Corynebacterium

corynebacterium
introduction
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. other corynebacteria are found in animals and plants. 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.
corynebacterium species and related bacteria tend to be clubbed or irregularly shaped, the term "coryneform bacteria" is a convenient one for denoting the group. these bacteria have a high guanosine plus cytosine content. actinomyces and propionibacterium are classified as anaerobes, but some isolates grow well aerobically (aerotolerant).
corynebacterium diphtheriae
morphology & identification
corynebacteria are 0.5–1 mm in diameter and several micrometers long. characteristically, they possess irregular swellings at one end that give them the "club-shaped" appearance. irregularly distributed within the rod (often near the poles) are granules staining deeply with aniline dyes (metachromatic granules) that give the rod a beaded appearance. individual corynebacteria in stained smears tend to lie parallel or at acute angles to one another.

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 brown-black halo because the tellurite is reduced intracellularly. four biotypes of c diphtheriae have been widely recognized: gravis, mitis, intermedius, and belfanti. these variants have been classified on the basis of growth characteristics such as colony morphology, biochemical reactions, and, severity of disease produced by infection. 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.
corynebacteria tend to pleomorphism in microscopic and colonial morphology. when some nontoxigenic diphtheria organisms are infected with bacteriophage from certain toxigenic diphtheria bacilli, the offspring of the exposed bacteria are lysogenic and toxigenic, and this trait is subsequently hereditary. when toxigenic diphtheria bacilli are serially subcultured in specific antiserum against the temperate phage that they carry, they tend to become nontoxigenic. thus, acquisition of phage leads to toxigenicity (lysogenic conversion). the actual production of toxin occurs perhaps only when the prophage of the lysogenic c diphtheriae becomes induced and lyses the cell. whereas toxigenicity is under control of the phage gene, invasiveness is under control of bacterial genes.

figure (1) pleomorphic shape
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 dropinglets 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 mg of iron per milliliter of medium. other factors influencing the yield of toxin in vitro are osmotic pressure, amino acid concentration, ph, and availability of suitable carbon and nitrogen sources.
diphtheria toxin is a heat-labile polypeptide (mw 62,000) that can be lethal in a dose of 0.1 mg/kg. if disulfide bonds are broken, the molecule can be split into two fragments. fragment b (mw = 38,000) has no independent activity but is required for the transport of fragment a into the cell. fragment a inhibits polypeptide chain elongation—provided nicotinamide adenine dinucleotide (nad) is present—by inactivating the elongation factor ef-2. this factor is required for translocation of polypeptidyl-transfer rna from the acceptor to the donor site on the eukaryotic ribosome. it is assumed that the abrupt arrest of protein synthesis is responsible for the necrotizing and neurotoxic effects of diphtheria toxin.
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.
wound or skin diphtheria occurs chiefly in the tropics. a membrane may form on an infected wound that fails to heal. however, absorption of toxin is usually slight and the systemic effects negligible. the small amount of toxin that is absorbed during skin infection promotes development of antitoxin antibodies. the "virulence" of diphtheria bacilli is due to their capacity for establishing infection, growing rapidly, and then quickly elaborating toxin that is effectively absorbed.
clinical findings
when diphtheritic inflammation begins in the respiratory tract, sore throat and fever usually develop. prostration and dyspnea soon follow because of the obstruction caused by the membrane. this obstruction may even cause suffocation if not promptly relieved by intubation or tracheostomy. irregularities of cardiac rhythm indicate damage to the heart. later, there may be difficulties with vision, speech, swallowing, or movement of the arms or legs. all of these manifestations tend to subside spontaneously.
diagnostic laboratory tests
these serve to confirm the clinical impression and are of epidemiologic significance. note: specific treatment must never be delayed for laboratory reports if the clinical picture is strongly suggestive of diphtheria.
dacron swabs from the nose, throat, or other suspected lesions must be obtained before antimicrobial drugs are administered. swabs should be collected from beneath any visible membrane. the swab should then be placed in semisolid transport media such as amies. smears stained with alkaline methylene blue or gram stain show beaded rods in typical arrangement.
inoculate a blood agar plate a loeffler slant, and a tellurite plate (eg, cystine-tellurite agar or modified tinsdale medium) and incubate all at 37 °c. in 12–18 hours,
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. 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. diphtheria antitoxin is produced in various animals (horses, sheep, goats, and rabbits) by the repeated injection of purified and concentrated toxoid. 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.
epidemiology, prevention, & control
active immunization in childhood with diphtheria toxoid yields antitoxin levels that are generally adequate until adulthood. young adults should be given boosters of toxoid, because toxigenic diphtheria bacilli are not sufficiently prevalent in the population of many developed countries to provide the stimulus of sub clinical infection with stimulation of resistance. levels of antitoxin decline with time, and many older persons have insufficient amounts of circulating antitoxin to protect them against diphtheria.
a filtrate of broth culture of a toxigenic strain is treated with 0.3% formalin and incubated at 37 °c until toxicity has disappeared. this fluid toxoid is purified and standardized in flocculating units (lf doses). fluid toxoids prepared as above are adsorbed onto aluminum hydroxide or aluminum phosphate. this material remains longer in a depot after injection and is a better antigen. such toxoids are commonly combined with tetanus toxoid (td) and sometimes with pertussis vaccine (dpt or dapt) as a single injection to be used in initial immunization of children. for booster injection of adults, only td toxoids are used these combine a full dose of tetanus toxoid with a tenfold smaller dose of diphtheria toxoid in order to diminish the likelihood of adverse reactions.

other coryneform bacteria
many other corynebacterium and propionibacterium species have been associated with disease in humans. the coryneform bacteria are classified as nonlipophilic or lipophilic depending upon enhancement of growth by addition of lipid to the growth medium. the lipophilic corynebacteria grow slowly on sheep blood agar, producing colonies < 0.5 mm in diameter after 24 hours of incubation. additional key reactions for the classification of the coryneform bacteria include but are not limited to the following tests: fermentative or oxidative metabolism, catalase production, motility, nitrate reduction, urease production, and esculin hydrolysis. corynebacterium species are typically nonmotile and catalase-positive. the coryneform bacteria are normal inhabitants of the mucous membranes of the skin, respiratory tract, urinary tract, and conjunctiva.
anaerobic corynebacteria
anaerobic corynebacteria (eg, propionibacterium species) reside in normal skin. propionibacterium acnes, however, is aerotolerant and grows aerobically. it participates in the pathogenesis of acne by producing lipases that split free fatty acids off from skin lipids. these fatty acids can produce tissue inflammation and contribute to acne. because p acnes is part of the normal skin flora, it occasionally appears in blood cultures and must be differentiated as a culture contaminant or a true cause of disease. p acnes occasionally causes infection of prosthetic heart valves and cerebrospinal fluid shunts.
actinomyces pyogenes, actinomyces neuii, and other actinomyces species are occasionally associated with clinically significant infections. actinomyces viscosis grows readily under aerobic conditions.

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.
morphology & identification
l monocytogenes is a short, gram-positive, non-spore-forming rod. it has a tumbling end-over-end motility at 22–28 °c but not at 37 °c the motility test rapidly differentiates listeria from diphtheroids that are members of the normal flora of the skin.
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 into bacteriologic media. 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.
antigenic classification
serologic classification is done only in reference laboratories and is primarily used for epidemiologic studies. serotypes ia, ib, and ivb make up more than 90% of the isolates from humans. serotype ivb was found to have caused an epidemic of listeriosis associated with cheese made from inadequately pasteurized milk.
pathogenesis & immunity
l monocytogenes enters the body through the gastrointestinal tract after ingestion of contaminated foods such as cheese or vegetables. it has a cell wall surface protein called internalin that interacts with e-cadherin, a receptor on epithelial cells, promoting phagocytosis into the epithelial cells. after phagocytosis, the bacterium is enclosed in a phagolysosome, where the low ph activates the bacterium to produce listeriolysin o. this enzyme lyses the membrane of the phagolysosome and allows the listeriae to escape into the cytoplasm of the epithelial cell. the organisms proliferate and acta, another listerial surface protein, induces host cell actin polymerization, which propels them to the cell membrane. pushing against the host cell membrane, they cause formation of elongated protrusions called filopods. these filopods are ingested by adjacent epithelial cells, macrophages, and hepatocytes, the listeriae are released, and the cycle begins again. l monocytogenes can move from cell to cell without being exposed to antibodies, complement, or polymorphonuclear cells. shigella flexneri and rickettsiae also usurp the host cells actin and contractile system to spread their infections.
iron is an important virulence factor. listeriae produce siderophores and are able to obtain iron from transferrin.
immunity to l monocytogenes is primarily cell-mediated, as demonstrated by the intracellular location of infection and by the marked association of infection and conditions of impaired cell-mediated immunity such as pregnancy, aids, lymphoma, and organ transplantation. immunity can be transferred by sensitized lymphocytes but not by antibodies.

figure (2) life cycle of listeria monocytogenes

clinical findings
there are two forms of perinatal human listeriosis. early onset-syndrome (granulomatosis infantiseptica) is the result of infection in utero and is a disseminated form of the disease characterized by neonatal sepsis, pustular lesions and granulomas containing listeria monocytogenes in multiple organs. death may occur before or after delivery. the late-onset syndrome causes the development of meningitis between birth and the third week of life it is often caused by serotype ivb and has a significant mortality rate.
adults can develop listeria meningoencephalitis, bacteremia, and (rarely) focal infections. meningoencephalitis and bacteremia occur most commonly in immunosuppressed patients, in whom listeria is one of the more common causes of meningitis. the diagnosis of listeriosis rests on isolation of the organism in cultures of blood and spinal fluid.
spontaneous infection occurs in many domestic and wild animals. in ruminants (eg, sheep) listeria may cause meningoencephalitis with or without bacteremia. in smaller animals (eg, rabbits, chickens), there is septicemia with focal abscesses in the liver and heart muscle and marked monocytosis.
many antimicrobial drugs inhibit listeria in vitro. clinical cures have been obtained with ampicillin, with erythromycin, or with intravenous trimethoprim-sulfamethoxazole. cephalosporins and fluoroquinolones are not active against l monocytogenes. ampicillin plus gentamicin is often recommended for therapy.