Development of bacterial resistance to antimicrobial botanical extracts Linda M. Sparks, Heather Harrington, Robert Waters, Yvan Rochon, Jeffrey Langland Arizona State University, Biodesign Institute, Tempe, Arizona Southwest College of Naturopathic Medicine, Tempe, Arizona
Staphylococcus aureus infections S. aureus is a common cause of skin infections (e.g. boils), respiratory disease (e.g. sinusitis), and food poisoning. Other more life-threatening diseases include as pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome (TSS), bacteremia, and sepsis. It is still one of the five most common causes of nosocomial infections and is often the cause of postsurgical wound infections. Each year, some 500,000 patients in American hospitals contract a staphylococcal infection.
Protocol to determine killing dose S. aureus 0 0.6 2 6 20 ul botanical extract/ml media Grow at 37 C Titer bacteria/colony forming units/ul every 8 hours
Colony forming units/ml Colony forming units/ml Antibiotic inhibition in Staphylococcus replication Tetracycline Vancomycin no treatment 0.6 ul/ml 2 ul/ml 6 ul/ml 20 ul/ml
Colony forming units/ml Colony forming units/ml Ineffective botanicals Schisandra chinensis Echinacea angustifolia Polygonum multiflorum Moderately effective botanicals: Berberis vulgaris Baptisia tinctoria Glycyrrhiza glabra no treatment 0.6 ul/ml 2 ul/ml 6 ul/ml 20 ul/ml
Colony forming units/ml Colony forming units/ml Highly effective botanicals Salvia officinalis Arctostaphylos uva-ursi Eucalyptus globulus Coptis chinensis no treatment 0.6 ul/ml 2 ul/ml 6 ul/ml 20 ul/ml Larrea tridentata
Development of antibiotic resistance Antibiotic resistance in S. aureus was uncommon when penicillin was first introduced in 1943. However, by 1950, 40% of hospital S. aureus isolates were penicillin-resistant; and, by 1960, this had risen to 80%. The emergence of antibiotic-resistant forms of pathogenic S. aureus (e.g. MRSA, methicillin-resistant S. aureus) is a worldwide problem in clinical medicine. Indeed, antibiotic-resistance in pathogenic bacteria as a whole is a significant concern to world health.
Minimum inhibitory concentrations: MIC Treatment MIC (mg/ml) MIC (ml/ml) Vancomycin 2 4.5 Tetracycline 0.06 7.0 Levofloxacin 1 4.0 Salvia officinalis 150 5.8 Eucalyptus globulus 120 4.2 Arctostaphylos uva-ursi 90 2.9 Coptis chinensis 120 4.5 Larrea tridentata 60 1.7 All botanical extracts had an average nonvolatile constituent concentration averaging 29.5 mg/ml (ranging from 25.9 35.3 mg/ml)
Selection protocol for developing resistant bacteria Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 75% MIC selection Test for increase in MIC level
Staphylococcus aureus treatment with standard antibiotics + indicates growth indicates no growth Vancomycin (stock 0.5 mg/ml) 4 6 8 10 12 Levofloxacin (stock 0.25 mg/ml) 3 4.5 6 7.5 9 10.5 Day 0 + - - - - Day 0 + - - - - - Day 15 + + + - - Day 15 + + + + - - ~ 1.5-fold MIC increase 2-fold MIC increase Tetracycline (stock 0.005 mg/ml) 5 15 25 35 45 55 65 Day 0 + - - - - - - Day 15 + + + + + + +/- ~ 4.5-fold MIC increase ml treatment/ml broth
Staphylococcus aureus treatment with antimicrobial botanicals + indicates growth indicates no growth Coptis (stock 26.7 mg/ml) 3 8 13 18 23 28 Day 0 + - - - - - Day 15 + + + + + - 3.5-fold MIC increase Arctostaphylos (stock 31.0 mg/ml) 2 4 6 8 10 Day 0 + - - - - Day 15 + + + - - 2.5-fold MIC increase Salvia (stock 25.9 mg/ml) 2.5 5 10 15 20 25 Day 0 + +/- - - - - Day 15 + + + + + - 2.5-fold MIC increase Larrea (stock 35.3 mg/ml) 1.5 2.5 3.5 4.5 5.5 6.5 Eucalyptus (stock 28.6 mg/ml) 3 4.5 6 7.5 9 Day 0 + - - - - - Day 0 + - - - - Day 15 + + + + +/- +/- Day 15 + + + + - ~ 3-fold MIC increase 3-fold MIC increase ml treatment/ml broth
Chemical mutagenesis protocol Pellet cells and resuspend in PBS Add Ethyl methanesulfonate (EMS) Bacteria culture 37 o C for 60 min Resuspend in media and grow overnight Pellet cells and wash
Staphylococcus aureus exposed to mutagen + indicates growth indicates no growth Vancomycin (stock 0.5 mg/ml) Eucalyptus (stock 28.6 mg/ml) 2 4 6 8 10 12 1.5 3 4.5 6 Normal + + - - - - Normal + + - - Mutant + + + + +/- - Mutant + + + +/- 2-fold MIC increase ~ 1.5-fold MIC increase Coptis (stock 26.7 mg/ml) 3 8 13 18 23 28 Salvia (stock 25.9 mg/ml) 2.5 5 10 15 20 Normal + - - - - - Normal + + - - - Mutant + + + + - - Mutant + + + + - ~ 3-fold MIC increase 2-fold MIC increase ml treatment/ml broth
Developing resistance Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Test for resistance Test for resistance Test for resistance
Staphylococcus aureus treatment with standard antibiotics + indicates growth indicates no growth Vancomycin (stock 0.5 mg/ml) 4 6 8 10 12 Levofloxacin (stock 0.25 mg/ml) 3 4.5 6 7.5 9 10.5 Day 0 + - - - - Day 0 + - - - - - Day 9 + + - - - Day 9 + - - - - - Day 12 + + - - - Day 12 + + - - - - Day 15 + + + - - Day 15 + + + + - - Tetracycline (stock 0.005 mg/ml) 5 15 25 35 45 55 65 Day 0 + - - - - - - Day 9 + + + + +/- - - Day 12 + + + + + +/- - Day 15 + + + + + + +/- ml treatment/ml broth
Staphylococcus aureus treatment with antimicrobial botanicals + indicates growth indicates no growth Coptis (stock 26.7 mg/ml) 3 8 13 18 23 28 Day 0 + - - - - - Day 9 + + + + - - Day 12 + + + + - - Day 15 + + + + + - Arctostaphylos (stock 31.0 mg/ml) 2 4 6 8 10 Day 0 + - - - - Day 9 + - - - - Day 12 + + - - - Day 15 + + + - - Salvia (stock 25.9 mg/ml) 2.5 5 10 15 20 25 Day 0 + +/- - - - - Day 9 + + - - - - Day 12 + + + + - - Day 15 + + + + + - ml treatment/ml broth
Proposed models of botanical resistance I. Multiple active constituents in the extract
Proposed models of botanical resistance I. Multiple active constituents in the extract II. Bacteria develops multiple mutations on same target
Proposed models of botanical resistance I. Multiple active constituents in the extract II. Bacteria develops multiple mutations on same target III. Bacteria develops multiple mechanisms of resistance
Conclusions 1. Several botanical extracts, including Salvia officinalis, Eucalyptus globulus, Larrea tridentata, Arctostaphylos uva-ursi, Coptis chinensis, were found to be highly effective in inhibiting S. aureus replication. 2. Sustained growth of S. aureus in the presence of these botanicals led to the development of resistance to the inhibitory effects of these extracts. 3. Chemical mutagenesis of S. aureus led to rapid development of resistance to these extracts. 4. Resistance levels continued to increase as selective pressure was maintained. 5. These results suggest that S. aureus, and likely other bacteria, can develop resistance to anti-bacterial botanical extracts.
Outcomes 1. The data demonstrates the anti-bacterial efficacy associated with several well-known botanical extracts, but suggests that proper use of these botanicals should be followed in order to reduce the development of bacterial resistance. 2. As has been standard practice, the use of botanical blends containing several different anti-bacterial extracts may help to reduce the development of bacterial resistance. This hypothesis is currently being investigated.
Future Research We are currently researching the botanicals we have found to inhibit bacterial growth in S. aureus. We are doing assays to find the mechanisms of action for each botanical and eventually each constituent. In the future we will further our resistance research by using tinctures with combinations of botanicals in the stead of the single tinctures used in this experiment.
Heather Harrington, ND Robert Waters, PhD Yvan Rochon, PhD Jeffrey Langland, PhD Acknowledgements Arizona State University, Biodesign Institute Southwest College of Naturopathic Medicine