Effect of the Essential Oil of Minthostachys verticillata (Griseb.) Epling and Limonene on Biofilm Production in Pathogens Causing Bovine Mastitis.

Bovine mastitis causes large annual economic losses around the world. Different microorganisms are associated with the disease. The capacity of pathogens to adhere to bovine mammary epithelial cells is associated with biofilm production which leads to antibiotic resistance. Research is now leading to search alternative control methods and medicinal plants constitute a natural, safe, effective and inexpensive option. Minthostachys verticillata is an autochthonous medicinal plant of Argentina with multiple ethnobotanical properties. In a previous study, we demonstrated that the essential oil (EO) of this species and limonene, one of its compounds, inhibited the growth of mastitis pathogens. The objective of the present work was to determine the inhibitory effect of the essential oil of M. verticillata and limonene, on biofilm formation and on mature biofilm produced by pathogens isolated from bovine mastitis. Time kill assay and bacterial lysis were also determined. Furthermore, RAPD-PCR assays were performed to determine changes in bacterial DNA after EO and limonene exposition. Bacterial isolates were identified as Escherichia coli (EC3 and EC9), Bacillus pumilus (BP5, BP6, and BP7) and Enterococcus faecium (EF1) by rRNA 16S sequencing and MALDI-TOF MS. All the strains were able to form biofilm. Addition of both lactose and sucrose did not affect biofilm production. MIC values for EO were 3.6 mg/ml for E. faecium; 0.9 mg/ml for E. coli (EC3), 14.5 mg/ml for E. coli (EC9), 1.8 mg/ml for B. pumilus (BP7), 3.63 mg/ml for B. pumilus (BP6) and 29.0 mg/ml for B. pumilus (BP7). MIC values for limonene were 6.6 mg/ml for B. pumilus (BP6) and 105 mg/ml for B. pumilus (BP5). These results demonstrated that EO was more effective than limonene, showing also bactericidal action against E. faecium (minimal inhibitory concentration (MBC) = 29.0 mg/ml). This result was corroborated by time of death assay, observing a cell decrease after at 6 h, and then by bacterial lysis assay. Both EO and limonene affected mature biofilm of isolated strains. The results contribute to the study of EO and limonene which may serve as a therapy against bovine mastitis pathogens inhibiting the development of pathogenic bacteria.

Evaluation of the antimicrobial efficacy of Minthostachys verticillata essential oil and limonene against Streptococcus uberis strains isolated from bovine mastitis.

Bovine mastitis is a disease that causes great economic losses per year, being Streptococcus uberis the main environmental pathogen involved. The aim of the present study was to determine the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of Minthostachys verticillata essential oil and limonene for S. uberis strains isolated from bovine mastitis. In addition, the effect of MIC on biofilm formation was analyzed. MIC values for the essential oil ranged from 14.3 to 114.5mg/ml (1.56-12.5%v/v) and MBC between 114.5 and 229mg/ml (12.5-25%v/v). MICs for limonene ranged from 3.3 to 52.5mg/ml (0.39-6.25%v/v) and MBC was 210mg/ml (25%v/v). Both compounds showed antibacterial activity and affected the biofilm formation of most of the strains tested. In conclusion, these compounds could be used as an alternative and/or complementary therapy for bovine mastitis caused by S. uberis.

Evaluación de la eficacia antimicrobiana del aceite esencial de Minthostachys verticillata y limoneno contra cepas de Streptococcus uberis aislados de mastitis bovina / Evaluation of the antimicrobial efficacy of Minthostachys verticillata essential oil and limonene against Streptococcus uberis strains isolated from bovine mastitis

Bovine mastitis is a disease that causes great economic losses per year, being Streptococcus uberis the main environmental pathogen involved. The aim of the present study was to determine the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of Minthostachys verticillata essential oil and limonene for S. uberis strains isolated from bovine mastitis. In addition, the effect of MIC on biofilm formation was analyzed. MIC values for the essential oil ranged from 14.3 to 114.5 mg/ml (1.56-12.5% v/v) and MBC between 114.5 and 229 mg/ml (12.5-25% v/v). MICs for limonene ranged from 3.3 to 52.5 mg/ml (0.39-6.25% v/v) and MBC was 210 mg/ml (25% v/v). Both compounds showed antibacterial activity and affected the biofilm formation of most of the strains tested. In conclusion, these compounds could be used as an alternative and/or complementary therapy for bovine mastitis caused by S. uberis.

La mastitis bovina es una enfermedad que causa grandes pérdidas económicas por año, Streptococcus uberis es el principal patógeno ambiental involucrado. El objetivo del presente estudio fue determinar la concentración inhibitoria mínima (CIM) y la concentración bactericida mínima (CBM) del aceite esencial de Minthostachys verticillata y del limoneno sobre cepas de S. uberis aisladas de mastitis bovina. Además, se analizó el efecto del aceite esencial y el limoneno en la CIM determinada en caso sobre la formación de biofilm de estas cepas. Los valores de CIM del aceite esencial oscilaron entre 14,3 y 114,5 mg/ml (1,56%-12,5% v/v) y los de CBM entre 114,5 y 229 mg/ml (12,5%-25% v/v). Las CIM del limoneno oscilaron entre 3,3 y 52,5 mg/ml (0,39% - 6,25% v/v) y la CBM fue de 210 mg/ml (25% v/v). Ambos compuestos mostraron actividad antibacteriana y afectaron la formación de biofilm de la mayoría de las cepas. En conclusión, estos compuestos podrían ser utilizados como terapia alternativa o complementaria para la mastitis bovina causada por S. uberis.

Evaluation of cytogenotoxic effects of cold aqueous extract from Achyrocline satureioides by Allium cepa L test.

Achyrocline satureioides ("marcela del campo") is native to America. Numerous investigations have reported several bioactive properties such as anti-inflammatory, hepatoprotective, immunomodulatory, antimicrobial and antiviral. Nowadays, few medicinal plants have been scientifically evaluated to test its safety, efficacy and potential benefits, despite the great public interest in these herbs. The aim of this work was to evaluate the cytotoxic and genotoxic activities of cold aqueous extract obtained from A. satureioides using Allium cepa L test. The results demonstrated the absence of genotoxicity of the extract. Only higher concentrations induced cytotoxicity but interestingly this effect was reversible and was not associated with mutagenicity. The contribution of this research provides assurance of safety in the application of Achyrocline satureioides in treatment of microbial diseases and other pathologies helping to define selective toxicity.