Journal of Medical Microbiology Papers in Press. Published September 26, 2014 as doi:10.1099/jmm.0.081711-0
1
Antibacterial
2
dehydroabietic acid, against multidrug-resistant strains
activity
Pinus
elliottii
and
its
major
compound,
3
4 5
Luís Fernando Leandro1, Miguel Jorge Oliveira Cardoso1, Sandro Donizeti Caetano Silva1;
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Maria Gorete Mendes Souza1, Rodrigo Cassio Sola Veneziani2, Sergio Ricardo Ambrosio2 and
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Carlos Henrique Gomes Martins1.
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Author affiliation
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1
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14404-600, São Paulo, Brazil
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2
13
600, São Paulo, Brazil
Laboratory of Research in Applied Microbiology, University of Franca - UNIFRAN, Franca,
Nucleus of Research in Sciences and Technology, University of Franca - UNIFRAN, 14404-
14 15 16 17 18 19 20 21 22 23 24 25 26
Correspondence Carlos Henrique G. Martins, Laboratory of Research in Applied Microbiology, University of Franca, Av. Dr. Armando Salles Oliveira, 201, 14404-600, Franca, São Paulo, Brazil Tel: +55 16 3711-8756; fax: +55 16 3711-8874 E-mail: [email protected] Abbreviations: MIC - minimum inhibitory concentration; MBC - minimum bactericidal concentration
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27 28 29 30
Abstract
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and have prompted the search for new sources of antimicrobial substances. Pinus species
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contain several bioactive compounds consisting mainly of terpenes, terpenoids, and some
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other aromatic and aliphatic constituents. These compounds exert important biological effects,
34
and pine oils have found wide application in the industry. In the present study, we have
35
evaluated the potential activity of the resin-oil of Pinus elliottii and its major compound
36
dehydroabietic acid against multiresistant bacteria by minimum inhibitory concentration
37
(MIC), minimum bactericidal concentration (MBC), and time-kill assays. The MIC of the
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resin-oil of Pinus elliottii varied between 25 and 100 μg mL-1. As for dehydroabietic acid,
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MIC and MBC varied between 6.25 and 50 and between 6.25 and 100 μg mL-1, respectively.
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The time-kill assay conducted with dehydroabietic acid at 6.25 μg mL-1 evidenced
41
bactericidal activity against S. epidermidis (ATCC 14990) within 24 hours. On the basis of
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these results, the resin-oil of Pinus elliottii and its major compound dehydroabietic acid play
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an important part in the search for novel sources of agents that can act against multiresistant
44
bacteria.
Antibiotic-resistant bacteria have emerged from the widespread use of antibiotics worldwide
45 46
Keywords: Pinus elliottii; dehydroabietic acid; multiresistant bacteria; Antibacterial activity
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2
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Introduction
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The widespread use of antibiotics worldwide has culminated in high prevalence of infections
53
by antibiotic-resistant bacteria. Indeed, exchangeable genetic elements such as plasmids,
54
transposons, and integons disseminate antibiotic resistance in many bacteria (Nikokar et al.,
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2013). The slower introduction of new chemotherapeutic drugs into the market and clinical
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practice over the last few years may yet be another reason for the growing number of resistant
57
bacteria (Russell, 2002; Coelho et al., 2013). This slowdown may have caused a number of
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antibiotics to lose their efficacy against antibiotic-resistant organisms, making the use of
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drugs that are often reserved for last-line treatment necessary (Coelho et al., 2013). The
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overall result of the emergence of human pathogenic organisms exhibiting multidrug
61
resistance has been the search for new antimicrobial substances from other sources, including
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plants (Ahmad & Beg, 2001).
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During the 1950s, exotic, fast-growing trees such as Pinus spp. were introduced in southern
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Brazil, to supply wood for the paper industry and cellulose for energy production. Today,
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Brazil holds one of the largest planted areas in the world (Pacheco et al., 2009). A number of
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compounds displaying anti-inflammatory, antiparasitic, antioxidant, antimutagenic, and
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antitumor activities as well as the ability to regulate cancer-related proteins have been isolated
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from different Pinus species (Tóro et al., 2003; Karonen et al., 2004; Kwak et al., 2006; Li et
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al., 2007; Politeo et al., 2011). Some species are frequently used for their medicinal benefits,
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such as diuretic and expectorant actions, to treat diseases that include pulmonary, urinary,
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hepatic, and hypertensive disorders (Lawless, 1992; Politeo et al., 2011). Moreover, pine oils
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are widely employed as fragrances in cosmetics, flavoring additives in food, and
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intermediates in the synthesis of perfume chemicals (Sticher, 1977; Politeo et al., 2011). They
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consist mainly of terpenes, terpenoids, and some other aromatic and aliphatic constituents, 3
75
which usually have strong smell and low molecular weight (Marriott et al., 2001; Burt et al.,
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2004; Politeo et al., 2011; Zeng et al., 2012).
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The current literature contains no reports on the biological activities of the resin-oil of Pinus
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elliottii and its bioactive compounds against antibiotic-resistant bacteria. Therefore, the
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present study aimed to investigate the in vitro antibacterial activity of the oleoresin of Pinus
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elliottii and its major compound dehydroabietic acid (DA) against multiresistant bacteria.
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Methods
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Tested microorganisms
84 85
The bacteria were acquired from the American Type Culture Collection (ATCC) and National
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Collection of Type Cultures (NCTC); Hospital das Clínicas de Ribeirão Preto (São Paulo
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State, Brazil) kindly supplied seven bacteria of clinical isolates (HCRP), which were
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maintained in the culture collection of the Laboratory of Research in Applied Microbiology
89
(LaPeMA) of the University of Franca, state of São Paulo, Brazil, at -80 oC. The following
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microorganisms were used: Staphylococcus epidermidis (ATCC 14990), Staphylococcus
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epidermidis (clinical isolate), Staphylococcus aureus (ATCC 29213), Staphylococcus aureus
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(clinical isolate), Enterococcus faecium (NCTC 717), Enterococcus faecium (clinical isolate),
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Staphylococcus capitis (ATCC 27840), Staphylococcus capitis (clinical isolate), Enterococcus
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faecalis (NCTC 14990), Enterococcus faecalis (clinical isolate), Staphylococcus haemolyticus
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(ATCC 29970), Staphylococcus haemolyticus (clinical isolate), Klebsiella pneumoniae
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(ATCC 700603) and Klebsiella pneumoniae (clinical isolate).
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Oil-resin and isolation of the major compound
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Associação dos Resinadores do Brasil - ARESB, located in the city of Avaré, state of São
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Paulo, Brazil, donated the original oil-resin of Pinus elliottii var. elliottii (OPe) tested here.
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First, the oil-resin (40.0 g) was partitioned by vacuum liquid chromatography using a solvent
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system of increasing polarity (n-hexane/ethyl acetate). This procedure furnished five fractions
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(2 L each) that were named OPe1 (100% n-hexane), OPe2 (20% ethyl acetate), OPe3 (40%
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ethyl acetate), OPe4 (60% ethyl acetate) and OPe5 (100% ethyl acetate). The minimum
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inhibitory concentration (MIC) values of the five fractions were determined against the tested
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bacteria. MIC results revealed that fraction OPe2 was the most active against multiresistant
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bacteria (Table 1).
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The major compound in OPe2 was isolated from 1.0 g of this fraction by classic
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chromatography using 500 mL of the mixture n-hexane/ethyl acetate 8:2 (v/v) as mobile
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phase. The obtained dehydroabietic acid (125.8 mg) was subjected to MIC and MBC assays
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in the presence of the bacteria evaluated in this study (Fig. 1). The chemical structure of
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dehydroabietic acid was confirmed based on comparison with nuclear magnetic resonance (1H
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and 13C NMR) literature data (Fraga et al., 1994; Gonzàles et al., 2010).
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Antimicriobial assays
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The minimal inhibitory concentration values (MIC; lowest concentration of the compound
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that was able to inhibit microorganism growth) and the minimal bactericidal concentration
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(MBC; lowest concentration of the compound at which 99.9% or more of the initial inoculum 5
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was killed) were determined in triplicate, by using the microdilution broth method in 96-well
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microtitre plates. Samples of the resin-oil of Pinus elliottii and its major compound DA were
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dissolved in dimethyl sulfoxide (DMSO; Synth) at 1.0 mg mL-1, followed by dilution in
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tryptic soy broth (Difco); concentrations ranging from 100.0 to 1.0 µg mL-1 were achieved.
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The final DMSO content was 5% (v/v), and this solution was used as negative control. The
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inoculums was adjusted for each organism, to yield a cell concentration of 5 x 105 colony
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forming units (CFU) per mL, according to previous standardization by the Clinical
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Laboratory Standards Institute (CLSI, 2009). One inoculated well, free of antimicrobial agent,
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was also employed, to ensure medium sterility. Vancomycin (VAN) and Imipenem were used
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as positive controls. The microtitre plates (96 wells) were sealed with plastic film and
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incubated at 37 ºC for 24 h. After that, resazurin (30µL) in aqueous solution (0.02 %) was
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added to the microplates, to indicate microorganism viability (Sarker et al., 2007; Ambrosio
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et al., 2008b; Porto et al., 2009; Porto et al., 2013). Before addition of resazurin and to
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determine MBC, an aliquot of the inoculum was aseptically removed from each well
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presenting no apparent growth and then plated onto tryptic soy agar. The plates were
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incubated as described previously.
137 138
Time-kill curves
139 140
Time-kill assays were performed in triplicate based on D’Arrigo et al. (2010). Dehydroabietic
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acid (DA) was chosen to conduct the time-kill curve assays because, according to Rios &
142
Recio, (2005) isolated compounds that inhibit the growth of the microorganisms at
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concentrations below 10 μg mL−1 are considered very promising in the search for new anti-
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infection agents, therefore the DA displayed the highest antibacterial activity (Table 1 - MIC
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and MBC 6.25 μg mL-1) only against Staphylococcus epidermidis (ATCC14990). This 6
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microorganism is considered as one of the most important pathogens involved in hospital-
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associated bloodstream infections related to vascular catheters, because of their virulence
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factors and ability to produce biofilm (Otto, 2009; Widerström et al., 2012). A tube
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containing 1 mL of tryptic soy broth sterile and DA at final concentration of 6.25 µg mL-1 (1
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x MBC of DA for S. epidermidis) was inoculated with the tested microorganism, resulting in
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an initial bacterial density of 13.3 x 105 CFU mL-1, and then incubated at 37 °C. Aliquots
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were removed (100 µL) for enumeration of viable colonies at 30 min and 6, 12, and 24 h after
153
incubation, followed by serially dilution, until final dilution (10-4) in tryptic soy broth sterile.
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The diluted samples (50 µL) were spread onto tryptic soy agar plate, incubated at 37 °C, and
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viable colonies were counted after 24 h. The data were analyzed, the time-kill curves were
156
constructed by plotting log10 CFU mL-1 versus time using Prism software (version 5.0;
157
GraphPad, Inc.). The assays were performed in triplicate and also for the positive (VAN) and
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negative controls (suspension of S. epidermidis without added DA). VAN was used at its
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MBC (0.74 µg mL-1).
160 161
Results
162 163
The resin-oil of Pinus elliottii furnished satisfactory MIC values (between 25 and 100 μg
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mL-1) when tested against the multiresistant bacteria S. epidermidis (ATCC 14990), S.
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epidermidis (clinical isolate), S. aureus (clinical isolate), E. faecium (NCTC 717), E. faecium
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(clinical isolate), S. capitis (ATCC 27840), E. faecalis (NCTC 14990), E. faecalis (clinical
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isolate), S. haemolyticus (ATCC 29970), and S. haemolyticus (clinical isolate). In the case of
168
the bacteria K. pneumoniae (ATCC 700603), K. pneumoniae (clinical isolate), S. capitis
7
169
(clinical isolate), and S. aureus (ATCC 29213), the MIC values were equal to 200 μg mL-1 or
170
higher (Table 1).
171
Assays revealed bactericidal activity against S. epidermidis (ATCC 14990) only–both MIC
172
and MBC were equal to 6.25 μg mL-1 (Table 1).
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The time-kill assay helped to assess the kinetic behavior of dehydroabietic acid. MBC values
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were lower than 10 μg mL-1. The best activity was obtained against S. epidermidis (ATCC
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14990).
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The results achieved with S. epidermidis (ATCC 14990) demonstrated that dehydroabietic
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acid at a concentration of 6.25 μg mL-1 started to progressively diminish the number of
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microorganisms at 12 h of incubation; bactericidal action was evident at 24 h. Vancomycin
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(0.74 μg mL-1), used as positive control, reduced the number of microorganisms by over 2
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log10 at 12 h and exhibited bactericidal activity at 24 h of incubation (Fig. 2).
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Discussion
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The development of antibiotic resistance by bacteria that threaten the human health has
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become a major clinical problem worldwide. This has prompted the search for new
186
antimicrobial substances from other sources, including plants (Ahmad & Beg, 2001; Bennett,
187
2008). In this context, the present study evaluated the antibacterial action of the resin-oil of
188
Pinus elliottii and its major compound dehydroabietic acid against multiresistant bacteria.
189
A study conducted by Zeng et al. (2012), the authors determined MIC and MBC for the
190
essential oil of Pine needles (Cedrus deodara) against the causative agents of food poisoning,
191
such as Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Bacillus
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subtilis (ATCC 21216), Bacillus cereus (ATCC 14579), Saccharomyces cerevisiae (ATCC
193
9763), Aspergillus niger (ATCC 16404), Pencicillium citrinum (ATCC 14994), Rhizopus 8
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oryzae (ATCC 9363), and Aspergillus flavus (ATCC 204304). The MIC and MBC values of
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the oil ranged from 0.2 to 1.56 μg mL-1 and from 0.39 to 6.25 μg mL-1, respectively. R. oryzae
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was the most susceptible bacteria among the tested microorganisms the essential oil of Pine
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needle inhibited the growth of R. oryzae at 0.2 μg mL-1 and exerted a sterilizing effect at 0.39
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μg mL-1. Among the assayed microorganisms, B. subtilis was the most susceptible to the
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essential oil of Pine needle; MIC and MBC values were 0.39 and 0.78 μg mL-1, respectively.
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The present study provided promising results: the MIC values of the resin-oil of Pinus elliottii
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varied from 25 to 100 μg mL-1 against the Gram-positive multiresistant bacteria S.
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epidermidis (ATCC 14990), S. epidermidis (clinical isolate), S. aureus (clinical isolate), E.
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faecium (NCTC 717), E. faecium (clinical isolate), S. capitis (ATCC 27840), E. faecalis
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(NCTC 14990), E. faecalis (clinical isolate), S. haemolyticus (ATCC 29970), and S.
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haemolyticus (clinical isolate). According to Rios & Recio (2005) a promising result arises
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when the crude extracts and essential oils of plants exhibit antibacterial activity at
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concentrations equal to or lower than 100 μg mL-1. Because the resin-oil of Pinus elliottii
208
contains many substances, the observed activity probably originated from a synergistic action
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among the resin-oil constituents. Concerning the tested Gram-negative multiresistant bacteria
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(K. pneumoniae ATCC 700603 and K. pneumoniae clinical isolate), MIC of the resin-oil of
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Pinus elliottii was 400 μg mL-1. In contrast to the Gram-negative bacteria, the Gram-positive
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microorganisms were more sensitive to certain compounds. Antimicrobials act on the cell
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wall, and the different composition of the cell walls of these classes of bacteria may account
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for these results. In fact, Gram-positive bacteria present a thick cell wall consisting mainly of
215
peptidoglycan, whereas Gram-negative bacteria display a stratified cell wall consisting of an
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outer membrane and a thin peptidoglycan layer (Beveridge, 1999; Navarre and Schneewind,
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1999; Araujo et al., 2010). The unique structural properties of the cell wall of the Gram-
218
negative bacteria may have hindered penetration of the resin-oil of Pinus elliottii the external 9
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membrane contains lypopolysaccharides, which determine surface properties and alter cell
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permeability and susceptibility to the resin-oil of Pinus elliottii.
221 222
Dehydroabietic acid was only active against S. epidermidis (ATCC14990); both MIC and
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MBC were 6.25 μg mL-1. According to Rios & Recio (2005), this is a promising result for a
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pure isolated compound, because it lay below 10 μg mL-1. MIC and MBC for the other tested
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microorganisms ranged from 25 to 50 μg mL-1 and from 50 to 100 μg mL-1, respectively.
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S. epidermidis is one of the species that is most frequently isolated from the human
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epithelium. It is an important opportunistic pathogen that often occurs in hospital infections
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(NNIS, 2004; Otto, 2009). This microorganism is the commonest source of infection in
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medical devices like intravenous catheters (Otto, 2009; Uckay et al., 2009; Widerström et al.,
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2012). The following factors complicate treatment: specific genes for antibiotic resistance,
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biofilm formation, multicellular agglomerations, and host defense mechanisms (Costerton et
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al., 1999; Otto, 2009). In the United States, annual costs involved in the treatment of hospital
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infections associated with catheters contaminated by S. epidermidis lie around US$ 2 billion
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(Kloos and Musselwhite, 1975; Costerton et al., 1999; Otto, 2009). Hence, it is essential to
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search for new substances that can fight and eliminate this pathogenic microorganism.
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Dehydroabietic acid proved to be a promising compound in this sense.
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We conducted time-kill assays for dehydroabietic acid only against S. epidermidis (ATCC
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14990) which, as already mentioned, is one of the most important pathogens involved in
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hospital-associated bloodstream infections related to vascular catheters for its ability to
240
produce a biofilm (Widerström et al., 2012).
241
A study conducted by Mun et al. (2013), the authors used time-kill assays to assess the action
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of curcumin isolated from the species Curcuma longa Linné against Staphylococcus aureus
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resistant to methicillin (MRSA) ATCC 33591. In isolation, curcumin was not active against 10
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MRSA. In combination with the antibiotic oxacillin, it presented activity. Our study
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demonstrated that dehydroabietic acid at 6.25 μg mL-1 no combination with antibiotics,
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exerted a bactericidal effect against S. epidermidis within 24 h.
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In the present scenario regarding the emergence of human pathogens that are resistant to
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multiple drugs, the resin-oil of P. elliottii and its major compound dehydroabietic acid have
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become important assets in the fight against multiresistant microorganisms. Further studies to
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unveil their action mechanisms are therefore essential.
251 252 253
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deodara). J Food Sci 77, C824-829.
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395 Table 1. In vitro antibacterial activity (MIC and MBC) of the resin-oil of P. elliottii, fraction OPe2 396 and dehydroabietic acid (DA) against multidrug-resistant bacteria. 397 Bacteria
Pinus elliottii resin-oil (μg mL-1)
Fraction OPe2 (μg mL-1)
Dehydroabietic acid
Positive controls
(μg mL-1)
(μg mL-1)
MIC MIC MIC MBC MIC MBC S. epidermidis 6.25 * 0.74 * 0.74 25 25 6.25 ATCC 14990 S. epidermidis 50 * 2.95 * 2.95 50 50 25 Clinical isolate S. aureus 25 * 1.50 * 1.50 200 100 25 ATCC 29213 S. aureus 25 * 0.74 * 0.74 100 50 25 Clinical isolate E. faecium 25 * 2.95 * 2.95 100 100 25 NCTC 717 E. faecium 50 * 1.50 * 1.50 100 100 25 Clinical isolate S. capitis 50 * 5.9 * 5.9 100 100 25 ATCC 27840 S. capitis 100 * 2.95 * 2.95 400 400 50 Clinical isolate E. faecalis * 0.74 * 0.74 100 200 50 NCTC 14990 E. faecalis * 5.9 * 5.9 100 200 25 Clinical isolate S. haemolyticus 50 * 0.74 * 0.74 100 100 25 ATCC 29970 S. haemolyticus 100 * 1.50 * 1.50 100 100 50 Clinical isolate K. pneumuniae 400 200 ** 5.9 ** 5.9 ATCC 700603 K. pneumonia ** 5.9 ** 5.9 400 400 Clinical isolate 398 --: > 400 µg mL-1 corresponds to lack of antibacterial activity; Positive controls µg mL-1 (*Vancomycin, ** Imipenem)
399 400 401 16
H
402 403
COOH
Fig. 1. Chemical structure of dehydroabietic acid.
404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419
17
S. epidermidis (ATCC 14990) 12
L og 1 0 UFC /m L
10
Inoculum Log = 13.3 x 105 = 6.12
8
Vancomycin (0.74 µg mL-1 )
6
Dehydroabietic acid (6.25 µg mL-1)
4 2 0 0.0
0.5
5
10
15
20
25
30
Time (h) 420 421
Fig. 2. Time-kill curve profiles for dehydroabietic acid against S.epidermidis (ATCC
422
14990). Positive control: Vancomycin.
423 424
18
1
Antibacterial
2
dehydroabietic acid, against multidrug-resistant strains
activity
Pinus
elliottii
and
its
major
compound,
3
4 5
Luís Fernando Leandro1, Miguel Jorge Oliveira Cardoso1, Sandro Donizeti Caetano Silva1;
6
Maria Gorete Mendes Souza1, Rodrigo Cassio Sola Veneziani2, Sergio Ricardo Ambrosio2 and
7
Carlos Henrique Gomes Martins1.
8 9
Author affiliation
10
1
11
14404-600, São Paulo, Brazil
12
2
13
600, São Paulo, Brazil
Laboratory of Research in Applied Microbiology, University of Franca - UNIFRAN, Franca,
Nucleus of Research in Sciences and Technology, University of Franca - UNIFRAN, 14404-
14 15 16 17 18 19 20 21 22 23 24 25 26
Correspondence Carlos Henrique G. Martins, Laboratory of Research in Applied Microbiology, University of Franca, Av. Dr. Armando Salles Oliveira, 201, 14404-600, Franca, São Paulo, Brazil Tel: +55 16 3711-8756; fax: +55 16 3711-8874 E-mail: [email protected] Abbreviations: MIC - minimum inhibitory concentration; MBC - minimum bactericidal concentration
1
27 28 29 30
Abstract
31
and have prompted the search for new sources of antimicrobial substances. Pinus species
32
contain several bioactive compounds consisting mainly of terpenes, terpenoids, and some
33
other aromatic and aliphatic constituents. These compounds exert important biological effects,
34
and pine oils have found wide application in the industry. In the present study, we have
35
evaluated the potential activity of the resin-oil of Pinus elliottii and its major compound
36
dehydroabietic acid against multiresistant bacteria by minimum inhibitory concentration
37
(MIC), minimum bactericidal concentration (MBC), and time-kill assays. The MIC of the
38
resin-oil of Pinus elliottii varied between 25 and 100 μg mL-1. As for dehydroabietic acid,
39
MIC and MBC varied between 6.25 and 50 and between 6.25 and 100 μg mL-1, respectively.
40
The time-kill assay conducted with dehydroabietic acid at 6.25 μg mL-1 evidenced
41
bactericidal activity against S. epidermidis (ATCC 14990) within 24 hours. On the basis of
42
these results, the resin-oil of Pinus elliottii and its major compound dehydroabietic acid play
43
an important part in the search for novel sources of agents that can act against multiresistant
44
bacteria.
Antibiotic-resistant bacteria have emerged from the widespread use of antibiotics worldwide
45 46
Keywords: Pinus elliottii; dehydroabietic acid; multiresistant bacteria; Antibacterial activity
47 48 49 50
2
51
Introduction
52
The widespread use of antibiotics worldwide has culminated in high prevalence of infections
53
by antibiotic-resistant bacteria. Indeed, exchangeable genetic elements such as plasmids,
54
transposons, and integons disseminate antibiotic resistance in many bacteria (Nikokar et al.,
55
2013). The slower introduction of new chemotherapeutic drugs into the market and clinical
56
practice over the last few years may yet be another reason for the growing number of resistant
57
bacteria (Russell, 2002; Coelho et al., 2013). This slowdown may have caused a number of
58
antibiotics to lose their efficacy against antibiotic-resistant organisms, making the use of
59
drugs that are often reserved for last-line treatment necessary (Coelho et al., 2013). The
60
overall result of the emergence of human pathogenic organisms exhibiting multidrug
61
resistance has been the search for new antimicrobial substances from other sources, including
62
plants (Ahmad & Beg, 2001).
63
During the 1950s, exotic, fast-growing trees such as Pinus spp. were introduced in southern
64
Brazil, to supply wood for the paper industry and cellulose for energy production. Today,
65
Brazil holds one of the largest planted areas in the world (Pacheco et al., 2009). A number of
66
compounds displaying anti-inflammatory, antiparasitic, antioxidant, antimutagenic, and
67
antitumor activities as well as the ability to regulate cancer-related proteins have been isolated
68
from different Pinus species (Tóro et al., 2003; Karonen et al., 2004; Kwak et al., 2006; Li et
69
al., 2007; Politeo et al., 2011). Some species are frequently used for their medicinal benefits,
70
such as diuretic and expectorant actions, to treat diseases that include pulmonary, urinary,
71
hepatic, and hypertensive disorders (Lawless, 1992; Politeo et al., 2011). Moreover, pine oils
72
are widely employed as fragrances in cosmetics, flavoring additives in food, and
73
intermediates in the synthesis of perfume chemicals (Sticher, 1977; Politeo et al., 2011). They
74
consist mainly of terpenes, terpenoids, and some other aromatic and aliphatic constituents, 3
75
which usually have strong smell and low molecular weight (Marriott et al., 2001; Burt et al.,
76
2004; Politeo et al., 2011; Zeng et al., 2012).
77
The current literature contains no reports on the biological activities of the resin-oil of Pinus
78
elliottii and its bioactive compounds against antibiotic-resistant bacteria. Therefore, the
79
present study aimed to investigate the in vitro antibacterial activity of the oleoresin of Pinus
80
elliottii and its major compound dehydroabietic acid (DA) against multiresistant bacteria.
81 82
Methods
83
Tested microorganisms
84 85
The bacteria were acquired from the American Type Culture Collection (ATCC) and National
86
Collection of Type Cultures (NCTC); Hospital das Clínicas de Ribeirão Preto (São Paulo
87
State, Brazil) kindly supplied seven bacteria of clinical isolates (HCRP), which were
88
maintained in the culture collection of the Laboratory of Research in Applied Microbiology
89
(LaPeMA) of the University of Franca, state of São Paulo, Brazil, at -80 oC. The following
90
microorganisms were used: Staphylococcus epidermidis (ATCC 14990), Staphylococcus
91
epidermidis (clinical isolate), Staphylococcus aureus (ATCC 29213), Staphylococcus aureus
92
(clinical isolate), Enterococcus faecium (NCTC 717), Enterococcus faecium (clinical isolate),
93
Staphylococcus capitis (ATCC 27840), Staphylococcus capitis (clinical isolate), Enterococcus
94
faecalis (NCTC 14990), Enterococcus faecalis (clinical isolate), Staphylococcus haemolyticus
95
(ATCC 29970), Staphylococcus haemolyticus (clinical isolate), Klebsiella pneumoniae
96
(ATCC 700603) and Klebsiella pneumoniae (clinical isolate).
97 4
98
Oil-resin and isolation of the major compound
99 100
Associação dos Resinadores do Brasil - ARESB, located in the city of Avaré, state of São
101
Paulo, Brazil, donated the original oil-resin of Pinus elliottii var. elliottii (OPe) tested here.
102
First, the oil-resin (40.0 g) was partitioned by vacuum liquid chromatography using a solvent
103
system of increasing polarity (n-hexane/ethyl acetate). This procedure furnished five fractions
104
(2 L each) that were named OPe1 (100% n-hexane), OPe2 (20% ethyl acetate), OPe3 (40%
105
ethyl acetate), OPe4 (60% ethyl acetate) and OPe5 (100% ethyl acetate). The minimum
106
inhibitory concentration (MIC) values of the five fractions were determined against the tested
107
bacteria. MIC results revealed that fraction OPe2 was the most active against multiresistant
108
bacteria (Table 1).
109
The major compound in OPe2 was isolated from 1.0 g of this fraction by classic
110
chromatography using 500 mL of the mixture n-hexane/ethyl acetate 8:2 (v/v) as mobile
111
phase. The obtained dehydroabietic acid (125.8 mg) was subjected to MIC and MBC assays
112
in the presence of the bacteria evaluated in this study (Fig. 1). The chemical structure of
113
dehydroabietic acid was confirmed based on comparison with nuclear magnetic resonance (1H
114
and 13C NMR) literature data (Fraga et al., 1994; Gonzàles et al., 2010).
115 116
Antimicriobial assays
117 118
The minimal inhibitory concentration values (MIC; lowest concentration of the compound
119
that was able to inhibit microorganism growth) and the minimal bactericidal concentration
120
(MBC; lowest concentration of the compound at which 99.9% or more of the initial inoculum 5
121
was killed) were determined in triplicate, by using the microdilution broth method in 96-well
122
microtitre plates. Samples of the resin-oil of Pinus elliottii and its major compound DA were
123
dissolved in dimethyl sulfoxide (DMSO; Synth) at 1.0 mg mL-1, followed by dilution in
124
tryptic soy broth (Difco); concentrations ranging from 100.0 to 1.0 µg mL-1 were achieved.
125
The final DMSO content was 5% (v/v), and this solution was used as negative control. The
126
inoculums was adjusted for each organism, to yield a cell concentration of 5 x 105 colony
127
forming units (CFU) per mL, according to previous standardization by the Clinical
128
Laboratory Standards Institute (CLSI, 2009). One inoculated well, free of antimicrobial agent,
129
was also employed, to ensure medium sterility. Vancomycin (VAN) and Imipenem were used
130
as positive controls. The microtitre plates (96 wells) were sealed with plastic film and
131
incubated at 37 ºC for 24 h. After that, resazurin (30µL) in aqueous solution (0.02 %) was
132
added to the microplates, to indicate microorganism viability (Sarker et al., 2007; Ambrosio
133
et al., 2008b; Porto et al., 2009; Porto et al., 2013). Before addition of resazurin and to
134
determine MBC, an aliquot of the inoculum was aseptically removed from each well
135
presenting no apparent growth and then plated onto tryptic soy agar. The plates were
136
incubated as described previously.
137 138
Time-kill curves
139 140
Time-kill assays were performed in triplicate based on D’Arrigo et al. (2010). Dehydroabietic
141
acid (DA) was chosen to conduct the time-kill curve assays because, according to Rios &
142
Recio, (2005) isolated compounds that inhibit the growth of the microorganisms at
143
concentrations below 10 μg mL−1 are considered very promising in the search for new anti-
144
infection agents, therefore the DA displayed the highest antibacterial activity (Table 1 - MIC
145
and MBC 6.25 μg mL-1) only against Staphylococcus epidermidis (ATCC14990). This 6
146
microorganism is considered as one of the most important pathogens involved in hospital-
147
associated bloodstream infections related to vascular catheters, because of their virulence
148
factors and ability to produce biofilm (Otto, 2009; Widerström et al., 2012). A tube
149
containing 1 mL of tryptic soy broth sterile and DA at final concentration of 6.25 µg mL-1 (1
150
x MBC of DA for S. epidermidis) was inoculated with the tested microorganism, resulting in
151
an initial bacterial density of 13.3 x 105 CFU mL-1, and then incubated at 37 °C. Aliquots
152
were removed (100 µL) for enumeration of viable colonies at 30 min and 6, 12, and 24 h after
153
incubation, followed by serially dilution, until final dilution (10-4) in tryptic soy broth sterile.
154
The diluted samples (50 µL) were spread onto tryptic soy agar plate, incubated at 37 °C, and
155
viable colonies were counted after 24 h. The data were analyzed, the time-kill curves were
156
constructed by plotting log10 CFU mL-1 versus time using Prism software (version 5.0;
157
GraphPad, Inc.). The assays were performed in triplicate and also for the positive (VAN) and
158
negative controls (suspension of S. epidermidis without added DA). VAN was used at its
159
MBC (0.74 µg mL-1).
160 161
Results
162 163
The resin-oil of Pinus elliottii furnished satisfactory MIC values (between 25 and 100 μg
164
mL-1) when tested against the multiresistant bacteria S. epidermidis (ATCC 14990), S.
165
epidermidis (clinical isolate), S. aureus (clinical isolate), E. faecium (NCTC 717), E. faecium
166
(clinical isolate), S. capitis (ATCC 27840), E. faecalis (NCTC 14990), E. faecalis (clinical
167
isolate), S. haemolyticus (ATCC 29970), and S. haemolyticus (clinical isolate). In the case of
168
the bacteria K. pneumoniae (ATCC 700603), K. pneumoniae (clinical isolate), S. capitis
7
169
(clinical isolate), and S. aureus (ATCC 29213), the MIC values were equal to 200 μg mL-1 or
170
higher (Table 1).
171
Assays revealed bactericidal activity against S. epidermidis (ATCC 14990) only–both MIC
172
and MBC were equal to 6.25 μg mL-1 (Table 1).
173
The time-kill assay helped to assess the kinetic behavior of dehydroabietic acid. MBC values
174
were lower than 10 μg mL-1. The best activity was obtained against S. epidermidis (ATCC
175
14990).
176
The results achieved with S. epidermidis (ATCC 14990) demonstrated that dehydroabietic
177
acid at a concentration of 6.25 μg mL-1 started to progressively diminish the number of
178
microorganisms at 12 h of incubation; bactericidal action was evident at 24 h. Vancomycin
179
(0.74 μg mL-1), used as positive control, reduced the number of microorganisms by over 2
180
log10 at 12 h and exhibited bactericidal activity at 24 h of incubation (Fig. 2).
181 182
Discussion
183 184
The development of antibiotic resistance by bacteria that threaten the human health has
185
become a major clinical problem worldwide. This has prompted the search for new
186
antimicrobial substances from other sources, including plants (Ahmad & Beg, 2001; Bennett,
187
2008). In this context, the present study evaluated the antibacterial action of the resin-oil of
188
Pinus elliottii and its major compound dehydroabietic acid against multiresistant bacteria.
189
A study conducted by Zeng et al. (2012), the authors determined MIC and MBC for the
190
essential oil of Pine needles (Cedrus deodara) against the causative agents of food poisoning,
191
such as Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Bacillus
192
subtilis (ATCC 21216), Bacillus cereus (ATCC 14579), Saccharomyces cerevisiae (ATCC
193
9763), Aspergillus niger (ATCC 16404), Pencicillium citrinum (ATCC 14994), Rhizopus 8
194
oryzae (ATCC 9363), and Aspergillus flavus (ATCC 204304). The MIC and MBC values of
195
the oil ranged from 0.2 to 1.56 μg mL-1 and from 0.39 to 6.25 μg mL-1, respectively. R. oryzae
196
was the most susceptible bacteria among the tested microorganisms the essential oil of Pine
197
needle inhibited the growth of R. oryzae at 0.2 μg mL-1 and exerted a sterilizing effect at 0.39
198
μg mL-1. Among the assayed microorganisms, B. subtilis was the most susceptible to the
199
essential oil of Pine needle; MIC and MBC values were 0.39 and 0.78 μg mL-1, respectively.
200
The present study provided promising results: the MIC values of the resin-oil of Pinus elliottii
201
varied from 25 to 100 μg mL-1 against the Gram-positive multiresistant bacteria S.
202
epidermidis (ATCC 14990), S. epidermidis (clinical isolate), S. aureus (clinical isolate), E.
203
faecium (NCTC 717), E. faecium (clinical isolate), S. capitis (ATCC 27840), E. faecalis
204
(NCTC 14990), E. faecalis (clinical isolate), S. haemolyticus (ATCC 29970), and S.
205
haemolyticus (clinical isolate). According to Rios & Recio (2005) a promising result arises
206
when the crude extracts and essential oils of plants exhibit antibacterial activity at
207
concentrations equal to or lower than 100 μg mL-1. Because the resin-oil of Pinus elliottii
208
contains many substances, the observed activity probably originated from a synergistic action
209
among the resin-oil constituents. Concerning the tested Gram-negative multiresistant bacteria
210
(K. pneumoniae ATCC 700603 and K. pneumoniae clinical isolate), MIC of the resin-oil of
211
Pinus elliottii was 400 μg mL-1. In contrast to the Gram-negative bacteria, the Gram-positive
212
microorganisms were more sensitive to certain compounds. Antimicrobials act on the cell
213
wall, and the different composition of the cell walls of these classes of bacteria may account
214
for these results. In fact, Gram-positive bacteria present a thick cell wall consisting mainly of
215
peptidoglycan, whereas Gram-negative bacteria display a stratified cell wall consisting of an
216
outer membrane and a thin peptidoglycan layer (Beveridge, 1999; Navarre and Schneewind,
217
1999; Araujo et al., 2010). The unique structural properties of the cell wall of the Gram-
218
negative bacteria may have hindered penetration of the resin-oil of Pinus elliottii the external 9
219
membrane contains lypopolysaccharides, which determine surface properties and alter cell
220
permeability and susceptibility to the resin-oil of Pinus elliottii.
221 222
Dehydroabietic acid was only active against S. epidermidis (ATCC14990); both MIC and
223
MBC were 6.25 μg mL-1. According to Rios & Recio (2005), this is a promising result for a
224
pure isolated compound, because it lay below 10 μg mL-1. MIC and MBC for the other tested
225
microorganisms ranged from 25 to 50 μg mL-1 and from 50 to 100 μg mL-1, respectively.
226
S. epidermidis is one of the species that is most frequently isolated from the human
227
epithelium. It is an important opportunistic pathogen that often occurs in hospital infections
228
(NNIS, 2004; Otto, 2009). This microorganism is the commonest source of infection in
229
medical devices like intravenous catheters (Otto, 2009; Uckay et al., 2009; Widerström et al.,
230
2012). The following factors complicate treatment: specific genes for antibiotic resistance,
231
biofilm formation, multicellular agglomerations, and host defense mechanisms (Costerton et
232
al., 1999; Otto, 2009). In the United States, annual costs involved in the treatment of hospital
233
infections associated with catheters contaminated by S. epidermidis lie around US$ 2 billion
234
(Kloos and Musselwhite, 1975; Costerton et al., 1999; Otto, 2009). Hence, it is essential to
235
search for new substances that can fight and eliminate this pathogenic microorganism.
236
Dehydroabietic acid proved to be a promising compound in this sense.
237
We conducted time-kill assays for dehydroabietic acid only against S. epidermidis (ATCC
238
14990) which, as already mentioned, is one of the most important pathogens involved in
239
hospital-associated bloodstream infections related to vascular catheters for its ability to
240
produce a biofilm (Widerström et al., 2012).
241
A study conducted by Mun et al. (2013), the authors used time-kill assays to assess the action
242
of curcumin isolated from the species Curcuma longa Linné against Staphylococcus aureus
243
resistant to methicillin (MRSA) ATCC 33591. In isolation, curcumin was not active against 10
244
MRSA. In combination with the antibiotic oxacillin, it presented activity. Our study
245
demonstrated that dehydroabietic acid at 6.25 μg mL-1 no combination with antibiotics,
246
exerted a bactericidal effect against S. epidermidis within 24 h.
247
In the present scenario regarding the emergence of human pathogens that are resistant to
248
multiple drugs, the resin-oil of P. elliottii and its major compound dehydroabietic acid have
249
become important assets in the fight against multiresistant microorganisms. Further studies to
250
unveil their action mechanisms are therefore essential.
251 252 253
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395 Table 1. In vitro antibacterial activity (MIC and MBC) of the resin-oil of P. elliottii, fraction OPe2 396 and dehydroabietic acid (DA) against multidrug-resistant bacteria. 397 Bacteria
Pinus elliottii resin-oil (μg mL-1)
Fraction OPe2 (μg mL-1)
Dehydroabietic acid
Positive controls
(μg mL-1)
(μg mL-1)
MIC MIC MIC MBC MIC MBC S. epidermidis 6.25 * 0.74 * 0.74 25 25 6.25 ATCC 14990 S. epidermidis 50 * 2.95 * 2.95 50 50 25 Clinical isolate S. aureus 25 * 1.50 * 1.50 200 100 25 ATCC 29213 S. aureus 25 * 0.74 * 0.74 100 50 25 Clinical isolate E. faecium 25 * 2.95 * 2.95 100 100 25 NCTC 717 E. faecium 50 * 1.50 * 1.50 100 100 25 Clinical isolate S. capitis 50 * 5.9 * 5.9 100 100 25 ATCC 27840 S. capitis 100 * 2.95 * 2.95 400 400 50 Clinical isolate E. faecalis * 0.74 * 0.74 100 200 50 NCTC 14990 E. faecalis * 5.9 * 5.9 100 200 25 Clinical isolate S. haemolyticus 50 * 0.74 * 0.74 100 100 25 ATCC 29970 S. haemolyticus 100 * 1.50 * 1.50 100 100 50 Clinical isolate K. pneumuniae 400 200 ** 5.9 ** 5.9 ATCC 700603 K. pneumonia ** 5.9 ** 5.9 400 400 Clinical isolate 398 --: > 400 µg mL-1 corresponds to lack of antibacterial activity; Positive controls µg mL-1 (*Vancomycin, ** Imipenem)
399 400 401 16
H
402 403
COOH
Fig. 1. Chemical structure of dehydroabietic acid.
404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419
17
S. epidermidis (ATCC 14990) 12
L og 1 0 UFC /m L
10
Inoculum Log = 13.3 x 105 = 6.12
8
Vancomycin (0.74 µg mL-1 )
6
Dehydroabietic acid (6.25 µg mL-1)
4 2 0 0.0
0.5
5
10
15
20
25
30
Time (h) 420 421
Fig. 2. Time-kill curve profiles for dehydroabietic acid against S.epidermidis (ATCC
422
14990). Positive control: Vancomycin.
423 424
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