The Control of Bacterial Growth ANTIBIOTICS & The Control of Bacterial Growth

Counting Chamber Makes thin microscopic preparation in a known volume of liquid

Plate Counts (keep track of the math)

Plate counts, continued

Plate counts, continued

Control of Bacterial Growth Natural control of bacterial growth

Control of Bacterial Growth Natural control of bacterial growth Commercial control of bacterial growth Nitrates, sulfur dioxide Temperature (eg., Pasteurization, dry heat) Salt, vinegar

Control of Bacterial Growth Natural control of bacterial growth Commercial control of bacterial growth Physical methods of disinfection Heat Filtration Radiation

Control of Bacterial Growth Natural control of bacterial growth Commercial control of bacterial growth Physical methods of disinfection Chemical methods of disinfection

Sites of action of germicidal chemicals Sterilization: removal or destruction of all microorganisms on or in a product Disinfection: elimination of most or all pathogens on or in a material Decontamination: reducing pathogens to levels which are safe to handle.

The story of Penicillin Late 1920s Alexander Fleming Scottish physician and bacteriologist Discovered that a fungal metabolite could be used to control bacterial growth Fungus: Penicillium notatum Penicillin prevented growth of Staphylococcus aureus

The story of Penicillin "the greatest contribution medical science ever made to humanity." Time magazine (1999) The Great Minds Of The Century “A spore that drifted into his lab and took root on a culture dish started a chain of events that altered forever the treatment of bacterial infections.”

Action of penicillin Overlay plate A colony of the fungus Penicillium notatum is allowed to grow on agar. The plate is overlaid with a thin film of molten agar containing bacteria. Penicillin production by the fungus creates a zone of growth inhibition of the bacteria. Penicillin rapidly became the "wonder drug“ One of the single most effective drugs of the last century

The extension of human lifespan Antibiotics Public health, Sanitation, Immunization

Antimicrobial Drugs Antimicrobial drugs: Low-molecular weight substances, natural or synthetic, which kill or inhibit the growth of microorganisms with relatively little harm to the host. Antibiotic: a compound naturally produced by molds or bacteria that kills or inhibits the growth of other microorganisms. Antiviral: a drug that interferes with the infection cycle of a virus. Bactericidal: kills bacteria. Bacteriostatic: inhibits the growth of bacteria.

Control of Bacterial Growth Natural control of bacterial growth Commercial control of bacterial growth Physical methods of disinfection Chemical methods of disinfection Features of antimicrobial drugs Selective toxicity Stability Access to targets

Effectiveness of individual antibiotics Varies with: the location of the infection the ability of the antibiotic to reach the site of infection sensitivity of the bacterial target Speed of action Side effect on the host the ability of the bacteria to resist or inactivate the antibiotic Access to the world-wide population: - should be inexpensive and easy to produce and administer - should be chemically-stable (have a long shelf-life)

Location of the infection Examples: Certain antibiotics including some of the -lactams do not function very well in the acidic environment of the stomach Some antibiotics such as bacitracin may be too toxic for internal use but can be used in topical creams for preventing wound infections

Ability of the antibiotic to reach the site of infection Oral antibiotics - simplest approach when effective Intravenous antibiotics - reserved for more serious cases Examples: Antibiotics vary in their ability to: be absorbed orally cross the blood brain barrier (BBB) Vancomycin cannot cross the intestinal lining; administer intravenously To overcome the BBB: inject large quantities of an antibiotic directly into a patient's bloodstream

Sensitivity of target Broad-spectrum (kill or inhibit a wide range of Gram-positive and Gram-negative bacteria) versus Narrow-spectrum (effective mainly against Gram-positive or Gram-negative bacteria) Limited spectrum (effective against a specific species)

Speed of action Generally faster: Bactericidal – drug kills bacteria (penicillin) Generally slower: Bacteriostatic – drug inhibits growth (tetracycline)

Side effects Antimicrobial therapy: Optimize the therapeutic index (ratio between the effective dose and the toxic dose) Stomach Pain/diarrhea Nausea   Yeast and other fungal infection  Allergic reactions Disrupt function of liver, kidneys and other organs

Control of Bacterial Growth Natural control of bacterial growth Commercial control of bacterial growth Physical methods of disinfection Chemical methods of disinfection Features of antimicrobial drugs Mechanisms of action of antibacterial drugs

Targets of antibiotic action Drug target is present in bacteria, but absent in host cells (preferred). Drug targets a step in metabolism that is essential in bacteria but not in host. Drug target is present in both bacteria and host, but the bacterial target is more sensitive to drug.

Targets of antibacterial medications

Peptidoglycan

Inhibition of peptidoglycan synthesis Penicillin interferes with peptidoglycan cross-linking through interaction with Penicillin-Binding Proteins (PBPs).

The effect of penicillin on a cell

Antibacterial medications that interfere with cell wall synthesis

The b-lactam rings of penicillin and cephalosporin

The penicillin family

Vancomycin Glycopeptide antibiotic Given intravenously – commonly used for nosocomial infections Vancomycin binds to the D-ala-D-ala terminal peptide on peptidoglycan precursors and prevents chain elongation - last resort

Antibiotics that inhibit prokaryotic protein synthesis Anti-ribosomal antibiotics – second largest class of antibiotics - selective; structural differences between ribosomes of bacteria and host cells - affect different stages of protein synthesis Block initiation Block chain elongation oxazolidinones

Control of Bacterial Growth Natural control of bacterial growth Commercial control of bacterial growth Physical methods of disinfection Chemical methods of disinfection Features of antimicrobial drugs Mechanisms of action of antibacterial drugs Determining susceptibility to antimicrobial drugs

Determining susceptibility to antimicrobial drugs Minimum inhibitory concentration (MIC) Minimum bactericidal concentration (MBC) Dilution assays and disc sensitivity assays

Determining the minimum inhibitory concentration (MIC) of an antimicrobial drug

The Kirby-Bauer method for determining drug susceptibility Size of the zone reflects sensitivity to the drug

Control of Bacterial Growth Natural control of bacterial growth Commercial control of bacterial growth Physical methods of disinfection Chemical methods of disinfection Features of antimicrobial drugs Mechanisms of action of antibacterial drugs Determining susceptibility to antimicrobial drugs Resistance to antimicrobial drugs

Antibiotic resistance Clatworthy et al Nature Chemical Biology, 2007

Antibiotic resistance Resistance to more than 15 drugs has become one of the world’s most pressing public health problems 70 percent of nosocomial bacteria - resistant to at least one of the drugs used to treat infections Some organisms - resistant to all approved antibiotics and must be treated with experimental and potentially toxic drugs Super bug

The selective advantage of drug resistance

Mechanisms of acquired antimicrobial resistance

Resistance to antibiotics For an antibiotic to exert its effect it must: be transported to the site of action associate with bacteria and penetrate their cell envelope bind to their specific molecular target Resistance to drug can occur at any step in this process. Intrinsic resistance Acquired resistance: resistance that develops through mutation or acquisition of new genes.

Mechanisms of resistance Synthesis of enzymes that break down the drug Chemical modification of the drug Prevention of access to the target site by blocking uptake Prevention of access to the target by increasing efflux of the drug Increased activity of an alternative pathway Modification of the target site

-lactams and resistance The principal mechanism of resistance to -lactams (penicillin) – inactivating enzymes called -lactamases. They hydrolyze the -lactam ring. Gram-positives produce extracellular -lactamases Gram-negatives make periplasmic -lactamases

Vancomycin

Resistance to anti-ribosomal antibiotics Enzymes modify antibiotics Pumps actively excrete antibiotics Methylation of ribosomal RNA Mutations in ribosomal RNA or ribosomal S50 subunit

Quinolones (nalidixic acid) Inhibit the action of topoisomerases. They trap these enzymes in the act of cutting DNA thus promoting the formation and persistence of double strand breaks in the bacterial chromosome. Resistance I: mutations in the topoisomerases that prevent/reduce the binding of the drugs. Resistance II: efflux pumps remove quinolones from the bacterial cells.

Rifampicin Binds to the RNA polymerase to prevent transcription One of the few drugs that can treat tuberculosis Resistance: point mutations in RNA polymerase

Multidrug therapy Advantage: The chance that a single bacterium becomes resistant to both antibiotics is small The effectiveness of the drugs together is greater than that of either drug alone (synergism) Disadvantage: The action of one drug reduces the efficiency of the other (antagonism). Indifference: Alone or in combination - no better no worse

Control of Bacterial Growth Natural control of bacterial growth Commercial control of bacterial growth Physical methods of disinfection Chemical methods of disinfection Features of antimicrobial drugs Mechanisms of action of antibacterial drugs Determining susceptibility to antimicrobial drugs Resistance to antimicrobial drugs New approaches to discovery of antimicrobial drugs

Efforts to identify and synthesize new antibiotics Continue modification of existing antibiotics Continue screening of soil isolates Rational drug design – use crystal structure of target molecule as guide for design and synthesis of chemicals that bind to and inactivate the target molecule Combinatorial chemistry – create an array of chemical derivatives and test this library against particular bacterial targets Screening of organisms from other environments

Inhibitor screen High-throughput screen of ~ 50,000-100,000 chemical compound libraries

New paradigm for antibiotic development Traditional antibiotics: kill or inhibit growth of bacteria New way: target virulence properties Target bacterial proteins that promote infection - Prevent colonization - Block the activity of toxins or other virulence factors Prevent their binding to receptors or intracellular targets Prevent their secretion

Target virulence properties

Three petri dishes

Inhibitors of folic acid metabolism