Characterization of the antibacterial activity from ethanolic extracts of the botanical, Larrea tridentata

Abstract

Background: β-lactam antibiotics are a class of broad-spectrum antibiotics consisting of all antibiotic agents that contain a β-lactam ring in their molecular structures. β-lactam antibiotics are only known to be isolated from fungi (e.g. Acremonium chrysogenum, Penicillium chrysogenum and Aspergillus nidulans) and bacteria (e.g. Streptomyces clavuligerus). We have shown that botanical extracts prepared from Larrea tridentata have strong antimicrobial activity against several bacteria, including members of Staphylococcus and Streptococcus genera.

Methods: Through resistance studies, inhibitor assays, and ELISA testing, we demonstrated L. tridentata extracts may contain a β-lactam type antibiotic activity.

Results: Based on the estimated β-lactam concentration within the extract, the antimicrobial activity of the L. tridentata extract was approximately 2000-8000-fold greater against Staphylococcus as compared to other β-lactams, penicillin or ampicillin. In the L. tridentata extract, this increased activity was found to be associated with the likely presence of a cofactor leading to increased potentiation of the β-lactam activity. This potentiation activity was also observed to enhance the activity of exogenously added natural penicillin antibiotics.

Conclusions: Although constituents were not isolated in this study, the results obtained strongly support the presence of β-lactam type antibiotic activity and antibiotic potentiation activity present in ethanolic extracts prepared from L. tridentata.

Keywords: Antibiotic; Antimicrobials; Bacteria; Larrea tridentata; Staphylococcus aureus; Tincture; β-Lactam.

Fig. 1

Antibacterial activity of L. tridentata extracts. Bacterial cultures were treated with increasing concentrations of the L. tridentata extract ranging from 1 to 1000 μg non-volatile constituents/ml media. The MBC was determined at 24 h. Part A was done with the indicated bacterial species using L. tridentata extracts prepared in 40% ethanol. Part B was done with S. aureus (ATCC 14775) using botanical extracts prepared with the indicated percentages of ethanol. Values shown with error bars represent the standard deviation from three separate experiments

Fig. 2

Activity of L. tridentata extract against botanical-resistant and penicillin-resistant strains of S. aureus. Bacterial cultures (Staph Pen^S, ATCC 14775; Staph Pen^R, ATCC 11632; and Staph Larrea^R) were treated with increasing concentrations of the L. tridentata extract ranging from 1 to 100 μg non-volatile constituents/ml media . The MBC was determined at 24 h. Values shown with error bars represent the standard deviation from three separate experiments. Statistical analysis was performed using a paired t-test. Samples with statistically significant differences are indicated with the calculated p-value

Fig. 3

Spectrum of antibiotic resistance of botanical-resistant and penicillin-resistant S. aureus strains. Bacterial cultures (Staph Pen^S, ATCC 14775; Staph Pen^R, ATCC 11632; and Staph Larrea^R) were tested for antibiotic susceptibility using the standard Kirby Bauer disc diffusion method. Standard antibiotic discs included penicillin 10 μg, tetracycline 30 μg, vancomycin 30 μg, and ciprofloxacin 5 μg. The diameters of the zones of complete inhibition were measured using calipers in mm. Values shown with error bars represent the standard deviation from three separate experiments. Statistical analysis was performed using a paired t-test. Samples with statistically significant differences are indicated with the calculated p-value

Fig. 4

Effect of clavulanic acid on the growth of the botanical-resistant S. aureus strain. Bacterial cultures (Staph Pen^S, ATCC 14775; and Staph Larrea^R) were treated with increasing concentrations of the L. tridentata extract ranging from 1 to 1000 μg non-volatile constituents/ml media in the presence or absence of clavulanic acid (6 μg/ml). The MBC was determined at 24 h. Values shown with error bars represent the standard deviation from three separate experiments. Statistical analysis was performed using a paired t-test. Samples with statistically significant differences are indicated with the calculated p-value

Fig. 5

Effect of β-lactamase on the antibacterial activity of penicillin and L. tridentata extract. A penicillin stock solution or L. tridentata extract were treated with 0.25 or 2.5 U/ml β-lactamase followed by inactivation of the β-lactamase. Bacterial cultures of non-antibiotic resistant S. aureus (ATCC 14775) were treated with increasing concentrations of the treated or untreated penicillin stock solution (20–200 μg/ml) or L. tridentata extract (ranging from 1 to 1000 μg non-volatile constituents/ml media). The MBC determined at 24 h. Values shown with error bars represent the standard deviation from three separate experiments. Statistical analysis was performed using a paired t-test. Samples with statistically significant differences are indicated with the calculated p-value

Fig. 6

Structural characteristics of β-lactam antibiotics related to L. tridentata extract potentiation effects. A) The molecular structures of β-lactam antibiotics separated based on levels of L. tridentata extract potentiation effects. B) Required structural features for L. tridentata extract potentiation effects on β-lactam antibiotics