Microbiology

Classification

Gram stain Ability to retain the crystal violet-iodine complex when treated with organic solvents such as acetone or alcohol. Gram-positive bacteria retain the stain, and hence appear purple or blue-black when visualised by bright-field microscopy. In contrast, Gram-negative bacteria cannot retain the dye complex, and need to be counterstained with a red dye such as carbol fuchsin before they can be seen in the bright-field microscope (that is why they are pink) Shape Bacteria display three basic shapes: o round (cocci, from the Greek kokkos - a berry), o rod shaped (bacilli, from the Latin bacillus - a stick or rod), o spiral. Gram-positive cocci two major groups of Gram-positive cocci that are of medical importance: the staphylococci and the streptococci . o staphylococci appear in clumps, like bunches of grapes. Staphule is Greek for grapes. staphylococci produce an enzyme, catalase and 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

o Streptococci form chains, and are named after streptos, the Greek word for twisted.

Staphylococci
To differentiate Staphylococcus aureus from other staphylococci, the coagulase test is used. o Coagulase is an enzyme that causes plasma to clot, and is elaborated by Staphylococcus aureus o NOT by the coagulase-negative staphylococci such as Staphylococcus epidermidis, Staphylococcus capitis and Staphylococcus saprophyticus S.saphrophyticus UTI in women. An alternative test to differentiate Staphylococcus aureus from the coagulase-negative staphylococci is the DNase test. Clinical Importance of Staph. Aureus Skin infections following surgical wounds or IV devices S.aureus pneumonia is rare but follows influenza (high mortality) S.aureus endocarditis is rapid and destructive o Follows IV drug use or colonisation of IV devices S.aureus is the most common agent of osteomyelitis or septic arthritis

Antibiotic susceptibility of Staph aureus = Flucloxacillin for MSSA. Vancomycin or teicoplanin for MRSA

Streptococci
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 nonhaemolytic streptococci. o Most important = are the enterococci such as Enterococcus faecalis and Enterococcus faecium The a-haemolytic streptococci cause partial breakdown of blood, o Their colonies are surrounded by a greenish halo. The green pigment is thought to comprise the metabolic degradation products of haem. o They are often referred to as "viridans" streptococci (viridis is Latin for green). NB CAUSE OF INFECTIVE ENDOCARDITIS o Streptococcus pneumoniae must be differentiated from the others viridans (Alpha-haemolytic) streptococci

the cause of pneumococcal pneumonia and meningitis, as well as less serious infections sensitive to optochin , 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 b-haemolytic streptococci cause the complete breakdown of blood in fresh blood agar plates. o The colonies are surrounded by haloes that are completely clear. Clinically, o the most important of the b-haemolytic streptococci is Streptococcus pyogenes. "Lancefield Group A" based upon its antigenic structure. Streptococcus pyogenes may be differentiated from other b-haemolytic streptococci on the basis of its sensitivity to the antibiotic bacitracin.

Clinical relevance of Strep. Pyogenes Most common cause of pharangitis (strep throat) Cellulites and nectrotizing fasciitis Toxin mediated: o erythrogenic toxins scarlet fever o Pyrogenic toxin-producing strains streptococcal shock (high mortality) Antibiotic Control Benzyl penicillin (no resistance ever reported

Gram positive bacilli

The Gram-positive rods can be divided according to their ability or otherwise to produce spores. Non spore forming G+ rods include the corynebacterium spp and listeria spp ( not very clinically relevant) Sporing Gram-positive rods that are confined to the (somewhat confusingly named) genus Bacillus. o Spores of Gram-positive rods are highly resistant structures that may add considerably to their pathogenic capacity. o 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). o Obligately anaerobic sporing Gram-positive rods are placed in the genus Clostridium. These include Clostridium perfringens, a principal cause of gangrene and food poisoning

Clostridium tetani, the cause of tetanus Toxin prevents release of GABA muscle spasms Clostridium botulinum the cause of the fatal food poisoning. Incomplete heat treatment in canning/bottling Clostridium difficile causes pseudomembranous colitis Tx is remove inciting antibiotic and replace with metronidazole (kills anaerobes)

Mycobacterium
a group of bacteria that are classified with other Gram-positive bacteria on the basis of their cellular architecture they rarely stain using conventional protocols such as the Gram stain. They require special staining techniques. o 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 . The former causes tuberculosis, and the latter is the cause of leprosy. It takes 6-8 weeks to grow Mycobacterium tuberculosis in artificial culture, and as yet Mycobacterium leprae cannot be grown in artificial culture.

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

Gram-negative bacilli (pretty dull)

The Enterobacteriaceae are a large family of medically important Gram-negative bacilli. grow in the presence or absence of oxygen, and are frequently found in the guts of humans and other animals

They are differentiated from one another largely on the basis of their metabolic behaviour and on their antigenic structure. Some, like Escherichia coli and members of the genus Klebsiella can ferment lactose to produce acid, Others including salmonellas , shigellas and proteus cannot and are thus known as non-lactose fermenters (NLF's). 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.

Pseudomonads Enterobacteriaceae do not elaborate the enzyme complex known as "oxidase", whereas many Gram-negative bacteria do. Pseudomonas aeruginosa is an oxidase-positive Gramnegative bacillus that is an obligate aerobe. o It cannot be grown in the absence of oxygen

Curved Gram-negative rods The vibrios and campylobacters are Gram-negative rods that appear curved or spiral in shape. o These bacteria are commonly found in natural waters, both fresh-water and marine. o Vibrio cholerae causes cholera, a waterborne infection.

The bacterium Helicobacter pylori, is gram negative. Other Gram-negative bacilli Some Gram-negative bacilli appear so short that they resemble cocci in the light microscope. Because of this they are sometimes called cocco-bacilli. o Moraxella, related to the neisserias, o Acinetobacter. Members of this genus are increasingly associated with hospital-acquired infection.

Some Gram-negative bacteria are very fastidious (fussy) in their nutritional requirements. Legionella, o cause atypical pneumonias like Legionnaires' disease, o require higher levels of iron and cysteine than are usually present in bacteriological media, grow best in media that incorporate activated charcoal to adsorb their toxic metabolic products. Bordetella also generate toxic metabolic products that inhibit their own artificial culture. o These bacteria also grow best on media that contain activated charcoal. o Bordetella pertussis, the cause of whooping cough, is an important member of this genus. Haemophilus influenzae requires both X- and V-factors for growth o The X-factor has now been identified as haem, o V-factor is nicotinamide adenine dinucleotide or NAD.