Microbiology Notes

Microbiology

1. Introduction

Gram negative bacteria have a cell wall and a further outer membrane. Gram negative bacteria stain pink/red with gram stain, as they have an outer membrane and a smaller amount of peptidoglycan in their cell wall.

Gram positive bacteria just a cell wall and have no outer membrane, although a capsule may be present. Gram positive bacteria stain blue with gram stain, as they have a thick cell wall composed of peptidoglycan and teichoic acids.

Gram staining procedure consists of:

• Primary stain (crystal violet)

• Mordant (Gram’s iodine)

• Alcohol as a decolouriser

• Counterstain (carbol fuchsin)

Acid fast bacteria are similar to gram positive bacteria, but have mycolic acids in the cell wall producing a ‘waxy coat’. This is assessed by the Ziehl-Neelsen stain using hot concentration carbol fuchsin. Acid fast bacteria stain red.

Mycoplasmas have triple layered limiting membranes but lack cell walls and have no gram stain.

To colonise and grow, pathogens need to replicate to avoid elimination or survive where they cannot be eliminated from. To replicate, pathogens must tolerate the host environment, evade host defence and compete successfully for nutrition. Iron availability is the most common limiting factor.

Avoidance of host defence mechanisms is essential for establishment of infections. Some examples include:

• O-antigen – lipopolysaccharide protein present on the outer membrane of gram negative bacteria. Protects the cell from some chemical attack.

• Capsule – a layer of extruded compound. Interferes with phagocytosis and associated with virulence.

• Fc binding proteins – bind to heavy chain Fc region of immunoglobulins. This disrupts opsonisation and phagocytosis.

• Leukotoxins – group of exotoxins that act against leukocytes.

• Inhibition of phagolysosome – survival inside the phagolysosome. Seen in Bacillus anthracis, Mycobacterium tuberculosis and Staphylococcus aureus.

• Escape from phagolysosome – employed by Rickettsiae species.

• Antigenic mimicry/variation – changing antigen expressed to avoid host defence.

• Coagulase – enables conversion of fibrinogen to fibrin.

Non-specific immunity to microorganisms include anatomical structures such as the skin and mucous membranes, inhibitory secretions such as hydrochloric acid in the stomach, antimicrobial factors such as lysozymes and interferons, and immunity cells such as macrophages and neutrophils.

Specific immunity includes T and B lymphocytes and secretions of cytokines and antibodies.

Bacterial infection may be

• Acute – short severe course of infection.

• Sub-acute – clinical effects but less intense than acute.

• Chronic – failure to eliminate pathogen.

• Persistent – infections where cell mediated and humoral defence are minimal.

• Latent or carrier – sub-clinical infections with sporadic shedding.

This is influenced by the host pathogen relationship. Factors that influence this include: pathogen related factors such as virulence, route of entry, infective dose and resistance to immunity; host factors such as species, breed, genetics, age, sex, physiological function and immune competence; and modifying risk factors such as nutrition, stress, weather, drugs and concurrent disease.

Endotoxins are components of the cell wall of gram negative bacteria released following cell death. They are lipopolysaccharide complexes and heat stable. They have moderate, non-specific generalised activity, and are weakly antigenic.

Exotoxins are produced by live bacteria, both gram positive and negative. They are proteins and typically heat labile. They are potent toxins usually with specific activity and are highly antigenic.

The difference in biochemical structure between bacteria (i.e. gram positive of negative, acid fast, mycoplasma etc.) results in different requirements for antibiotics, as targets and resistance may be different.

Common organisms known from previous teaching:

2. Antimicrobials

Antibiotics reduce the effects of infection and allow host mechanisms to clear the infection. Some antibiotics may be chosen to directly clear the infection, or may be chosen for use prophylactically where risk of infection is high. Antibiotics have also been used as growth promoters. Antibiotic use is regulated by the same rules as for other pharmaceuticals.

An antibiotic is defined as a low molecular weight microbial metabolite which can kill or inhibit the growth of susceptible bacteria. Antibiotics include chemical substances produced by various species of microorganisms which may suppress or kill other organisms.

‘Magic bullets’ target unique features of a pathogen compared to the host (eukaryotic versus prokaryotic). However, some antibiotics can affect the host either directly or through responses to the structure. It is also important to consider the impact of killing pathogens – for example, gram negative bacteria release Lipid A (an endotoxin component) as they die, which has a...

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