Identification of the Antimicrobial Effect of Liquorice Extracts on Gram-Positive Bacteria: Determination of Minimum Inhibitory Concentration and Mechanism of Action Using a luxABCDE Reporter Strain
Natural preservatives have been used as alternatives to traditional chemical preservatives; however, a limited number have been commercially developed and many remain to be investigated as sources of safer and effective antimicrobials. In this study, we have been investigating the antimicrobial activity of an extract of Glycyrrhiza glabra (liquorice) that was provided as a waste material from the production of liquorice flavourings for the food industry, and to investigate if this retained the expected antimicrobial activity so it could be used as a natural preservative. Antibacterial activity of liquorice extract was screened for evidence of growth inhibition against eight species of Gram-negative and Gram-positive bacteria, including Listeria monocytogenes, Listeria innocua, Staphylococcus aureus, Enterococcus faecalis and Bacillus subtilis. The Gram-negative bacteria tested include Pseudomonas aeruginosa, Escherichia coli and Salmonella typhimurium but none of these were affected by the extract. In contrast, for all of the Gram-positive bacteria tested, growth was inhibited as monitored using optical density. However parallel studies using viable count indicated that the cells were not killed meaning that the extract was bacteriostatic rather than bacteriocidal. The Minimum Inhibitory Concentration [MIC] and Minimum Bactericidal Concentration [MBC] of the extract was also determined and a concentration of 50 µg ml-1 was found to have a strong bacteriostatic effect on Gram-positive bacteria. Microscopic analysis indicated that there were changes in cell shape suggesting the cell wall was affected. In addition, the use of a reporter strain of Listeria transformed with the bioluminescence genes luxABCDE indicated that cell energy levels were reduced when treated with either 12.5 or 50 µg ml-1 of the extract, with the reduction in light output being proportional to the concentration of the extract used. Together these results suggest that the extract is inhibiting the growth of Gram-positive bacteria only by damaging the cell wall and/or membrane.
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 Organization, W. H. WHO estimates of the global burden of foodborne diseases: foodborne disease burden epidemiology reference group 2007–2015 (cited 2016 Feb 8).
 WTO. International trade statistics 2015. Geneva: World Trade Organization.
 WHO. Food safety & food-borne illness. Fact sheet no. 237 (reviewed March 2007). Geneva: World Health Organization; 2007a.
 WHO. The world health report, 2007. Global public health security in the 21st century. Geneva: World Health Organization; 2007b.
 Farber, J., Coates, F. & Daley, E. 1992. Minimum water activity requirements for the growth of Listeria monocytogenes. Letters in Applied Microbiology, 15, 103-105.
 Mclinden, T., Sargeant, J. M., Thomas, M. K., Papadopoulos, A. & Fazil, A. 2014. Component costs of foodborne illness: a scoping review. BMC Public Health, 14, 1.
 Alocilja, E. C. & Radke, S. M. 2003. Market analysis of biosensors for food safety. Biosensors and Bioelectronics, 18, 841-846.
 Chemburu, S., Wilkins, E. & Abdel-Hamid, I. 2005. Detection of pathogenic bacteria in food samples using highly-dispersed carbon particles. Biosensors and Bioelectronics, 21, 491-499.
 Thomas, M. K., Vriezen, R., Farber, J. M., Currie, A., Schlech, W. & Fazil, A. 2015. Economic cost of a Listeria monocytogenes outbreak in Canada, 2008. Foodborne Pathogens and Disease, 12, 966-971.
 Olszewska, M. A., Panfil‐Kuncewicz, H. & Łaniewska‐Trokenheim, Ł. 2015. Detection of Viable but Non-culturable cells of Listeria monocytogenes with the use of direct epifluorescent filter technique. Journal of Food Safety, 35, 86-90.
 RAGHU, R. 2013. Listeria monocytogenes: An interesting pathogen. Microbiology Focus 5, 1–2.
 Montañez-Izquierdo, V. Y., Salas-Vázquez, D. I. & Rodríguez-Jerez, J. J. 2012. Use of epifluorescence microscopy to assess the effectiveness of phage P100 in controlling Listeria monocytogenes biofilms on stainless steel surfaces. Food Control, 23, 470-477.
 Vazquez-Boland, J. A., Kuhn, M., Berche, P., Chakraborty, T., Dominguez-Bernal, G., Goebel, W., et al. (2001). Listeria pathogenesis and molecular virulence determinants. Clinical Microbiology Reviews, 14(3), 584e640.
 Nocker, A., Caspers, M., Esveld-Amanatidou, A., Van der Vossen, J., Schuren, F., Montijn, R. & Kort, R. 2011. A multiparameter viability assay for stress profiling applied to the food pathogen Listeria monocytogenes F2365. Applied and Environmental Microbiology, 77, 6433-40.
 Hill, P. J. & Stewart, G. S. 1994. Use of lux genes in applied biochemistry. Journal of Bioluminescence and Chemiluminescence, 9, 211-215.
 Meighen, E. 1993. Bacterial bioluminescence: organization, regulation, and application of the lux genes. The FASEB Journal, 7, 1016-1022.
 Perehinec, T. M., Qazi, S. N., Gaddipati, S. R., Salisbury, V., Rees, C. E. & Hill, P. J. 2007. Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria. BMC Molecular Biology, 8, 1.
 Robinson, G. M., Tonks, K. M., Thorn, R. M. & Reynolds, D. M. 2011. Application of bacterial bioluminescence to assess the efficacy of fast-acting biocides. Antimicrobial Agents and Chemotherapy, 55, 5214-5219.
 Lorang, J., Tuori, R., Martinez, J., Sawyer, T., Redman, R., Rollins, J., Wolpert, T., Johnson, K., Rodriguez, R. & Dickman, M. 2001. Green fluorescent protein is lighting up fungal biology. Applied and Environmental Microbiology, 67, 1987-1994.
 Zimmer, M. 2002. Green fluorescent protein (GFP): applications, structure, and related photophysical behavior. Chemical reviews, 102, 759-782.
 Lowder, M., Unge, A., Maraha, N., Jansson, J. K., Swiggett, J. & Oliver, J. D. 2000. Effect of Starvation and the Viable-but-Nonculturable State on Green Fluorescent Protein (GFP) Fluorescence in GFP-Tagged Pseudomonas fluorescens A506. Applied and Environmental Microbiology, 66, 3160-3165.
 Qazi, S., Rees, C., Mellits, K. & Hill, P. 2001. Development of gfp vectors for expression in Listeria monocytogenes and other low G+C Gram-positive bacteria. Microbial Ecology, 41, 301-309.
 Qazi, S. N., Counil, E., Morrissey, J., Rees, C. E., Cockayne, A., Winzer, K., Chan, W. C., Williams, P. & Hill, P. J. 2001. agr expression precedes escape of internalized Staphylococcus aureus from the host endosome. Infection and Immunity, 69, 7074-7082.