- Email: [email protected]
Myrcia ovata Cambessedes essential oils: A proposal for a novel natural antimicrobial against foodborne bacteria
Accepted Manuscript Myrcia ovata Cambessedes essential oils: A proposal for a novel natural antimicrobial against foodborne bacteria Isabela Cristina de Jesus, Gladslene Góes Santos Frazão, Arie Fitzgerald Blank, Luciana Cristina Lins de Aquino Santana PII:
S0882-4010(16)30331-X
DOI:
10.1016/j.micpath.2016.08.023
Reference:
YMPAT 1922
To appear in:
Microbial Pathogenesis
Received Date: 16 June 2016 Revised Date:
18 July 2016
Accepted Date: 19 August 2016
Please cite this article as: de Jesus IC, Santos Frazão GG, Blank AF, de Aquino Santana LCL, Myrcia ovata Cambessedes essential oils: A proposal for a novel natural antimicrobial against foodborne bacteria, Microbial Pathogenesis (2016), doi: 10.1016/j.micpath.2016.08.023. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Myrcia ovata Cambessedes essential oils: A proposal for a novel natural
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antimicrobial against foodborne bacteria
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Isabela Cristina de Jesusa, Gladslene Góes Santos Frazãoa, Arie Fitzgerald Blankb,
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Luciana Cristina Lins de Aquino Santanaa*
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a
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Rondon, S/N, São Cristóvão, Sergipe CEP 49100-000, Brazil.
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b
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Department of Food Technology, Federal University of Sergipe, Av. Marechal
Department of Agronomy, Federal University of Sergipe, Av. Marechal Rondon, S/N,
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São Cristóvão, Sergipe CEP 49100-000, Brazil.
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*Corresponding author at: Federal University of Sergipe, Department of Food
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Technology, Laboratory of Food Microbiology and Bioengineering, Avenida Marechal
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Rondon s/n, CEP: 49100-000, São Cristóvão, Sergipe.
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Tel.: + 55 79 2105 7420; Fax: +55 79 2105 6903.
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Email address: [email protected]
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ABSTRACT
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This paper reports the innovative antibacterial activity of essential oils (EOs) from nine
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Myrcia ovata Cambessedes plants against eight foodborne bacteria. Staphylococcus
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aureus, Bacillus cereus, Bacillus subtilis, Enterococcus faecalis and Pseudomonas
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aeruginosa were the most susceptible bacteria to EOs. In particular, the P. aeruginosa,
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which is usually resistant to antimicrobials agents, was extremely sensitive to some
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EOs. The gram-positive and gram-negative bacteria were inhibited and eliminated with
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minimum EOs concentrations ranging from 0.78 to 25 µL/mL. The Serratia marcensces
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and Escherichia coli were less susceptible to EOs alone. Consequently, some EOs
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combinations were investigated by checkerboard method against these bacteria and a
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synergistic effect was obtained. Myrcia ovata Cambessedes EOs showed high inhibitory
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and bactericidal effects against foodborne bacteria might be an interesting alternative
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for future applications as natural antimicrobials in food systems.
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Keywords: antibacterial activity, essential oils, Myrcia ovata, antimicrobials.
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1. Introduction
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Essential oils (EOs) are natural antimicrobials found in many plants and are
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capable of decreasing growth and survival of microorganisms including bacteria and
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fungi [1]. The antimicrobial activity of EOs is directly correlated with the presence of
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their bioactive volatile components such as terpene compounds (mono-, sesqui- and
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diterpenes), alcohols, acids, esters, epoxides, aldehydes, ketones, amines and sulphides
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[2,3]. The antimicrobial action of EOs against bacteria may be attributed to their ability
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to penetrate through bacterial membranes and inhibit functional and lipophilic
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properties of the cell. Phenolic compounds found in EOs may alter microbial cell
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permeability, damage cytoplasmic membranes, interfere with cellular energy (ATP)
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generation systems and disrupt the proton motive force, resulting in cell death [1,2,4-6].
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Over the years, a variety of EOs, such as oregano, thyme, cinnamon, orange,
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marjoram, clove bud, lemon grass and others have shown antimicrobial activity against
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bacteria and fungi [1]. Some essential oils as Satureja montana L., Thymus vulgaris L.
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and Rosmarinus officinalis L. have been used in food industry as flavouring agents,
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antioxidants and antimicrobials and in cosmetics industries [7,8]. The Myrcia ovata
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Cambessedes, popularly known as “laranjinha do-Mato”, is a shrub about eight meters
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tall and its leaves are commonly used in tea, folk medicin and treatment of gastric
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illnesses, including gastritis and diarrhoea. Myrcia, comprising around 377 species, is
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one of the largest genera in the Myrtaceae family recognized in the Brazilian cerrado
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and Atlantic forests [9,10]. The antimicrobial activity of Myrcia ovata EO from plants
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cultivated in Guaramiranga at Ceará State (Brazil) against microorganisms related to
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gastric and intestinal disorders was studied by Cândido et al. [11]. In previous study,
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Sampaio et al. [12] also have reported the antifungal potential of some Myrcia ovata
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EOs against Fusarium solani, a phytopathogenic fungi of importance in agriculture. The EOs have attracted interest for use as natural food additives in order to
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prevent the growth of foodborne pathogens or to delay the onset of food spoilage in
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substitution to chemical preservatives [7]. Bacteria such as Salmonella sp. constitutes a
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major public health problem in many countries and millions of cases of salmonellosis
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are noticed worldwide [13]. The Listeria monocytogenes and Escherichia coli are
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among the major foodborne bacteria implicated in produce outbreaks [14]. The S.
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aureus is one of the most important foodborne pathogen responsible for two-thirds of
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global foodborne disease outbreaks [15]. Besides, this bacterium produces heat stable
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enterotoxins responsible for foodborne intoxications [16]. The Bacillus cereus is a
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spore-forming, opportunistic Gram-positive bacterium, which produces emetic toxin
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and enterotoxins and causes food poisoning, including vomiting and diarrhea,
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particularly after the consumption of rice-based dishes [17]. The genus Bacillus
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including Bacillus cereus or Bacillus subtilis may be present in fresh and pasteurised
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food products due to their ability to generate heat-resistant spores under adverse
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environmental conditions [18]. Despite, the B. subtilis has not been considered a human
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pathogen, some strains of this species may occasionally cause food poisoning, such as
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the 2005 outbreak in a kindergarten caused by milk powder [19]. The E. faecalis is a
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microorganism of the normal intestinal flora in humans and animals, ranked second or
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third in frequency among bacteria isolated from hospitalized patients. This bacterium
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has resistance to pasteurization temperatures and ability to adapt to different substrates
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and environmental conditions. As consequence, it can be found in food products
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manufactured from raw materials (milk or meat) and in heat-treated food products.
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[20,21]. The S. marcescens is a Gram-negative bacterium that belongs to
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infections [22]. Whereas the need for new methods of reducing or eliminating
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foodborne pathogens, this is the first study that investigated the in vitro antimicrobial
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activity of Myrcia ovata Cambessedes EOs alone or in combination from plants
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collected in the state of Sergipe (Brazil) against eight foodborne bacteria.
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2. Materials and Methods
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2.1 Essential oils
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Nine Myrcia ovata Cambessedes (MYRO) essential oils (MYRO-154, MYRO-
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155, MYRO-156, MYRO-157, MYRO-158, MYRO-159, MYRO-173, MYRO-174 and
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MYRO-175) were provided by Department of Agronomy of Federal University of
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Sergipe. The plants were collected in the municipality of Japaratuba, State of Sergipe,
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Brazil, in November 2013. The locality presents sandy coastal dune vegetation, tropical
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rainy climate, with annual average temperature of 25.3°C, and average annual rainfall of
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1628.8 mm [23]. Exsiccates of all plants were deposited in the herbarium of the Federal
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University of Sergipe, and identification of the species was done by the Myrtaceae
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taxonomist Dr. Marcos Eduardo Guerra Sobral according Table 1. The essential oils
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were extracted by hydrodistillation using a modified Clevenger apparatus and the
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chemical composition was determined by Sampaio et al. [12] as shown in Table 2.
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2.2 Microorganisms
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Pseudomonas aeruginosa (INCQS 00025), Staphylococcus aureus (INCQS
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00014), Bacillus cereus (INCQS 00003), Bacillus subtilis (INCQS 00002),
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Enterecoccus faecalis (INCQS 00531), Serratia marcescens (INCQS 00131),
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Escherichia coli (INCQS 00032) and Salmonella enteritidis (INCQS 00258) were
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purchased from the National Institute of Health and Quality Control/Oswaldo Cruz 5
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Foundation (Manguinhos, Rio de Janeiro, Brazil). The strains were stored in Brain
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Heart Infusion (BHI) broth with 20% glycerol in a -80°C Ultrafreezer. All culture media
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were purchased from Oxoid (Brazil).
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2.3 Antibacterial activity by disk diffusion assay The antibacterial activity of EOs was determined using the disk diffusion
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method (in triplicate) as described by the Clinical and Laboratory Standards Institute
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[24]. Solutions containing 1.5 x 108 of each type of bacteria per mL were prepared to
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0.5 standard of McFarland’s tube. Petri plates containing 20mL of Muller-Hinton agar
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were inoculated with bacterial solutions. Sterile filter paper disks (6 mm) with 10 µL of
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EOs were placed on the agar surface using sterile forceps (four disks on each plate). The
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antibiotics, gentamicin (30 µg/disk) and chloramphenicol (30 µg/disk), were used as
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positive controls. Plates were incubated at 37°C for 24 h. Following incubation, the
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diameter (in mm) of the inhibition zone, including the disk diameter, was measured with
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a calliper. Sensitivity of bacteria to EOs was assessed by measuring the diameter of
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inhibition zone and bacteria were classified into four groups: not sensitive (diameter < 8
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mm); sensitive (diameter between 9.0 and 14.0mm); very sensitive (diameter between
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15.0 and 19.0 mm); and extremely sensitive (diameter > 20 mm) [25].
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2.4 Determination of minimum inhibitory concentration (MIC) and minimum
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bactericidal concentration (MBC)
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The determination of the minimum inhibitory concentration (MIC) and
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minimum bactericidal concentration (MBC) was performed using the broth
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microdilution method according to Clinical and Laboratory Standards Institute [24].
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EOs were diluted with 1% dimethyl sulfoxide solution to obtain concentrations from
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0.78 to 400 µL/mL. Cultures of each bacterial strain were obtained from 24 h broth 6
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suspensions were then diluted to 1 x 106 CFU/mL in Mueller-Hinton broth. 100 µL of
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EO dilutions were placed into a 96‐well microplate inoculated with 100 µL of each
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bacterial strain. The microplate was incubated aerobically for 24 h at 37°C. Mueller-
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Hinton medium incubated with a target bacterium (without EO) was used as a positive
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control. The MIC was defined as the lowest concentration of EO required for preventing
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visible bacterial growth. Muller-Hinton broth and bacterial suspension were used as
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negative and positive controls, respectively. The MBC was determined by sub-culturing
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100µL aliquots from MIC wells with no visible growth onto Muller-Hinton agar plates
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and incubating at 37ºC for 24h. The lowest concentration that yielded no visible
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colonies was considered the MBC.
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2.5 The checkerboard method
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The checkerboard method was performed using 96-well microtitre plates to
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obtain the fractional inhibitory concentration index (FICI) [26, 27]. The combinations
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were performed between two EOs (MYRO A and MYRO B) with stronger and weaker
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potential of inhibition against less susceptible bacteria. The assay was arranged as
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follows: MYRO-A was diluted two-fold in vertical orientation, while MYRO B was
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diluted two-fold in horizontal orientation. The EOs concentrations were prepared
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according to 1, 1/2, 1/4, 1/8, 1/16 and 1/32 of the MIC values. The final volume in each
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well was 100 µl comprising 50 µl of each EO dilution. After this, 100 µl of media
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containing 1 × 106 CFU/ml of the indicator strain were added to all wells. The plates
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were then incubated at 37°C for 24 h. The FICI were calculated as FICMYRO
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FICMYRO B, where FICMYRO A = MICMYRO A of the combination/MICMYRO A alone and
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FICMYRO
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B
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alone. The results were
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interpreted as synergy (FICI<0.5), addition (0.5≤FIC≤1), indifference (1
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antagonism (FIC>4).
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2.6 Statistical analysis One-way analysis of variance (ANOVA) was applied to the data to determine
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differences (p<0.05). Statistical analyses were performed using Assistat program 7.7
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Beta.
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3. Results and Discussion
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3.1 Antibacterial activity by disk diffusion assay
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The in vitro antibacterial activities of EOs were evaluated by the disk diffusion
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method against eight foodborne bacteria. The diameters of inhibition zone of EOs
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ranged from 6.0 to 32.0 mm (Table 3). Among the gram-positive bacteria, S. aureus was
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sensitive to the majority of EOs, very sensitive to MYRO-156 and extremely sensitive
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to MYRO-154 and MYRO-157 EOs. The MYRO-154 EO showed a similar inhibition
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to the antibiotic chloramphenicol without any significant difference (p<0.05), and was
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significantly higher than that obtained by MYRO-157 (p<0.05).
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B. cereus was extremely sensitive to MYRO-157 EO and to antibiotics.
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However, the antibiotics were more effective to inhibit the bacterial activity. Moreover,
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the bacterium was also very sensitive to five EOs (MYRO-158, MYRO-173, MYRO-
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159, MYRO-174 and MYRO-175). B. subtilis was extremely sensitive to MYRO-154,
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MYRO-157 and MYRO-174 EOs and antibiotics. However these EOs showed
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statistically significant differences in antibacterial activity (p<0.05). The MYRO-157
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EO
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chloramphenicol) to inhibit B. subtilis. This bacterium was also very sensitive to
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MYRO-158 EO. For E. faecalis, the EOs showed significant differences in
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antimicrobial activity (p<0.05). The bacteria was extremely sensitive to MYRO-154,
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MYRO-175 and to antibiotics, very sensitive to MYRO-157 and sensitive to MYRO-
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156, MYRO-173 and MYRO-174 EOs.
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From gram-negative bacteria group, P. aeruginosa was the most susceptible to
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EOs with diameters of the inhibition zone ranging from 11.0 to 32.0 mm. The
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antimicrobial activities of the EOs did differ statistically (p<0.05), except to MYRO-
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155 and MYRO-156. This bacterium was sensitive, very sensitive and extremely
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sensitive to two (MYRO-154 and MYRO-158), five (MYRO-155, MYRO-156,
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MYRO-159, MYRO-173 and MYRO-175) and two (MYRO-157 and MYRO-174)
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EOs, respectively. Moreover, the P. aeruginosa showed only a soft sensitivity to
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chloramphenicol antibiotic. This bacterium is known to have a high level of intrinsic
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resistance to virtually all known antimicrobials and antibiotics, due to a very restrictive
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outer membrane barrier, highly resistant even to synthetic drugs [4,28]. The S.
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marcensces was only sensitive to MYRO-175 EO. However, the antibiotics were
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effective against this bacterium, which was very sensitive and extremely sensitive to
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gentamicin and chloramphenicol, respectively. E. coli was sensitive to MYRO-154,
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MYRO-155 and MYRO-157 EOs, which did differ statistically in antimicrobial activity
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(p<0.05) and extremely sensitivity to chloramphenicol. The S. enteritidis was sensitive
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to five EOs (MYRO-154, MYRO-155, MYRO-156, MYRO-157 and MYRO-174).
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However, this bacterium showed more sensitivity to antibiotics (p<0.05), being
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extremely sensitive to gentamicin. The results showed that Gram-positive bacteria were
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the most susceptible to EOs that Gram-negative bacteria in accordance with the
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literature [1]. The Gram-negative bacteria should be more resistant to EOs due the
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hydrophilic cell wall which prevents the penetration of hydrophobic compounds
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[8,29,30]. Particularly, three (S. aureus, B. subtilis and E. faecalis) and four bacteria (S.
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aureus, B. cereus, B. subtilis and P. aeruginosa) were extremely sensitive to MYRO-
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154 and MYRO-157 EOs, respectively. The strong antibacterial activity of these EOs
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may be due to the presence of monoterpenes geranial and neral, and sesquiterpene (E)-
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nerolidol. Tyagi et al. [31] correlated the highest antifungal activity of lemon grass EO
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to the presence of high level of oxygenated monoterpenes (78.2%) constituted by
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geranial (α-citral) and neral (β-citral) as its major components. The antimicrobial action
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of monoterpenes suggests that they diffuse into and damage cell membrane structures
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[32,33]. Nerolidol is also well known for their antibacterial activity [34,35]. Bonikowisk
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et al. [36] have shown the antimicrobial activity of this compound against
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Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Escherichia coli,
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Klebsiella pneumoniae and Acinetobacter baumannii.
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Also, two (B. subtilis and P. aeruginosa) and one bacterium (E. faecalis) were
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extremely sensitive to MYRO-174 and MYRO-175 EOs, respectively. The MYRO-174
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EO contains the major compounds linalool, isopulegol and iso-isopulegol and MYRO-
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175 linalool and nerolic acid. Others 3 EOs (MYRO-155, MYRO-158 and MYRO-159)
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also contains nerolic acid as major compounds. EOs of Myrtaceae family such as
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Eucalyptus citriodora, which possess citronellal, citronellol and isopulegol as major
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compounds, showed fungicidal activity against Candida albicans and bacteriostatic
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against E. coli and S. aureus [37]. Sampaio et al. [12] published the first report about
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the presence of the nerolic acid compound in M. ovata EOs. The authors attributed the
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antifungal activity of these oils against Fusarium solani due to presence of this one and
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others major compounds such as linalool, geraniol, neral, geranial, (E)-nerolidol, 1,8-
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cineole and isopulegol. Cândido et al. [11] have showed similar potential of Myrcia ovata EO, from
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plants cultivated in Guaramiranga at Ceará State, to inhibit S. aureus and E. faecalis.
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However, differences were observed for inhibit E. coli and P. aeruginosa, the latter
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being more susceptible to EOs of this work. These results may be attributed to variation
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on chemical composition of EOs. Researchers have reported that the great diversity in
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the chemical composition of essential oils of a single species may be caused by genetic
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and/or environmental factors [12,38,26]. Environmental factors such as soil, lumi-
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nosity, and precipitation may affect the availability of nutrients for plants in different
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locations within the same population and, consequently, influence the content and
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quality of secondary metabolites within a community or population [39,40]. The
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reproductive biology of M. ovata is another factor to be considered. It is believed that
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the reproduction between plants of this species occurs by cross pollination as detected in
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other species of the same genus, such as Myrcia tomentosa and Myrcia rostrata [41].
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Cross pollination enables M. ovata plants, in a natural environment, to generate a wide
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variety of compounds for having received genetic information of plants which are
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genetically distinct regarding their ability to synthesize certain substances [12].
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3.2 Minimum inhibitory concentrations (MIC) and minimum bactericidal
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concentrations (MBC) of essential oils
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The MICs and MBCs of MYRO EOs are shown in Tables 4 and 5, respectively.
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The results show that gram-positive bacteria were more susceptible to essential oil than
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gram-negative bacteria in accordance with disk diffusion tests. P. aeruginosa was the
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bacterium most susceptible to MYRO-154, MYRO-159, MYRO-174 and MYRO-175 11
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of 0.78 µL/mL. Also, B. cereus, B. subtilis and E. faecalis were inhibited with 0.78
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µL/mL of MYRO-154, MYRO-174 and MYRO-156 EOs, respectively. S. marcensces,
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E. coli and S. enteritidis were more susceptible to MYRO-174 EO with MIC values of
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12.5, 6.25 and 6.25 µL/mL, respectively.
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Regarding to bactericidal activity, the MYRO-154 EO showed efficiency to
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eliminate B. cereus and S. aureus with the lowest MBC values of 3.13 and 6.25 µL/mL,
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respectively. The MYRO-154, MYRO-157 and MYRO-174 EOs showed potential to
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eliminate B. subtilis with 12.5 µL/mL without any significant difference (p>0.05). The
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MYRO-174 EO was more effective to eliminate E. faecalis, E. coli and P. aeruginosa
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with the lowest bactericide concentrations of 25 and 6.25 µL/mL, respectively. These
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results proved the greater antibacterial potential of MYRO EOs in comparison with the
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oil used by Cândido et al. [11], which MIC and MBC values were 31, 1000 µg/mL and
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>1000 µg/mL against E. faecalis, E. coli and P. aeruginosa, respectively. On the other
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hand, both MYRO-174 and MYRO-175 EOs showed similar ability to eliminate S.
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marcensces and S. enteritidis (p>0.05) with MBC of 25 µL/mL. All bacteria were less
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susceptible to MYRO 159 EO since the MBC values were >400 µL/mL.
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There is no unanimity on the MIC value acceptable for natural products when
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compared with standard antibiotics. Some researchers consider only products with MIC
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values similar to that of antibiotics, while others consider as good antimicrobial agents
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even those with higher levels of inhibitions [42]. Aligiannis et al. [43] proposed a
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classification for plant material antimicrobial activity on the basis of MIC results: strong
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inhibition for MIC values < 500 µg/mL; moderate inhibition for MIC values between
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600-1500 µg/mL; and weak inhibition for MIC values > 1600 µg/mL. According to this
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classification system, all MYRO EOs had strong inhibition against tested bacteria in this
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study.
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3.3. Synergistic effect between essential oils The bacteria S. marcensces and E. coli were less susceptible to seven EOs. Then
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the synergistic effect between the EOs was investigated by checkerboard test (Table 6),
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where the MYRO-174 was used in all combinations since that inhibited alone these
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bacteria with the lowest MIC values. All tested combinations displayed a synergism
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(FICI value ≤ 0.50) for S. marcensces and for E. coli, except the combination of
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MYRO-174 and MYRO-158 that was indifferent for E. coli. In addition, the test strains
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were inhibited with lower concentrations of EOs in the combinations than those used
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with them alone. These results could be explained as reported by Burt [4] and Ultee et
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al. [44] which have suggested that the minor components present in the EOs are more
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critical to the activity than EOs main components mixed, and the combination of major
317
components with other minor components that have a weaker activity may achieve a
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synergistic effect.
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4. Conclusions
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The present study showed the antimicrobial potential of nine MYRO EOs
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against eight foodborne bacteria. The pathogenic bacteria were from sensitive to
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extremely sensitive to EOs and gram-positive were more susceptible than gram-negative
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bacteria. The EOs showed to be effective bactericides, since all bacteria could be
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eliminated with MBC values ranging from 3.13 to 25 µL/mL. It was interesting to show
325
that the majority of EOs were able to inhibit and eliminate P. aeruginosa, since that this
326
bacterium has been resistant to others EOs and antibiotics. Furthermore, EOs
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combinations promoted a synergistic effect against S. marcensces and E. coli, which
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showed less susceptible to EOs alone. The Myrcia ovata Cambessedes EO alone or in
329
combination showed potential as natural antimicrobial agent for future studies in food
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systems.
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Acknowledgments
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The authors thank the Fundação de Apoio à Pesquisa e à Inovação Tecnológica
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do Estado de Sergipe (FAPITEC, Sergipe, Brazil) for financial support, and the
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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil) for
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providing a scholarship for the first author.
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ACCEPTED MANUSCRIPT Table 1 Geographic data of Myrcia ovata Cambessedes plants collected in Japaratuba, Sergipe State Brazil (Sampaio et al., 2016) Plant code Geographic date Herbarium code o o MYRO-154 10 37' 38.1"S; 36 53' 16.8"W 33.830 MYRO-155
10o 37' 38.0" S; 36o 53' 17.4" W
MYRO-156
10o 37' 38.7" S; 36o 53' 19.6" W
MYRO-157
10o 37' 38.8" S; 36o 53' 19.9" W
MYRO-158
10o 37' 37.6" S; 36o 53' 18.5" W
MYRO-159
10o 37' 37.2" S; 36o 53' 17.5" W
33.845
MYRO-173
10o 37' 38.5" S; 36o 53' 21.9" W
33.828
MYRO-174
10o 38' 45.4" S; 36o 52' 16.9" W
35.709
MYRO-175
10o 38' 44.8" S; 36o 52' 17.7" W
33.827
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33.835 33.839
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33.842
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Table 2 Chemical composition (%) of Myrcia ovata Cambessedes essential oils (MYRO) obtained by hydrodistillation. Composition (%) MYROMYRO157 158 0.35 0.65 0.50 0.98 0.28 0.30 4.36 5.11 5.77 2.89 1.76 1.00 1.38 5.02 0.86 18.21 1.87 0.70 0.92 36.96 2.90 50.02 1.12 1.29 0.43 1.53 20.24 0.80 0.97 1.10 16.65 3.03 93.76 96.18
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MYROMYRO155 156 α-Pinene 932 933 0.44 0.30 β-Pinene 974 974 0.32 0.95 0.29 p-Cymene 1020 1020 0.23 1,8-Cineole 1026 1026 0.75 4.65 1.38 Linalool 1095 1095 0.53 1.20 7.56 Isopulegol 1145 1145 2.30 Citronellal 1148 1148 9.19 Iso-Isopulegol 1155 1155 1.40 Terpinen-4-ol 1174 1174 0.79 1.41 α-Terpineol 1186 1186 1.01 1.26 2.24 Citronellol 1223 1223 3.27 Neral 1235 1235 0.34 0.11 28.39 Geraniol 1249 1249 1.33 74.37 Methyl citronellate 1257 1257 1.33 Geranial 1264 1265 0.16 1.93 40.10 Methyl nerolate 1280 1280 1.74 Citronellic acid 1312 1315 Nerolic acid 1347 1346 67.87 Geronic acid 1375 1376 1.88 (E)-Caryophyllene 1417 1422 0.51 0.18 0.74 β-Selinene 1489 1490 1.61 0.94 α-Selinene 1498 1498 1.43 0.91 (E)-Nerolidol 1561 1562 1.02 Caryophyllene oxide 1582 1585 0.93 0.35 1.47 β-Bisabolene 1674 1673 3.38 (2E, 6Z)-Farnesol 1714 1715 1.30 (2Z, 6E)-Farnesol 1722 1723 0.44 3.32 (2E, 6E)-Farnesol 1742 1742 0.55 (2E, 6E)-Farnesal 1740 1745 Total identified 95.36 90.87 93.90 RRIlit: Relative Retention Index-Literature; RRIexp.: Relative Retention Index – Experimental
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MYRO159 0.26 3.98 1.40 0.72 1.64 1.28 73.97 1.63 1.29 0.50 2.05 0.27 5.38 94.37
MYRO173 3.89 2.01 3.13 33.03 0.44 7.77 7.42 1.31 1.86 25.62 0.82 2.44 1.36 0.88 91.98
MYRO174 0.86 1.04 0.66 2.88 19.61 27.50 10.29 2.71 2.33 2.71 0.14 0.45 0.10 0.10 0.71 6.33 1.19 1.68 1.40 0.29 1.92 5.69 0.43 91.03
MYRO175 0.82 0.81 0.21 8.68 14.97 1.17 4.60 0.23 1.61 1.21 52.61 0.88 3.61 3.51 0.66 95.58
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Table 3 Diameters of inhibition zone (including paper disc diameter of 6.0 mm) of Myrcia ovata Cambessedes (MYRO) EOs against foodborne bacteria and antibiotics used as a positive control. Diameters of inhibition zone (mm) (Mean ± Standard Deviation) Bacterium Essential oils Standard antibiotics MYROMYROMYROMYRO- MYROMYROMYROMYROMYROGEN CHLOR 154 155 156 157 158 159 173 174 175 27.5±3.5a S. aureus 28.5±1.2a 13.0±1.5f 16.0±1.0d 20.0±2.0c 11.5±0.5g 12.5±0.5fg 11.0±0.0g 14.0±2.5e 12.5±0.7f 26.5±3.5b B. cereus 13.5±0.5f 10.0±0.8g 13.0±0.8f 20.0±0.0c 16.5±0.9d 15.0±1.4e 17.0±1.0d 15.5±0.7e 15.0±1.4e 24.0±2.3b 25.5±0.7a b i g a j f h d e B. subtilis 25.0±2.0 16.0±0.0 17.0±2.0 30.0±2.5 13.5±0.5 17.5±1.5 16.5±0.7 20.0±1.4 18.5±2.0 25.0±0.0b 24.0±0.0c d i f e i j g h a b E. faecalis 20.0±0.0 8.5±0.5 13.0±0.8 17.0±2.0 8.5±0.5 7.5±0.5 12.5±0.7 10.5±0.7 22.0±1.4 21.3±1.2 20.6±0.6c h e e b g f c a d P. aeruginosa 11.0±0.0 16.5±0.5 16.5±1.2 30.0±0.9 13.0±0.5 15.5±0.5 19.0±1.4 32.0±2.5 17.5±0.7 8.0±2.8j 10.5±6.4i d f d e f f e g c b S. marcensces 8.0±0.2 7.0±0.0 8.0±0.4 7.6±0.2 7.0±0.0 7.0±0.2 7.5±0.5 6.0±0.0 11.0±1.4 17.0±3.2 27.0±2.8a b c f d e e f f f e E. coli 10.5±1.2 10.0±0.0 8.0±0.0 9.5±0.5 8.5±0.5 8.6±0.5 8.0±0.0 8.0±0.5 8.0±0.0 8.5±3.5 24.0±1.4a S. enteritidis 13.0±0.8c 9.5±1.2e 9.6±0.5e 10.5±0.5d 8.0±0.0g 8.5 ±0.5f 7.5±0.5h 13.0±3.0c 6.0±0.0i 26.5±2.5a 14.5±1.0b a–j
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For each bacterium, different letters in the same line indicate significant differences (p < 0.05) between the mean values according to Tukey’s test. GEN: gentamicin antiobiotic (20 µg/disc) CHLOR: Chloramphenicol antibiotic (20 µg/disc)
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MYRO157 12.5c 12.5c 6.3d 200b 6.25c 200b 200a 25c
MYRO158 25b 50a 12.5c 400a 12.5b 400a 100b 50b
MYRO159 6.25d 25.0b 25.0b 3.13e 0.78e 25.0d 12.5d 6.25e
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MYRO156 25b 50a 50a 0.78f 3.13d 50c 100b 12.5d
MYRO173 50a 25b 12.5c 200b 3.13d 400a 100b 25c
MYRO174 6.25d 3.13d 0.78e 12.5d 0.78e 12.5e 6.25e 6.25e
MYRO175 25b 12.5c 12.5c 25c 0.78e 25d 25c 12.5d
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MYRO155 50a 50a 6.25d 400a 50a 200b 200a 200a
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MYRO154 3.13e 0.78e 1.56e 400a 0.78e 50c 12.5d 12.5d
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Table 4 Minimum inhibitory concentrations (MIC) of Myrcia ovata Cambess. (MYRO) essential oils (µL/mL)
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Table 5 Minimum bactericidal concentrations (MBC) of Myrcia ovata Cambess. (MYRO) essential oils (µL/mL) MYRO- MYRO- MYRO- MYRO- MYRO- MYRO- MYRO- MYRO154 155 156 157 158 159 173 174 d a c b a b S. aureus 6.25 200. 25 50 200 >400 50 25c B. cereus 3.13e 50c 200a 50c 25d >400 100b 25d B. subtilis 12.5d 25c 400a 12.5d 100b >400 25c 12.5d a b E. faecalis 100 >400 >400 >400 25d >400 >400 400 P. aeruginosa 25b 100.0a >400 >400 100a >400 >400 6.25c S. marcensces >400 400a >400 400a >400 >400 25c 200b E. coli 50c >400 400a >400 >400 >400 >400 25d a a S. enteritidis >400 >400 >400 >400 >400 100 25b 100 a–e
MYRO175 25c 25d 25c 50c 25b 25c 100b 25b
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For each bacterium, different letters in the same line indicate significant differences (p < 0.05) between the mean values according to Tukey’s test. Values MBC >400 µL/mL were not considered in the statistical analysis.
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Combinations
MYRO 174 – MYRO 158
FICI value
Interaction
0.28
Synergism
0.50
Synergism
0.15
Synergism
0.37
Synergism
0.06
Synergism
-
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0.06
Synergism
1.03
Indifferent
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MYRO 174 – MYRO 173
Interaction
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MYRO 174 – MYRO 157
FICI value
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MYRO 174 – MYRO 155
E. coli
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HIGHLIGHTS
Paper : “Myrcia ovata Cambessedes essential oils: A proposal for a novel natural
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antimicrobial against foodborne bacteria “
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- Myrcia ovata Cambessedes essential oil alone or in combination, a potent natural antibacterial. - Foodborne bacteria were from sensitive to extremely sensitive to EOs.
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- Essential oil effective to inhibit P. aeruginosa, usually resistant to antimicrobials.
S0882-4010(16)30331-X
DOI:
10.1016/j.micpath.2016.08.023
Reference:
YMPAT 1922
To appear in:
Microbial Pathogenesis
Received Date: 16 June 2016 Revised Date:
18 July 2016
Accepted Date: 19 August 2016
Please cite this article as: de Jesus IC, Santos Frazão GG, Blank AF, de Aquino Santana LCL, Myrcia ovata Cambessedes essential oils: A proposal for a novel natural antimicrobial against foodborne bacteria, Microbial Pathogenesis (2016), doi: 10.1016/j.micpath.2016.08.023. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Myrcia ovata Cambessedes essential oils: A proposal for a novel natural
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antimicrobial against foodborne bacteria
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Isabela Cristina de Jesusa, Gladslene Góes Santos Frazãoa, Arie Fitzgerald Blankb,
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Luciana Cristina Lins de Aquino Santanaa*
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a
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Rondon, S/N, São Cristóvão, Sergipe CEP 49100-000, Brazil.
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b
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Department of Food Technology, Federal University of Sergipe, Av. Marechal
Department of Agronomy, Federal University of Sergipe, Av. Marechal Rondon, S/N,
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São Cristóvão, Sergipe CEP 49100-000, Brazil.
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*Corresponding author at: Federal University of Sergipe, Department of Food
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Technology, Laboratory of Food Microbiology and Bioengineering, Avenida Marechal
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Rondon s/n, CEP: 49100-000, São Cristóvão, Sergipe.
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Tel.: + 55 79 2105 7420; Fax: +55 79 2105 6903.
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Email address: [email protected]
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ABSTRACT
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This paper reports the innovative antibacterial activity of essential oils (EOs) from nine
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Myrcia ovata Cambessedes plants against eight foodborne bacteria. Staphylococcus
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aureus, Bacillus cereus, Bacillus subtilis, Enterococcus faecalis and Pseudomonas
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aeruginosa were the most susceptible bacteria to EOs. In particular, the P. aeruginosa,
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which is usually resistant to antimicrobials agents, was extremely sensitive to some
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EOs. The gram-positive and gram-negative bacteria were inhibited and eliminated with
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minimum EOs concentrations ranging from 0.78 to 25 µL/mL. The Serratia marcensces
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and Escherichia coli were less susceptible to EOs alone. Consequently, some EOs
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combinations were investigated by checkerboard method against these bacteria and a
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synergistic effect was obtained. Myrcia ovata Cambessedes EOs showed high inhibitory
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and bactericidal effects against foodborne bacteria might be an interesting alternative
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for future applications as natural antimicrobials in food systems.
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Keywords: antibacterial activity, essential oils, Myrcia ovata, antimicrobials.
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1. Introduction
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Essential oils (EOs) are natural antimicrobials found in many plants and are
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capable of decreasing growth and survival of microorganisms including bacteria and
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fungi [1]. The antimicrobial activity of EOs is directly correlated with the presence of
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their bioactive volatile components such as terpene compounds (mono-, sesqui- and
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diterpenes), alcohols, acids, esters, epoxides, aldehydes, ketones, amines and sulphides
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[2,3]. The antimicrobial action of EOs against bacteria may be attributed to their ability
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to penetrate through bacterial membranes and inhibit functional and lipophilic
65
properties of the cell. Phenolic compounds found in EOs may alter microbial cell
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permeability, damage cytoplasmic membranes, interfere with cellular energy (ATP)
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generation systems and disrupt the proton motive force, resulting in cell death [1,2,4-6].
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Over the years, a variety of EOs, such as oregano, thyme, cinnamon, orange,
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marjoram, clove bud, lemon grass and others have shown antimicrobial activity against
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bacteria and fungi [1]. Some essential oils as Satureja montana L., Thymus vulgaris L.
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and Rosmarinus officinalis L. have been used in food industry as flavouring agents,
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antioxidants and antimicrobials and in cosmetics industries [7,8]. The Myrcia ovata
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Cambessedes, popularly known as “laranjinha do-Mato”, is a shrub about eight meters
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tall and its leaves are commonly used in tea, folk medicin and treatment of gastric
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illnesses, including gastritis and diarrhoea. Myrcia, comprising around 377 species, is
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one of the largest genera in the Myrtaceae family recognized in the Brazilian cerrado
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and Atlantic forests [9,10]. The antimicrobial activity of Myrcia ovata EO from plants
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cultivated in Guaramiranga at Ceará State (Brazil) against microorganisms related to
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gastric and intestinal disorders was studied by Cândido et al. [11]. In previous study,
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Sampaio et al. [12] also have reported the antifungal potential of some Myrcia ovata
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EOs against Fusarium solani, a phytopathogenic fungi of importance in agriculture. The EOs have attracted interest for use as natural food additives in order to
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prevent the growth of foodborne pathogens or to delay the onset of food spoilage in
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substitution to chemical preservatives [7]. Bacteria such as Salmonella sp. constitutes a
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major public health problem in many countries and millions of cases of salmonellosis
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are noticed worldwide [13]. The Listeria monocytogenes and Escherichia coli are
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among the major foodborne bacteria implicated in produce outbreaks [14]. The S.
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aureus is one of the most important foodborne pathogen responsible for two-thirds of
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global foodborne disease outbreaks [15]. Besides, this bacterium produces heat stable
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enterotoxins responsible for foodborne intoxications [16]. The Bacillus cereus is a
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spore-forming, opportunistic Gram-positive bacterium, which produces emetic toxin
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and enterotoxins and causes food poisoning, including vomiting and diarrhea,
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particularly after the consumption of rice-based dishes [17]. The genus Bacillus
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including Bacillus cereus or Bacillus subtilis may be present in fresh and pasteurised
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food products due to their ability to generate heat-resistant spores under adverse
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environmental conditions [18]. Despite, the B. subtilis has not been considered a human
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pathogen, some strains of this species may occasionally cause food poisoning, such as
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the 2005 outbreak in a kindergarten caused by milk powder [19]. The E. faecalis is a
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microorganism of the normal intestinal flora in humans and animals, ranked second or
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third in frequency among bacteria isolated from hospitalized patients. This bacterium
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has resistance to pasteurization temperatures and ability to adapt to different substrates
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and environmental conditions. As consequence, it can be found in food products
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manufactured from raw materials (milk or meat) and in heat-treated food products.
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[20,21]. The S. marcescens is a Gram-negative bacterium that belongs to
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infections [22]. Whereas the need for new methods of reducing or eliminating
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foodborne pathogens, this is the first study that investigated the in vitro antimicrobial
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activity of Myrcia ovata Cambessedes EOs alone or in combination from plants
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collected in the state of Sergipe (Brazil) against eight foodborne bacteria.
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2. Materials and Methods
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2.1 Essential oils
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Nine Myrcia ovata Cambessedes (MYRO) essential oils (MYRO-154, MYRO-
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155, MYRO-156, MYRO-157, MYRO-158, MYRO-159, MYRO-173, MYRO-174 and
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MYRO-175) were provided by Department of Agronomy of Federal University of
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Sergipe. The plants were collected in the municipality of Japaratuba, State of Sergipe,
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Brazil, in November 2013. The locality presents sandy coastal dune vegetation, tropical
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rainy climate, with annual average temperature of 25.3°C, and average annual rainfall of
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1628.8 mm [23]. Exsiccates of all plants were deposited in the herbarium of the Federal
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University of Sergipe, and identification of the species was done by the Myrtaceae
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taxonomist Dr. Marcos Eduardo Guerra Sobral according Table 1. The essential oils
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were extracted by hydrodistillation using a modified Clevenger apparatus and the
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chemical composition was determined by Sampaio et al. [12] as shown in Table 2.
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2.2 Microorganisms
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Pseudomonas aeruginosa (INCQS 00025), Staphylococcus aureus (INCQS
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00014), Bacillus cereus (INCQS 00003), Bacillus subtilis (INCQS 00002),
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Enterecoccus faecalis (INCQS 00531), Serratia marcescens (INCQS 00131),
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Escherichia coli (INCQS 00032) and Salmonella enteritidis (INCQS 00258) were
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purchased from the National Institute of Health and Quality Control/Oswaldo Cruz 5
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Foundation (Manguinhos, Rio de Janeiro, Brazil). The strains were stored in Brain
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Heart Infusion (BHI) broth with 20% glycerol in a -80°C Ultrafreezer. All culture media
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were purchased from Oxoid (Brazil).
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2.3 Antibacterial activity by disk diffusion assay The antibacterial activity of EOs was determined using the disk diffusion
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method (in triplicate) as described by the Clinical and Laboratory Standards Institute
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[24]. Solutions containing 1.5 x 108 of each type of bacteria per mL were prepared to
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0.5 standard of McFarland’s tube. Petri plates containing 20mL of Muller-Hinton agar
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were inoculated with bacterial solutions. Sterile filter paper disks (6 mm) with 10 µL of
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EOs were placed on the agar surface using sterile forceps (four disks on each plate). The
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antibiotics, gentamicin (30 µg/disk) and chloramphenicol (30 µg/disk), were used as
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positive controls. Plates were incubated at 37°C for 24 h. Following incubation, the
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diameter (in mm) of the inhibition zone, including the disk diameter, was measured with
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a calliper. Sensitivity of bacteria to EOs was assessed by measuring the diameter of
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inhibition zone and bacteria were classified into four groups: not sensitive (diameter < 8
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mm); sensitive (diameter between 9.0 and 14.0mm); very sensitive (diameter between
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15.0 and 19.0 mm); and extremely sensitive (diameter > 20 mm) [25].
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2.4 Determination of minimum inhibitory concentration (MIC) and minimum
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bactericidal concentration (MBC)
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The determination of the minimum inhibitory concentration (MIC) and
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minimum bactericidal concentration (MBC) was performed using the broth
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microdilution method according to Clinical and Laboratory Standards Institute [24].
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EOs were diluted with 1% dimethyl sulfoxide solution to obtain concentrations from
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0.78 to 400 µL/mL. Cultures of each bacterial strain were obtained from 24 h broth 6
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suspensions were then diluted to 1 x 106 CFU/mL in Mueller-Hinton broth. 100 µL of
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EO dilutions were placed into a 96‐well microplate inoculated with 100 µL of each
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bacterial strain. The microplate was incubated aerobically for 24 h at 37°C. Mueller-
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Hinton medium incubated with a target bacterium (without EO) was used as a positive
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control. The MIC was defined as the lowest concentration of EO required for preventing
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visible bacterial growth. Muller-Hinton broth and bacterial suspension were used as
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negative and positive controls, respectively. The MBC was determined by sub-culturing
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100µL aliquots from MIC wells with no visible growth onto Muller-Hinton agar plates
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and incubating at 37ºC for 24h. The lowest concentration that yielded no visible
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colonies was considered the MBC.
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2.5 The checkerboard method
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The checkerboard method was performed using 96-well microtitre plates to
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obtain the fractional inhibitory concentration index (FICI) [26, 27]. The combinations
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were performed between two EOs (MYRO A and MYRO B) with stronger and weaker
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potential of inhibition against less susceptible bacteria. The assay was arranged as
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follows: MYRO-A was diluted two-fold in vertical orientation, while MYRO B was
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diluted two-fold in horizontal orientation. The EOs concentrations were prepared
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according to 1, 1/2, 1/4, 1/8, 1/16 and 1/32 of the MIC values. The final volume in each
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well was 100 µl comprising 50 µl of each EO dilution. After this, 100 µl of media
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containing 1 × 106 CFU/ml of the indicator strain were added to all wells. The plates
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were then incubated at 37°C for 24 h. The FICI were calculated as FICMYRO
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FICMYRO B, where FICMYRO A = MICMYRO A of the combination/MICMYRO A alone and
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FICMYRO
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B
= MICMYRO
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of the combination/MICMYRO
B
A
+
alone. The results were
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interpreted as synergy (FICI<0.5), addition (0.5≤FIC≤1), indifference (1
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antagonism (FIC>4).
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2.6 Statistical analysis One-way analysis of variance (ANOVA) was applied to the data to determine
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differences (p<0.05). Statistical analyses were performed using Assistat program 7.7
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3. Results and Discussion
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3.1 Antibacterial activity by disk diffusion assay
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The in vitro antibacterial activities of EOs were evaluated by the disk diffusion
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method against eight foodborne bacteria. The diameters of inhibition zone of EOs
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ranged from 6.0 to 32.0 mm (Table 3). Among the gram-positive bacteria, S. aureus was
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sensitive to the majority of EOs, very sensitive to MYRO-156 and extremely sensitive
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to MYRO-154 and MYRO-157 EOs. The MYRO-154 EO showed a similar inhibition
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to the antibiotic chloramphenicol without any significant difference (p<0.05), and was
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significantly higher than that obtained by MYRO-157 (p<0.05).
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B. cereus was extremely sensitive to MYRO-157 EO and to antibiotics.
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However, the antibiotics were more effective to inhibit the bacterial activity. Moreover,
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the bacterium was also very sensitive to five EOs (MYRO-158, MYRO-173, MYRO-
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159, MYRO-174 and MYRO-175). B. subtilis was extremely sensitive to MYRO-154,
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MYRO-157 and MYRO-174 EOs and antibiotics. However these EOs showed
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statistically significant differences in antibacterial activity (p<0.05). The MYRO-157
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EO
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chloramphenicol) to inhibit B. subtilis. This bacterium was also very sensitive to
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was
particularly
more
effective
than
the
antibiotics
(gentamicin
and
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MYRO-158 EO. For E. faecalis, the EOs showed significant differences in
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antimicrobial activity (p<0.05). The bacteria was extremely sensitive to MYRO-154,
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MYRO-175 and to antibiotics, very sensitive to MYRO-157 and sensitive to MYRO-
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156, MYRO-173 and MYRO-174 EOs.
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From gram-negative bacteria group, P. aeruginosa was the most susceptible to
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EOs with diameters of the inhibition zone ranging from 11.0 to 32.0 mm. The
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antimicrobial activities of the EOs did differ statistically (p<0.05), except to MYRO-
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155 and MYRO-156. This bacterium was sensitive, very sensitive and extremely
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sensitive to two (MYRO-154 and MYRO-158), five (MYRO-155, MYRO-156,
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MYRO-159, MYRO-173 and MYRO-175) and two (MYRO-157 and MYRO-174)
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EOs, respectively. Moreover, the P. aeruginosa showed only a soft sensitivity to
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chloramphenicol antibiotic. This bacterium is known to have a high level of intrinsic
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resistance to virtually all known antimicrobials and antibiotics, due to a very restrictive
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outer membrane barrier, highly resistant even to synthetic drugs [4,28]. The S.
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marcensces was only sensitive to MYRO-175 EO. However, the antibiotics were
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effective against this bacterium, which was very sensitive and extremely sensitive to
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gentamicin and chloramphenicol, respectively. E. coli was sensitive to MYRO-154,
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MYRO-155 and MYRO-157 EOs, which did differ statistically in antimicrobial activity
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(p<0.05) and extremely sensitivity to chloramphenicol. The S. enteritidis was sensitive
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to five EOs (MYRO-154, MYRO-155, MYRO-156, MYRO-157 and MYRO-174).
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However, this bacterium showed more sensitivity to antibiotics (p<0.05), being
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extremely sensitive to gentamicin. The results showed that Gram-positive bacteria were
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the most susceptible to EOs that Gram-negative bacteria in accordance with the
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literature [1]. The Gram-negative bacteria should be more resistant to EOs due the
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hydrophilic cell wall which prevents the penetration of hydrophobic compounds
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[8,29,30]. Particularly, three (S. aureus, B. subtilis and E. faecalis) and four bacteria (S.
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aureus, B. cereus, B. subtilis and P. aeruginosa) were extremely sensitive to MYRO-
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154 and MYRO-157 EOs, respectively. The strong antibacterial activity of these EOs
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may be due to the presence of monoterpenes geranial and neral, and sesquiterpene (E)-
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nerolidol. Tyagi et al. [31] correlated the highest antifungal activity of lemon grass EO
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to the presence of high level of oxygenated monoterpenes (78.2%) constituted by
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geranial (α-citral) and neral (β-citral) as its major components. The antimicrobial action
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of monoterpenes suggests that they diffuse into and damage cell membrane structures
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[32,33]. Nerolidol is also well known for their antibacterial activity [34,35]. Bonikowisk
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et al. [36] have shown the antimicrobial activity of this compound against
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Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Escherichia coli,
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Klebsiella pneumoniae and Acinetobacter baumannii.
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Also, two (B. subtilis and P. aeruginosa) and one bacterium (E. faecalis) were
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extremely sensitive to MYRO-174 and MYRO-175 EOs, respectively. The MYRO-174
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EO contains the major compounds linalool, isopulegol and iso-isopulegol and MYRO-
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175 linalool and nerolic acid. Others 3 EOs (MYRO-155, MYRO-158 and MYRO-159)
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also contains nerolic acid as major compounds. EOs of Myrtaceae family such as
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Eucalyptus citriodora, which possess citronellal, citronellol and isopulegol as major
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compounds, showed fungicidal activity against Candida albicans and bacteriostatic
251
against E. coli and S. aureus [37]. Sampaio et al. [12] published the first report about
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the presence of the nerolic acid compound in M. ovata EOs. The authors attributed the
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antifungal activity of these oils against Fusarium solani due to presence of this one and
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others major compounds such as linalool, geraniol, neral, geranial, (E)-nerolidol, 1,8-
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cineole and isopulegol. Cândido et al. [11] have showed similar potential of Myrcia ovata EO, from
257
plants cultivated in Guaramiranga at Ceará State, to inhibit S. aureus and E. faecalis.
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However, differences were observed for inhibit E. coli and P. aeruginosa, the latter
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being more susceptible to EOs of this work. These results may be attributed to variation
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on chemical composition of EOs. Researchers have reported that the great diversity in
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the chemical composition of essential oils of a single species may be caused by genetic
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and/or environmental factors [12,38,26]. Environmental factors such as soil, lumi-
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nosity, and precipitation may affect the availability of nutrients for plants in different
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locations within the same population and, consequently, influence the content and
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quality of secondary metabolites within a community or population [39,40]. The
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reproductive biology of M. ovata is another factor to be considered. It is believed that
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the reproduction between plants of this species occurs by cross pollination as detected in
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other species of the same genus, such as Myrcia tomentosa and Myrcia rostrata [41].
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Cross pollination enables M. ovata plants, in a natural environment, to generate a wide
270
variety of compounds for having received genetic information of plants which are
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genetically distinct regarding their ability to synthesize certain substances [12].
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3.2 Minimum inhibitory concentrations (MIC) and minimum bactericidal
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concentrations (MBC) of essential oils
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The MICs and MBCs of MYRO EOs are shown in Tables 4 and 5, respectively.
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The results show that gram-positive bacteria were more susceptible to essential oil than
277
gram-negative bacteria in accordance with disk diffusion tests. P. aeruginosa was the
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bacterium most susceptible to MYRO-154, MYRO-159, MYRO-174 and MYRO-175 11
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of 0.78 µL/mL. Also, B. cereus, B. subtilis and E. faecalis were inhibited with 0.78
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µL/mL of MYRO-154, MYRO-174 and MYRO-156 EOs, respectively. S. marcensces,
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E. coli and S. enteritidis were more susceptible to MYRO-174 EO with MIC values of
283
12.5, 6.25 and 6.25 µL/mL, respectively.
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Regarding to bactericidal activity, the MYRO-154 EO showed efficiency to
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eliminate B. cereus and S. aureus with the lowest MBC values of 3.13 and 6.25 µL/mL,
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respectively. The MYRO-154, MYRO-157 and MYRO-174 EOs showed potential to
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eliminate B. subtilis with 12.5 µL/mL without any significant difference (p>0.05). The
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MYRO-174 EO was more effective to eliminate E. faecalis, E. coli and P. aeruginosa
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with the lowest bactericide concentrations of 25 and 6.25 µL/mL, respectively. These
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results proved the greater antibacterial potential of MYRO EOs in comparison with the
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oil used by Cândido et al. [11], which MIC and MBC values were 31, 1000 µg/mL and
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>1000 µg/mL against E. faecalis, E. coli and P. aeruginosa, respectively. On the other
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hand, both MYRO-174 and MYRO-175 EOs showed similar ability to eliminate S.
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marcensces and S. enteritidis (p>0.05) with MBC of 25 µL/mL. All bacteria were less
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susceptible to MYRO 159 EO since the MBC values were >400 µL/mL.
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There is no unanimity on the MIC value acceptable for natural products when
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compared with standard antibiotics. Some researchers consider only products with MIC
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values similar to that of antibiotics, while others consider as good antimicrobial agents
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even those with higher levels of inhibitions [42]. Aligiannis et al. [43] proposed a
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classification for plant material antimicrobial activity on the basis of MIC results: strong
301
inhibition for MIC values < 500 µg/mL; moderate inhibition for MIC values between
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600-1500 µg/mL; and weak inhibition for MIC values > 1600 µg/mL. According to this
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classification system, all MYRO EOs had strong inhibition against tested bacteria in this
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study.
305 306
3.3. Synergistic effect between essential oils The bacteria S. marcensces and E. coli were less susceptible to seven EOs. Then
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the synergistic effect between the EOs was investigated by checkerboard test (Table 6),
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where the MYRO-174 was used in all combinations since that inhibited alone these
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bacteria with the lowest MIC values. All tested combinations displayed a synergism
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(FICI value ≤ 0.50) for S. marcensces and for E. coli, except the combination of
312
MYRO-174 and MYRO-158 that was indifferent for E. coli. In addition, the test strains
313
were inhibited with lower concentrations of EOs in the combinations than those used
314
with them alone. These results could be explained as reported by Burt [4] and Ultee et
315
al. [44] which have suggested that the minor components present in the EOs are more
316
critical to the activity than EOs main components mixed, and the combination of major
317
components with other minor components that have a weaker activity may achieve a
318
synergistic effect.
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4. Conclusions
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The present study showed the antimicrobial potential of nine MYRO EOs
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against eight foodborne bacteria. The pathogenic bacteria were from sensitive to
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extremely sensitive to EOs and gram-positive were more susceptible than gram-negative
323
bacteria. The EOs showed to be effective bactericides, since all bacteria could be
324
eliminated with MBC values ranging from 3.13 to 25 µL/mL. It was interesting to show
325
that the majority of EOs were able to inhibit and eliminate P. aeruginosa, since that this
326
bacterium has been resistant to others EOs and antibiotics. Furthermore, EOs
327
combinations promoted a synergistic effect against S. marcensces and E. coli, which
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showed less susceptible to EOs alone. The Myrcia ovata Cambessedes EO alone or in
329
combination showed potential as natural antimicrobial agent for future studies in food
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systems.
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Acknowledgments
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The authors thank the Fundação de Apoio à Pesquisa e à Inovação Tecnológica
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do Estado de Sergipe (FAPITEC, Sergipe, Brazil) for financial support, and the
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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil) for
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providing a scholarship for the first author.
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ACCEPTED MANUSCRIPT Table 1 Geographic data of Myrcia ovata Cambessedes plants collected in Japaratuba, Sergipe State Brazil (Sampaio et al., 2016) Plant code Geographic date Herbarium code o o MYRO-154 10 37' 38.1"S; 36 53' 16.8"W 33.830 MYRO-155
10o 37' 38.0" S; 36o 53' 17.4" W
MYRO-156
10o 37' 38.7" S; 36o 53' 19.6" W
MYRO-157
10o 37' 38.8" S; 36o 53' 19.9" W
MYRO-158
10o 37' 37.6" S; 36o 53' 18.5" W
MYRO-159
10o 37' 37.2" S; 36o 53' 17.5" W
33.845
MYRO-173
10o 37' 38.5" S; 36o 53' 21.9" W
33.828
MYRO-174
10o 38' 45.4" S; 36o 52' 16.9" W
35.709
MYRO-175
10o 38' 44.8" S; 36o 52' 17.7" W
33.827
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33.833
33.835 33.839
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33.842
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Table 2 Chemical composition (%) of Myrcia ovata Cambessedes essential oils (MYRO) obtained by hydrodistillation. Composition (%) MYROMYRO157 158 0.35 0.65 0.50 0.98 0.28 0.30 4.36 5.11 5.77 2.89 1.76 1.00 1.38 5.02 0.86 18.21 1.87 0.70 0.92 36.96 2.90 50.02 1.12 1.29 0.43 1.53 20.24 0.80 0.97 1.10 16.65 3.03 93.76 96.18
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MYROMYRO155 156 α-Pinene 932 933 0.44 0.30 β-Pinene 974 974 0.32 0.95 0.29 p-Cymene 1020 1020 0.23 1,8-Cineole 1026 1026 0.75 4.65 1.38 Linalool 1095 1095 0.53 1.20 7.56 Isopulegol 1145 1145 2.30 Citronellal 1148 1148 9.19 Iso-Isopulegol 1155 1155 1.40 Terpinen-4-ol 1174 1174 0.79 1.41 α-Terpineol 1186 1186 1.01 1.26 2.24 Citronellol 1223 1223 3.27 Neral 1235 1235 0.34 0.11 28.39 Geraniol 1249 1249 1.33 74.37 Methyl citronellate 1257 1257 1.33 Geranial 1264 1265 0.16 1.93 40.10 Methyl nerolate 1280 1280 1.74 Citronellic acid 1312 1315 Nerolic acid 1347 1346 67.87 Geronic acid 1375 1376 1.88 (E)-Caryophyllene 1417 1422 0.51 0.18 0.74 β-Selinene 1489 1490 1.61 0.94 α-Selinene 1498 1498 1.43 0.91 (E)-Nerolidol 1561 1562 1.02 Caryophyllene oxide 1582 1585 0.93 0.35 1.47 β-Bisabolene 1674 1673 3.38 (2E, 6Z)-Farnesol 1714 1715 1.30 (2Z, 6E)-Farnesol 1722 1723 0.44 3.32 (2E, 6E)-Farnesol 1742 1742 0.55 (2E, 6E)-Farnesal 1740 1745 Total identified 95.36 90.87 93.90 RRIlit: Relative Retention Index-Literature; RRIexp.: Relative Retention Index – Experimental
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MYRO154
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Compounds
AC C
N°
MYRO159 0.26 3.98 1.40 0.72 1.64 1.28 73.97 1.63 1.29 0.50 2.05 0.27 5.38 94.37
MYRO173 3.89 2.01 3.13 33.03 0.44 7.77 7.42 1.31 1.86 25.62 0.82 2.44 1.36 0.88 91.98
MYRO174 0.86 1.04 0.66 2.88 19.61 27.50 10.29 2.71 2.33 2.71 0.14 0.45 0.10 0.10 0.71 6.33 1.19 1.68 1.40 0.29 1.92 5.69 0.43 91.03
MYRO175 0.82 0.81 0.21 8.68 14.97 1.17 4.60 0.23 1.61 1.21 52.61 0.88 3.61 3.51 0.66 95.58
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Table 3 Diameters of inhibition zone (including paper disc diameter of 6.0 mm) of Myrcia ovata Cambessedes (MYRO) EOs against foodborne bacteria and antibiotics used as a positive control. Diameters of inhibition zone (mm) (Mean ± Standard Deviation) Bacterium Essential oils Standard antibiotics MYROMYROMYROMYRO- MYROMYROMYROMYROMYROGEN CHLOR 154 155 156 157 158 159 173 174 175 27.5±3.5a S. aureus 28.5±1.2a 13.0±1.5f 16.0±1.0d 20.0±2.0c 11.5±0.5g 12.5±0.5fg 11.0±0.0g 14.0±2.5e 12.5±0.7f 26.5±3.5b B. cereus 13.5±0.5f 10.0±0.8g 13.0±0.8f 20.0±0.0c 16.5±0.9d 15.0±1.4e 17.0±1.0d 15.5±0.7e 15.0±1.4e 24.0±2.3b 25.5±0.7a b i g a j f h d e B. subtilis 25.0±2.0 16.0±0.0 17.0±2.0 30.0±2.5 13.5±0.5 17.5±1.5 16.5±0.7 20.0±1.4 18.5±2.0 25.0±0.0b 24.0±0.0c d i f e i j g h a b E. faecalis 20.0±0.0 8.5±0.5 13.0±0.8 17.0±2.0 8.5±0.5 7.5±0.5 12.5±0.7 10.5±0.7 22.0±1.4 21.3±1.2 20.6±0.6c h e e b g f c a d P. aeruginosa 11.0±0.0 16.5±0.5 16.5±1.2 30.0±0.9 13.0±0.5 15.5±0.5 19.0±1.4 32.0±2.5 17.5±0.7 8.0±2.8j 10.5±6.4i d f d e f f e g c b S. marcensces 8.0±0.2 7.0±0.0 8.0±0.4 7.6±0.2 7.0±0.0 7.0±0.2 7.5±0.5 6.0±0.0 11.0±1.4 17.0±3.2 27.0±2.8a b c f d e e f f f e E. coli 10.5±1.2 10.0±0.0 8.0±0.0 9.5±0.5 8.5±0.5 8.6±0.5 8.0±0.0 8.0±0.5 8.0±0.0 8.5±3.5 24.0±1.4a S. enteritidis 13.0±0.8c 9.5±1.2e 9.6±0.5e 10.5±0.5d 8.0±0.0g 8.5 ±0.5f 7.5±0.5h 13.0±3.0c 6.0±0.0i 26.5±2.5a 14.5±1.0b a–j
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For each bacterium, different letters in the same line indicate significant differences (p < 0.05) between the mean values according to Tukey’s test. GEN: gentamicin antiobiotic (20 µg/disc) CHLOR: Chloramphenicol antibiotic (20 µg/disc)
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MYRO157 12.5c 12.5c 6.3d 200b 6.25c 200b 200a 25c
MYRO158 25b 50a 12.5c 400a 12.5b 400a 100b 50b
MYRO159 6.25d 25.0b 25.0b 3.13e 0.78e 25.0d 12.5d 6.25e
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MYRO156 25b 50a 50a 0.78f 3.13d 50c 100b 12.5d
MYRO173 50a 25b 12.5c 200b 3.13d 400a 100b 25c
MYRO174 6.25d 3.13d 0.78e 12.5d 0.78e 12.5e 6.25e 6.25e
MYRO175 25b 12.5c 12.5c 25c 0.78e 25d 25c 12.5d
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AC C
a–f
MYRO155 50a 50a 6.25d 400a 50a 200b 200a 200a
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S. aureus B. cereus B. subtilis E. faecalis P. aeruginosa S. marcensces E. coli S. enteritidis
MYRO154 3.13e 0.78e 1.56e 400a 0.78e 50c 12.5d 12.5d
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Table 4 Minimum inhibitory concentrations (MIC) of Myrcia ovata Cambess. (MYRO) essential oils (µL/mL)
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Table 5 Minimum bactericidal concentrations (MBC) of Myrcia ovata Cambess. (MYRO) essential oils (µL/mL) MYRO- MYRO- MYRO- MYRO- MYRO- MYRO- MYRO- MYRO154 155 156 157 158 159 173 174 d a c b a b S. aureus 6.25 200. 25 50 200 >400 50 25c B. cereus 3.13e 50c 200a 50c 25d >400 100b 25d B. subtilis 12.5d 25c 400a 12.5d 100b >400 25c 12.5d a b E. faecalis 100 >400 >400 >400 25d >400 >400 400 P. aeruginosa 25b 100.0a >400 >400 100a >400 >400 6.25c S. marcensces >400 400a >400 400a >400 >400 25c 200b E. coli 50c >400 400a >400 >400 >400 >400 25d a a S. enteritidis >400 >400 >400 >400 >400 100 25b 100 a–e
MYRO175 25c 25d 25c 50c 25b 25c 100b 25b
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For each bacterium, different letters in the same line indicate significant differences (p < 0.05) between the mean values according to Tukey’s test. Values MBC >400 µL/mL were not considered in the statistical analysis.
ACCEPTED MANUSCRIPT Table 6 FICI values of EOs combinations against S. marcensces and E. coli S. marcensces
Combinations
MYRO 174 – MYRO 158
FICI value
Interaction
0.28
Synergism
0.50
Synergism
0.15
Synergism
0.37
Synergism
0.06
Synergism
-
-
0.06
Synergism
1.03
Indifferent
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MYRO 174 – MYRO 173
Interaction
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MYRO 174 – MYRO 157
FICI value
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MYRO 174 – MYRO 155
E. coli
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HIGHLIGHTS
Paper : “Myrcia ovata Cambessedes essential oils: A proposal for a novel natural
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antimicrobial against foodborne bacteria “
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- Myrcia ovata Cambessedes essential oil alone or in combination, a potent natural antibacterial. - Foodborne bacteria were from sensitive to extremely sensitive to EOs.
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- Essential oil effective to inhibit P. aeruginosa, usually resistant to antimicrobials.