Non forming spore Gram positive bacilli Lecture 4

Corynebacterium spp Gram positive bacilli, with characteristic morphology (club shaped and beaded) Non motile Non spore forming Non capsulated Facultative anaerobic Breakdown glucose by oxidative and fermentative i.e. O+/F+ C. diphtheriae is fastidious while diphtheriods are non-fastidious Catalase positive Oxidase negative

Species of Corynebacteria It caused diphtheria C. diphtheriae is the only pathogenic members of this genus Normal flora of RT, urethra, vagina, Skin

Gram Positive Bacilli Gram positive rods Spore forming Non spore forming Aerobic Anaerobic Corynebacterium Bacillus spp Clostridium spp

The non-spore-forming gram-positive bacilli are a diverse group of bacteria. Many members of the genus corynebacterium and their anaerobic equivalents, propionibacterium species, are members of the normal flora of skin and mucous membranes of humans.

Corynebacterium diphtheriae is the most important member of the group, as it can produce a powerful exotoxin that causes diphtheria in humans. Listeria monocytogenes and Erysipelothrix rhusiopathiae are primarily found in animals and occasionally cause severe disease in humans

Based upon Gram stain morphology, formation of spores, and catalase reaction  Spore-forming, catalase positive (aerobic): Bacillus sp.  Regularly-shaped, non-spore forming  Catalase positive: Listeria monocytogenes  Catalase negative: Erysipelothrix rhusiopathiae, Lactobacillus sp., Gardnerella vaginalis  Irregularly-shaped, non-spore forming, catalase positive: Corynebacterium sp. (“diphtheroids”)  Branching: Nocardia sp., Actinomyces sp., & Streptomyces sp.

Corynebacterium species “coryneform bacteria” have a high guanosine plus cytosine content and include the genera corynebacterium, arcanobacterium, brevibacterium, mycobacterium, and others.

Actinomyces and propionibacterium are classified as anaerobes, but some isolates grow well aerobically (aerotolerant) and must be differentiated from the aerobic coryneform bacteria.

species—than they are to the coryneform bacteria. Other nonspore- forming gram-positive bacilli have more regular shapes and a lower guanosine plus cytosine content. The genera include listeria and erysipelothrix; these bacteria are more closely related to the anaerobic lactobacillus species, which sometimes grow well in air, to the sporeforming bacillus and clostridium species—and to the gram-positive cocci of the staphylococcus and streptococcus species—than they are to the coryneform bacteria.

There is no unifying method for identification of the gram-positive bacilli. Few laboratories are equipped to measure guanosine plus cytosine content. Growth only under anaerobic conditions implies that the isolate is an anaerobe, but many isolates of lactobacillus, actinomyces, and propionibacterium species and others are aerotolerant.

CORYNEBACTERIUM DIPHTHERIAE Morphology & Identification Corynebacteria are 0.5–1 μm in diameter and several micrometers long. Characteristically, they possess irregular swellings at one end that give them the “clubshaped” appearance. Irregularly distrib uted within the rod (often near the poles) are granules staining deeply with aniline dyes (metachromatic granules) that give the rod a beaded appearance.

On blood agar, the C diphtheriae colonies are small, granular, and gray, with irregular edges, and may have small zones of hemolysis. On agar containing potassium tellurite, the colonies are brown to black with a brownblack halo because the tellurite is reduced intracellularly (staphylococci and streptococci can also produce black colonies).

C diphtheriae and other corynebacteria grow aerobically on most ordinary laboratory media. Propionibacterium is an anaerobe. On Loeffler’s serum medium, corynebacteria grow much more readily than other respiratory organisms, and the morphology of organisms is typical in smears.

Pathogenesis The principal human pathogen of the group is C diphtheriae. In nature, C diphtheriae occurs in the respiratory tract, in wounds, or on the skin of infected persons or normal carriers. It is spread by droplets or by contact to susceptible individuals; the bacilli then grow on mucous membranes or in skin abrasions, and those that are toxigenic start producing toxin.

All toxigenic C diphtheriae are capable of elaborating the same disease-producing exotoxin. In vitro production of this toxin depends largely on the concentration of iron. Toxin production is optimal at 0.14 μg of iron per milliliter of medium but is virtually suppressed at 0.5 μg/mL. Other factors influencing the yield of toxin in vitro are osmotic pressure, amino acid concentration, pH, and availability of suitable carbon and nitrogen sources. The factors that control toxin production in vivo are not well understood.

Diphtheria toxin is a heat-labile polypeptide that can be lethal in a dose of 0.1 μg/kg.

Pathology Diphtheria toxin is absorbed into the mucous membranes and causes destruction of epithelium and a superficial inflammatory response. The necrotic epithelium .

becomes embedded in exuding fibrin and red and white cells, so that a grayish “pseudomembrane” is formed—commonly over the tonsils, pharynx, or larynx. Any attempt to remove the pseudomembrane exposes and tears the capillaries and thus results in bleeding. The regional lymph nodes in the neck enlarge, and there may be marked edema of the entire neck. The diphtheria bacilli within the membrane continue to produce toxin actively. This is absorbed and results in distant toxic damage, particularly parenchymatous degeneration, fatty infiltration, and necrosis in heart muscle, liver, kidneys, and adrenals, sometimes accompanied by gross hemorrhage. The toxin also produces nerve damage, resulting often in paralysis of the soft palate, eye muscles, or extremities

Diagnostic Laboratory Tests Swabs from the nose, throat, or other suspected lesions must be obtained before antimicrobial drugs are administered. Smears stained with alkaline methylene blue or Gram’s stain show beaded rods in typical arrangement.

Laboratory diagnosis of case Specimen: A throat swap Culture: The swap is inoculated on Loeffler's serum medium and/or on blood tellurite agar aerobically at 37C for 24. On Loeffler's serum medium: Corynebacteria grow much more readily than other respiratory pathogens Deep blue or red metachromatic granules (accumulated inorganic polyphosphates) by methylene blue stain Loefflers serum

The colonies of C. diphtheriae are small, granular, grey, smooth, and creamy with irregular edges

Cultural characteristics On blood tellurite agar It is selective medium for isolation of C. diphtheriae (Potassium tellurite)

3 biotypes of C. diphtheriae are characterized on BTA i.e. Gravis, mitis and intermedius biotypes The most severe disease is associated with the gravis biotype Colony of gravis biotype is large, non-hemolytic & grey. Colonies of mitis biotype are small, hemolytic and black Colonies of intemedius biotype are intermediate in size, non-hemolytic with black center & grey margin.

Biochemical Reaction All Corynebacterium species are catalase positive (Also, Staphylococcus and Bacillus species are catalase positive)

2- Carbohydrate Fermentation Test: Principle: Each species of corynebacteria has its specific carbohydrate fermentation pattern C.diphtheriae can be differentiated from other Corynebacterium species by fermentation of glucose and maltose but not sucrose with production of acid only

Procedure: Inoculate three tubes of carbohydrate fermentation medium (broth containing one type of sugar and phenol red as the pH indicator) with the test organism Incubate the tubes at 37oC for 24 hrs. Glucose Maltose Sucrose

2- Carbohydrate Fermentation Test: Principle: Each species of corynebacteria has its specific carbohydrate fermentation pattern C.diphtheriae can be differentiated from other Corynebacterium species by fermentation of glucose and maltose but not sucrose with production of acid only Procedure: Inoculate three tubes of carbohydrate fermentation medium (broth containing one type of sugar and phenol red as the pH indicator) with the test organism Glucose Maltose Sucrose Incubate the tubes at 37oC for 24 hrs.

Results: Sugar fermentation can be indicated by change of color of the medium from red to yellow due to formation of acid which decrease the pH Glucose Maltose Sucrose Glucose Maltose Sucrose +ve +ve +ve +ve +ve - ve C. diphtheriae C. xerosis

Detection of toxin: Elek’s Test Principle: It is toxin/antitoxin reaction Toxin production by C.diphtheriae can be demonstrated by a precipitation between exotoxin and diphtheria antitoxin Procedure: A strip of filter paper impregnated with diphtheria antitoxin is placed on the surface of serum agar The organism is streaked at right angels to the filter paper Incubate the plate at 37C for 24 hrs

Filter paper saturated with diphtheria antitoxin Resuls: After 48 hrs incubation, the antitoxin diffusing from filter paper strip and the toxigenic strains produce exotoxin, which diffuses and resulted in lines four precipitation lines radiating from intersection of the strip and the growth of organism Lines of precipitations Inoculated M.O. Positive Elek’s Test

1-4 positve 2-3 negative

Resistance & Immunity Since diphtheria is principally the result of the action of the toxin formed by the organism rather than invasion

by the organism, resistance to the disease depends largely on the availability of specific neutralizing antitoxin in the bloodstream and tissues. It is generally true that diphtheria occurs only in persons who possess no antitoxin (or less than 0.01 Lf unit/mL). Assessment of immunity to diphtheria toxin for individual patients can best be made by review of documented diphtheria toxoid immunizations and primary or booster immunization if needed

Treatment The treatment of diphtheria rests largely on rapid suppression of toxin-producing bacteria by antimicrobial drugs and the early administration of specific antitoxin against the toxin formed by the organisms at their site of entry and multiplication.

Antimicrobial drugs (penicillin, erythromycin) inhibit the growth of diphtheria bacilli. Although these drugs have virtually no effect on the disease process, they arrest toxin production. They also help to eliminate coexistent streptococci and C diphtheriae from the respiratory tracts of patients or carriers.

Anaerobic Corynebacteria Propionibacterium species) reside in normal skin. Propionibacterium acnes, is aerotolerant and grows aerobically. It participates in the pathogenesis of acne Arcanobacterium haemolyticum produces betahemolysis on blood agar. It is occasionally associated with pharyngitis

LISTERIA MONOCYTOGENES There are several species in the genus listeria. Of these, L monocytogenes is important as a cause of a wide spectrum of disease in animals and humans.

Culture & Growth Characteristics Listeria grows on media such as Mueller-Hinton agar. Identification is enhanced if the primary cultures are done on agar containing sheep blood, because the characteristic small zone of hemolysis can be observed around and under colonies. Isolation can be enhanced if the tissue is kept at 4 °C for some days before inoculation bacteriologic media.

produces acid but not gas in a variety of carbohydrates. The organism is a facultative anaerobe and is catalase-positive and motile. Listeria produces acid but not gas in a variety of carbohydrates. The motility at room temperature and hemolysin production are primary findings that help differentiate listeria from coryneform bacteria.

ACTINOMYCETES Actinomycetes are a large, diverse group of gram-positive bacilli with a tendency to form chains or filaments. They are related to the corynebacteria and mycobacteria as well as the stepomycete

The most famous species are Actinomyces israelii Actinomyces naeslundii

Diagnostic Laboratory Tests: Pus from draining sinuses, sputum, or specimens of tissue are examined for the presence of sulfur granules. The granules are hard, lobulated, and composed of tissue and bacterial filaments, which are club-shaped at the periphery .Specimens are cultured in thioglycolate broth and on brain-heart infusion blood

NOCARDIA Causing Nocardiosis wich is caused by infection withNocardia asteroides complex or, less frequently, Nocardia brasiliensis or Nocardia otitidiscaviarum, and only rarely by other species of nocardia. The Nocardia asteroides complex includes Nocardia abscessus, Nocardia farcinia, Nocardia nova, and others

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