14A10: Exam Report

Classify bacteria according to the Gram stain system and their shape. Give two examples for each classification. (40% of marks) List with examples the mechanisms of bacterial antibiotic resistance. (60% of marks)

63% of candidates passed this question.

Generally candidates provided an accurate classification of bacteria based upon Gram staining and  shape.  None or  poorly  staining  organisms  were  often  overlooked.  Mechanism  of resistance was also well covered, with examples. Areas of weakness were a lack of descriptive detail  and/or  omission  of  an  example  for  each  mechanism. Mechanisms  such  as  metabolic blockade  of  essential  pathways  for  antibacterial  action  and  active  removal  of  intracellular antibiotic were more commonly omitted.

T1i / 14A10: Classify bacteria according to Gram stain system and their shape. Give two examples for each classification (40% marks). List with examples the mechanisms of bacterial antibiotic resistance (60% marks).

Definitions

Bacteria = Prokaryote – single cell organism lacking a nucleus or any membrane-bound organelles

Gram Stain

  • Bacterial slides are first stained w Crystal Violet and Iodine
  • Then destained w alcohol
  • Then counter-stained w safranin

Gram +ve

  • Blue purple
  • Large peptidoglycan wall
  • Crystal violet precipitates in wall and not affected by alcohol wash

Gram –ve

    • Pink
    • From the safranin stain
    • Small peptidoglycan wall with w additional outer PM layer

Gram Indeterminate

  • Bacteria which do not respond predictably to Gram staining or have a variable pink/purple pattern
  • Cannot be classified as G +/-
  • Myocabacterium, Clostridium

Morphology & Growth Requirements

Gram -ve

Anaerobic

Rods

Baceteriods

Cocci

Veillonella

Aerobic

Rods

Pseudomonas

E Coli

Klebsiella

Hemophilus

Cocci

Neisseria

Gram +ve

Anaerobic

Rods

Clostridium

Cocci

Staphylococci

Streptococci

Enterococci

Aerobic

Rods

Listeria

Cocci

Peptococcus

Additional Classification

Catalase

    • Hydrogen peroxide added to bacterial sample
    • Presence of catalase produced oxygen
      • Catalase+ = Staphylococci
      • Catalase- = Streptococci & Enterococci

Coagulase

  • Performed on Staphylococcals
  • Coagulase is an enzyme wh cleaves fibrinogen → fibrin
  • Rabbit plasma added to bacterial sample
  • If coagulase is present, fibrin will form
  • Coag+ = S Aureus
  • Coag- = S Epidermidis

Hemolytic

    • Performed on Streptococcal
    • Streptococcal colonies are added to blood agar
    • Colour change = hemolysis
      • α haemolytic
        • dark green agar
        • due to metHb produced by organism
        • Strep pneumonia
      • β haemolytic
        • yellow agar
        • complete hemolysis
        • Strep pyogenes
      • ϒ haemolytic
        • Agar unchanged
        • E faecalis
        • E faecium

Resistance = when the maximal level of the agent tolerated is insufficient to inhibit growth

  1. Natural – bacteria do not possess molecular target for that drug
  2. Acquired:

1. Modification of antimicrobial molecule

a. Chemical Alteration of the Antibiotic

    • Production of enzymes capable of introducing chemical changes to the antimicrobial molecule
    • Example: Aminoglycoside Modifying Enzymes (AMEs) which modify the hydroxyl/amino groups of the aminoglycoside, enabling resistance E Faecium

b. Destruction of the Antibiotic Molecule

    • b-lactamases produced by bacteria hydrolyse the b-lactam ring
    • \negates penicillin
    • still sensitive to cephalosporins
    • ESBLs = even more widespread resistance
    • R to cephalosporins/penicillin/b-lactamase inhibitors (Clav Acid)
    • Only Mero is effective

2. Prevention of antibiotic reaching target

a. Decreased permeability

  • Many a/b have targets inside bacteria or on their membrane
  • Example: Vancomycin is not active against Gm- bacteria as it cannot penetrate the outer membrane
  • Example: P aeruginosa is very good at altering porin fn/expression to limit permeability of a/b

b. Efflux pumps

  • P aeruginosa possess an efflux system to pump a/b out of the cell

3. Change/Bypass of target site

    • Changing of target site = ¯affinity of drug
    • Example: VRE
      • Glycopeptides bind to D-Ala-D-Ala
      • Bacterial remodelling of cell wall to provide new substrate D-Ala-D-Lactate prevents Vancomycin binding
      • Especially common in enterococci (E Faecium)

4. Resistance due to global cell adaptive processes

  • Example: Daptomycin resistance
  • Altered cell wall metabolism resulting in changes in surface charge of the bacteria and produces an electrostatic ‘repulsion’ of Daptomycin from the cell wall of enterococci
Antibiotics

Pseudomonas Aeruginosa has been shown to possess a high level of intrinsic resistance to most a/b via

  1. Restricted outer membrane permeability
  2. Efflux systems
  3. Production of inactivating enzyme like b-lactamases

Methods of Resistance Spread

1.     Transfer of bacteria between people

2.     Transfer of R genes between bacteria

a.     Conjugation – bacteria connect through tubing & transmit plasmid

b.     Transduction – plasmid enclosed in ‘phage’ and transferred to another bacterium

c.     Transformation – bac take up DNA from enviro and incorporate into their genome