Antibacterial in vitro effects of preparations from Anthroposophical Medicine.

BACKGROUND: Medications from Anthroposophical Medicine (AM) are clinically used for the treatment of infections within a whole medical system but have not yet been evaluated regarding antibacterial effects. The aims of this study was to investigate antibacterial activity of AM medications in cell culture. METHODS: Screening of AM drug registers for preparations used to treat any kind of infection and being available in dilutions ≤ D2 and without alcoholic content. Selected medications were screened for antimicrobial activity against Bacillus subtilis, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa using the agar diffusion method. For antimicrobial active preparations growth kinetics (drop plate method) and minimal inhibitory concentrations (MIC, macrodilution method) were determined. RESULTS: Thirty-three preparations matched the selection criteria and were chosen for own experiments. One of them (Berberis Decoctum D2) exhibited bactericidal activities against Bacillus subtilis and Staphylococcus aureus, including methicillin resistant strains. The MIC could be determined as 5 mg/ml. The effects could be related to the content of berberine in the extract. No activity towards gram-negative bacteria was found. The other tested extracts had no antibacterial effects. CONCLUSION: Berberis Decoctum D2 which is used in AM to treat infections exhibits bactericidal effects on Staphylococcus aureus, including methicillin resistant strains.

Evaluation of an aqueous-ethanolic extract from Pelargonium sidoides (EPs® 7630) for its activity against group A-streptococci adhesion to human HEp-2 epithelial cells.

AIM OF THE STUDY: The root extract of Pelargonium sidoides DC (Geraniaceae), EPs® 7630, is currently used to treat respiratory tract infections. The therapeutic benefits are largely related to the modulation of the non-specific immune system. The present study was designed to investigate the anti-adhesive activity of this herbal medicine with Streptococcus pyogenes as model microorganism and to identify the underlying biologically active principle. MATERIALS AND METHODS: Adherence of fluorescent-labelled group A-streptococci (GAS) to human epithelial (HEp-2) cells was assessed by flow cytometry. Anti-adhesive properties of the parent extract as well as a methanol-soluble (MSF) and a methanol-insoluble fraction (MIF) derived thereof were examined. Treatment with skin powder produced polyphenol-free samples which were included for comparison. Anti-adherence studies were extended to a series of highly purified proanthocyanidins including homogenous epicatechin- and catechin-based polyflavans, a 'mixed' procyanidin sample, an A-type proanthocyanidin mixture as well as a prodelphinidin test substance. RESULTS: After pre-treatment of GAS with EPs® 7630 or its subfractions MIF and MSF at concentrations of 30 µg/ml, adhesion of the pathogen to HEp-2 cells was inhibited by ca. 45%, ca. 35% and ca. 30%, respectively. However, following preincubation of cells with the extract and the fractions no effect was observed. This finding indicates that the anti-adhesive effects are due to interactions with binding factors on the bacterial surface. Since polyphenol-free samples proved to be inactive, proanthocyanidins appear to represent the anti-adhesive principle. Comparative studies with chemically defined proanthocyanidins revealed that the prodelphinidin nature, i.e. the pyrogallol B-ring elements of constituent flavanyl units, represented an important structural feature of the anti-adhesive potential of this herbal medicine. CONCLUSIONS: The current data provide strong evidence for a potent anti-adhesion principle of the Pelargonium sidoides root extract related to specific proanthocyanidins. This finding suggests an interaction with bacterial binding sites in a specific rather than non-specific manner. However, the blocked adhesion molecules remain to be identified. The anti-adhesive mechanism may well contribute to the anti-infective activity of EPs® 7630 at an early time point of a bacterial infection.

Extract of Pelargonium sidoides (EPs 7630) inhibits the interactions of group A-streptococci and host epithelia in vitro.

EPs 7630 is an extract of the root of the South African geranium Pelargonium sidoides. Clinical data have shown that this herbal drug preparation can be used to treat upper respiratory tract infections (URTI). The objective of this study was to investigate the impact of EPs 7630 on group A-streptococci (GAS) adhering to and invading host epithelial cells in vitro. Adhesion was assessed by a flow cytometric adhesion assay using calcein-AM-stained S. pyogenes (DSM 2071) as test organism, and HEp-2 cells and buccal epithelial cells (BEC) as substrata. For cell invasion, HEp-2 cell monolayers were infected with S. pyogenes. Intracellular bacteria were determined using a penicillin/gentamicin-protection assay. EPs 7630 was applied in therapeutically relevant concentrations between 0 and 30 microg/ml in both test systems. Compared to controls, EPs 7630 significantly reduced GAS adhesion to HEp-2 cells in a concentration-dependent manner by up to 46% (p<0.001). This was semi-quantitatively confirmed by fluorescence microscopy. Adhesion kinetics additionally indicated a specific antagonistic effect on GAS adhesion. Pre-treatment of epithelial cells or GAS with EPs 7630 showed that it targets GAS rather than HEp-2 cells. However when using BEC as substrata, GAS adhesion increased 7-fold under the influence of EPs 7630 (p<0.001). In viability assays, HEp-2 suspension contained over 90% vital cells, whereas only 10% of the BEC were viable. EPs 7630 reduced GAS invasion of HEp-2 cells significantly when the data taken at time points 60, 120, and 180 min (p=0.026) were analyzed. In conclusion, EPs 7630 develops complementary anti-infective properties: Firstly, EPs 7630 reduces bacterial adhesion to intact epithelial cells and thus protects the organism from bacterial colonization and infection/super-infection. Secondly, EPs 7630 enhances the attachment of bacteria to decaying BEC. In this way pathogens may be trapped and rendered inactive. Thirdly, the inhibition of GAS invasion of epithelial cells protects the host from microorganisms that may have evaded host defences and antibiotic treatment, and therefore prevents recurrent infections. Thus, our investigations provide a rational basis for the treatment of URTI with EPs 7630.

Extract of Pelargonium sidoides (EPs 7630) improves phagocytosis, oxidative burst, and intracellular killing of human peripheral blood phagocytes in vitro.

Clinical data show that EPs 7630, an aqueous ethanolic extract from the roots of Pelargonium sidoides, can be used for the treatment of upper respiratory tract infections (URTI). The biological effects of the preparation have not been fully investigated. The objective of this study was to examine the impact of EPs 7630 on the activity of human peripheral blood phagocytes (PBP). A whole blood-based, flow cytometric assay was used to simultaneously assess phagocytosis and oxidative burst. Calcein-AM stained Candida albicans (DSM 1386) were used as target organisms. Oxidative burst was measured by addition of dihydroethidium (DHE). Target organisms and whole blood were co-incubated and analyzed after 0, 2, 4, 6, 10, and 30 min. Intracellular killing of the target organisms was evaluated by determining the number of surviving yeast cells after co-incubation of C. albicans and human whole blood. EPs 7630 was applied in therapeutically relevant concentrations between 0 and 30 microg/ml. Compared with controls EPs 7630 increased the number of phagocytosing PBP during the observed time points between 2 and 10 min in a concentration-dependent manner, with a maximum enhancement of 56% at 2 min (p=0.002). The application of EPs 7630 also led to a significant increase in the number of burst-active PBP for all time points observed beyond 2 min (p<0.001). The maximum augmentation was 120% after application of 30 microg/ml EPs 7630 at 4 min. Using a microbiological assay, intracellular killing was also enhanced by EPs 7630. This was expressed by a significant reduction in the number of surviving target organisms (p<0.001). The maximum reduction in viable yeast cells (-31%) was observed after co-incubation for 120 min with the highest concentration of EPs 7630 (30 microg/ml). In conclusion, the positive effects of EPs 7630 on phagocytosis, oxidative burst, and intracellular killing of yeast cells as test organisms are important components of the compound's biological activity. Our findings constitute a valuable contribution to understanding the clinical effects of EPs 7630.

[In vitro study to evaluate the antibacterial activity of a combination of the haulm of nasturtium (Tropaeoli majoris herba) and of the roots of horseradish (Armoraciae rusticanae radix)]. / In-vitro-Untersuchungen zur antibakteriellen wirksamkeit einer Kombination aus Kapuzinerkressenkraut (tropaeoli majoris herba) und meer- rettichwurzel (armoraciae rusticanae radix).

OBJECTIVE: To evaluate the in-vitro antimicrobial properties of a commercialized preparation (Angocin Anti-Infekt N) containing a combination of the haulm of nasturtium (Tropaeoli majoris herba; N) and of the roots of horseradish (Armoraciae rusticanae radix; H). This preparation can be used to treat upper respiratory tract (URTI) and urinary tract infections (UTI). The active ingredients are volatile mustard oils, which are activated in the gastrointestinal tract after oral intake. Previous research has shown mustard oils derived from either N or H to possess antibacterial activity. METHODS: In order to assess the antimicrobial capacity of phytotherapeutic compounds containing volatile mustard oils, a modified gas-test was used. Native preparations of N and H were applied to the lids of Columbia agar plates (ratio N:H = 2.5:1) and mixed with sterile H20. Thirteen different bacterial species including Haemophilus influenzae, Moraxella catarrhalis, Escherichia coli, Pseudomonas aeruginosa, Streptococcus pyogenes, methicillin-susceptible and resistant Staphylococcus aureus (MSSA, MRSA) were tested (20 isolates each). The test organisms were plated onto the blood agar plates and placed above the native preparations. The plates were sealed with adhesive tape and incubated at 37 degrees C. Following incubation of 24 h and 92 h, colony forming units (CFU) were counted and the minimal inhibitory concentrationg (MIC90) was determined for each bacterial species. RESULTS: Relevant antimicrobial activities of the combined native preparations were found against H. influenzae (MIC90 50 mg N / 20 mg H), M. catarrhalis (100 mg N / 40 mg H), E. coli (400 mg N / 160 mg H), P aeruginosa (400 mg N / 160 mg H), MSSA (400 mg N 1 160 mg H), MRSA (400 mg N / 160 mg H), and S. pyogenes (400 mg N / 160 mg H). CONCLUSION: Antimicrobial testing of a combination of N and H revealed broad antibacterial activities against clinically relevant pathogens covering both gram-positive and gram-negative organisms, thus confirming previous reports of the antibacterial properties of mustard oils. Additionally, this study demonstrated that the combination of N and H leads to synergistic activity in terms of improved Pseudomonas-susceptibility compared to the previous reported activities of the single compounds. Thus, these results prove that there is a rational basis for treatment of URTI and UTI with a combination of N and H.

Development and mechanism of fluoroquinolone resistance in Legionella pneumophila.

The potential for selection in vitro of Legionella pneumophila mutants resistant to fluoroquinolones was investigated. Six distinct clinical isolates of L. pneumophila were subcultured in subinhibitory concentrations of ciprofloxacin, levofloxacin, clinafloxacin, trovafloxacin and moxifloxacin until MICs increased at least eight-fold. The numbers of serial passages required in microbroth dilution series were determined. The gyrA gene of the six parental strains, and 12 selected mutant strains, was sequenced. The five quinolones differed markedly in their ability to select mutants with decreased susceptibility. The average number of serial passages required was low in the cases of clinafloxacin (n = 10.6), ciprofloxacin and levofloxacin (both n = 13), but notably higher for trovafloxacin (n = 26.6) and moxifloxacin (n = 22.5). Five mutants treated with ciprofloxacin and three treated with moxifloxacin showed Thr83-->Lys or Thr83-->Ile amino acid changes in the gyrA gene. In conclusion, different quinolones lose their antimicrobial effect after a varying number of passages. This study demonstrated, for the first time to our knowledge, that gyrA in L. pneumophila is a possible target of fluoroquinolones.