BIOL0510: Antibiotics and Resistance

Define chemotherapy

the use of drugs to treat a disease

define antimicrobial drugs

interfere with the growth of microbes within a host

define antibiotic

a substance produced by a microbe that, in small amounts, inhibits another microbe

What is the difference between a bactericidal and a bacteriostatic drug?

bactericidal: kill microbes directly

bacteriostatic: prevent microbes from growing (pause of growth until it’s removed again)

Where do we get antibiotics? (the sources from nature)

fungi and bacteria (especially gram +); often soil-derived organisms

List the compounds/natural antibiotics. Describe their qualities and how we modify them for our use

all organic, structures differ, sometimes are modified in the lab based on what we need them for; most antimicrobial drugs are derivative of natural compounds

What are the 3 major targets of antibiotics?

1. Nucleic acid synthesis

2. cell wall/membrane synthesis

3. protein synthesis

Describe the work Gehard Domagk did prior to antibiotic establishment in 1932

worked with coal-tar and isolated prontosil from it; prontosil is used to treat a streptococcus pyogenes infection in the wound of his daughter’s hand; today, it treats scoriasis

Why is prontosil an example of a prodrug? What are those drugs called today?

prontosil only gets its microbial properties when metabolized by the liver inside an infected host; it won’t kill bacteria in a dish

prodrug: a drug that needs to be processed in some way to gain its antimicrobial properties

they are now called sulfa drugs (sulfonamide)

How do sulfa drugs block bacterial synthesis to folic acid?

• inhibit folic acid synthesis

• acts as a competitive inhibitor of PABA (PABA is needed by bacteria to make folic acid)

What is PABA?

an intermediate in the bacterial synthesis of folate (folate/folic acid is a substance needed to process food)

How do sulfa drugs work?

humans don’t have the enzyme that converts PABA to folate, so we must ingest folate in our food; sulfa drugs work to kill bacteria by preventing nucleotides from being processed and DNA transcription from happening

What is rifampicin? What is its function in preventing bacterial growth?

• isolated from a bacteria growing in France soil

• treats TB and leprosy

• inhibits RNA progression and exit by binding to RNA polymerase (no transcripts or proteins are made, stopping bacterial growth)

What is the mechanism of action of quinolones as antibiotics to inhibit DNA replication?

• inhibit bacterial DNA replication by binding to gyrase and topoisomerase IV (which uncoil DNA)

• these enzymes create temporary double strand breaks in DNA to allow strand passage and then reseal the breaks

• However, the binding of quinolones to those enzymes prevents re-ligation (resealing) of the strands, causing double-strand breaks to accumulate

• leads to replication fork stalling and DNA fragmentation of chromosomes

What are the targets of antibiotics that inhibit cell surface molecules?

1. cell wall

   • B-lactams (penicillins)

   • polypeptide antibiotics (bacitracin)

   • glycopeptide antibiotic (vancomycin)

2. mycolic acid

   • isoniazid and ethambutol

3. cell membrane

   • polymyxin

What did Alexander Fleming contribute to antibiotic history?

natural microbial ecosystems are competitive environments, antibiotics are toxins, signalling molecules, or byproducts produced in microbial ecosystems; Fleming discovered Penicillium through microbial competition between common microbes

Who perfected Penicillin to expand its distribution as an antibiotic?

Ernst Chain and Howard Florey

• 1 dose in 1941

• 2 million doses in 1942

Discuss the qualities of cell wall synthesis inhibitors

B-lactam antibiotics (ex. penicillins) inhibit the enzyme transpeptidase (aka penicillin-binding proteins) that forms peptide cross bridges. This inhibits peptidoglycan polymerization and formation; moenomycin inhibits glycosyltransferases, blocking the elongation of the sugar side chains of peptidoglycan

Why aren’t penicillins prescribed anymore? 2 ways

1. bacteria now have an enzyme called penicillinase which breaks the B-lactam group, forming penicilloic acid, and rendering the penicillin completely unuseful (bacteria developed resistance)

2. PBPs change their structure such that b-lactams can’t bind to and inhibit them

How did humans combat bacterial resistance to penicillin? How have bacteria responded?

built semisynthetic antibiotics that prevent penicillinase from chopping the B-lactam group; bacteria evolved to change their shape

What are the virulence factors that bacteria use to cause disease?

formation of biofilms, panton-valentine leucocidin (PVL) toxin which kills human leukocytes and inflict severe tissue damage

What are the new penicillins?

they’re semi-synthetic —>

1. penicillinase-resistant penicillins (ex. oxacillin)

2. penicillins + B-lactamase inhibitors (ex. amoxicillin + potassium clavulanate)

Describe another cell wall synthesis inhibitor: polypeptide antibiotics

ex. bacitracin

• isolated from Bacillus subtilis

• applied to surface wounds

• it binds to lipid carrier (bactoprenol) on gram + bacteria that transports NAG and NAM across the membrane

Describe vancomycin

• a glycopeptide antibiotic

• a “worst case scenario” antibiotic

• identified from amycolatopsis orientalis (gram +) bacteria in soil from forests on Borneo

• made via nonribosomal peptide synthesis

How does vancomycin work? What type of bacteria does it work on?

• binds to the D-Ala-D-Ala end of NAM peptide side chains to prevent transpeptidase activity

• is bacteriostatic and only active against gram + bacteria (because it is large)

Name 2 antibiotics that inhibit mycolic acid (MA). What types of bacteria can they treat?

isoniazid (INH): inhibits MA synthesis

ethambutol: inhibits incorporation of MA

treat indeterminant bacteria (ex. tuberculosis)

Describe a type of antibiotic that ruptures cell membranes. What type of bacteria does it mainly treat?

polymyxins: bind to LPS and disrupt the structure of the bacterial cell membrane by interacting with its phospholipids (punctures holes in membranes); treats gram - bacteria (ex. pink eye infections)

Describe an antibiotic that inhibit protein synthesis

streptomycin: changes shape of 30s portion of the ribosome (the small subunit), causing code on mRNA to be read incorrectly

What type of antibiotic is streptomycin?

aminoglycoside; derived from streptomyces

What did Selman Waskman do?

• identified streptomysin

• Founded the Waksman method: put bacteria of interest on a plate and surrounded it with different types of bacteria; looked for a zone of inhibition

What is the difference between broad and narrow spectrum antibiotics? Include the pros and cons of each

broad:

• kill off multiple types of bacteria

• used to treat people with sepsis

• con: disrupt the natural microbiome, leading to superinfections

narrow:

What is a superinfection? 2 definitions

• a reinfection after already being ill with an infectious agent

  OR

• a secondary infection that develops during (coinfection) or immediately after an infection

How do we test if an antibiotic is effective? 2 ways

disk-diffusion assay

broth dilution test

E test

Describe how to understand the broth dilution test

• dilute an antibiotic in different concentrations from left to right in the plates

• if bacterial growth appears across all concentrations = its resistant to that antibiotic (doxycyclin)

• if bacterial growth appears only in high concentrations = its less resistant to that antibiotic

• if bacterial growth doesn’t appear at all = its sensitive to that antibiotic (ex. streptomycin)

Describe the E (epsilometer) test

like a disk diffusion; determine the specific concentrations of certain antibiotics bacteria are most resistant/sensitive to

What is an antibiogram?

a table that shows the percentage of bacteria from an institution that are susceptible to the antibiotics normally used to treat them; informs treatment plans of bacterial infections based on geographic area

When might it be worth treating an infection with two different antibiotics?

if there is synergistic inhibition between two antibiotics in a disk-diffusion assay

What is antibiotic resistance?

ability of microbes to grow in the presence of chemicals that would normally kill them or limit their growth

What is minimum inhibitory concentration? (MIC)

the lowest concentration of an antimicrobial agent that prevents the visible growth of a microorganism after incubation/exposure

What are the 4 major categories of antibiotic resistance?

1. prevention of penetration of drug

2. enzymatic destruction of drug

3. alteration of the drug’s target site

4. rapid ejection of the drug

Where are antibiotic resistant genes found?

they’re often encoded in plasmids (R-factors)

Describe MRSA and B-lactam resistance

MRSA encodes PBP2a (MecA gene), a transpeptidase with no B-lactam binding site; does 3 of the 4 functions of resistance

What is the clinical significance of antibiotic resistance?

• 2.8 million resistance infections/year in US

• 5 million deaths/year globally

• expected to keep rising

Where does resistance come from?

• plasmids, transposons, phages that exchange through:

  • transduction

  • transformation

  • conjugation

What is heteroresistance? What is the con it creates in the medical community?

a single bacterial strain harbors both susceptible and resistant cells

• susceptible cells grow faster and have a fitness cost (take more energy to make)

• con: sample collecting and appropriate treatment can be difficult

How can we reduce antibiotic resistance?

by using antibiotics thoughtfully

List ways antibiotics are misused

• using antibiotics in animal feed

• using outdated antibiotics

• using antibiotics for the common cold/other inappropriate conditions

• failing to complete prescribed regimens

• use in animals (ex. livestock) to prevent them from sickness

Describe the natural selection process contributing to bacterial resistance and how reducing antibiotic use will lower antibiotic resistance

• most resistance mechanisms have an associated fitness reduction/cost to maintain them when not in the presence of antibiotics

• but, continuous selection (presence of the antibiotic) is important to maintain a plasmid in a homogenous bacterial population or a genetic trait in a mixed population

• So, reducing antibiotic use (selection) should reduce antibiotic resistance rates

What are future chemotherapeutic agents?

• other animals (ex. cockroaches never get bacterial infections despite living in germ-filled environments; tapping into that ability can be effective)

• HINS light

• Phage therapy: selective way to kill off bacteria

• Block quorum sensing

• siRNA (complementary segements to genes of interest; function: bind to RNA and signal it to be broken down if deemed harmful by us)