basic microbiology

Cell structure

cell wall structure

· differs between Gram-positive and Gram-negative

· Gram-positive

· 3 layers

· Gram-negative

· complex multilayered structure

Function

· maintains shape of cell

· corsets cell because of high internal osmotic pressure

· injury to cell wall or inhibition of formation leads to lysis

· in hypertonic environment, cell forms

· protoplasts (G+)

· spheroplasts (G-)

· these explode when placed in isotonic environment

Layers

Cytoplasmic membrane

· innermost layer

· present in both G+ and G-

· functions

· permeability barrier

· transport system

Cell wall

· intervening layer

· gives osmotic protection

· is difference between G+ and G-

· in G+, consists of thick peptidoglycan layer

· in G-, consists of

· thin peptidoglycan layer

· lipoprotein

· outer membrane

· lipopolysaccharide

Capsule and glycocalyx

· extracellular polymer

· if forms condensed, well-defined layer, called capsule

· if forms loose meshwork of fibrils, called glycocalyx

· contributes to invasiveness and adherence

Spores

· produced by G+ rods Bacillus and Clostridium

· sporulation occurs when nutrional conditions become unfavourable

· spore liberated when mother cell undergoes autolysis

· sopre is resting cell

· highly resistant to dessication, heat and chemical agents

· spore germinates to produce single vegetative cell when favourable nutritional conditions occur

staining

Gram-stain

· response to Gram’s stain correlates with many morphological properties of bacteria

· basis is difference in cell wall

· reason unclear

Process

· smear prepared and fixed with heat

· crystal violet applied and washed

· iodine then applied and washed

· all bacteria will be stained blue at this point

· cells then treated with alcohol and acetone

· G+ cells retain crystal violet-iodine complex (blue)

· G- cells completely decolorised

· saffron counterstain applied and wahed

· G- cells take on contrasting red

· G+ cells become purple

Acid-fast stain

· Ziehl-Neelsen

· acid-fast bacteria retain carbol-fuschin stain even when decolorised with acid-alchol

· most commonly mycobacterium

Process

· smear prepared

· flooded with red carbolfuschin

· heated on steam bath

· decolorised with hydrochloric acid in alcohol

· contrasting blue counterstain applied

· acid-fast bacteria appear red

· others are blue

Antimicrobial therapy

history

· 1929 - Fleming discovers penicillin

· 1935 - Dogmagk discovers sulfonamides

· 1940 - Chain & Florey use penicillin

mechanism

· 4 basic mechanisms of action

1. cell wall synthesis

2. cell membrane permeability

3. protein synthesis

4. nucleic acid synthesis

Cell wall synthesis

· examples are

· b-lactam drugs (penicillins and cephalosporins)

· vancomycin

b-lactam drugs

· bind to penicillin receptors (PBPs, penicillin-binding proteins)

· block transpeptidation

· inhibit petidoglycan synthesis

· inhibitor of autolytic enzymes in wall removed

· cell wall lysed

· if isotonic environment, lysis occurs

· if hypertonic environment, protoplast or spherolast may be produced

· difference in susceptibility of G+ and G- to different penicillins due to structural difference in cell walls

Resistance

· insusceptibility to b-lactams dependent on production of penicillin-destroying enzymes

· called b-lactamases

· open b-lactam ring of penicillins and cephalosporins

· flucloxacillin and clavulinic acid resistant to effect

· have high affinity for b-lactam

· bind it but are not hydrolysed by it

· result is

1. efficacy

· not inactivated by b-lactamase

2. synergism

· protects simultaneously present hydrolysable penicillins (eg. amoxycillin)

· other forms of resistance exhibited by staphylococci

· absence of PBPs due to chromosomal mutation

· failure of drug to activate autolytic enzymes in cell wall

Vancomycin

· inhibits early stage of pepdidoglycan synthesis

· these stages intracellular

· requires cell wall penetration

Inhibition of cell membrane function

· examples are

· polymyxins and G- cells

· amphoterecin B and fungi

Inhibition of protein synthesis

· examples are

· gentamicin

· erythromycin

· tetracyclines

Gentamicin

· attaches to specific receptor protein on bacterial ribosome (30S)

· then blocks initiation complex of peptide formation

· results in misreading of mRNA and nonfunctional protein production

· ribosome broken up into fragments

· result is cell death

Resistance

· due to

1. lack of protein receptor on ribosome (chromosomal)

2. production of enzymes that destroy drug (plasmid-induced)

3. permeability defect (plasmid-induced)

Inhibition of nucleic acid synthesis

· examples are

· rifampicin

· sulfonamides

· trimethoprim

· quinolones (ciprofloxacin)

Rifampicin

· binds to DNA-dependent RNA polymerase

· thus inhibits RNA synthesis

Ciprofloxacin

· inhibits DNA gyrase

resistance

· many mechanisms

· two important for orthopaedics

Enzymes

· b-lactamase produced by staphylococcus to resist penicillin G

· under plasmid control

· enzymes induced by plasmids produced by some gram-negatives to resist aminoglycosides

Altered target

· some gram-negatives alter 30S binding site of bacterial ribosome

antimicrobial activity

· influenced by numerous factors

Environment

Metabolic activity

· dormant organisms have low level of biosynthetic activity

· relatively insusceptible to drug action

Distribution

· drug unequally distributed in body

· may be poor penetration into

· blood-brain barrier

· abscess

Location

· bacteria may be intracellular

· drug may not penetrate cell well

Interfering substances

· drug may be bound by blood and tissue proteins

· may be adsorbed onto exudates and necrotic debris

· may be inhibited by acid pH

Concentration

Absorption

· irregular, esp. from GIT

Distribution

· varies with different tissues

· some drugs penetrate certain tissues poorly

· CNS

· bone

· joint

Variability

· fluctuations in concentration from

· intermittent administration

· irregular absorption and excretion