In Vitro Antibacterial Effects of Five Volatile Oil Extracts Against Intramacrophage Brucella Abortus 544
Abstract
Background: Brucellaabortus is a gram-negative facultative intracellular bacterium that can cause a highly contagious disease in sheep, goats, cattle and one-humped camels. It is responsible for one of the most important zoonosis in human. The aim of this study was to evaluate the role of Mentha piperita, Origanum majorana, Citrus lemon, Cinnamomum verum and Myristica fragrans essential volatile oil extracts on human macrophages infected by B. abortus 544.
Methods: Essential volatile oil extracts from M. piperita, O. majorana, C. lemon, C. verum and M. fragrans were extracted. Human macrophages were cultured at a density of 2×105 cells per well in sterile 96-well microtiter plates, and infected with B. abortus 544 at a ratio of 1:100 bacteria/cell. Then essential volatile oil extracts were added at a concentration of 1%. At specified times; cells were washed, lysed with 0.1% Triton, and plated on 2YT agar to determine the number of intracellular bacteria.
Results:Cinnamomum verum volatile oil at a concentration of 1% had the highest antibacterial activity against B. abortus 544 inside human macrophages. Its inhibitory effect observed from 24 h and continued till 144 h after the infection. Moreover, C. verum (0.1%) in combination with 1% concentration of M. piperita, O. majorana, C. lemon or M. fragrans volatile oil extracts produced a synergistic inhibitory effect against B. abortus 544.
Conclusion: The results indicate that, among the five selected oil extracts, C. verum volatile oil applied either separately or in combination with other oil extracts had the most effective antimicrobial activity against Brucella.
Introduction
Brucellosis is a zoonotic disease with a worldwide distribution that is endemic in the world. Brucella abortus remains a major cause of morbidity in humans and domestic animals.1 After invasion of the lymphoid system, the bacteria are developed within mononuclear phagocytes, and the infected cells play a crucial role in the dissemination of the bacteria in specific locations of the body such as spleen, brain, heart, and bones. 2Brucella species virulence and chronic infections are thought to be due to their ability to escape killing mechanisms within macrophages, such as lysosomal enzymes and products of the oxidative burst.3 Food and pharmaceutical industries still need to find new and improved antimicrobial agents capable of being effective against brucellosis.
In spite of the improvements in food hygiene and food production techniques, food safety is an increasingly important public health issue.4 For this reason, to produce safe foods new methods are still needed, to possibly in combination with the existing methods, reduce or inhibit foodborne pathogens.5 Because of increasing pressure from consumers and legal authorities, food industry has tended to reduce the use of chemical preservatives in their products to either completely nil or to adopt more natural alternatives for the maintenance or extension of product shelf life.
Plants extracts have been traditionally used worldwide in the treatment of some diseases from long time ago, but more recently plants essential volatile oil extracts are becoming more important due to their proved antimicrobial effects. Consequently, they are extensively used in medicine, and food and cosmetic industries. In addition to their role as antimicrobial agents,6 they have a role as antioxidant agents.7 For instance, lemon Citrus lemon (L.) Burm. has been used as an antimicrobial,8 anticoccidial,9 and antifungal agent,10 whereas, cinnamon Cinnamomum verum J. Presl has been used only an antimicrobial agent.11 However, nutmeg Myristica fragrans Houtt., peppermint Mentha piperita L., and sweet marjoram Origanum majorana L. have been used as stimulating agents against bacteria,6 and fungus.12 There is, however, no information about the role of essential volatile oil extracts against intracellular bacteria such as B. abortus 544 inside the human macrophages. Thus, the aim of this study was to assess the efficacy of several essential volatile oil extracts from C. verum, M. fragrans, M. piperita, C. Lemon or O. Majorana. Such oil extracts arelargely used in Syrian traditional medicine for the treatment of respiratory and gastrointestinal diseases, against B. abortus 544, inside the human macrophages.
Materials and Methods
Bacterial Culture
For infection experiments, B. abortus 544 was grown for 48 h in 2YT (peptone; 16 g, sodium chloride; 5 g, meat extract; 10 g, distilled water; 1 litre, (Difco, BD, Spars, MD) with 5% sterile horse serum. Bacteria were suspended in a sterile phosphate-buffered saline (PBS). Abundance of B. abortus 544 in PBS was monitored by recording optical density (OD) at 590 nm. The exact number of bacteria colony forming units (CFU) was assessed by viable count on 2YT agar (20 g/L) plates. Plates were placed in an incubator for 48 h at 37°C with 10% CO2 tension adjusted automatically. During the contact with the organism, laboratory personnel were wearing impermeable protective clothes, gloves, and face masks.
Plant Samples Collection
Leaves samples of M. piperita (Lamiaceae) and O. majorana (Lamiaceae), and peel samples of C. lemon (Rutaceae) were collected from their native growing regions in Syria, while C. verum (Lauraceae) bark samples and M. fragrans (Myristicaceae) fruit samples were purchased from the local markets. Plants characterizations were consigned in table 1.
Scientific name | Plant family | Collection site | Altitude | Collection time | Extracted part | Essential oils content (%) |
---|---|---|---|---|---|---|
Mentha piperia | Lamiaceae | Latakia | 350 m | May | leaves | 1.5 |
Origanum majorana | Lamiaceae | Kafr Nobol- Idlib | 750 m | June | leaves | 2.08 |
Myristica fragrans | Myristicaceae | Market | – | – | fruits | 9.5 |
Cinnamomun verum | Lauraceae | Market | – | – | Bark | 0.5 |
Citrus limon | Rutaceae | Latakia | 100 m | September | peel of ripe fruit | 2.5 |
Essential Volatile Oil Extraction
Aerial parts of M. piperita and O. majorana were cleaned and dried prior to steam distillation in a glass apparatus using double distilled water. The plant leaves, which were collected from one station, were separated from the steams and mixed thoroughly to ensure a good homogeneity. Seventy five grams of the dried leaves and 700 ml of water were placed into a distillation flask of one litre capacity, and were extracted for three hours. This process was applied on all plants collection. Citrus lemon peels of ripe fruit were separated from the whole sample and hydrodistilled directly (without drying) using the same steam distillation extraction method. Dried fruit of M. fragrans and the barks of C. verum were grounded, and the powdered materials were hydrodistilled into steam distillation apparatus, as mentioned above. Isolated volatile oil extracts collected from each distillation process were added to each other and dried over anhydrous sodium sulphate and stored in dark glass bottles in a fridge at 4°C until use.
Macrophage Infection
Healthy human macrophage cells were collected and cultured in RPMI. medium supplemented with 10% heat-inactivated fetal calf serum. For macrophage growth assays, 96-well microtiter plates were seeded with 2×105 macrophages/well and infected with B. abortus 544 at a ratio of 1:100 bacteria/macrophage. Cells were incubated for one h at 37°C in 5% CO2. Extracellular bacteria were removed by three washes with PBS, followed by treatment with 25 µg/ml of gentamicin for 30 min. Then, the cells were maintained by the addition of medium containing 5 µg/ml of gentamicin. To evaluate the effect of plants volatile oil extracts on the ability of Brucella to invade human macrophage, 1% concentration of the five studied volatile oil extracts, or 0.1% of C. verum plus 1% of the other four volatile oil extracts, were added after 2, 4, 24, 48, 72, 96, 120 and 144 h of infection, the cells were washed three times by PBS, and lysed with 0.1% Triton. Five minutes after the incubation at room temperature, the lysates were plated on 2YT agar and incubated at 37°C for 48 h; in order to determine the intracellular bacterial count. All experiments were performed in triplicate. Macrophages infected with B. abortus 544 at a ratio of one bacteria/100 macrophage without adding any oil extract as a control.
Statistical Methods
Antibacterial properties of oil extracts were analyzed with one-way repeated measures analysis of variance (ANOVA) fallowed by Tukey’s multiple comparison test to compare the difference between each pair of means. Data were transformed into log10 CFU prior to analysis to homogenize the variance. All analyses were conducted by using GraphPad Prism Statistical Software V5.03. Differences were considered statistically significant at P<0.05.
Results
Brucella abortus 544 log10 counts in human macrophages were significantly suppressed (F5,35=22.7; P<0.0001) by volatile oil extracts treatments compared with the untreated control. For example, the inhibitory effect of C. verum at a concentration of 1% was started 24 h and continued till 144 h after the infection, and the log10 counts increased only from 3.11 to 4.9. The repeated measures ANOVA followed by Tukey’s test of multiple comparisons revealed that C. verum volatile oil possessed the strongest antibacterial effect compared to all the other essential oil extracts (figure 1). It is worth pointing out that no significant difference occurred between the antibacterial activity of lemon, peppermint, sweet marjoram and nutmeg volatile oil extracts.
Cinnamon oil extract, when applied at a concentration of 0.1%, did not show any significant inhibitory effect against B. abortus 544 compared to the control group. In contrast, strong and statistically significant inhibitory effect was observed when 0.1% concentration of cinnamon volatile oil was applied in combination with 1% concentration of the other plants volatile oil extracts (F5,35=34.6; P<0.0001). For instance, the log10 CFU did not exceed 4.6 and 4.9 for cinnamon (0.1%) and sweet marjoram (1%) or lemon (1%) mixture after 144 h of incubation, respectively (figure 2). Based on Tukey’s multiple comparison test, we did not observe any significant differences between the four oil extracts used in combination with cinnamon volatile oil (0.1%) and when the above-mentioned oil mixtures were compared to cinnamon volatile (1%) oil used separately. However, a significant reduction in bacterial counts was recorded for each oil mixture and the cinnamon at 1% treatments compared to the untreated control (F5,35=31.4; P<0.001).
Discussion
Nowadays, people worldwide try to avoid chronic stress, pollution and synthetic drugs. It is well documented that the number of pathogenic bacteria resistant to current antibiotics increases progressively, and the infections due to resistant strains of bacteria pose a serious clinical problem. All these negativities have brought natural agents to the fore and have brought alternative and complementary medicine up to date.13
Malta fever or Brucellosis is a disease found in the Middle East.1 Most of the cases are not usually recognized, and fail to be classified. A low efficiency therapy system to eliminate Brucella is currently in use. That is the reason why there are lots of relapses and chronic infections.14 Patients are usually medicated, thus, as being infected with other diseases; this would increase the odds of having some chronic cases.15 With all in mind, it seems difficult to provide accurate estimates and numbers of Brucella infected patients. Estimates are usually lower than reality, especially in the case of children.16 For all these reasons, good and new treatment regimens against B. abortus are urgently needed.
Our results showed that at a concentration of 1% C. verum volatile oil exhibited strong inhibitory effect against B. abortus 544 strain inside the human macrophages. This result concords with that found by Mayaud et al.17 who reported that the C. verum bark volatile oil had an excellent antimicrobial activity against Staphylococcus aureus and Escherichia coli at concentrations ranging from 0.31% to 10% (v/v). The antimicrobial effect of cinnamon against gram negative bacteria was also reported by Ooi et al.11 who concluded that C. verum was effective against a broad spectrum of bacteria, such E. coli, Enterobacter aerogenes, Proteus vulgaris, Pseudomonas aeruginosa, Vibrio cholerae, V. parahaemolyticus and Salmonella typhymurium. It seems that the efficacy of C. verum oil related directly with the presence of active components, such as cinnamaldehyde, cinnamyl acetate, and cinnamyl alcohol, plus a wide range of other volatile substances.11 On the other hand, C. verum volatile oil at concentration of 0.1% revealed a minimum to nil inhibitory effect against B. abortus 544. Also, Ouafae et al.18 also reported that viable bacterial counts decreased from 107 to 104 CFU/mL when E. coli O157:H7 cells were incubated at 37°C for 2 h in the presence of 0.025% concentration of cinnamon essential volatile oil. However, this bacteria was almost completely eliminated after 30 min of incubation in the presence of 0.05% concentration of cinnamon oil.
Our results revealed that M. fragrans, C. lemon, O. majorana and M. piperita volatile oil extracts had significant activities against B. abortus 544. Dabbah et al.19 found that terpineol and terpeneless fractions of Citrus volatile oil extracts to have greater inhibitory effects on gram-positive than gram-negative bacteria. However, Waikedre et al.8 reported that the essential oil extracts from the leaves of Citrus macroptera and C. hystrix were inactive against 5 species of bacteria. Moreover, Baik et al.20 reported that essential volatile oil extracted from 14 kinds of Korean endemic Citrus species did not have any activity against S. epidermidis, whereas O’Bryan et al.21 found that Citrus essential volatile oil extracts at minimum inhibitory concentrations (MIC) ranging from 0.125% to 0.5% had a good inhibitory activity against the Salmonella spp. Barbosa et al.22 found that the MIC90 of oregano (Origanum vulgare L.) essential volatile oil extracts was 5% and 0.46% v/v against gram-positive (S. aureus and Listeria monocytogenes) and gram-negative (E. coli and S. Enteritidis) bacteria, respectively. Whereas, López et al.23 found that 8-10% (v/v) concentrations of O. vulgare essential volatile oil completely inhibited the growth of E. coli, Y. enterocolitica, P. aeruginosa, and S. choleraesuis bacteria.
Firouzi et al.24 mentioned that the M. fragrans volatile oil extract had a moderate effect against Y. enterocolitica; while Mahady et al. 25 reported that the MIC of methanol extracts of M. fragrans seeds was 12.5 µg/mL against Helicobacter pylori. Al-Bayati,26 reported that Mentha longifolia L. volatile oil had an antimicrobial activity against some gram positive pathogenic like as S. aureus, Streptococcus mutans but did not have any activity against P. aeruginosa bacteria. Mkaddem et al.27 found that the Mentha essential volatile oil extracts were very active against L. monocytogenes and Klebsiella pneumoniae bacteria; whereas they were less effective against E. coli. Celikel and Kavas,28 reported that essential oil of sweet orange (Citrussinensis L.) at a concentration of 3% (v/v) exhibited the lowest antibacterial and bacteriostatic activity against E. coli, L. monocytogenes, and S. aureus. The results of Soković et al.29 demonstrated that the essential oil of M. piperita possessed good activity against human pathogenic bacteria such as E. coli O157:H7, S. typhimurium, S. aureus, P. aeruginosa and P. mirabilis. Whereas, C. lemon was effective against only E. coli O157:H7, S. typhimurium, and S. aureus. These conflicting results are in agreement with reports indicating that essential oil extracts antimicrobial activities varied depending on the species, subspecies, variety or geo-ecological regions. Thus, it is not surprising to find that essential volatile oil extracts of some plants pertaining to the same species that were collected from different agricultural areas showed different levels of antimicrobial properties.30-32
Our data showed that the mixture of concentrations 1% of individual essential oil extracts and small amount of cinnamon oil (0.1%) was associated with enhanced antibacterial activity. In other words, the antibacterial property of the volatile oil extracts was apparently strengthened through the combination between cinnamon oil at low concentration and all the other essential oil extracts at high concentration. Thus, the results presented herein provide positive evidence regarding the synergism between different percentages of essential oil extracts as antibacterial agents against B. abortus 544. Our finding is in accordance with report of Probst et al.33 findings, which showed that combinations of cinnamon with peppermint, ginger (Zingiberofficinale Roscoe) and clove (Syzygiumaromaticum L.) essential oil extracts produced synergistic antibacterial effects against gram-positive and gram-negative microorganisms. Moreover they are in agreement with Nanasombat and Wimuttigosol’s,34 results, which revealed that cinnamon oil in combination with nutmeg or makaen (Zanthoxylumlimonella Alston) oil extracts showed a synergistic effect against S.aureus, Pseudomonasfluorescens, and Salmonella Rissen bacteria.
Conclusion
The goal of this study was to develop an effective and inexpensive therapy against Brucella inside human macrophages. Cinnamomum verum verum bark essential oil at a concentration of 1% used separately, or at a concentration of 0.1% in combination with a concentration of 0.1% C. verum with 1% M. fragrans, M. piperita, C. Lemon or O. majorana represents an alternative source of natural antimicrobial substances, and may replace conventional chemical antimicrobials. The high specific activity of cinnamon at low and non-toxic concentrations suggests that it could be used in clinical practice for the treatment of Brucellosis in animals and humans. More specific studies are recommended to examine this suggestion.
Acknowledgment
The authors would like to thank the Director General of AECS, and the head of the Molecular Biology and Biotechnology Department for their support. The authors would also like to thank Dr. M. Safi for his critical reading of this manuscript.
Conflict of Interest: None declared
References
- 1. Brucellosis: an overview Emerg Infect Dis1997321321doi: 10.3201/eid0302.970219. PubMed PMID: 9204307; PubMed Central PMCID: PMC2627605[PubMed][Google Scholar]
- 2. Brucella intracellular life: from invasion to intracellular replication Vet Microbiol20029028197doi: 10.1016/S0378-1135(02)00214-6. PubMed PMID: 12414149[PubMed][Google Scholar]
- 3. Brucella evades macrophage killing via VirB-dependent sustained interactions with the endoplasmic reticulum J Exp Med200319854556doi: 10.1084/jem.20030088. PubMed PMID: 12925673; PubMed Central PMCID: PMC2194179[PubMed][Google Scholar]
- 4. World Health OrganizationFood Safety and Foodborne Illness2002Fact Sheet 237Geneva
- 5. Synergistic bactericidal effect of carvacrol, cinnamaldehyde or thymol and refrigeration to inhibit Bacillus cereus in carrot broth Food Microbiol2006236873doi: 10.1016/j.fm.2005.01.016. PubMed PMID: 16942988[PubMed][Google Scholar]
- 6. Studies on essential oil extracts: part 10; antibacterial activity of volatile oil extracts of some spices Phytother Res2002166802doi: 10.1002/ptr.951. PubMed PMID: 12410554[PubMed][Google Scholar]
- 7. Effect of dietary administration of oil extracts from rosemary and sage on lipid oxidation in broiler meat Br Poult Sci19983923540doi: 10.1080/00071669889187. PubMed PMID: 9649877[PubMed][Google Scholar]
- 8. Chemical composition and antimicrobial activity of the essential oil extracts from New Caledonian Citrus macroptera and Citrus hystrix Chem Biodivers201078717PubMed PMID: 20397222[PubMed][Google Scholar]
- 9. Effect of dietary supplementation with oregano essential oil on performance of broilers after experimental infection with Eimeria tenella Arch Tierernahr20035799106PubMed PMID: 12866780[PubMed][Google Scholar]
- 10. Antifungal activity of Thymus oil extracts and their major compounds J Eur Acad Dermatol Venereol200418738doi: 10.1111/j.1468-3083.2004.00886.x. PubMed PMID: 14678536[PubMed][Google Scholar]
- 11. Antimicrobial activities of Cinnamon oil and cinnamaldehyde from the Chinese medicinal herb Cinnamomum cassia Blume Am J Chin Med20063451122PubMed PMID: 16710900[PubMed][Google Scholar]
- 12. Effect of oil extracted from some medicinal plants on different mycotoxigenic fungi Food Chem Toxicol200240166975doi: 10.1016/S0278-6915(02)00120-5. PubMed PMID: 12176092[PubMed][Google Scholar]
- 13. Antimicrobial agents from plants: antibacterial activity of plant volatile oil extracts J Appl Microbiol20008830816doi: 10.1046/j.1365-2672.2000.00969.x. PubMed PMID: 10736000[PubMed][Google Scholar]
- 14. Multivariate model for predicting relapse in human brucellosis J Infect1998368592doi: 10.1016/S0163-4453(98)93342-4. PubMed PMID: 9515675[PubMed][Google Scholar]
- 15. Human brucellosis: do we need to revise our therapeutic policy J Rheumatol200330266672PubMed PMID: 14719211[PubMed][Google Scholar]
- 16. Brucellosis in children: clinical observations in 115 cases Int J Infect Dis200261826doi: 10.1016/S1201-9712(02)90108-6. PubMed PMID: 12718832[PubMed][Google Scholar]
- 17. Comparison of bacteriostatic and bactericidal activity of 13 essential oil extracts against strains with varying sensitivity to antibiotics Lett Appl Microbiol20084716773doi: 10.1111/j.1472-765X.2008.02406.x. PubMed PMID: 19552780[PubMed][Google Scholar]
- 18. Inactivation of Escherichia coli O157:H7 by essential oil from Cinnamomum zeylanicum Braz J Infect Dis2007112346PubMed PMID: 17625768[PubMed][Google Scholar]
- 19. Antimicrobial action of some citrus fruit oil extracts on selected food-borne bacteria Appl Microbiol1970192731PubMed PMID: 4905947; PubMed Central PMCID: PMC376603[PubMed][Google Scholar]
- 20. Chemical composition and biological activities of essential oil extracted from Korean endemic citrus species J Microbiol Biotechnol200818749PubMed PMID: 18239420[PubMed][Google Scholar]
- 21. Orange essential oil extracts antimicrobial activities against Salmonella spp J Food Sci2008732647PubMed PMID: 19241555[Google Scholar]
- 22. Essential oil extracts against foodborne pathogens and spoilage bacteria in minced meat Foodborne Pathog Dis200967258doi: 10.1089/fpd.2009.0282. PubMed PMID: 19580445; PubMed Central PMCID: PMC3145167[PubMed][Google Scholar]
- 23. Development of flexible antimicrobial films using essential oil extracts as active agents J Agric Food Chem200755881424doi: 10.1021/jf071737b. PubMed PMID: 17880148[PubMed][Google Scholar]
- 24. Effects of essential oil extracts of oregano and nutmeg on growth and survival of Yersinia enterocolitica and Listeria monocytogenes in barbecued chicken J Food Prot200770262630PubMed PMID: 18044446[PubMed][Google Scholar]
- 25. In vitro susceptibility of Helicobacter pylori to botanical extracts used traditionally for the treatment of gastrointestinal disorders Phytother Res20051998891PubMed PMID: 16317658[PubMed][Google Scholar]
- 26. Isolation and identification of antimicrobial compound from Mentha longifolia L leaves grown wild in Iraq Ann Clin Microbiol Antimicrob2009820doi: 10.1186/1476-0711-8-20. PubMed PMID: 19523224; PubMed Central PMCID: PMC2707363[PubMed][Google Scholar]
- 27. Chemical composition and antimicrobial and antioxidant activities of Mentha (longifolia L. and viridis) essential oil extracts J Food Sci20097435863doi: 10.1111/j.1750-3841.2009.01272.x. PubMed PMID: 19895481[Google Scholar]
- 28. Antimicrobial Properties of Some Essential Oil extracts against Some Pathogenic Microorganisms Czech J Food Sci20082617481[Google Scholar]
- 29. Antibacterial effects of the essential oil extracts of commonly consumed medicinal herbs using an in vitro model Molecules201015753246PubMed PMID: 21030907[PubMed][Google Scholar]
- 30. The in vitro antimicrobial activities of the essential oil extracts of some Lamiaceae species from Turkey J Med Food2009129027PubMed PMID: 19735193[PubMed][Google Scholar]
- 31. Antimicrobial effect of essential oil extracts from plants collected from different localities Acta Fytotechnica et Zootechnica2011142931[Google Scholar]
- 32. Variation in antibacterial activity and phenolic content of Hypericum scabrum L. populations J Med Plant Res20115411925[Google Scholar]
- 33. Antimicrobial activity of propolis and essential oil extracts and synergism between these natural products J Venom Anim Toxins incl Trop Dis20111715967[Google Scholar]
- 34. Antimicrobial and antioxidant activity of spice essential oil extracts Food Sci Biotechnol2011204553[Google Scholar]