Abstract
The emergence of resistance to antibiotics has been favored by abuse in the application of antimicrobials in human and animal medicine. Essential oils are a great resource to deal with this crisis. Melaleuca armillaris belongs to the family of Myrtaceae, rich in species with essential oils. Plant extracts has shown antimicrobial activity in many investigations. Cloxacillin (CLOX) is an antibiotic widely used in veterinary medicine against Staphylococcus aureus. Our aim was to assess pharmacodynamic interaction established by combining essential oil of M. armillaris (EO) with CLOX in search of a synergistic effect that maximizes the antibacterial activity against S. aureus. The EO was obtained by steam distillation and its composition was analyzed by a GC–FID–MS. The most abundant components in the EO were 1.8 cineole (72.3%), limonene (7.8%). and α-pinene (6%). We worked with wild type S. aureus strains (n = 3) isolated from Holstein cows, and S. aureus ATCC 29213 as the reference strain. The Minimum Inhibitory Concentration (MIC) of CLOX, EO and the combination was determined by microdilution in broth at pH 7.4; 6.5 and 5.0. The checkerboard method was applied to evaluate the interaction between CLOX and EO. The Fractional Inhibitory Concentration index (FIC) was established. From those combinations that yielded the lowest FIC values, we evaluated the index of antibacterial activity (E), established as the difference between the Log10 values of the number of viable bacteria at the initial (nt0) and at the end of the test (nt24). So, time-killing curves with CLOX and EO/CLOX combination at 0.5, 1, 2, 4, and 8 fold the MIC in broth at pH 7.4; 6.5 and 5.0 were prepared. We considered Bacteriostatic effect (E = 0) Bactericidal effect (E = −3) and Effect of virtual eradication of bacteria (E = −4). A clear synergic activity between the EO and the CLOX was demonstrated, which allows reducing the MIC of β-lactam against S. aureus. This interaction was favored by acidification of the medium, where lower concentrations of CLOX achieved a bactericidal effect, close to virtual eradication, in the presence of small amounts of EO.
Highlights
Bovine mastitis is one of the most prevalent problem which affects dairy herd production worldwide, and antimicrobial therapy is the primary tool for the treatment
The strains used in this study were identified as members of S. aureus specie after characterizing them biochemically, resulting in Gram positive, coagulase positive, catalase positive, β-hemolytic, glucose fermenting, and growth in saline (7.5%) strains
The Minimum Inhibitory Concentration (MIC) of the CLOX obtained for S. aureus ATCC 29213 at pH 7.4 was 0.125 μg/mL, which is in accordance with the recommendations of the CLSI 2009 [32]
Summary
Bovine mastitis is one of the most prevalent problem which affects dairy herd production worldwide, and antimicrobial therapy is the primary tool for the treatment. This disease is responsible for a negative effect in the economy of several countries, because of the decrease results in the production level and quality of milk. The most frequent pathogen which caused subclinical mastitis in dairy cows is Staphylococcus aureus [1] This microorganism is characterized by its capacity of select resistance against traditional antimicrobials and some virulence factors as its ability to form biofilms and to invade and survive inside epithelial cells [2]. The excessive and irrational usage of antimicrobials favored the emergence of resistance to multiple antibiotic families
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