Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas ISSN: 0717-7917 [email protected] Universidad de Santiago de Chile Chile

MONTENEGRO, Iván; MADRID VILLEGAS, Alejandro; ZAROR, Luis; MARTÍNEZ, Rolando; WERNER, Enrique; CARRASCO-ALTAMIRANO, Hector; CUELLAR FRITIS, Mauricio; PALMA- FLEMMING, Hernán Antimicrobial activity of ethyl acetate extract and essential oil from bark of Laurelia sempervirens against multiresistant bacteria Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas, vol. 11, núm. 4, julio- agosto, 2012, pp. 306-315 Universidad de Santiago de Chile Santiago, Chile

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Artículo Original | Original Article

Antimicrobial activity of ethyl acetate extract and essential oil from bark of Laurelia sempervirens against multiresistant bacteria

[Actividad antimicrobiana del extracto acetato de etilo y aceite esencial de corteza de Laurelia sempervirens contra bacterias multirresistentes]

Iván MONTENEGRO1, Alejandro MADRID VILLEGAS1, Luis ZAROR2, Rolando MARTÍNEZ3*, Enrique WERNER4, Hector CARRASCO-ALTAMIRANO3, Mauricio CUELLAR FRITIS5 & Hernán PALMA-FLEMMING6.

1Departamento de Química, Universidad Técnica Federico Santa María, Av. España 1680, Valparaíso, Chile 2Instituto de Microbiología Clínica, Facultad de Medicina, Universidad Austral de Chile. 3Departamento de Química, Facultad de Ecología y Recursos Naturales. Universidad Andrés Bello, Viña del Mar, Chile 4Departamento De Ciencias Básicas, Universidad del Bio-Bio, Chillán, Chile 5Facultad de Farmacia, Universidad de Valparaíso, Valparaíso, Chile. 6Instituto de Química, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile Contactos | Contacts: Iván MONTENEGRO - E-mail address: [email protected]

Abstract Context: Laurelia sempervirens (R. et P.) Tul., is an evergreen tree that growths in southern Chile, its leaves and bark are used in folk medicine as an infusion. Objective: The antimicrobial activities of the essential oil and ethyl acetate extract obtained from the bark of Laurelia sempervirens were investigated. Materials and methods: Ethyl acetate extract and essential oil were analyzed by GC- mass and the antimicrobial activity was investigated against gram positive and gram negative bacteria. Results: The extract and essential oil showed a strong antimicrobial activity against bacteria such as Acinetobacter baumanii, a relevant world nosocomial pathogen Discussion and conclusion: These findings demonstrate that the ethyl acetate extract and essential oil of L.sempervirens bark have excellent antimicrobial activities and thus have great potential as a source for natural health products.

Keywords: Laurelia sempervirens, inhibitory activity, bacteria.

Resumen Contexto: Laurelia sempervirens (R. et P.) Tul., es un árbol de hoja perenne que crece en el sur de Chile, sus hojas y corteza se utilizan en medicina popular como infusión. Objetivo: Investigar la actividad antimicrobiana del aceite esencial y el extracto de acetato de etilo obtenido de la corteza de Laurelia sempervirens. Materiales y métodos: El extracto de acetato de etilo y el aceite esencial se analizaron por GC-masa y la actividad antimicrobiana se analizó contra bacterias gram positivas y gram negativas. Resultados: El extracto y aceite esencial evidenció una fuerte actividad antimicrobiana frente a la bacteria, Acinetobacter baumanii patógeno que causa infecciones nosocomiales de relevancia mundial. Discusión y conclusiones: Estos hallazgos demuestran que el extracto de acetato de etilo y aceite esencial de corteza de L.sempervirens tienen excelentes actividades antimicrobianas y por lo tanto tienen un gran potencial como fuente de productos naturales para la salud.

Palabras Clave: Laurelia sempervirens, actividad inhibitoria, bacterias.

Recibido | Received: December 20, 2011. Aceptado en versión corregida | Accepted in revised form: March 12, 2012. Publicado en línea | Published online: July 30, 2012. Declaración de intereses | Declaration of interests: This work was supported by DI-Universidad Andres Bello. Grant N° DI-19-08/R and DID UACH-s-2005-41. PIIC 2009-2010, PAC 2011-2012 and DGIP of Universidad Técnica Federico. Santa María for financial support. Este artículo puede ser citado como / This article must be cited as: Iván Montenegro, Alejandro Madrid Villegas, Luis Zaror, Rolando Martínez, Enrique Werner, Hector Carrasco-Altamirano, Mauricio Cuellar Fritis, Hernán Palma-Flemming. 2012. Antimicrobial activity of ethyl acetate extract and essential oil from bark of Laurelia sempervirens against multiresistant bacteria. Bol Latinoam Caribe Plant Med Aromat 11(4): 306 – 315.

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INTRODUCTION marcescens 1332-2; Pseudomonas aeruginosa1020- Considering the knowledge of Chilean indigenous 1,1020-2,1048-2; Acinetobacter baumannii 999, cultures on the use of medicinal plants, we conducted Staphylococcus aureus 2312 (not ATCC bacteria was the research on those that present obvious biological isolated from clinical samples at Regional Hospital, activities, being Laurelia sempervirens one of the most Valdivia, Chile) cultures were obtained from the used. Laurelia sempervirens, also called Chilean culture collections of Clinical Microbiology Laurel, is a species of evergreen tree endemic to Chile. Department of Universidad Austral de Chile. Leaves of Laurelia sempervirens were used by the Mapuche amerindians for treating headache and as a Essential oil extraction diuretic. Intravenous administration of a Fresh bark of Laurelia sempervirens (4 kg) was steam- hydroalcoholic L. sempervirens extract to rats, elicited distilled for 5 h using a Clevenger type apparatus. a hypotensive response. Bioassay-guided isolation of Essential oil yielded 1.0%. The essential oil obtained the active Laurel metabolites led to the alkaloid was stored at 4º C and used in biological assays laurotetanine as the main hypotensive principle of L. without further purification. sempervirens leaves (Schmeda-Hirschmann et al., 1994). Bittner et al., 2009 demonstrate the fungistatic Preparation of ethyl acetate extract activity of essential oil of Laurelia sempervirens To extract bioactive compounds, 2.7 kg of Laurelia leaves. Cassels and Urzúa in 1985 reported the sempervirens bark with 20 L of ethyl acetate were isolation alkaloids (obaberine, thalrugosine, and processed twice. The resulting solution was brought to oxyacanthine) of the stem bark. Liriodenine and dryness in a Büchi R-114 rotary evaporator with Büchi oxonantenina present in bark extracts are powerful Waterbath thermoregulator bath B-480 at a broad spectrum antimicrobial agents (Hufford et al., temperature of 60º C. The sample was treated with hot 1980). In recent studies by Mølgaard et al., 2011 water to remove chlorophyll and then it was dried. A demonstrate the antimicrobial activities of aqueous fraction was used for chromatographic analysis. 28.6 g and ethanolic extracts of leaves, these results support of this extract was suspended in 100 mL of ethyl the previous comprehensive reports regarding the acetate providing in this way a solution of 0.286 g/mL, antimicrobial and/or cytotoxic effects of Laurelia which was used for biological tests. sempervirens. Based on these data, we carried out the study Antimicrobial Assays of compounds through joined gas chromatography To evaluate the antibacterial activity, the hole-plate present in the phase of ethyl acetate and in the agar diffusion method was implemented (Van den essential oil of L. sempervirens bark. In addition, we Berghe and Vlietinck, 1991) 4-5 colonies were taken perform tests on inhibitory activity in bacterial from the microorganisms studied. The colonies were development considered a problem for its frequency re-suspended in physiological saline solution, in and ability to present resistance to commonly used duplicate, to 0.5 Mac-Farland concentration (108 antibiotics like Acinetobacter baumanni, Pseudomona CFU/mL). The bacterial suspension was uniformly aeruginosa and Staphylococcus aureus (Storti et al., spread by using sterile cotton swab on a sterile Petri 2005; Akers et al., 2010; Perazzi et al., 2010). dish MH agar. After letting it dry for 10-15 minutes, 6 mm diameter holes were cut in the agar at 24 mm EXPERIMENTAL SECTION distance from each other. 20 μL of serial dilutions Plant Material obtained from the initial extract were placed in each The samples of L. sempervirens were collected well. These dilutions had concentrations ranging from in September 2003, from trees growing in plantations 7200 μg/mL to 0.72 μg/mL. Plates were incubated located at the Arboretum of the Universidad Austral de overnight at 35º C, under aerobic conditions. After Chile, Valdivia, Chile. Number of herbal 17472. incubation, confluent bacterial growth was observed. The inhibition zone around the hole was measured Microorganisms with a caliper. Minimum inhibition concentration Staphylococcus aureus ATCC29583, Pseudomonas (MIC) was defined as the lowest concentration that aeruginosa ATCC 27853, Bacillus subtilis inhibited visible growth. ATCC6633, Salmonella sp108,109; Serratia

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Essential oil was assayed to each Program and a NIST/EPA/NIH Mass Spectral Library microorganism in the same way that ethyl acetate (NIST, 1998). extract. 20 µL of serial dilutions in n-hexane 1:10; 1:100; 1:1000; 1:10000 were assayed. Negative controls were performed with pure solvent. All the Data analysis experiments were repeated five times. Results were expressed as mean value ± standard error Inhibition assays were performed by adding of growth inhibition halo diameters were obtained antibiotic clinical samples as described in NCCLS with ethyl acetate extract and essential oil of Laurelia 2003 and CLSI, 2009 in order to compare with the sempervirens bark. To determine significant results obtained after addition of ethyl acetate extract differences among inhibitory activity of Antibiotic and and essential oil from bark. 20 uL of either 70 ug/mL essential oil or antibiotic and ethyl acetate extract, a gentamicin, amikacin and ciprofluoxacine clinical one way ANOVA was carried out using GraphPad samples were added. Prism V5 software. P values less than 0.05 (p < 0.05) were considered as significant. Antibiotics assay To determine the sensitivity of microorganism to RESULTS AND DISCUSSION antibiotics (MICs) of amikacin (AKN), gentamicin Composition analysis of the ethyl acetate extract and (GEN) and ciprofloxacine were obtained (CIPRO) in essential oil by gas chromatography coupled to a vitro through the inhibition zone determination mass selective detector method, as stated by microdilution NCCLS guidelines Tables I and II show the data obtained from gas (National Committee for Clinical Laboratory chromatographic analysis of the ethyl acetate extract Standards) and CLSI (Clinical and Laboratory and essential oil of Laurelia sempervirens bark. Standards Institute) Salmonella sp. 108,109; Serratia According to Cespedes et al., (2006) and Zapata marcescens 1332-2; Pseudomonas aeruginosa 1020- (2010), the high antimicrobial activity of the essential 1,1020-2,1048-2; Acinetobacter baumannii 999, oil and ethyl acetate extract from bark of L. Staphylococcus aureus 2312. Isolates from clinical sempervirens are not that surprising. This is because samples and the ATCC species Staphylococcus aureus safrole (63.03 and 65.03% respectively) was one of the ATCC29583, Pseudomonas aeruginosa ATCC 27853, main metabolites in these samples. Safrole is also the Bacillus subtilis ATCC6633. It has been reported that main constituent (74.29%) present in the essential oil these bacterias are sensitive to these antibiotics of Piper auritum. It has shown high Antimicrobial (Fernandéz-Cuenca et al., 2004 and Zambrano et al., activity when was tested against Xanthomonas 2004; Simberkoff et al., 1986). albilineans and Acidovorax avenae subsp. avenae (Sánchez et al., 2009) The other component of the Chromatographic analysis ethyl acetate extract is (E)-5-prop-1-enyl) benzo [d] The essential oil and ethyl acetate extract were diluted [1,3] dioxolan, better known as isosafrole (12.94%), a with n-hexane, 1 μL was sampled for the gas carcinogenic aromatic compound (Tulp and Bohlin, chromatography and analyzed in a chromatograph HP 2004; Delaforge et al., 1982). The isosafrole has two 6890 equipped with a programmable temperature that types of isomers and it is the precursor to the MDMA vaporizes inlet (PTV). In addition, it was coupled to an drug of abuse (known as ecstasy) and Spathulenol HP 5973 mass selective detector (Hewlett-Packard, (6.60%) this sesquiterpene presents activity against Palo Alto, CA, USA) in scan mode in order to Escherichia coli, Salmonella typhimurium and determine the composition of extracts fractions. The Staphylococcus aureus (de Souza et al., 2011). In injector temperature was maintained at 280º C in a essential oils we found 3-Carene and β-Caryophyllene, pulsed splitless mode. A GC program temperature described in essential oils that present antimicrobial ramp was set at 70º C for 3 min. and then at a rate of activity (Pichette et al., 2006; Oztürk et al., 2009). α- 10 ºC/min. up to 300º C was used to afford the best Gurjunene was identified in the essential oil of tubers separation by using a capillary HP-5 MS column. The of Cyperus rotundus Linn. (Bisht et al., 2011), and transfer line was maintained at 300º C. Mass spectra showed antifungal activity against the pathogenic was obtained at 70 eV and compared through direct Trichophyton menthagrophytes (Flach et al., 2002). matching by using a NIST Mass Spectral Search Finally, we note the presence of 3-Allyl-6-

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Table I Volatile compounds identified in ethyl acetate extract from bark of Laurelia sempervirens.

Compound RI Area (%) Identification Isosafrole 1177.5 12.94 RI, MS, Co-I Safrole 1292.6 63.03 RI,MS, Co-I Spathulenol 1457.2 6.60 RI, MS

Table II Mayor compounds identified in essential oil from bark of Laurelia sempervirens.

Compound RI Area (%) Identification α-Pinene 941,11 1.48 RI, MS Camphene 957,37 0.11 RI, MS α-Phellandrene 1009,80 0.33 RI, MS p-Cymene 1031,75 0.12 RI, MS β-Phellandrene 1037.66 1.08 RI, MS 3-Carene 1070.4 0.42 RI, MS Isosafrole 1177.5 11.90 RI, MS, Co-I Safrole 1292,6 65.03 RI,MS, Co-I Piperonal 1358.2 0.37 RI, MS 3-Allyl-6-methoxyphenol 1359.6 1.13 RI, MS β-Caryophyllene 1384.9 0.74 RI, MS α-Gurjunene 1396.6 0.24 RI, MS α-Humelene 1440.9 0.35 RI, MS Germacrene D 1444.8 2.33 RI, MS Spathulenol 1457.2 11.16 RI, MS α-Cadinol 1615,0 1.10 RI, MS Adams RP. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry; 4th Ed.; Allured Publishing Corporation: Carol Steam, 2007.

Bacterial Development Inhibition sample is much less likely to interfere with the The ethyl acetate extract and essential oil from bark of diffusion of the antimicrobial substance into the agar Laurelia sempervirens show strong and efficient than in the filter paper disc (Valgas et al., 2007). antimicrobial activity against different types of bacteria. As shown in table III and IV, the extract and Minimum Inhibitory concentration (MIC) essential oil were tested against Acinetobacter Previously defined as the minimum inhibitory baumanii, Staphylococcus aureus, Pseudomonas concentration as the lowest concentration of pure aeruginosa, Salmonella spp, Bacillus subtilis ATCC compounds or greater dilution of essential oil that 9799 and Serratia marcensens, High efficiency against inhibited visible bacterial growth using as control the these resistant and persistent microorganisms causing pure solvent. In all cases we considered as bacterial nosocomial infections was observed. The hole plate growth inhibition when the inhibition halo was 1 mm method was chosen because is a suitable diffusion in diameter at least. The results are shown in tables III technique for testing suspensions of plants extracts. In and IV. this method, the presence of particulate matter in the

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Gram positive bacteria Staphylococcus aureus multidrug resistant. In the hospital, they may be and Bacillus subtillis, and gram negatives: Salmonella isolated from equipment and accessories, air spp, Serratia marcescens, Pseudomonas aeruginosa conditioner ducts as well as the skin of workers and and Acinetobacter baumannii. An extensive literature rarely cause infection in healthy persons (Harbarth et describes the structural and physiological differences al., 2008; McGowan, 2006; Uduman et al., 2002; between gram (-) and gram (+) bacteria. However, Hejazi and Falkiner, 1997). Staphylococcus aureus, Serratia marcescens, Multi-resistance to methicillin and other Pseudomonas aeruginosa and Acinetobacter antibiotics by Staphylococcus aureus as reported, baumannii, represents a challenge for healthcare as a representing a high risk of infection in surgical cause of hospital acquired infections, difficult to treat patients (Harbarth et al., 2008). because they represent opportunistic pathogens

Table III Minimun Inhibitory concentration (μg/mL) of ethyl acetate extract from bark of Laurelia sempervirens

Species Concentration (µg/ml) Average of inhibition halo (mm) Salmonella sp 72 12

Acinetobacter baumanni 7 28

Pseudomonas aeruginosa 1020 72 23

Serratia marcescens 7200 14

Bacillus subtilis 0.72 25

Staphylococcus aureus 2312 72 17

Serratia marcescens, classified as a member compromised patients associated with ineffective of the enterobacteriaceae, has been recognized because empirical therapy in these infections (Van Eldere, of resistance to a variety of antibiotics, including 2003). ampicillin, cephalosporins, quinolones and Acinetobacter baumannii is a multidrug aminoglycosides (Hejazi and Falkiner, 1997) and is resistant bacteria (Akers et al., 2010) able to develop involved in various types of infection such as microcolonies and microbial biofilms on materials for respiratory tract infections, urinary tract infections, medical use such as catheters being the cause of neonatal sepsis and meningitis (Uduman et al., 2002). bloodstream infections (Storti et al., 2005). The high resistance to fluoroquinolones In the last two decades Acinetobacter (ciprofloxacin) that nosocomial Pseudomonas shows baumanni has emerged as a major relevant world is a worldwide problem in many hospitals and health nosocomial pathogen can be a causal agent of diseases services as reported by Van Eldere 2003. like pneumonia, bacteremia, meningitis, soft tissue and Pseudomonas aeruginosa has been isolated from urinary tract infections, associating to high mortality. nosocomial pneumonia, primary bacteraemias, (Diomedi, 2005). with high mortality, particularly in immune-

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Table IV Minimun Inhibitory concentration of essential oil from bark of Laurelia sempervirens.

Species Dilution in n-hexane Average of inhibition halo (mm) Salmonella sp 1/10000 12

Acinetobacter baumanni 1/10000 25

Pseudomonas aeruginosa 1020 1/10000 23

Serratia marcescens 1/10000 16

Bacillus subtilis 1/10000 17

Staphylococcus aureus 2312 1/10000 14

The minimum inhibitory concentration of Our results show that the phytochemical study ethyl acetate extract among bacteria could be due to of native plants and subsequent biological tests are a the strains origin. Those with greater susceptibility are way to get products with antimicrobial activity. It has those with greater possibility of presenting multi been possible to characterize components of the resistance activity due to its nosocomial origin. essential oil through the method of GC-MS We can not compare the results of bacterial compounds, but this is not possible in ethyl acetate growth inhibition among ethyl acetate extract and extract of Laurelia sempervirens, because GC-MS essential oil, it is not possible because we could not technique used for the volatile and nonvolatile be determine the minimum inhibitory concentration for retained in the column and/or decomposed in injection. essential oil since in all cases inhibition was achieved Biological activity of essential oil and ethyl acetate with the maximum dilution tested 1/10000 (Table IV). extract depends on their chemical composition and However, we compare these results with the interaction among structural components. Even minor inhibitory capacity of clinically used antibiotics tested compounds can have a critical function (Zapata et al., (Table V), calculating the ratios among the inhibition 2010), therefore we are not in a position to explain the halos of ethyl acetate extract, essential oil and different mechanisms of action. However, antibiotics (Graphic 1). antimicrobial activity of essential oil and ethyl acetate From these results, highlight the activity of extract obtained from Laurelia sempervirens bark was ethyl acetate extract (ratio inhibition halo/antibiotic demonstrated against a multidrug resistant bacteria inhibition halo = 4.7) and essential oil (ratio inhibition isolated from clinical specimens, nevertheless, toxic halo/antibiotic inhibition halo = 4.2) from bark of effects to humans should be evaluated in future Laurelia sempervirens against Acinetobacter studies. baumannii (Graphic 1). Results based on Salmonella spp. and Bacillus subtilis are significant but these are minor clinic significance.

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Table V The susceptibility of the microorganisms to selected antibiotics.

Species Concentration (µg/ml) Average of inhibition halo Antibiotics (mm) Salmonella sp (CIPRO)a 70 14

Acinetobacter baumanni (AKN)b 70 6

Pseudomonas aeruginosa 1020 ( GEN)c 70 15

Serratia marcescens (CIPRO)a 70 14

Bacillus subtilis (GEN)c 70 21

Staphylococcus aureus 2312 (GEN)c 70 9

a; Ciprofuoxacine, b; Amikacin, c; Gentamicin

* The inhibition rate was calculated dividing the ethyl acetate extract and essential oil inhibition halo (mm) with respect to the antibiotic inhibition halo (mm) (table V).

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CONCLUSIONS (Uttarakhand). Int J Res Pharm Biomed Sci Antibiotics are an essential part of medicine. However, 2: 661 - 665. bacterial resistance emerges and reduce efficacy of Barrero A, Quilez del Moral J, Lara A, Herrador M. antibiotics in the human population. At this point, the 2005. Antimicrobial activity of sesquiterpenes search of new compounds with potential effects from the essential oil of Juniperus thurifera”. against pathogenic bacteria is required (Morales et al., Planta Med 71: 67 - 71. 2003). Chilean flora is a potential source of bioactive Bittner M, Aguilera M, Hernández V, Albert C, compounds, including some with antimicrobial Becerra J, Casanueva ME. 2009. Fungistatic activity. activity of essential oil extracted from Peumus These preliminary studies are highly boldus Mol., Laureliopsis philippiana interesting as they open new pathways for further (Looser) Schoddle and Laurelia sempervirens studies, which would allow the validation of the (Ruiz & Pav.) (Chilean Monimiaceae). Chil J traditional use of plants in the treatment of infections. Agric Res 69: 30 - 37. The next step in our research is the isolation of pure Böhm L, Arismendi N, Böhm L, Arismendi N, compounds to determine their antibiotic activity and Ciampi L. 2009. Nematicidal activity of leaves their mechanisms of action. Therefore, our study of common shrub and tree species from serves as a basis for new targets on molecular Southern Chile against Meloidogyne hapla. mechanisms of action for natural compounds. Our Cien Inv Agr 36: 249 - 258. findings provide with new data to fight this pathogen Boyom FF, Ngouana V, Zollo PH, Menut C, Bessiere and with new drugs that can be developed from natural JM, Gut J, Rosenthal PJ. 2003. Composition products. and anti-plasmodial activities of essential oils from some Cameroonian medicinal plants. ACKNOWLEDGMENTS Phytochemestry 64: 1269 - 1275. This work was supported by DI-Universidad Andres Cassels B, Urzúa A. 1985. Bisbenzylisoquinoline Bello. Grant N° DI-19-08/R and DID UACH-s-2005- alkaloids of Laurelia sempervirens. J Nat 41. PIIC 2009-2010, PAC 2011-2012 and DGIP of Prod 48: 671. Universidad Técnica Federico. Santa María for Céspedes CL, Marín JC, Domínguez M, Ávila JG, financial support. Serrato B. 2006. Plant growth inhibitory activities by secondary metabolites isolated REFERENCES from Latin American flora. Adv Phytomed 2: Abegaz V, Dagne E, Bates C, Waterman PG. 1989. 373 - 410. Chemistry of the Burseaceae. Part 12, Cheng SS, Wu HT, Chang YT, Kao S. 2004. monoterpene-rich resins from two Ethiopian Antitermitic and antifungal activities of species of Commiphora. Flavour Fragr J 4: essential oil of Calocedrus formosana leaf and 99 - 101. its composition. J Chem Ecol 30: 1957 - Adams RP. 2007. Identification of Essential Oil 1967. Components by Gas Chromatography/Mass CLSI (Clinical and Laboratory Standards Institute). Spectrometry. 4th Ed. Allured Publishing 2009. Method for dilution antimicrobial Corporation. Carol Steam, Illinois, USA. susceptibility tests for bacterial that grow Akers KS, Chaney C, Barsoumian. A, Beckius M, aerobically; approved standard. 8th Edition. ZeraW, Yu. X, Guymon C, Keen EF, CLSI document M07-A8, Wayne, PA, USA. Robinson BJ, Mende K, Murray C. 2010 Delaforge M, Koop D, Coon R, Minor J. 1982 “Role Aminoglycoside Resistance and susceptibility of isosafrole as complexing agent and inducer testing errors in Acinetobacter baumanni- of p-450lm4 in rabbit liver microsomes”. calcoaceticus complex. J Clin Microbiol 48: Biochem Biophys Res Comm 108: 59-65. 1132 - 1138. De Souza L, Damé L, Hörnke G, Ziemann M, Alves Bisht A, Bisht GRS, Singh M, Gupta R, Singh V. M, Araújo M. 2011. Evaluation of the 2011. Chemical composition and antimicrobial bactericidal action of essential oils from activity of essential oil of tubers of Cyperus guava, Surinamese cherry and strawberry rotundus Linn. collected from Dehradun guava. Rev Cub Plant Med 16: 324 - 330.

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Montenegro et al. Antimicrobial activity of Laurelia sempervirens against multiresistant bacteria

Diomedi A. 2005. Infecciones por Acinetobacter Serratia marcescens. Infect Immun 11: 4870 baumannii pan-resistente. Consideraciones - 4881. epidemiológicas y de manejo antimicrobiano McGowan JE Jr. 2006. Resistance in nonfermenting actualizado. Rev Chil Infect 22: 298 - 320. gram-negative bacteria: multidrug resistance Edrada RA, Wray V, Witte L, van Ofwegen L, to the maximum. Am J Med 119: 62 - 70. Proksch P. 2000. Bioactive terpenes from the Mølgaard P, Holler J, Asar B, Liberna I, Bakkestrøm soft coral Heteroxenia sp. from Mindoro, L, Ploug C, Jørgensen L, Lauritzen J, Guzman Philippines. Z Naturforsch 55: 82 - 86. A, Adsersen A, Toft Simonsen H. 2011. Fernandez-Cuenca F, Pascual A, Ribera A, Vila J, Bou Antimicrobial evaluation of Huilliche plant G, Cisneros JM, Rodriguez-Baño J, Pachón J, medicine used to treat wounds. J Martinez L. 2004. Diversidad clonal y Ethnopharmacol 138: 219 - 227. sensibilidad a los antimicrobianos de Morales G, Sierra P, Mancilla A, Paredes A, Loyola L Acinetobacter baumannii aislados en A, Gallardo O. Borquez J. 2003. Secondary hospitales españoles. Estudio multicéntrico metabolites from four medicinal plants from nacional: proyecto GEIH-Ab 2000. Enferm northern Chile: Antimicrobial activity and Infecc Microbiol Clin. 22: 267 - 271. biotoxicity against Artemia salina. J Chil Flach A, Gregel B, Simionatto E, da Silva UF, Zanatta Chem Soc 48: 13 - 18. N, Morel AF, Linares CE, Alves SH. 2002. NCCLS (National Committee for Clinical Laboratory Chemical analysis and antifungal activity of Standards). 2003. Methods for dilution the essential oil of Calea clematidea. Planta antimicrobial susceptibility tests for Med 68: 836 - 838. bacteria that grow aerobically. Approved González A, Alvarenga N, Ravelo A, Jiménez I, Standard - NCCLS document M7-A6, 6th Ed. Bazzocchi I, Canela N, Moujir L. 1996. Wayne, PA, USA. . Antibiotic phenol nor-triterpenes from Newman DJ, Cragg GM, Snader KM. 2000. The Maytenus canariensis. Phytochemistry 43: influence of natural products upon drug 129 - 132. discovery. Nat Prod Rep 17: 215 - 234. Harbarth S, Fankhauser C, Schrenzel J, Christenson J, Oztürk M, Duru ME, Aydoğmuş-Oztürk F, Harmandar Gervaz P, Bandiera-Clerc C, Renzi G, Vernaz M, Mahliçli M, Kolak U, Ulubelen A. 2009. N, Sax H, Pittet D. 2008. Universal screening GC-MS analysis and antimicrobial activity of for methicillin-resistant Staphylococcus essential oil of Stachys cretica subsp. aureus at Hospital Admission and nosocomial Smyrnaea. Nat Prod Commun 4: 109 - 114. infection in surgical patients. JAMA 10: 1149 Pichette A, Larouche PL, Lebrun M, Legault J. 2006. - 1157. Composition and antibacterial activity of Hejazi A, Falkiner FR. 1997. Serratia marcescens. J Abies balsamea essential oil. Phytother Res Med Microbiol 46: 903 - 912. 20: 371 - 373. Hussein MA, Wada S, HaTai K, Yamamoto A. 2000. Perazzi B, Camacho M, Bombicino K, Flores Z, Vay Antimycotic activity of eugenol against C, Famiglietti A. 2010. Staphylococcus selected water molds. J Aquat Anim Health aureus: nuevos y antiguos antimicrobianos. 12: 224 - 229. Rev Argent Microbiol 42: 199 - 202. Hufford C, Sharma A, Oguntimein B. 1980. Phutdhawong W, Kawaree R, Sanjaiya S, Sengpracha Antibacterial and antifungal activity of W, Buddhasukh, D. 2007. Microwave-assisted liriodenine and related oxoaporphine isolation of essential oil of Cinnamomum iners alkaloids. J Pharm Sci. 69: 1180 - 1183. Reinw. ex Bl: Comparison with conventional León L, Beltrán B; Moujir L. 2005. Antimicrobial hydrodistillation. Molecules 12: 868 - 877. activity of 6-oxophenolic triterpenoids. Mode Sánchez Y, Pino O, Correa T, Naranjo E, Iglesia A. of action against Bacilus subtilis. Planta Med 2009. Estudio químico y microbiológico del 71: 313 - 319. aceite esencial de Piper auritum Kunth Horng L, Yang J, Tsai Ch, Su Y, Wei L, Chen Ch, (caisimón de anís). Rev Protección Veg 24: Hsieh Ch, Lu S, Lai H. 2010. RssAB-FlhDC- 39 - 46. ShlBA as a major pathogenesis pathway in Schmeda-Hirschmann G, Loyola J, Rodríguez J, Dutra-Behrens M. 1994. Hypotensive effect of

Boletin Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas/314

Montenegro et al. Antimicrobial activity of Laurelia sempervirens against multiresistant bacteria

Laurelia sempervirens (Monimiaceae) on Antofagasta, Chile, Rev Chil Infect 21: 117 - normotensive rats. Phytother Res 8: 49 - 51. 124. Schmeda-Hirschmann G, Dutra-Behrens M, Habermeh Zapata M, Lognay G, Smagghe G. 2010. Bioactivity G, Jakupovic J. 1996. Seco-isotetrandrine of essential oil from leaves and bark of from Laurelia sempervirens. Phytochemistry Laurelia sempervirens and Drimys winteri 41: 339 - 341. against Acyrthosiphon pisum. Pest Manag Sci Seo EK, Kinghorn AD. 2000. In: Studies in Natural 66: 1324 - 1331. Products Chemistry; Atta-ur-Rahman, Ed.; Elsevier Science BV, Amsterdam, The Netherland. Simberkoff M, Rahal J jr. 1986. Bactericidal Activity of ciprofloxacin against Amikacin- and Cefotaxime-Resistant Gram-Negative Bacilli and Methicillin-Resistant Staphylococci. Antimicrob Agents Chemother 29: 1098 - 1100. Storti A, Pizzolitto AC, Pizzolitto EL. 2005. Detection of mixed microbial biofilms on central venous catheters removed from intensive care unit patients. Braz J Microbiol 36: 275 - 280. Tulp M, Bohlin L. 2004. Unconventional natural sources for future drug discovery. Drug Discov Today 9: 450 - 458. Uduman SA, Farrukhy AS, Nathz KNR, Zuhairz MYH, Ifrahy A, Khawlaz AD, Sunitaz P. 2002. An outbreak of Serratia marcescens infection in a special-care baby unit of a community hospital in United Arab Emirates: the importance of the air conditioner duct as a nosocomial reservoir. J Hosp Infection 52: 175 - 180 Valgas C. Machado de Souza S, Smania E, Smania A. 2007. Screening methods to determine antibacterial activity of natural products. Braz J Microbiol 38: 369 - 380. Van Eldere J. 2003. Multicenter surveillance of Pseudomonas aeruginosa susceptibility patterns in nosocomial infections. J Antimicrob Chemother 51: 347 - 352. Van den Berghe DA, Vlietinck AJ. 1991. Screening methods for antibacterial and antiviral agents from higher plants. In Methods in Plant Biochemistry, Assays for Bioactivity, Hostettman K, Ed., Academic Press: London, San Diego, New York, Boston, Sydney, Tokyo and Toronto. Zambrano AF, Herrera N. 2004. Susceptibilidad antimicrobiana de cepas de Pseudomonas aeruginosa aisladas en el laboratorio del Hospital Regional Dr. Leonardo Guzmán de

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