Inhibition of Bacillus anthracis growth by Australian native plants used traditionally as antibacterial medicines

Introduction: Anthrax is a zoonotic disease caused by the bacterium Bacillus anthracis. It is often fatal if left untreated. Many Australian plants have documented therapeutic properties as general antiseptics, inhibiting the growth of a wide variety of bacterial species. This study examines the ability of selected Australian plant extracts to inhibit B. anthracis growth. Methods: Solvent extracts were prepared using plants with documented ethnobotanical usage to treat bacterial infections, or published antibacterial activity. The extracts were investigated by disc diffusion assay for the ability to inhibit the growth of an environmental strain of B. anthracis. Their MIC values were determined to quantify and compare their efficacies. Toxicity was determined using the Artemia franciscana nauplii bioassay. Results: Methanolic and aqueous extracts of Eucalyptus baileyana and Eucalyptus major displayed potent antibacterial activity in the disc diffusion assay against B. anthracis. The methanolic extracts were particularly potent with MIC values as low as 290 μg/mL (E. major methanolic extract). Tasmannia insipidia and Tasmannia stipitata extracts also inhibited B. anthracis growth, albeit with low efficacy. The E. baileyana and E. major methanolic leaf extracts as well as the E. baileyana aqueous leaf extract induced significant mortality in the Artemia fransiscana bioassay, with LC50 values substantially <1000 μg/mL, indicating the toxicity of these extracts. Conclusion: The potent inhibitory bioactivity of the E. baileyana and E. major extracts against B. anthracis demonstrate their potential as medicinal agents in the treatment and prevention of anthrax. However, their toxicity indicates that their use may be limited to the treatment of the cutaneous form of the disease, or for sterilisation of infected sites.


SUMMARY
• Methanolic and aqueous Eucalyptus spp.extracts were potent inhibitors of Bacillus anthracis growth.• The E. baileyana and E. major methanolic leaf extracts were particularly potent growth inhibitors with MIC's of 386 and 287 µg/mL respectively.
• Tasmannia spp.extracts had low growth inhibitory activity, with high MIC values.
• Melaleuca alternifolia and Scaevola spinescens were completely devoid of Bacillus anthracis growth inhibitory activity.• The methanolic and aqueous E. baileyana and methanolic E. major leaf extracts displayed significant toxicity in the Artemia nauplii assay.• All other extracts were non-toxic in the Artemia nauplii assay.

INTRODUCTION
Bacillus anthracis is a facultative, spore-forming member of the Bacillaceae family and the etiological agent of the zoonotic disease anthrax. 1 The bacterium has recently gained notoriety as a biological weapon, highlighting it as a focal point of bacteriological exploration as a matter of international urgency.B. anthracis has an extensive history in both modern warfare and in terrorism attacks, with the intentional infection and subsequent deaths of humans documented for over a century. 2,3owever, aside from its potential applications to bioterrorism, B. anthracis is simply one of many Bacillus spp.found ubiquitously in terrestrial environments which indiscriminately infects animals and humans alike.Indeed, anthrax is well known within the agricultural community as the causative agent responsible for the indiscriminate, haphazard death of livestock and wildlife. 4As a result, extensive research has investigated the treatment and prevention of anthrax, as well as understanding the mechanisms and nature of the bacterium responsible for causing the lethal disease.
Human infection of anthrax can be divided into three forms: inhalation (pulmonary), cutaneous (direct contact to open wounds) and gastrointestinal (ingestion). 5Dissimilar to typical bacterial infections, anthrax is contracted through exposure to endospores rather than vegetative cells.Under environmental stresses, such as extreme surroundings or when deprived of necessary nutrients, B. anthracis produces spores that place it in a dormant-like state as a protection mechanism until conditions are once again favourable for cellular proliferation. 6,7Once internalised, the endospores resume normal metabolic functions and proliferation within the host ensues.Symptoms vary between disease types and if untreated anthrax has an extremely high mortality rate. 8Signs of anthrax infection range from flu-like symptoms (gastrointestinal and inhalational) to visible eschars (cutaneous), although any form can progress to fatal systemic anthrax. 9Anthrax meningitis may also develop and is indicated by the presence of blood in cerebrospinal fluid, shortly followed by loss of consciousness and ultimately death.The treatment and prevention of anthrax is principally achieved through vaccinations and antibiotic intervention.Vaccination against B. anthracis offers an effective means of disease prevention.However it is not routinely provided unless an individual is at risk of being exposed to the disease.Those at particular risk such as military personnel (in biological warfare), are regularly vaccinated to avoid infection. 11While considered the best method of prevention, vaccination is only 92.5% effective and must be administered well before infection occurs. 12Once the disease is contracted, the course of treatment is large doses of oral and/or intravenous antibiotics.However, due to the inherent risk that antibiotic treatment will result in resistant strains, the development and discovery of effective drugs is important in the long term management of the disease.The search is ongoing for new antimicrobials to inhibit B. anthracis growth, either by (a) the design and synthesis of new agents, or (b) researching the repertoire of natural resources for as yet unrecognised or poorly characterised antimicrobial agents. 13A re-examination of traditional medicines for the treatment of bacterial pathogens is an attractive prospect as the antiseptic qualities of medicinal plants have been long recognised and recorded.Furthermore, there has recently been a revival of interest in herbal medications due to a perception that there is a lower incidence of adverse reactions to plant preparations compared to synthetic pharmaceuticals.Probing of natural resources for compounds with known antimicrobial properties offers a novel approach to the treatment of anthrax.5][16][17] Furthermore, many Australian plants have well known antiseptic properties. 18Several plant species were selected for B. anthracis growth inhibitory activity screening based on their usage in traditional medicine systems and/or their reported antibacterial activities (Table 1).9][20][21] The first Australians crushed the leaves and inhaled the volatiles to treat coughs and colds. 18,19Fresh leaves or decoctions prepared from the leaves were also used as wound antiseptics and to treat skin and throat infections.Several in vitro studies have demonstrated the growth inhibitory properties of Eucalyptus baileyana and Eucalyptus major extracts towards a panel of bacterial species. 20,21Indeed, these studies reported both species to strongly inhibit the growth of the bacterium Bacillus cereus, which shares a close taxonomic relationship with B. anthracis (>99% 16S rRNA sequence homology), indicating their potential to inhibit B. anthracis growth.Essential oils prepared from Eucalyptus leaves remain a popular antiseptic agent, not only in Australia, but are also commonly sold in pharmacies internationally.Melaleuca spp.also have well established antibacterial properties (Table 1).'Tea-tree' essential oils are used as antiseptics in a similar manner as the Eucalyptus essential oils. 18,19Melaleuca spp.essential oils are rich also in 1,8-cineol, as well as other mono and sesquiterpenoids. 18Laboratory studies have reported broad spectrum antibacterial activity for Melaleuca quinquenervia and Melaleuca alternifolia, although neither species inhibited the growth of B. cereus in those studies. 20Scaevola spinescens is also used in several Australian Aboriginal medicinal systems (Table 1). 18,19Infusions of the roots were used to treat stomach pain and urinary disorders.A decoction of the stems material is applied externally to treat boils, rashes and skin disorders.Burning the whole plant produces fumes which are inhaled to treat coughs and colds.The leaves and twigs may also be steamed and sores and skin disorders treated by exposure to the steam.Bacterial infections are responsible for many of these disorders.Recent studies reported potent broad spectrum bacterial growth inhibitory activity for S. spinescens extracts, 22 however also reported that the extracts were ineffective inhibitors of B. cereus growth.Plants from the genus Tasmannia (family Winteraceae) have culinary uses, although there is no record of their usage in traditional Australian medicinal systems (Table 1). 23Recent studies have reported potent broad spectrum antimicrobial activity for Tasmannia lanceolata 24 and Tasmannia stipitata extracts in vitro. 14Interestingly, moderate to good B. cereus growth inhibitory activity was reported for both Tasmannia species.Furthermore, T. lanceolata 25 and T. stipitata 14 have also been reported to inhibit the proliferation of the gastrointestinal protozoal parasite Giardia duodenalis. 14,25To our knowledge, there no similar studies reporting therapeutic properties for T. insipidia.Despite this relative wealth of information documenting antibacterial Australian plants, many are yet to be tested for the ability toinhibit B. anthracis growth.The current study examines the growth inhibitory activity of extracts of selected Australian

Bailey's stringy bark
The leaves and kinos of many Eucalyptus spp.are used to prepare extracts, decoctions or as essential oils.
leaves Inhalation of the volatiles from essential oils or from crushed leaves is used to treat coughs and colds.The leaves, oils or infusions produced from the leaves are also used as wound antiseptics, or to treat skin or throat infections.
This species has been shown to have potent broadspectrum antibacterial activity in vitro.

Queensland grey gum
The leaves and kinos of many Eucalyptus spp.are used to prepare extracts, decoctions or as essential oils.

leaves and flowers
Inhalation of the volatiles from essential oils or from crushed leaves is used to treat coughs and colds.The leaves, oils or infusions produced from the leaves are also used as wound antiseptics, or to treat skin or throat infections.
This species has been shown to have potent broadspectrum antibacterial activity in vitro.

Melalueca alternifolia
narrow leaved paperbark, narrow leaved teatree, narrow leaved ti-tree, snow in summer The leaves of many Melaleuca spp.are used to prepare extracts, decoctions or as essential oils.
leaves Inhalation of the volatiles from essential oils or from crushed leaves is used to treat coughs and colds.The leaves, oils or infusions produced from the leaves are also used as wound antiseptics, or to treat skin or throat infections.
This species has been shown to have potent broadspectrum antibacterial activity in vitro.
plants which prevent the growth of other bacteria against B. anthracis with the aim of determining new leads for the prevention and treatment of anthrax.

Plant source and extraction
Air dried Tasmannia insipidia and Tasmannia stipitata leaves and berries were supplied and verified by the Queensland Bushfoods Association, Australia.Scaevola spinescens was supplied by Jeannie Crago of Outback Books Australia (a commercial supplier of S. spinescens tea) as a predried and course milled whole plant material.Eucalyptus baileyana, Eucalyptus major and Melaleuca alternifolia plant materials were collected from Toohey Forest, Brisbane and were identified with reference to a taxonomic key to Toohey Forest plants. 29Voucher samples of all plant specimens have been stored in the School of Natural Sciences, Griffith University.The plant materials were thoroughly dried in a Sunbeam food dehydrator and the dried plant materials were stored at -30 o C. Prior to use, the plant materials were thawed and freshly ground to a coarse powder.Individual 1 g quantities of the ground plant material were weighed into separate tubes and 50 mL of methanol or water were added.All solvents were obtained from Ajax and were AR grade.The ground plant materials were individually extracted in each solvent for 24 hours at 4 o C with gentle shaking.The extracts were subsequently filtered through filter paper (Whatman No. 54) under vacuum, followed by drying by rotary evaporation in an Eppendorf concentrator 5301.The resultant dry extract was weighed and redissolved in 10 mL deionised water (containing 1% DMSO).

Qualitative phytochemical studies
[32] Antibacterial screening Environmental Bacillus anthracis strain An environmental strain of Bacillus anthracis was isolated and used in these studies.The bacterium was originally isolated from a water sample taken from Paralana hot springs (30°17'49"S, 139°44'15"E), South Aus-tralia.Isolation was achieved through successive culturing steps using a modified Peptone/Yeast Extract (PYE) agar as previously described. 13equence analysis of the environmental isolate generated a contig of 1428bp which was revealed to be 99.92% similar to B. anthracis by Ez-Taxon and designated as Bacillus anthracis strain PMO.The GenBank accession number for the 16S rRNA gene sequence for the isolate is KR003287.

Evaluation of antimicrobial activity
1][32] Briefly, 100 µL of the test bacteria were grown in 10 mL of fresh nutrient broth media until they reached a count of approximately 10 8 cells/mL.An amount of 100 µL of bacterial suspension was spread onto nutrient agar plates.The extracts were tested for antibacterial activity using 6 mm sterilised filter paper discs.Discs were impregnated with 10 µL of the test sample, allowed to dry and placed onto inoculated plates.The plates were allowed to stand at 4 o C for 2 hours before incubation at 30 o C for 24 hours.The diameters of the inhibition zones were measured in millimetres.All measurements were to the closest whole millimetre.Each assay was performed in at least triplicate.Mean values (± SEM) are reported in this study.Standard discs of penicillin-G (2 µg) and chloramphenicol (10 µg) were obtained from Oxoid Australia Ltd. and served as positive controls for antibacterial activity.Filter discs impregnated with 10 µL of distilled water were used as a negative control.

Minimum inhibitory concentration (MIC) determination
1][32] Briefly, the plant extracts were diluted in deionised water and tested across a range of concentrations.Discs were impregnated with 10 µL of the test dilutions, allowed to dry and placed onto inoculated plates.The assay was performed as outlined above and graphs of the zone of inhibition versus concentration were plotted for each extract.Linear regression was used to calculate the MIC values.

Toxicity screening
Reference toxin for toxicity screening Potassium dichromate (K 2 Cr 2 O 7 ) (AR grade, Chem-Supply, Australia) was prepared as a 4 mg/mL solution in distilled water and was serially diluted in artificial seawater for use in the Artemia franciscana nauplii bioassay.

Scaevola spinescens R.Br.
currant bush, maroon bush, fanflower, prickly fanflower The whole plant was used medicinally by the first Australians.
leaves An infusion of the roots was used to treat stomach pain and urinary disorders; a decoction of the stem was used to treat boils, rashes and skin disorders; the whole plant was burnt and fumes were inhaled to treat colds; leaves and twigs were steamed and sores and skin disorders were treated by exposure to the steam.4][35] Briefly, 400 µL of seawater containing approximately 43 (mean 43.2, n=155, SD 14.5) A. franciscana nauplii were added to wells of a 48 well plate and immediately used for the bioassay.A volume of 400 µL of diluted plant extracts or the reference toxin were transferred to the wells and incubated at 25 ± 1 o C under artificial light (1000 Lux).A negative control (400 µL seawater) was run in triplicate for each plate.All treatments were performed in at least triplicate.The wells were checked at regular intervals and the number of dead counted.The nauplii were considered dead if no movement of the appendages was observed within 10 seconds.After 24 h, all nauplii were sacrificed and counted to determine the total % mortality per well.The LC 50 with 95% confidence limits for each treatment was calculated using probit analysis.

Statistical analysis
Data are expressed as the mean ± SEM of at least three independent experiments.

Liquid extraction yields and qualitative phytochemical screening
Extraction of 1 g of the various dried Australian plant materials with the solvents yielded dried plant extracts ranging from 116 mg (S. spinescens methanolic extract) to 324 mg (E.major methanolic flower extract) (Table 2).With the exception of the S. spinescens extracts, the methanolic extracts generally gave higher yields of dried extracted material compared with the corresponding aqueous extracts.The dried extracts were resuspended in 10 mL of deionised water (containing 1% DMSO) resulting in the extract concentrations shown in Table 2.
Qualitative phytochemical studies showed that the methanolic and aqueous extracts generally had a wide range of phytochemicals (Table 2).Both methanol and water extracted high levels of phenolics (water soluble) for all plant materials.Moderate to high flavonoid contents were present in most extracts.Similar levels of tannins were evident in all extracts except the Tasmannia spp.extracts, which were devoid of detectable tannins.Low to moderate levels of saponins were seen in all Eucalyptus spp.extracts as well as the M. alternifolia and S. spinescens extracts.All extracts were devoid of detectable levels of alkaloids, polysterols, cardiac glycosides and athraquinones.

Antimicrobial activity
To determine the ability of the crude plant extracts to inhibit the growth of B. anthracis, aliquots (10 µL) of each extract were screened using a disc diffusion assay.The bacterial growth was inhibited by 11 of the 16 extracts screened (68.8%) (Figure 1).The E. baileyana and E. major methanolic leaf extracts were the most potent inhibitor of B. anthracis growth (as judged by zone of inhibition), with inhibition zones of 15.7 ± 0.6 mm and 15.3 ± 0.6 mm respectively.This compares favourably with the penicillin and chloramphenicol controls, which had zones of inhibition of 8.3 ± 0.6 and 15.6 ± 0.6 mm respectively.All of the Eucalyptus spp.extracts displayed potent growth inhibition with >10 mm zones of inhibition (with the ex-   -indicates no bacterial growth inhibition was evident, or that an LC 50 value could not be obtained as the mortality did not reach 50% for any dose tested. ception of E. baileyana aqueous extract, with a zone of inhibition of 8.3 ± 0.6 mm).The methanolic and aqueous T. stipitata extracts also displayed moderate to good growth inhibitory activity (7.7-10.3mm).The M. alternifolia and S. spinescens methanolic and aqueous extracts were completely devoid of B. anthracis growth inhibitory activity.
The antimicrobial efficacy was further quantified by determining the MIC values (Table 3).The methanolic E. baileyana and E. major leaf extracts were particularly effective at inhibiting microbial growth, with MIC values against B. anthracis <400 µg/mL (<4 µg impregnated in the disc).The aqueous and methanolic E. major flower and the aqueous E. baileyana and E. major leaf extracts, whilst less potent B. anthracis growth inhibitors, also displayed good growth inhibition with MIC's <2000 µg/mL (<20 µg impregnated in the disc).All other plant extracts were either inactive or showed only low inhibitory activity with MIC values >10,000 µg/mL.

Quantification of toxicity
All extracts were initially screened at 2000 µg/mL in the assay (Figure 2).For comparison, the reference toxin potassium dichromate (1000 µg/ mL) was also tested in the bioassay.The potassium dichromate reference toxin was rapid in its onset of mortality, inducing nauplii death within the first 3 hours of exposure and 100% mortality was evident following 4-5 hours (results not shown).With the exception of the M. alternifolia aqueous extract and the aqueous and methanolic S. spinescens extracts, the plant extracts displayed >50% mortality rates at 24 h.To further quantify the effect of toxin concentration on the induction of mortality, the extracts were serially diluted in artificial seawater to test across a range of concentrations in the Artemia nauplii bioassay.

DISCUSSION
22]28 S. spinescens leaves 22 and the leaves and berries of several Tasmannia spp. 14,16,24,28Have been reported to have inhibitory activity against extensive panels of pathogenic bacteria.Each of these species was equally effective at inhibiting the growth of Gram positive and Gram negative bacteria.In contrast, the bacterial growth inhibitory properties of the Eucalyptus spp.and of M. alternifolia have been reported against a narrower range of pathogenic bacteria. 20,21nterestingly, whilst B. anthracis was not tested in any of the previous studies, E. baileyana and E. major extracts were reported to strongly inhibit the growth of the related bacterial species B. cereus.Whilst an investigation of the phytochemistry of the Eucalyptus spp.extracts was beyond the scope of our study, plants of the genus Eucalyptus are well known for their high terpenoid contents.In particular, high 1, 8-cineol contents was reported for several Eucalyptus spp. 18Potent bacterial growth inhibitory activity has been reported for 1, 8-cineol against a panel of pathogenic bacteria, including B. cereus. 40Another study reported MIC values for 1, 8-cineol against Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli of between 16 and 256 µg/mL. 41hat study did not screen 1, 8-cineol against Bacillus spp.Eucalyptus spp.are also rich in a variety of other mono and sesquiterpenoids. 18Some of these terpenoids have been previously reported to have potent broad spectrum antibacterial activity 23 and therefore may contribute to the inhibitory activity against B. anthracis.Another commonality between the inhibitory Eucalyptus spp.extracts was that all contained relatively high levels of flavonoids and tannins.Many studies have reported potent antibacterial activities for a wide variety of flavonoids. 42Whilst we were unable to find any reports of B. anthracis growth inhibitory activity of flavonoids, they have been reported to inhibit growth of the closely related species B. cereus. 43Similarly, a number of tannin compounds have bacterial growth inhibitory activity.Gallo tannins have been reported to inhibit the growth of a broad spectrum of bacterial species 44 through a variety of mechanisms including binding cell surface molecules including lipotoichoic acid and proline-rich cell surface proteins, 45,46 and by inhibiting glucosyl transferase enzymes. 47Elligitannins are also highly potent inhibitors of bacterial growth, with MIC values as low as 62.5 µg/ml. 44,46Ellagitannins have also been reported to function via several antibiotic mechanisms including interaction with cytoplasmic oxidoreductases and by disrupting bacterial cell walls. 44,46Thus, it is likely that multiple compounds within the Eucalyptus spp.extracts are contributing to the growth inhibition of B. anthracis.
The findings reported here demonstrate that the majority of the Australian plant extracts tested in our study were nontoxic towards Artemia franciscana nauplii.However, the most promising B. anthracis growth inhibitory extracts (methanolic and aqueous E. baileyana and methanolic E. major leaf extracts) displayed substantial toxicity, with LC 50 values as low as 455 µg/mL.Extracts with LC 50 values <1000 µg/ml towards Artemia nauplii are defined as being toxic, 34 which may impact on their therapeutic potential.As the LC 50 values are within the therapeutic ranges that would be required for B. anthracis growth inhibition (determined by MIC), studies using human cell lines are required to further evaluate the safety of these extracts.However, even if the Eucalyptus spp.extracts are subsequently deemed unsafe for ingestion, they may still be useful B. anthracis growth inhibitory agents.The most prevalent form of anthrax is cutaneous infection via skin cuts and abrasions. 48Topical application of the extracts may prove effective in treating this form of the disease.Alternatively, the Eucalyptus spp.extracts may be useful for disinfecting contaminated sites (e.g.where infected livestock have perished), or for sterilising surfaces that have been in contact with B. anthracis.Furthermore, whilst the results of our study are promising, it must be noted that the growth inhibitory studies screened against vegetative cells.As Bacillus spp.are spore formers, further studies are required to determine whether extracts with B. anthracis growth inhibitory activity also affect bacterial growth from the spores.

CONCLUSION
The results of this study demonstrate the potential of the Eucalyptus spp.extracts to block the growth of B. anthracis.However, the toxicity of these extracts may limit their clinical usage.Further studies aimed at the purification of the bioactive components are needed to examine the mechanisms of action of these agents.

Figure 1 :
Figure 1: Growth inhibitory activity of plant extracts against the B. anthracis environmental isolate measured as zones of inhibition (mm).L=leaf; F=flower; B=berry; M=methanolic extract; W=aqueous extract Results are expressed as mean zones of inhibition ± SEM

Figure 2 :
Figure 2: The lethality of the Australian plant extracts (2000 µg/mL) and the potassium dichromate (1000 µg/mL) and seawater controls towards Artemia franciscana nauplii after 24 hours exposure.L=leaf; F=flower; B=berry; M=methanolic extract; W=aqueous extract Results are expressed as mean zones of inhibition ± SEM 10

Table 3 : Minimum inhibitory concentration (µg/mL) of the plant extracts and LC 50 values (µg/mL) in the Artemia nauplii bioassay. Species Part Extract MIC (µg/mL) LC 50 (µg/mL)
Numbers indicate the mean MIC and LC 50 values of triplicate determinations.

Table 3
34ows the LC 50 values of the extracts towards A. franciscana.No LC 50 values are reported for S. spinescens extracts or for the M. alternifolia aqueous extract as <50% mortality was seen for all concentrations tested.Significant toxicity was noted for both E. baileyana extracts as well as the E. major leaf methanolic extract with LC 50 values substantially <1000 µg/ mL.All other extracts were determined to be nontoxic, with LC 50 values substantially greater than 1000 µg/mL following 24 h exposure.Extracts with an LC 50 of greater than 1000 µg/mL towards Artemia nauplii have been defined as being nontoxic.34