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Microbiology Notes

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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: Organism Streptococc us spp. Staphyloco ccus spp. Escherichia coli.

Bacteri a Bacteri a Bacteri a

Gram stain Gram positive Gram positive Gram negative

Salmonella sp.

Bacteri a

Gram negative

Morpholo gy Cocci chains Cocci clusters Rods

Aerobe/anae robe Facultative anaerobes Facultative anaerobes Facultative anaerobes

Straight rods

Facultative anaerobes

Notes Catalase negative Catalase positive Catalase positive Oxidase negative Catalase positive Oxidase

negative Campylobac ter sp. Enterobact er sp.

Bacteri a Bacteri a

Gram negative Gram negative

Curved rods Rods

Microaerophili c Faculative anaerobe

Neiseria sp.

Bacteri a

Gram negative

Malassezia sp. Proteus sp.

Yeast fungal Bacteri a

-

Diplococca Aerobes l- resemble kidney bean Paired or short chains Hyphae
-

Gram negative

Rods

Facultative anaerobe

Mycoplasm a sp.

Bacteri a

No stain

Pleomorph ic

Mostly facultative anaerobes

Catalase positive Oxidase negative Catalase positive Oxidase positive

Catalase positive Opportunistic Lack cell walls Triple layered cell membranes

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 link to sepsis and toxic shock. Bacteriocidal antimicrobials cause death of the organism.

Bacteriostatic antibiotics are drugs that temporarily inhibit the growth of an organism, but the effect is reversible. If the drug is removed the bacteria can regrow. Some bacteriocidal drugs require the bacteria to be growing to be effective as they affect cell wall components during growth. Therefore if used in conjunction with bacteriostatic drugs they would be ineffective. Bacteriocidal antibiotics are indicated where infections cannot be controlled by or eradicated by host mechanisms, for example as a result of immunosuppression. Bacterial sensitivity to a minimum inhibitory concentration (MIC) is required at the site of infection to achieve bacterial inhibition. Pharmacological properties will affect how the antimicrobials distribute in different tissue groups. Tissue localisation, damage to tissue, changes to host tissue and changes of bacterial metabolism in tissue are important factors. Some factors to consider include:




Intracellular organisms will not be affected by drugs that stay in the extracellular phase. Milk proteins may bind some antibiotics Local pH may reduce dissociation of some antibiotics and reduce distribution Poor blood supply and good epithelial barriers may reduce drug access. Factors may restrict access or concentration of antimicrobials at the site of infection, such as abscess formation, pus, foreign bodies, oedema fluid etc. Presence or absence of food may improve or decrease uptake of orally administered drugs.

For drugs with time dependent activity, increasing the serum concentration above MIC will not increase killing of bacteria. For drugs with concentration dependent activity, rate of killing increases with concentration increases above the MIC. Client compliance is a major issue with antimicrobial administration. The pharmacokinetics of antibiotics must also be considered, such as absorption, half-life, protein binding, Vd, metabolism and excretion. Highly lipid soluble drugs can penetrate cells and will have a large volume of distribution. Water soluble drugs remain confined to blood and extracellular fluids. There are limitations on the use of antimicrobials in food producing animals regarding maximum residue levels (MRL). Residues can persist in food. Withdrawal periods are therefore used.

Adverse reactions to antimicrobicals include direct host toxicity, toxic interference with other drugs, interference with protective host flora, tissue necrosis at injection site, impairment of host immune function, hypersensitivity and enzyme induction/inhibition. Examples include nephrotoxicity as a result of aminoglycoside use, tendon damage as a result of quinolones and liver disturbances as a result of rifampicins.

3. Antibiotics and bacteria Antibiotics can be sourced from bacteria, such as polymyxin B, bacitracin or tetracycline or from fungi, such as penicillin or cephalosporins. They work by targeting many different areas.

3.1

Antibiotics that target the cell wall

Peptidoglycan is unique to bacteria, and so provides a good target for antibiotics. It is a polymer of sugars and amino acids that forms a mesh like cell wall. Beta-lactam antibiotics interfere with the production of peptidoglycan by binding bacterial enzymes known as penicillin-binding proteins (PBPs). This stops the formation of bonds between oligopeptides in peptidoglycan and leads to failure in cell wall formation. Penicillins and cephalosporins are the largest and most important class of antibiotics that target cell wall synthesis. Other antibiotics that inhibit cell wall synthesis include bet-lactamase inhibitors and other beta-lactam antibiotics. Mutations in the PBPs that lead to reduced interactions with an antibiotic are a significant source of emerging antibiotic resistance. Other resistance is from enzymes that degrade the beta-lactams. Beta-lactam antibiotics circulate in blood and extracellular fluids without entering cells. They have short half-lives, are weak acids and non-lipid soluble. They are largely excreted in the urine.

For gram negative bacteria, porin is required to access the cell wall through the outer membrane.

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