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Clinical bacteriology2. Dr. Dalia Mohsen Dalia.mohsen@riyadh.edu.sa. CULTURE AND IDENTIFICATION OF INFECTIOUS AGENTS .
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Clinical bacteriology2 Dr. Dalia Mohsen Dalia.mohsen@riyadh.edu.sa
Isolation and identification of bacteria from patients aids treatment since infectious diseases caused by different bacteria have a variety of clinical courses and consequences. • Susceptibility testing of isolates can help in selection of antibiotics for therapy. • Recognizing that certain species (or strains) are being isolated atypically may suggest that a disease outbreak has occurred e.g. from contaminated hospital supplies or poor aseptic technique on the part of hospital personnel.
When patients are suspected of having a bacterial infection, it is usual to isolate visible colonies of the organism in pure culture (on agar plates). • The identification is based on taxonomic principles applied to the clinical microbiological situation • In the diagnostic laboratory, many samples must be characterized each day and results obtained as quickly as possible.
These classical methods for speciation of bacteria are based on morphological and metabolic characteristics. • The diagnostic tests have been selected on the basis that they provide discriminating information. • There are numerous different tests for each of the many target pathogens. • Additionally, molecular biology techniques (for characterization of specific genes or gene segments) are now commonplace in the clinical laboratory.
Taxonomic terms (classification) • Family: a group of related genera. • Genus: a group of related species. • Species: a group of related strains. • Type: sets of strain within a species (e.g. biotypes, serotypes). • Strain: one line or a single isolate of a particular species.
The most commonly used term is the species name (e.g. Streptococcus pyogenes - abbreviation S.pyogenes). There are always two parts to the species name, one defining the genus in this case "Streptococcus" and the other the species (in this case "pyogenes"). • The genus name is always capitalized but the species name is not. Both species and genus are underlined or in italics
Steps in diagnostic isolation and identification of bacteria
Step 1 • Samples of body fluids (e.g. blood, urine, cerebrospinal fluid) • streaked on culture plates and isolated colonies of bacteria (which are visible to the naked eye) • Appear after incubation for one to several days • Each colony consists of millions of bacterial cells. • Observation of these colonies for size, texture, color, and (if grown on blood agar) hemolysis reactions, is highly important as a first step in bacterial identification. • Whether the organism requires oxygen for growth is another important differentiating characteristic.
Step 2 • Colonies are Gram stained and individual bacterial cells observed under the microscope.
Step 3 • The bacteria are speciated using these isolated colonies. • This often requires an additional 24 hours of growth
Gram-positive cocci • 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.
Staphylococci • 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)
DIAGNOSIS • Specimen processing No special considerations are required for processing of the organisms • Direct detection methods: Gram stain of patient specimens, gram positive cells irregular clusters
Coagulase test • 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
DNase test • An alternative test to differentiate Staphylococcus aureus from the coagulase-negative staphylococci is the DNase test. Staphylococcus aureus produces a DNase that can diffuse from a colony and hydrolyse DNA within a plate.
DIAGNOSIS • cultivation: Media of choice: Grow on 5% sheep blood and chocolate agars Do not grow in MacConkey agar Grow well in broth blood culture system and common nutrient broths, such as thioglycollate
DIAGNOSIS • Selective media: Used to isolate staph from clinical material • Mannitol salt agar is commonly used • Contains high concentration of salt(10%),suger manitol and phenol red as pH indicator • S.aureus grow in the presence of salt and ferment mannitol
DIAGNOSIS • Incubation conditions and duration: Visible growth on 5%sheep blood and chocolate agars incubated at 35C within 24 hours In Mannitol salt agarrequire 48 to 72 hours
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 non-haemolytic streptococci • 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).
Streptococci • 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.
Streptococci • 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.
Streptococci • 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
Streptococci • 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.
Diagnosis • Specimen processing No special considerations are required for processing of the organisms • Direct detection methods: Gram stain of patient specimens, gram positive cells irregular chains
Diagnosis • cultivation: Media of choice: Grow on 5% sheep blood and chocolate agars Do not grow in MacConkey agar Grow well in broth blood culture system and common nutrient broths, such as thioglycollate
Lancefield grouping • Beta-haemolytic streptococci can be identified using group specific antiserum • The main species and groups of medical importance are : Streptococcus pyogenes - Lancefield group A Streptococcus agalactiae-Lancefield group B Streptococcus Enterococci-Lancefield group D
Alpha-haemolytic streptococci • alpha-haemolytic streptococci are resistant to optochin(5 µg disc) • The main specie of medical importance is Streptococcus viridans
Alpha-haemolytic streptococci • Bile Solubility - rapid lysis of the pneumococcus(Streptococcus pneumoniae) in the presence bile caused by theactivation of cell wall autolytic enzyme as a consequence of the detergentaction of bile optochin - pneumococci are sensitive (i.e. growthinhibition) to low concentrations of the chemical, ethylhydroxycupreine; other alpha streptococci such asStreptococcus viridans are not
Bacillus • The main species of medical importance is anthrax bacillus Specimens: • include pus, fluid , sputum and blood • Must marked high risk
Bacillus • Culture Mannitol egg yolk phenol-red polymyxin agar (MYPA) is recommended as a selective medium for the isolation of B.cereus from faeces, vomit, or food - B.cereus is beta haemolytic B.cereus
Bacillus • Microscopy: large 5-8 µm ,capsulated (contains polypeptide) gram positive bacillus without spores • Culture -Grows aerobically on blood agar -Produce large 2-5 mm in diameter grey white irregular colonies with wavy edges -the colonies are non-haemolytic species
Corynebacterium • The main species of medical importance is C.diphtheriae Specimens: • include throat or nasopharyngeal swabs
Corynebacterium • Microscopy: Gram positive non capsulated without spores and non motile rod • Culture -Grows aerobically on blood agar -the selective medium is: Tellurite whole blood agar -blood agar +potassium tellurite -Produces grey-black colonies measuring 0.5-2mm after overnight incubation
throat culture on blood agar and tellurite medium. black colonies on tellurite medium are Corynebacterium diphtheriae
Listeria • The main species of medical importance is listeria monocytogenes • Microscopy: large 5-8 µm , non capsulated, gram positive curved rod or coccobacillus without spores and has a very characteristic tumbling and rotating motility at low temperatures (18-22C)at 35-37 it is only weakly motile or non motile • Culture -Grows aerobically and anaerobically on blood agar And tryptose agar - Temperature range for growth is 3-45 with optimum of 30 C
Listeria • On blood agar Listeria monocytogenes produces small grey droplet like colonies surrounded by a small zone of beta haemolysis • On tryptose agar the colonies apper pale blue green • The organism is catalase positive
Gardenella • The main species of medical importance is Gardenella vagainalis • Microscopy • gram positive , non capsulated , thin small rod or coccobacillus ,non motile • Culture • Anaerobic or a carbon dioxide atmosphere are necessary for the reliable isolation • The organism is slow growing require 48 hours to produce colonies • The colonies are white with dark centers and surrounded by clear area due to starch utilization • Catalase negative
Clostridia • Obligately anaerobic sporing Gram-positive rods are placed in the genus Clostridium. • These include Clostridium perfringens, a principal cause of gangrene non motile and non capsulated • Clostridium tetani, the cause of tetanus, motile and non capsulated • Clostridium botulinum motile and plemorphic rods • the cause of the fatal food poisoning - botulism (Botulus is the Latin for a sausage). Clostridium • In Gram films prepared from cultures it is very rare to see the spores of Clostridium perfringens, • but Clostridium tetani produces large spores at the ends of the bacilli, giving them the characteristic appearance of drumsticks.
