Chapter - 7
Anthelmintic activity of Bacillus
cereus and Bacillus pumilus
metabolites
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7 .1 . I n tr o d u c tio n
7.1.1. H elm inthes (w orm s)
Helminthes infections are among the most common infections in humans
affecting a large population o f world.
About
o f world’s population harbors
helminthes o f one species or another (Bundy, 1994). Nematodes can attack and
destroy a wide variety of organisms, including animals, microorganisms and plants.
7.1.2. A nim al infections
Internal parasitic infection is a great threat not only to humans but also to the
productivity o f the sheep and goat industry (Ayers et a l, 2010). Parasitic worms can
infect livestock and cause diseases and death. It has a great impact on economy of
domesticated live stock throughout the world due to their adverse effect on
productivity (Jabbar et a l, 2006). Also o f importance is the infection o f domestic
pets. Indeed, the companion animal market is a major economic consideration for
animal health companies undertaking drug discovery programs.
7.1.3. Infections in plants
Parasitic worms also infect crops affecting food production with a resultant
economic impact. Among the species susceptible to nematode infections, plants with
significant agricultural and forestry importance have probably attracted the most
attention. This is because plant parasitic nematodes can cause serious damages to
agriculture and forestry (Siddiqui and Mahmood, 1996). For example, it has been
estimated that plant root-knot nematodes cause around US $ 80 billion worth of
damages to the global agricultural crops annually (Rodriguez-Kabana and Canullo,
1992). As a result, there have been significant financial and scientific investments to
find effective solutions to control parasitic nematodes.
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7.1.4. H u m a n in fectio n s
There are two clinically important types o f worm infectionsA. The worms living in the host’s alimentary canal.
The main examples o f worms that lives in the host’s alimentary canal are;
Tapeworms (eg:- Taenia saginata. Taenia solium. Hymenolepis nana....Qio. ) and
intestinal roundworms ( Qg\-Ascaris lumbricoides, Enterobius vermicularis (thread
worm), Trichuris trichiura (whipworm), Ankylostoma duodenale (Hookworm) etc.
It is estimated that 1000 million people harbor A. lumbricoides, 500 million T.
trichiura and 500 million E. vermicularis while atleast 800 million have hookworm
infection.
B. The worms living in other tissues o f the host’s body.
The main examples o f worms that live in the tissues o f the host are;
Trematodes or Flukes (eg:- Schistosoma haematobium, Schistosoma mansoni etc).
Tissue roundworms (eg:- Dracunculus medinensis (Guinea-worm) Filariae, etc) and
Hydatid tapeworms (eg:- Echinococcus species) (Rang et al„ 1999).
The intestinal roundworms including hookworms and whipworms harm the
host by depriving him o f food, causing blood loss, injury to organs, intestinal or
lymphatic obstruction and by secreting the toxins (Tripati, 2003). These infections
have led to lowering o f immune systems for HIV, malaria and tuberculosis and
debilitating the individual both physically and cognitively (Fang, 2010). Majority of
infections are generally limited to tropical regions and contribute to the prevalence
o f malnutrition, anemia, eosinophilia and pneumonia (Dikasso et a l, 2006).
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7.1.5. A nthelm intics
Anthelmintics are drugs that are used to treat infections with parasitic
worms. This includes both flat worms, e.g., flukes, tapeworms and round worms,
i.e., nematodes. Anthelmintics can kill (Vermicide) or expel (Vermifuge) infesting
helminthes. Tremendous progress has been made in the development o f anthelmintic
drugs in the past 50 years. During this period, the current classes o f synthetic drugs
were developed including the benzimidazoles and imidazothiazoles. Another major
step was achieved with the introduction o f the avermectin class o f macrolactones in
the early 1980’s. The discovery o f this compound class led to anthelmintics drugs,
such as ivermectin and doramectin, which have excellent broad spectrum activity
and superior potency (Ayers et a l, 2010). Anthelmintic drugs are o f huge
importance for humans and for veterinary as medicine. The World Health
Organization estimates that a staggering 2 billion people harbor parasitic worm
infections (http://www.who.int/wormcontrol/statistics/).
An anthelmintic drug can act by causing paralysis o f the worm, or by damaging
its cuticle, leading to partial digestion or to rejection by immune mechanisms.
Anthelmintic drugs can also interfere with the metabolism o f the worm. Some
important examples o f anthelmintic drugs with their mode o f action are discussed
below in brief
A. Piperazine
Introduced in 1950, it is highly active against Ascaris and Enterobius. It
reversibly inhibits neuromuscular transmission in the worm, probably by acting like
GABA; the inhibitory neurotransmitter on GABA-gated chloride channels in
nematode muscle. The paralyzed worms are expelled alive.
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B. Benzimidazoles
The benzimidazole anthelmintics include mebendazole, thiabendazole and
albendazole. These compounds are board spectrum agents and constitute one o f the
main groups o f anthelmintics used clinically. They bind to free P-tubulin, inhibiting
its polymerization and thus interfering with microtubule-dependant glucose uptake.
C. Iverm ectin
Ivermectin is a semisynthetic agent derived from a group o f natural substances
obtained from an Actinomycete. It is thought to paralyse the worm by opening
chloride channels and increasing chloride conductance. Its binding site is different
from that in mammalian species and distinct from that o f all other effector molecules
o f the chloride charmel (Rang et al., 1999).
7.1.6. R esistance to anthelm intic drugs
Though many drugs are currently available in the market, still nematodes
represent a serious threat to both plants and animals.
Resistance to all o f these
classes o f drugs has been observed. New methods o f control o f these parasites are
being sought since a number o f soil applied commercial nematocides are being
phased out and resistance to anthelmintics is an increasing problem (Ghisalberti,
2002). The resistance to nematodes has been reported from several countries
(Jackson, 1993; Rolfe, 1997). Hookworms and some other parasitic nematodes have
shown signs of resistance to albendzole, the current treatment approved by the world
health organization (Fang, 2010). The increasing prevalence o f helminth parasites
that are resistant to convectional anthelmintics has been the spur for different
research programs exploring alternative approaches to control the parasites and to
discover new classes o f anthelmintics, especially those with novel mode o f action
(Jackson et al., 2000; McKellar et al., 2004; Ayers et a l, 2010).
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7.1.7. H erbel products as anthelm intics
Herbal medicines are in great demand in the developed as well as in
developing countries for primary health care because o f their wide biological and
medicinal activities, higher safety margin and lower costs. Since the time
immemorial, our folklore and traditional system o f medicine are claiming that
medicinal plants as a whole or their parts are being used in all types o f diseases
successfully including antibacterial, anthelmintic and anti-inflammatory etc. Plants
are generally known to produce many secondary metabolites which constitute an
important source o f microbicides, pesticides and many pharmaceutical drugs. The
medicine practiced by our forefathers has been the precursor o f modem
pharmaceutical medicine. This includes herbal medicine and phytotherapy, which is
prevalent in Chinese, Ayurvedic (Indian), and Greek medicine (Ahmad, 1992). They
still remain the principal source of pharmaceutical agents (Ibrahim, 1997; Ogundipe
et al., 1998). Many potential phytotherapeutics have been revealed that some plant
species, when grazed or fed as conserved material may reduce the degree o f internal
parasite infection in sheep (Niezen etal, 1995; Marley et a l, 2003; Paolini et a l,
2003). Because o f this resurgence o f interest the research on plants o f medicinal
importance is growing phenomenally at the international level, often to the detriment
o f natural habitats and mother populations in the countries o f origin (Farnsworth and
Soejarto, 1991)
Though the development o f avermectin class o f compounds obtained from a
soil bacterium Streptomyces avermitilis initiated more research to look for microbial
metabolites for potent anthelmintic compounds, less literature are available
regarding microbial metabolites as anthelmintics. Hence in the present study, the
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metabolic extracts o f two bacteria B. cereus and B. pumilus were tested for
anthelmintic activity in vitro.
7 .2 . R e v ie w o f L ite r a tu r e
Most o f the literatures available on anthelmintic activity are medicines o f
plant derived products. Since the discovery o f avermectins in 1979 many
investigations have reported bacterial and fungal metabolites with anthelminic
activity. In the present survey, though we looked for metabolites o f both, bacteria
and fungi for anthelmintic activity, more emphasis was given to literatures on
bacterial metabolites because the present study was also on bacterial metabolites for
anthelminic activity.
7.2.1. B acterial m etabolites w ith anthelm intic activity
Burg et al. (1979) reported a complex o f chemically related compounds
called the “avermectins” which exhibit extraordinarily potent anthelmintic activity.
They were produced by a novel species o f Actinomycete, NRRL 8165, which they
named Streptomyces avermitilis. The morphological and cultural characteristics
which differentiate the producing organism from other species were described. The
avermectins have been identified as a series o f macrocyclic lactone derivatives
which in contrast to the macrolide or polyene antibiotics, lack significant
antibacterial or antifungal activity. The avermectin complexes were fully active
against the gastrointestinal nematode Nematospiroides dubius when fed to infected
mice for 6 days at 0.0002% o f the diet.
Campbell et al. (1983) reported a compound called “Ivermectin”. It is a 22,
23-dihydro derivative o f avermectin B l, a macrocyclic lactone produced by an
Actinomycete, Streptomyces avermitilis. It was active at extremely low dosage
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against a wide variety of nematode and arthropod parasites, apparently by virtue of
its action on the mediation o f neurotransmission by gamma-aminobutyric acid.
Hood et al. (1989) reported a series o f milbemycin antibiotics produced by
soil isolate strain E225 which was found to be a Streptomyces species. The
antibiotics displayed anthelmintic activity against Trichostrongylus colubriformis.
Two o f the compounds, VM 44857 and VM 44866 were shown to be potent
anthelmintics against mixed nematode infections in sheep.
Haber et al. (1991) reported four morphologically similar cultures that
produced avermectin-like activities. Isolation o f the active components from one of
these cultures (strain UC
8984) followed by nuclear magnetic resonance
spectroscopy resulted in the identification o f milbemycins alpha 1 and alpha 3.
These metabolites are members o f a large family o f milbemycins produced by
Streptomyces hygroscopicus subsp. aureolacrimosus NRRL 5739. Systematic
studies revealed that strain UC 8984 is also a S. hygroscopicus strain, but which is
taxonomically distinct from NRRL 5739.
Heins et al. (1995) reported a novel strain o f Bacillus pumilus AQ 717 that
produces a metabolite that exhibits pesticidal activity against com rootworms and
protecting the plant from com rootworm, nematode and beet armyworm infestations.
Kopcke et al. (2002) isolated eight secondary metabolites from submerged
cultures o f the Ascomycete A ll 1-95 during a search for new nematicidal
metabolites. Compounds 1, 3, 4, 5, 6 and 8 (3 and 4 were obtained as an unseparable
mixture). Galiellalactone (7) and compound 2 were metabolites previously obtained
from cultures o f Galiella rufa. Compound 2, pregaliellalactone (5) and the mixture
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o f 3 and 4 showed nematicidal activities towards Caenorhahditis elegans and
Meioidogyne incognita.
Hu, (2010) reported isolation o f a soil bacterium Bacillus thuringiensis (Bt)
with nematicidal activity. This bacterium produced “Bt toxin” and was found to be a
natural soil predator o f nematodes. Bt toxin, produced by bacteria, could help battle
roundworm infections.
7.2.2. Fungal m etabolites w ith anthelm intic activity
Anke et al. (1995) reported screening o f nematode-trapping fungi for
antimicrobial and nematicidal activities. From trap-forming submerged cultures of
Arthrobotrys conoides, linoleic acid was isolated as a nematicidal principle. Its
production increased with the number o f traps formed in both Arthrobotrys
oligospora and Arthrobotrys conoides. Several strains o f Ascomycetes had
nematicidal activities; linoleic acid was responsible for the activity in cultures o f a
Chlorosplenium species, 14-epicochlioquinone B in cultures o f Neobulgaria pura,
and two naphthalenes derived from the melanin biosynthetic pathway in Daldinia
concentrica. 5-Pentyl-2-furaldehyde, previously known as a metabolite from a
Basidiomycete, was produced by an unidentified Australian Ascomycete. More than
30 mostly new metabolites have been isolated from cultures o f Lachnum
papyraceum, many being chlorinated. Under different conditions the fungus
incorporated bromine instead o f chlorine.
Omura et al. (2001) reported “Naafuredin”, an epoxy-6-lactone with a
methylated olefmic side chain isolated from the culture broth o f Aspergillus niger
which exerted anthelmintic activity against the ruminant parasite worm Haemonchus
contours and the dwarf tapeworm Hymenolepsis nana in mice.
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Yanai et al. (2004) reported isolation o f a filamentous fungus Rosellinia sp.
The fungus was reported to produce a cyclooctadepsipeptide PF1022A possessing
strong anthelmintic properties.
The broad-spectrum anthelmintic cyclooctadepsipeptide PF1022A is a fungal
metabolite from Rosellinia sp. PF1022, which is found on the leaves o f Camellia
japonica. A broad range o f structurally related cyclooctadepsipeptides has been
characterized and tested for anthelmintic activities. These metabolites have been
used as starting points to generate semisynthetic derivatives with varying
nematocidal capacity (Krucken et al., 2012).
Guohong et al. (2007) in their review article have summarized 179
compounds from fungi that have shown to possess nematicidal activities. These
compounds belong to diverse chemical groups and they were mainly isolated from a
variety o f Deuteromycete, Ascomycete and Basidiomycete fungi. Some o f them have
been patented as nematicidal agents.
7 .3 . M a te r ia ls a n d M e th o d s
7.3.1. T est sam ples
The samples prepared by successive solvent extraction with petroleum ether,
ethyl acetate and methanol from metabolites o f Bacillus cereus (BC-1 to BC-3) and
Bacillus pumilus (BP-1 to BP-3) were used as test samples.
7.3.2. D eterm ination o f paralysis and death tim e o f earthw orm
Pherethima postuma
Adult Indian earthworms (Department o f Pharmacology, National college of
Pharmacy, Shimoga, Karnataka) were used for the initial screening o f anthelmintic
compounds in vitro (Sollmarm 1918, Dash et a l, 2002). The assay was performed
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on earthworm pherethima postuma due to its anatomical and physiological
resemblance with the intestinal roundworm parasite o f human beings and also
because o f its easy availability (Chatterjee, 1967; Vigar, 1984; Thom et a l, 1997).
Indian adult earthworms were washed with normal saline to remove all dirt
and fecal matter before use for the anthelmintic study. The earthworm o f 8-12 cms
in length and 0.3-0.5 cm in width were used. The anthelmintic assay was carried as
per the method o f Ajaiyeoba et al (2001).
The earthworms were divided into eight groups o f six worms each. The
worms o f groups 1, 2, and 3 were released into separate plates containing a solution
o f 50 ml o f the test samples of B.cereus BC-1, BC-2 and BC-3 respectively.
Similarly, groups 4, 5 and 6 received the test samples o f B.pumilus BP-1, BP-2 and
BP-3 respectively. All the samples were prepared in sterile water at a dose o f 20
mg/ml. The group 7 worms were released into 50 ml o f standard drug albendazole
solution (GlaxoSmithKline Pharmaceuticals Limited, Nashik, Maharashtra, India) of
20 mg/ml concentration. The control group was placed in plain sterile water o f 50 ml
(group eight).
Observations were made for the time taken to paralysis and death of
individual worms. Time for paralysis was noted when no movement o f any sort
could be observed except when the worms were shaken vigorously and failed to
revive even after transfer to normal saline. Death was concluded when the worms
lost their motility completely and failed to respond even after a touch with the
needle followed by fading away o f their body colors.
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7 .4 . R e su lts
7.4.1. Bacillus cereus
The sample extracts BC-1, BC-2 and BC-3 paralyzed the worms in 15
minutes, 17 minutes and 15 minutes respectively at 20 mg/ml concentration. The
time taken by test samples for paralysis o f worms was significantly better when
compared to the standard drug albendazole which took 30 minutes for paralysis at 20
mg/ml.
The time taken for death o f worms by test samples BC-1, BC-2 and BC-3
was 120 minutes, 130 minutes and 150 minutes respectively. The time taken for
death o f worms by all the test samples was more when compared to standard drug
which brought the death in 70 minutes.
7.4.2. Bacillus pumilus
The sample extracts BP-1, BP-2 and BP-3 paralyzed the worms in 13
minutes, 16 minutes and 10 minutes respectively at 20 mg/ml concentration. This
time response by test samples for paralysis was much better than the standard drug
albendazole which took 30 minutes for paralysis at 20 mg/ml.
The time taken for death o f worms by the test samples BP-1, BP-2 and BP-3
was 90 minutes, 85 minutes and 95 minutes respectively. The time taken for death
by all the sample extracts was more when compared to standard drug which killed
the worms in 70 minutes.
The time taken for paralysis o f worms and subsequent death in presence of
different test samples and standard drug are shown in table 7.1. The histogram
showing the paralysis o f worms in minutes o f BC and BP test samples in
comparison with standard is shown in figure 7.1. The histogram showing the death
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o f worms in minutes o f BC and BP test samples in comparison with standard is
shown in figure 7.2.
Table 7.1. The time taken for paralysis and death o f worms in presence of
various test samples of Bacillus cereus and Bacillus pumilus
metabolites
Test Samples
Concentration
(mg/ml)
Time taken for
paralysis (in
minutes)
Time taken for
death (in minutes)
-
240±0.16
300±0.35
20 mg/ml
30±0.12
70±0.28
20
20
20
20
20
20
15±016
17±0.15
15±0.18
13±0.28
16±0.05
10±0.25
120±0.83
130±0.93
150±0.15
90±0.66
85±0.88
95±0.43
Control
(Sterile water)
Standard
(Albendazole)
BC-1
BC-2
BC-3
BP-1
BP-2
BP-3
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
Readings are average of six trials (±SEM)
BC - Bacillus cereus; BC-1 Petroleum ether extract; BC-2 Ethyl acetate extract;
BC-3 Methanol extract
BP - Bacillus pumilus; BP-1 Petroleum ether extract; BP-2 Ethyl acetate extract;
BP-3 Methanol extract
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_
300
^
250
I
200
150
U 100
a,
u
50
£
c
0«
03
i
c /
Figure 7.1. Paralysis of w orm s in m inutes in presence of Bacillus cereus and
Bacillus pum ilus metabolites in com parison with stan d ard
BC - Bacillus cereus', BC-I Petroleum ether extract; BC-2 Ethyl acetate extract;
BC-3 Methanol extract
BP - Bacillus pumilus', BP-1 Petroleum ether extract; BP-2 Ethyl acetate extract;
BP-3 Methanol extract
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350
3
c
300
1 250
fi/5 200
E
U
o
150
o
100
50
rs
0
I
III
1 1 1
Figure 7.2. Death of worms in m inutes in presence of Bacillus cereus and
Bacillus pum ilus m etabolites in com parison with stan d ard
BC - Bacillus cereus; BC-1 Petroleum ether extract; BC-2 Ethyl acetate extract;
BC-3 Methanol extract
BP
Bacillus pumilus; BP-1 Petroleum ether extract; BP-2 Ethyl acetate extract;
BP-3 Methanol extract
7 .5 . D isc u ssio n
The objective o f the present study w as to test the potential o f B. cereus and
B. p u m ilu s metabolites for anthelmintic activity. The motive behind to carryout
anthelmintic activity w as the toxicity shown by the extracts during cytotoxicity
studies on various cell lines (Both, normal and cancerous cells) during anticancer
studies.
M any bacteria and fungi have been reported to have antinem atode activity.
A m ong bacteria the metabolites o f genera P asteuria. P seudom onas. Serratia, and
am ong fungi A rthrobotrys. C atenaria and C ephalosporium are reported to have
antinem atode activity (Rodgers, 1989). “ L achnum on B1 and 8 2 ” , “ Mycorrhizin 81
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andB2”, both brominated
metabolites obtained
from Ascomycete
Lachnum
papyraceum with nematicidal and antimicrobial activities have been reported by
Stadler et al (1995).
“3-hydroxypropionic acid” obtained from endophytic fungi
Phomopsis phaseoli and Melanconium betulinum with nematicidal activity against
Caenorhabditis elegans and Meloidogyne incognita has been reported by Schwarz et
al. (2004).
“Avermectins”, a series o f highly potent anthelmintic and insecticidal
compounds o f the macrolide structural class produced by the soil bacterium
''Streptomyces avermitilis" was reported by Burg et al., (1979). Avermectin A, a
macrolide, molecular weight 873 is a commercially available compound with
anthelmintic and insecticides activity. Since the discovery o f the avermectins,
members of the structurally and functionally similar milbemycin family have been
isolated from Streptomyces hygroscopicus subsp. aureolacrinosus (Takaguchi et al.,
1980), Streptomyces cyaneogriseus subsp. moncyogenus (Carter et a l, 1988) and
Streptomyces sp. strain E225 (Hood et al., 1989).
Among the genera Bacillus, two species have been reported to have
anthelmintic activity. A solvent-extractable metabolite with a small molecular
weight, less than 10,000 dolton produced by a novel strain o f B. pum ilus with
pesticidal activity against rootworm, beet armyworm and nematodes have been
reported by Heins et al (1995). A crystal protein isolated from soil bacterium
Bacillus thuringiensis has been reported to kill the nematode worms and it is said to
be safe for vertebrates (Hu et al., 2010).
The present investigations have shown that both the bacterial extracts have
significant anthelmintic activity. The paralyzing capacities o f the sample extracts are
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comparatively better than the standard drug albendazole. Among the entire test
samples the methanol extract o f B. pumilus (BP-3) was good enough to paralyze the
worms in 10 minutes when compared to standard drug time o f 30 minutes. However,
regarding time taken for death o f worms, the sample extracts o f B. cereus took more
time when compared to sample extracts o f B. pumilus. The time taken by B. pumilus
extracts for death o f worms (between 85-95 minutes) was close to that o f the
standard drug time o f 70 minutes.
Since the sample extracts o f B. cereus showed early paralysis but delayed
death time o f the worms, the effect o f the extracts may be due to chloride ion
conductance o f worm muscle membrane, producing hyperpolarization and reduced
excitability that leads to muscle relaxation and flaccid paralysis. The B. pum ilus test
samples showed not only early paralysis but also the early death o f worms which
was close to the time o f death exhibited by standard drug. Hence, the metabolic
compounds o f B. pumilus responsible for anthelmintic activity appear to be different
with a different mechanism o f action from that o f B. cereus metabolites.
Though the Bacillus pumilus strain (15.1) with antinematodal activity has
been reported by Heins et al (1995), Bacillus cereus strain with anthelmintic activity
has not been reported in the past. Hence, as per the available literature and to the
best o f our knowledge, this may be the first report on Bacillus cereus bacteria with
anthelmintic activity.
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7.6. C onclusion
The petroleum ether extract, ethyl acetate extract and methanol extract of
both bacteria B. cereus and B. pumilus have exhibited anthelmintic activity
indicating potential metabolites in them capable o f controlling the worms. The
future scope involves the need o f isolation o f active constituents responsible for the
activity. Further studies are required to establish the mechanism o f action.
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