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Antimicrobial activity of Amazonian oils against Paenibacillus species
Roberto Christ Vianna Santos
a,
⇑
, Camilla Filippi dos Santos Alves
a
, Taiane Schneider
a
,
Leonardo Quintana Soares Lopes
a
, Carlos Aurich
b
, Janice Luehring Giongo
b
, Adriano Brandelli
c
,
Rodrigo de Almeida Vaucher
a,b
a
Laboratório de Microbiologia, Ciências da Saúde, Centro Universitário Franciscano, UNIFRA, Santa Maria, Rio Grande do Sul, Brazil
b
Laboratório de Tecnologia Farmacêutica, Centro de Ciências da Saúde, Universidade da Região da Campanha, URCAMP, Bagé, Rio Grande do Sul, Brazil
c
Laboratório de Bioquímica e Microbiologia Aplicada, Instituto de Ciência e Tecnologia de Alimentos, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
article info
Article history:
Received 28 September 2011
Accepted 8 December 2011
Available online 17 December 2011
Keywords:
Andiroba
Copaíba
Apis mellifera
Paenibacillus larvae
abstract
The Gram-positive, spore-forming bacterium Paenibacillus larvae is the primary bacterial pathogen of
honeybee brood and the causative agent of American foulbrood disease (AFB). One of the feasible alter-
native treatments being used for their control of this disease is essential oils. In this study in vitro anti-
microbial activity of Andiroba and Copaíba essential oils against Paenibacillus species, including P. larvae
was evaluated. Minimal inhibitory concentration (MIC) in Mueller–Hinton broth by the microdilution
method was assessed. Andiroba registered MIC values of 1.56–25%, while the MICs values obtained for
Copaíba oil were of 1.56–12.5%. In order to determine the time-response effect of essential oils on P. lar-
vae, this microorganism was exposed to the oils for up to 48 h. After 24 h treatment with Andiroba oil and
after 48 h treatment with Copaíba oil no viable cells of P. larvae ATCC 9545 were observed. The possible
toxic effect of essential oils were assessed by the spraying application method of the same concentrations
of MICs. Bee mortality was evident only in treatment with Andiroba oil and the Copaíba oil shows no
toxic effects after 10 days of observation. Taking together ours results showed for the first time that these
oils presented a high activity against Paenibacillus species showing that Copaíba oil may be a candidate
for the treatment or prevention of AFB.
Ó2011 Elsevier Inc. All rights reserved.
1. Introduction
American foulbrood (AFB) is among the economically most
important honeybee diseases. The etiological agent of AFB is the
Gram-positive, spore-forming bacterium Paenibacillus larvae (Gen-
ersch, 2010). The clinical symptoms of AFB are typical, with the
brown, viscous larval remains forming a ropy thread when drawn
out with a matchstick. The decaying larvae desiccate into hard
scales, consisting of millions of bacterial spores (Genersch et al.,
2005).
Copaíba oils are produced by exudation from the trunks of trees
belonging to the genus Copaifera. The medicinal properties of
Copaíba oils were known among American Indians, who probably
observed that animals rubbed themselves on Copaíba tree trunks
to heal their wounds (Veiga-Junior et al., 2006). The effects attrib-
uted to Copaíba oils in folk medicine include anti-inflammatory,
anti-tetanus, anti-tumour, anti-blenorrhagea, and urinary antisep-
tic activities. In addition, it has been used to treat bronchitis, skin
diseases, ulcers, and syphilis, as well as for healing wounds.
Pharmacological studies have demonstrated its properties as an
anti-inflammatory; gastroprotective; analgesic; wound-cicatrisa-
tial (Paiva et al., 2004; Araújo-Junior et al., 2005; Brito et al.,
2005; Carvalho et al., 2005); anti-nociceptive; anti-tumour (Lima
et al., 2003); and antimicrobial (Costa-Lotufo et al., 2002; Tincusi
et al., 2002).
Carapa guaianensis is a tall tree that grows wild throughout
South America, West India and South Africa. In Brazil, it can be
found prevalently in areas of the Amazon rainforest that are rich
in soils and swamps. From the nuts of this plant is extracted an
oil, called Andiroba oil, which has a long history of traditional
use in South America, such as analgesic, anti-inflammatory, anti-
bacterial, anti-parasitic and as an anti-cancer remedy (Gilbert
et al., 1999; Moura et al., 2002). Andiroba oil has shown great
interest in pharmaceutical and cosmetic industries. Among their
properties stand out the anti-inflammatory (Penido et al., 2006a),
anti-allergic and analgesic actions (Penido et al., 2006b).
Treatments for AFB involve a variety of chemicals, which have
been applied in a continuous and excessive way, so that strains
may develop resistance to antibiotics, besides generating residues
in honey, thus affecting quality and commercialization. Therefore,
0022-2011/$ - see front matter Ó2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.jip.2011.12.002
⇑
Corresponding author. Address: Laboratório de Microbiologia, Ciências da
Saúde, Centro Universitário Franciscano, UNIFRA, Rua dos Andradas 1614, Sala
115, 97010-032 Santa Maria, Brazil.
E-mail addresses: robertochrist@gmail.com,roberto@unifra.br (R.C.V. Santos).
Journal of Invertebrate Pathology 109 (2012) 265–268
Contents lists available at SciVerse ScienceDirect
Journal of Invertebrate Pathology
journal homepage: www.elsevier.com/locate/jip
during the last years it has been appealed to natural substances,
such as essential oils, to treat infected beehives (Sabaté et al.,
2009; Benitez et al., 2011). This work evaluates for the first time
the antimicrobial activity of Amazonian oils Andiroba and Copaíba
oils against Paenibacillus species. Toxicity against honey bees Apis
mellifera was also investigated.
2. Materials and methods
Andiroba oil (C. guaianensis) code RF3150 and Copaíba oil (Copa-
ifera officinalis) code RF3350 were purchased from Beraca Sabará
Químicos e Ingredientes S/A (São Paulo, Brazil). In this study, eight
isolates of Paenibacillus species from the collection of Ministry of
Agriculture (LANAGRO/RS) Brazil were used. The test organisms in-
cluded isolates of Paenibacillus alginolyticus,Paenibacillus pabuli,
Paenibacillus azotofixans,Paenibacillus borealis,Paenibacillus glucon-
olyticus,Paenibacillus validus,Paenibacillus thiaminolyticus and P.
larvae (ATCC9545). The microorganisms were grown in Mueller–
Hinton broth (Difco, Sparks, Maryland, USA) at 37 °C for 24 h and
maintained on slopes of nutrient agar (Difco).
The minimum inhibitory concentrations (MIC) of Andiroba and
Copaíba oils were determined by microdilution techniques in
Mueller–Hinton broth (Difco) for Paenibacillus species (CLSI,
2008). The assay was carried out in 96-well microtitre plates. Each
oil was mixed with an inoculum prepared in the same medium at a
density adjusted per tube to 0.5 of the McFarland scale
(1.5 10
8
CFU/mL) and diluted 1:10 for the broth microdilution
procedure. Microtitre trays were incubated at 37 °C and the MICs
were recorded after 24 h of incubation. The MIC was defined as
the lowest concentration of compounds that inhibits bacterial
growth. This test was performed in triplicate on separate occa-
sions. The 2,3,5-triphenyltetrazolium chloride was used as an indi-
cator of bacterial growth.
The antibacterial activity of oils against P. larvae (ATCC9545)
was studied over the MICs (1.56% to Copaíba and 25% to Andiroba).
Tests were performed in triplicate at 37 °C. At predetermined time
points (0, 3, 6, 9, 12, 24, 48 h), a 100
l
L sample was removed from
each test suspension, serially diluted in sterile saline, and plated on
Muller–Hinton Agar plates for colony count determination. Data
from triplicate runs were averaged and plotted as log CFU/mL ver-
sus time (h) for each time point.
The viscous extracts were dissolved in DMSO to reach the final
concentrations of 25% for Andiroba oil and 1.56% for Copaíba oil.
These concentrations used in the toxicity test were determined
from the determination of MIC values (Fig. 1A). The spraying appli-
cation method was performed according to Damiani et al. (2009).
Petri dishes (150 15 mm) padded with absorbent filter paper
on the inner bottom and with an extra lid of plastic mesh were
used. Six adult worker bees were placed in every modified Petri
dish. Then, 1 ml of each concentration (for both botanical extracts)
was individually sprayed on the bees throughout the plastic lid
using a hand sprayer. A device with candy and water was placed
inside each unit as food for the bees. Six bees in a modified Petri
dish sprayed with DMSO were included as negative control, and
six bees in a modified Petri dish sprayed with 0.07% Deltamethrin
(DTT) (Pirisa-Piretro Industrial Ltda, Brazil) were included as posi-
tive death control. Four replicates for each experimental group
were run. Bioassay dishes were placed in incubators at 28 ± 1 °C
and 60% relative humidity. Mortality of bees was evaluated by vi-
sual inspection daily up to 10 days.
Differences in survival after 10 days of observation were as-
sessed by Kaplan–Meier analysis followed by the Logrank test. A
Pvalue <0.05 was considered statistically significant. One-way
analysis of variance (ANOVA) was used to determine the significant
differences between treatment groups and Newman–Keuls test
was used for multi-group comparisons in Time-course inhibition
of P. larvae. All statistical analyses were performed with the soft-
ware package GraphPad Prism 4.00 for Windows (GraphPad Soft-
ware, San Diego, CA, USA).
3. Results
In this study, the antimicrobial activity of Andiroba and Copaíba
oils against Paenibacillus species was determined. As shown in
Fig. 1A, the MICs of these oils range 1.56–25%. All species of Paeni-
bacillus were susceptible to Copaíba oil, showing MIC values of
1.56% in most cases, excepting the higher MIC value observed for
P. azotofixans. Similar results were obtained with Andiroba oil,
although in this case both P. azotofixans and P. larvae showed a
MIC of 25%. In order to determine the time-response effect of Andi-
roba and Copaíba oils on P. larvae, this microorganism was exposed
Fig. 1. Determine the time-course effect of Andiroba and Copaíba oils, after
exposition of P. larvae (ATCC 9545). For further details, see Section 2.
Fig. 2. Effects of spraying applications on bees. For further details, see Section 2.
266 R.C.V. Santos et al. / Journal of Invertebrate Pathology 109 (2012) 265–268
to the oils for up to 48 h. After 24 h treatment with Andiroba oil
(25%) and after 48 h treatment with Copaíba oil (1.56%) no viable
cells of P. larvae ATCC 9545 were observed (Fig. 1B). Furthermore,
a steep decline in CFU/mL was observed after incubation with
Andiroba and Copaíba oils for 12 and 24 h, respectively.
The bees treated with Copaíba oil showed similar result to that
observed in the negative control group throughout observation
time. No mortality of bees was observed during 10 days of treat-
ment (Fig. 2). Bee mortality was evident only in treatment with
DTT (positive control group) and Andiroba oil. At 24 h after treat-
ment, all bees were death in positive control group. Bees treated
with Andiroba oil showed a mortality index of about 20% per day
until the day 4, with 20% survival at the end of the experiment
(Fig. 2).
4. Discussion
Efforts toward drug discovery and prudent use of antimicrobial
agents are the mainstay for overcoming the worldwide problem of
microbial resistance. One resort for drug discovery is natural prod-
ucts (Santos et al., 2010), crude or isolated from medicinal plants.
In this work, the antimicrobial activity of Andiroba and Copaíba
oils were evaluated against various Paenibacillus species, showing
an important antimicrobial effect in all strains tested.
Recently, some researchers have proved the biological activity
of essential oils against bee pathologies. Pimpinella anisum and
Foeniculum vulgare essential oils have a considerable potential for
controlling P. larvae. These oils were found to be especially rich
in (E)-anethole (96.3% and 92.7%, respectively) and presented high
MICs (300 mg/mL and 250 mg/mL) (Gende et al., 2009).
Each individual extract comprises a complex unique mixture of
different phytochemicals (plant secondary metabolites). The
chemical nature of these constituents varies considerably between
species (Paul et al., 2009). Andiroba oil is rich in fatty acids such as
oleic, palmitic, stearic and linoleic acids, together with 2–5% of
unsaponificable material. In addition have been isolated limonoids,
triterpenes, steroids, coumarins, flavonoids and diglycerides (Lavie
et al., 1972; Marcelle and Moto, 1975). The main constituents of
Copaíba oil are sesquiterpenes and diterpenes (Braga et al.,
1998). Sesquiterpenes comprise about 80% of the oils; the most
common are
a
-copaene, b-cariofilene, b-bisabolene,
a
and b-selin-
ene,
a
-humulene, and dand
c
-cadidene (Veiga-Junior and Pinto,
2002).
Infectious diseases are a component of such an environment
that must be critically controlled in the dense population struc-
tures of a social organism. This is no exception with the honey
bee and important pathogens such as P. larvae can be devastating
to a colony (Genersch, 2010). Our results showed that Andiroba
(1.56–25%), and Copaíba (1.56–12.5%) oils presented a high activity
against Paenibacillus species showing that these oils may be candi-
dates for the treatment or prevention of AFB.
In this research, the Amazonian essential oils were sprayed on
A. mellifera to verify the possible toxic effects. Copaíba oil pre-
sented similar results to the control group, causing no toxic effects
or animal death. On contrast, Andiroba oil caused the death of bees
at the same concentration that exhibit antimicrobial activity,
resulting in a survival rate of approximately 20% after 10 days of
observation. Andiroba has been used in traditional medicine as
an insect repellent and some studies verified antifeedant proper-
ties of the Andiroba that protects against bites of Aedes aegypti
(Fradin and Day, 2002). Andiroba also induced mortality of Aedes
albopictus larvae after 24 h (Silva et al., 2004, 2006). This important
insecticide effect of Andiroba oil reported in other insects, may ex-
plain our findings, since after exposure to oil only 20% of the ani-
mals remained alive. Copaíba oil appears to be non-toxic to A.
mellifera adults at tested concentration (same concentration that
inhibits the growth of P. larvae), showing that this oil can be used
for the treatment of AFB.
Brazil, and specially Amazonia, has a very rich biological diver-
sity with numerous medicinal and pharmacological properties that
should be explored. In the area of honeybee health, there is an
unexplored way for future research involving alternative natural
substances to control the AFB. Elimination of P. larvae in A. mellifera
colonies involves treatments with acceptable antimicrobial activity
with no side effects on A. mellifera that minimize residues in honey
and wax and that constitute a viable alternative to reduce antimi-
crobial resistance.
Acknowledgments
Authors thank the Laboratory of Microbiology of LANAGRO/RS,
Ministry of the Agriculture (Brazil) by isolates of Paenibacillus spe-
cies. This work received financial support of PRPGPE/UNIFRA-PRO-
BIC, Brazil.
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