Biological control of sheep parasite nematodes by nematode-trapping fungi: In vitro activity and after passage through the gastrointestinal tract

The main method used for the control of gastrointestinal nematodes in sheep production is the application of chemotherapeutic agents, which often lead to the selection of parasites resistant to given active principles. Biological control can be considered a promising alternative, contributing to an increase in the efficacy of verminous control. We determined the in vitro activity and in situ survival of the predatory fungi Arthrobotrys musiformis and Arthrobotrys conoides during passage through the gastrointestinal tract of sheep after oral administration of conidia in microencapsulated form and as a liquid in natura. Initial in vitro tests showed that both fungi were efficient in the predation of trichostrongylid L3 larvae present in the faeces of sheep naturally infected with gastrointestinal nematodes. The fungi presented high nematophagous activity, which was 99.3% for A. conoides and 73.7% for A. musiformis. A. conoides did not survive passage through the gastrointestinal tract under the conditions of the present experiment. On the other hand, A. musiformis was reisolated after administration in either microencapsulated or liquid form, suggesting that this species is a promising alternative for the control of nematodes in sheep since it survives without any protection (in natura). © Springer 2005.

In vitro activity of pineapple extracts (Ananas comosus, Bromeliaceae) on Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

Measures to control the cattle tick, Rhipicephalus (Boophilus) microplus, based only on chemical products are becoming unsustainable, mainly because of the development of resistance. The objective of this study was to test the effect of the aqueous extract of pineapple skin (AEPS) and bromelain extracted from the stem (Sigma-Aldrich®, B4882) on engorged females and larvae of R. (B.) microplus in vitro. These substances were diluted in water and evaluated at eight concentrations. Engorged females were collected and distributed in groups of 10, with three repetitions for each treatment. After immersion in the solutions, the females were placed in an incubator for observation of survival, oviposition and larval hatching. The larval packet method was used, also with three repetitions with about 100 larvae each. The packets were incubated and the readings were performed after 24h. The estimated reproduction and efficacy of the solutions were calculated. The LC50 and LC90 were estimated using the Probit procedure of the SAS program. The eight concentrations were compared within each treatment by the Tukey test. For the experiment with engorged females, the most effective concentrations were 125, 250 and 500mg/mL: 33%, 48% and 59% for the AEPS and 27%, 51% and 55% for the bromelain. The LC50 and LC90 values were, respectively, 276 and 8691mg/mL for AEPS and 373 and 5172mg/mL for bromelain. None of the dilutions tested was effective against the larvae of R. (B.) microplus. This is the first report of the action of pineapple extracts or their constituents on cattle ticks. The results demonstrate that further studies regarding composition of tick cuticle, with evaluation of other solvents and formulations, should be conducted seeking to enhance the effect of pineapple extracts and compounds against this ectoparasite. © 2013 Elsevier Inc.

Evaluation of the in vitro Activity of Dermaseptin 01, a Cationic Antimicrobial Peptide, against Schistosoma mansoni

Schistosomiasis is a neglected tropical disease that remains a considerable public health problem worldwide. Since the mainstay of schistosomiasis control is chemotherapy with a single drug, praziquantel, drug resistance is a concern. Here, we examined the in vitro effects of dermaseptin 01 (DS 01), an antimicrobial peptide found in the skin secretion of frogs of the genus Phyllomedusa, on Schistosoma mansoni adult worms. DS 01 at a concentration of 100 mg/ml reduced the worm motor activity and caused the death of all worms within 48 h in RPMI 1640 medium. At the highest sublethal concentration of antimicrobial peptide (75 mg/ml), a 100% reduction in egg output of paired female worms was observed. Additionally, DS 01 induced morphological alterations on the tegument of S. mansoni, and a quantitative analysis carried out by confocal microscopy revealed extensive destruction of the tubercles in a dose-dependent manner over the concentration range of 50-200 mu g/ml. It was the first time that an anthelmintic activity towards schistosomes has been reported for a dermaseptin.

In vitro activity of extracts and isolated polyphenols from West African medicinal plants against Plasmodium falciparum

The aim of the study was to screen 11 selected traditional medicinal plants from West Africa for their in vitro antiplasmodial activity in order to determine the activity of single and of combination of plant extracts and to examine the activity of isolated pure compounds. Ethanolic and aqueous extracts of the 11 selected plants and pure compounds from Phyllanthus muellerianus and Anogeissus leiocarpus were tested in vitro against Plasmodium falciparum 3D7. Proliferation inhibitory effects were monitored after 48 h. Among the plants and pure compounds investigated in this study, geraniin from P. muellerianus, ellagic, gentisic, and gallic acids from A. leiocarpus, and extracts from A. leiocarpus, P. muellerianus and combination of A. leiocarpus with P. muellerianus affected the proliferation of P. falciparum most potently. Significant inhibitory activity was observed in combination of A. leiocarpus with P. muellerianus (IC50 = 10.8 mu g/ml), in combination of A. leiocarpus with Khaya senegalensis (IC50 = 12.5 mu g/ml), ellagic acid (IC50 = 2.88 mu M), and geraniin (IC50 = 11.74 mu M). In general growth inhibition was concentration-dependent revealing IC50 values ranging between 10.8 and -40.1 mu g/ml and 2.88 and 11.74 mu M for plant extracts and pure substances respectively. Comparison with literature sources of in vivo and in vitro toxicity data revealed that thresholds are up to two times higher than the determined IC50 values. Thus, the present study suggests that geraniin from P. muellerianus; ellagic acid, gallic acid, and gentisic acid from A. leiocarpus; and combination of extracts from A. leiocarpus with either P. muellerianus or K. senegalensis could be a potential option for malaria treatment.

In vitro activity of clinically implemented β-lactams against Aerococcus urinae: presence of non-susceptible isolates in Switzerland.

We analyzed the in vitro susceptibility to several ?-lactams and vancomycin of 80 Aerococcus urinae isolates col- lected during 2011-2012 in Switzerland. MICs were determined by Etest (bioMérieux) on Müller-Hinton agar with 5% sheep blood and interpreted according to the CLSI and EUCAST criteria set for viridans streptococci. MIC50/90 for penicillin, amoxicillin, ceftriaxone and vancomycin were 0.016/0.064 mg/l, 0.032/0.064 mg/l, 0.125/0.5 mg/l and 0.38/0.5 mg/l, respectively. Three (3.8%) isolates were resistant to ceftriaxone regardless of the criteria used (MICs ?2 mg/l); one of them was also non-susceptible to penicillin (MIC of 0.25 mg/l) according to CLSI. ?-lactam resis- tance in A. urinae is a concern and suggests that more studies are needed to determine the molecular mechanisms of such resistance.

In vitro Activity of Fosfomycin Alone and in Combination with Ceftriaxone or Azithromycin Against Clinical Neisseria gonorrhoeae Isolates.

New therapeutic strategies are needed to combat the emergence of infections due to multidrug-resistant Neisseria gonorrhoeae (Ng). In this study, fosfomycin (FOS) was tested against 89 Ng using the Etest method and showing MIC50/90s of only 8/16 μg/ml (range ≤ 1-32 μg/ml). FOS in combination with ceftriaxone (CRO) or azithromycin (AZT) was then evaluated using the checkerboard method for eight strains, including F89 (CRO-resistant) and AZT-HLR (high-level AZT-resistant). All combinations including FOS gave indifferent effects (fractional inhibitory concentration [FIC] index values between 1.2-2.3 for FOS plus CRO and between 1.8-3.2 for FOS plus AZT). Time-kill experiments for FOS, CRO, AZT and their combinations (at concentrations of 0.5×, 1×, 2× and 4× MIC) were performed against ATCC 49226, one Ng of NG-MAST ST1407, F89 and AZT-HLR. For all strains, at 24 hours results indicated that: i) FOS was bactericidal at 2× MIC concentrations but after >24 hours there was re-growth of bacteria; ii) CRO was bactericidal at 0.5× MIC; iii) AZT was bactericidal at 4× MIC; iv) CRO plus AZT was less bactericidal than CRO alone; v) FOS plus AZT was bactericidal at 2× MIC; vi) CRO plus AZT and FOS plus CRO were both bactericidal at 0.5× MIC, but the latter had more rapid effects. FOS is appealing for the management of Ng infections because of its single and oral formulation. However, our results suggest its use in combination with CRO. This strategy could, after appropriate clinical trials, be implemented for the treatment of infections due to isolates possessing resistance to CRO and/or AZT.

In vitro activity of photoactivated disinfection using a diode laser in infected root canals

OBJECTIVE To investigate the lethal activity of photoactivated disinfection (PAD) on Enterococcus faecalis (ATCC 29212) and mixed populations of aerobic or anaerobic bacteria in infected root canals using a diode laser after the application of a photosensitizer (PS). MATERIALS AND METHODS First, the bactericidal activity of a low power diode laser (200 mW) against E. faecalis ATCC 29212 pre-treated with a PS (toluidine blue) for 2 min were examined after different irradiation times (30 s, 60 s and 90 s). The bactericidal activity in the presence of human serum or human serum albumin (HSA) was also examined. Second, root canals were infected with E. faecalis or with mixed aerobic or anaerobic microbial populations for 3 days and then irrigated with 1.5% sodium hypochlorite and exposed to PAD for 60 s. RESULTS Photosensitization followed by laser irradiation for 60 s was sufficient to kill E. faecalis. Bacteria suspended in human serum (25% v/v) were totally eradicated after 30 s of irradiation. The addition of HSA (25 mg/ml or 50 mg/ml) to bacterial suspensions increased the antimicrobial efficacy of PAD after an irradiation time of 30 s, but no longer. The bactericidal effect of sodium hypochlorite was only enhanced by PAD during the early stages of treatment. PAD did not enhance the activity of sodium hypochlorite against a mixture of anaerobic bacteria. CONCLUSIONS The bactericidal activity of PAD appears to be enhanced by serum proteins in vitro, but is limited to bacteria present within the root canal.

In vitro-activity of oily calcium hydroxide suspension on microorganisms as well as on human alveolar osteoblasts and periodontal ligament fibroblasts.

BACKGROUND Findings from animal and human studies have indicated that an oily calcium hydroxide suspension (OCHS) may improve early wound healing in the treatment of periodontitis. Calcium hydroxide as the main component is well known for its antimicrobial activity, however at present the effect of OCHS on the influence of periodontal wound healing/regeneration is still very limited. The purpose of this in vitro study was to investigate the effect of OCHS on periodontopathogenic bacteria as well as on the attachment and proliferation of osteoblasts and periodontal ligament fibroblasts. METHODS Human alveolar osteoblasts (HAO) and periodontal ligament (PDL) fibroblasts were cultured on 3 concentrations of OCHS (2.5, 5 and 7.5 mg). Adhesion and proliferation were counted up to 48 h and mineralization was assayed after 1 and 2 weeks. Furthermore potential growth inhibitory activity on microorganisms associated with periodontal disease (e.g. Porphyromonas gingivalis, Tannerella forsythia, Aggregatibacter actinomycetemcomitans) as well as the influence of periodontopathogens and OCHS on the HAO and PDL fibroblasts counts were determined. RESULTS More than a 2-fold increase in adherent HAO cells was observed at 4 h following application of OCHS when compared to the control group (p = 0.007 for 2.5 mg). Proliferation of HAO cells at 48 h was stimulated by moderate concentrations (2.5 mg; 5 mg) of OCHS (each p < 0.001), whereas a high concentration (7.5 mg) of OCHS was inhibitory (p = 0.009). Mineralization was observed only for HAO cells treated with OCHS. OCHS did not exert any positive effect on attachment or proliferation of PDL fibroblasts. Although OCHS did not have an antibacterial effect, it did positively influence attachment and proliferation of HAO cells and PDL fibroblasts in the presence of periodontopathogens. CONCLUSIONS The present data suggests that OCHS promotes osteoblast attachment, proliferation and mineralization in a concentration-dependent manner and results are maintained in the presence of periodontal pathogens.

In-vitro activity of taurolidine on single species and a multispecies population associated with periodontitis.

The antimicrobial activity of taurolidine was compared with minocycline against microbial species associated with periodontitis (four single strains and a 12-species mixture). Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs), killing as well as activities on established and forming single-species biofilms and a 12-species biofilm were determined. The MICs of taurolidine against single species were always 0.31 mg/ml, the MBCs were 0.64 mg/ml. The used mixed microbiota was less sensitive to taurolidine, MIC and the MBC was 2.5 mg/ml. The strains and the mixture were completely killed by 2.5 mg/ml taurolidine, whereas 256 μg/ml minocycline reduced the bacterial counts of the mixture by 5 log10 colony forming units (cfu). Coating the surface with 10 mg/ml taurolidine or 256 μg/ml minocycline prevented completely biofilm formation of Porphyromonas gingivalis ATCC 33277 but not of Aggregatibacter actinomycetemcomitans Y4 and the mixture. On 4.5 d old biofilms, taurolidine acted concentration dependent with a reduction by 5 log10 cfu (P. gingivalis ATCC 33277) and 7 log10 cfu (A. actinomycetemcomitans Y4) when applying 10 mg/ml. Minocycline decreased the cfu counts by 1-2 log10 cfu independent of the used concentration. The reduction of the cfu counts in the 4.5 d old multi-species biofilms was about 3 log10 cfu after application of any minocycline concentration and after using 10 mg/ml taurolidine. Taurolidine is active against species associated with periodontitis, even within biofilms. Nevertheless a complete elimination of complex biofilms by taurolidine seems to be impossible and underlines the importance of a mechanical removal of biofilms prior to application of taurolidine.

In Vitro Activity of the Novel Antimicrobial Peptide Dendrimer G3KL against Multidrug-Resistant Acinetobacter baumannii and Pseudomonas aeruginosa.

The in vitro activity of the novel antimicrobial peptide dendrimer G3KL was evaluated against 32 Acinetobacter baumannii (including 10 OXA-23, 7 OXA-24, and 11 OXA-58 carbapenemase producers) and 35 Pseudomonas aeruginosa (including 18 VIM and 3 IMP carbapenemase producers) strains and compared to the activities of standard antibiotics. Overall, both species collections showed MIC50/90 values of 8/8 μg/ml and minimum bactericidal concentrations at which 50% or 90% of strains tested are killed (MBC50/90) of 8/8 μg/ml. G3KL is a promising molecule with antibacterial activity against multidrug-resistant and extensively drug-resistant A. baumannii and P. aeruginosa isolates.

Amino ozonides exhibit in vitro activity against Echinococcus multilocularis metacestodes.

Artemisinin is an antimalarial sesquiterpene lactone that contains a 1,2,4-trioxane heterocycle. Dihydroartemisinin and artesunate demonstrated activity against Echinococcus multilocularis metacestodes in vitro but were not effective in a mouse model. In this study, the in vitro effects of a small library of synthetic ozonides (1,2,4-trioxolanes) were investigated. Initial compound screening against E. multilocularis metacestodes was performed at 20μM, and selected ozonides were further assessed in dose-response studies in metacestode cultures and mammalian cells. Transmission electron microscopy (TEM) was employed to characterise compound-induced structural alterations. At 20μM, the most potent ozonides (OZ401, OZ455, OZ491 and OZ494) led to death of ca. 60-100% of the parasites. Subsequent dose-response experiments demonstrated that OZ401, OZ455 and OZ491, which contain an aminopropylether substructure, were the most potent, with 50% inhibitory concentrations ranging from 11μM to 14μM. Cytotoxicity for these three ozonides, assessed in human foreskin fibroblasts, rat hepatoma cells and green monkey epithelial kidney (Vero) cells, was evident only at high concentrations. TEM demonstrated that OZ401 and OZ491 treatment induced considerable metabolic impairment in metacestodes at 1 day post exposure. At Day 3 post exposure, the germinal layer was severely distorted, although some intact cells were still visible, demonstrating that not all cell types in the parasite tissue were equally affected. Complete destruction of the germinal layer was noted at 5 days post exposure. Synthetic ozonides could represent interesting leads that will be further investigated in a suitable in vivo model of E. multilocularis infection.

Similarity between the in vitro activity and toxicity of two different fungizone™ / lipofundin™ admixtures

Amphotericin B (AmB), an antifungal agent that presents a broad spectrum of activity, remains the gold standard in the antifungal therapy. However, sometimes the high level of toxicity forbids its clinical use. The aim of this work was to evaluate and compare the efficacy and toxicity in vitro of Fungizon™ (AmB-D) and two new different AmB formulations. Methods: three products were studied: Fungizon™, and two Fungizon™ /Lipofundin™ admixtures, which were diluted through two methods: in the first one, Fungizon™ was previously diluted with water for injection and then, in Lipofundin™ (AmB-DAL); the second method consisted of a primary dilution of AmB-D as a powder in the referred emulsion (AmB-DL). For the in vitro assay, two cell models were used: Red Blood Cells (RBC) from human donors and Candida tropicallis (Ct). The in vitro evaluation (K+ leakage, hemoglobin leakage and cell survival rate-CSR) was performed at four AmB concentrations (from 50 to 0.05mg.L-1). Results: The results showed that the action of AmB was not only concentration dependent, but also cellular type and vehicle kind dependent. At AmB concentrations of 50 mg.L-1, although the hemoglobin leakage for AmB-D was almost complete (99.51), for AmB-DAL and AmB-DL this value tended to zero. The p = 0.000 showed that AmB-D was significantly more hemolytic. Conclusion: The Fungizon™- Lipofundin™ admixtures seem to be the more valuable AmB carrier systems due to their best therapeutic index presented

Similarity between the in vitro activity and toxicity of two different fungizone™ / lipofundin™ admixtures

Amphotericin B (AmB), an antifungal agent that presents a broad spectrum of activity, remains the gold standard in the antifungal therapy. However, sometimes the high level of toxicity forbids its clinical use. The aim of this work was to evaluate and compare the efficacy and toxicity in vitro of Fungizon™ (AmB-D) and two new different AmB formulations. Methods: three products were studied: Fungizon™, and two Fungizon™ /Lipofundin™ admixtures, which were diluted through two methods: in the first one, Fungizon™ was previously diluted with water for injection and then, in Lipofundin™ (AmB-DAL); the second method consisted of a primary dilution of AmB-D as a powder in the referred emulsion (AmB-DL). For the in vitro assay, two cell models were used: Red Blood Cells (RBC) from human donors and Candida tropicallis (Ct). The in vitro evaluation (K+ leakage, hemoglobin leakage and cell survival rate-CSR) was performed at four AmB concentrations (from 50 to 0.05mg.L-1). Results: The results showed that the action of AmB was not only concentration dependent, but also cellular type and vehicle kind dependent. At AmB concentrations of 50 mg.L-1, although the hemoglobin leakage for AmB-D was almost complete (99.51), for AmB-DAL and AmB-DL this value tended to zero. The p = 0.000 showed that AmB-D was significantly more hemolytic. Conclusion: The Fungizon™- Lipofundin™ admixtures seem to be the more valuable AmB carrier systems due to their best therapeutic index presented

In Vitro Antifungal Activity and Toxicity of Itraconazole in DMSA-PLGA Nanoparticles

Itraconazole (ITZ) is a drug used to treat various fungal infections and may cause side effects. The aim of this study was to develop and evaluate the in vitro activity of DMSA-PLGA nanoparticles loaded with ITZ against Paracoccidioides brasiliensis, as well as their cytotoxicity. Nanoparticles were prepared using the emulsification-evaporation technique and characterized by their encapsulation efficiency, morphology (TEM), size (Nanosight) and charge (zeta potential). Antifungal efficacy in P brasiliensis was determined by minimal inhibition concentration (MIC), and cytotoxicity using MU assay. ITZ was effectively incorporated in the PLGA-DMSA nanoparticles with a loading efficiency of 72.8 +/- 3.50%. The shape was round with a solid polymeric structure, and a size distribution of 174 +/- 86 nm (Average +/- SD). The particles were negatively charged. ITZ-NANO presented antifungal inhibition (MIC = 6.25 ug/mL) against P brasiliensis and showed lower in vitro cytotoxicity than free drug (ITZ).