BACKGROUND

1. untreated 1 days 105 days Progression of healing (clockwise) 28 days Good Grip Hand Mobility Restored Evaluating the oxidation state of antimicrobial minerals DAVID W METGE1, RONALD W. HARVEY1, DENNIS EBERL1, Alex Blum1, LAURA WASYLENKI2, LYNDA WILLIAMS2 1U.S. Geological Survey, 3215 Marine St. Boulder, CO 80303 2Arizona State University, Tempe, AZ 85287-1404 USA Chemical and mineralogical properties of antibacterial and nonantibacterial clays used in experiments ABSTRACT Understanding the natural mechanism of antimicrobial clays is desirable due to their potential as alternatives to commercially available antimicrobial compounds and due to the emergence of multiple antimicrobial-resistant bacterial strains such as multiple-resistant Staphylococcus aureus (MRSA). Our past research on French green clays used in the treatment of Buruli ulcer (a mycobacterial skin infection), showed that one green clay type killed a broad spectrum of human pathogens including antimicrobial resistant strains. Recently we identified two domestic US clay deposits that exhibit similar antimicrobial characteristics. During evaluation, there was no physical interaction of clays and bacterial surfaces; therefore our investigation focuses on chemical interactions. A comparison of the mineralogical and chemical properties of these deposits shows that: (1) each is dominated by expandable clay minerals, but in detail are mineralogically different, (2) each sample contains minerals with reduced Fe and other transition metals that may produce reactive oxygen or nitrogen radicals capable of degrading DNA, (3) the antibacterial agent(s) are water soluble, and leachates display extreme (<4 or >10) pH and low oxidation state and (4) the minerals and leachate lose their bactericidal capacity when oxidized. Differences in redox between antibacterial and nonantibacterial clays Mineralogical composition of clays (in weight %) used in antibacterial experiments. Clays were analyzed by quantitative X-ray diffraction using the RockJock computer program (Eberl, 2003). ABC = antibacterial clay; NABC = non-antibacterial clay. Bactericidal clays produce changes in pH, redox and dissolved oxygen Results:Significant changes in oxidation state and pH of aqueous leachate for pyroclay, an antibacterial clay, occurred over 24 h period. A drop in pH of ~2 units was accompanied by a ~150mV reduction in redox potential (ORP) and dissolved oxygen. Conclusions: The killing curve for E. coli (upper right) compared to a buffer (pH 3.8) suggests a minimal role for pH and points to the more important role of oxidation state in the bactericidal process. Results:Stability plots for different clays (ABC and NABC) further show that there are chemical equilibria differences between the therapeutic and nontherapeutic clays. Conclusions: Redox and pH differences suggest that the aqueous chemistry produced by these clays would be difficult for pathogenic bacteria to maintain either virulence or pathogenicity. Assessing Chemical Effects of Clays Upon Bacterial Viability Oxidation-Reduction Potential (ORP) of Clays Affected E. coli Bacterial Viability Overview A dialyses tube system which physically separated clay suspensions and bacterial cultures was devised. Following 24 incubations of bacterial isolates within the dialyses tubing in the presence of clay suspensions, bacteria were removed and cells differentially labeled with cell membrane-specific and DNA-specific fluorochromes. This procedure allows evaluation of viable and nonviable cells by either flow cytometry or epifluorescence microscopy. Although viability was related to plate counts, often pathogenic organisms are difficult to culture (ie, viable but unculturable. The oxidation-reduction potential, (ORP), pH and dissolved oxygen levels for each clay suspension. Nonantibacterial clays and cell culture control (No significant cell viability loss BACKGROUND Natural clay minerals have been used to heal skin infections since humanity’s earliest recorded history. Recent documentation of therapeutic clays that heal Buruli ulcer, (BU, a skin infection by Mycobacterium ulcerans; http://www.burulibuster.com), drew our attention to investigate the antibiotic mechanism. Clay treatments could provide an inexpensive alternative. Due to the rising incidence of this disease, the World Health Organization (WHO) declared Buruli ulcer an emerging public health threat (2000). The WHO and NIH supported research to create an understanding of scientific basis of the clay antibacterial activity and before suggesting clay as an antibiotic treatment.The pictures below are a progression of Buruli ulcer healing occurring as a result of daily wetted clay treatment on a hand infection. This was just one of several dozen documented cases of clay treatment of skin infections caused by Mycobacterium ulcerans. Antibacterial clays and Nonantibacterial clays with added reducing agents which reduced cell viability RESULTSExperiments with antibacterial and nonantibacterial clays show lowered ORP levels were accompanied by reduced bacterial viability as assessed by epifluorescence microscopy and/or flow cytometry. Additionally, reduced viability was often accompanied by significant pH changes (> 2pH units) and lowered dissolved oxygen. pH buffers did appear to reduce the effect of antibacterial clay suspensions. Finally, chemical radicals in clay suspensions were detected and may be involved with reduced bacterial viability. CONCLUSIONS Several therapeutic clays were tested- all ABC clay suspensions created significant reducing conditions. Thus, changes in redox chemistry accom-panied reduced bacterial viability within the bacterial isolates tested. However, the degree of viability loss differed between bacterial type. Chemical radicals in ABC suspensions (but not in NABC suspensions) were detected and which might provide a mechanism for antibacterial activity. Bacteria incubated in dialyses tube systems were removed and subjected to fluorescent viability stains. The picture above shows results from a cell culture in which relative abundances of cells were established and placed on an epifluorescent light source which excited the viable-specific and nonviable specific flurorescent dyes. The photo at right, taken with an epifluorescent microscope system designed to simultaneously diffentiate viable (green) and nonviable (reddish) cells. Nonviable Viable Photomicrograph of differentially-stained E coli culture after 24 h exposure to clay suspension in dialysis tube system (788x total magnification). Bactericidal Clay Heals Mycobacterium ulcerans infection Results We observed reduced bacterial cell viability without physical contact of bacteria with clay suspensions, especially with E. coli, Staphylococcus, Streptococcus bacteria. Experiments showed that ABC clays were more effective than other minerals (ie, quartz sand) at reducing cell viability. Some clay types were more antibacterial than others and nonantibacterial clays could be made antibacterial. Conclusions Experiments suggested and confirmed that a chemical transfer or a chemically-mediated mechanism is involved. Also, reduced bacterial viability did not require association or physical contact with clay surfaces. • CONCLUSIONS • The antibacterial clays we tested contain minerals with reduced Fe and other transition metals that may play a role in the antibacterial process by producing reactive oxygen or nitrogen species that potentially degrade organic components critical to cell survival. • The antibacterial agent is soluble at extreme pH and low ORP conditions. There is no physical attraction of the clay to bacteria, and, without water, there is no antibacterial effect. Therefore the antibacterial mechanism involves solution components and chemical reactions affecting the cell wall or metabolic functions. • Clay leachates (with water) are antibacterial initially, but lose their effect on bacteria as the solution becomes oxidized. This points to an important role of the clay in buffering the solution chemistry to conditions that promote an antibacterial reaction. Bactericidal clay degrades biomolecules Methods: Gel electrophoresis was performed on products of 4 hour incubations of bacterial plasmids and linear DNA fragments AMT clay or pyrite (figures at right). Gel conditions: (1.0 % agarose, 1 x TBE buffer, 1 hr, 75 V, 50mA, 300 W, post stained with Sybr Green) RESULTS: AMT-clay or pyrite-treated linear l-ladder DNAs were significantly degraded, particularly in the 100-400 bp lengths (bands not visible or faint; (Lanes : linear DNA + AMT, linear DNA + pyrite). Non AMT-treated linear DNA bands appear unaltered bands and in appropriate sizes for DNA size standards. BACTERIAL CULTURE TESTING Methods: The effect of a soluble component within clays being responsible for clay bactericidal activity was assessed using a dialyses tube system which physically separated bacteria and clay suspesions. Log phase cultures of Staphylococcus epidermidis, and Escherichia coli were loaded to Spectrum dialysis tubes (Float-A-Lyzer 25000 MWCO) and then placed in dialysis sleeves containing clay suspensions. Separate dialysis replicate vials for antbacterial (ABC) and nonantbacterial (NABC) clays suspensions were placed in polystyrene sleeves. These dialysis tube-sleeve systems were then gently agitated (24h, 35C) with gentle agitation. Following a 24 hour exposure to the dialyzed clay suspension chemistry, the bacteria were removed from the dialysis tubes. Bacterial viability was then determined using differential staining (Molecular Probes, Eugene, OR, Live/Dead stain) for viable and nonviable cells. The cultures were enumerated and viability evaluated using epifluorescence microscopy and flow cytometry. Plasmid (pLS588) circular DNA of ~4000-5000 bp are degraded with the AMT clay and, in the presence of pyrite, was nearly completely degraded. Note: no band. Non AMT-treated circular (plasmid) DNA bands appeared as unaltered bands and in appropriate sizes as would be expected. Dialysis tube containingbacteria Dialysis sleeve with clay suspension Antibacterial Clays Clay suspensions Healthy E coli After Clay Treatment Antibacterial and Non-antibacterial clays in suspension Dialysis tube system separated clay suspension from bacterial cells Our results were similar to Cohn et al. (2006), whose group treated ribosomal RNA and plasmid DNA with pyrite. Their study found that reactive oxygen species produced hydroxyl radicals and other products that attack cell walls. Nonviable cells Viable cells Loading bacterial culture to dialysis tube and within biosafety level II hood E. coli bacteria evaluated for viability by using differential eplifluorescent staining