Activity of essential oils from spices against Staphylococcus spp. isolated from bovine mastitis

Atividade de óleos essenciais de plantas condimentares frente Staphylococcus spp. isolados de mastite bovina

medicinal plants; phytotherapy; antimicrobials; plantas medicinais; fitoterapia; antimicrobianos

COMMUNICATION

Activity of essential oils from spices against Staphylococcus spp. isolated from bovine mastitis

Atividade de óleos essenciais de plantas condimentares frente Staphylococcus spp. isolados de mastite bovina

M. Dal Pozzo^I; D.F. Santurio^II; L. Rossatto^II; A.C. Vargas^III; S.H. Alves^II; E.S. Loreto^II; J. Viegas^I

^IAluno de pós-graduação - Universidade Federal de Santa Maria, Santa Maria, RS

^IIUniversidade Federal de Santa Maria, Santa Maria, RS

^IIIUniversidade Federal de Santa Maria, Santa Maria - Campus/Camobi, Pr

Keywords: medicinal plants, phytotherapy, antimicrobials

RESUMO

Avaliou-se a atividade antimicrobiana dos óleos essenciais (OE) de Origanum vulgare (orégano), Thymus vulgaris (tomilho), Lippia graveolens (lipia), Zingiber officinale (gengibre), Salvia officinalis (sálvia), Rosmarinus officinalis (alecrim) e Ocimum basilicum (manjericão), e de suas frações majoritárias, carvacrol e timol, frente a 32 isolados de Staphylococcus spp, oriundos de rebanhos leiteiros bovinos. A concentração inibitória mínima (CIM) e a concentração bactericida mínima foram determinadas por meio da técnica de microdiluição em caldo. Orégano, tomilho e lípia (Orégano Mexicano) apresentaram atividade antimicrobiana similar, médias geométrica de CIM de 1600µg mL^-1; 1564µg mL^-1; 1562µg mL^-1, respectivamente, no entanto menos ativos que carvacrol, 584µg mL^-1 e thymol, 427µg mL^-1. Isolados com diferentes perfis de susceptibil idade aos antimicrobianos usados no tratamento de mastite bovina, quando subagrupados, foram inibidos por concentrações semelhantes de OE . Estes resultados confirmam a atividade antimicrobiana de OE e algumas frações majoritárias.

Palavras-chave: plantas medicinais, fitoterapia, antimicrobianos

Worldwide, economic losses due to mastitis have been estimated at $35 billion (Wellenberg et al., 2002). Staphylococcus spp. is the main causative agent of bovine mastitis, with higher prevalence in cases of clinical and subclinical manifestations (Fagundes et al., 2010). The most common treatment is based on intramammary infusion of antibacterial agents. However, cure rates obtained with such drugs are not always effective, because it may determine the emergence of resistant bacteria (Zafalon et al., 2007) as well increase amounts of antibiotic residues in milk (Fagundes et al., 2010). Nevertheless, the treatment of bovine subclinical mastitis caused by S. aureus in the lactation can be economically unviable (Zafalon et al., 2007).

Alternative treatments to bovine mastitis with bacteriocins (Pieterse et al., 2010) and plant derived compounds (Baskaran et al., 2009; Mubarack et al., 2011) have been described. Essential oils (EO) are classified as GRAS (generally regarded as safe), show antibacterial proprieties and resistance has not been reported after prolonged exposure. Then, the investigation of their antimicrobial activity against bacterial agents of mastitis is justifiable. In this context, this study aimed to: a) evaluate the in vitro antimicrobial activity of EO from spices against Staphylococcus spp.; b) compare the activity of EO against Staphylococcus spp.isolates with multiple profiles of susceptibility and resistance to penicillin, erithromicin and tetracycline antibiotics.

Thirty two isolates from bovine mastitis, from the Laboratório de Bacteriologia Veterinária, Universidade Federal de Santa Maria, Brazil, were studied. The isolates were identified as coagulase positive Staphylococcus spp. The standard strain S. aureus ATCC 29213 was employed as reference strain. The antimicrobial susceptibility tests were performed according to CLSI M31-A3 agar diffusion method (CLSI, 2008). Based on these tests, the isolates were divided into subgroups according to the following resistance profiles: 1) susceptible; 2) penicillin-resistant; 3) tetracycline and penicillin-resistant; 4) erythromycin and tetracycline-resistant, and 5) erythromycin, penicillin and tetracycline-resistant.

The EO were purchased from Essential7.com Roewell, NM, USA. The majority constituents were previously determined by Pozzatti et al. (2010).

The essential oil of Origanum vulgare (oregano) contains 92% of carvacrol; Thymus vulgaris (thyme), 64% of γ-terpinene; Lippia graveolens (Mexican oregano), 56.8% of carvacrol and 32.2% of o-cymene; Rosmarinus officinalis L. (rosemary), 28.6% of 1,8-cineole and 26.31% of camphor; Salvia officinalis L. (sage), 40.6% of cis-thujone; Ocimum basilicum L. (Basil), 32.2% of linalool and 23.6% of 1,8-cineole; Zingiber officinale (ginger), 20.8% of zingiberene. The majority constituents were purchased from Acros Organics, Fair Lawn, NJ, USA.

Carvacrol (99.5% carvacrol), thymol (thymol 99.0%) and cineole (cineole 97.0%) EO were first diluted with methanol to achieve the concentration of 640mg/mL (Solution I) and then diluted to 1:100 in Mueller-Hinton broth, resulting in the concentration of 6,400µg mL^-1 (Solution II).

The minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined based on the document M31-A3 (CLSI, 2008). Briefly, the inoculum was prepared from cultures grown in Muller-Hinton agar, resulting in a bacterial suspension in saline equivalent to the 0.5 McFarland standard (1x108 CFU mL^-1). The solution II was distributed into wells of a microtiter plate and serial dilutions were prepared to obtain final concentrations from 6,400 to 100µg mL^-1. Inoculum volumes of 10µL were distributed in each well containing EO incorporated into Mueller-Hinton broth. The MICs were determined after incubation at 35ºC/24h under aerobic conditions. These tests were performed in triplicate. To determine the MBC, volumes of 10µL from wells without visible bacterial growth after 24h incubation were transferred to the surface of Mueller-Hinton agar, which was incubated at 35ºC/24h. Then, bacterial growth was observed, registering the lowest concentration of EO or majority compounds that did not show bacterial growth. These tests were performed in triplicate.

The Mann Whitney test was used to compare two independent samples in order to observe whether the different study groups had similar patterns of susceptibility or not.

The geometric means (GM) of the MICs and MBCs of EO against Staphylococcus spp. were [GM-MIC/GM-MBC]: Origanum vulgare [1600/2288µg mL^-1], Lippia graveolens [1562/2707µgmL^-1], Thymus vulgaris [1564/2370µg mL^-1], carvacrol [584/732µg mL^-1] and thymol [427/856µg mL^-1]. In this study, ginger, basil, rosemary and sage EO, and the major compound cineole showed no antibacterial activity at the tested concentrations against the microorganisms evaluated. The GM of MICs and MBCs indicated that oregano, thyme and Mexican oregano EO were equally active (P>0.05) but less active than carvacrol and thymol (P<0.001) (Table 1). Based on the antimicrobial susceptibility, significant differences were not observed between susceptibilities profile of subgroups (Table 2).

The remarkable antimicrobial activity of O. vulgare, L. graveolens and T. vulgaris against Staphylococcus spp. from multiple sources has been previously confirmed (Smith-Palmer et al., 1998; Hoferl et al., 2009). These studies reported significant antimicrobial activity of oregano EO against Gram positive and Gram negative bacteria, including Staphylococcus aureus. Arana-Sanchez et al. (2010) showed antimicrobial activity of L. graveolens against E. coli ATCC 11229 and Staphylococcus species. The phenolic compounds carvacrol and thymol as major constituents ensure such activities. This activity had also been reported against sensible and resistant yeasts to fluconazole (Pozzatti et al., 2010).

Rosemary, sage, basil and ginger EO, and the major constituent cineole did not show antimicrobial activity at the studied concentrations. However, Viuda-Martos et al. (2008) and Delamare et al. (2007) reported antimicrobial activity of sage and rosemary EO against Staphylococcus spp., Enterobacter gergoviae, E. amnigenus, Lactobacillus sakei, and Lactobacillus curvatus. The disparity between the results of the present study and those reported by these authors can be attributed to the composition of the EO or the techniques employed. The lack of an internationally standardized technique to evaluate antimicrobial activity of EO and plant extracts invalidate the comparison of some studies.

In this study, the antimicrobial activities of EO against the subgroups of Staphylococcus species with different antibiotic susceptibility profiles were partially independent of the pattern of antibacterial resistance which is in agreement with other authors. Mahboubi et al. (2010) reported that the Zataria multiflora EO - Iranian condiment similar to thyme) consisting of thymol (38%), carvacrol (15.3%) and p-cymene (10.2%), showed similar antimicrobial activity against methicillin resistant Staphylococcus aureus as well to sensitive susceptible stains. Against fluconazole-sensible and resistant Candida spp., Pozzatti et al. (2010) reported significant antifungal activities of EO regardless of the previous susceptibility of those yeasts to antifungal agents.

The finding that the activities of oregano, thyme and Mexican oregano EO, and majority constituents carvacrol and thymol are independent of resistance to antibacterials tested, reinforces the status of EO as alternative candidates to be considered in the therapy of bovine mastitis.

In conclusion, this study demonstrated that essential oils of oregano, thyme, Mexican oregano, as well as the major constituents thymol and carvacrol showed antimicrobial activity against Staphylococcus spp. and that the EO of oregano, thyme and Mexican oregano exhibited similar antimicrobial activities (MICs and MBCs), but lower than major constituents thymol and carvacrol. The activities of EO were similar against subgroups of isolates, independently of the resistance to antimicrobials used for treatment of mastitis.

Recebido em 15 e junho de 2011

Aceito em 1 de setembro de 2011

E-mail: [e-mail]

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Publication Dates

• Publication in this collection
  16 Nov 2011
• Date of issue
  Oct 2011

History

• Received
  15 June 2011
• Accepted
  01 Sept 2011