Development of bacterial resistance to antimicrobial botanical extracts

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1 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

2 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.

3 Protocol to determine killing dose S. aureus ul botanical extract/ml media Grow at 37 C Titer bacteria/colony forming units/ul every 8 hours

4 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

5 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

6 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

7 Development of antibiotic resistance Antibiotic resistance in S. aureus was uncommon when penicillin was first introduced in 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.

8 Minimum inhibitory concentrations: MIC Treatment MIC (mg/ml) MIC (ml/ml) Vancomycin Tetracycline Levofloxacin Salvia officinalis Eucalyptus globulus Arctostaphylos uva-ursi Coptis chinensis Larrea tridentata All botanical extracts had an average nonvolatile constituent concentration averaging 29.5 mg/ml (ranging from mg/ml)

9 Selection protocol for developing resistant bacteria Day % MIC selection Test for increase in MIC level

10 Staphylococcus aureus treatment with standard antibiotics + indicates growth indicates no growth Vancomycin (stock 0.5 mg/ml) Levofloxacin (stock 0.25 mg/ml) Day Day Day Day ~ 1.5-fold MIC increase 2-fold MIC increase Tetracycline (stock mg/ml) Day Day /- ~ 4.5-fold MIC increase ml treatment/ml broth

11 Staphylococcus aureus treatment with antimicrobial botanicals + indicates growth indicates no growth Coptis (stock 26.7 mg/ml) Day Day fold MIC increase Arctostaphylos (stock 31.0 mg/ml) Day Day fold MIC increase Salvia (stock 25.9 mg/ml) Day 0 + +/ Day fold MIC increase Larrea (stock 35.3 mg/ml) Eucalyptus (stock 28.6 mg/ml) Day Day Day /- +/- Day ~ 3-fold MIC increase 3-fold MIC increase ml treatment/ml broth

12 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

13 Staphylococcus aureus exposed to mutagen + indicates growth indicates no growth Vancomycin (stock 0.5 mg/ml) Eucalyptus (stock 28.6 mg/ml) Normal Normal Mutant /- - Mutant /- 2-fold MIC increase ~ 1.5-fold MIC increase Coptis (stock 26.7 mg/ml) Salvia (stock 25.9 mg/ml) Normal Normal Mutant Mutant ~ 3-fold MIC increase 2-fold MIC increase ml treatment/ml broth

14 Developing resistance Day Test for resistance Test for resistance Test for resistance

15 Staphylococcus aureus treatment with standard antibiotics + indicates growth indicates no growth Vancomycin (stock 0.5 mg/ml) Levofloxacin (stock 0.25 mg/ml) Day Day Day Day Day Day Day Day Tetracycline (stock mg/ml) Day Day /- - - Day /- - Day /- ml treatment/ml broth

16 Staphylococcus aureus treatment with antimicrobial botanicals + indicates growth indicates no growth Coptis (stock 26.7 mg/ml) Day Day Day Day Arctostaphylos (stock 31.0 mg/ml) Day Day Day Day Salvia (stock 25.9 mg/ml) Day 0 + +/ Day Day Day ml treatment/ml broth

17 Proposed models of botanical resistance I. Multiple active constituents in the extract

18 Proposed models of botanical resistance I. Multiple active constituents in the extract II. Bacteria develops multiple mutations on same target

19 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

20 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.

21 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.

22 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.

23 Heather Harrington, ND Robert Waters, PhD Yvan Rochon, PhD Jeffrey Langland, PhD Acknowledgements Arizona State University, Biodesign Institute Southwest College of Naturopathic Medicine

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