Microbiology Lab Review: Chapter 18

Lab Review

Chapter 18: DNA Gel Electrophoresis

1. [CH-18-01] In what way are restriction enzymes specific?

Restriction enzymes cut DNA at specific palindromic sequences (same DNA sequence in 3’ to 5’ direction). The palindromic sequences vary depending on the specific restriction enzyme.

2. [CH-18-02] If you cut the same DNA with two different restriction enzymes separately would you get the same banding pattern? Explain the answer.

NO, the restriction enzymes each have their own specific recognized palindromic sequence. The resulting banding patterns would vary because of the different sizes of the fragments.

3. [CH-18-03] Explain how electrophoresis works –

DNA is separated in fragments by restriction enzymes. The fragments are then placed on a gel submerged in a buffer solution to be separated based on size using an electrical field (cathode – negative to anode – positive).

4. [CH-18-04] In your lab class, you have used gel electrophoresis to separate fragments of DNA. Can you use gel electrophoresis to separate proteins? Yes, or No, explain your answer –

YES, BUT since proteins are not always negatively charged they need to be treated with sodium dodecyl sulfate (unfolds proteins into linear and coats them with negative charges) so that they will move with the electrical field.

5. [CH-18-05] If the banding pattern from two DNA samples is the same, does it mean that the samples are identical? If your answer is no, then what would you have to do to show that they are identical? –

NO, the banding patterns rely heavily on the type of restriction enzyme used and the sample DNA. To get more accurate confirmations, the DNA samples have to be tested with multiple restriction enzymes and the banding patterns resulting form the multiple fragments have to be compared.

6. [CH-18-06] DNA electrophoresis is used for genomic typing of bacterial strains. What are serotyping and phage typing of bacterial strains? –

Bacterial serotyping – Classification of bacteria into specific strains depending on their DNA banding patterns.
Phage typing – Determining a specific strain based on the bacteriophage that targets and kills that strain.

7. [CH-18-07] What is the difference between sticky and blunt ends of DNA fragments formed during digestion by restriction enzymes? –

Sticky ends – Forms overhang (stretch of unpaired nucleotides), the orientation of new inserted DNA is specific b/c overhangs.
Blunt ends – NO overhang, DNA strand ends at a base pair, the potential for inserting DNA in wrong orientation.

8. [CH-18-08] Why is DNA moving toward anode during gel electrophoresis? –

DNA is negatively charged, because of oxygen bonded to phosphate, and move towards the (+) anode. Opposites attract (-) → (+).

9. [CH-18-09] Explain how the molecules are separated during gel electrophoresis.

Fragments of the DNA formed by restriction enzymes move towards the (+) anode. The larger fragments are slowed by resistance as they move through the pores of the cell, while the smaller fragments move more easily and get farther.

10. [CH-18-10] Why is DNA sample mixed with sample loading buffer before it is loaded on the gel?
    Sample loading buffer combines with DNA to increase the density of the sample and ensures that the DNA stays within the wells of the gel. Loading buffer also contains dyes that combine with the DNA fragments and allow us to visually “see” the fragments moving.

Microbiology Lecture 9 Review: Chapter 21

Lecture Review

Chapter 21

1.    [21-01] Compare the properties of bacterial genera Staphylococcus and Streptococcus Name principal species that are human pathogens and diseases they cause – Lecture 9, Slides 5, 6 –
Similarities: Both genera belong to phylum Firmicutes that includes Gram-positive non-motile cocci with low GC content that do NOT form endospores. Both genera include some species that can produce exotoxins and are human pathogens.

Differences: Genus Staphylococcus includes salt-tolerant, catalase-positive facultative anaerobic cocci growing in clusters. Streptococci are part of human normal flora. Principal human pathogen is Staphylococcus aureus, which is also a part of human normal flora. Strains of S. aureus can cause in humans wide variety of infections, from boils to scalded skin syndrome. Genus Streptococcus includes salt-sensitive, catalase-negative aerotolerant cocci that are growing in chains. Streptococci are not part of human flora, but they are often carried asymptomatically. Principal human pathogen is Streptoccocus pyogenes that can cause various infections in humans, from streptococcal pharyngitis (strep throat) to necrotizing fasciitis (flesh-eating disease).

2.    [21-02] Compare cellular properties of Staphylococcus aureus and Streptococcus pyogenes. Name diseases they cause – Lecture 9, Slides 7 – 
Staphylococcus aureus is salt-tolerant, coagulase-positive, catalase-positive cocci growing in clusters. They are facultative anaerobes that can produce various virulence factors, like capsule, adhesins (clumping factor A, fibronectin-binding protein A), proteins covering bacterial cells with human proteins (coagulase, protein A), various enzymes and toxins. It can form biofilm. Depending on set of virulence factors produced, strains of S. aureus can cause various infectious diseases in humans, from boils to food poisoning, to pneumonia and to scalded skin syndrome.

Streptococcus pyogenes is salt-sensitive, coagulase-negative, catalase-negative cocci growing in chains. They are aerotolerant bacteria that can produce various virulence factors, like non-antigenic capsule, adhesins (M protein, protein F), protein covering bacterial cells with human proteins (protein G), various enzymes and toxins. It can form biofilm. Depending on set of virulence factors produced, strains of S. pyogenes can cause various infectious diseases in humans, from impetigo, to streptococcal pharyngitis, to necrotizing fasciitis. Due to frequent very serious complications, even mild infection caused by S. pyogenes MUST BE TREATED with antibiotics.

3.    [21-03] Besides capsules, what are other virulence factors of S. aureus and S. pyogenes preventing killing of bacterial cells by phagocytes? – Lecture 9, Notes of Slides 5, 6, 8 –
Virulence factors of S. aureus: Protein A binds IgG inverted; Leukocidin is secretory toxin that kills phagocytes by forming pores in their plasma (cytoplasmic) membrane. Staphyloxantin neutralizes hydrogen peroxide produced by attacking phagocyte; Biofilm interferes with ingestion of bacteria by phagocytes.

Virulence factors of S. pyogenes: Protein G binds IgG inverted; M protein inhibit activity of C3 convertase thus preventing activation of compliment system and interfering with attraction of phagocytes to the site of infection.

4.    [21-04] Describe the functions of virulence factors involved in covering the cells of S. aureus and S. pyogenes by human proteins – Lecture 9, Slides 5, 6, 8 –
Virulence factors of Staphylococcus aureus – Protein A binds IgG inverted thus interfering with phagocytosis (prevents ingestion); Clumping factor A binds to fibrin, fibrinogen, plastic devices; FnBPA (fibronectin-binding protein A) binds to acellular tissue substances and plastic devices.; Coagulase deposit human proteins on bacterial cell and, thus interfering with phagocytosis. It is found in the most virulent strains.

Virulence factors of Streptococcus pyogenes – Protein G binds IgG inverted thus interfering with phagocytosis (prevents ingestion); M protein acts as adhesin but it also inhibits the activity of C3 convertase, thus preventing activation of compliment system and interfering with attraction of phagocytes to the site of infection.; Protein F is fibronectin-binding protein.

5.    [21-05] Diphtheria – describe signs and symptoms; describe etiological agent and its virulence factors – Lecture 9, Slides 12, 13, –

Signs & symptoms: Mild sore throat → fatigue, malaise, barking cough → Bull neck, whitish pseudo-membrane formed on tonsils/pharynx, bluish color of skin, difficulty breathing

Etiological agent: Corynebacterium diphtheriae (Gram-positive bacillus, noncapsulated, nonmotile)
Virulence factors: Diphthin – IgA protease, Diphtheria toxin – A-B toxin inhibits protein synthesis in targets cells by binding to EF-2.

6.    [21-06] Diphtheria – describe pathogenesis and epidemiology – Lecture 9, Slides 13, 14
Pathogenesis: Corynebacterium diphtheriae is part of normal body flora → Undergoes lysogenic conversion with ß-virus (bacteriophage) → transformed bacteria produces diphtheria toxin → Inactive A-B toxin enters bloodstream and spreads → B part of toxin binds to receptor on target cell → cell uptakes toxin via endocytosis → A part cleaved from A-B → A part of toxin enters cytosol and inhibits protein synthesis by binding to elongation factor 2 (EF2) → inhibits ribosome movement along mRNA (Targets: kidneys, heart muscle, nervous system).

Epidemiology: disease is contagious, notifiable disease
Reservoir – humans (asymptomatic cases, people with active disease, people recovering from disease (still carrying bacteria), contaminated fomites
Transmission – Respiratory droplets, direct contacts (Handshake), indirect contact (fomites)
Number of cases reported – From 1980-2018 only 60 cases
Mortality rate –20% death rate even if promptly treated

7.    [21-07] Diphtheria – describe prevention and treatment – Lecture 9, Slides 15, 16 –

Prevention: C. diphtheriae is part or normal microbiota → DTaP vaccine (Diphtheria toxoid + tetanus toxoid + acellular pertussis) → body forms antibodies to neutralize weakened diphtheria toxoid → later diphtheria toxin also neutralize.
DTaP part of US immunization schedule, booster every 10 years

Treatment: Diphtheria Antitoxin (Neutralize toxin in bloodstream, requires early application) + Antibiotics (Kill pathogen to prevent production of new toxin, usually penicillin/erythromycin).

8.    [21-08] Pertussis – describe signs and symptoms; describe etiological agent – Lecture 9, Slide 20 –

Signs & Symptoms: 3 stages of symptoms – (1) Catarrhal stage = Similar to common cold w/ runny nose and no fever (2) Paroxysmal stage = Severe cough spasms w/ forceful inspiration, fever, vomiting and possible seizure (3) Convalescence stage = Recovery from disease
Etiological agent: Bordetella pertussis (Gram-negative coccobacillus, obligate aerobe)
Virulence factors: Capsule – Interferes with phagocytosis, Toxins – (1) Pertussis toxin (PTx) = Adhesin, A-B toxin leads to increased production of cAMP → increases mucus production, decrease phagocytes natural killers (2) Invasive adenylate cyclase = Membrane damaging toxin and enzyme → lysis of accumulated leukocytes, increased intracellular cAMP → increase mucus production, inhibit T cell function (3) Tracheal cytotoxin = cillostasis, ciliated cells stop beating → cell death, pyrogenic (fever inducing).

9.    [21-09] Pertussis – describe pathogenesis and epidemiology – Lecture 9, Slides 21, 22
Pathogenesis: Bordetella pertussis enters respiratory tract through inhaled droplets → attaches to ciliated cells using adhesins: pertussis toxin (PTx) and filamentous hemagglutinin (FHA) → colonizes nasopharynx, trachea, bronchi, bronchioles and forms dense masses → production of toxins: pertussis toxin & invasive adenylate cyclase (IAC) increase mucous production, IAC kills leukocytes, tracheal cytotoxin causes ciliostasis and kills ciliated cells → mucous build up causes whooping cough reflex (Pneumonia as secondary infection common)

Epidemiology: disease is highly contagious, notifiable disease
Reservoir – humans only
Transmission – Airborne disease transported via respiratory droplets
Number of Cases Reported – 50% of cases occur in infants, # of cases on the rise in US from 1,700 (1980) to 12,000 (2009).
Mortality rate – 0.5% of hospitalize children

10. [21-10] Pertussis – describe prevention and treatment – Lecture 9, Slide 23 –
Prevention: DTaP vaccine (Diphtheria toxoid + tetanus toxoid + acellular pertussis) → body products antibodies to acellular portions of pertussis.
Antibiotic prophylaxis – Antibiotics given to anyone in contact with infected person.
Quarantine – Up to 6 weeks to prevent spread of disease.

Treatment: Antibiotics ONLY effective during catarrhal stage of disease (erythromycin/sulfa drugs preferred). Antibiotics ineffective during paroxysmal stage.

11. [21-11] Tuberculosis – describe signs, symptoms; describe etiological agent – Lecture 9, Slide 24 –
Signs & symptoms: Loss of appetite & fatigue → chest pain, slight fever with night sweats, progressive weight loss, chronic productive cough (if sputum has blood TB is active form, individual is infective to others).

Etiological agent: Mycobacterium tuberculosis and other mycobacterium species (M. bovis, M. africanum, M. canetti, M. microtti.

Virulence factor: Components of cell wall → acid-fast, mycolic acid (extremely hydrophobic cell wall)

12. [21-12] Tuberculosis – describe pathogenesis and diagnostics – Lecture 9, Slide 25, 29
Pathogenesis: Damage done via Type IV Delayed allergy reaction.
Pathogen enters lung alveoli → Ingested by pulmonary macrophage → TB cell wall prevents fusion of phagosome and lysosome → Mycobacterium survive and multiply inside macrophage, forming primary site of infection → More macrophages are attracted to site of infection, fuse with already infected macrophages, BUT fail to kill bacteria → more macrophages are attracted and form protective layer, tubercle → formation of tubercle results in chronic inflammation reaction → Slow maturation of tubercle and calcification → IF tubercle breaks up, leaks blood and bacteria into lungs → Microbe can spread to other parts of body forming secondary site of infection.

Diagnostics: (Tuberculin/Montoux/PPD test) Standard dose of tuberculin antigen injected intradermally → reaction read in 48-72 hrs for induration diameter → IF NO induration or <5mm = negative, IF induration ≥5mm = positive → X-ray of chest take for positive confirmation of TB.
(QuantiFERON®-TB Gold test) Blood test → Good for patients who have taken BCG vaccine.

13. [21-13] Tuberculosis – describe epidemiology – Lecture 9, Slides 26, 27, 28 –

Epidemiology: Contagious (active form), notifiable disease
Reservoir – Humans ONLY
Transmission – Respiratory droplets (Inhaled microbes → ID₅₀ = 10), animals (cow milk)
Number of cases reported – Worldwide TB cases have doubled to 6 million (80% of new cases are from Brazil, China, India, Malaysia, and Russia), US decrease from 25,000 (1993) to 9,400 (2014)

Mortality rate – Asymptomatic in 90% cases →10% cases develop into active TB (IF secondary site of infection untreated, almost 100% mortality)

14. [21-14] Tuberculosis – describe prevention, and treatment – Lecture 9, Slides 29 –

Prevention: BCG (Bacillus Calmette–Guérin) vaccine widely used in many parts of world, 50%-71% effective (NOT in US) → US uses Tuberculin test to detect those previously exposed to pathogen.
Direct Observed Therapy Short Course (DOTS) –Method to insure medication compliance from patient, healthcare worker watches patient take each dose of medication.

Treatment: After positive confirmation of tuberculosis → First 2 months: Rifampin, Isoniazid, Pyrazinamide, Ethambutol → Next 4-7 months: Isoniazid and Rifampin.

Bactericidal antibiotics over long period (6 months+), cocktail of drugs to reduce hydrophobic cell wall (Ethambutol, Isoniazid, Pyrazinamide) and bactericidal antibiotic (Rifampin – Inhibit nucleic acid synthesis) or (Streptomycin – inhibit protein synthesis).

15. [21-15] Influenza – describe etiological agent. What are antigenic drift and antigenic shift? Why is influenza virus undergoing antigenic drift and antigenic shift? – Lecture 9, Slides 36-37 –
Signs & Symptoms: Short incubation period (1-2days) → headache, fever, muscle pain, dry cough → acute symptoms abate within a week, though cough and fatigue may linger
Etiological agent: Influenza virus types A, B, C – Single-stranded, segmented RNA genome (A & B – 8 segments, C – 7 segments). Envelop has two essential proteins: hemmagglutinin (viral adhesin) can agglutinate RBC & neuraminidase an enzyme that is involved In budding of newly assembled viral particles from host cells.

Virulence factors: Hemmagglutinin and neuraminidase proteins.
Antigens of the virus constantly changing due to antigenic drift and antigenic shift.
Antigenic drift – Characteristic of RNA viruses, mutation to genes by RNA polymerase because no proof reading capability = multiple errors. Viral constantly changing at slow pace.
Antigenic shift – Result of infection of cell by two different viral strains at the same time → mixing of viral genes (RNA segments) → new recombinant viral strains.

Pathogenesis: Virus enters respiratory track → attaches to epithelial cells via hemagglutinin → Virus is temperate and slowly replicates in host cell → New virions released via budding → infected cells eventually die → inflammation reaction (sets off signs and symptoms of disease) → Humoral immune response (antibodies) quickly suppress viral replication.
Epidemiology: Contagious, notifiable disease, zoonosis
Reservoir –Humans, animals, birds
Transmission –Respiratory droplets, contaminated fomites (touching face after touching fomite)
Number of cases reported –Outbreaks occur annually worldwide (~500,000 deaths reported worldwide, 10,000-40,000 in USA)
Mortality rate – 1.6 deaths/100,000

Prevention: Vaccine produced from attenuated viruses grown in embryonic eggs.
Quadrivalent (4 strains), Trivalent (3 strains). (80-90% effective)

Treatment: Various antiviral drugs available (70-90% effective if taken early)
Neurominidase inhibitors –Prevent budding of viral particales off the cells (Ex: Tamuflu, Relenza, Inavir)
M2 proton channel inhibitors – Prevent replication of virus inside the cell (Ex: Amantidine, Rimantidine)

Microbiology Lecture 8 Review: Chapter 18, 20

Lecture Review

Chapter 18 - Application of Immune Responses

1.    [18-01] Compare attenuated vaccine versus inactivated vaccine. Give examples of each – Lecture 8, Slides 3 and 4. –
Attenuated vaccine –Weakened, but still alive pathogen. Can still grow and cause disease in immune-compromised individuals. Usually, one injection is sufficient.
Ex: Polio (Sabin), Measles, Mumps, Rubella (MMR), Chickenpox (Varicella-Zoster), Smallpox (Vaccinia virus)
Inactivated vaccine –Killed pathogen/parts of the pathogen. Cannot cause disease in immune-compromised individuals. Usually, need multiple injections to develop strong immunity.
Ex: Polio (Stalk), Hepatitis, Rabies, Diphtheria, Tetanus, Pertussis (DTap), Meningitis (MCN-4, MPSV-4, Hib), Pneumonia (PCV-7, PCV-13).

2.    [18-02] Describe the differences between subunit vaccine, toxoid and conjugated vaccines. Give example of each – Lecture 8, Slide 4 (notes). –
Subunit vaccine: Contain purified pathogen’s key protein antigens OR antigenic fragments. Ex: Acellular pertussis (part of DTap vaccine)
Toxoid: Toxins from a pathogen that is inactivated. Ex: Diphtheria and Tetanus toxoids (part of DTap vaccine)
Conjugated vaccine: Contains polysaccharides linked to carrier proteins à Turns polysaccharides into T-cell dependent antigens. Ex: Hib vaccine, PCV-7, PCV-13 (against S.pneumoniae)

3.    [18-03] Using poliomyelitis as examples, explain the importance of immunization in prevention of infectious diseases – Lecture 8, Slides 5, 6 –
Poliomyelitis – Viral disease transferred via fecal-oral route (mainly via contaminated water). 99% of infected NO signs & symptoms or mild ones. 1% develops paralytic polio.
Before vaccine ~ 50,000 cases every year in US.
After the introduction of Salk and Sabin vaccines, the number of cases fell dramatically. Leading to last indigenous case was in 1980. NO cases reported in US since 1998.

4.    [18-04] Describe DTaP and MMR vaccines used in the USA in the prevention of infectious diseases. – Lecture 8, Slide 8 –
DTap – Contains diphtheria toxoid, tetanus toxoid, and acellular portion of Bordetella pertussis cell wall.

MMR – Three attenuated viruses: measles, mumps, rubella.
*Both are part of the immunization schedule.

5.    [18-05] Describe the properties of polyclonal and monoclonal antibodies. Which antibody (polyclonal or monoclonal) is more likely to be suited for medical use? Explain why. – Lecture 8, Slides 10, 11. –
Properties of polyclonal antibody. They are called polyclonal as they are mixture of antibodies produced by different clones of plasma cells descended from various naïve B-cells. These antibodies can recognize various epitopes. Therefore, are more likely cross-react with other antigens.

Properties of monoclonal antibody. They are called monoclonal as they contain the antibody produced by single clone of plasma cells descended from single naïve B-cell. Produced by specific cell cultures called hybridomas. These antibodies can recognize only single epitope. Therefore, they are less likely to cross-react with other antigens, including human cells and tissues.

Medical use of antibodies. Monoclonal antibodies are better suited for medical use as they have higher specificity than polyclonal antibodies and they are less likely to cross-react with human antigens. Out of all monoclonal antibodies, humanized antibodies are the best for medical use, as they do not trigger an immune response in patients and can be used multiple times in the same patient.

6.    [18-06] Compare chimerical and humanized monoclonal antibodies. Which one is better suited for medical use in humans and why? – Lecture 8, Slide 12. –
Chimerical antibody is monoclonal antibody produced in hybridomas derived from genetically modified mice, when constant region of mouse antibody is replaced with constant region of human antibody. Chimeric antibody has reduced antigenic activity in humans (immune response against them is muted) and it can be injected into patient more than once.

Humanized antibody is monoclonal antibody produced in hybridomas derived from genetically modified mice, when constant and some of variable regions of mouse antibody are replaced with the human ones. If the humanized antibody is injected, it will not trigger the immune response in humans.

Both chimerical and humanized monoclonal antibodies can be used as immunotoxins, as they both can deliver the toxin specifically to targeted cell and kill it. Humanized antibodies are better suited for use in humans than chimerical antibody as they as less likely to case immune response in humans. Nine monoclonal antibodies have been approved for cancer therapy.

7.    [18-07] Describe fluorescent antibody test – Lecture 8, Slide 13 –
Two pathways: Direct labeling of antibody OR Indirect labeling of antibody
Direct labeling = Fluorescent dye is attached to antibody directed against microbe.

Indirect labeling = Involved secondary antibody that binds to antibody directed against microbe. (Better labeling b/c same-tagged secondary antibody can be used to label any antibody produced in the same biological species). 

Chapter 20 - Antimicrobial Medications

8.    [20-01] Define the terms “selective toxicity”, “bactericidal”, “bacteriostatic”, “narrow-spectrum”, “broad-spectrum” as they are applied to antibiotics. Give examples – Lecture 8, Slide 14 –
Selective toxicity –Ability to interfere with microbial growth without causing damage to patient. Therapeutic index = [lowest toxic to human] / [lowest toxic to microbe]

Bactericidal –Kills bacteria (Used against acid-fast bacteria). Ex: penicillin, streptomycin.
Minimal bactericidal concentration = [lowest] that kills 99.9% of microbes.
Bacteriostatic –Stops bacterial growth (Used against Gram-negative bacteria). Microbes resume growth after drug withdrawal. Ex: Tetracycline, erythromycin

Narrow-spectrum– Active against a specific group of microbes. Ex: penicillin, erythromycin (Gram positive bacteria only).
Broad-spectrum– Active against a wide range of various microbes. Ex: Tetracycline (Gram+/-), streptomycin (Gram+/-, acid-fast).

9.    [20-02] Compare three groups of antibiotics that inhibit the cell wall synthesis – Lecture 8, Slide 18 –

ß-lactum drugs – Competing with side chain of peptidoglycan for binding to active site of penicillian-binding protein (PBP). Binds to PBP preventing cross-linking of NAM-NAG polymeric fibers. Ex: Pencillian, Cephalosporins.

Vancomycin – Competing with penicillin-binding protein for binding to peptide side chain. Binds to side chains of NAM in peptidoglycan, preventing NAM-NAG cross-linking. Ex: Vancomycin. CANNOT cross outer membrane of Gram-negative bacteria, only used against gram-positive bacteria. Poor absorption from GI tract, so much is given intravenously.

Bacitracin –Interferes with the delivery of peptidoglycan precursors across the membrane. The drug inhibits de-phosphorylation of bactoprenol-phosphate (carrier for precursors). Highly toxic so limited to topical applications. Ex: Bacitracin.

10. [20-03] Mechanisms of bacterial resistance to b (beta) lactam drugs and vancomycin – Lecture 8, Slide 18 –
Bacterial resistance to ß lactam drugs:
1 – Mutation in penicillin-binding protein so it no longer binds penicillin, but still functions in cross-linking peptidoglycan fibers.
2 – Microbe produces enzyme (penicillases/ß-lactamases that break down ß-lactum ring of penicillins/cephahlosporins).

Bacterial resistance to vancomycin:
Genetic mutation that leads to replacement of terminal amino acid in pentapeptide side chain of peptidoglycan. NO longer binds vancomycin, but still can be cross-linked by penicillin binding protein.

11. [20-04] Compare the properties of Streptomycin and Tetracycline – Lecture 8, Slide 19 –
Streptomycin
Class = Aminoglycosides
Broad-spectrum, Bactericidal
Binds irreversibly to 30S ribosomal subunit blocking initiation of translation.
Effective Gram+/-, acid-fast. NOT effective against Enterococcus and Streptococcus.
Side effects: Extended use may lead to nephrotoxicity.
Resistance: Enzymes to modify antibiotic.

Tetracyline
Class = Tetracyclines
Broad-spectrum, Bacteriostatic
Stops growth by reversible binding to 30S ribosomal subunit + blocking tRNA binding to ribosome.
Effective Gram+/-.
Side effects: Discoloration of teeth when used by young children.
Resistance: Reduce uptake/increase excretion.

12. [20-05] Compare the properties of Erythromycin and Chloramphenicol – Lecture 8, Slide 19 –
Erythromycin
Class = Macrolides
Narrow-spectrum, Bacteriostatic
Inhibits bacterial growth by reversible binding to 50S ribosomal subunit + blocking translocation of ribosome → blocks elongation of polypeptide chain during protein synthesis.
Effective Gram+.
Resistance: Enzyme that chemically modifies 23S ribosomal RNA via methylation.

Chloramphenicol
Class = Chloramphenicol
Broad-spectrum, Bacteriostatic
Inhibits bacterial growth through reversible binding to 50S ribosomal subunit + blocking peptide bond formation → blocks elongation of polypeptide chain during protein synthesis.
Effective Gram+/-.
Side effects: Toxic → last resort. Can cause fatal aplastic anemia, increase risk of leukemia.
Resistance: Enzyme that acetylates antibiotic.

13. [20-06] Compare the properties of Ciprofloxacin and Rifampin (Rifampicin) – Lecture 8, Slide 20 –

Ciprofloxacin

Class = Fluoroquinolones
Broad-spectrum, Bactericidal
Irreversibly inhibits DNA gyrase and other enzymes involved in DNA synthesis.
Effective Gram+/-.
Side effects: Tendon, muscle, central nervous system issues.
Resistance: Genetic mutations changes targeted enzymes.

Rifampin
Class = Rifamycins
Broad spectrum, Bactericidal
Irreversibly inhibits RNA polymerase and RNA synthesis.
Effective against Gram+/-. Used in TB treatment.
Side effects: Hepatotoxicity.
Resistance: Genetic mutations changes targeted enzymes.

14. [20-07] Describe the strategy used in antibiotic treatment of infections caused by acid-fast bacteria – Lecture 8, Slide 22 –

1 – Reduce hydrophobicity of cell wall
Ethambutol– Prevents attachment of mycolic acids to cell wall.
Isoniazid– Inhibits synthesis of mycolic acid.
Pyrazinamide– Action NOT clear, ≈ inhibits fatty acid synthesis.

2 – Bactericidal kill microbe
Streptomycin – Irreversibly binds to ribosomes + inhibits protein synthesis.
Rifampin– Irreversibly binds to RNA polymerase + inhibits RNA synthesis.
Ciprofloxacin– Irreversibly binds to DNA gyrase + inhibits DNA synthesis.

Directly Observed Therapy – Health care professional makes sure patient takes the right dose of medication (watches them swallow every pill).

15. [20-08] Describe the mechanisms of microbial resistance to antibiotics. Give examples – Lecture 8, Slides 24 –  
Microbes can become resistant to antibiotics either through mutations in existing genes or through acquisition of new genes.

Mutations in existing genes may lead to alteration of target molecule so that antibiotic no longer binds to it or alteration of the molecule responsible in the transport of antibiotic into the cell. Examples: Resistance to vancomycin is due to replacement of terminal D-Ala by lactic acid in peptidoglycan precursor’s side chain; Resistance to penicillin is due to modification of penicillin-binding protein – PBP type 2a in S. aureus; Resistance to tetracyclines may occur due to either reduced drug uptake or Increased excretion rate by microbial cell; Resistance to ciprofloxacin, rifampin, sulfa drugs, and trimethoprim occur due to mutations in the genes encoding corresponding bacterial enzymes targeted by these antibiotics.

Acquisition of new genes may lead to alteration of target molecule so that antibiotic no longer binds to it or alteration of antibiotic structure and antibiotic inactivation. Examples: Acetylation of chloramphenicol by newly acquired bacterial acetyltransferase; Breakdown of β-lactam ring of penicillins or cephalosporins by newly acquired bacterial penicillinase or β-lactamase; Resistance to erythromycin due to methylation of 23S rRNA by newly acquired methylase.