The ecology and bioactivity of some Greco-Roman medicinal minerals: the case of Melos earth pigments.

Mineral compounds, as pigments and therapeutics, appeared regularly in the technical and medical texts of the Greco-Roman (G-R) world. We have referred to them as 'G-R medicinal minerals' and we suggest that despite their seeming familiarity, there are actually many unknowns regarding their precise nature and/or purported pharmacological attributes. Earth pigments are part of that group. This paper presents a brief overview of our work over the past twenty years relating to: a. the attempt to locate a select number of them in the places of their origin; b. their chemical/mineralogical characterization; c. the study of their ecology via the identification of the microorganisms surrounding them; d. their testing as antibacterials against known pathogens. In the process, and to fulfil the above, we have developed a novel methodological approach which includes a range of analytical techniques used across many disciplines (mineralogy, geochemistry, DNA extraction and microbiology). This paper focuses on a select number of earth pigments deriving from the island of Melos in the SW Aegean, celebrated in antiquity for its Melian Earth, a white pigment, and asks whether they might display antibacterial activity. We demonstrate that some (but not all) yellow, green and black earth pigments do. We also show that the manner in which they were dispensed (as powders or leachates) was equally important. The results, although preliminary, are informative. Given their use since deep time, earth pigments have never lost their relevance. We suggest that the study of their ecology/mineralogy and potential bioactivity allows for a better understanding of how our perception of them, as both pigments and therapeutics, may have evolved.

On metal and 'spoiled' wine: analysing psimythion (synthetic cerussite) pellets (5th-3rd centuries BCE) and hypothesising gas-metal reactions over a fermenting liquid within a Greek pot.

A Pb-based synthetic mineral referred to as psimythion (pl. psimythia) was manufactured in the Greek world at least since the 6th c BCE and routinely by the 4th c BCE. Theophrastus (On Stones, 56) describes its preparation from metallic Pb suspended over a fermenting liquid. Psimythion is considered the precursor of one of western art's most prominent white pigments, i.e. lead white (basic lead carbonate or synthetic hydrocerussite). However, so far, and for that early period, published analyses of psimythia suggest that they consisted primarily of synthetic cerussite. In this paper, we set out to investigate how it was possible to manufacture pure cerussite, to the near exclusion of other phases. We examined the chemical and mineralogical composition (pXRF/XRD) of a small number of psimythion pellets found within ceramic pots (pyxis) from Athens and Boeotia (5th-4th c BCE) in the collection of the National Archaeological Museum (NAM), Athens. Analyses showed that the NAM pellets consisted primarily of Pb/cerussite with small amounts of Ca (some samples) and a host of metallic trace elements. We highlight the reference in the Theophrastus text to 'spoiled wine' (oxos), rather than 'vinegar', as has been previously assumed, the former including a strong biotic component. We carried out DNA sequencing of the pellets in an attempt to establish presence of microorganisms (Acetic Acid Bacteria). None was found. Subsequently, and as a working hypothesis, we propose a series of (biotic/abiotic) reactions which were likely to have taken place in the liquid and vapour phases and on the metal surface. The hypothesis aims to demonstrate that CO2 would be microbially induced and would increase, as a function of time, resulting in cerussite forming over and above hydrocerussite/other Pb-rich phases. Psimythion has for long been valued as a white pigment. What has perhaps been not adequately appreciated is the depth of empirical understanding from the part of psimythion manufacturers of the reactions between abiotic and biotic components within 'oxos'/pot, as key drivers of minerals synthesis. Ultimately, psimythion manufacture may rest in understanding the nature of 'oxos', antiquity's relatively little researched strongest acid.

The interweaving roles of mineral and microbiome in shaping the antibacterial activity of archaeological medicinal clays.

ETHNOPHARMACOLOGICAL RELEVANCE: Medicinal Earths (MEs), natural aluminosilicate-based substances (largely kaolinite and montmorillonite), have been part of the European pharmacopoeia for well over two millennia; they were used generically as antidotes to 'poison'. AIM OF THE STUDY: To test the antibacterial activity of three Lemnian and three Silesian Earths, medicinal earths in the collection of the Pharmacy Museum of the University of Basel, dating to 16th-18th century and following the methodology outlined in the graphical abstract. To compare them with natural clays of the same composition (reference clays) and synthetic clays (natural clays spiked with elements such as B, Al, Ti and Fe); to assess the parameters which drive antibacterial activity, when present, in each group of samples. MATERIALS AND METHODS: a total of 31 samples are investigated chemically (ICP-MS), mineralogically (both bulk (XRD) and at the nano-sized level (TEM-EDAX)); their organic load (bacterial and fungal) is DNA-sequenced; their bioactivity (MIC60) is tested against Gram-positive, S. aureus and Gram-negative, P. aeruginosa. RESULTS: Reference smectites and kaolinites show no antibacterial activity against the above pathogens. However, the same clays when spiked with B or Al (but not with Ti or Fe) do show antibacterial activity. Of the six MEs, only two are antibacterial against both pathogens. Following DNA sequencing of the bioactive MEs, we show the presence within of a fungal component, Talaromyces sp, a fungus of the family of Trichocomaceae (order Eurotiales), historically associated with Penicillium. Talaromyces is a known producer of the exometabolite bioxanthracene B, and in an earlier publication we have already identified a closely related member of the bioxanthracene group, in association with one of the LE samples examined here. By linking fungus to its exometabolite we suggest that this fungal load may be the key parameter driving antibacterial activity of the MEs. CONCLUSIONS: Antibacterial activity in kaolinite and smectite clays can arise either from spiking natural clays with elements like B and Al, or from an organic (fungal) load found only within some archaeological earths. It cannot be assumed, a priori, that this organic load was acquired randomly and as a result of long-term storage in museum collections. This is because, at least in the case of medicinal Lemnian Earth, there is historical evidence to suggest that the addition of a fungal component may have been deliberate.

Greco-Roman mineral (litho)therapeutics and their relationship to their microbiome: The case of the red pigment miltos.

This paper introduces a holistic approach to the study of Greco-Roman (G-R) lithotherapeutics. These are the minerals or mineral combinations that appear in the medical and scientific literature of the G-R world. It argues that they can best be described not simply in terms of their bulk chemistry/mineralogy but also their ecological microbiology and nanofraction component. It suggests that each individual attribute may have underpinned the bioactivity of the lithotherapeutic as an antibacterial, antifungal or other. We focus on miltos, the highly prized, naturally fine, red iron oxide-based mineral used as a pigment, in boat maintenance, agriculture and medicine. Five samples (four geological (from Kea, N. Cyclades) and one archaeological (from Lemnos, NE Aegean)) of miltos were analyzed with physical and biological science techniques. We show that: a. Kean miltos and Lemnian earth/miltos must have been chemically and mineralogically different; b. Lemnian miltos must have been more effective as an antibacterial against specific pathogens (Gram + and Gram - bacteria) than its Kean counterpart; c. two samples of Kean miltos, although similar, chemically, mineralogically and eco-microbiologically (phylum/class level), nevertheless, displayed different antibacterial action. We suggest that this may constitute proof of microbial ecology playing an important role in effecting bioactivity and, interestingly, at the more specific genus/species level. From the perspective of the historian of G-R science, we suggest that it may have been on account of its bioactivity, rather than simply its 'red-staining' effect, that miltos gained prominent entry into the scientific and medical literature of the G-R world.

Increased waterborne blaNDM-1 resistance gene abundances associated with seasonal human pilgrimages to the upper ganges river.

Antibiotic resistance (AR) is often rooted in inappropriate antibiotic use, but poor water quality and inadequate sanitation exacerbate the problem, especially in emerging countries. An example is increasing multi-AR due to mobile carbapenemases, such as NDM-1 protein (coded by blaNDM-1 genes), which can produce extreme drug-resistant phenotypes. In 2010, NDM-1 positive isolates and blaNDM-1 genes were detected in surface waters across Delhi and have since been detected across the urban world. However, little is known about blaNDM-1 levels in more pristine locations, such as the headwaters of the Upper Ganges River. This area is of particular interest because it receives massive numbers of visitors during seasonal pilgrimages in May/June, including visitors from urban India. Here we quantified blaNDM-1 abundances, other AR genes (ARG), and coliform bacteria in sediments and water column samples from seven sites in the Rishikesh-Haridwar region of the Upper Ganges and five sites on the Yamuna River in Delhi to contrast blaNDM-1 levels and water quality conditions between season and region. Water quality in the Yamuna was very poor (e.g., anoxia at all sites), and blaNDM-1 abundances were high across sites in water (5.4 ± 0.4 log(blaNDM-1·mL(-1)); 95% confidence interval) and sediment (6.3 ± 0.7 log(blaNDM-1·mg(-1))) samples from both seasons. In contrast, water column blaNDM-1 abundances were very low across all sites in the Upper Ganges in February (2.1 ± 0.6 log(blaNDM-1·mL(-1))), and water quality was good (e.g., near saturation oxygen). However, per capita blaNDM-1 levels were 20 times greater in June in the Ganges water column relative to February, and blaNDM-1 levels significantly correlated with fecal coliform levels (r = 0.61; p = 0.007). Given that waste management infrastructure is limited in Rishikesh-Haridwar, data imply blaNDM-1 levels are higher in visitor's wastes than local residents, which results in seasonally higher blaNDM-1 levels in the river. Pilgrimage areas without adequate waste treatment are possible "hot spots" for AR transmission, and waste treatment must be improved to reduce broader AR dissemination via exposed returning visitors.

Legionella spp. in UK composts--a potential public health issue?

Over the past 5 years, a number of cases of legionellosis in Scotland have been associated with compost use; however, studies investigating sources of infection other than water systems remain limited. This study delivers the first comprehensive survey of composts commonly available in the UK for the presence of Legionella species. Twenty-two store-bought composts, one green-waste compost and one home-made compost were tested for Legionella by culture methods on BCYE-α medium, and the findings were confirmed by macrophage infectivity potentiator (mip) speciation. Twenty-two of the samples were retested after an enrichment period of 8 weeks. In total, 15 of 24 composts tested positive for Legionella species, a higher level of contamination than previously seen in Europe. Two isolates of Legionella pneumophila were identified, and Legionella longbeachae serogroup 1 was found to be one of the most commonly isolated species. L. longbeachae infection would not be detected by routine Legionella urinary antigen assay, so such testing should not be used as the sole diagnostic technique in atypical pneumonia cases, particularly where there is an association with compost use. The occurrence of Legionella in over half of the samples tested indicates that compost could pose a public health risk. The addition of general hygiene warnings to compost packages may be beneficial in protecting public health.

A comparative assessment of molecular biological and direct microscopic techniques for assessing aquatic systems.

Molecular techniques have clear value for community characterization; however, almost all previous datasets are based upon non-molecular measurements and it is hard to compare "old" data with "new" data because few correlations have been made. Therefore, the purpose of this evaluation was to simultaneously use molecular and non-molecular methods within the same sampling program to determine how data compare. Three methods were used for characterizing microbial populations in Lake 260 (L260) at Experimental Lakes Area (Ontario, Canada) during a whole-lake exposure study. Methods included whole-cell microscopic counts (for bacteria, cyanobacteria, algae and zooplankton), chlorophyll a, and small sub-unit (ssu)-rRNA hybridization using EUB-338, EUB-785, CYAN-785-a/b, EUCA-1379 and UNIV-1390 gene probes. Strong correlations were found between the EUB-785 probe signal and "bacteria minus cyanobacteria" direct counts, and the EUB-338 probe signal and "bacteria plus cyanobacteria" counts. Furthermore, the difference in probe signal between EUB-338 and EUB-785 (a presumptive signature for cyanobacteria and plastids) correlated with cyanobacterial direct counts and also with chlorophyll a. However, EUCA-1379 probe signal did not correlate with algae counts, and UNIV-1390 probe signal only correlated with total bacteria counts. Results suggest that, although ssu-rRNA methods are fast, reproducible, and specifically detect "viable" organisms, their use may be limited to non-eukaryotic populations unless new probes are developed that are more specific.

Field assessment of oxytetracycline exposure to the freshwater macrophytes Egeria densa Planch. and Ceratophyllum demersum L.

In a microcosm study, two aquatic macrophytes, Egeria densa and Ceratophyllum demersum were exposed to nominal concentrations of 0, 5, 20, 50, and 250 microg/L oxytetracycline (n=3), plus 20 microg/L oxytetracycline amended with additional nitrogen (N) and phosphorus (P). Responses were monitored bi-weekly over a six-week exposure period. Both plant species exhibited a significant decline in growth in the 250 microg/L oxytetracycline and the N- and P-amended units. Decreased light penetration resulting from accumulating oxytetracycline by-products appears to be the primary modifier in the growth of these plants. Increased susceptibility to oxytetracycline exposure was noted in some paired plantings (e.g., E. densa root development), relative to individual plants in these treatments, however, no clear explanation for this response is available. Based on the toxicity data generated in this study, we estimate that current concentrations of oxytetracycline in freshwater environments do not pose a direct risk to E. densa and C. demersum.

Fate and effects of enrofloxacin in aquatic systems under different light conditions.

The fate and effects of fluoroquinolone antibacterials (FQ) in the environment is of significance because of apparent increased FQ resistance in environmental and clinical organisms. Here we simultaneously assessed the fate and effects of enrofloxacin (enro), an FQ often used in agriculture, on the chemistry and in situ microbial communities in receiving waters. We added enro to 25 microg/L in nine outdoor mesocosms maintained under three light conditions (in triplicate): full sunlight typical of the upper epilimnion (100% full-light exposure, FLE), partial shading typical of the lower epilimnion (28% FLE), and near-complete shading typical of the hypolimnion (0.5% FLE). Enro disappearance and ciprofloxacin (cipro) formation were monitored over time using LC/MS, and water chemistry and ambient microbial communities (using denaturing gradient gel electrophoresis; DGGE) were characterized. Enro half-lives were 0.8, 3.7, and 72 days for the 100%, 28%, and 0.5% FLE treatments, respectively, creating three distinct FQ exposure scenarios. Although FQ exposures ranged from approximately 6 microg/L for 24 h to approximately 21 microg/L for 30 days, no statistically significant exposure effects were noted in water quality or microbial communities (as indicated by whole-community 16S rDNA DGGE analysis and specific amplification of the QRDR region of gyrase A). Small changes in water chemistry were noted over time; however, changes could not be specifically attributed to FQs. In general, enro addition had minimal effect on water column conditions at the levels and durations used here; however, further investigation is needed to assess effects in aquatic sediments.

The acquisition and maintenance of behavioral skills: a response to Michael.

A response to Michael's (1980) presidential address to the Association for Behavior Analysis is presented. The position is taken that in many instances we have failed to adopt a behavioral approach to dealing with problems within our field concomitant with the shift from an emphasis on behaviorism and a science of behavior to technology. It is argued that we need to be sensitive to the data and consider the contingencies that are operating within the culture. A general strategy and some tactics are presented to acquire and maintain behavioral skills.