Gerald Müller

Biocompatibility index of antiseptic agents by parallel assessment of antimicrobial activity and cellular cytotoxicity

OBJECTIVES To assess the suitability of an antiseptic agent, both the microbicidal activity and the cytotoxic effect must be taken into consideration to derive biocompatible antibacterial agents. METHODS We defined the biocompatibility index (BI) by measuring the antibacterial activity against the test organisms Escherichia coli and Staphylococcus aureus and, in parallel, the cytotoxicity on cultured murine fibroblasts. The antiseptic agents tested were benzalkonium chloride (BAC), cetylpyridinium chloride (CPC), chlorhexidine digluconate (CHX), mild silver protein (MSP), octenidine dihydrochloride (OCT), polyhexamethylene biguanide (PHMB), povidone iodine in solution [PVP-I(s)], povidone iodine in ointment [PVP-I(o)], silver nitrate (AgNO(3)), silver (I) sulfadiazine (SSD) and triclosan (TRI). Assays were carried out for 30 min of exposure at 37 degrees C in the presence of cell culture medium containing 10% fetal bovine serum. The resulting dimensionless BI was defined as the ratio of the concentration at which 50% of the murine fibroblasts are damaged and the microbicidal effect producing at least 3 log(10) (99.9%) reduction. RESULTS The resulting rank ordering of BI for the ratio of fibroblast cytotoxicity to E. coli toxicity was OCT > PHMB > CHX > PVP-I(o) > PVP-I(s) > BAC > CPC > TRI > MSP and that to S. aureus was OCT > PHMB > CHX > CPC > PVP-I(o) > BAC > PVP(s) > TRI > MSP. OCT and PHMB were the most suitable agents with a BI greater than 1. CONCLUSIONS The BI presented may be a useful tool to evaluate antiseptic agents for use in clinical practice.

Risk assessment of a former military base contaminated with organoarsenic-based warfare agents: uptake of arsenic by terrestrial plants.

Organoarsenic-based chemical warfare agents (CWAs) such as the sternutators diphenylchloroarsine (CLARK I), diphenylcyanoarsine (CLARK II) or phenyldichloroarsine (PFIFFIKUS) still pose a notable risk in countries where former military bases that have stored these weapons have not yet been reclaimed. In fact, this is the case for many countries of Eastern Europe and the CIS. One of the most important military bases of the former Third Reich, the Heeresmunitionsanstalt I and II, is situated close to the German-Polish border at Loecknitz (Fig. 1). The German army stored and decanted different compounds of CWAs at this military base until 1945. When the Soviet Army destroyed the base in 1946, large amounts of CWAs and other organoarsenic compounds polluted the soil. Today up to 250 g (!) of arsenic may be found in 1 kg of soil at some places in this area. Since 1991, a Government Working Group has been working on the risk assessment in order to define the scope of reclamation measures. This study investigates the contamination and the uptake of arsenic by plants because little is known about the bioavailability and metabolism of sternutators and their constituents. The total arsenic concentration of nine different species of terrestrial plants with at least six samples per species is presented. In spite of the considerable arsenic contamination of the soil (mean value 923 mg arsenic/kg soil) the plant contamination remained comparably low. The median value of arsenic contamination of the above-ground organs of velvet grass, Holcus lanatus, was 0.7 mg/kg dry wt. and the mean value was 4.3 mg/kg dry wt. due to some highly contaminated samples. The highest arsenic concentration registered was 26 mg/kg dry wt. in a sample of H. lanatus, which was most probably caused by soil particles adhering to the plant. The chemical structure of the arsenic compounds carried by the above-ground plant organs has been determined by gas chromatographic investigations and showed an uptake of triphenyl arsine by the plants.

Comparative Study of in vitro Cytotoxicity of Povidone-Iodine in Solution, in Ointment or in a Liposomal Formulation (Repithel®) and Selected Antiseptics

The cytotoxicity of povidone-iodine in Repithel®, Betaisodona® ointment and Betaisodona® solution was investigated using CHO-K1 cells. To estimate the vitality of test cells after 30 min contact time using vital dye neutral red, the following IC50 were determined: 16–18% Repithel, 8–9% Betaisodona ointment and 1.8–2% Betaisodona solution; using MTT for detecting vitality, the IC50 were: 5–10% Repithel, 1.3–2.5% Betaisodona ointment and 0.6–1.3% Betaisodona solution. Therefore, the first attack of the antiseptic agent iodine to mammalian cells is carried out on enzymes, perhaps by oxidation, followed by membrane attack. Murine fibroblasts were used to compare the cytotoxic impact of povidone-iodine with those of chlorhexidine digluconate (CHex), octenidine dihydrochloride (Oct) and polyhexamethylene biguanide (PHMB). On the base of molecular concentration, povidone-iodine is more than 20 times better tolerated by L929 cells than CHex, Oct or PHMB. Moreover, after 30 min contact of L929 cells with povidone-iodine, there is a temporarily cytotoxic reaction, which leads after 24 h culture to an unexpected revitalisation of murine fibroblasts. This phenomenon was not detected using CHex, Oct or PHMB. Povidone-iodine seems to be the most tolerated antiseptic in comparison with CHex, Oct or PHMB.

Reduced cytotoxicity of polyhexamethylene biguanide hydrochloride (PHMB) by egg phosphatidylcholine while maintaining antimicrobial efficacy

Liposomes or oil-in-water emulsions containing egg yolk phosphatidylcholine (EPC) were combined with aqueous polyhexamethylene biguanide hydrochloride (PHMB). The bactericidal activity of these preparations against Pseudomonas aeruginosa and Staphylococcus aureus as well as their cytotoxicity on cultured murine fibroblasts (L929 cells) was then assayed for either 30 min or 60 min in the presence of cell culture medium containing 10% fetal bovine serum as surrogate for wound fluid. We used two assay designs: in the first bactericidal activity and cytotoxicity were determined in separate experiments; in the second both were determined in one experiment. Combining PHMB and EPC containing o/w emulsions or liposomes protects mammalian cells without neutralizing the antiseptic effect. From all tested combinations the o/w emulsions containing 0.05% PHMB proved to be superior in this respect to the aqueous preparation.

In vitro action of a combination of selected antimicrobial agents and chondroitin sulfate.

Chondroitin sulfate (CS), a highly anionic polymer and the most predominant sulfated glycosaminoglycan in connective tissues, was investigated regarding to its interaction with cationic disinfectants, which are used as antiinfectives in humans. Combinations of cetylpyridiniumchloride (CPC), chlorhexidine (CHex), and polyhexamethylene biguanide (PHMB) with CS, respectively, were prepared and the resulting microbicidal activity of the mixtures was tested in the quantitative suspension test without organic matter. Polyvidone-iodine and Ringers solution were used as controls. Even precipitated, the resulting test combinations behave differently against Staphylococcus aureus, Enterococcus faecium, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans. CPC/CS demonstrated only microbicidal activity against Gram-positive bacteria, and CHex/CS was more active against Gram-negative bacteria and C. albicans. PHMB/CS, especially in combination with CS-A, only revealed an antimicrobial effect against P. aeruginosa after 60 min action. The interaction of cationic disinfectants with CS showed depending on the investigated microorganism a more or less controlled sustained release manner of the microbicidal agent from the precipitated complex, with the only exception of PHMB in combination with CS-C, which is completely neutralized. Polyvidone-iodine and Ringers solution were not affected by CS.

Interaction of polyhexamethylene biguanide hydrochloride (PHMB) with phosphatidylcholine containing o/w emulsion and consequences for microbicidal efficacy and cytotoxicity

Oil-in-water (o/w) emulsions containing egg yolk phosphatidylcholine (EPC) were combined with aqueous polyhexamethylene biguanide hydrochloride (PHMB). The PHMB concentration in the aqueous phase was estimated by filtration centrifugation experiments. In parallel, PHMB concentration was assessed utilizing cytotoxicity assays (neutral red) on cultured murine fibroblasts (L929 cells) and tests of bactericidal efficacy on either Pseudomonas aeruginosa or Staphylococcus aureus. Biological tests were performed in cell culture medium. Filtration centrifugation experiments demonstrated much higher aqueous PHMB concentrations than did the assays for biologically effective PHMB. Therefore, biological test systems should preferably be used to verify effective PHMB concentrations. Tests of microbicidal efficacy in which the same 0.05% PHMB o/w emulsion was re-used 8 times revealed a drug delivery system activated by the presence of test bacteria.

In vitro action of combinations of selected antimicrobial agents and adult bovine articular cartilage (sesamoid bone)

Anatomically intact articular cartilage in form of sesamoid bones from metacarpophalangeal joints of 2-year-old cows was tested for its influence on the microbicidal effect of the iodophore Betaisodona, the bispyridinamine Octenisept, and the biguanide Lavasept. Comparisons were carried out in Hams F12 medium with and without 0.2% bovine serum albumin as organic matter loading. The expected abolition of the microbicidal effect of these antiseptics against the test organisms Escherichia coli or Staphylococcus aureus in the presence of sesamoid bone was not evident. Furthermore, sesamoid bone alone demonstrated antibacterial activity against Staphylococcus aureus, which may involve adherence of bacteria to surface constituents of articular cartilage. Final concentrations of 2.5-5% Betaisodona, 5% Octenisept as well as 0.025% Lavasept are effective in killing of 10(8)-10(9) cfu/ml Escherichia coli or Staphylococcus aureus in the presence of sesamoid bone without the reduction of antimicrobial activity expected from binding to CS, which has previously been demonstrated for CS in solution.

Arsenaufnahme von Wildpflanzen auf einem kampfstoffkontaminierten Rüstungsaltlastenstandort

ZusammenfassungDie Gefährdungsabschätzung von Rüstungsaltlasten, auf denen chemische Kampfstoffe der sog. “Blaukreuzgruppe” hergestellt, abgefüllt oder gelagert wurden, gestaltet sich schwierig, da über die Bioverfügbarkeit und Metabolisierung von Phenylarsinverbindungen nur wenig bekannt ist. Zur Beurteilung des Eintrags altlastentypischer Schadstoffe in die Nahrungskette wurden auf dem Gelände der ehemaligen Heeresmunitionsanstalt Löcknitz die Gesamtarsengehalte von insg. 186 Blütenpflanzenproben bestimmt. Es zeigte sich, daß bei den Pflanzen hohen Futterwerts die Arsenkonzentrationen nicht über 1 ppm anstiegen, während sich bei beprobten Pflanzen mittleren und geringen Futterwerts Arsenkonzentrationen von über 1 ppm bei mehr als 15% der Proben fanden. Insgesamt erscheint die Arsenaufnahme in Wildpflanzen in Anbetracht der nachhaltigen Bodenkontaminationen des Standorts eher gering. Eine endgültige toxikologische Beurteilung des Risikopotentials ist allerdings nicht möglich, da sie die genaue Charakterisierung der (an) organischen Arsenverbindungen in den Pflanzen voraussetzen würde.AbstractThe risk assessment of former ammunition factories, where chemical weapons consisting of diphenylarsenic compounds have been produced or stored is difficult because of the lack of data concerning bioavailability and metabolism of the residues of these sternutators. In order to estimate the contamination of fodder plants by typical pollutants of the former Heeresmunitionsanstalt Löcknitz, the total concentration of arsenic was determined in 186 samples of wild plants. The total arsenic concentration in plants of high feed value did not exceed 1 ppm, whereas 15% of the samples of plants of middle and little value in regard to animal fodder showed arsenic concentrations above 1ppm. The uptake of arsenic by wild plants, however, appears to be comparably low taken the high-grade contamination of the soil into account. A final toxicological evaluation of the risk potential is not yet possible because it requires the characterization of the anorganic and organic arsenic compounds in these plants.

Efficiency of Emulsified Particle-Associated Polyhexamethylenbiguanid-Hydrochlorid (Polihexanide) for Peritoneal Lavage in a Murine Sepsis Model

BACKGROUND Peritoneal lavage is often used for peritonitis, however, the volume and type of lavage fluid varies. Saline or Ringers solution are used most often and lavage is performed until the fluid is clear. However, at present there is no irrigation fluid for peritoneal lavage with residual antiseptic activity. Because the combination of aqueous polyhexamethylenbiguanid-hydrochlorid (PHMB) and egg phosphatidylcholine containing oil/water emulsions (Lipofundin® MCT 20%, B. Braun AG, Melsungen, Germany) protect mammalian cells without neutralizing the antiseptic effect of PHMB, it seemed promising to investigate such human cell protecting, yet antibacterial combination for peritoneal lavage in a murine sepsis model. METHODS After induction of colon ascendens stent peritonitis (CASP) in mice, the foci were eradicated by re-laparotomy, followed by twofold lavage with 2 × 3 mL of the tested emulsion. The following lavage fluids were investigated blindly: 10% Lipofundin/0.05% PHMB, 100% Lipofundin, 0.05% PHMB, and 0.9% saline. After 24 hours the animals were euthanized and organs, blood, and lavage fluid were examined for cytokine levels (tumor necrosis factor [TNF]-α, interferon [IFN]-γ, interleukin [IL]-6, IL-10), liver enzymes (alanine aminotransferase [ALT], aspartate aminotransferase [AST], gamma-glutamyltransferase [gamma-GT], glutamate dehydrogenase [GLDH]), creatinine, and bacterial density. RESULTS Only the combination of Lipofundin/PHMB (n = 23) increased the survival rate. Compared with saline alone, PHMB alone decreased the survival rate. Twenty-four hours after induction of peritonitis, the lowest number of colony forming units (CFU) was observed after lavage with PHMB/Lipofundin in all examined organs, blood, and lavage fluid (p < 0.01). Alanine aminotransferase, AST, and creatinine levels were increased after lavage with PHMB compared with the other lavage fluids (p < 0.05). CONCLUSIONS Peritoneal lavage using 0.05% aqueous PHMB alone resulted in no survival benefit in a CASP murine model. The increase of liver enzymes and creatinine seem to be a toxic side effect of PHMB. However, an emulsion of 0.05% PHMB/10% Lipofundin decreased cytotoxicity while maintaining antiseptic efficiency. The advantage for survival was explained by decrease of bacterial load in organs, blood, and lavage fluid. The results provide a new option for the treatment of peritonitis using peritoneal lavage with the combination of PHMB/Lipofundin.

Impact of the cosmetic mouthwash "Jack Pro Spülung plus" ("rheodol-Spülung plus") on the oral cavity flora, tested in a monocentric, controlled, randomized, blind, cross-over comparative study.

Aim: Jack Pro Spülung Plus (also available as “rheodol-Spülung plus”) is recommended to mechanically maintain oral hygiene as part of an overall oral hygiene concept. Because Jack Pro Spülung Plus contains the active agents polihexanide and tosylchloramide sodium in concentrations below microbicidal efficacy, this study tested the hypothesis that the combination of mechanical rinsing and bacteriostatic effect surpasses the effect of mechanical rinsing alone. Method: The study was performed with 30 volunteers as a monocentric, controlled, randomized, blind, cross-over comparative study. The efficacy of the test product (active agents polihexanide 0.02–0.03% and tosylchloramide sodium 0.004–0.006%) was compared to an aqueous solution of polihexanide (0.02–0.03%) and to Ringer solution as negative control. The efficacy was measured as the reduction of colony forming units (cfu) on buccal mucosa after aerobic and anaerobic cultivation. After determination of pre-values, the volunteers performed mouthrinsing for 30 sec with each of the 3 tested solutions. After 1, 10 and 60 minutes, cfu numbers were determined again. The reduction factor was calculated as the difference between log10 of the measured cfu before and after mouthrinsing with the test solution. The sampling was performed using a template with a smear area of 1 x 1 cm. Results: Using Ringer solution led to a slight mechanically-induced reduction of cfu in the oral cavity 1 min after rinsing the mouth cavity with the solution. After 10 min and 60 min, no influence on the cfu number could be detected. Using Jack pro Spülung Plus led to a bacteriostatic effect up to 60 min after mouthrinsing; 10 min and 60 min after rinsing the efficacy of Ringer solution was also significantly surpassed. The aqueous solution of polihexanide was less effective than Jack pro Spülung Plus after 10 and 60 min. Conclusion: Based on these observations, we conclude that Jack pro Spülung Plus is suitable for improvement of oral hygiene if patients have sensitive oral mucosa, e.g., in cases of aggressive cancer therapy or for patients with tracheostoma.