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Applied and Environmental Microbiology, October 2008, p. 5986-5990, Vol. 74, No. 19
0099-2240/08/$08.00+0     doi:10.1128/AEM.00981-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Antibacterial Activities of Naturally Occurring Compounds against Mycobacterium avium subsp. paratuberculosis

Stella Y. Y. Wong,^1* Irene R. Grant,¹ Mendel Friedman,² Christopher T. Elliott,¹ and Chen Situ¹

Institute of Agri-Food and Land Use, School of Biological Sciences, Queen's University Belfast, Belfast, Northern Ireland, United Kingdom,¹ Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Albany, California²

Received 30 April 2008/ Accepted 22 July 2008

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The antibacterial activities of 18 naturally occurring compounds (including essential oils and some of their isolated constituents, apple and green tea polyphenols, and other plant extracts) against three strains of Mycobacterium avium subsp. paratuberculosis (a bovine isolate [NCTC 8578], a raw-milk isolate [806R], and a human isolate [ATCC 43015]) were evaluated using a macrobroth susceptibility testing method. M. avium subsp. paratuberculosis was grown in 4 ml Middlebrook 7H9 broth containing 10% oleic acid-albumin-dextrose-catalase, 0.05% Tween 80 (or 0.2% glycerol), and 2 µg/ml mycobactin J supplemented with five concentrations of each test compound. The changes in the optical densities of the cultures at 600 nm as a measure of CFU were recorded at intervals over an incubation period of 42 days at 37°C. Six of the compounds were found to inhibit the growth of M. avium subsp. paratuberculosis. The most effective compound was trans-cinnamaldehyde, with a MIC of 25.9 µg/ml, followed by cinnamon oil (26.2 µg/ml), oregano oil (68.2 µg/ml), carvacrol (72.2 µg/ml), 2,5-dihydroxybenzaldehyde (74 µg/ml), and 2-hydroxy-5-methoxybenzaldehyde (90.4 µg/ml). With the exception of carvacrol, a phenolic compound, three of the four most active compounds are aldehydes, suggesting that the structure of the phenolic group or the aldehyde group may be important to the antibacterial activity. No difference in compound activity was observed between the three M. avium subsp. paratuberculosis strains studied. Possible mechanisms of the antimicrobial effects are discussed.

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Mycobacterium avium subsp. paratuberculosis is the causative agent of paratuberculosis, also known as Johne's disease, in wild and domestic ruminants, especially dairy cattle (7, 27, 31, 37, 40, 52). M. avium subsp. paratuberculosis may also have some roles in the development of Crohn's disease and diabetes in humans (25, 32, 41, 43, 50). Johne's disease is a chronic granulomatous enteropathy (3), the clinical signs of which include chronic diarrhea, progressive weight loss, decreased milk production, and infertility (8). There is currently no approved drug or treatment for Johne's disease (34). Once farm animals are infected by M. avium subsp. paratuberculosis, they are culled early (39). The disease leads to economic loss of livestock and dairy producers due to reduced productivity, animal wasting, and, in severe cases, mortalities (3, 39). Because Johne's disease occurs worldwide (7), its global impact should not be underestimated. Transmission of M. avium subsp. paratuberculosis is through the fecal-oral route between animals (34). Animals usually become infected with M. avium subsp. paratuberculosis early in their lives as neonates, when their immune systems are not fully developed (8). Before the onset of the clinical symptoms, there is a latency period that lasts for 2 to 5 years. During the latency period, the carrier animals can infect surrounding animals and the farm environment through the shedding of M. avium subsp. paratuberculosis into their feces (7). Farmers can realize the infection of their animals with M. avium subsp. paratuberculosis only when clinical signs are observed. At that stage, the farm and other animals within the herd will have already been exposed to the M. avium subsp. paratuberculosis pathogen.

Milk can be contaminated with M. avium subsp. paratuberculosis, as it is systemic (48), or by feces from infected animals during milking (21). Recent studies have shown that M. avium subsp. paratuberculosis survives pasteurization (18-22), suggesting a possible risk to human health. Other attempts to inactivate M. avium subsp. paratuberculosis include the uses of UV irradiation (2), pulsed electric fields (38), and hydrostatic pressure (30). Another possible route of transmission of M. avium subsp. paratuberculosis from cattle to humans is via contaminated water (53). This animal pathogen could be a hidden food safety threat (45), and further study is necessary to help prevent or control the spread of the organisms to both farm animals and humans.

Many antimicrobial compounds, mainly synthetic antibiotics, had been tested for their anti-M. avium subsp. paratuberculosis effects (4, 6, 33, 34, 54, 57). As antibiotics are associated with the development of antibiotic-resistant bacteria (35) that can pass to humans via the food chain (55), the use of antibiotics in food production is restricted and must be minimized (56). Many naturally occurring compounds, like essential oils and phenolic compounds, have been evaluated extensively for antimicrobial activity and chemically characterized (10, 12-15). They are secondary plant metabolites and can be obtained naturally from different parts of plant materials of common herbs, spices, and teas, including flowers, buds, seeds, leaves, twigs, bark, wood, fruits, and roots, by steam distillation, expression, fermentation, or extraction (36) or can be synthesized. They are ideal for study as most of them are "generally regarded as safe" (28). Most studies have focused on food-borne pathogens and food spoilage bacteria. However, some studies have involved Mycobacterium species, like the human pathogen M. tuberculosis (9, 17), and other fast-growing mycobacteria, including M. aurum, M. fortuitum, M. phlei, M. smegmatis (42, 46), and M. abscesus (42). Previous studies of mycobacteria have investigated the effects of crude plant extracts and their constituents (23), isolated from either the aerial part (23) or the root (44) of the plant, and some of these studies have shown promising results. To our knowledge, this study represents the first evaluation of the activities of naturally occurring compounds against M. avium subsp. paratuberculosis, an important animal pathogen and a potential human pathogen. There is enormous scope for exploring these naturally occurring compounds as potential nutraceuticals (as replacements for discontinued standard antibiotics in feeds for food animals) and as potential food additives.

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Test compounds.
The following compounds were obtained from Sigma (St. Louis, MO): 2,4,6-trihydroxybenzaldehyde, 2,5-dihydroxybenzaldehyde, 2-hydroxy-5-methoxybenzaldehyde, caffeic acid, capsaicin (natural), carvacrol, chlorogenic acid hemihydrates, trans-cinnamaldehyde, trans-cinnamic acid, citral (cis plus trans), gallic acid, geraniol, methyl cinnamate, and vanillic acid. The purity levels of these compounds ranged from 95 to 99.9% according to the manufacturer. Cinnamon (cassia) oil was obtained from Yerba Buena Co. (Berkeley, CA). Oregano (origanum) oil was obtained from Lhasa Karnak Herb Co. (Berkeley, CA). Their purity levels were not specified. Apple E (concentrated apple polyphenols) was obtained from Apple Poly LLC (Morrill, NE); the purity level was approximately 82%. Green tea polyphenols were obtained from LKT Laboratories, Inc. (St. Paul, MN); the purity level was not specified. Rifampin (Sigma) was used as a positive control antibiotic as it is known to be active against M. avium subsp. paratuberculosis.

Preparation of test compounds for susceptibility testing.
A stock solution, 50 mg/ml for solid compounds or 50 µl/ml for oil compounds, was prepared by suspending each test compound in absolute ethanol. Rifampin was prepared as a 10-mg/ml stock suspension in absolute ethanol. The stock solutions were stored in aluminum foil-wrapped bottles at 4°C. As a high percentage of ethanol could be bactericidal, the amount of ethanol added to the growth medium was kept as low as possible in order to minimize the potential effect on growth of M. avium subsp. paratuberculosis. A preliminary experiment was carried out to determine the maximum percentage of ethanol which could be included in the growth medium without growth inhibition of M. avium subsp. paratuberculosis. This was found to be 0.7% (vol/vol) (data not shown). The final concentration of ethanol present in the growth medium was standardized at 0.4% (vol/vol) in this study.

Before the test, each stock solution was serially diluted (threefold) in absolute ethanol, and 17.8 µl of each dilution was then added to sterile screw-cap test tubes (Sterilin; Barloworld Scientific, Staffordshire, United Kingdom) containing 3.9 ml of Middlebrook 7H9 broth to yield final concentrations of 74, 24.7, 8.2, 2.7, and 0.9 µg/ml for solid compounds and the same values in nl/ml for oil compounds. The concentrations of the oil compounds were expressed later in µg/ml according to the density of each oil compound. For rifampin, the final concentrations were 10, 5, 2.5, 1.3, and 0.6 µg/ml. For apple E and green tea polyphenols, precipitations were observed after addition to the growth medium due to the presence of Tween 80. The complete growth medium used for testing these two compounds was therefore supplemented with 0.2% (vol/vol) glycerol instead of 0.05% (wt/vol) Tween 80. Ethanol at 0.4% (vol/vol) was added to the growth medium to serve as a negative control.

M. avium subsp. paratuberculosis strains.
Three M. avium subsp. paratuberculosis strains (ATCC 43015, NCTC 8578, and 806R) were tested in this study. ATCC 43015 is a human isolate obtained from the American Type Culture Collection. NCTC 8578 is a bovine isolate obtained from the National Collection of Type Cultures, Colindale, London, United Kingdom. 806R was isolated from raw cow's milk by Grant et al. (21). The M. avium subsp. paratuberculosis strains were prepared for testing as described by Whan et al. (53), with slight modification. Briefly, the strains were maintained in Cryobank vials (Mast Group Ltd., Merseyside, United Kingdom) at –70°C. Two cryobeads were inoculated into 10 ml Middlebrook 7H9 broth (Difco Laboratories, Detroit, MI) supplemented with 0.05% (wt/vol) Tween 80 (Sigma), 10% (vol/vol) Middlebrook oleic acid-albumin-dextrose-catalase supplement (Difco), and 2 µg/ml mycobactin J (Synbiotics Europe SAS, Lyon, France), pH 6.6 ± 0.2, and the broths were incubated at 37°C with gentle shaking (100 rpm) for 3 to 4 weeks until the optical density at 600 nm (OD₆₀₀) was between 0.7 and 0.9.

Preparation of M. avium subsp. paratuberculosis for susceptibility testing.
The M. avium subsp. paratuberculosis culture was declumped by adding 10 sterile 3-mm glass beads and then vortexing the culture at high speed for 2 min, resting it for 2 min, and repeating the cycle three times. The culture was transferred to a sterile test tube, and the OD was determined using a cell density meter (WPA Biowave CO8000) at 600 nm. The OD was then adjusted to 0.10 by addition of maximum-recovery diluent (Oxoid, Unipath Ltd., Basingstoke, United Kingdom) to give a concentration of approximately 4.8 x 10⁷ CFU/ml, which was later verified by diluting and plating the culture onto Herrold's egg yolk medium supplemented with 2 µg/ml mycobactin J. The agar plates were sealed with Duraseal laboratory sealing film (Diversified Biotech, Boston, MA) and incubated at 37°C for 4 to 6 weeks before colonies were counted.

Macrobroth susceptibility testing method.
Sterile test tubes containing Middlebrook 7H9 broth with the test compounds added (as described above) were inoculated with 100 µl of the adjusted bacterial suspension. Thus, the concentration of M. avium subsp. paratuberculosis in each test tube was approximately 1.2 x 10⁶ CFU/ml. The tubes were incubated at 37°C without shaking, with the caps screwed on tightly, and OD₆₀₀ was monitored at regular intervals for a period of 42 days. The OD₆₀₀ results were recorded, and growth curves were plotted for all the test compounds at all concentrations in order to compare the results with those of the negative-control culture. Some of the test compounds are colored, which made reading OD₆₀₀ impossible, so these test tubes were observed daily to check for the presence of culture settled at the bottom, if any, compared to that in the negative control. The entire experiment was repeated twice for the three M. avium subsp. paratuberculosis strains.

MIC estimation.
The lowest concentration of the test compound in the test tubes with no visible or detectable bacterial growth was considered to represent the MIC. For test tubes that showed bacterial growth, samples were taken from the tubes containing the highest concentrations of test compounds that showed no inhibitory effect for acid-fast staining to confirm that the cultures had not become contaminated and were still pure M. avium subsp. paratuberculosis cultures.

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Six of the 18 compounds tested were found to inhibit the growths of all three M. avium subsp. paratuberculosis strains during incubation at 37°C for up to 42 days. The most effective compound was trans-cinnamaldehyde, with a MIC of 25.9 µg/ml, followed by cinnamon oil (26.2 µg/ml), oregano oil (68.2 µg/ml), carvacrol (72.2 µg/ml), 2,5-dihydroxybenzaldehyde (74 µg/ml), and 2-hydroxy-5-methoxybenzaldehyde (90.4 µg/ml) (Table 1). Three of these compounds were aldehydes (trans-cinnamaldehyde, 2-hydroxy-5-methoxybenzaldehyde, and 2,5-dihydroxybenzaldehyde) (Fig. 1). trans-Cinnamaldehyde is the main active ingredient (81%) in cinnamon cassia oil (15). The phenolic compound carvacrol (Fig. 1) is the major component in oregano oil (51). These results suggest that the aldehyde group of the three compounds, the phenolic group of carvacrol, and/or its hydrophobic-hydrophilic nature may be important for antimicrobial activity. The MICs of these six compounds were the same for all three strains of M. avium subsp. paratuberculosis. The chemical structures of trans-cinnamaldehyde, 2-hydroxy-5-methoxybenzaldehyde, 2,5-dihydroxybenzaldehyde, and carvacrol show slight similarity to isoniazid and pyrazinamide, drugs shown to be effective against M. tuberculosis (47).

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TABLE 1. MICs of naturally occurring compounds tested for activity against three M. avium subsp. paratuberculosis strains

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FIG. 1. Structures of 2,5-dihydroxybenzaldehyde, 2-hydroxy-5- methoxybenzaldehyde, cavacrol, and trans-cinnamaldehyde.

The macrobroth susceptibility testing method adopted for this study essentially mimics the Bactec 12B or MGIT culture approach for antibiotic susceptibility testing of mycobacteria (57). The macrobroth method was used because we had no access to the Bactec 460 or MGIT 960 instruments. A growth curve for M. avium subsp. paratuberculosis was plotted for each test compound at different concentrations based on the OD₆₀₀ values in order to compare differences in growth characteristics, i.e., length of lag phase and maximum OD₆₀₀ achieved by culture. The growth curves for M. avium subsp. paratuberculosis NCTC 8578 in Middlebrook 7H9 broth with the addition of trans-cinnamaldehyde at different concentrations and the negative control of 0.4% (vol/vol) ethanol are shown in Fig. 2. This figure shows a concentration-dependent inhibitory effect of trans-cinnamaldehyde on the growth of M. avium subsp. paratuberculosis NCTC 8578, with complete inhibition of growth at and above the MIC of 25.9 µg/ml. Figure 3 illustrates growth of M. avium subsp. paratuberculosis NCTC 8578 in Middlebrook 7H9 broth supplemented with MICs of cinnamon oil (26.2 µg/ml), trans-cinnamaldehyde (25.9 µg/ml), oregano oil (68.2 µg/ml), carvacrol (72.2 µg/ml), 2,5-dihydroxybenzaldehyde (74 µg/ml), and 2-hydroxy-5-methoxybenzaldehyde (90.4 µg/ml). The noninhibitory concentrations of ethanol (0.4%, vol/vol) (negative control), vanillic acid (74 µg/ml), and citral (65.7 µg/ml) are also shown in Fig. 3. This figure illustrates the complete inhibition of M. avium subsp. paratuberculosis growth by the six inhibitory compounds, whereas growth of M. avium subsp. paratuberculosis was not inhibited by vanillic acid or citral at the highest concentrations tested or by the negative control (0.4% ethanol).

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FIG. 2. Effects of different concentrations (µg/ml) of trans-cinnamaldehyde and noninhibitory concentrations of ethanol (0.4%) (negative control) on the growth of M. avium subsp. paratuberculosis NCTC 8578 in Middlebrook 7H9 broth.

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FIG. 3. Growth of M. avium subsp. paratuberculosis NCTC 8578 in Middlebrook 7H9 broth supplemented with the six active compounds at their MICs (trans-cinnamaldehyde [25.9 µg/ml], cinnamon oil [26.2 µg/ml], carvacrol [72.2 µg/ml], oregano oil [68.2 µg/ml], 2-hydroxy-5-methoxybenzaldehyde [90.4 µg/ml], and 2,5-dihydroxybenzaldehyde [74 µg/ml]), ethanol (0.4%) (negative control), vanillic acid (74 µg/ml), and citral (65.7 µg/ml). 2-Hydroxy-5-methoxybenzaldehyde and 2,5-dihydroxybenzaldehyde had slightly higher initial OD values as they produced colored solutions.

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There is a long history of using essential oils as antimicrobials, beginning as early as 1910 (26). According to a recent series of studies carried out by Friedman and colleagues, five out of the six compounds that were found to be active against M. avium subsp. paratuberculosis in this study—cinnamon oil, trans-cinnamaldehyde, oregano oil, 2-hydroxy-5-methoxybenzaldehyde, and 2,5-dihydroxybenzaldehyde—have been shown to inhibit the growth of food-borne pathogens such as Campylobacter jejuni, Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica (14, 15), while carvacrol, cinnamon oil, and oregano oil were also found to be active against antibiotic-resistant Staphylococcus aureus and Bacillus cereus vegetative cells and spores as well as against resistant Micrococcus luteus (10, 11). The published MICs for the five compounds active against Campylobacter jejuni, expressed in terms of reduction of 50% of CFU, ranged from 6.6 to 190 µg/ml: 430 to 1,100 µg/ml for Escherichia coli O157:H7, 80 to 1,900 µg/ml for Listeria monocytogenes, and 330 to 1,900 µg/ml for Salmonella enterica (14, 15). In a separate study (10), the antimicrobial activities of the test compounds carvacrol, cinnamon oil, and oregano oil were expressed as the numbers of CFU recovered from the bactericidal assay performed with different compounds compared to the number of CFU recovered from the negative control. Upon adoption of the definition of MIC from the previous studies, i.e., a 50% CFU reduction or more (as some compounds showed either no inhibition or inhibition of over 50% of the CFU at the next higher concentration), the MICs of carvacrol, cinnamon oil, and oregano oil for Staphylococcus aureus were 66.7 µg/ml or above, and those for vegetative Bacillus cereus cells ranged from 0.0667 to 66.7 µg/ml. No inhibition against Bacillus cereus spores was observed. One similarity between our findings and those of the above-mentioned studies is that the MICs of the six compounds that were found to be active against M. avium subsp. paratuberculosis fell within the same range as those for the other food-borne pathogens studied. However, due to the slow-growing nature of M. avium subsp. paratuberculosis and its fastidious nutritional requirements, the assay methods and way of determining the MICs in this study are different from the methods used by Friedman et al. (10, 12-15). It is therefore difficult to directly compare the degrees of effectiveness of the six compounds against M. avium subsp. paratuberculosis and the other pathogens mentioned above.

The mechanism of antimicrobial activity of naturally occurring compounds is thought to be specific rather than nonspecific since it is concentration dependent (15). Possible modes of action of the essential oils include disruption of cell membranes, inhibition of essential enzymes, chelation of essential trace elements (such as iron), and targeting of cell membranes (15). Ultee et al. (49) found that after treatment of B. cereus, a gram-positive bacterium, with carvacrol, cell membrane permeability was increased for essential ions, like potassium ions and hydrogen ions, consequently causing leakage of essential ions out of the cells. As a result of this leakage, enzymes might not be able to function properly, which could affect turgor pressure, DNA synthesis, and other metabolic activities. They also found that after the treatment with carvacrol, the ATP concentration inside the cells declined but there was no leakage of ATP to the external environment. It is possible that carvacrol might have reduced the rate of ATP synthesis or increased ATP hydrolysis. In a related study, Helander et al. (24) also found that carvacrol exerted an effect on cell membranes. They observed leakage of cellular material and also of ATP. These results differ from the finding of Ultee et al. (49). This difference may be due to the fact that Helander et al. (24) used gram-negative bacteria. Lambert et al. (29) found that besides leakage of potassium and phosphate ions, changes in the internal pHs of the treated cells also occurred. There was a reduction of pH gradient across the cytoplasmic membrane, affecting pH homeostasis.

In conclusion, because currently there is no drug approved for treatment of Johne's disease, the naturally occurring anti-M. avium subsp. paratuberculosis compounds identified in this study may have the potential to be included in therapeutic drugs for treatment of Johne's disease in farm animals and possibly also in human medicine against Crohn's disease. Carvacrol, the major component of widely consumed oregano oil, and trans-cinnamaldehyde, the major constituent of cinnamon oil, are already designated "generally regarded as safe" (1, 5, 16). However, we have no information about the safety of 2,5-dihydroxybenzaldehyde or 2-hydroxy-5-methoxybenzaldehyde. Further research will focus on the modes of action of the six compounds effective against M. avium subsp. paratuberculosis, and toxicity testing will be performed in order to confirm the potential of these compounds for use as additives in animal feedstuffs and human foods.

 
* Corresponding author. Mailing address: Institute of Agri-Food and Land Use, School of Biological Sciences, Queen's University Belfast, David Keir Building, Stranmillis Road, Belfast BT9 5AG, United Kingdom. Phone: 44 28 9097 2065. Fax: 44 28 9097 6513. E-mail: ywong04{at}qub.ac.uk

Published ahead of print on 1 August 2008.

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Applied and Environmental Microbiology, October 2008, p. 5986-5990, Vol. 74, No. 19
0099-2240/08/$08.00+0     doi:10.1128/AEM.00981-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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