Evidence-Based Complementary and Alternative Medicine

Evidence-Based Complementary and Alternative Medicine / 2016 / Article

Research Article | Open Access

Volume 2016 |Article ID 1643762 | 4 pages | https://doi.org/10.1155/2016/1643762

Antimicrobial Susceptibility of Escherichia coli Strains Isolated from Alouatta spp. Feces to Essential Oils

Academic Editor: Thierry Hennebelle
Received28 Mar 2016
Accepted09 May 2016
Published30 May 2016

Abstract

This study evaluated the in vitro antibacterial activity of essential oils from Lippia graveolens (Mexican oregano), Origanum vulgaris (oregano), Thymus vulgaris (thyme), Rosmarinus officinalis (rosemary), Cymbopogon nardus (citronella), Cymbopogon citratus (lemongrass), and Eucalyptus citriodora (eucalyptus) against Escherichia coli () strains isolated from Alouatta spp. feces. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined for each isolate using the broth microdilution technique. Essential oils of Mexican oregano (MIC mean = 1818 μg mL−1; MBC mean = 2618 μg mL−1), thyme (MIC mean = 2618 μg mL−1; MBC mean = 2909 μg mL−1), and oregano (MIC mean = 3418 μg mL−1; MBC mean = 4800 μg mL−1) showed the best antibacterial activity, while essential oils of eucalyptus, rosemary, citronella, and lemongrass displayed no antibacterial activity at concentrations greater than or equal to 6400 μg mL−1. Our results confirm the antimicrobial potential of some essential oils, which deserve further research.

1. Introduction

The indiscriminate use of antibacterial agents has led to one of the largest recent global health problems which is the emergence of bacterial resistance. Several bacteria genera have developed multidrug resistance, including Escherichia coli [1]. E. coli are Gram-negative, nonsporulating facultative anaerobes, found primarily in the gastrointestinal tract of different species of domestic and wild animals and environments such as soil, water, and plants [2]. This pathogen can cause mild to severe infection, possibly leading to death from septicemia depending on the bacterial strain and its virulence, as well as host-related factors such as age and immunity [3].

In nonhuman primates, enteric infection by E. coli and the isolation of pathogenic strains from healthy animals have been documented [4]. Moreover, there are reports of the isolation of resistant and multidrug-resistant E. coli strains from wild animals [57]. This finding is important given the increased contact between wild animals and humans, enabling cross-species transmission (CRT) of these bacteria. Additionally, the synthesis of new antimicrobials has declined in recent years. As such, new treatment options are needed to overcome bacterial resistance.

Essential oils (EOs) are volatile and complex natural products derived from the secondary metabolism of plants and can be found in different plant parts, including the leaves and stalk. EOs probably consist of 20 to 60 different compounds, in which at least two or three are in higher concentrations, depending on the EOs [8]. These compounds exhibit significant therapeutic and pharmacological potential as well as antimicrobial properties, already established for Gram-positive bacteria and Gram-negative bacteria found in different animals species, including humans [716]. The main compounds with possible antimicrobial activity are terpinenes, cymenes, thymol, and carvacrol [1720]. This antimicrobial effect is mainly related to changes on bacterial cell membrane permeability and integrity [21].

The Howler monkey is a primate from the family Atelidae and genus Alouatta. It is widely distributed, occurring from the states of Bahia to Rio Grande do Sul, and is listed as an endangered species [22]. Howlers are an arboreal species and their diet consists mainly of fruit, leaves, seeds, and flowers [23]. In Brazil, it is common practice to keep monkeys and other wild animals in captivity, where they are generally treated like humans. The animals are fed and medicated indiscriminately, largely receiving antibacterial agents. In addition, direct owner-animal contact is a public health problem, given the possible transmission of infectious zoonotic microorganisms as well as drug-resistant bacteria.

The aim of our study was to evaluate the susceptibility of 22 Escherichia coli strains isolated from captive Howler monkeys (Alouatta) to seven essential oils, in order to assess their potential use as an alternative treatment for E. coli infection.

2. Materials and Methods

2.1. Escherichia coli Strains Tested

We studied 22 strains of E. coli, isolated from the feces of Howler monkeys (Alouatta spp.) with diarrhea from the Anaerobe Laboratory of Universidade Federal de Santa Maria (UFSM).

2.2. Essential Oils

The essential oils tested were Mexican oregano (Lippia graveolens), oregano (Origanum vulgare), rosemary (Rosmarinus officinalis), eucalyptus (Eucalyptus citriodora), citronella (Cymbopogon nardus), lemongrass (Cymbopogon citratus), and thyme (Thymus vulgaris). Mexican oregano essential oil was purchased from Agroindustrial Don Pablo (Chihuahua, CHIH, Mexico). Oregano, rosemary, eucalyptus, citronella, lemongrass, and thyme essential oils were purchased from Essential 7 (Roswell, New Mexico, USA), and all of them came in sealed amber glass bottles. EOs selection was based on previous studies from our laboratory and other studies [1012, 19, 24, 25].

2.3. Minimum Inhibitory Concentration (MIC)

The essential oils were weighed (1 g), diluted in methanol to a concentration of 640 mg mL−1 (solution I), and then diluted in Müller-Hinton broth at a proportion of 1 : 100, obtaining a concentration of 6400 μg mL−1 (solution II). Based on standard M7-A7 of the Clinical and Laboratory Standards Institute (CLSI) (2006) [26], 100 μL volumes of Müller-Hinton broth were distributed on a microtiter plate. Next, serial dilution was performed with solution II, obtaining final concentrations of 3200, 1600, 800, 400, 200, and 100 μg mL−1. The E. coli were cultivated in Müller-Hinton agar and the colonies were then suspended in 0.085% saline solution, producing turbidity equivalent to McFarland Standard number 0.5 (1 × 108 UFC mL−1). Each well containing essential oils was then inoculated with 10 μL (1 × 105 UFC mL−1) of this suspension. The microplates were incubated aerobically at 35°C/24 h. The MIC is the lowest concentration of essential oil that will inhibit bacterial growth. Positive controls for inocula growth as well as solvent and negative (medium alone) controls were included. All experiments were performed in triplicate.

2.4. Minimum Bactericidal Concentration (MBC)

Minimum bactericidal concentration is the lowest concentration of essential oils required to kill the inoculum and was determined by the wells with no visible bacterial growth after 24 h of incubation. A 10 μL aliquot was transferred from these wells to the surface of the Müller-Hinton agar. Essential oil concentration declined after 24 h incubation at 35°C, with no bacterial growth observed. Experiments were performed in triplicate.

3. Results and Discussion

In recent years, research has been conducted on the susceptibility of essential oils and their chemical compounds to different bacteria species isolated from domestic animals, humans, and food [9, 11, 12, 20, 27, 28]. The present study is the first to present results on the susceptibility of 22 E. coli strains from Howler monkeys (Alouatta spp.) to seven essential oils.

The oil from Lippia graveolens was the most effective essential oil against the 22 E. coli strains tested, of which 27.2% were inhibited at a concentration of 800 μg mL−1 and 100% were inhibited by a concentration of 3200 μg mL−1 (mean MIC = 1818 μg mL−1; mean MBC = 2618 μg mL−1) (Table 1). This study found similar results to those reported in a previous investigation showing a moderate antimicrobial effect of Mexican oregano against E. coli isolated from poultry and cattle [12]. However, based on the MIC and MBC values obtained here, it can be inferred that the E. coli strains isolated from Howler monkeys showed greater susceptibility to essential oil of Mexican oregano than those isolated from poultry and cattle [12].


Essential oilsMIC (g mL−1)MBC (g mL−1)
BandMIC50MIC90MeanBandMBC50MBC90Mean

Lippia graveolens800–3200160032001818800–3200320032002618
Thymus vulgaris1600–32003200320026181600–3200320032002909
Origanum vulgare1600–32003200320034183200–6400320064004800
Rosmarinus officinalis>6400>6400>6400ND000ND
Eucalyptus citriodora>6400>6400>6400ND000ND
Cymbopogon nardus>6400>6400>6400ND000ND
Cymbopogon citratus>6400>6400>6400ND000ND

MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration; ND: not determined.

Results obtained for thyme (Thymus vulgaris) indicated a lower bactericidal effect and susceptibility than L. graveolens, with only 36.36% (8/22) of the 22 E. coli strains tested inhibited by a concentration of 600 μg mL−1 and 63.63% (14/22) inhibited at 3200 μg mL−1 (mean MIC = 2618 μg mL−1; mean MBC = 2909 μg mL−1) (Table 1). Other studies also show the antibacterial activity of T. vulgaris against E. coli strains isolated from different animal species [12, 18, 20]. However, Sartoratto et al. (2004) [18] found no effect for T. vulgaris against E. coli CCT0547.

The major compounds of L. graveolens and T. vulgaris EOs are -cymene, γ-terpinene, thymol, and carvacrol [17, 19]. These compounds have shown antimicrobial activity against some bacteria, especially E. coli strains [8, 10, 17, 27]. Although the chemical compounds from the EOs used in our study were not analyzed, it could be suggested that -cymene, γ-terpinene, thymol, and carvacrol were responsible for the antimicrobial effect against E. coli strains isolated from Howler monkeys, since those EOs were already evaluated in a previous study of our group [19]. However, further studies evaluating these constituents separately are necessary to assess their individual activity against E. coli strains.

Oregano (Origanum vulgare) is widely used as seasoning in several countries and its antimicrobial activity has also been demonstrated. In our experiment, essential oil of oregano showed lower antimicrobial activity than that of L. graveolens and T. vulgaris against 100% of the E. coli used (mean MIC = 3418 μg mL−1; mean MBC = 4800 μg mL−1) (Table 1). MIC values (between 1600 and 3200 μg mL−1) were lower than those recorded against E. coli from poultry and cattle [12]. However, our findings differed significantly from those reported in another study [18], where the authors tested an E. coli standard strain (CCT0547) and found no antimicrobial effect. By contrast, Salmonella enterica strains from poultry were highly susceptible to antibacterial treatment with oregano when compared to thyme [11]. It is important to underscore that the main components of oregano essential oil are carvacrol (at 66%–92.6% concentration), cymene (4.6%–9.2%), and thymol (1.0%–1.9%) [19, 20], suggesting that E. coli strains from Howler monkeys may be more susceptible to thymol than carvacrol or the thymol and carvacrol combination. However, further research is needed to confirm this hypothesis.

The other four essential oils tested, namely, rosemary (Rosmarinus officinalis), eucalyptus (Eucalyptus citriodora), citronella (Cymbopogon nardus), and lemongrass (Cymbopogon citratus), showed no antibacterial effect against the 22 E. coli strains studied. Our results differed from those found by other researchers, who observed antimicrobial activity in these oils against Gram-positive and Gram-negative bacteria [10, 13, 14, 16]. Studies indicate that how essential oils are obtained, the season, and geographic distribution are factors that can change the composition of these oils and alter their antimicrobial properties [28, 29], thus explaining the different results obtained in a number of studies on plant essential oils.

In conclusion, the essential oils of Lippia graveolens, Thymus vulgaris, and Origanum vulgare used in the present study show potential for use as antibacterial agents against E. coli strains. Moreover, based on our findings, it can be assumed that -cymene, γ-terpinene, thymol, and carvacrol were the active ingredients with the highest antimicrobial effect in vitro against E. coli strains.

Competing Interests

The authors declare no competing interests.

Acknowledgments

The authors would like to thank the Criadouro Conservacionista São Braz (Santa Maria, RS, Brazil) for technical support.

References

  1. L. D. Högberg, A. Heddini, and O. Cars, “The global need for effective antibiotics: challenges and recent advances,” Trends in Pharmacological Sciences, vol. 31, no. 11, pp. 509–515, 2010. View at: Publisher Site | Google Scholar
  2. A. R. Manges and J. R. Johnson, “Reservoirs of extraintestinal pathogenic Escherichia coli,” Microbiology Spectrum, vol. 3, no. 5, Article ID UTI-0006-2012, 2005. View at: Publisher Site | Google Scholar
  3. L. Beutin, “Escherichia coli as a pathogen in dogs and cats,” Veterinary Research, vol. 30, no. 2-3, pp. 285–298, 1999. View at: Google Scholar
  4. V. M. Carvalho, C. L. Gyles, K. Ziebell et al., “Characterization of monkey enteropathogenic Escherichia coli (EPEC) and human typical and atypical EPEC serotype isolates from neotropical nonhuman primates,” Journal of Clinical Microbiology, vol. 41, no. 3, pp. 1225–1234, 2003. View at: Publisher Site | Google Scholar
  5. S. E. Jobbins and K. A. Alexander, “From whence they came—antibiotic-resistant Escherichia coli in African wildlife,” Journal of Wildlife Diseases, vol. 51, no. 4, pp. 811–820, 2015. View at: Publisher Site | Google Scholar
  6. C. Marinho, G. Igrejas, A. Gonçalves et al., “Azorean wild rabbits as reservoirs of antimicrobial resistant Escherichia coli,” Anaerobe, vol. 30, pp. 116–119, 2014. View at: Publisher Site | Google Scholar
  7. N. Silva, G. Igrejas, N. Figueiredo et al., “Molecular characterization of antimicrobial resistance in enterococci and Escherichia coli isolates from European wild rabbit (Oryctolagus cuniculus),” Science of the Total Environment, vol. 408, no. 20, pp. 4871–4876, 2010. View at: Publisher Site | Google Scholar
  8. D. Thapa, R. Losa, B. Zweifel, and R. J. Wallace, “Sensitivity of pathogenic and commensal bacteria from the human colon to essential oils,” Microbiology, vol. 158, no. 11, pp. 2870–2877, 2012. View at: Publisher Site | Google Scholar
  9. T. Hernández, M. Canales, J. G. Avila et al., “Ethnobotany and antibacterial activity of some plants used in traditional medicine of Zapotitlán de las Salinas, Puebla (México),” Journal of Ethnopharmacology, vol. 88, no. 2-3, pp. 181–188, 2003. View at: Publisher Site | Google Scholar
  10. A. F. Millezi, N. N. Baptista, D. S. Caixeta, D. F. Rossoni, M. G. Cardoso, and R. H. Piccoli, “Chemical characterization and antibacterial activity of essential oils from medicinal and condiment plants against Staphylococcus aureus and Escherichia coli,” Revista Brasileira de Plantas Medicinais, vol. 16, no. 1, pp. 18–24, 2014. View at: Publisher Site | Google Scholar
  11. J. M. Santurio, D. F. Santurio, P. Pozzatti, C. Moraes, P. R. Franchin, and S. H. Alves, “Antimicrobial activity of essential oils from oregano, thyme and cinnamon against Salmonella enterica sorovars from avian source,” Ciencia Rural, vol. 37, no. 3, pp. 803–808, 2007. View at: Publisher Site | Google Scholar
  12. D. F. Santurio, M. M. da Costa, G. Maboni et al., “Antimicrobial activity of spice essential oils against Escherichia coli strains isolated from poultry and cattle,” Ciencia Rural, vol. 41, no. 6, pp. 1051–1056, 2011. View at: Publisher Site | Google Scholar
  13. R. Scherer, R. Wagner, M. C. T. Duarte, and H. T. Godoy, “Composition and antioxidant and antimicrobial activities of clove, citronella and palmarosa essential oils,” Revista Brasileira de Plantas Medicinais, vol. 11, no. 4, pp. 442–449, 2009. View at: Publisher Site | Google Scholar
  14. M. T. N. Silva, P. I. Ushimaru, L. N. Barbosa, M. L. R. S. Cunha, and A. Fernandes Jr., “Antibacterial activity of plant essential oils against Staphylococcus aureus and Escherichia coli strains isolated from human specimens,” Revista Brasileira de Plantas Medicinais, vol. 11, no. 3, pp. 257–262, 2009. View at: Publisher Site | Google Scholar
  15. N. Thosar, S. Basak, R. N. Bahadure, and M. Rajurkar, “Antimicrobial efficacy of five essential oils against oral pathogens: An in vitro study,” European Journal of Dentistry, vol. 7, no. 5, supplement 1, pp. S71–S77, 2013. View at: Publisher Site | Google Scholar
  16. J. A. A. Zago, P. I. Ushimaru, L. N. Barbosa, and A. Fernandes Jr., “Synergism between essential oils and antimicrobial drugs against Staphylooccus aureus and Escherichia coli strains from human infections,” Brazilian Journal of Pharmacognosy, vol. 19, no. 4, pp. 828–833, 2009. View at: Publisher Site | Google Scholar
  17. M. Marino, C. Bersani, and G. Comi, “Antimicrobial activity of the essential oils of Thymus vulgaris L. measured using a bioimpedometric method,” Journal of Food Protection, vol. 62, no. 9, pp. 1017–1023, 1999. View at: Google Scholar
  18. A. Sartoratto, A. L. M. Machado, C. Delarmelina, G. M. Figueira, M. C. T. Duarte, and V. L. G. Rehder, “Composition and antimicrobial activity of essential oils from aromatic plants used in Brazil,” Brazilian Journal of Microbiology, vol. 35, no. 4, pp. 275–280, 2004. View at: Publisher Site | Google Scholar
  19. P. Pozzatti, L. A. Scheid, T. B. Spader, M. L. Atayde, J. M. Santurio, and S. H. Alves, “In vitro activity of essential oils extracted from plants used as spices against fluconazole-resistant and fluconazole-susceptible Candida spp.,” Canadian Journal of Microbiology, vol. 54, no. 11, pp. 950–956, 2008. View at: Publisher Site | Google Scholar
  20. M. Höferl, G. Buchbauer, L. Jirovetz et al., “Correlation of antimicrobial activities of various essential oils and their main aromatic volatile constituents,” Journal of Essential Oil Research, vol. 21, no. 5, pp. 459–463, 2009. View at: Publisher Site | Google Scholar
  21. R. J. W. Lambert, P. N. Skandamis, P. J. Coote, and G.-J. E. Nychas, “A study of the minimum inhibitory concentration and mode of action of oregano essential oil, thymol and carvacrol,” Journal of Applied Microbiology, vol. 91, no. 3, pp. 453–462, 2001. View at: Publisher Site | Google Scholar
  22. Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio), “Lista Oficial da Fauna Brasileira ameaçada de extinção,” 2015, http://www.icmbio.gov.br/portal/biodiversidade/fauna-brasileira/lista-de-especies.html. View at: Google Scholar
  23. R. M. Silva, J. C. B. Marques, and H. D. Ferreira, “Dados preliminares sobre a dieta do Bugio Alouatta caraya (Primates, Cebidae) na área do Parque Zoológico de Goiânia-PZG,” in Proceedings of the 21st Congresso Brasileiro de Zoologia, p. 216, Anais, Porto Alegre, Brazil, 1996. View at: Google Scholar
  24. A. Chaibi, L. H. Ababouch, K. Belasri, S. Boucetta, and F. F. Busta, “Inhibition of germination and vegetative growth of Bacillus cereus T and Clostridium botulinum 62A spores by essential oils,” Food Microbiology, vol. 14, no. 2, pp. 161–174, 1997. View at: Publisher Site | Google Scholar
  25. R. Santos Pereira, T. C. Sumita, M. R. Furlan, A. O. Cardoso Jorge, and M. Ueno, “Antibacterial activity of essential oils on microorganisms isolated from urinary tract infection,” Revista de Saúde Pública, vol. 38, no. 2, pp. 326–328, 2004. View at: Publisher Site | Google Scholar
  26. Clinical and Laboratory Standards, “Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically,” Approved Standard M7-A7, CLSI, Wayne, Pa, USA, 2006. View at: Google Scholar
  27. A. Arana-Sánchez, M. Estarrón-Espinosa, E. N. Obledo-Vázquez, E. Padilla-Camberos, R. Silva-Vázquez, and E. Lugo-Cervantes, “Antimicrobial and antioxidant activities of Mexican oregano essential oils (Lippia graveolens H. B. K.) with different composition when microencapsulated in β-cyclodextrin,” Letters in Applied Microbiology, vol. 50, no. 6, pp. 585–590, 2010. View at: Publisher Site | Google Scholar
  28. C. A. Marco, R. Innecco, S. H. Mattos, N. S. Borges, and E. O. Nagao, “Características do óleo essencial de capim-citronela em função de espaçamento, altura e época de corte,” Horticultura Brasileira, vol. 25, no. 3, pp. 429–432, 2007. View at: Publisher Site | Google Scholar
  29. A. F. Blank, A. G. Costa, M. D. F. Arrigoni-Blank et al., “Influence of season, harvest time and drying on Java citronella (Cymbopogon winterianus Jowitt) volatile oil,” Brazilian Journal of Pharmacognosy, vol. 17, no. 4, pp. 557–564, 2007. View at: Google Scholar

Copyright © 2016 Valéria Maria Lara et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


More related articles

1884 Views | 443 Downloads | 5 Citations
 PDF  Download Citation  Citation
 Download other formatsMore
 Order printed copiesOrder

Related articles

We are committed to sharing findings related to COVID-19 as quickly and safely as possible. Any author submitting a COVID-19 paper should notify us at help@hindawi.com to ensure their research is fast-tracked and made available on a preprint server as soon as possible. We will be providing unlimited waivers of publication charges for accepted articles related to COVID-19. Sign up here as a reviewer to help fast-track new submissions.