Antibiotic Resistance: How and why did this

happen?

Jon R. Wiener, Ph.D. Science Friday

March 20, 2015

CRE outbreak: You're due to go in for a procedure. Should you be worried?

By Elizabeth Cohen, Senior Medical Correspondent, CNN February 20, 2015

(CNN) “You're due to go in for a medical procedure. But after what happened at a UCLA hospital, you're a little apprehensive. Two patients died at the Ronald Reagan UCLA Medical Center in a superbug CRE outbreak, caused by two medical scopes that still carried the bacteria even after they were disinfected. In addition to the two victims, seven hospital patients were infected with the deadly superbug between October and January. The medical center has contacted 179 others who had endoscopic procedures between October and January and is offering them home tests to screen for the bacteria. The superbug, carbapenem-resistant Enterobacteriaceae, or CRE, can kill up to half the patients who contract them…. “

What are Antibiotics

and How do They Function?

DEFINITION: An Antibiotic is a molecule, originally developed by one microorganism to

limit the growth of, or kill, another microorganism, to better compete in the

environment for food or space.

So, antibiotics limit the growth of, or kill, competitor microorganisms. They were not

evolved to be used by humans….but we were smart enough to figure out how to use them.

However, antibiotic resistance was present before

we started using them!

Some Antibiotic Chemical Structures

Tetracycline

Erythromycin Teixobactin

Common Penicillin Derivatives

Salvarsan, aka Compound 606, or Arsphenamine, was

the first antibiotic. A completely synthetic arsenical compound

developed in the laboratory of Dr. Paul Ehrlich in 1907 to kill

Treponema pallidum, a spirochete which

causes Syphilis.

Dr. Paul Ehrlich, the father of Antibiotic

Chemotherapy

Antibiotics generally work in two ways:

1. Bacteriocidal, i.e. Kill bacteria (cidal = killing) 2. Bacteriostatic, i.e. Limit bacterial growth/reproduction (static = keep same) …and they usually work by: 1. Interfering with synthesis of the bacterial cell wall 2. Interfering with synthesis of bacterial cellular proteins 3. Interfering with metabolic processes, e.g. blocks folic

acid synthesis (‘metabolic antagonism’; next slide) 4. Blocking synthesis of DNA or RNA

Penicillin Family Activity

In 1940 Penicillin usage was initiated. By 1942 resistance was observed. So

synthetic penicillins were made, one of which, Methicillin, was resistant to

Beta-Lactamases. Methicillin usage was initiated in 1959. By 1962 resistance

was observed…but this method of resistance was not about Beta-

Lactamase inactivation of Penicillin.

Penicillin works by binding bacterial transpeptidase enzymes called penicillin

binding proteins (PBP1-4) which are involved in cell wall synthesis. Penicillins block these enzymes from cross-linking peptide chains in the cell wall, lessening cell wall integrity….leakage occurs…cell

integrity falters….bacteria dies.

How sulfa drugs work….Humans can take in Folic Acid exogenously, bacteria can’t.

Sir Alexander Fleming and the Discovery of Penicillin, 1929

Penicillin has a high ‘therapeutic

index’, i.e. it attacks a target

human cells don’t have (cell wall), so

large quantities can be given safely

to humans.

Spectrum of Use for some Commonly Used Antibiotics

Example of Novel Antibiotics Antibiotic Peptides, e.g. Magainins

Magainin 2: GLY-ILE-GLY-LYS-PHE-LEU-HIS-SER-ALA-LYS-LYS-PHE-GLY-LYS-ALA-PHE-VAL-GLY-GLU-ILE-MET-ASN-SER

“Thousands of antimicrobial peptides, similar to magainin, have been found throughout the plant and animal kingdoms. Many exhibit broad-spectrum antimicrobial activity, and can inhibit growth of numerous species of bacteria and fungi, as well as induce osmotic lysis of protozoa. These antimicrobial peptides are components of the "innate immune system" of animals and provide an important first line of antimicrobial defense.” (Wiki)

Antimicrobial Peptides and Peptidomimetics - Potent Therapeutic Allies for Staphylococcal Infections.

Mohammad H, Thangamani S, Seleem MN

The pervasiveness of bacterial resistance to conventional antibiotics, particularly those associated with staphylococcal infections, has become a global epidemic. However, research involving antimicrobial peptides (AMPs) and their synthetic analogues has unearthed a potentially novel class of antibacterials for the treatment of an array of diseases caused by pathogenic bacteria, such as staphylococci. AMPs have several unique advantages over traditional antibiotics such as the projected slow emergence of bacterial resistance to these agents and their capability to modulate the host immune response to infection. Unfortunately, their susceptibility to proteolytic degradation, loss of antimicrobial activity due to serum binding or physiological concentration of salts, and toxicity to host tissues has limited their use as systemic agents thus far….

What does antibiotic testing look like in the lab?

Kirby-Bauer Disc

Diffusion Assay

Antibiotic Resistance:

How and wHy…

And one additional antibiotic-

related issue..

So how would bacteria defend themselves, i.e. become ‘resistant’:

They could: 1. Slow or prevent antibiotic from getting into cell (or pumping it out with an

efflux pump) or to its target 2. Change the target (Note: when bacteria alter themselves to evade the

antibiotic, it costs them, metabolically or elsewise, but they live) 3. Destroy the antibiotic, e.g. beta-lactamases 4. Grow in a biofilm which slows diffusion of drugs and may also slow

metabolism of bacteria 5. Generate a false target, e.g. in M. tuberculosis Generally, antibiotic resistance is acquired by bacteria by 1. Mutating a gene and thus altering the target, or 2. acquiring new genes from other bacteria that are already resistant. This can

happen via salvage of DNA from destroyed bacteria, or through mechanisms whereby bacteria exchange genetic information, e.g. transformation, transduction, or conjugation.

An example of how bacteria inactivate an antibiotic: penicillinase

Digestion of the β-lactam ring of penicillin

The Penicillin variants are designed to prevent this, but….

Methicillin-Resistant bacteria (e.g. Staphylococcus aureus) have developed a new Penicillin-bind protein (PBP) called PBP2a which has a very low affinity

for Penicillin but still is enzymatically active in cell wall cross-linking…no killing.

Penicillin G Methicillin

Primary Reasons for Antibiotic Resistance (i.e. WHY?)

1. Given out by physicians for infections that will self-limit. Why?

Pressure by patients, self imposed pressure by physicians… 2. Used every day in thousands of agribusiness applications to

‘enhance’ growth of animals… 3. When used by humans, they don’t complete the course, then

used for infections when unnecessary… 4. Capability of bacteria to share genetic information… 5. Speed of production of new antibiotics wayyyyyy behind

speed/capability of bacteria to mutate or share antibiotic resistance…

From the start, it has been a losing battle against the bacteria in terms of ‘molecular warfare’. Let’s take a

look…

What does antibiotic resistance look like in the lab?

Kirby-Bauer Disc

Diffusion Assay

Kirby-Bauer Measurements of Antibiotic Sensitivity and

Resistance

What are the numbers regarding antibiotic resistance? A common occurrence? ..a rare occurrence?

It is generally stated that one out of every 100 million (1 in 108) bacteria in a culture is resistant to an antibiotic. This is before exposure to an antibiotic.

Antibiotic stress kills off susceptible bacteria, while resistant bacteria survive and reproduce, coming to dominate the population. So….antibiotics don’t

‘produce’ resistant cells….they were there to begin with.

Common Resistance Patterns (CDC) (RED=URGENT, YELLOW=SERIOUS, BLUE= OF CONCERN)

Carbapenem-Resistant Enterobacteriaceae DR (Drug Resistant) Neisseria gonorrhea

Clostridium difficile MDR (Multiple Drug Resistant) Acinetobacter

DR Campylobacter Fluconazole-Resistant Candida (fungi) Vancomycin-Resistant Enterococcus

MDR Pseudomonas aeroginosa DR Salmonella and Shigella

Methicillin-Resistant Staphylococcus aureus (MRSA) DR Streptococcus pneumoniae

DR Mycobacterium tuberculosis Vancomycin-Resistant Staphylococcus aureus (VRSA)

Erythromycin-Resistant Group A Streptococcus Clindamycin-Resistant Group B Streptococcus

Another Man-Made Problem due to Antibiotic Usage

Clostridium difficile The Clostridium genus are anaerobic bacteria that can form spores in suboptimal conditions, e.g. temperature, loss of nutrients, etc.

Examples: Botulism, Gas Gangrene, Tetanus… C. difficile is not normally a pathogen, but is an opportunistic

pathogen.

Clostridium difficile colitis or pseudomembranous colitis is inflammation of the large intestine resulting from infection with Clostridium difficile, a spore-forming bacteria. The colitis occurs when this bacteria replaces normal gut flora that has been compromised, usually following antibiotic treatment for an unrelated infection. The disturbance of normal healthy bacteria provides C. difficile an opportunity to overrun the intestinal microbiome. It is an antibiotic-associated diarrhea.

C. diff., continued

Treatment is problematic: spores resistant to antibiotics.

Metronidazole , Oral Vancomycin work for mild to moderate cases

Drugs that slow or stop diarrhea may worsen illness, not recommended.

Evidence to support use of probiotics for treatment is insufficient

Fecal bacteriotherapy, known as a stool transplant (the ‘yellow’ treatment), is

approximately 90% effective in those for whom antibiotics have not worked. It

involves infusion of bacterial flora acquired from the feces of a healthy donor

to reverse the bacterial imbalance responsible for the recurring nature of the

infection. The procedure replaces normal, healthy colonic flora that had been

wiped out by antibiotics, and reestablishes resistance to colonization by

Clostridium difficile. There is growing evidence that it may be an effective

treatment (possibly in pill form) for severe or recurrent CDI.

In those with severe C. difficile colitis, colectomy may improve the outcomes.

How serious is the issue? How many antibiotic-resistance cases seen per year? Organism Annual # Cases Annual # Deaths CRE 9,300 600 N. Gonorrhea 246,000 <5 Acinetobacter (‘Iraqibacter’) 7,300 500 Campylobacter 310,000 28 Candida 3,400 220 ESBL (Extended Spec B-Lactamase) 26,000 1,700 VRE (Vanco-resist Enterococci) 20,000 1,300 P. Aeruginosa 6,700 440 Non typhoid Salmonella 100,000 40 S. Typhi 3,800 <5 Shigella 27,000 <5 MRSA 80,000 11,000 S. Pneumoniae 1,200,000 7,000 TB 1,050 50 VRSA <5 <5 Group A,B Strepts 9000 600 Total 2,050,000 23,500 C. Difficile 250,000 14,000 (CDC, 2013)

Some Facts….

1. 70% of antibiotic usage in the U.S. is to livestock in subclinical dosages. Given to animals quickens their growth for unknown reasons.

2. 50% of Staphylococcus (‘Staph’) in the U.S. are resistant to Penicillin, Methicillin, Tetracycline, and Erythromycin. 85% of hospital Staph are resistant to everything but Vancomycin.

3. In the 1990s Staph began showing up with resistance to Vancomycin, but a Vancomycin variant now is generally effective (Thank Goodness!!).

4. Antibiotic resistance is as old as antibiotics, i.e. some resistant organisms have been found that are hundreds of years old….AR is probably millions of years old.

5. Antibiotic resistant bacteria cause >2 million illnesses and 23,000 deaths per year in the U.S. “Many more die from other complications that were complicated by an antibiotic-resistant infection” (CDC, 2013).

6. Total economic cost to the U.S. economy—estimates of 20-35 Billion$/yr (2008).

7. Estimates are ~50% of all antibiotics prescribed for humans is unnecessary.

What to Do? How to Avoid? 1. Use only when prescribed by a physician. 2. Finish complete course of antibiotic when prescribed. 3. Never share antibiotics with other people. 4. Physicians should only give them out when absolutely

necessary. Many other therapies work for a majority of illnesses as well. Do not ‘doctor-shop’ and try to force physicians to distribute. (850 prescriptions/1000 NC residents!)

5. Reduce use in animals (and thereby ingestion by humans). 6. Old/unused antibiotics.. do not flush them down toilet. 7. As a patient, consider antibiotics to be the ‘last resort’. 8. Controversial….but think about: consider natural antibiotics that

frequently work but don’t generate resistance, e.g. thyme, oregano, garlic, honey.

Consider something…. Antibiotics have been around in the medical community since

WWII…75 years.

The ‘golden age of antibiotics’ (1940 to present) has saved hundreds of millions of human lives.

The effectiveness of antibiotics is compromised by overuse and

subsequent resistance.

Someday in the future, you or a loved one may contract an illness that was once so easily treated it was an afterthought….now that

illness may seriously damage or cause fatality.