Non-Spore-Forming Gram-Positive Bacilli: Corynebacterium, Propionibacterium, Listeria, Erysipelothrix, Actinomycetes, & Related Pathogens

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1 4010_1-16 2/11/04 9:27 AM Page 212 Non-Spore-Forming Gram-Positive Bacilli: Corynebacterium, Propionibacterium, Listeria, Erysipelothrix, Actinomycetes, & Related Pathogens 13 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; although not all isolates have the irregular shapes, the term coryneform bacteria is a convenient one for denoting the group. These bacteria have a high guanosine plus cytosine content and include the genera corynebacterium, arcanobacterium, brevibacterium, mycobacterium, and others (Table 13 1). Actinomyces and propionibacterium are classified as anaerobes, but some isolates grow well aerobically (aerotolerant) and must be differentiated from the aerobic 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. The medically important genera of gram-positive bacilli are listed in Table 13 1 and include some sporeforming and anaerobic genera. Anaerobic bacteria are discussed briefly in this chapter and in Chapter 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. Most isolates of the rapidly growing mycobacterium species and of the nocardia and rhodococcus species are acid-fast and, therefore, readily distinguished from the coryneform bacteria. Many but not all genera of bacillus and clostridium produce spores, and the presence of spores readily distinguishes the isolate from the coryneform bacteria; however, Clostridium perfringens and other filamentous clostridia generally do not produce spores on laboratory media. Determination that an isolate is a lactobacillus (or propionibacterium) may require gas-liquid chromatography to measure lactic acid (or propionic acid) metabolic products, but this is generally not practical. Other tests that are used to help identify an isolate of non-sporeforming gram-positive bacilli as a member of a genus or species include catalase production, indole production, nitrate reduction, and fermentation of carbohydrates, among others. CORYNEBACTERIUM DIPHTHERIAE Morphology & Identification Corynebacteria are µm in diameter and several micrometers long. Characteristically, they possess irregular swellings at one end that give them the clubshaped appearance (Figure 13 1). Irregularly distrib-

2 4010_1-16 2/23/04 3:25 PM Page 213 NON-SPORE-FORMING GRAM-POSITIVE BACILLI / 213 Table Some of the more common gram-positive bacilli of medical importance. Aerobic Gram-Positive Bacilli With High G + C Content and Irregular Shape 1 Aerobic Gram-Positive Bacilli With Lower G + C Content and More Regular Shape Genera Common Corynebacterium Uncommon Arcanobacterium Rhodococcus Rothia Many other genera of skin and environmental flora Aerotolerant anaerobes Actinomyces Propionibacterium Major pathogen: Corynebacterium diphtheriae Common or clinically important isolates of the genus corynebacterium C amycolatum C minutissimum C jeikeium C pseudodiphtheriticum C striatum C urealyticum C xerosis G + C = guanine plus cytosine base. 1 The medically important coryneform bacteria. Genera Common Listeria Erysipelothrix Gardnerella Aerotolerant anaerobes/strict anaerobes Lactobacillus Clostridium (spore-forming) (Chapter 12) Aerobes Bacillus (spore-forming) (Chapter 12) Major pathogens Listeria monocytogenes Erysipelothrix rhusiopathiae uted 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 Figure Corynebacterium diphtheriae from Pai medium and stained with methylene blue. Arrows indicate clubbed ends on some of the bacteria. acute angles to one another. True branching is rarely observed in cultures. 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). 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. Very few reference laboratories provide the biotype characterization; the incidence of diphtheria has greatly decreased and the association of severity of disease with biovar is not important to clinical or public health management of cases or outbreaks. If necessary in the setting of an outbreak, immunochemical and molecular methods can be used to type the C diphtheriae isolates. C diphtheriae and other corynebacteria grow aerobically on most ordinary laboratory media. Propionibacterium is an anaerobe. On Loeffler s serum medium,

3 4010_1-16 2/11/04 9:27 AM Page / CHAPTER 13 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. 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 (MW 62,000) that can be lethal in a dose of 0.1 µg/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. Toxin fragment A inactivates EF-2 by catalyzing a reaction that yields free nicotinamide plus an inactive adenosine diphosphateribose-ef-2 complex. It is assumed that the abrupt arrest of protein synthesis is responsible for the necrotizing and neurotoxic effects of diphtheria toxin. An exotoxin with a similar mode of action can be produced by strains of Pseudomonas aeruginosa. 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. C diphtheriae does not need to be toxigenic to establish localized infection in the nasopharynx or skin, for example but nontoxigenic strains do not yield the localized or systemic toxic effects. C diphtheriae does not actively invade deep tissues and practically never enters the bloodstream. 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. In general, var gravis tends to produce more severe disease than var mitis, but similar illness can be produced by all types. Diagnostic Laboratory Tests These serve to confirm the clinical impression and are of epidemiologic significance. Note: Specific treatment

4 4010_1-16 2/11/04 9:27 AM Page 215 NON-SPORE-FORMING GRAM-POSITIVE BACILLI / 215 must never be delayed for laboratory reports if the clinical picture is strongly suggestive of diphtheria. 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. Inoculate a blood agar plate (to rule out hemolytic streptococci), a Loeffler slant, and a tellurite plate (eg, cystine-tellurite agar or modified Tinsdale medium) and incubate all at 37 C. Unless the swab can be inoculated promptly, it should be kept moistened with sterile horse serum so the bacilli will remain viable. In hours, the Loeffler slant may yield organisms of typical diphtheria-like morphology. In hours, the colonies on tellurite medium are sufficiently definite for recognition of C diphtheriae. A presumptive C diphtheriae isolate should be subjected to testing for toxigenicity. Such tests are performed only in reference public health laboratories. There are several methods, as follows: (1) A filter paper disk containing antitoxin is placed on an agar plate. The cultures to be tested for toxigenicity are streaked across the plate next to the disk. After 48 hours of incubation, the antitoxin diffusing from the paper disk has precipitated the toxin diffusing from toxigenic cultures and has resulted in precipitate bands between the disk and the bacterial growth. This is the modified Elek method described by the WHO Diphtheria Reference Unit. (2) Polymerase chain reaction-based methods have been described for detection of the diphtheria toxin gene (tox). PCR assays for tox can also be used directly on patient specimens before culture results are available. A positive culture confirms a positive PCR assay. A negative culture following antibiotic therapy along with a positive PCR assay suggests that the patient probably has diphtheria. (3) Enzyme-linked immunosorbent assays can be used to detect diphtheria toxin from clinical C diphtheriae isolates. (4) An immunochromographic strip assay allows detection of diphtheria toxin in a matter of hours. Historically, toxigenicity of a C diphtheriae isolate has been demonstrated by injecting two guinea pigs with the emulsified isolate. If the guinea pig protected with diphtheria antitoxin survives while the unprotected one dies, the isolate is considered to be toxigenic. This test has largely been replaced by more modern technology. 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. Diphtheria antitoxin is produced in various animals (horses, sheep, goats, and rabbits) by the repeated injection of purified and concentrated toxoid. Treatment with antitoxin is mandatory when there is strong clinical suspicion of diphtheria. From 20,000 to 100,000 units are injected intramuscularly or intravenously after suitable precautions have been taken (skin or conjunctival test) to rule out hypersensitivity to the animal serum. The antitoxin should be given on the day the clinical diagnosis of diphtheria is made and need not be repeated. Intramuscular injection may be used in mild cases. 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. Epidemiology, Prevention, & Control Before artificial immunization, diphtheria was mainly a disease of small children. The infection occurred either clinically or subclinically at an early age and resulted in the widespread production of antitoxin in the population. An asymptomatic infection during adolescence and adult life served as a stimulus for maintenance of high antitoxin levels. Thus, most members of the population, except children, were immune. By age 6 8 years, approximately 75% of children in developing countries where skin infections with C diphtheriae are common have protective serum antitoxin levels. Absorption of small amounts of diphtheria toxin from the skin infection presumably provides the antigenic stimulus for the immune response; the amount of absorbed toxin does not produce disease. 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

5 4010_1-16 2/11/04 9:27 AM Page / CHAPTER 13 sufficiently prevalent in the population of many developed countries to provide the stimulus of subclinical 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. The principal aims of prevention are to limit the distribution of toxigenic diphtheria bacilli in the population and to maintain as high a level of active immunization as possible. To limit contact with diphtheria bacilli to a minimum, patients with diphtheria should be isolated. Without treatment, a large percentage of infected persons continue to shed diphtheria bacilli for weeks or months after recovery (convalescent carriers). This danger may be greatly reduced by active early treatment with antibiotics. 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) 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. All children must receive an initial course of immunizations and boosters. Regular boosters with Td are particularly important for adults who travel to developing countries, where the incidence of clinical diphtheria may be 1000-fold higher than in developed countries, where immunization is universal. 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. Nonlipophilic Corynebacteria Corynebacterium ulcerans and Corynebacterium pseudotuberculosis are closely related to Corynebacterium diphtheriae and may carry the diphtheria tox gene; they probably are not separate species from C diphtheriae. The toxigenic C ulcerans can cause disease similar to clinical diphtheria, while C pseudotuberculosis rarely causes disease in humans. Other species in the nonlipophilic fermentative group include Corynebacterium xerosis, Corynebacterium striatum, Corynebacterium minutissimum, and Corynebacterium amycolatum. These are among the most commonly isolated coryneform bacteria. Many isolates previously identified as C xerosis may have been misidentified and were really C amycolatum. There are few well-documented cases of disease caused by C minutissimum, though the organism is frequently isolated from clinical specimens. Historically, C xerosis and C striatum have caused a variety of infections in humans. The group of nonlipophilic nonfermentative corynebacteria includes multiple species. Corynebacterium auris has been associated with ear infections in children, and Corynebacterium pseudodiphtheriticum has been associated with respiratory tract infections. Corynebacterium glucuronolyticum is often urease-positive and is a urinary tract pathogen. Lipophilic Corynebacteria Corynebacterium jeikeium is the coryneform bacterium most commonly isolated from acutely ill patients. It can cause disease in immunocompromised patients and is important because it produces infections, including bacteremia, that have a high mortality rate and because it is resistant to many commonly used antimicrobial drugs. Corynebacterium urealyticum is a slowly growing species that is multiply resistant to antibiotics. As its name implies, it is urease-positive. It has been associated with acute or chronic encrusted urinary tract infections manifested by alkaline urine ph and crystal formation. 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

6 4010_1-16 2/11/04 9:27 AM Page 217 NON-SPORE-FORMING GRAM-POSITIVE BACILLI / 217 clinically significant infections. Actinomyces viscosis grows readily under aerobic conditions. Other Coryneform Genera There are many other genera and species of coryneform bacteria. Arcanobacterium haemolyticum produces betahemolysis on blood agar. It is occasionally associated with pharyngitis and can grow in media selective for streptococci. A haemolyticum is catalase-negative, like group A streptococci, and must be differentiated by Gram stain morphology (rods versus cocci) and biochemical characteristics. Most of the coryneform bacteria in the other genera are infrequent causes of disease and are not commonly identified in the clinical laboratory. Rhodococcus equi (formerly Corynebacterium equi) may appear to be a bacillus after a few hours of incubation in broth, but with further incubation it becomes coccoid in shape. The organisms are generally weakly acid-fast when stained by the modified Kinyoun method. R equi occasionally causes infections such as necrotizing pneumonia in immunosuppressed patients with abnormal cell-mediated immunity (eg, AIDS patients). R equi is present in soil and in dung of herbivores. The organism is an occasional cause of disease in cattle, sheep, and swine and can cause severe lung infections in foals. Other species of the diverse genus rhodococcus are present in the environment but rarely cause disease in humans. Rothia dentocariosa is a gram-positive rod that forms branching filaments. It has been associated with abscesses and endocarditis, presumably following entry into the blood from the mouth. 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-sporeforming rod. It has a tumbling end-over-end motility at 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 induce 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. Clinical Findings Perinatal human listeriosis (granulomatosis infantiseptica) may be an intrauterine infection. The early-onset syndrome results in intrauterine sepsis and death before

7 4010_1-16 2/11/04 9:27 AM Page / CHAPTER 13 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. Clinical presentation of listeria meningitis in these patients varies from insidious to fulminant and is nonspecific. 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 trimethoprimsulfamethoxazole. Ampicillin plus gentamicin is often recommended for therapy, but gentamicin does not enter host cells and may not help treat the listeria infection. ERYSIPELOTHRIX RHUSIOPATHIAE Erysipelothrix rhusiopathiae (also called Erysipelothrix insidiosa) is a gram-positive bacillus that produces small, transparent glistening colonies. It may be alphahemolytic on blood agar. On Gram stains it sometimes looks gram-negative because it decolorizes easily. The bacteria may appear singly, in short chains, randomly, or in long nonbranching filaments. The colony morphology and Gram stain appearance vary depending upon the growth medium, incubation temperature, and ph. Erysipelothrix is catalase-, oxidase-, and indole-negative. When erysipelothrix is grown on triple sugar iron agar, hydrogen sulfide is produced, turning the TSI butt black. E rhusiopathiae must be differentiated from L monocytogenes, Arcanobacterium pyogenes, and Arcanobacterium haemolyticum, but these three species are betahemolytic and do not produce hydrogen sulfide when grown on TSI medium. It is more difficult to differentiate E rhusiopathiae from aerotolerant lactobacilli; both may be alpha-hemolytic. They are catalase-negative and vancomycin-resistant (80% of lactobacilli). In addition, some strains of lactobacilli produce H 2 S much like E rhusiopathiae. E rhusiopathiae is distributed in land and sea animals worldwide, including a variety of vertebrates and invertebrates. It causes disease in domestic swine, turkeys, ducks, and sheep. The most important impact is in swine, where it causes erysipelas. In humans, erysipelas is caused by group A beta-hemolytic streptococci and is much different from erysipelas of swine. People obtain E rhusiopathiae infection by direct inoculation from animals or animal products. Persons at greatest risk are fishermen, fish handlers, abattoir workers, butchers, and others who have contact with animal products. The most common E rhusiopathiae infection in humans is called erysipeloid. It usually occurs on the fingers by direct inoculation at the site of a cut or abrasion (and has been called seal finger and whale finger ). After 2 7 days incubation, pain, which can be severe, and swelling occur. The lesion is raised, and violaceous in color. Pus is usually not present at the infection site, which helps differentiate it from staphylococcal and streptococcal skin infections. Erysipeloid can resolve after 3 4 weeks, or more rapidly with antibiotic treatment. Additional clinical forms of infection (both rare) are a diffuse cutaneous form and bacteremia with endocarditis. Erysipelothrix is highly susceptible to penicillin G, the drug of choice for severe infections. The organism is intrinsically resistant to vancomycin. 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 streptomycetes. As the bacilli grow, the cells remain together after division to form elongated chains of bacteria (1 µm in width) with occasional branches. The extent of this process varies in different taxa. It is rudimentary in some actinomycetes the chains are short, break apart after formation, and resemble diphtheroids; others develop extensive substrate or aerial filaments (or both); and either may produce spores or fragment into coccobacillary forms. Most are saprophytes that live in soil, but members of this group of bacteria are responsible for three human infections: actinomycosis, nocardiosis, and actinomycetoma. ACTINOMYCOSIS Actinomycosis is a chronic suppurative and granulomatous infection that produces pyogenic lesions with interconnecting sinus tracts that contain granules composed of microcolonies of the bacteria embedded in tissue elements. The etiologic agents are several closely related members of the normal flora of the mouth and gastrointestinal tract. Most cases are due to Actinomyces israelii, Actinomyces naeslundii, and related anaerobic or facultative bacteria. Based on the site of involvement, the three common forms are cervicofacial, thoracic, and abdomi-

8 4010_1-16 2/23/04 3:25 PM Page 219 NON-SPORE-FORMING GRAM-POSITIVE BACILLI / 219 nal actinomycosis. Regardless of site, infection is initiated by trauma that introduces these endogenous bacteria into the mucosa. Often, in addition to the primary agent of actinomycosis, there are concomitant bacteria present. Some of these are relatively fastidious gram-negative bacilli such as Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Eikenella corrodens, and capnocytophaga species. Occasionally, staphylococci, streptococci, or enteric gram-negative bacilli are found. Morphology & Identification Most strains of A israelii and the other agents of actinomycosis are facultative anaerobes that grow best in an atmosphere with increased carbon dioxide. On enriched medium, such as brain-heart infusion agar, young colonies (24 48 hours) produce gram-positive substrate filaments that fragment into short chains, diphtheroids, and coccobacilli. After a week, these spider colonies develop into white, heaped-up molar tooth colonies. In thioglycolate broth, A israelii grows below the surface in compact colonies. Species are identified based on cell wall chemotype and biochemical reactions. The sulfur granules found in tissue are yellowish in appearance, up to 1 mm in size, and are composed of macrophages, other tissue cells, fibrin, and the bacteria. Eosinophilic club-shaped enlargements of the bacterial cells often project from the periphery of the granule. Pathogenesis & Pathology Regardless of the body site, the natural history is similar. The bacteria bridge the mucosal or epithelial surface of the mouth, respiratory tract, or lower gastrointestinal tract associated with dental caries, gingivitis, surgical complication, or trauma. Aspiration may lead to pulmonary infection. The organisms grow in an anaerobic niche, induce a mixed inflammatory response, and spread with the formation of sinuses, which contain the granules and may drain to the surface. The infection causes swelling and may spread to neighboring organs, including the bones. There is often superinfection with other endogenous bacteria. Clinical Findings Cervicofacial disease presents as a swollen, erythematous process in the jaw area. With progression, the mass becomes fluctuant, producing draining fistulas. The disease will extend to contiguous tissue, bone, and lymph nodes of the head and neck. The symptoms of thoracic actinomycosis resemble those of a subacute pulmonary infection: mild fever, cough, and purulent sputum. Eventually, lung tissue is destroyed, sinus tracts may erupt to the chest wall, and invasion of the ribs may occur. Abdominal actinomycosis often follows a ruptured appendix or an ulcer. In the peritoneal cavity, the pathology is the same, but any of several organs may be involved, including the kidneys, vertebrae, and liver. Genital actinomycosis is a rare occurrence in women that results from colonization of an intrauterine device with subsequent invasion. 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 (Figure 13 2). Specimens are cultured in thioglycolate broth and on brain-heart infusion blood A B C Figure Actinomyces israelii. A: Sulfur granule in pus. B: Gram-positive filaments in broth culture. C: Diphtheroid-like and branching bacilli in agar culture.

9 4010_1-16 2/11/04 9:27 AM Page / CHAPTER 13 agar plates, which are incubated anaerobically or under elevated carbon dioxide conditions. Growth is examined for typical morphology and biochemical reactions. The main agents of actinomycosis are catalase-negative, whereas most other actinomycetes are catalase-positive. Surface lesions may also contain other bacterial species. Treatment Prolonged administration (6 12 months) of a penicillin is effective in many cases. Clindamycin or erythromycin is effective in penicillin-allergic patients. However, drugs may penetrate the abscesses poorly, and some of the tissue destruction may be irreversible. Surgical excision and drainage may also be required. Epidemiology Because A israelii and the related agents of actinomycosis are endogenous members of the bacterial flora, they cannot be eliminated. Some individuals with recurrent infections are given prophylactic penicillin, especially prior to dental procedures. NOCARDIOSIS Nocardiosis is caused by infection with Nocardia 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. The importance of the complex is that its members tend to have variable antimicrobial susceptibility, which can influence treatment. The pathogenic nocardiae, like many nonpathogenic species of nocardia, are found worldwide in soil and water. Nocardiosis is initiated by inhalation of these bacteria. The usual presentation is as a subacute to chronic pulmonary infection that may disseminate to other organs, usually the brain or skin. Nocardiae are not transmitted from person to person. Morphology & Identification Nocardia species are aerobic and grow on a variety of media. Over the course of several days to a week or more, they develop heaped, irregular, waxy colonies. Strains vary in their pigmentation from white to orange to red. These bacteria are gram-positive, catalase-positive, and partially acid-fast bacilli. They produce urease and can digest paraffin. Nocardiae form extensive branching substrates and aerial filaments that fragment after formation, breaking into coccobacillary cells. The cell walls contain mycolic acids. They are considered to be weakly acid-fast, but if they are stained with the routine acid-fast reagent (carbol-fuchsin) but decolorized with 1 4% sulfuric acid instead of the stronger acidalcohol decolorant, most isolates will stain acid-fast. The species of nocardia are identified by routine physiologic tests. Pathogenesis & Clinical Findings In most cases, nocardiosis is an opportunistic infection associated with several risk factors, most of which impair the cell-mediated immune responses: corticosteroid treatment, immunosuppression, organ transplantation, AIDS, tuberculosis, and alcoholism. Nocardiosis begins as chronic lobar pneumonia, and a variety of symptoms may occur, including fever, weight loss, and chest pain. The clinical manifestations are not distinctive and mimic tuberculosis and other infections. Pulmonary consolidations may develop, but granuloma formation and caseation are rare. The usual pathologic process is abscess formation. Spread from the lung often involves the central nervous system, where abscesses develop in the brain, leading to a variety of clinical presentations. Some patients have subclinical lung involvement and present with brain lesions. Dissemination may also occur to the skin, kidney, or elsewhere. Diagnostic Laboratory Tests Specimens consist of sputum, pus, spinal fluid, and biopsy material. Gram-stained smears reveal gram-positive bacilli, coccobacillary cells, and branching filaments. With the modified acid-fast stain, most isolates will be acid-fast. Nocardia species grow on most laboratory media. Serologic tests are unreliable at present. Treatment The treatment of choice is trimethoprim-sulfamethoxazole. If patients fail to respond, a number of other antibiotics have been used with success, such as amikacin, imipenem, and cefotaxime. Surgical drainage or resection may be required. ACTINOMYCETOMA Mycetoma (Madura foot) is a localized, slowly progressive, chronic infection that begins in subcutaneous tissue and spreads to adjacent tissues. It is destructive and often painless. In many cases the cause is a soil fungus that has been implanted into the subcutaneous tissue by minor trauma. This form of mycetoma is discussed in Chapter 45. An actinomycetoma is a mycetoma caused by filamentous branching bacteria. The actinomycetoma granule is composed of tissue elements and grampositive bacilli and bacillary chains or filaments (1 µm in diameter). The most common causes of actinomycetoma are Nocardia brasiliensis, Streptomyces somaliensis,

10 4010_1-16 2/11/04 9:27 AM Page 221 NON-SPORE-FORMING GRAM-POSITIVE BACILLI / 221 and Actinomadura madurae. N brasiliensis may be acidfast. These and other pathogenic actinomycetes are differentiated by biochemical tests and chromatographic analysis of cell wall components. Actinomycetomas respond well to various combinations of streptomycin, trimethoprim-sulfamethoxazole, and dapsone if therapy is begun early before extensive damage has occurred. REVIEW QUESTIONS 1. Three months ago, a 53-year-old woman had surgery and chemotherapy for breast cancer. Four weeks ago, she developed a cough occasionally productive of purulent sputum. About 2 weeks ago, she noted a slight but progressive weakness of her left arm and leg. On chest examination, rales were heard over the left upper back when the patient breathed deeply. Neurologic examination confirmed weakness of the left arm and leg. Chest x-ray showed a left upper lobe infiltrate. Brain scan showed two lesions in the right hemisphere. Gram stain of a purulent sputum specimen showed branching gram-positive rods that were partially acid-fast. Which of the following organisms is the cause of this patient s current illness? (A) Actinomyces israelii (B) Corynebacterium pseudodiphtheriticum (C) Aspergillus fumigatus (D) Nocardia asteroides (E) Erysipelothrix rhusiopathiae 2. The drug of choice to treat this patient s infection (Question 1) is (A) Penicillin G (B) Trimethoprim-sulfamethoxazole (C) Gentamicin (D) Amphotericin B (E) A third-generation cephalosporin 3. It is particularly difficult to differentiate Erysipelothrix rhusiopathiae from (A) Corynebacterium diphtheriae (B) Bacillus cereus (C) Actinomyces israelii (D) Nocardia asteroides (E) Lactobacillus species 4. Movement of Listeria monocytogenes inside of host cells is caused by (A) Inducing host cell actin polymerization (B) The formation of pili (fimbriae) on the listeriae surface (C) Pseudopod formation (D) The motion of listeriae flagella (E) Tumbling motility 5. An 8-year-old boy develops a severe sore throat. On examination, a grayish exudate (pseudomembrane) is seen over the tonsils and pharynx. The differential diagnosis of severe pharyngitis such as this includes group A streptococcal infection, Epstein-Barr virus (EBV) infection, Neisseria gonorrhoeae pharyngitis, and diphtheria. The cause of the boy s pharyngitis is most likely (A) A gram-negative bacillus (B) A single-stranded positive-sense RNA virus (C) A catalase-positive gram-positive coccus that grows in clusters (D) A club-shaped gram-positive bacillus (E) A double stranded RNA virus 6. The primary mechanism in the pathogenesis of the boy s disease (Question 5) is (A) A net increase in intracellular cyclic adenosine monophosphate (B) Action of pyrogenic exotoxin (a superantigen) (C) Inactivation of acetylcholine esterase (D) Action of enterotoxin A (E) Inactivation of elongation factor 2 7. Corynebacterium jeikeium is (A) Catalase-negative (B) Gram-negative (C) Often resistant to commonly used antibiotics (D) Motile (E) Common but clinically unimportant 8. Priopionibacterium acnes is (A) Catalase-negative (B) A strict anaerobe (C) Part of the normal gastrointestinal flora (D) Not a coryneform bacterium (E) So named because its major metabolic product is propionic acid 9. Skin diphtheria as occurs in children in tropical areas typically (A) Does not occur in children who have been immunized with diphtheria toxoid (B) Is clinically distinct from skin infections (pyoderma, impetigo) caused by Streptococcus pyogenes and Staphylococcus aureus (C) Is also common in northern latitudes (D) Results in protective antitoxin levels in most of the children by the time they are 6 8 years old (E) Yields toxin-mediated cardiomyopathy

11 4010_1-16 2/11/04 9:27 AM Page / CHAPTER Actinomycosis (A) Usually requires 6 12 months of antimicrobial therapy (eg, a penicillin, erythromycin) (B) Is typically associated with only one pathogen on laboratory investigation (C) Is rarely caused by Actinomyces israelii (D) Is caused by a microorganism that is not part of the normal flora in humans Answers 1. D 6. E 2. B 7. C 3. E 8. E 4. A 9. D 5. D 10. A REFERENCES Funke G et al: Clinical microbiology of coryneform bacteria. Clin Microbiol Rev 1997;10:125. Funke G, Bernard KA: Coryneform gram-positive rods. In: Manual of Clinical Microbiology, 8th ed. Murray PR et al (editors). ASM Press, Lorber B: Listeriosis. Clin Infect Dis 1997;24:1. Reboli AC, Farrar WE: Erysipelothrix rhusiopathiae: an occupational pathogen. Clin Microbiol Rev 1989;2:354. Russo TA: Agents of actinomycosis. In: Mandell, Douglas, and Bennett s Principles and Practice of Infectious Diseases, 5th ed. Mandell GL, Bennett JE, Dolin R (editors). Churchill Livingstone, Sorrell TC, Iredell JR, Mitchell DH: Nocardia species. In: Mandell, Douglas, and Bennett s Principles and Practice of Infectious Diseases, 5th ed. Mandell GL, Bennett JE, Dolin R (editors). Churchill Livingstone, Southwick FS, Purich DL: Intracellular pathogenesis of listeriosis. N Engl J Med 1996;334:770.