Novel approaches to antibiotic resistance

1. Novel approaches to antibiotic resistance Gili Regev-Yochay Harvard School of Public Health Children’s Hospital

2. We are reaching/have reached the post-antibiotic era • Past 20 years number of new drugs that reached the marked has fallen to less than 50% the previous level. • Past 40 years only 2 novel Ab classes (lipopeptide- Daptomycin, Oxazolidinone- linezolid) • Rate of loss of efficacy of old Ab is outstripping their replacement with new ones • Particularly worrisome for Gram-negative Ab.

3. Who is to blame? • Ab industry? • Grant funding agencies? Less funding for Ab development? • Financial reward to develop new Ab is unfavorable (restricting Ab to control resistance will paradoxically reduce rate of development). • 10/15 major pharma reduced or eliminated Ab R&D • Reason: Ab less valuable: short course therapies, curable disease.

4. Novel approaches • Ab in non-culturable bacteria • Bacteriophage • Bacterial interference • Don’t kill the pathogen – kill the virulence factors • Immunomodulation • Drug interactions (Kishony group)

5. Novel methods using natural sources • The original source of antibiotics: bacteria (aimed to killl or inhibit the replication of competitors). • Most marketed Ab -derivatives from bacterial Ab. • All from culturable bacteria. • What about non culturable bacteria (clone large fragments of non-culturable bacterial genome)

6. Natural compounds: non-culturable bacteria/ Lee et al. Biotechnol. Lett 2007, Garcia et al. nat. Biotechnol. 2006

7. Bacteriophages • Bacteriophages = bacterial viruses • Estimated: every 2 days 50% of the world’s population is destroyed by bacteriophages. • Initially discovered in 1915 by Twort and independetly in 1917 by d’Herelle • Used before introduction of penicillin even in US. Was abandoned since Ab use. • During Ab era considered “non-conventional” medicine. • Continued use in former USSR: Eliave Institute in Tbilisi (http://www.evergreen.edu/phage/home.htm). • FDA approved use of bacteriophages in poultry and cattle for Listeria monocytogenes contamination of meat. • Recent year: 90 papers on Bacteriophage treatment!

8. Phage Therapy • Appealing: • Specific, does not disrupt flora • Either use cocktail of phages or have initial identification of bacteria to treat • Marketed phage: ListexTM P100 for controlling Listeria in cheese & meat • Option: use of lysis-deficient phages that still kill bacteria. • Can be used as such to induce immunity (WCV).

10. Bacteriophage – other optional uses • Therapy delivery systems • Lytic enzymes (Fischetti et al. 2005. Trends Microbiol) • Phage use lysins which are host specific • Active also on non-replicating bacteria or biofilms. • Resistance is rare • Combined. • Phage or phage lysins + Ab: available comercially in Georgia “PhagoBioDerm” biogradable polymer impregnated with lytic phage cocktail + Cipro (Markoishvili et al. 2002. Int. J. Dermatology).

11. Phage treatment – potential problems • Quality control and standardization. • Highly immunogenic and induce neutralizing ab -> single use per patient? Or only external use? • Safety: • Massive bacterial lysis may lead to toxic shock. • lysogeny, transduction of virulent/Ab resistance genes… • Another reason for reluctance in the West: • Difficult to obtain IP rights (public for many years)

12. Bacterial interference • Exchange of “bad” bugs with “good” bugs • 1909 Danish physician Schiotz: sprayed S. aureus in throats of diphteria carriers and eliminated carriage of diphteria. • Initially Shinefield et al. 1960-1970s • 1965 Bacterial interference; protection of adults against nasal S. aureus infection after colonization with a heterologous S. aureus strain. Boris et al. • 1974 Bacterial interference between strains of S. aureus Shinefield et al. Ann NY Acad Sci.

13. Bacterial interference – Shinefield et al. • Direct inoculation of infants, medical students and prisoners with a low virulence S. aureus (502A) • Controlling S. aureus outbreaks in neonatal units. Neonates colonized with 502A (at birth) completely protected from epidemic S. aureus. • 4,000 neonates artificially colonized with 502A (nose & umbilical stump). • Adverse events: 5-15% local infection in the umbilical area (vesicles). One nursery 34% vesicles. • One case of conjuctivitis • Sepsis and death at 84h. (colonized at 3h), cathetrized into ubmilical vein (area of colonization).

14. Bacterial interference - Probiotics • Bacterial vaginosis • Med. J. Aust. 2007 Treatment of VRE carriage with lactobacillus

15. Don’t aim at the bacteria - aim at its virulence factors • S. aureus golden pigment impairs neutrophil killing and promotes virulence through its antioxidant activity/ Liu GY et al. JEM 2005 • “white” S. aureus, are not as pathogenic. • Can we target this virulence factor? • Biosynthesis of the staphyloxanthin is similar to biosynthesis of cholesterol • SKF-525A drug in the pipeline of cholesterol Rx. But not as good as statins. • Indeed if added to growing culture of SA-> “white” SA

16. S. aureus golden pigment impairs neutrophil killing and promotes virulence through its antioxidant activity/ Liu GY et al. JEM 2005

18. Immunomodulation • Induce the immune system to eliminate infections: • Vaccines • Antimicrobial peptides • Other: • Hypoxia-induced factor (HIF) -1

19. Vaccines • Vaccination can reduce Ab resistance in several ways: • As in the case of PCV7 – reduction in the most popular strains which were also the main burden of resistance.

20. Vaccine types associated with drug resistanceUS, invasive disease, pre-PCV Conjugate vaccine Not in vaccine

21. 39.0% 37.4% General decline in VT + VT association with drug resistance = modest decline in proportion resistant Kyaw et al. 2006, NEJM

22. Resistance is now creeping upwards within NVT Kyaw et al. 2006, NEJM Based on Kyaw et al. 2006, NEJM

23. Serotype replacement in Massachusetts USA:broad range of clones carried; these maintained their resistance profiles NVT or 19F/A Hanage et al. JID 2007

24. Vaccination -> reduced resistance • VT association with drug resistance => vaccine disproportionately reduced resistant strains • After 2-3 years, resistance began creeping up again • Mainly outgrowth of previously existing, resistant clones • Some serotype switching • Stay tuned: likely evolution of resistance in previously susceptible clones

25. Other ways by which vaccines can reduce ab resistance: • Indirect – by reduction of Ab use • Less pneumococcal infections • Less fear of physicians to “miss” pneumococcal infections. • Both directed at the disease • Empiric treatment.

26. Antimicrobial peptides • Cationic host defense peptides (innate immunity) • Small , highly basic cystein-rich peptides. • Initial antimicrobial barrier for mucosal surfaces. • Broad spectrum, non specific.

27. Antimicrobial peptides - mechanism • Mechanism of antimicrobial action is unclear. • Involves targeting membranes whose composition includes negatively charged phospholipids (in contrast to mammalian – mostly neutral). • In addition modulate components of the innate immune system by activating MO (mechanism unknown, but not through TLR). But activation of TLR4 can lead to up-regulation of β-defensins by epithelial cells. • Recent studies have also suggested that they have a role in modulating the adaptive immune system through activation of immature dendritic cells.

28. Studies of potential uses: • Display antibacterial, antifungal and antiviral activity, non-cytotoxic • From plant and insect – antifungal defensins. • TB – α + β defensins possess anti-TB activity • Anthrax – inactivate the enzymatic activity of anthrax lethal factor. • Can serve as a valuable resource as a template for designing small synthetic peptides • Problems: unknown PD and toxicology • Phase IIIa trials as topical direct antimicrobial treatment

29. Hypoxia-induced factor (HIF) -1 / Nizet et al. JID 2008 • A transcriptional regulator (HIF-1a) plays a role in supporting the inflammatory and bactericidal activity of neutrophils & MO (murine model). • Use of HIF-1a agonist (Developed for angiogenesis and cancer). • Check this in vitro for S. aureus and human neutrophil cells. • HIF-1a boosts capacity of phagocyts to kill SA