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.

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.

Single stage treatment of saline wastewater with marine bacterial-microalgae consortia in a fixed-bed photobioreactor.

Currently, the treatment of aquaculture-origin effluents is mainly performed through land-based recirculating aquaculture systems (RAS). In this study, we evaluate and introduce a novel immobilized/packed bed bioreactor which uses a synthetic textile as the support medium. A marine microbial consortium was developed on the textile by its inoculation with the microalgae Picochlorum sp. The bioreactor was tested with variable loadings of C and N and showed outstanding performance approaching removal rates up to 95% within a few hours (4-5h) of operation. Pyrosequencing analysis revealed a novel microbial consortium consisting mainly of chitrinomycetes, Pseudomonas sp. and the absence of ß-proteobacteria, which is the Class encompassing autotrophic nitrifiers. Quantitative polymerase chain reaction further confirmed these findings suggesting heterotrophic nitrification and aerobic denitrification as the principal mechanisms of N-removal from the bioreactor. Overall our findings reveal the potential of the AdvanTex System for the treatment of marine aquaculture effluents-COD reduction and N-removal, in a single stage.

Substantial underreporting of tuberculosis in West Greece: implications for local and national surveillance.

In order to estimate the reliability of the officially reported national tuberculosis (TB) incidence rates we performed a retrospective review of data collected in regional and national public health framework. TB notifications for the period 2000-2003 were obtained from two major hospitals and three relevant Public Health Departments (PHDs) in the region of West Greece, and subsequently compared with the data reported to the Hellenic Centre for Diseases Control (KEELPNO). During the four-year study period a total of 161 cases of TB were reported to the PHDs in West Greece; 70% of these cases were reported to the KEELPNO. Furthermore only 72 (38.7%) out of the 186 cases of TB identified in the two hospitals were notified to the PHDs. Assuming that the degree of undernotification observed for the two hospitals is the same throughout the region, we estimated that the case detection rate was 14 cases per 100,000 persons per year, i.e. 3.7 times higher than the rate officially reported for the period 2000-2003. Male predominance (2.1, male/female ratio) and an increased incidence in the elders (older than 60 years) and adolescents (10-14 years old) were also evident. The study demonstrated a substantial underestimation of TB burden in West Greece. In the face of the massive influx of immigrants and refugees coming from regions with high TB incidence and the increase of the number of drug-resistant cases a reliable and complete notification of TB is crucial in the planning of programs and development of appropriate control policies.

Hybridisation of F+ RNA coliphages detected in shellfish samples with oligonucleotide probes to assess the origin of microbiological pollution of shellfish.

Current measures for controlling the public health risks associated with bivalve molluscan shellfish consumption rely on the use of Escherichia coli to indicate the sanitary quality of shellfish harvesting areas. However, it has been demonstrated that E. coli is an inadequate indicator of the viral risk associated with shellfish. An alternative indicator, male-specific B+ coliphages, have been investigated as viral indicators of faecal contamination that may provide source-specific information for impacted environmental waters. This study compared the distribution of E. coli and F+ RNA bacteriophages in shellfish grown in harvesting areas of Greece and also examined the presence and proportions of the different subgroups of F+ RNA coliphages in shellfish. F+ RNA bacteriophages were present in shellfish at higher concentrations than E. coli. Elevated numbers of F+ RNA bacteriophages observed in the winter concur with the known increased viral risk associated with shellfish harvested at that time of year in Greece. The majority of F+ RNA coliphages detected in shellfish samples belonged to group IV which indicated the possible presence of animal faecal material in sample harvesting areas. Phages of groups II and III (human waste and human faecal material, respectively) were present at low levels. Finally, 8% of the phages hybridised were found to belong to group I. The presence of group IV showed seasonal distribution (more in winter, less in summer) whereas the other groups did not show any difference. Monitoring of F+ coliphage subgroups may indicate the presence and major sources of microbial inputs to surface waters; however, environmental effects on the relative occurrence of different groups need to be considered.

Differentiation of faecal Escherichia coli from humans and animals by multiple antibiotic resistance analysis.

AIMS: Multiple antibiotic resistance (MAR) was performed on 128 Escherichia coli isolates, recovered from faecal samples of humans and animals (cattle, goat, sheep) to determine and compare their antibiotic resistance patterns and to evaluate them statistically in order to specify the source of the faecal material. METHODS AND RESULTS: Disk diffusion method was applied with a selection of antibiotics. Statistical approach was performed with hierarchical cluster analysis (CA), discriminant analysis (DA) and principal component analysis. Comparing human and animal isolates there was significant difference in levels of resistance to all antibiotics tested (P<0.05) with 46 and 24 distinct resistance patterns for human and animal isolates respectively. CA and DA separated human and animal isolates with a high average rate of correct classification (99.2%), when all animal isolates were pooled together. CONCLUSIONS: MAR analysis compared with appropriate statistical evaluation may provide a useful tool for differentiating the human or animal origin of E. coli isolates derived from environmental samples. Subsequently, determination of the source of faecal pollution becomes possible. SIGNIFICANCE AND IMPACT OF THE STUDY: Determining the source of faecal pollution enables the prediction of possible risk for public health and the application of appropriate management plans for prevention of further contamination.

Microbiological evaluation of bottled non-carbonated ("still") water from domestic brands in Greece.

The microbiological quality of 1,527 samples of bottled non-carbonated ('still') mineral water, purchased from retail outlets and derived from 10 manufacturing companies in Greece, was investigated during the period 1995-2003. Applying the membrane filter technique, the aliquots of water samples (250 ml) were analyzed for the presence and enumeration of total coliforms, Escherichia coli, Enterococcus spp. and Pseudomonas aeruginosa. Also, aerobic bacteria were counted as Heterotrophic Plate Count (HPC) ml(-1) at 22 and 37 degrees C. Positive samples for the parameters tested varied significantly among brands with an overall percentage of 13.95% bottled water samples noncompliant with the Greek water regulation. Microorganisms isolated from the samples tested were identified as species of Pseudomonas, Aeromonas, Pasteurella, Citrobacter, Flavobacterium, Providencia and Enterococcus. The most frequent isolated microorganism during the period of the study was P. aeruginosa. Generally, bacterial load of the samples tested ranged in low levels. The purpose of the current study was to evaluate the microbiological quality of the bottled water provided by domestic brands in the Greek market during the period 1995-2003.

Evaluation of microbiological quality of coastal waters in Greece.

To evaluate the microbiological water quality of bathing sites along the Achaia coastline (south western Greece), a survey was conducted to determine the concentration of faecal bacterial and phage indicators as well as the presence of human viruses. Seawater samples (234) were collected from nine bathing sites on the Achaia coastline and were analysed for the presence of: total coliforms, faecal coliforms, faecal streptococci, Escherichia coli, somatic coliphages, F-RNA bacteriophages, bacteriophages infecting Bacteroides fragilis, enteroviruses, adenoviruses and hepatitis A viruses. Most of the bacteriological analysis results were in accordance with the European Union standards. In all sites, bacteriophages were detected occasionally. Enteroviruses and adenoviruses were detected in 24 samples (10.26%) and 37 samples (15.81%) respectively. No samples were positive for the presence of hepatitis A virus. The overall data indicates that bathing sites are impacted by human faecal material. Both bacterial indicators and phages have low predictive capability for the presence of human viruses in coastal waters. None of the environmental parameters analysed was strongly related to the presence of the indicator organisms and viruses. Appropriate and effective administrative measures that should be taken into account may be considered in order to improve water quality and reduce public health risk.

Differentiation of faecal Escherichia coli from human and animal sources by random amplified polymorphic DNA-PCR (RAPD-PCR).

In this study the assessment of randomly amplified polymorphic DNA (RAPD) analysis was established as a molecular epidemiological tool. RAPD analysis was performed to differentiate faecal Escherichia coli isolates from human and animal sources. E. coli strains (128) were isolated from human and animal faeces (from cattle and sheep). Genomic DNA was extracted and randomly amplified polymorphic DNA-PCR (RAPD-PCR) fingerprinting was performed. Seven arbitrary primers were tested with a view to discriminating between E. coli isolates from humans and E. coli isolates from animals. RAPD profiles were analysed with hierarchical cluster analysis using an unweighted pair group method. RAPD profiles obtained with three of the tested primers (1247, 1290 and 1254) established a distinct differentiation between E. coli isolates from humans and E. coli from animals. Low levels of misclassification and high levels of specificity make RAPD a sensitive, efficient and reliable means of distinguishing closely related strains.

Evaluation of virological quality of sewage from four biological treatment plants by a nested-PCR technique.

In order to determine the virological quality of sewage from four biological treatment plants in Greece (two in the city of Athens and two in the city of Patras), 92 raw sewage samples were analysed for the presence of enteroviruses and adenoviruses during the period from October 2000 to February 2003. A nested-PCR method was used in order to increase the sensitivity of virus detection. Enteroviruses were detected in 43 samples (47%) and adenoviruses in 75 samples (81.5%) of raw sewage by nested PCR. The more frequent isolation of adenoviruses in raw sewage indicated their stability as virological indicators of the pollution of the environment and their increased persistence in sewage.

Development of a multiplex PCR detection of Salmonella spp. and Shigella spp. in mussels.

Multiplex PCR amplification of invA and virA genes was developed enabling simultaneous detection in mussels of Salmonella spp. and Shigella spp., respectively. Simultaneous amplification of products of 215 and 275 bp was obtained either by using mixtures of individual strains of Sh. dysenteriae and Salm. typhimurium or spiked contaminated mussels with both bacteria. In the case of the mussels, 10-100 cells of Salmonella spp. and Shigella per millilitre of homogenate were detected by the multiplex PCR following a pre-enrichment step to increase sensitivity and to ensure that detection was based on the presence of cultivable bacteria. Also, the sensitivity and specificity of this method was evaluated. Multiplex PCR amplification was shown to be an effective, sensitive and rapid method for the simultaneous detection of pathogens in mussels.