1. Combating the rising global threat of antimicrobial resistance with clay minerals: A study on two major hospital superbugs
E. C. Myles1*, I. A. Centeleghe1**, L. Baillie2, K. A. BéruBé1, T. Jones3, J. A. Blaxland2, J. Thwaite4
1School of Biosciences, Cardiff University, UK 2School of Pharmacy and Pharmaceutical Sciences, Cardiff University, UK 3School of Earth and Ocean Sciences, Cardiff University, UK 4DSTL, Ministry of Defence, Porton Down, UK
Introduction
Results
Clostridium difficile is an anaerobic, spore forming bacillus responsible for ≈13,897 cases of infectious diarrhoea within hospitals in England per year1.
Methicillin-resistant Staphylococcus aureus is a coccal bacterium, associated with mortality rates of ≈20% in hospitals worldwide2.
Historically, clays have often been used to treat intestinal ailments and mild skin conditions3.
Recently, it has been shown that certain clays possess antimicrobial properties effective against Escherichia coli4.
Clays which are high in iron, particularly those found in basaltic terrains, have been found to be bioreactive due to the production of reactive oxygen species (ROS)5.
ROS are known to induce oxidative stress and damage bacterial DNA6.
We aim to assess the antimicrobial potential of seven different clays against C. difficile and MRSA and by characterisation, we aim to elucidate the underlying mechanism of the clays bioreactivity. A. B. C. D.
Methods
Clay added to PBS (0.15 g/mL) and autoclaved at 121ºC for 15 minutes
Figure 1. Mineral composition of clays (A) Kaolinite, (B) French green, (C) Madeira red clay and (D) Premium red clay. Mineralogy obtained through X-Ray Diffraction, using powdered samples Philips PW1710 Automated Powder Diffractometer using Cu K radiation at 35kV and 40mA.
Vegetative C. difficile (DS1813) cultured in BHI broth under anaerobic conditions for 48 hours
MRSA (ATCC 49775) and UHW clinical isolate cultured in LB overnight at 37ºC and shaken at 200 rpm
Cultures standardised to ≈1x106 cfu/mL and added to each clay suspension in a 1:1 ratio
Cultures standardised to ≈1x107 cfu/mL and added to French green and Premium red clay in a 1:1 ratio
Inoculated clay suspensions incubated anaerobically for 48 hours
Inoculated clay suspensions incubated at 37ºC and shaken at 200 rpm, for 1, 2, 4, 8 and 19 hours
Figure 2. The effect of clay samples on Clostridium difficile (DS1813) growth after a 48hr incubation period. French green clay exhibits a significant decrease (p<0.001) in cfu/mL compared to the control.
Miles and Misra drop counts used to assess antibacterial activity
Conclusions
Only French green clay has shown antibacterial properties amongst the two superbugs tested.
High level of quartz (91%) in French green clay may contribute to its antibacterial properties.
Greater percentage of iron oxide minerals found in red clays does not seem to have an impact on antibacterial potential of the clay as previously hypothesised.
Figure 3. Comparison of the effect of Premium red clay and French green clay on a clinical isolate MRSA over a 1, 2, 4, 8 and 19 hour period.
Future Work
Transmission electron microscopy to observe clay-bacteria interactions, as well as metal ion localisations within the clays
Haemolysis assays to determine bioreactivity of the clays with human cells
Via the use of 3D intestinal and skin tissue models, to assess the feasibility of French green clay as a therapeutic agent (nutraceutical)
References
1. CCG Public Health England
2. Stefani, S. et al Int J Antimicrob Ag 39:
3. Choy, J-H. et al Appl Clay Sci 36:
4. Williams, L. B. et al Environ Sci Technol 45:
5. Hassan, Y Podoconiosis: Geotoxicology of Soils in Basalt Terrain. MRes Thesis, CU.
6. Park, S. and Imlay, J. A J Bacteriol 185:1942–1950.
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