ORIGINAL PAPER
Flow cytometric assessment of the antimicrobial properties of an essential oil mixture against Escherichia coli
C. Girard 1,   K. Fayolle 2,   S. Kerros 1,   F. Leriche 2  
 
More details
Hide details
1
Phytosynthese Innovation Centre, 63200 Mozac, France
2
Université Clermont-Auvergne, VetAgro Sup, INRA, UMR–Fromage UMRF, 15000 Aurillac, France
CORRESPONDING AUTHOR
F. Leriche   

Université Clermont-Auvergne, VetAgro Sup, INRA, UMR–Fromage UMRF, 15000 Aurillac, France
Publication date: 2019-06-13
 
J. Anim. Feed Sci. 2019;28(2):187–198
 
KEYWORDS
TOPICS
ABSTRACT
Essential oils are increasingly being used in human health and animal farming as alternatives to antibiotics. The aim of this study was to better understand the mode of antimicrobial action of a natural essential oil mix (EO mix) by comparison with the colistin, as an antibiotic. The growth inhibitory concentration (GIC) of the EO mix and colistin was determined by turbidimetry. Escherichia coli exposed to EO mix and colistin were analysed by flow cytometry using the fluorescent dyes 3,3-diethyloxacarbocyanine iodide(DiOC2 (3)) to assess membrane potential, and propidium iodide (PI) and SYTO9 to assess membrane integrity following treatment at the GIC and ½-GIC of the EO mix every h for 4 h. At 1 h, treatment with EO mix and colistin resulted in significant cell membrane alteration and depolarization. Membrane integrity measurements identified four sub-populations that were not distributed in the same way between EO mix and antibiotic treated cells. Colistin at GIC and ½-GIC drastically disintegrated the cells that appeared as debris (69.8% of cells were lysed after 1 h of treatment) whereas the EO mix at GIC altered membrane of a majority of cells (67.4 ± 1.3% of cells were partially altered). Contact with ½-GIC EO mix led to sub-populations that persisted or recovered a physiological state with intact membrane (from 1 h to 4 h of treatment, intact cells increased from 23 to 33%). So, it was demonstrated that the EO mix presented antibacterial action against E. coli. It altered membrane properties by decreasing its polarity and integrity which were reversible phenomena here.
 
REFERENCES (37)
1.
Alagawany M., Abd El-Hack M.E., Farag M.R., Tiwari R., Dhama K., 2015. Biological effects and modes of action of carvacrol in animal and poultry production and health – a review. Adv. Anim. Vet. Sci. 3(2s), 73–84, https://doi.org/10.14737/journ....
 
2.
Baranyi J., McClure P.J., Sutherland J.P., Roberts T.A., 1993. Modeling bacterial growth responses. J. Ind. Microbiol. 12, 190–194, https://doi.org/10.1007/BF0158....
 
3.
Bassolé I.H.N., Juliani H.R., 2012. Essential oils in combination and their antimicrobial properties. Molecules 17, 3989–4006, https://doi.org/10.3390/molecu....
 
4.
Berney M., Hammes F., Bosshard F., Weilenmann H.-U., Egli T., 2007. Assessment and interpretation of bacterial viability by using the LIVE/DEAD BacLight kit in combination with flow cytometry. Appl. Environ. Microbiol. 73, 3283–3290, https://doi.org/10.1128/AEM.02....
 
5.
Bouhdid S., Abrini J., Zhiri A., Espuny M.J., Manresa A., 2009. Investigation of functional and morphological changes in Pseudomonas aeruginosa and Staphylococcus aureus cells induced by Origanum compactum essential oil. J. Appl. Microbiol. 106, 1558–1568, https://doi.org/10.1111/j.1365....
 
6.
Burt S., 2004. Essential oils: their antibacterial properties and potential applications in foods –a review. Int. J. Food Microbiol. 94, 223–253, https://doi.org/10.1016/j.ijfo....
 
7.
Demir E., Sarica Ş., Özcan M.A., Suiçmez M., 2003. The use of natural feed additives as alternatives for an antibiotic growth promoter in broiler diets. Br. Poult. Sci. 44, Suppl. 2, 44–45, https://doi.org/10.1080/713655....
 
8.
Deris Z.Z., Akter J.., Sivanesan S., Roberts K.D., Thompson P.E., Nation R.L., Li J., Velkov T., 2014. A secondary mode of action of polymyxins against Gram-negative bacteria involves the inhibition of NADH-quinone oxidoreductase activity. J. Antibiot. 67, 147–151, https://doi.org/10.1038/ja.201....
 
9.
Di Pasqua R., Hoskins N., Betts G., Mauriello G., 2006. Changes in membrane fatty acids composition of microbial cells induced by addiction of thymol, carvacrol, limonene, cinnamaldehyde, and eugenol in the growing media. J. Agric. Food Chem. 54, 2745–2749, https://doi.org/10.1021/jf0527....
 
10.
Fairbrother J.M., Nadeau É., Gyles C.L., 2005. Escherichia coli in postweaning diarrhea in pigs: an update on bacterial types, pathogenesis, and prevention strategies. Anim. Health Res. Rev. 6, 17–39, https://doi.org/10.1079/AHR200....
 
11.
Gill A.O., Holley R.A., 2004. Mechanisms of bactericidal action of cinnamaldehyde against Listeria monocytogenes and of eugenol against L. monocytogenes and Lactobacillus sakei. Appl. Environ. Microbiol. 70, 5750–5755, https://doi.org/10.1128/AEM.70....
 
12.
Grela E.R., Krusiński R., Matras J., 1998. Efficacy of diets with antibiotic and herb mixture additives in feeding of growingfinishing pigs. J. Anim. Feed Sci. 7, Suppl. 1, 171–175, https://doi.org/10.22358/jafs/....
 
13.
Hashemi S.R., Davoodi H., 2010. Phytogenics as new class of feed additive in poultry industry. J. Anim. Vet. Adv. 9, 2295–2304, https://doi.org/10.3923/javaa.....
 
14.
Hayouni E.A., Bouix M., Abedrabba M., Leveau J.-Y., Hamdi M., 2008. Mechanism of action of Melaleuca armillaris (Sol. Ex Gaertu) Sm. essential oil on six LAB strains as assessed by multiparametric flow cytometry and automated microtiterbased assay. Food Chem. 111, 707–718, https://doi.org/10.1016/j.food....
 
15.
Helander I.M., Alakomi H.-L., Latva-Kala K., Mattila-Sandholm T., Pol I., Smid E.J., Gorris L.G.M., von Wright A., 1998. Characterization of the action of selected essential oil components on Gram-negative bacteria. J. Agric. Food Chem. 46, 3590–3595, https://doi.org/10.1021/jf9801....
 
16.
Kantas D., Papatsiros V.G., Tassis P.D., Athanasiou L.V., Tzika E.D., 2015. The effect of a natural feed additive (Macleaya cordata), containing sanguinarine, on the performance and health status of weaning pigs. Anim. Sci. J. 86, 92–98, https://doi.org/10.1111/asj.12....
 
17.
Khattak F., RonchiA., CastelliP., SparksN., 2014. Effects of natural blend of essential oil on growth performance, blood biochemistry, cecal morphology, and carcass quality of broiler chickens. Poult. Sci. 93, 132–137, https://doi.org/10.3382/ps.201....
 
18.
Ku Y.-H., Lee M.-F., Chuang Y.-C., Chen C.-C., Yu W.-L., 2015. In vitro activity of colistin sulfate against Enterobacteriaceae producing extended-spectrum β-lactamases. J. Microbiol. Immunol. Infect. 48, 699–702, https://doi.org/10.1016/j.jmii....
 
19.
Leclerc S., Boerlin P., Gyles C., Dubreuil J.D., Mourez M., Fairbrother J.M., Harel J., 2007. paa, originally identified in attaching and effacing Escherichia coli, is also associated with enterotoxigenic E. coli. Res. Microbiol. 158, 97–104, https://doi.org/10.1016/j.resm....
 
20.
Linder K., Oliver J.D., 1989. Membrane fatty acid and virulence changes in the viable but nonculturable state of Vibrio vulnificus. Appl. Environ. Microbiol. 55, 2837–2842.
 
21.
Manini E., Danovaro R., 2006. Synoptic determination of living/dead and active/dormant bacterial fractions in marine sediments. FEMS Microbiol. Ecol. 55, 416–423, https://doi.org/10.1111/j.1574....
 
22.
Mikulášová M., Chovanová R., Vaverková Š., 2016. Synergism between antibiotics and plant extracts or essential oils with efflux pump inhibitory activity in coping with multidrug-resistant staphylococci. Phytochem. Rev. 15, 651–662, https://doi.org/10.1007/s11101....
 
23.
Nazzaro F., Fratianni F., De Martino L., Coppola R., De Feo V., 2013. Effect of essential oils on pathogenic bacteria. Pharmaceuticals 6, 1451–1474, https://doi.org/10.3390/ph6121....
 
24.
Novo D., Perlmutter N.G., Hunt R.H., Shapiro H.M., 1999. Accurate flow cytometric membrane potential measurement in bacteria using diethyloxacarbocyanine and a ratiometric technique. Cytometry 35, 55–63, https://doi.org/10.1002/(sici)...<55::aid-cyto8>3.0.co;2-2
 
25.
Oliver J.D., 2010. Recent findings on the viable but nonculturable state in pathogenic bacteria. FEMS Microbiol. Rev. 34, 415–425, https://doi.org/10.1111/j.1574....
 
26.
Paparella A., Taccogna L., Aguzzi I., Chaves-López C., Serio A., Marsilio F., Suzzi G., 2008. Flow cytometric assessment of the antimicrobial activity of essential oils against Listeria monocytogenes. Food Control 19, 1174–1182, https://doi.org/10.1016/j.food....
 
27.
Regulation (EC) No 1831/2003 of the European Parliament and of the Council of 22 September 2003 on additives for use in animal nutrition. O. J. L268, 18.10.2003, pp. 29–43.
 
28.
Shen S., Zhang T., Yuan Y., Lin S., Xu J., Ye H., 2015. Effects of cinnamaldehyde on Escherichia coli and Staphylococcus aureus membrane. Food Control 47, 196–202, https://doi.org/10.1016/j.food....
 
29.
Somolinos M., García D., Condón S., Mackey B., Pagán R., 2010. Inactivation of Escherichia coli by citral. J. Appl. Microbiol. 108, 1928–1939, https://doi.org/10.1111/j.1365....
 
30.
Stiefel P., Schmidt-Emrich S., Maniura-Weber K., Ren Q., 2015. Critical aspects of using bacterial cell viability assays with the fluorophores SYTO9 and propidium iodide. BMC Microbiol. 15, 36, https://doi.org/10.1186/s12866....
 
31.
Stocks S.M., 2004. Mechanism and use of the commercially available viability stain, BacLight. Cytometry 61A, 189–195, https://doi.org/10.1002/cyto.a....
 
32.
Tiihonen K., Kettunen H., Bento M.H.L., Saarinen M., Lahtinen S., Ouwehand A.C., Schulze H., Rautonen N., 2010. The effect of feeding essential oils on broiler performance and gut microbiota. Br. Poult. Sci. 51, 381–392, https://doi.org/10.1080/000716....
 
33.
Velkov T., Thompson P.E., Nation R.L., Li J., 2010. Structure–activity relationships of polymyxin antibiotics. J. Med. Chem. 53, 1898–1916, https://doi.org/10.1021/jm9009....
 
34.
Velkov T., Roberts K.D., Nation R.L., Thompson P.E., Li J., 2013. Pharmacology of polymyxins: new insights into an ‘old’ class of antibiotics. Future Microbiol. 8, 711–724, https://doi.org/10.2217/fmb.13....
 
35.
Xu J., Zhou F., Ji B.-P., Pei R.-S., Xu N., 2008. The antibacterial mechanism of carvacrol and thymol against Escherichia coli. Lett. Appl. Microbiol. 47, 174–179, https://doi.org/10.1111/j.1472....
 
36.
Yahav D., Farbman L., Leibovici L., Paul M., 2012. Colistin: new lessons on an old antibiotic. Clin. Microbiol. Infect. 18, 18–29, https://doi.org/10.1111/j.1469....
 
37.
Yang C., Chowdhury M.A., Huo Y., Gong J., 2015. Phytogenic compounds as alternatives to in-feed antibiotics: potentials and challenges in application. Pathogens 4, 137–156, https://doi.org/10.3390/pathog....
 
 
CITATIONS (4):
1.
1,2-Diarylethanols—A New Class of Compounds That Are Toxic to E. coli K12, R2–R4 Strains
Paweł Kowalczyk, Damian Trzepizur, Mateusz Szymczak, Grzegorz Skiba, Karol Kramkowski, Ryszard Ostaszewski
Materials
 
2.
δ-Lactones—A New Class of Compounds That Are Toxic to E. coli K12 and R2–R4 Strains
Paweł Kowalczyk, Barbara Gawdzik, Damian Trzepizur, Mateusz Szymczak, Grzegorz Skiba, Stanisława Raj, Karol Kramkowski, Rafał Lizut, Ryszard Ostaszewski
Materials
 
3.
Effectiveness of Two Plant-Based In-Feed Additives against an Escherichia coli F4 Oral Challenge in Weaned Piglets
Daniel Montoya, Matilde D’Angelo, Susana Martín-Orúe, Agustina Rodríguez-Sorrento, Mireia Saladrigas-García, Coralie Araujo, Thibaut Chabrillat, Sylvain Kerros, Lorena Castillejos
Animals
 
4.
Chemical composition and biological activity of Peucedanum dhana A. Ham essential oil
Sarunpron Khruengsai, Teerapong Sripahco, Narawadee Rujanapun, Rawiwan Charoensup, Patcharee Pripdeevech
Scientific Reports
 
ISSN:1230-1388