Introduction to Antibacterial Therapeutics (antibiotics)Diphtheria (Corynebacterium diphtheriae) Cholera (Vibrio cholerae) • Mortaility due to infectious disease was ~20x higher

MEDCH 561P

Introduction to Antibacterial Therapeutics (antibiotics)

http://botit.botany.wisc.edu/toms_fungi/nov2003.html

April 8, 2013Kelly Lee, Ph.D.

[email protected]

Penicillium chrysogenum + Staph. Aureus

“It's not often that our scientists come to me to say that we have a very serious problem, and we need to sound an alarm. But that's exactly what we're doing today...What I’m talking about today is CRE, carbapenem-resistant enterobacteriaceae. CRE are nightmare bacteria. They pose a triple threat. First, they're resistant to all or nearly all antibiotics. Even some of our last-resort drugs. Second, they have high mortality rates. They kill up to half of people who get serious infections with them. And third, they can spread their resistance to other bacteria.”

-Thomas Frieden, Director CDCMarch 5, 2013

• CRE at present has only been detected in healthcare institutions (nosocomial), and not in the community, yet...

• 4.6% of hospitals, but 17.8% of long-term care facilities in 2012

• Widespread over 42 states, but not yet prevalent

• Incidence has risen 4-fold over the past decade

Carbapenem-resistant enterobacteriaceae (CRE)

Antibiotic resistance is a huge and growing problem

• CRE at present has only been detected in healthcare institutions (nosocomial), and not in the community, yet...

• 4.6% of hospitals, but 17.8% of long-term care facilities in 2012

• Widespread over 42 states, but not yet prevalent

• Incidence has risen 4-fold over the past decade

• Enterobacteriaceae include some of the most common bateria:

• E. coli

• Klebsiella

• Salmonella

• Shigella

Carbapenem-resistant enterobacteriaceae (CRE)

• CRE introduced via a lung transplant patient transferred from a New York facility

• Hospital tried to quarantine infected patients, walled off in a separate ICU, ripped out plumbing, scrubbed surfaces, imposed patient screening and infection-control rules

• Over more than a year, the CRE bug (KPC Klebsiella) spread to 19 patients

• New patients even beyond the new ICU continued to become infected, even without direct contact or coming into proximity

• Even more stringent disinfection of surfaces used, hand washing monitors hired

• 12 patients died, 7 attributed directly to CRE

Example: CRE outbreak at the NIH hospital 2011

Carbapenem-resistant Acinetobacter

Center for Disease Dynamics, Economics and Policyhttp://www.cddep.org/research/2

Much of modern medical care relies upon the ability to effectively treat microbial infections

“...a catastrophic threat...a ticking time bomb. [Unless resistance is curbed] we will find ourselves in a health system not dissimilar to the early 19th century...”

-Dame Sally Davies, United Kingdom, Chief Medical OfficerMarch 11, 2013

Much of modern medical care relies upon the ability to effectively treat microbial infections

Surgery?

Organ transplants?

Catheterization?

Joint replacement?

Cancer treatment?

AIDS?

Wounds?

• For example, hip and knee replacements (~800,000 per year in US):• Antibiotic prophylaxis is commonly used to prevent hospital acquired infections

• Currently, infection rates ~0.5-2%

• Without antibiotics, the likely postoperative infection rate would be 40-50%, and ~30% of those infections would likely be fatal

Much of modern medical care relies upon the ability to effectively treat microbial infections

R. Smith, J. Coast, “The true cost of antimicrobial resistance”, BMJ 2013, vol 346

Overview for Week 1

• Impact of antimicrobials on health and health care

• History and background

• Mechanisms of action

• Resistance

Brett Finlay, HHMI

E. coli growing under nutrient-rich conditions

Armstrong GL et al., (1999) JAMA 281:61-66

Infectious disease mortality rate 1900-1996

๏ A combination of improved sanitation, access to clean water, hygiene, vaccination, and antibiotics led to a decrease in mortality starting around the industrial revolution (late 19th century)

1918 influenza pandemic~50-100 million deaths (~3% of world population)~30% of world population infected

Life in a pre-antimicrobial world

Tuberculosis (Mycobacterium tuberculosis)

Typhus (Rickettsia prowazekii)

Scarlet Fever (Streptococcus pyogenes)

Pneumonia (Streptococcus pneumonia, Haemophilus influenzae)

Diphtheria (Corynebacterium diphtheriae)

Cholera (Vibrio cholerae)

• Mortaility due to infectious disease was ~20x higher in 1900 than now

• Before antibiotics Streptococcus pyogenes responsible for ~50% of infant mortality

• S. pyogenes also frequently caused death in infected burn wounds

• Staphylococcus aureus fatal in nearly 80% of infected wounds

• Prior to antibiotics, wound infection killed more soldiers than weapons in warfare

Impact on health care

• In 2004, total global trade in antibiotics > $27 billion

• Beta-lactams accounted for ~45%

• 6 antibiotics topped $1 billion in sales:

• ceftriaxone (beta-lactam)

• amoxicillin/clavulanate (beta-lactam+beta-lactamase inhibitor)

• azithromycin (macrolide)

• clarithromycin (macrolide)

• ciprofloxacin (fluoroquinolone)

• levafloxacin (fluoroquinolone)

Taylor JB and Triggle DJ (eds), Comprehensive Medicinal Chemistry II, vol7Walsh CT, Antibiotics: Actions, origins, resistance, (2003) ASM Press, Washington DC, USA

Coates A, et al., Nature Rev. Drug Discovery (2002) 1:895-910

Impact on health care

• Antibiotics make up a substantial fraction of prescriptions:• ~42% of patients admitted to hospitals receive antibiotics• ~50% of drug prescription costs go to antibiotics, where prescription costs amount to 10-15%

of total health care costs

• Most likely place for an individual to acquire an antibiotic-resistant infection is the ICU

• U. Michigan Health System study: In 2002, 41 million antibiotic prescriptions for people suffering from colds (viral), more than one-third of patients who saw a doc about a cold (Feb 24, 2003 edition of Archives of Internal Medicine)

Selective Toxicity

Antimicrobial: Microbial secondary metabolites or synthetic compounds that in

small doses inhibit the growth and survival of microorganisms without serious

toxicity to the host (us)

Antibiotic: Natural product subset of antimicrobials

We are >90% bacterial

For our 10 trillion cells, ~100 trillion bacterial cells make their home in and on us.

Impact on our microbial flora

• The antimicrobials are not so specifically targeted that they knock out only the primary pathogen, they can act potently against other species of the flora.

• Can compromise the balanced bacterial ecology, especially of the gut

• e.g. leading to diarrhea, Clostridium difficile overgrowth

• The flora can also be reservoirs for transferable resistance factors (R-factors).

• R-factors can be detected even during the course of the therapy, and persist for years after antibiotic therapy

Ways commensal bacteria impact our health

• In many cases the flora-host interaction is mutualistic. Commensal bacteria provide:

• Aid in digestion of food and production of vitamins, link to obesity

• Processing of nutrients and drugs in our gut

• Overall metabolite profile (metabolome) of host with natural bacterial flora is significantly different from those that are germ-free (tested in animal models)

• Prevent establishment of pathogenic competitors

• Immunity

• Imbalance can impact asthma

• Cancer

• Is the impact on flora transient or long-lasting? Some evidence suggests the affect can persist and lead to long-term health consequences.

Organs and internal tissues are normally sterile. Commensal bacteria do colonize

“exterior” including skin, gut, respiratory tract, mouth, eyes, urogenital tract, etc.

Normal flora

Growing appreciation of enterotypesNative flora is specific to an individual, impact of treatment may differ significantly

• Appears human microbiome of the gut may be categorizable into 3 main “enterotypes”, each dominated by a main genus

• Not related to nation, ethnicity, gender, or age

• May be linked to long-term diet

• There may be a link between the enterotype found in an individual and susceptibility to disorders/diseases

“Enterotypes of the human gut microbiome,” Arumugam et al., Nature (2011) 472:7343

Sources of pathogenic bacterial infections

• Some of the commensal bacteria may become pathogens if they gain access to normally sterile internal sites through wounds, medical device insertion, etc. OPPORTUNISTIC PATHOGENS.

• Compromised immune systems also can create opportunities for pathogenesis.

• Some pathogens are extrinsic and not related to our commensal flora.

Organs and internal tissues are normally sterile. Commensal bacteria do

colonize “exterior” including skin, gut, respiratory tract, mouth, eyes,

urogenital tract, etc.

Some non-commensal pathogens

• Mycobacterium tuberculosis (“acid-fast”; waxy mycolic acid cell surface)

• Acinetobacter baumannii (gram-, highly resistant to most antimicrobials; significant source of hospital-acquired infections)

• Stenotrophomonas maltophilia (gram-, naturally resistant to broad spectrum antimicrobials; medical devices)

• Burkholderia cepacia (gram-; naturally resistant to many antimicrobials including polymyxin; transmissible, pathogenic; cystic fibrosis)

• Neisseria gonorrhoeae

• Treponema pallidum

• Chlamydia trachomatis

• Bacillus anthracis (gram+; environmental exposure, bioweapon)

• Salmonella typhi

History๏ 1877 concept of “antibiosis” first documented when Louis Pasteur observed that a “common bacterium”

could inhibit the growth of Bacillus anthracis

๏ 1908 Paul Erlich (w/ Sahachiro Hata) developed Salvarsan a synthetic organoarsenical treatment for syphilis and trypanosomiasis (sleeping sickness). Erlich developed the “magic bullet concept”, proposing that one could screen chemical compounds for selective anti-microbial activity.

๏ 1928 Alexander Fleming observed Penicillium notatum (now classified P. chrysogenum) killing staphylococci

๏ 1931-1936 Gerhard Domagk with others at Bayer Lab discovered and tested Prontosil, the first sulfonamide drug, effective against gram+ bacteria

๏ 1939 Rene Dubos isolated gramicidin (Bacillus brevis), one of the first commercially produced antibiotics to prove highly effective as a topical treatment for wounds and ulcers; used in World War II

๏ 1940 Flory, Chain, Heatley led industrialization of penicillin, increased yields 100-fold

๏ 1945 Benjamin Duggar discovered the first of the tetracyclines, the first broad-spectrum antibiotics from streptomyces a soil bacterium

๏ 1948 Guiseppe Brotzu isolated first cephalosporins from Cephalosporium acremonium active against Salmonella typhi that expresses beta-lactamases (knock out penicillins).

๏ 1952 Selman Waksman (and Albert Schatz) streptomycin from actinomycetes against TB; aminoglycosides from soil bacteria; coined the term “antibiotics”

๏ In late 1999 the development of a drug called Zyvox was finally announced. It’s in a new class of antibiotics called the Oxazolidinones. It’s claimed to be effective against multi-resistant strains of bacteria.

๏ 1943 Mary Hunt, a lab worker in Peoria, Illinois, found a moldy cantaloupe at a market sporting a strain of P. chrysogenum that combined with improvements in fermentation recipes, increased yields so dramatically that 2.3 million doses were available by D-Day, 1944.

๏ By 1942, the first patient was treated for septicemia, using up half of the entire world’s supply of penicillin

History

Wright GD (2007) Nature Reviews Microbiology 5:175

History

Future?๏ Many fewer antibiotics are in development and being approved now than in previous decades due

to economic and regulatory factors

๏ 1983-1987: 16 approved๏ 1988-1992: 14๏ 1993-1997: 10๏ 1998-2002: 7๏ 2003-2007: 4๏ 2007-present: 2

๏ But bacterial resistance to existing therapeutics is increasing

History๏ 2000 linezolid (Zyvox; Pfizer) approved by FDA. Represents a new class of synthetic antimicrobials called

the oxazolidinones (discovered in the 1980s). Effective against multi-resistant strains of gram+ bacteria.

๏ 2003 daptomycin (Cubicin; Cubist and Novartis) from Streptomyces roseosporus approved by FDA (discovered in 1980s). A lipopeptide that acts by depolarizing the membrane of gram+ bacteria.

๏ 2005 tigecycline approved (Tygacil; Wyeth). Glycylcycline class anti-microbial with broad spectrum activity including against MRSA. Structurally similar to tetracycline.

What Fleming saw

A Kirby-Bauer disc test

Don Stanlons; phil.cdc.gov

disc saturated with antibioticplated with

Staph. Aureus

Where do antimicrobials come from?

www.flickr.com/photos/ ajc1/2902232380/

ANTIBIOTICS:Natural products ofmicrobial warfare

ANTIBACTERIALS:Synthetic products from

chemical screens

Arsphenamine (Salvarsan, compound 606)

1910 used to treat syphilis

the first chemotherapeutic drug

Penicillium chrysogenum

Streptomyces

Where do antimicrobials come from?

ANTIBIOTICS:Natural products ofmicrobial warfare

ANTIBACTERIALS:Synthetic products from

chemical screens

• Beta-lactams:• Penicillins

• Cephalosporins

• Carbapenems

• Monobactams

• Aminoglycosides

• Macrolides

• Tetracyclines

• Daptomycin (Cubicin)

• Vancomycin

• Chloramphenicol

• Bacitracin

• Phosphomycin

• Polymyxin

• Sulfa drugs (sulfonamides)

• Quinolones

• Linezolid (Zyvox)

Production of pigmented secondary metabolites by Streptomyces colonies. (a) Typical colonial morphologies of Streptomyces isolated from the soil. Colonies often excrete colored pigments, providing a visual recording of secondary metabolite biosynthesis. The chemically diverse compounds represent a vast array of bioactive compounds that often have pharmaceutical applications. (b) A panoramic view of Streptomyces coelicolor colonial morphology. Both

Actinobacteria: especially genus Streptomyces

• Erythromycin (Saccharopolyspora erythrea)

• Neomycin (S. fradiae)

• Streptomycin (S. griseus)

• Tetracycline (S. rimosus)

• Vancomycin (S. orientalis)

• Daptomycin (S. roseosporus)

• Rifamycin (Amycolatopsis rifamycinica/S. mediterranei)

• Chloramphenicol (S. venezuelae)

• Puromycin (S. alboniger)

• Lincomycin (S. lincolnensis)

• Cefoxitin (S. lactamdurans)

•Approximately two-thirds of the antibiotics (natural products) in use are from Streptomyces, including:

Thompson CJ et al. (2002) Genome Biology 3:reviews1020

• Medicinal chemistry starting from the natural products to produce derivatives with greater potency, broader antimicrobial spectrum, lower toxicity, and improved pharmacokinetic profile, and to overcome resistance

• 1950s and ‘60s much work along these lines, especially for the beta-lactams (penicillins, cephalosporins, etc).

Semi-synthetic derivatives

Penicillin G (natural product)

General concepts

Therapeutic Control of Infection

Bacteriostatic: some antimicrobials do not necessarily kill the bacteria• Break the logarithmic growth phase, allowing the immune system to deal with the

infection. Tend to involve inhibition of protein synthesis.

• Examples: Tetracyclines, Sulfonamides, Chloramphenicol, Macrolides, Lincosamides

Bactericidal: kill the bacterium• Examples: Beta-lactams (penicillins, cephalosporins, carbapenems), Glycopeptides

(vancomycin), Aminoglycosides, Fluoroquinolones, Metronidazole

• Weaken the cell wall, leading to lysis (e.g. penicillins)

• Disrupt DNA replication (quinolones: DNA gyrase and topoisomerase IV)

• Disrupt RNA synthesis (rifampin: RNA polymerase)

• Disrupt protein synthesis (some however are bacteriostatic)

• Some drugs that are bacteriostatic at lower concentrations can be -cidal at higher concentrations

Therapeutic Control of Infection

Bacteriostatic: some antimicrobials do not necessarily kill the bacteria• Break the logarithmic growth phase, allowing the immune system to deal with the

infection. Tend to involve inhibition of protein synthesis.

• Examples: Tetracyclines, Sulfonamides, Chloramphenicol, Macrolides, Lincosamides

Bactericidal: kill the bacterium• Examples: Beta-lactams (penicillins, cephalosporins, carbapenems), Glycopeptides

(vancomycin), Aminoglycosides, Fluoroquinolones, Metronidazole

• Weaken the cell wall, leading to lysis (e.g. penicillins)

• Disrupt DNA replication (quinolones: DNA gyrase and topoisomerase IV)

• Disrupt RNA synthesis (rifampin: RNA polymerase)

• Disrupt protein synthesis (some however are bacteriostatic)

• Some drugs that are bacteriostatic at lower concentrations can be -cidal at higher concentrations

MIC: Minimum Inhibitory ConcentrationMBC: Minimum Bactericidal Concentration

medium drug+

medium drug-

MBC

MIC

[drug]

KILLED partially inhibited

notinhibited

Minimum Inhibitory Concentration: lowest concentration of drug that gives no visible growth after 24h incubation

Minimum Bactericidal Concentration: concentration of drug that gives no visible growth even in the absence of drug

Bioavailability• Before the anti-microbials such as penicillin, arsenicals, and sulfa drugs, topical

antiseptics were the only tools available for treating infection.

• Penicillin in particular provided low host toxicity, high potency that could get to the site of infection and permeate it.

• The drug must get to its target:

• Tissue penetration

• Penetrate biofilms

• Bacterial cell penetration to bind to the target

•Attain adequate concentrations to occupy a sufficient number of target active sites to produce desired effect, but without toxicity to host

•Must remain bound for sufficient time to inhibit the biological/metabolic process that will lead to bacterial cell death

Concentration-time curve: measured in blood

MIC

CMAX

time post-administration

AUC (Area Under the Curve)

๏ MIC: minimum inhibitory concentration in vitro

๏ AUC: a measure of the total exposure to the drug

๏ Cmax: maximum concentration attained

conc

entr

atio

n fr

ee d

rug

Time-dependent killing

๏ For example, beta-lactams

๏ Duration of time concentration>MIC is most important, slow bactericidal action

๏ So long as concentration>MIC, absolute concentration does not matter too much

๏ Related to number of target enzymes for the drug and their saturation

๏ Frequent, lower doses

conc

entr

atio

n

MIC

t>MIC

CMAX

time post-administration

Concentration-dependent killing

๏ For example, aminoglycosides and fluoroquinolones

๏ AUC/MIC or CMAX/MIC are the critical index for effective control

๏ Concentration-dependent mechanism of killing, fast bactericidal action

๏ Relates to barriers in drug getting to target site.

๏ Less frequent, higher doses

conc

entr

atio

n

MIC

CMAX

time post-administration

Time versus concentration-dependent activity

Craig WA, “Pharmacokinetic/pharmacodynamic parameters: rationale for antibacterial dosing of mice and men,” (1998) Clin. Infect. Dis. 26:1-12

beta-lactamaminoglycoside fluoroquinolone

Time-depConc-dep

Post-antibiotic effect (PAE)

๏ Post-antibiotic effect: after initial dosing, even when concentration in the blood drops below the MIC, microbial growth may be inhibited

๏ Believed to be due to either drugs that remain bound or are concentrated in/near infection or due to damage to the bacteria that they must repair before they can recommence their growth

AUC/MIC but not concentration-dependent killing

๏ Example: Vancomycin, macrolides (e.g. azithromycin)๏ AUC/MIC is the critical index for effective control, but higher concentrations not

necessarily more effective above a certain point

๏ Strong post-antibiotic effect (PAE), likely reflects antibiotic-induced cell damage

๏ The duration of the PAE is increased by increasing the AUC/MIC

๏ When MIC is relatively high and AUC/MIC is low, these drugs may act similarly to time-dependent agents

conc

entr

atio

n

MIC

CMAX

time post-administration

Time versus concentration-dependent activity

Antimicrobialclass Goal of therapy index

type Iconc-dependent

killing long PAE

fluoroquinolonesaminoglycosides

daptomycinketolides (telithromycin)

maximize concentrationAUC/MICCmax/MIC

type IItime-dependent

killing minimal PAE

beta-lactamslinezolid

macrolides (e.g. erythromycin, clarithromycin)

maximize duration of exposure

type IIItime-dependent

killingmoderate to

long PAE

tetracyclinesglycopeptide (vancomycin)

clindamycinmaximize total exposure AUC/MIC

t>MIC