Revision Aids for Medical Microbiology

Dr Heritage's lectures

Introduction to microorganisms

Infections may be caused by:

• viruses,
• microfungi,
• eukaryotic parasites,
• bacteria.

Viruses

Viruses are simple structures; they may not even be considered to be living organisms.

Around the nucleic acid core of a virus lies a protein coat, made up of units called capsomeres.

Some but not all viruses have an envelope.

Viruses have a nucleic acid core, either DNA or RNA but not both. 

Retroviruses have an RNA genome in the virus particle, which, upon infection, is converted into a cDNA copy that integrates into its host's genome.

Microfungi

All fungi are eukaryotic; they have cells that carry a membrane-bound nucleus and organelles.

Moulds grow as mats of filaments known either as mycelia (singular:  mycelium) or hyphae (singular: hypha).

Moulds are multicellular; yeasts are unicellular fungi (although the daughter cells are often seen budding off mother cells).

Moulds cause a variety of common, superficial infections such as ringworm and athlete's foot.

In compromised individuals they can cause much more severe infections but these are rare.

The most common yeast infection is "thrush" caused by Candida albicans.

Other eukaryotic parasites

There are various groups of eukaryotic parasites.

The simplest are the protozoa which include:

• sporozoa (such as parasites of the genus Plasmodium. These cause malaria);
• flagellate protozoa;
• ciliate protozoa;
• amoebae.

Infections caused by protozoa include, for example, toxoplasmosis, amoebic meningitis, malaria, trypanosmiasis, leishmaniasis (Kala-Azar) and amoebic dysentery as well as diarrhoea caused by Cryptosporidium spp. or Giardia intestinalis (lamblia)

Vaginal infections may be caused by the protozoan Trichomonas vaginalis.

The microbe Pneumocystis jiroveci (carinii) was for many years considered to be a protozoan because of its microscopic appearance and its behaviour, but molecular biological studies have shown this organism to be a fungus!

Helminths are multicellular parasites.  They include nematodes (roundworms), cestodes (tapeworms) and trematodes (flukes).

Bacteria

Bacteria are simple structures.

They lack a membrane-bound nucleus and so are known as prokaryotyes.

They are either round, rod-shaped or spiral.

Round bacteria are known as cocci (singular: coccus).

Rod-shaped bacteria are known as bacilli (singular: bacillus - not to be confused with the genus Bacillus).

Bacterial cell envelopes

Almost all bacteria have a cell wall made of peptidoglycan.

The bacterial cell wall is important in defining the shape of the cell, and giving the cell mechanical strength.

The bacterial cell wall is a unique biopolymer in that it contains both D- and L-amino acids, cross liking chains of sugars.

Bacteria may be divided into two groups depending on their ability to retain a  complex of crystal violet and iodine when treated with acetone or alcohol.  This is the Gram stain.

Gram-positive bacteria retain the complex when treated with solvent and have many layers of peptidoglycan in their envelope.

Gram-negative bacteria fail to retain the complex and have just one or two layers of peptidoglycan in their envelopes.

As well as the cell membrane, Gram-negative bacteria have an "outer membrane" lying outside their peptidoglycan layer(s). 

The Gram-negative outer membrane is not a conventional phospholipid bilayer; the outer leaflet comprises lipopolysaccharide (LPS).

LPS acts as an endotoxin, causing symptoms of endotoxic shock.

A few bacteria have very waxy envelopes that make them acid-fast, meaning that, once stained with strong carbol fuchsin dye, they resist decolourisation with acid (and also with alcohol).

Important acid-fast bacteria include Mycobacterium tuberculosis, the cause of tuberculosis, and Mycobacterium leprae, the cause of leprosy.

Bacterial genetic structures

The bacterial chromosome is a single circle of DNA located in a region of e cell known as the nucleoid.

Additional genetic information in bacteria may be carried on plasmids.

Plasmids code for useful information that is not essential for bacterial survival under normal conditions. 

An important group of plasmids code for antibiotic resistance - the R-plasmids or R-factors.

Plasmids are mobile genetic elements and  may transfer between strains, species and genera of bacteria.  In some cases some plasmids may transfer between bacterial families.

Toxins are sometimes coded for by plasmid genes.

Lysogens are bacteria that have been stably infected with a bacteriophage (a bacterial virus) and that carry the virus as a 'prophage'.

The gene for diphtheria toxin is encoded by prophage.

Other bacterial structures

The cytoplasm of bacteria contains polysomes - a range of ribosomes actively translating messenger RNA into proteins.

Some bacteria also have inclusion bodies within the cytoplasm. These are often energy storage resources.

Flagella are responsible for the motility of bacteria.

Gram-negative pathogenic bacteria may be covered in fine hairs called fimbriae (singular: fimbria) these help to stick to body surfaces.

Pili can attach two bacterial cells together: sex pili are necessary for the transfer of certain plasmids between bacteria.

Some bacteria are enclosed within a capsule. This protects the bacterium, even within phagocytes, helping to prevent the cell from being killed.

Some bacteria produce slime to help them to stick to surfaces.

A few species of bacteria have the ability to produce highly resistant structures known as endospores (or simply spores).

Bacteria and oxygen

Obligate aerobes have an absolute requirement for oxygen.

Facultative anaerobes can survive in the absence as well as the presence of oxygen.

Obligate anaerobes are killed by traces of oxygen.

A small group of bacteria the microaerophiles, are killed by normal atmospheric levels of oxygen, but yet require traces of oxygen to grow.

Bacteria and temperature

Bacteria that grow at very low temperatures are known as psychrotrophs.

Bacteria found to grow at high temperatures are known as thermophiles.

Those that grow at moderate temperatures are known as mesophiles.

Medically important bacterial species

Pathogenic Gram-positive cocci

There are two major groups of Gram-positive cocci that are of medical importance: the STAPHYLOCOCCI and the STREPTOCOCCI.  Micrococci  are not considered to cause disease. When viewed microscopically, staphylococci appear in clumps, like bunches of grapes.  Staphule is Greek for grapes.  Streptococci form chains, and are named after streptos, the Greek word for  twisted.  These groups of bacteria can be distinguished because staphylococci produce an enzyme, CATALASE: streptococci do not.  Catalase  causes the conversion of hydrogen peroxide to water with the concomitant release of oxygen gas, seen as bubbles in the reaction tube.  The catalase test is a more reliable test to differentiate staphylococci from streptococci than microscopic observation.

The staphylococcus that causes most of the serious clinical problems associated with this group is Staphylococcus aureus.  This is so named because the classical description of the species is that its colonies appear golden yellow  (aurum - Latin for gold).  However, clinically significant isolates are made that do not conform to this description.  To differentiate Staphylococcus aureus from other staphylococci, the COAGULASE test is used.  Coagulase is an enzyme that causes plasma to clot, and is elaborated by Staphylococcus aureus but not by the coagulase-negative staphylococci such as Staphylococcus epidermidis, Staphylococcus capitis  and Staphylococcus saprophyticus.  Coagulase-negative staphylococci are resistant to drying, but Staphylococcus aureus is less so.

STREPTOCOCCI are classified according to their ability to break down blood in fresh blood agar plates.  Some streptococci have no effect on blood.  These are the NON-HAEMOLYTIC STREPTOCOCCI (see below).  The a-HAEMOLYTIC STREPTOCOCCI cause partial breakdown of blood, and their colonies are surrounded by a greenish halo.  The green pigment is thought to comprise the metabolic degradation products of haem.  Because of the colour of halo that surrounds a-haemolytic streptococci, they are often referred to as "viridans" streptococci (viridis is Latin for green).  There is one a-haemolytic streptococcus that must be differentiated from the others.  This is Streptococcus pneumoniae.    This is the cause of pneumococcal pneumonia and meningitis, as well as less serious infections.  Streptococcus pneumoniae  is sensitive to optochin, an antimicrobial agent, and lyses when suspended in a solution of bile salts.  All other viridans streptococci are resistant to optochin and are also insoluble in bile salts.  The viridans streptococci are a large and heterogeneous group of bacteria that are poorly differentiated, but they include organisms that play a role in tooth decay and those that can cause endocarditis and brain abscesses.  The viridans streptococci are not often differentiated in diagnostic laboratories.

The b-HAEMOLYTIC STREPTOCOCCI cause the complete breakdown of blood in fresh blood agar plates.  The colonies are surrounded by haloes that are completely clear.  Clinically, the most important of the b-haemolytic streptococci is Streptococcus pyogenes.  This belongs to the "Lancefield Group A", based upon its antigenic structure.  Streptococcus pyogenes may be differentiated from other b-haemolytic streptococci by its sensitivity to the antibiotic bacitracin.

The most important of the non-haemolytic streptococci are the ENTEROCOCCI such as Enterococcus faecalis   and  Enterococcus faecium.  Until the early 1990's these bacteria were classified in the genus Streptococcus but  molecular biological techniques have shown that they are sufficiently distant from other streptococci to warrant being placed in their own genus.  As their names imply, these bacteria can be found in the gut, and can grow in the presence of bile salts. 

Pathogenic Gram-positive Bacilli

The Gram-positive rods can be divided according to their ability or otherwise to produce spores.  Spores of Gram-positive rods are highly resistant structures that may add considerably to their pathogenic capacity.  Sporing Gram-positive rods that are confined to the (somewhat confusingly named) genus Bacillus.  Important members of this genus include Bacillus anthracis the cause of anthrax, and Bacillus cereus a cause of food poisoning.  (Ceres was a Roman goddess of the harvest).  The genus Bacillus also has members that produce clinically useful antibiotics, like Bacillus polymyxa, the source of polymyxin.

Obligately anaerobic sporing Gram-positive rods are placed in the genus Clostridium.  These include Clostridium perfringens, a principal cause of gangrene, Clostridium tetani, the cause of tetanus, and Clostridium botulinum the cause of the fatal food poisoning botulism.  (Botulus is the Latin for a sausage).  Clostridium perfringens used to be known as Clostridium welchii, and you may find reference to this name in older text books.

The motility of the non-sporing Gram-positive rods is an important attribute in distinguishing CORYNEFORM BACTERIA  and LACTOBACILLI from LISTERIA.  Listeria monocytogenes is an important human pathogen, and it is capable of a characteristic tumbling motility seen at 25^oC but not at 37^oC.  Lactobacilli appear microscopically as long, slender rods that often grow in chains.  They may appear "Gram-variable" with some parts of the cell appearing blue-black and other portions looking red.  They tend to make their immediate environment too acid for other bacteria to tolerate.  Some lactobacilli are important members of the vaginal commensal flora of women of child-bearing age.  These are sometimes referred to as Döderlein bacilli.  The lactobacilli are catalase-negative, and can thus be distinguished from the coryneform bacteria that do produce catalase. 

The most infamous of the coryneform bacteria is Corynebacterium diphtheriae,  toxigenic strains of which cause diphtheria.  This gives the coryneform bacteria their alternative name - diphtheroids.  They appear somewhat irregular in shape, and tend to cluster in Gram films.  Some microbiologists think that this gives them the microscopic appearance of Chinese letters.  PROPIONIBACTERIA  are coryneforms that cannot grow in the presence of air.  A notable example is Propionibacterium acnes, associated with acne.

The MYCOBACTERIA are a group of bacteria that are classified with other Gram-positive bacteria on the basis of their cellular architecture, but they possess a very waxy cell wall, and they rarely stain using conventional protocols such as the Gram stain.  They require special staining techniques to be observed easily under the microscope.  In the Ziehl Neelsen technique.  Because mycobacteria resist decolourisation with acids and alcohol they are sometimes called ACID ALCOHOL-FAST BACILLI.  Important examples include Mycobacterium tuberculosis and  Mycobacterium leprae.  Other mycobacteria are opportunist pathogens, particularly in patients with AIDS.

Pathogenic Gram-negative cocci

Medically, the most important of the Gram-negative cocci belong to the genus Neisseria.  Neisseria meningitidis  is an important cause of bacterial meningitis, and Neisseria gonorrhoeae causes gonorrhoea.  Members of the genus NEISSERIA are most often seen in pairs, and are hence sometimes referred to as diplococci.  They are very vulnerable to drying, and can only be cultivated in an atmosphere where the concentration of carbon dioxide is greater than that found in air.  In the laboratory, CO₂ incubators are used that maintain a moist environment with a CO₂ concentration of 5-10%.

Pathogenic Gram-negative bacilli

The ENTEROBACTERIACEAE are a large family of medically important Gram-negative bacilli.  They can grow in the presence or absence of oxygen, and are frequently found in the guts of humans and other animals, and hence their name.  They are differentiated from one another largely by their metabolic behaviour and their antigenic structure.  Some, like Escherichia coli and members of the genus Klebsiella can ferment lactose to produce acid, whereas others including salmonellas, shigellas and proteeae cannot and are thus known as NON-LACTOSE FERMENTERS (NLF's).  Members of the genus Proteus are so highly motile that a single colony can grow to swarm over the entire surface of a Petri dish after overnight incubation.  The family Enterobacteriaceae include Yersinia pestis, the cause of plague, Salmonella typhi, the cause of typhoid, Shigella dysenteriae, the cause of bacillary dysentery, and  Salmonella enteritidis implicated in many cases of food poisoning.

Enterobacteriaceae do not elaborate the enzyme complex known as "OXIDASE", whereas many Gram-negative bacteria do.  Pseudomonas aeruginosa is an oxidase-positive Gram-negative bacillus that is an obligate aerobe.  It cannot be grown in the absence of oxygen.  It is responsible for wound infections, and the bacteria in this species produce a soluble pigment. 

The VIBRIOs  and CAMPYLOBACTERs are Gram-negative rods that appear curved or spiral in shape.  These bacteria are commonly found in natural waters, both fresh-water and marine.  Vibrio cholerae causes cholera, a waterborne infection.  Campylobacters have only been recognised as human pathogens since the late 1970's, although they have been long considered to be animal pathogens.  Campylobacters are now responsible for more cases of bacterial enteritis annually than salmonellas.

Some Gram-negative bacilli appear so short that they resemble cocci in the light microscope.  Because of this they are sometimes called COCCO-BACILLI.  These include members of the genus Moraxella, related to the neisserias, and also members of the genus Acinetobacter.  Members of this genus are increasingly associated with hospital-acquired infection.

Some Gram-negative bacteria are very fastidious in their nutritional requirements.  Members of the recently recognised genus Legionella, some of which cause atypical pneumonias like Legionnaires' disease, require higher levels of iron and cysteine than are usually present in bacteriological media, and they grow best in media that incorporate activated charcoal to adsorb their toxic metabolic products.  Similarly, species of the genus Bordetella also generate toxic metabolic products that inhibit their own artificial culture.  These bacteria also grow best on media that contain activated charcoal Bordetella pertussis, the cause of whooping cough, is an important member of this genus.

At one time, species of the genus Bordetella were classified in the genus Haemophilus,  but they were re-classified.  This is partly because they require neither the X- nor the V-factor for growth such as required by members of the genus Haemophilus.  The X-factor has now been identified as haem, and the V-factor is nicotinamide adenine dinucleotide or NAD.  Haemophilus influenzae requires both X- and V-factors for growth whereas Haemophilus parainfluenzae requires just the V-factor to support its growth, since it can elaborate its own supply of haem. 

The most important group of OBLIGATELY ANAEROBIC GRAM-NEGATIVE BACILLI are the BACTEROIDES.  This is a heterogeneous group that form part of the human commensal flora, and that are also implicated in anaerobic infections.  The taxonomy of the anaerobic Gram-negative rods is currently undergoing radical revision.

Antibiotics

Those agents that kill bacteria are said to be bactericidal and those whose effects are reversible upon removal of the drug are bacteriostatic.

Those agents that kill bacteria are said to be bactericidal and those whose effects are reversible upon removal of the drug are bacteriostatic.

In the laboratory, susceptibility is most often measured using a disk diffusion test.

An alternative measure of susceptibility is to determine the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) of a drug.

Cidal drugs have MBC values that are close to the MIC value for particular organisms.

With static agents, the MIC is much lower than the MBC.

When antibiotics are given in combination, they may interact. 

If the overall effect is to reduce the efficacy of therapy, the drugs are said to be antagonistic.

Synergy is seen where the activity of one drug in a mixture enhances the activity of the other.

Mode of action of antibiotics

Bacterial cell wall inhibitors

b-lactam antibiotics

These antibiotics include the penicillins, cephalosporins, monobactams and carbapenems.

The principal targets are the enzymes that are responsible for the turnover of peptidoglycan - the penicillin binding proteins.

Clavulainc acid is not a potent antibacterial agent but it inhibits enzymes that degrade  b-lactam antibiotics, preventing resistance.
 

Vancomycin

This is has a bulky molecular structure and cannot penetrate the outer membrane of Gram-negative bacteria.

It prevents new peptidoglycan subunits binding to the peptidoglycan polymer by binding to the monomer while it is still attached to the bacterial cell membrane.
 

Fosfomycin

Acts at a very early stage in the synthesis of the peptidoglycan monomer - easy selection of resistance limits its clinical applications.
 

Cycloserine

Prevents the conversion of L-alanine to D-alanine and also stops the formation of D-alanyl-D-alanine. 

This drug may be neurotoxic so is only used for  treatment of mycobacterial infection where other drugs have failed.
 

Bacitracin

This acts to prevent recycling of  lipid carrier that takes peptidoglycan monomers across the bacterial membrane.

It is too toxic for human clinical use but it is very useful for bacterial identification.

Group A streptococci (Streptococcus pyogenes) may be distinguished from other  b-haemolytic streptococci because the Group A streptococci are susceptible to bacitracin.

Antibiotics affecting cell membranes

These include polymyxins and gramicidins - old drugs that are relatively toxic.

Metronidazole may act on membranes as part of its mode of action.

Antibiotics interfering with bacterial DNA

Sulphonamides and trimethoprim

Both act on enzymes involved in the synthesis of folic acid - a nucleotide precursor.

Sulphonamides act by inhibition of dihydropteroate synthetase.

Trimethoprim inhibits dihydrofolate reductase, the next step in the folic acid biosynthetic pathway.
 

Quinolones

Quinolones are synthetic drugs that interfere with DNA supercoiling.
 

Metronidazole

Metronidazole has a similar mode of action to the quinolones but also acts on bacterial membranes.
 

Antibiotics interfering with bacterial RNA

Rifampicin and the nitrofurans

Rifampicin and the nitrofuran compounds inhibits DNA-dependent RNA polymerase, stopping the production of mRNA.

Antibiotics interfering with bacterial protein synthesis

Aminoglycosides

The aminoglycosides  are broad-spectrum agents that include streptomycin, gentamicin, tobramycin, kanamycin, amikacin and netilmicin.

Closely related to the aminoglycosides is the aminocyclitol spectinomycin.

They have multiple modes of action but principally they prevent initiation of protein synthesis.

Aminoglycosides are toxic to humans, causing problems with kidney function and damage to the eighth cranial nerve.
 

Tetracyclines

The tetracyclines are a family of antibiotics that have a four-ring structure.

Tetracyclines are broad-spectrum agents that inhibit binding of the aminoacyl tRNA to the 30S ribosomal subunit in bacteria.

The clinical use of tetracyclines is generally confined to adults.

Tetracyclines affect bone development and can cause staining of teeth in children.

Tigecycline is a new tetracycline that is active against meticillin-resistant Staphylococcus aureus.

Chloramphenicol

Chloramphenicol acts by binding to the 50S ribosomal subunit and blocking the formation of the peptide bond by inhibiting peptidyl transferase activity.

Chloramphenicol may cause aplastic anaemia.

Macrolides and lincosamides

Macrolides include erythromycin, azithromycin and claritromycin.

These drugs have bulky structures and are mostly active against Gram-positive bacteria.

Macrolides bind to the 50S ribosomal subunit and inhibit either peptidyl transferase activity or translocation of the growing peptide.

Lincosamides such as clindamycin and lincomycin have a similar mode of action.
 

Fusidic acid

Fusidic acid is a steroid antibiotic active against Gram-positive bacteria.

It acts by preventing translocation of peptidyl tRNA.

Resistant mutants may easily be selected, even during therapy and therefore fusidic acid is usually administered in combination with another antibiotic.

Streptogramins

Group A strptogramins distort the ribosome to prevent binding of the t-RNA

Group B streptogramins are thought to block translocation of the growing peptide.

A combination of dalfopristin and quinupristin has recently been introduced for use in the treatment of meticillin-resistant Staphylococcus aureus. Dalfopristin is a Type A streptogramin and quinupristin is a Type B streptogramin. In combination, these drugs show a synergistic effect.

Mupirocin

Muprocin is an analogue of iso-leucine.

It inhibits the iso-leucyl-transfer RNA synthetase.

It is not toxic to humans but can only be used topically for skin infections. This is because humans rapidly metabolise the drug to an inactive form.

Linezolid

Linezolid prevents the initiation of protein synthesis.

It does this by interfering with the interaction between mRNA and the two ribosomal subunits .

It is active against Gram-positive cocci, including meticillin-resistant Staphylococcus aureus and vancomycin resistant enterococci.
 

Antibiotics acting against mycobacteria

Mycobacteria have very waxy cell walls, making penetration of many antimicrobial drugs difficult.

Mycobacteria are also very slow growing.

Combinations of drugs are used to treat mycobacterial infections such as leprosy and tuberculosis to prevent selection of resistance.

Antimycobacterial drugs are frequently associated with unpleasant side effects

Many health authorities are recommending Daily Observed Therapy: DOTs for short.

Streptomycin was the first drug used successfully to treat tuberculosis.

Rifampicin is also used as an antimycobacterial agent.

Isoniazid inhibits the formation of very long chain fatty acids such as those found in the cell walls of mycobacteria.

Ethambutol is a first-line antimycobacterial drug that inhibits cell wall synthesis although its mode of action remains to be elucidated fully.

Pyrazinamide is another first-line antimycobacterial drug that inhibits mycobacterial metabolism. Again, its mode of action remains to be elucidated fully.

Antibiotic resistance

Some antimicrobial resistance genes have only ever been found located on the bacterial chromosome.

Others have been found to lie on plasmids.

Plasmids encoding antibiotic resistance are often called resistance factors, R-factors or R-plasmids.

R-plasmids may encode resistance to several unrelated antibiotics.

Some R-plasmids are self-transmissible and can move from strain to strain, even between different bacterial genera.

Antibiotic resistance genes are frequently located within transposons.

Genes encoded by transposons may spread very easily because many transposable elements may become associated with transmissible plasmids.

Control of antimicrobial resistance in pathogenic microbes is one of the greatest challenges currently facing medical microbiology.
 

If we are unsuccessful, we will surely enter the post-antibiotic era.