ARTÍCULOS

Antibacterial activity of avocado extracts (Persea americana Mill.) against Streptococcus agalactiae

Actividad antibacteriana de extractos de aguacate (Persea americana Mill.) sobre Streptococcus agalactiae

 

Cardoso PF¹, JA Scarpassa¹, LG Pretto-Giordano², ES Otaguiri³, SF Yamada-Ogatta³, G Nakazato³, MRE Perugini⁴, IC Moreira⁵, GT Vilas-BÔas¹*

¹ Departamento de Biologia Geral, Centro de Ciências Biológicas, Universidade Estadual de Londrina.
² Departamento de Medicina Veterinária Preventiva, Centro de Ciências Agrárias, Universidade Estadual de Londrina.
³ Departamento de Microbiologia, Centro de Ciências Biológicas, Universidade Estadual de Londrina.
⁴ Departamento de Patologia, Análises Clínicas e Toxicológicas, Centro de Ciências da Saúde, Universidade Estadual de Londrina.
⁵ Universidade Tecnológica Federal do Paraná- Campus Londrina, Brazil.

Address correspondence to: Gislayne Trindade Vilas-BÔas, e-mail: gvboas@uel.br

Received 17.X.2014.
Accepted 17.IV.2015.

 

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Abstract. Plants contain numerous constituents and are valuable sources of new biologically active molecules. Avocado (Persea americana Mill.) is cultivated and used as food in most tropical and subtropical countries. Its high nutritional value and biological activities, as antioxidant, antimicrobial and analgesic properties, have been thoroughly investigated. Interest in plant extracts with antimicrobial properties has increased as a result of the indiscriminate use of antibiotics, leading to the emergence of resistant bacterial strains. Among bacterial species with clinical importance to multiple hosts, Streptococcus agalactiae is outstanding, as it can cause infections especially in humans, fsh and cattle. Te current study aimed to evaluate the antimicrobial activity of two extracts (ethanol and dichloromethane) from avocado seeds, ‘Margarida’ variety, against isolates of S. agalactiae. Extracts were diluted in ethanol / water (1:1) at a concentration of 100 mg/mL. Antimicrobial activity was tested by the disk difusion method (antibiogram) against isolates of S. agalactiae of human and fsh origin.Te ethanol extract showed antimicrobial activity only for some isolates of S. agalactiae of human origin. Te dichloromethane extract showed activity against all isolates of S. agalactiae of both origins. A comparison of the results obtained with dichloromethane extract from isolates of S. agalactiae of human or fsh origin demonstrated the existence of phenotypic variability among isolates from the same host. However, when comparing measurements obtained in each of the groups, they were statistically similar, showing a lack of interpopulation variability. Tus, it can be verifed that the resistance profle of isolates of S. agalactiae was independent of host origin and typical of the species.

Keywords: Plant extracts; Disk difusion method.

Resumen. Las plantas contienen numerosos constituyentes y son fuentes ricas de nuevas moléculas biológicamente activas. El aguacate (Persea americana Mill.) es cultivado y utilizado como alimento en la mayoría de los países tropicales y subtropicales, ya que tiene alto valor nutricional, y sus actividades biológicas han sido muy investigadas, entre las cuales la actividad antioxidante, analgésica o antimicrobiana. El interés en extractos vegetales con propiedades antimicrobianas se ha intensifcado como consecuencia de la utilización indiscriminada de antibióticos, que llevó a la selección de cepas bacterianas resistentes. Entre las especies bacterianas de relevancia clínica para variados hospederos, se puede destacar la bacteria Streptococcus agalactiae, que puede causar infecciones, principalmente en humanos, peces y ganado. El objetivo de ese trabajo fue evaluar la actividad antimicrobiana de dos extractos (etanólico y diclorometanico) de hueso de aguacate variedad margarita en relación a aislados de S. agalactiae. Los extractos fueron resuspendidos en etanol/agua en la concentración de 100 mg/mL. La actividad antibacteriana de los extractos fue comprobada usando el método de difusión en discos frente a aislados de S. agalactiae de origen humano y peces. El extracto etanólico presentó actividad antimicrobiana solamente para algunos aislados de S. agalactiae de origen humano. El extracto diclorometanico presentó actividad antimicrobiana para todos los aislados de S. agalactiae de ambos orígenes. La comparación de los resultados obtenidos con el extracto diclorometanico enfrente a los aislados de S. agalactiae de origen humano y peces mostró la existencia de variabilidad fenotípica entre aislados del mismo hospedero. Sin embargo, la comparación de las medias obtenidas en cada uno de los grupos fue estadísticamente semejante, demostrando la ausencia de variabilidad interpoblacional. De esta manera, se pudo observar que el perfl de resistencia de aislados de S. agalactiae fue independiente del hospedero de origen y característico de la especie.

Palabras clave: Extractos vegetales; Método de difusión en disco.

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INTRODUCTION

Te control of bacterial infections is mostly carried out with antibiotics. However, the emergence of resistant bacterial strains has become more frequent, leading to the need of new sources of molecules with antimicrobial activity, which have been found mainly in microorganisms and plants (Cowan, 1999; Mlynarczyk et al., 2010). Natural plant products have been used since ancient times for medicinal purposes as they comprise numerous components and valuable sources of new biologically active molecules (Cowan, 1999; Gupta et al., 2004).
Many plants synthesize antimicrobial secondary metabolites as part of their normal growth and development, often keeping them in tissues that need protection against microbial attack (Gupta et al., 2004). Te antimicrobial activity of plant extracts may reside in a variety of diferent phytochemical constituents, namely terpenoids, essential oils, alkaloids, lectins, polypeptides and polyphenolics and phenolic substances (simple phenols, phenolic acids, quinones, favones, favonols and favonoids, tannins and coumarins) (Gonçalves et al., 2005). Te antibacterial activity of these extracts may be ascribable to the combined efects of the polyphenols adsorption on bacterial membrane, leading to its rupture and subsequent leakage of cellular content, and the generation of hydroperoxides (Negi, 2012).
Among plants, avocado (Persea americana Mill), originated from Central America, presents a high nutritional value and is cultivated and used as food in most tropical and subtropical countries. Its peel, fruit and leaves are commonly used in America, Antilles and Africa for the treatment of various diseases such as menorrhagia, hypertension, stomach pain, bronchitis, diarrhea and diabetes (Adeyemi et al., 2002). However, avocado seeds are usually discarded during consumption or industrial processes generating residues that could be an economical alternative for treatment of some diseases.
Te avocado leaf, stem, fruit and peel have biological activities scientifcally proven (Miranda et al., 1997; Adeyemi et al., 2002; Quing-Yi et al., 2005; Gomez-Flores et al., 2008; Castro et al., 2010; Rodríguez-Carpena et al., 2011). Studies with seed demonstrated antioxidant activity and antimicrobial activity against Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, Pseudomonas spp. and Yarrowia lipolytica. Te Gram-positive bacteria are more sensitive than Gram-negative bacteria (Rodríguez-Carpena et al., 2011). Other seed properties already studied are larvicidal (in Aedesaegypti), antifungal (Candida spp., Cryptococcus neoformans and Malassezia pachydermatis) (Leite et al., 2009) and antimicrobial activities against several species including S. aureus, Enterococcus faecalis, Salmonella Enteritidis, Citrobacter freundii, Pseudomonas aeruginosa, Salmonella Typhimurium, Enterobacter aerogenes and Zygosaccharomyces bailii (Chia & Dykes, 2010).
Phytochemical studies of the avocado seed allowed the identifcation of several classes of active compounds such as favonoids, anthocyanins, condensed tannins, alkaloids and triterpenoids in methanolic extracts, while sterols and triterpenes were detected in the hexane extract (Leite et al., 2009).
Several bacterial species are considered of clinical importance because they cause a number of diseases in various hosts. Among these, the species Streptococcus agalactiae, a Gram positive, catalase negative and facultatively anaerobic bacteria is remarkable.Tis species can cause infections in cattle, humans and fsh. Furthermore, it can occasionally infect mice, cats, dogs, camels and frogs (Elliot et al., 1990; Figueiredo, et al., 2006; Pereira et al., 2010).
Streptococcus agalactiae is one of the most common causes of perinatal bacterial infections in humans. It is also an opportunistic pathogen of the elderly and immunocompromised people, and may cause pneumonia, meningitis, bacteremia and skin or soft tissue infections (Gibbs et al., 2004; Nakamura et al., 2011). Penicillin is the treatment of choice. However, for patients allergic to β-lactam, erythromycin or clindamycin are prescribed. Mammal isolates are preferably β-hemolytic, but some nonhemolytic have been isolated, and are usually cultivated at 37 °C (Evans et al., 2002; Gibbs et al., 2004).
Besides humans, S. agalactiae can infect freshwater and marine fsh, either in fsh farming or free in the environment (Figueiredo et al., 2006). It is considered the main pathogenic bacteria of diferent species of fsh with high mortality. Naturally or experimentally infected fsh exhibit symptoms, such as unilateral or bilateral exophthalmia, corneal opacity, erratic swimming, changes in skin color, skin lesions and ascites (Figueiredo et al., 2006; Pretto-Giordano et al., 2010a). Fish isolates of S. agalactiae are usually not hemolytic and are cultivated at 30°C, which may indicate phenotypic adaptations to host (Elliot et al., 1990; Evans et al., 2002; Castro et al., 2008).
S. agalactiae isolates of human source present resistance to tetracycline, clindamycin, erythromycin, chloramphenicol, rifampicin, norfoxacin, levofoxacin, ciprofoxacin, moxifoxacin (Borger et al., 2005; Correa et al., 2011; Nakamura et al., 2011; Ki et al, 2012; Usein et al., 2012). Fish isolates may be resistant to nalidixic acid, gentamicin, neomycin, norfoxacin and streptomycin (Evans et al., 2002; Figueiredo et al., 2006).
Te susceptibility of S. agalactiae to natural extracts was analyzed in diferent works. According to Cueva et al. (2012), S. agalactiae presented sensitivity to phenolic compounds isolated from wine, epicatechin and gallic acid, and was not sensitive to oenological extracts. It was also sensitive to extracts of wild mushrooms (Alves et al., 2012) and to the essential oil from eight eucalyptus species (Elassi et al., 2012). Leaf extracts of Calyptranthes clusiifolia, Croton foribundus, Heisteria silvianii, Merremia tomentosa and Zanthoxylum riedelianum also inhibited S. agalactiae growth (Castro et al., 2008).
Tus, the aim of this study was to investigate the antibacterial activity of avocado (P. americana Mill) seed extracts against S. agalactiae isolates of human and fsh origin. Terefore, comparison of intra- and inter-population variability of resistance profles was evaluated and indicated the potential therapeutic use of the avocado seed against this bacterial species.

MATERIALS AND METHODS

Plant extracts. In order to obtain seed extracts of avocado (P. americana Mill., ‘Margarida’ variety), the seed was initially separated from the pulp, fragmented, dried and ground into powder. Te seed powder was then exposed to a maceration process for a period of seven days, either using ethyl alcohol as solvent, resulting in an extract termed “ethanolic extract”, or using dichloromethane as solvent, yielding an extract termed “dichloromethane extract”. Subsequently, the extracts were fltered and concentrated in a rotary evaporator. Procedures of maceration, fltration and concentration were repeated once more with both extracts. In order to measure the efciency of extraction, the obtained extracts were weighed and the ratio between 500g of the initial seed powder and the fnal weight calculated. Extracts were dissolved in ethanol / water (1:1), stored at room temperature and protected from light until use.

Bacterial strains and culture conditions. Te evaluation of 29 S. agalactiae isolates recovered from vaginal-rectal swabs and urine of female patients at the University Hospital of Universidade Estadual de Londrina (originally used by Otaguiri et al., 2013) was performed. Tese isolates had already been characterized for bacterial species confrmation by phenotypic tests (CAMP, KEA, NaCl, hippurate, bacitracin, trimethoprim-sulfamethoxazole, Gram staining and catalase). Tese isolates were incubated for 24 hours at 37 °C in Muller Hinton blood agar plates (supplemented with 5% sheep blood).
Te assessment of 26 isolates of S. agalactiae obtained from the Nile tilapia (Oreochromis niloticus) with bacterial infection symptoms was conducted. Te isolates were collected from diferent organs, including eyes, brain, liver, heart, blood, visceral fuid and kidney fsh collected at fsh farming properties located in the northern region of Paraná state and northwest region of São Paulo state, Brazil.Te strains had been previously identifed as S. agalactiae by Gram stain and biochemical assays, and confrmed by more accurate tests, such as API 20 Strep Microtest (BioMerieux) and SlidexStrepto-kit (BioM-erieux) (Pretto-Giordano et al., 2010b). Tese isolates were incubated for 48 hours at 30 °C in Muller Hinton blood agar plates.

Antibiograms. Te antibacterial activity of avocado seed extracts against S. agalactiae was evaluated by the Disc diffusion method on Muller Hinton blood agar plates, as recommended by CLSI (Clinical Laboratory Standard Institute, 2010). For this purpose, bacteria concentration followed the 0.5 MacFarland scale, yielding an inoculum density of approximately 10⁸ CFU/mL (Ostrosky et al., 2008) which was homogeneously distributed over the plates using sterile swabs.
Discs of 6 mm diameter (Laborclin, Brazil) received the application of 10 µL of 100 mg/mL ethanol or dichloromethane extracts. Additionally, other discs received 10 µL of solvents and were used as a negative control. All discs were kept for an hour under a laminar fow for solvent evaporation (Ostrosky et al., 2008).
Biplates were used, forming a duplicate of each isolate per plate. Tree discs were placed on each plate side: control, ethanol and dichloromethane extract. In other words, two disks were tested for each extract per strain. Samples of human source were incubated at 37 °C for 24 hours. Strains of fsh origin were maintained at 30 °C for 48 hours. At the end of this time, the inhibition zone diameter was measured.

Statistical analysis. Te susceptibility test results were analyzed using the Analysis of Variance (ANOVA) followed by the Tukey test or the Mann-Whitney test for interpopulation analysis, at 95% confdence level. Tests were performed with the GraphPad InStat program, version 3.05.

RESULTS

After the extraction procedures, the fnal weight of extracts was 12.76 g for the ethanolic and 7.48g for the dichloromethane extract. Bacterial inhibition by extracts was evaluated visually by measuring the inhibition zone diameters around disks (disk diameter included) recorded in millimeters. Te antimicrobial activity was classifed into three levels: low activity (inhibition zone ≤12 mm), moderate activity (inhibition zone between 12 and 20 mm) and strong activity (inhibition zone ≥20 mm), following the criteria adopted in other studies with plant extracts (Rota et al., 2008; Fei et al, 2011).
Antibiogram results of S. agalactiae isolates are shown in Table 1 and exemplifed in Figures 1 and 2. Both human and fsh isolates showed statistical variability in intra-group analysis, exhibiting an inhibition zone between 7 mm e 13 mm for human isolates, and between 9 mm and 12 mm for fsh isolates.

Table 1. Antimicrobial activity of avocado seed extracts against S. agalactiae strains.
Tabla 1. Actividad antimicrobiana de extractos de semilla de aguacate contra cepas de S. agalactiae.

a: statistically differs from strain 14; b: statistically differs from strain 52; c: statistically differs from strain 40; d: statistically differs from strain 42; e: statistically differs from strain 48 (P<0.05).
a: difiere estadísticamente de la cepa 14; b: difiere estadísticamente de la cepa 52; c: difiere estadísticamente de la cepa 40; d: difiere estadísticamente de la cepa 42; e: difiere estadísticamente de la cepa 48 (P<0,05)

Fig. 1. Antibiogram of S. agalactiae isolates of human source: a) strain 6, b) strain 14 and c) strain 25. Disc 1: ethanol/water control; Disc 2: ethanolic extract (100 mg/mL); Disc 3: dichloromethane extract (100 mg/mL).
Fig. 1. Antibiograma de aislados humanos de S. agalactiae: a) cepa 6, b) cepa 14 y c) cepa 25. Disco 1: control etanol/agua; Disco 2: extracto etanólico (100 mg/mL); Disco 3: extracto diclorometanico (100 mg/mL).

Fig. 2. Antibiogram of S. agalactiae isolates of fish source: (a) strain 26, (b) strain 34 and (c) strain 55. Disc 1: ethanol/water control; Disc 2: ethanolic extract (100 mg/mL); Disc 3: dichloromethane extract (100 mg/mL).
Fig. 2. Antibiograma de aislados de peces de S. agalactiae: (a) cepa 26, (b) cepa 34 y (c) cepa 55. Disco 1: control etanol/agua; Disco 2: extracto etanólico (100 mg/mL); Disco 3: extracto diclorometanico (100 mg/mL).

For the intergroup analysis, the average of inhibition zones obtained for each group (human and fsh origin) was compared. Statistical analysis for ethanolic extract could not be performed, since inhibition zones on plates with fsh isolates were not observed. However, diferences in susceptibility between strains of human and fsh could be observed, given that the frst show some susceptible isolates, while in the latter, no susceptible isolates were found (Table 1). Te antimicrobial activity of the ethanolic extract, when present, was considered weak, with an inhibition zone between 7 mm and 9.5 mm.
Te mean ± standard deviation of the inhibition zone diameter for the isolates of human origin observed for the dichloromethane extract was 10.93 ± 0.62 mm. On the other hand, fsh isolates presented a mean ± standard deviation of 10.61 ± 0.56 mm. Te comparison between means was not statistically signifcant, with p = 0.0897. Te dichloromethane extract antibacterial activity was considered weak.

DISCUSSION

Plant extracts are sources of a variety of biotechnology products. Terefore, countless studies have been conducted in order to evaluate characteristics of these extracts, which can be used for the treatment of diseases, due to their antimicrobial, antifungal, analgesic, anti-infammatory and antitumor activities (Miranda et al., 1997; Adeyemi et al., 2002; Qing-Yi et al., 2005; Leite et al., 2009; Rodríguez-Carpena et al., 2011). Among the commonly evaluated properties, the antimicrobial activity has received special attention, and numerous studies have been conducted, including diferent avocado extracts (Gomez-Flores et al., 2008; Castro et al., 2010; Chia & Dykes, 2010; Rodríguez-Carpena et al., 2011).
However, although widely used, there are not yet any standardization methods to analyze the antimicrobial activity of extracts of natural products (Ostrosky et al., 2008). Te Disk difusion test is indicated by the FDA (Food and Drug Administration / USA) and established as standard by the CLSI (Clinical Laboratory Standard Institute / USA, 2010), and, therefore, was the method chosen to conduct this study.
Several S. agalactiae isolates of human and fsh origin were used in this work, aiming to comprise diferent phenotypic variations found in isolates from each of the two sources, as well as verify what kind of host presents isolates more susceptible to the evaluated extracts.
Human source isolates used in this study have already been analyzed for capsular type, genotyping by MLVA, antibiotics susceptibility and genetic virulence determinants. Te results suggest that even commensal S. agalactiae isolates have high potential for virulence and are susceptible to most antimicrobial agents tested (penicillin, ampicillin, vancomycin, etc.) (Otaguiri et al., 2013). However, they presented moderate resistance to erythromycin (19%) and clindamycin (13%) which demands the search for new treatment alternatives, especially for patients allergic to β-lactam antibiotics.
Te diference in efciency of the two extracts can be explained by the diference in polarity of solvents. During the extraction process, polarity infuences solubility of the main active substance, leading to diference in their chemical composition and consequently, in their biological activity (Idris et al., 2009). Te yield of extraction and concentration of the extract solution can also intervene in the results.
Te antimicrobial activity of avocado extracts may be ascribable to its chemical composition. Phytochemical screening highlighted the presence of phenolic compounds in avocado tissues, whose antimicrobial activity is well documented (Idris et al., 2009; Rodriguez-Carpena et al., 2011).
Avocado seed extracts showed low antimicrobial activity against of S. agalactiae isolates. Tis can probably be overcome by increasing extract concentration. Te results indicate that the avocado seed is a potential source of antimicrobial substances and arouses considerable interest in new research with more purifed extracts for the identifcation of compounds responsible for the antimicrobial activity.

ACKNOWLEDGEMENTS

Tis work was supported by grants from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científco e Tecnológico (CNPq).

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