B alone and combined with betalactams not only in treating Gram-negative infections but also in neutralizing the biologic effect of the endotoxin.

The present study was designed to investigate the antiendotoxin activity and therapeutic efficacy of sheep myeloid antimicrobial peptide (SMAP)-29, a cathelicidin-derived peptide. The in vitro ability of SMAP-29 to bind LPS from Escherichia coli 0111:B4 was determined using a sensitive limulus chromogenic assay. Two rat models of septic shock were performed: (1) rats were injected intraperitoneally with 1 mg E. coli 0111:B4 LPS and (2) intraabdominal sepsis was induced via cecal ligation and single puncture. All animals were randomized to receive parenterally isotonic sodium chloride solution, 1 mg/kg SMAP-29, 1 mg/kg polymyxin B or 20 mg/kg imipenem. The main outcome measures were: abdominal exudate and plasma bacterial growth, plasma endotoxin and tumor necrosis factor-alpha concentrations, and lethality. The in vitro study showed that SMAP-29 completely inhibited the LPS procoagulant activity at approximately 10 microM peptide concentration. The in vivo experiments showed that all compounds reduced the lethality when compared with control animals. SMAP-29 achieved a substantial decrease in endotoxin and tumor necrosis factor-alpha plasma concentrations when compared with imipenem and saline treatment and exhibited a slightly lower antimicrobial activity than imipenem. No statistically significant differences were noted between SMAP-29 and polymyxin B. SMAP-29, because of its double antiendotoxin and antimicrobial activities, could be an interesting compound for septic shock treatment.

A rat model was used to investigate the efficacy of linezolid, alone or in combination with levofloxacin and vancomycin, in the prevention of vascular prosthetic graft infection resulting from methicillin-resistant Staphylococcus epidermidis with intermediate resistance to glycopeptides.Graft infections were established in the subcutaneous tissue of adult male Wistar rats by implantation of Dacron prostheses, followed by topical inoculation with S. epidermidis. The study comprised: one group without inoculation; one inoculated group without prophylaxis; six inoculated groups that received intraperitoneal linezolid (8 mg/kg), levofloxacin (7 mg/kg) or vancomycin (7 mg/kg) alone or in combination at the dosages mentioned above. Each group included 20 animals. The grafts were removed after 7 days and evaluated by quantitative culture.Quantitative graft cultures from animals treated with a single drug showed a significant efficacy only for linezolid. The efficacy of levofloxacin was similar to that of vancomycin. Combination studies demonstrated that only the treatments that included linezolid produced no evidence of staphylococcal infection.Linezolid as perioperative prophylaxis can be useful for the prevention of graft infections caused by multiresistant staphylococcal strains.

The prevalence of intestinal protozoans and helminths in stool samples of individuals with allergic cutaneous symptoms was evaluated to study a possible link between parasites and allergy. Altogether, 218 patients who had chronic urticaria, atopic dermatitis, or pruritus of unknown origin were included in the study. Standard laboratory tests for the detection of allergic etiology were performed for all patients. The presence of intestinal parasites was investigated using microscopy, immunofluorescence, and immunoenzymatic assays. Overall, protozoans and helminths were recovered from the stools of 48 subjects (P = 0.004), 18 of whom were affected with intestinal symptoms (P = 0.023). The presence of Giardia lamblia in the stools was significantly associated with allergic cutaneous manifestations (P = 0.030). In addition, patients with allergy were significantly more likely to haveor = 5 Blastocystis hominis organisms per field (P = 0.046). There was a set of patients with allergic cutaneous diseases in whom the presence of intestinal parasites may not be incidental.

Staphylococcus aureus and S. epidermidis are major causes of infection related to biofilm formed on indwelling medical devices. Such infections are common causes of morbidity and mortality and, because of biofilm resistance to antibiotics, are difficult to treat. The RNAIII-inhibiting peptide (RIP) (YSPWTNF-NH2) inhibits the pathogenesis of staphylococci by disrupting bacterial cell-cell communication (known as "quorum sensing"). Using a vascular-graft rat model, we show that RIP, applied locally and systemically, can completely inhibit drug-resistant S. aureus and S. epidermidis biofilms. The present study provides the first direct demonstration that interfering with cell-cell communication by use of a quorum-sensing inhibitor can eliminate medical device-associated staphylococcal infections. We suggest that medical devices could be coated with RIP to prevent infections, including those by antibiotic-resistant staphylococcal strains.

The therapeutic efficacy of protegrin peptide IB-367 was investigated in three rat models of septic shock: (i) rats injected intraperitoneally with 1 mg Escherichia coli 0111:B4 lipopolysaccharide, (ii) rats given an intraperitoneal injection of 2×10 10 CFU of E. coli ATCC 25922, and (iii) rats in which intra-abdominal sepsis was induced via cecal ligation and puncture. All animals were randomized to receive parenterally isotonic sodium chloride solution, 1 mg/kg of IB-367, 60 mg/kg piperacillin and 1 mg/kg of IB-367 plus 60 mg/kg piperacillin. The peptide demonstrated lower level of antimicrobial activity than piperacillin, nevertheless it exhibited the dual properties of antimicrobial and antiendotoxin agent. Finally IB-367 and piperacillin association showed to be the most effective therapeutic approach.

An animal study was performed to investigate the efficacy of two glycopeptides and two cationic peptides in the prevention of lethality in a septic shock rat model. Adult Wistar rats were given an intraperitoneal injection of 2x10(10) CFU of Escherichia coli ATCC 25922, with the exception of an uninfected control group (C0). Animals were randomized to receive, immediately after bacterial challenge, intraperitoneally isotonic sodium chloride solution (control group C1), 3 mg/Kg teicoplanin (group 1), 7 mg/Kg vancomycin (group 2), 1 mg/Kg colistin (group 3), 1 mg/Kg buforin II (group 4), or 60 mg/Kg piperacillin (group C(PIP)). In addition, four groups (1a, 2a, 3a, and 4a) received the above mentioned drugs in combination with piperacillin. All compounds and combinations significantly reduced the lethality and the number of E. coli in abdominal fluid compared with C1 group, with the exception of the glycopeptides. Colistin and buforin II combined with piperacillin significantly decreased the lethality compared with piperacillin alone. Finally, colistin, buforin II, and teicoplanin significantly reduced plasma endotoxin concentration in comparison with piperacillin and saline treatment. Antimicrobial peptides and teicoplanin act as antiendotoxin agents and enhance the efficacy of piperacillin.

Two laboratory methods, a cell culture system and double fluorogenic staining, were used to study the viability and infective ability of Cryptosporidium parvum sporozoites and oocysts after short-term exposure to four cathelicidin peptides. The compounds, SMAP-29, BMAP-28, PG-1 and Bac7(1-35), exerted a strong cytotoxic effect on sporozoites, but did not affect the viability and function of oocysts consistently. Overall, in the sporozoite series, a percentage of the viable population decreased rapidly to less than detectable levels after 15 and 60 min exposure to the peptides at concentrations of 100 and 10 micro g/mL, respectively. In the oocyst series, no compound produced complete inhibition of parasite growth: 60-85% of the oocyst population was viable after 180 min exposure at 100 micro g/mL. SMAP-29 exerted the highest activity against both sporozoites and oocysts.

A rat model was used to investigate the efficacy of quinupristin-dalfopristin (Q-D) in the prevention of vascular prosthetic graft infection due to methicillin-resistant Staphylococcus epidermidis with intermediate resistance to glycopeptides. The in vitro activity of the compound was compared to that of vancomycin by MIC determination and time-kill study. Moreover, the efficacy of collagen-sealed Q-D-soaked Dacron was evaluated in a rat model of graft infection. Graft infections were established in the subcutaneous tissue of the backs of 120 adult male Wistar rats. The in vivo study included a control group, one contaminated group that did not receive any antibiotic prophylaxis, two contaminated groups that received grafts soaked with 10 and 100 μg of Q-D per ml, respectively, and two contaminated groups that received grafts soaked with 10 and 100 μg of vancomycin per ml, respectively. Rats that received Dacron grafts soaked with 100 μg of Q-D per ml showed no evidence of infection (2 ± 1.3 × 10 2 , 2.2 × 10 6 ± 1.9 × 10 5 , and 5.6 × 10 2 ± 0.3 × 10 2 CFU/ml, respectively. Taken together the results of the study demonstrate that the use of Dacron grafts soaked with Q-D can result in significant bacterial growth inhibition and show that this compound is potentially valuable for prevention of vascular prosthetic graft infection.

The viability of Cryptosporidium parvum after exposure to peptide antibiotics was studied by two different methods, a cell culture system and a double fluorogenic staining. The peptides KFFKFFKFF and IKFLKFLKFL exerted high cytotoxic effects on sporozoites, as demonstrated by cell cultures (complete inhibition after 60 min at 100 microg/ml) and flow cytometry (30% after 20 min at 100 microg/ml), but did not affect consistently the oocysts. Clarithromycin and rifabutin demonstrated less activity against sporozoites but higher activity against oocysts (30% after 180 min at 10 microg/ml). The combination between peptides and azithromycin or rifabutin exerted the highest activities.

Staphylococcus epidermidis is a frequent cause of infections associated with foreign bodies and indwelling medical devices. The bacteria are capable of surviving antibiotic treatment through encapsulation into biofilms. RNAIII-inhibiting peptide (RIP) is a heptapeptide that inhibits S. aureus pathogenesis by disrupting quorum-sensing mechanisms. In this study, RIP inhibited drug-resistant S. epidermidis biofilm formation through a mechanism similar to that evidenced for S. aureus. RIP is synergistic with antibiotics in eliminating 100% of graft-associated in vivo S. epidermidis infections, which suggests that RIP may be used to coat medical devices to prevent staphylococcal infections. Disruption of cell-cell communication can prevent infections associated with antibiotic-resistant strains.

Oriana Simonetti *, Guendalina Lucarini , Oscar Cirioni , Antonio Zizzi , Fiorenza Orlando , Mauro Provinciali , Roberto Di Primio , Andrea Giacometti , Annamaria Offidani a Department of Dermatology, Universita Politecnica delle Marche, Ancona, Italy Department of Clinic and Molecular Sciences – Histology, Universita Politecnica delle Marche, Ancona, Italy c Institute of Infectious Diseases and Public Health, Universita Politecnica delle Marche, Ancona, Italy d Institute of Pathology, Universita Politecnica delle Marche, Ancona, Italy Experimental Animal Models for Aging Unit, Scientific Technological Area, I.N.R.C.A., I.R.C.C.S., Ancona, Italy

The therapeutic efficacies of three polycationic peptides selected among the class of the magainins (magainin I, magainin II, and magainin II amide), alone and combined with piperacillin, were investigated in a rat model of septic shock. Rats were given an intraperitoneal injection of 2 × 1010CFU ofEscherichia coliand randomized to receive intraperitoneally isotonic sodium chloride solution, 60 mg of piperacillin per kg of body weight, and 1 mg of each magainin per kg alone and combined with 60 mg of piperacillin per kg. The main outcome measures were bacterial growth in abdominal exudate and plasma, endotoxin and tumor necrosis factor alpha (TNF-α) concentrations in plasma, and lethality. Treatments with the magainins achieved significant reductions of bacterial growth and plasma endotoxin and TNF-α concentrations. In general, treatments with the combinations of magainins and piperacillin demonstrated the highest efficacies.

Background. A rat model was used to investigate the efficacy of a polycationic peptide, the polymyxin-like ranalexin, in the prevention of lethality in a rat model of septic shock. The effect of ranalexin was compared with those of polymyxin B and imipenem. Methods. Adult male Wistar rats (weight range: 250–300 g) were used for all the experiments. The study included five groups: an uninfected control group C 0 , an untreated control group C 1 , and three drug-treated groups that received 1 mg/kg ranalexin (group 2), 20 mg/kg imipenem (group 3), and 3 mg/kg polymyxin B (group 4). Rats, with the exception of the uninfected control group (C 0 ), were given an intraperitoneal injection of 2 × 10 10 colony-forming units of Escherichia coli . Each group included 15 animals. Bacterial growth in abdominal exudate and plasma; endotoxin and tumor necrosis factor α (TNF-α) concentrations in plasma, and mortality were evaluated. Results. Results were evaluated 48 h after inoculation. Ranalexin, imipenem, and polymyxin B significantly reduced the lethality (survival was 93.3, 80.0, and 93.3%, respectively) and the growth of E. coli both in abdominal fluid and plasma compared with saline treatment. Ranalexin showed higher antimicrobial activity than polymyxin B and imipenem and, at the same time, exhibited an antiendotoxin activity similar to that of polymyxin B (≤0.015 EU/mL). Finally, ranalexin and polymyxin B significantly reduced plasma TNF-α levels (≤4 pg/mL). Conclusion. Monodose ranalexin treatment prevents bacterial growth, endotoxemia, and mortality in rats with septic shock.

Objective To investigate the efficacy of three cecropins, cecropin A, cecropin B, and cecropin P1, in preventing lethality in a rat model of septic shock. Design Prospective, randomized, controlled animal study. Setting Research laboratory in a university hospital. Subjects Adult male Wistar rats. Interventions Rats were given an intraperitoneal injection of 2 × 1010 colony forming units of Escherichia coli, with the exception of the uninfected control group (C0). Animals were randomized to receive, immediately after bacterial challenge, intraperitoneally isotonic sodium chloride solution (untreated control group C1), 1 mg/kg cecropin A (group 2), 1 mg/kg cecropin B (group 3), 1 mg/kg cecropin P1 (group 4), 20 mg/kg imipenem (group 5), or 60 mg/kg piperacillin (group 6). Each group included 15 animals. Measurements and Main Results We measured bacterial growth (quantitative agar culture) in abdominal exudate and plasma, endotoxin and tumor necrosis factor-α concentration in plasma, and mortality. Results were evaluated at 48 hrs after inoculation. Cecropins, piperacillin, and imipenem significantly reduced the lethality and the number of E. coli in abdominal fluid compared with saline treatment. In addition, cecropin B significantly decreased the lethality compared with piperacillin treatment. Finally, only cecropins significantly reduced plasma endotoxin concentration. Conclusions Mono-dose cecropin treatment prevents bacterial growth, endotoxemia, and mortality in rats with septic shock. Cecropin B was the most effective compound in reducing all variables measured.

A rat model was used to investigate the efficacy of mupirocin in the prevention of vascular prosthetic graft infections. The effect of mupirocin-soaked Dacron was compared with the effect of rifampin-soaked, collagen-sealed Dacron in the rat model of graft infection caused by methicillin-susceptible Staphylococcus aureus and methicillin-resistant S. aureus . Graft infections were established in the back subcutaneous tissue of 195 adult male Wistar rats by implantation of 1-cm 2 Dacron prostheses followed by topical inoculation with 5 × 10 7 colony-forming units of S. aureus . The study included a control group (no graft contamination), two contaminated groups that did not receive any antibiotic prophylaxis, two contaminated groups in which perioperative intraperitoneal amoxicillin clavulanate prophylaxis (50 mg/kg) was administered, four contaminated groups that received mupirocin- or rifampin-soaked graft, and four contaminated groups that received mupirocin- or rifampin-soaked graft and perioperative intraperitoneal amoxicillin clavulanate prophylaxis (50 mg/kg). The grafts were sterilely removed 7 days after implantation and the infection was evaluated by using sonication and quantitative agar culture. Data analysis showed that the efficacy of mupirocin against both strains was significantly different from that of the untreated control. In addition, mupirocin was more effective than rifampin against the methicillin-resistant strain. Finally, only the combination of mupirocin and amoxicillin clavulanate produced complete suppression of growth of all strains.

The in vitro anti-cryptosporidial activity of buforin II alone and in combination with azithromycin and minocycline was investigated. Buforin II showed moderate activity, which increased with increasing concentration to 55.7% suppression of growth at 20 microM. Moreover, its activity was enhanced when it was combined with either azithromycin or minocycline with >90% parasite reduction at the highest concentration tested. Buforin II may be active in inhibiting Cryptosporidium parvum growth in vitro upon combination with either azithromycin or minocycline.

A cell culture system and double fluorogenic staining were used to study the susceptibility of Cryptosporidium parvum to membrane-active antibiotics. Buforin II and magainin II exerted a cytotoxic effect on sporozoites but did not consistently affect oocyst viability. Lasalocid and nigericin demonstrated less activity against sporozoites but reduced the infectivity of oocysts.

The in vitro interaction between five polycationic peptides, buforin II, cecropin P1, indolicidin, magainin II, and ranalexin, and several clinically used antimicrobial agents was evaluated against several clinical isolates of Gram-positive and Gram-negative aerobic bacteria, using the microbroth dilution method. The combination studies showed synergy between ranalexin and polymyxin E, doxycycline and clarithromycin. In addition, magainin II was shown to be synergic with betalactam antibiotics.

The in vitro activity of nitazoxanide alone and in combination with azithromycin and rifabutin was investigated against four clinical isolates of Cryptosporidium parvum. The susceptibility tests were performed by inoculation of the isolates on toe cell monolayers and determination of the parasite count after 48 h incubation at 37 degrees C. The culture medium was supplemented with Dulbecco's modified Eagle's medium containing serial dilutions of each agent. Antibiotic-free plates were used as controls. Experiments were performed in triplicate. Nitazoxanide showed moderate anticryptosporidial activity: it suppressed the growth of parasites by >50% at 8 mg/L. A parasite reduction of 79.8-83.9% was observed when nitazoxanide 8 mg/L was combined with azithromycin 8 mg/L and rifabutin 8 mg/L. The study suggests that nitazoxanide may be active in inhibiting C. parvum growth in vitro upon combination with azithromycin or rifabutin.

The in vitro activity of six polycationic peptides, buforin II, cecropin P1, indolicidin, magainin II, nisin, and ranalexin, were evaluated against several clinical isolates of gram-positive and gram-negative aerobic bacteria, yeasts, Pneumocystis carinii and Cryptosporidium parvum, by using microbroth dilution methods. The peptides exhibited different antibacterial activities and rapid time-dependent killing. The gram-negative organisms were more susceptible to buforin II and cecropin P1, whereas buforin II and ranalexin were the most active compounds against the gram-positive strains. Similarly, ranalexin showed the highest activity against Candida spp., whereas magainin II exerted the highest anticryptococcal activity. Finally, the peptides showed high anti-Pneumocystis activity, whereas no compound had strong inhibitory effect on C. parvum.

The in-vitro activity of cecropin P1, indolicidin, magainin II, nisin and ranalexin alone and in combination with nine clinically used antimicrobial agents was investigated against a control strain, Pseudomonas aeruginosa ATCC 27853 and 40 clinical isolates of P. aeruginosa. Antimicrobial activities were measured by MIC, MBC and viable count. In the combination study, the clinically used antibiotics were used at concentrations close to their mean serum level in humans in order to establish the clinical relevance of the results. To select peptide-resistant mutants, P. aeruginosa ATCC 27853 was treated with consecutive cycles of exposure to each peptide at 1 x MIC. The peptides had a varied range of inhibitory values: all isolates were more susceptible to cecropin P1, while ranalexin showed the lowest activity. Nevertheless, synergy was observed when the peptides were combined with polymyxin E and clarithromycin. Consecutive exposures to each peptide at 1 x MIC resulted in the selection of stable resistant mutants. Cationic peptides might be valuable as new antimicrobial agents. Our findings show that they are effective against P. aeruginosa, and that their activity is enhanced when they are combined with clinically used antimicrobial agents, particularly with polymyxin E and clarithromycin.

The in-vitro activity of magainin II, indolicidin and ranalexin against 14 clinical isolates of eukaryotic microorganisms was evaluated. Antifungal susceptibility testing was performed by broth microdilution, and activity against Pneumocystis carinii and Cryptosporidium parvum was determined by inoculation on to cell monolayers. For yeasts, peptide MICs and MFCs ranged from 6.25 to > 50 mg/L. Ranalexin showed the highest activity against Candida spp., while magainin II demonstrated greatest anticryptococcal activity. The peptides suppressed the growth of P. carinii by > or = 50% and > or = 90% at 5 and 50 microM, respectively, with the exception of indolicidin. Ranalexin, the most effective compound against C. parvum, suppressed its growth by > or = 40% at 50 microM.