Novel approaches to antibiotic resistance

Gili Regev-Yochay

Harvard School of Public Health

Children’s Hospital

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.

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.

Novel approaches• Ab in non-culturable bacteria

• Bacteriophage

• Bacterial interference

• Don’t kill the pathogen – kill the virulence factors

• Immunomodulation

• Drug interactions (Kishony group)

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)

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

2006

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!

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).

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).

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)

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.

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).

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

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

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

JEM 2005

Immunomodulation

• Induce the immune system to eliminate infections:– Vaccines

– Antimicrobial peptides

– Other:• Hypoxia-induced factor (HIF) -1

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.

Vaccine types associated with drug resistance

US, invasive disease, pre-PCV

0

5

10

1520

25

30

35

4014

19F

6B 18C 4

23F

9V 6A 19A

9A 7F 22F 3

12F

Oth

ers

Serotype

Per

cen

t o

f al

l cas

es

Nonsusceptible Susceptible

Conjugatevaccine

Not in vaccine

General decline in VT + VT association with drug resistance = modest decline in

proportion resistant

39.0%37.4%

Kyaw et al. 2006, NEJM

Resistance is now creeping upwards within NVT

Kyaw et al. 2006, NEJM

Penicillin Nonsusceptibility Among NVT, ABCs USA

0%

2%

4%

6%

8%

10%

12%

14%

<2 65+ All

Pro

po

rtio

n P

en

-NS

1999

2004

Based on Kyaw et al. 2006, NEJM

Serotype replacement in Massachusetts USA:broad range of clones carried; these maintained

their resistance profiles

NVT or 19F/A

Hanage et al. JID 2007

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

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.

Antimicrobial peptides • Cationic host defense peptides (innate

immunity)

• Small , highly basic cystein-rich peptides.

• Initial antimicrobial barrier for mucosal surfaces.

• Broad spectrum, non specific.

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.

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

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