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Biology Dept

CSU, Stanislaus

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Diagnostics: Gram Positive pathogens

Evaluation of Cocci and Gram-Positive Bacilli Pathogens

OBJECTIVE

p To learn clinical microbiology methods for the identification of potentially pathogenic Gram positive microorganisms

p 90 percent of hospital infections caused by bacteria.

p Isolation and identification of bacteria from patients informs:

n Treatment

n origin of pathogen

n whether an outbreak has occurred.

p In the diagnostic laboratory

n many samples processed and results obtained as quickly as possible.

n Tests must be easily learned, low in cost and rapidly performed.

Gram Positive Pathogens

p This laboratory will focus on biochemical and morphological tests for Gram-positive organisms.

n Cocci

n Rods (bacillus)

Gram Positive Cocci

p grouped together based on their:

n Gram-stain reaction

n thick cell wall composition

n spherical shape

p genus Staphylococcus

n pathogens of man and other mammals.

p genus Streptococcus

n part of normal microbial flora of animals and humans

n some also cause diseases

p scarlet fever, rheumatic heart disease, glomerulonephritis, and pneumococcal pneumonia.

Catalase test

p oxygen à hydrogen peroxide (toxic to bacteria).

p Some bacteria possess enzymes to break down hydrogen peroxide.

n Catalase

p Convert hydrogen peroxide to water and oxygen

p Procedure:

n Using a sterile loop, spread a small amount of organism onto a dry slide.

p If bacteria produce catalase, oxygen gas produced (bubble).

n Add 1 drop of 3% hydrogen peroxide and evaluate for production of bubbles.

Staphylococci

S. aureus

n leading cause of soft tissue infections

n toxic shock syndrome (TSS)

n scalded skin syndrome.

p pathogenic effects of Staph due to toxins.

n enterotoxin

p causes quick onset food poisoning.

n Leukocidin

p destroys white blood cells and leads to the formation of pus and acne.

p Also causative agent in:

n pneumonia, meningitis, boils, arthritis, and osteomyelitis (chronic bone infection).

p Most S. aureus are penicillin resistant à MRSA.

S. epidermis:

n normal resident of human skin à opportunistic pathogen

n Those susceptible to infection

p IV drug users, newborns, elderly, and those using catheters or other artificial appliances.

n easily treatable with vancomycin or rifampin.

p Bacterium Morphology

n Gram-positive (purple) cocci

n Non-motile

n Occurs in pairs, short chains or in irregular grape-like clusters

p Colony Morphology

n Usually opaque, circular, and smooth

n May produce a golden-yellow pigment, but white or colorless colonies are also seen

p LABORATORY INDICATIONS:

n Anaerobic glucose fermentation with acid production

n Catalase +

n Nitrate +

n Coagulase +

n Hemolytic activity

p May be present, but its variability makes it a poor means of identification

Tests for Identification

Coagulase Test for Staphylococcus aureus

p test for production of coagulase

n secreted extracellularly

n reacts with coagulase-reacting factor present in plasma à Clot formation in the plasma.

p used to distinguish between:

n Staphylococcus aureus and other coagulase-negative staphylococci

Coagulase test

p measures "free" coagulase and is the best single test for identifying pathogenic staphylococci.

p To do this test, inoculate heavily 0.5 ml of 25% rabbit plasma and incubate at 37°C (overnight).

n Observe for clot formation by gently tipping the tube.

p Complete or partial coagulation is interpreted as "positive."

Mannitol Salt Agar

p differential and selective media.

n selective because its high salt concentration (7.5 %).

p Staphylococcus is able to tolerate this high salinity.

n differential because it contains the sugar mannitol and phenol red, a pH indicator.

p When mannitol is fermented, acid products are produced and the pH drops.

p Phenol red is yellow in color below pH 6.8.

p Result:

Streptococci

p Examples: Strep pyogenes (Group A), Strep group D (Enterococcus), and Strep viridans (Alpha Strep)

p Bacterium Morphology

• Gram-positive cocci (purple, but may turn Gram-negative or variable with age)

• Normally occurring in chains of varying lengths

• Cells are about 0.5 μm to 1 μm in diameter depending on the growth condition and age of culture.

p Colony Morphology

• Grow well on most enriched media

• Produce grayish, translucent to slightly opaque, circular, small colonies

• Colonies may vary from the mucoid, or smooth type, to the matte or rough form.

p Four different classification systems exist for this important microorganism:

n CLINICAL

p Pyogenic Streptococci

p Oral Streptococci

p Enteric Streptococci,

n HEMOLYSYS

p alpha-hemolysis

p beta-hemolysis

p gamma-hemolysis

n SEROLOGICAL-Lancefield (A-H), (K-U)

n BIOCHEMICAL(physiological)

GROUP A

p only one species, S. pyogenes.

n responsible for about 90% of all cases of pharyngitis

p "Strep throat" - characterized by inflammation and swelling of the throat, pus-filled regions on the tonsils.

p Penicillin is usually administered to patients ASAP.

p Once in lungs à pneumonia. Some cases also develop into rheumatic fever if left untreated.

p Other diseases linked to S. pyogenes

§ skin infections such as impetigo, cellulitis, and erysipelas.

p LABORATORY INDICATIONS:

n Catalase -

n Beta-hemolysis

n Bacitracin sensitive

GROUP B

p only one bacterium, S. agalactiae.

p cause of sexually transmitted urogenital infections in females.

p 1/3 of women are asymptomatic vaginal carriers

n women nearing labor screened for organism

n infection can easily spread to child via the birth canal

p cause severe neonatal infection including pneumonia, meningitis, and septicemia of the newborn.

p infection is easily treated with penicillin

p LABORATORY INDICATIONS:

n CAMP +

n Beta-hemolysis

The CAMP Test for Group B Streptococci

p identification simplified by implementation of the CAMP test.

p PRINCIPLE:
The hemolytic activity of staphylococcal ß-hemolysin on RBC is enhanced by an extracellular factor produced by Group B streptococci, called the CAMP factor. Therefore, wherever the two reactants overlap in sheep BAP, an accentuation of the ß-hemolytic reaction will be noted.

p PROCEDURE:
The CAMP test is performed by making a single streak of the Streptococcus perpendicular to a strain of Staphylococcus aureus that is known to produce ß-hemolysin. The two streak lines must not touch one another.

Camp Factor

GROUP D

p many are harmless, pathogenic strains cause complications of the human digestive tract.

p recently reclassified into two divisions: Enterococcus and non-Enterococcus.

n Enterococci include E. faecalis, a cause of urinary tract infections, and E. faecium, a bacterium resistant to many common antibiotics.

n Diseases: septicemia, endocarditis, and appendicitis.

n Fecal matter from infected individuals is a source for isolation and identification techniques.

n treated with ampicillin alone or in combination with gentamicin.

p LABORATORY INDICATIONS:

n Hydrolysis of bile esculin (dark brown medium)

p indicates the ability of the bacteria to tolerate bile from the liver

n Growth in high salt conc.

Tests for Identification

Hemolytic Activity

p Streptococci are usually isolated on blood agar.

n Streak plate for colony isolation and incubate overnight.

n evaluate area surrounding colony for α, ß, or γ hemolysis.

n Hemolysis - lysis of erythrocytes (red blood cells) and the release of hemoglobin from the cells.

p 3 Types

a-Hemolysis

p greenish zone around colonies is due to incomplete lysis of red blood cells

p characteristic of Streptococcus pneumoniae

b-Hemolysis

p clearing around growth due to complete lysis of red blood cells.

p characteristic of Streptococcus pyogenes as well as some strains of Staphylococcus aureus

g-Hemolysis

p actually a lack of hemolysis in the area surrounding a bacterial colony

p Staphylococcus epidermidis on a Sheep Blood Agar illustrates no hemolysis.

Bacitracin Test

p Group A Streptococci - sensitive to Bacitracin, but most other Streptococci are not inhibited.

p Procedure:

n colonies are thickly subcultured on a blood agar plate and a paper disc containing 0.04 units of bacitracin ("A" disc) is carefully applied to the center of the subculture.

n a zone of inhibition around the disc indicates sensitivity

n Rapid identification of Group A Streptococci in clinical laboratories is now performed by the fluorescent antibody method and other serological tests.

Bile-Esculin Slant Test

p aids in the differentiation of group D Streptococci from other "not group D Streptococci."

p purpose of this test:

n to determine ability of an organism to hydrolyze glycoside esculin to esculatin and glucose in the presence of bile (10 - 40%).

p Group D Streptococci are capable of hydrolyzing esculin to 6,7-dihydroxycoumarin which reacts with an iron salt in the medium to form a dark brown of black compound.

p Procedure:

n Inoculate the organism to be tested into the bile esculin medium by streaking the surface of the slant

n Incubate at 37oC for 24 hours.

n A result is positive if a black to dark brown color develops on the slant.

n A negative result will not have blackening of the medium OR blackening of less than half the tube after 72 hours of incubation.

Salt Tolerance Test

p based on the ability of the Enterococci to grow in 6.5% NaCl and separates them from the "Group-D streptococci, not Enterococci".

p Group D Enterococci are capable of growth in salt broth while the nonenterococcal Group D strains, S. bovis and S. equinus are not.

p Group D streps may produce alpha, beta, or gamma hemolysis, but the usual isolate is gamma hemolytic.

p Procedure:

n Inoculate a pure culture of the suspect organism into the 6.5% NaCl broth.

n Incubate overnight.

n A change in turbidity 24-48 hours after inoculation is interpreted as ‘positive’. A negative result is indicated by broth that has not changed in appearance.

OTHER IMPORTANT STREP

p Pneumococci

n Viridans Group

p The Viridans Streptococci, consisting of S. mutans and S. mitis, are alpha-hemolytic bacteria.

p part of the normal flora of the upper respiratory tract ("viridans Strep", e.g. Streptococcus mitis).

§ These bacteria inhabit the mouth.

§ In fact, a large percentage of tooth decay can be attributed to S. mutans.

n Streptococcus pneumoniae

p discussed separately because its surface carbohydrate antigens do not correspond to a specific Lancefield group.

p Example: Streptococcus pneumoniae

n major cause of community-acquired bacterial pneumonia

n most frequent cause of otitis media (middle ear infections) and bacteremia in infants and children.

n Pyogenic (pus-producing) strain of Strep.

n distinguished from other Pyogenic bacteria by its high sensitivity to Optochin (no growth zone of inhibition).

p Cell Morphology

• Gram-positive (purple) diplococci

• Lancet-shaped organism occurring as a diplococcus although single organisms and short chains can be seen

• Organisms may become Gram-negative and lyse spontaneously as the culture is allowed to age.

p Colony Morphology

• Small, shiny, flattened and transparent

• May appear green on blood agar because of induced discoloration in the agar

• The center of the colonies may be depressed due to autolysis.

Tests for Identification

p important to distinguish Streptococcus pneumoniae from other alpha-hemolytic streptococci of normal flora.

p Two tests are commonly employed for distinguishing Streptococcus pneumoniae from the viridans strep

n optochin test and bile solubility test.

n distinguished from other Pyogenic bacteria by its high sensitivity to Optochin (no growth zone of inhibition).

n demonstrates alpha-hemolytic growth on blood agar - partial lysing (bursting) of red blood cells.

Optochin Test

p Pneumococci (but not other alpha-hemolytic Streptococci) are inhibited by optochin.

p Optochin test is performed on a blood-agar medium using a disk diffusion principle.

p Procedure:

n Inoculate a blood agar plate with the test organism from the primary isolation plate.

n Using aseptic technique, place a bacitracin or optochin disk onto the inoculated media using sterile forceps.

n Lightly touch the disk with the sterile forceps to ensure complete contact between the disk and agar surface.

n Invert and incubate plates at 35-37°C in CO2 for 18-24 hours. Observe for a zone of inhibition around the disks.

n A zone of inhibition (area with no growth) of 14 mm. or more in diameter will be seen around the disk after incubation if the organism is Streptococcus pneumoniae.

n Other alpha-hemolytic streptococci are resistant to (not killed by) optochin. Their colonies will thus grow right up to the disk of optochin or have zones of inhibition less than 14 mm. in diameter.

The Bile Solubility Test

p determines the ability of an organism to lyse in the presence of bile salts (sodium deoxycholate).

p Pneumococci are identified by solubility in bile.

n Bile will selectively lyse colonies of Streptococcus pneumoniae while other strep are immune to bile's activity.

p Other alpha-hemolytic streptococci are resistant to (not lysed by) bile and will stay visible or turbid (cloudy).

p Procedure:

n The bile solubility test is performed by adding a bile-salt solution to an established broth or blood-agar culture of the organism in question.

n A positive result in broth culture is obtained by noting visible clearing of the culture's turbidity, as compared to a control tube, after addition of the bile salt solution and re-incubation for up to 3 hours.

n This test is most easily performed by placing a drop of bile solution (10% sodium deoxycholate) on suspected colonies on solid media.

n In 5-10 minutes, colonies of Pneumococci will lyse while those of alpha hemolytic Streptococci will not.

n The test can be performed by adding the reagent to an actively growing broth culture and determining whether it will clear in 5-10 minutes.

Gram-Positive Bacilli

p Corynebacteria Sp: "Diphtheroids“

p Corynebacteria (kŏ-ri’ne-bak-tēr’ĭ-um) are widely distributed in nature and are part of the normal flora of man. They are isolated from the normal respiratory tract and conjunctivae and also occur as saprophytes in various types of skin lesions such as acne.

p Examples: Corynebacterium diphtheriae, Listeria monocytogenes

p Cell Morphology

• Gram-positive (purple) rods

• Frequently stain irregularly, giving a banded or beaded appearance; this may lead to the incorrect conclusion that spores are present

• Usually non-motile cells that characteristically tend to lie parallel to one another in "palisades" or at acute angles forming "Chinese letters."

• Often club-shaped

p Colony Morphology

• Variable; may appear as tiny dust-like colonies or may be hemolytic and resemble streptococcal colonies

Tests for Identification

p Gram Stain

p "Diphtheroids" may be distinguished by colony characteristics but should be Gram stained to distinguish them from Streptococci.

p Corynebacteria stains as Gram-positive pleiomorphic rods arranged in perpendicular, parallel, and palisade formations.

p Colony morphology

p Grow on most laboratory media, but on Loeffler’s coagulated medium colonies are small, granular and gray with irregular edges.

p On blood agar, the colonies are gray to black in color.

p Catalase Test

p Touch top of colony with needle and smear onto slide.

p Add 1 drop of hydrogen peroxide. The reaction is positive if there is a rapid evolution of gas bubbles.

n This is difficult when applied to cultures grown on blood agar because catalase is present in red blood cells.

n Diphtheroids and Staphylococci are catalase-positive;

n Streptococci and Pneumococci are catalase-negative.

GRAM-POSITIVE RODS

p The Gram-positive rods in this section will be divided into three distinct varieties based upon their ability to produce endospores and their morphological appearance:

p ENDOSPORE-FORMING

n BACILLUS

p REGULAR, NON-ENDOSPORE-FORMING

n LISTERIA

p IRREGULAR, NON-ENDOSPORE-FORMING

n CORYNEBACTERIUM

p These bacteria are ubiquitous in nature and most are aerobic or facultatively anaerobic.

BACILLUS

p genus of Gram-positive bacteria which are ubiquitous in nature (soil, water, and airborne dust).

p Some species are natural flora in the human intestines.

p When grown on blood agar, Bacillus produces large, spreading, gray-white colonies with irregular margins.

p A unique characteristic of this bacterium is its ability to produce endospores when environmental conditions are stressful.

p The only other known spore-producing bacterium is Clostridium.

p Although most species of Bacillus are harmless saprophytes, two species are considered medically significant: B.anthracis and B. cereus.

B. anthracis

p B. anthracis is the bacterium which causes anthrax in cows, sheep, and sometimes humans.

p Anthrax is transmitted to humans via direct contact with animal products or inhalation of endospores.

p Under the microscope, B. anthracis cells appear to have square ends and seem to be attached by a joint to other cells.

p The spores are best observed when the bacterium is cultured on artificial media.

p Sources of infection are usually industrial or agricultural and the infection is classified as one of three types:

n CUTANEOUS INFECTION (95% of human cases)

n INHALATION ANTHRAX (rare)

n GASTROINTESTINAL ANTHRAX (very rare!)

p LABORATORY INDICATIONS:

n Nonhemolytic (sheep blood agar)

n Non-motile

n Gel hydrolysis -

n Catalase +

B. cereus

p Unlike B. anthracis, B.cereus is a motile bacterium which can cause toxin-mediated food poisoning.

p It is known to inhabit many kinds of food including stew, cereal, and milk.

p Most recently, however, it has been found in fried rice.

p The two toxins released by the bacterium lead to vomiting and diarrhea, symptoms similar to those of Staphylococcus food poisoning.

p Because toxin production usually takes place after the infected foods are cooked, proper cold storage of food is recommended immediately after preparation.

p LABORATORY INDICATIONS:

n Hemolytic (sheep blood agar)

n Motile

n Gel hydrolysis +

n Glucose, maltose, & salicin fermentative

n Catalase +

LISTERIA

p Listeria is a Gram-positive rod which is not capable of forming endospores.

p L. monocytogenes

n human pathogen has been implicated in several food poisoning epidemics.

n This normal inhabitant of the gastrointestinal tract and of animal feces led to a 1986 outbreak in Massachusetts hospital patients.

n Those infected suffered from vomiting, nausea, and diarrhea. Apparently, the hospital patients contracted the microbe from the infected hospital food and were at high risk of infection.

n Those at high risk include newborns, pregnant women and their fetuses, the elderly, and persons lacking a healthy immune system.

n The bacterium usually causes septicema and meningitis in patients with supressed immune function.

n It also causes listeriosis which is an inflammation of the brain.

n Antibiotics are recommended for treatment of infection because most strains of Listeria are sensitive to ampicillin and gentamicin.

p LABORATORY INDICATIONS:

n Catalase +

n Motile at room temperature

n Growth at 4 degrees Celsius

n Bile esculin hydrolysis

n Beta-hemolysis

CORYNEBACTERIA

p non-spore-forming rods which are ubiquitous in nature.

p clinically significant: Corynebacterium.

n comprised of facultatively anaerobic bacteria which are normally saprophitic and harmless to humans.

n An exception is the bacterium C. diphtheriae which produces the exotoxin that causes diphtheria, a disease of the upper respiratory system in humans.

n Although other species of Corynebacterium can inhabit the mucous membrane, C.diphtheria is unique in its exotoxin formation.

n Treatment for the disease usually consists of administration of an antitoxin with penicillin.

p LABORATORY INDICATIONS:

n Catalase +

n Nitrate +

n Glucose fermentation

n Non-motile

GRAM POSITIVE ANAEROBES

p CLOSTRIDIUM

n Members of genus Clostridium are Gram-positive, spore-forming rods that are anaerobic.

n These motile bacteria are ubiquitous in nature and are especially fond of soil.

n Under the microscope, they appear as long drumsticks with a bulge located at their terminal ends.

n A Gram-stain is a good method for identifying Clostridium because the cell incorporates the dye while the spore remains unstained.

n Clostridium shows optimum growth when plated on blood agar at human body temperatures.

n When the environment becomes stressed, however, the bacteria produce spores that tolerate the extreme conditions that the active bacteria cannot.

n In their active form, these bacteria secrete powerful exotoxins that are responsible for such diseases as tetanus, botulism, and gas gangrene.

n The four clinically important species of Clostridium will be discussed here: C. tetani, C. difficile, C. perfringens, and C. botulinum.

C. tetani

p bacterium that causes tetanus (lockjaw) in humans.

p spores acquired from any type of skin trauma involving an infected device.

p If an anaerobic environment is present, the spores will germinate and eventually form active C. tetani cells.

p At the tissue level, the bacterium then releases an exotoxin called tetanospasmin that causes certain nervous system irregularities by means of retrograde transmission through neurons to the brain.

p One of the toxin's effects includes constant skeletal muscle contraction due to a blockage of inhibitory interneurons that regulate muscle contraction.

p Prolonged infection eventually leads to respiratory failure, among other things.

p If not treated early, the mortality rates of this disease are high.

p Immunization is the best way to prevent C. tetani infections in children and adults.

p The process is started early with the first four shots being administered within two years of birth.

p The initial shots are then followed up with periodic booster shots given every ten years.

p LABORATORY INDICATIONS:

n Motile

n Terminal spores

n Non-aerotolerant

C. botulinum

p produces one of the most potent toxins in existence and the cause of the deadly botulism food poisoning.

p Because Clostridium spores can be airborne, they sometimes find their way into foods that will be placed in anaerobic storage such as cans or jars.

p Once the jars are sealed, the spores germinate and the bacteria release their potent toxin.

p Patients will experience muscular paralysis as well as blurred vision.

p Immmediate treatment with an anti-toxin must take place for the patient to have a chance at survival.

p Infantile botulism is acquired in a similar manner but is much milder than the adult version.

p Honey, however, is the most common source of the spores which germinate in the child's intestinal tract.

p Bacterial proliferation and subsequent toxin production cause symptoms which last a few days and then subside without the use of an antitoxin.

p LABORATORY INDICATIONS:

n Motile

n No terminal spores

n Non-aerotolerant

n Lipase +

C. perfringens

p non-motile bacterium - invasive pathogen that can be contracted from dirt via large cuts are wounds.

p cells proliferate after spore germination occurs and they release their exotoxin.

p toxin causes necrosis of the surrounding tissue (Clostridial myonecrosis destroys muscular tissues).

p The bacteria themselves produce gas which leads to a bubbly deformation of the infected tissues.

p capable of necrotizing intestinal tissues and can release an enterotoxin that may lead to severe diarrhea.

p Treatment of infection can consist of penicillin G (to kill the organism), hyperbaric oxygen, and administration of an antitoxin.

p LABORATORY INDICATIONS:

n Non-motile

n No terminal spores

n Non-aerotolerant

n Double zone hemolysis

C. difficile

p motile bacterium that can be part of the natural intestinal flora.

p Infection can occur through the use of broad-spectrum antibiotics which lower the relative amount of other normal gut flora.

p When this situation occurs, C. difficile proliferates and infects the large intestine.

p The bacterium then releases two enterotoxins that destroy the intestinal lining and cause diarrhea.