At the time of this writing, the genus Corynebacterium comprises 66 species; of these, 38 are thought to be clinically significant. This large group of bacteria includes animal and human pathogens as well as free-living saprophytes and plant pathogens. The majority of the species are found as normal flora on skin and mucous membranes of humans and animals. Some species are found worldwide in the environment. The corynebacteria are closely related to the mycobacteria and nocardiae; these three groups collectively may be referred to as the CMN group. The cell walls of corynebacteria contain meso-diaminopimelic acid (m-DAP) as the diamino acid, as well as short-chained mycolic acids.
Upon Gram staining, corynebacteria demonstrate slightly curved, gram-positive rods with unparallel sides and slightly wider ends, producing the described “club shape” or coryneform. The term diphtheroid, meaning diphtheria-like, is sometimes used in reference to this Gram staining morphology. The classification of the diphtheroids is not well characterized. Consequently, there is a low rate of identification for clinical isolates.
Even when sent to a reference laboratory, 30% to 50% of the coryneform-like isolates are unable to be identified to the species level.
Although they are frequently dismissed as contaminants, the non diphtheria corynebacteria are commonly isolated from clinical specimens. As with many previously deemed nonpathogenic organisms, however, the coryneforms are being isolated from a variety of body sites, especially in immunocompromised patients. The most significant pathogen of the group is C. diphtheriae, which has been extensively studied and is well characterized.
Other species that produce disease in humans are C. bovis, C. ulcerans, C. xerosis, C. jeikeium, C. pseudodiphtheriticum, and C. pseudotuberculosis.
The major virulence factor associated with C. diphtheria is the diphtheria toxin. This toxin is produced by strains of C. diphtheriae infected with a temperate bacteriophage, which carries the tox gene for diphtheria toxin. Nontoxigenic strains can be converted to tox by infection with the appropriate bacteriophage.
Only toxin-producing C. diphtheriae causes the disease diphtheria; however, C. ulcerans and C. pseudotuberculosis, which belong to the “c. diphtheria group,” may also produce the toxin when they become infected with the tox-carrying bacteriophage.
Diphtheria toxin is a protein of 62,000 daltons. It is exceedingly potent and is lethal for humans in amounts of 130 ng per kg of body weight. The toxicity of the toxin is due to its ability to block protein synthesis in eukaryotic cells. The toxin is secreted by the bacterial cell and is nontoxic until exposed to trypsin.
The trypsinization results in two polypeptide fragments, A and B, which are linked together by a disulfide bridge. Both fragments are necessary for cytotoxicity. Fragment A is responsible for the cytotoxicity; fragment B binds to receptors on the eukaryotic cells and mediates the entry of fragment A into the cytoplasm. On reaching the cytoplasm, fragment A disrupts protein synthesis.
Fragment A splits nicotinamide adenosine dinucleotide (NAD)to form nicotinamide and adenosine diphospharibose (ADPR).ADPR binds to and inactivates elongation factor 2 (EF-2),an enzyme required for elongation of polypeptide chains on ribosomes. The reaction can be summarized as follows:
NAD’ + EF-2 = ADPR-EF-2 + nicotinamide + H+
active inactive Production of the toxin in vitro depends on a number of environmental conditions: an alkaline pH (7.8 to 8.0), oxygen, and, most importantly, the iron concentration in the medium. The amount of iron needed for optimal toxin production is less than the amount needed for optimal growth. The toxin is released in significant amounts only when the available iron in the culture medium is exhausted.
Clinical Infections Diphtheria, which occurs in two forms (respiratory and cutaneous), is found worldwide but is uncommon in North America and Western Europe. Those cases that occur are invariably in nonimmunized populations as diphtheria has been uncommon in the United States since universal vaccination began in the 1940s. Epidemic diphtheria emerged in the New Independent States of the former Soviet Union in the 1990s, most probably due to inadequate mass vaccination strategies.
To prevent the spread of diphtheria, the Centers for Disease Control and Prevention (CDC)recommends that international travelers ensure that they are upto- date on all vaccinations. Individuals vaccinated as children who have not been revaccinated as adults are susceptible to infection. Humans are the only natural hosts for C. diphtheriae.
The organism is carried in the upper respiratory tract and spread by droplet infection or hand-ta-mouth contact. The incubation period averages 2 to 5 days.
The illness begins gradually and is characterized by low-grade fever, malaise, and a mild sore throat. The most common site of infection is the tonsils or pharynx.
The organisms rapidly multiply on the epithelial cells, and the toxigenic strains of C. diphtheriae produce toxin locally, causing tissue necrosis and exudates formation triggering an inflammatory reaction. This combination of cell necrosis and exudate forms a tough gray to white pseudomembrane, which attaches to the tissues. It may appear on the tonsils and then spread downward into the larynx and trachea. There is the potential for suffocation if the membrane spreads and blocks the air passage or if it is dislodged, perhaps as the result of sampling for a throat culture.
The toxin also is absorbed and can produce a variety of systemic effects involving the kidneys, heart, and nervous system, although all tissues possess the receptor for the toxin and may be affected. Death often is a result of cardiac failure. Another effect of the toxin is a demyelinating peripheral neuritis, which may result in paralysis following the acute illness.
Other nonrespiratory sites may be infected, although much less often than the upper respiratory tract. In the cutaneous form of diphtheria, which is prevalent in the tropics, the toxin also is absorbed systemically, but systemic complications are less common than from upper respiratory infections with C. diphtheriae. Diphtheria is treated by prompt administration of antitoxin. Commercial diphtheria antitoxin is produced in horses. Approximately 10%of patients who receive the antitoxin develop an allergic reaction to the horse serum. Consequently, hypersensitivity to horse serum precludes its administration. Antimicrobial agents have no effect on the toxin that is already circulating, but they do serve to eliminate the focus of infection as well as prevent the spread of the organism. The drug of choice is penicillin; erythromycin is used for penicillinsensitive individuals.
Corynebacterium diphtheriae is a gram-positive, nonsporulating, bacillus. The organism is highly pleomorphic and appears in palisades or as individual cells lying at sharp angles to another in Vand L formations.
This particular arrangement associated with C. diphtheriae has been described by westerners as “Chinese characters” (Figure 16-1), although it may be demonstrated by other Corynebacterium spp. Clubshaped swellings and beaded forms are common.
The organisms often stain irregularly, especially when stained with methylene blue, giving them a beaded appearance. The metachromatic areas of the cell, which stain more intensely than other parts, are called Babes-Ernst granules. They represent accumulation of polymerized polyphosphates. The presence of the Babes-Ernst granules indicates the accumulation of nutrient reserves and varies with the type of medium and the metabolic state of the individual cells.
The diphtheria bacillus, like most other corynebacteria, is a facultative anaerobe. It grows best under aerobic conditions and has an optimal growth temperature of 37° C, although multiplication occurs within the range of 15° to 40° C. Growth requirements are complex, requiring eight essential amino acids.
Although C. diphtheriae will grow on nutrient agar, better growth is usually obtained on a medium containing blood or serum, such as Loeffler serum or Pai agars.
Characteristic microscopic morphology is demonstrated well when organisms are grown on Loeffler medium. On SBA (Figure 16-2), the organism may have a very small zone of I3-hemolysis
Cystine-tellurite blood agar (CTBA) is a modification of Tinsdale medium; it contains sheep red blood cells, bovine serum, cystine, and potassium tellurite.
CTBA is both a selective and differential medium.
The potassium tellurite inhibits many non-coryneform bacteria. When grown on CTBA, corynebacteria form black or brownish colonies from the reduction of tellurite. This appearance, however, is not unique for C. diphtheriae, so care must be taken not to presumptively identify other genera that produce black colonies (e.g., Staphylococcus and Streptococcus) as Corynebacterium. A brown halo surrounding the colony, due to cystinase activity, is a useful differentiating feature, because only C. diphtheriae, C. ulcerans, and C. pseudotuberculosis produce a brown halo on CTBA. Identification The biochemical identification of medically important corynebacteria is outlined in Table 16-1. CTBA medium is useful for differentiations because only C. diphtheriae, C. ulcerans, and C. pseudotuberculosis form a brown halo. C. diphtheriae is distinguished from the other two species by its lack of urease production. C. diphtheria ferments glucose and maltose, producing acid but not gas, and is catalase positive. It reduces nitrate to nitrite and is nonmotile. A schematic diagram to presumptively identify C. diphtheriae is shown in Figure 16-3.
Test for Toxigenicity
The identification of an isolate as C. diphtheriae does not mean that the patient has diphtheria. Diagnosis of diphtheria depends on showing that the isolate produces diphtheria toxin. The growth medium and conditions markedly affect toxin production. The iron content of the medium must be growth rate-limiting
for full toxin production. The addition of iron to ironstarved cultures inhibits toxin production very quickly.
Toxin detection can be done either by in vivo or in vitro testing. In vivo testing is rarely done because the in vitro methods are reliable, less expensive, and free from the need to use animals.
The in vitro diphtheria toxin detection procedure is an immunodiffusion test first described by Elek. In the Elek test, organisms (controls and unknowns) are
streaked on media of low iron content to optimize toxin production. Each organism is streaked in a single straight line parallel to each other and 10 mm apart on an Elek plate. A filter paper strip impregnated with diphtheria antitoxin is laid along the center of the plate on a line at right angles to the lines of control
and unknown organisms (Figure 16-4). The plate is then incubated at 35° C and examined after 18, 24, and 48 hours. Lines of precipitation are best seen by transmitted light against a dark background. The white precipitin lines start about 4 to 5 mm from the filter paper strip and are at an angle of about 45 degrees to the line of growth. If an isolate is positive for toxin production,
and it is placed next to the positive control, the toxin line of the positive control should join the toxin line of the positive unknown to form an arch of identity.
The Elek test requires that reagents and antisera be carefully controlled and titrated. For this reason, and because of the difficulty of the test, it is performed
only in certain reference laboratories.
Rapid, in about 3 hours, enzyme-linked immunosorbent assays and immunochromatographic strip assays are also available for the detection of diphtheria toxin.
In addition, procedures for detecting the C. diphtheriaetox gene by the polymerase chain reaction (PCR) have been developed. The PCR assay can also be applied directly to clinical specimens.
Cornybacterium jeikeium, named after Johnson and Kaye who first linked this organism with human infections, appears to be part of the usual skin flora.
Infections have been limited to patients who are immune compromised, have undergone invasive procedures, or have a history of intravenous drug abuse. The presence of catheters or prosthetic devices also contributes to infection with C. jeikeium, and in fact, it is the most common cause of diphtheroid prosthetic valve endocarditis in adults. C. jeikeium is a strict aerobe that has been reported to be resistant to a wide range of antimicrobials. Most strains are susceptible to
vancomycin but show variable susceptibility to tetracycline and erythromycin. Multidrug resistance cannot be used as the only identifying characteristic because CDC group G may also demonstrate this characteristic.
Part of the normal flora of the human nasopharynx, C. pseudodiphtheriticum very rarely causes infection, but when infection occurs, it often takes the form of endocarditis. Respiratory and urinary tract infections (UTls) as well as cutaneous wound infections resulting from this organism have been seen in immunosuppressed patients, including those with acquired immunodeficiency
syndrome (AIDS). C. pseudodiphtheriticum, unlike other corynebacteria, does not show the characteristic pleomorphic morphology. The cells stain evenly and often lie in parallel rows (palisades). The species grows well on standard laboratory media, reduces nitrate, and hydrolyzes urea.
Like C. ulcerans discussed later, C. pseudotuberculosis is primarily a veterinary pathogen. Human infections usually have been associated with contact with sheep.
C. pseudotuberculosis produces a dermonecrotic toxin that causes death of a variety of cell types, and it can produce diphtheria toxin. Like C. diphtheriae, C. pseudotuberculosis also produces a brown halo on CTBA. On sheep blood agar (SBA), C. pseudotuberculosis forms small yellowish-white colonies. It is urease positive and gelatin negative. The organism is susceptible to penicillin and erythromycin.
As well as being found in the nasopharynx, C. striatum is normal human skin flora and is a rare cause of infection.
Nosocomial infections have been reported. The organism is a slow-growing, pleomorphic species that often produces small shiny, convex colonies in about
A veterinary pathogen causing mastitis in cattle and other domestic and wild animals, C. ulcerans has been isolated from patients with diphtheria-like illness. A significant number of isolates produce the diphtheria toxin, although the amount of toxin elaborated is much less than by C. diphtheriae. Human infections usually are acquired through contact with animals or by ingestion
of unpasteurized dairy products. The organism has been isolated from skin ulcers and exudative pharyngitis.
It grows well on Loeffler agar and CTBA, producing a brown halo around the colonies on CTBA. The organisms grow well on SBAand show a narrow zone
of B-hemolysis. Unlike C. diphtheriae, C. ulcerans does not reduce nitrate. It is gelatin positive at room temperature.
c. urealyticum is one of the most frequently isolated clinically significant corynebacteria. It has been described as primarily a urinary pathogen. This species is a slow-growing strict aerobe, so cultures must be incubated at least 48 hours before growth is detected. Urine isolates with pinpoint, nonhemolytic, white colonies that have characteristic diphtheroid microscopic morphology
are likely C. urealyticum. For a presumptive identification, the isolate should be catalase positive and rapidly urease positive, within minutes following inoculation on a Christensen urea slant. In addition, C. urealyticum does not ferment glucose. It also has shown resistance to a wide variety of antimicrobials, such as the B-Iactams, aminoglycosides, and macrolides.
c. xerosis is commonly found on skin and mucocutaneous sites. Opportunistic infections associated with this organism include prosthetic valve endocarditis,
bacteremia associated with intravenous OV)catheters, postsurgical wound infections, and pneumonia. Human infection with C. xerosis is rare, and affected patients are invariably immunosuppressed. This organism grows well on SBAand forms dry, pigmented (yellow to tan) colonies.
Identification of Coryneform Bacteria
Coryneform bacteria should be identified to the species level (1) if they are isolated from normally sterile sites, particularly from two or more blood cultures; (2) if they are the predominant organism from properly collected
clinical material; (3) from urine samples if they are the predominant organism and the total bacterial count is >105/mL in urine samples or if they are the sole isolate with a bacterial count of >104/mL.
In addition to conventional biochemical testing, commercial identification systems presently available include the RapID CB Plus system (Remel, Lenexa,
Kan), API Rapid Coryne System (bioMerieux, Durham, NC), the Microscan Panel (Dade Behring, Sacramento,Calif), and the BBL Crystal GP System (BD, Sparks, Md), among others. Although these systems have short incubation times ranging from a few hours to 48 hours, users should keep in mind their limitations.
The test systems perform best with species that grow rapidly in air and do not require nutritional supplements.
Despite the small number of substrates in the panels and infrequently updated databases, commercial systems coupled with Gram stain and colonial morphologies can provide relevant data for identification.
Clinically significant, unidentifiable coryneform bacteria should be sent to a reference laboratory for complete characterization. Thin-layer chromatography,
gas chromatography, mass spectrometry, or high-performance liquid chromatography can be used to further characterize coryneform bacteria. More recently, molecular genetic-based identification including restriction fragment length polymorphism analysis of the 16SrRNAgene has been used to identify species of corynebacteria. Arcanobacterium The genus Arcanobacterium contains five species, three of which are medically important: A. haemolyticum (formerly Corynebacterium haemolyticum), A. pyogenes, and A. bernardiae. All three species are catalase positive. A. haemolyticum has been recovered from 10- to 20-yearold patients with pharyngitis and, therefore, must be
distinguished from C. diphtheriae and C. ulcerans as well as group A streptococcus. A pruritic, scarlatiniform rash and desquamation of the skin of the hands and feet may occur in infections with A. haemolyticum.
A. haemolyticum produces small colonies on SBA that demonstrate a narrow zone of B-hemolysis after 24 to 48 hours of incubation similar in appearance to the f)-hemolytic streptococci. Frequently, a black opaque dot is observed on the agar when the colony is scraped away. Pitting of the agar beneath the colony has also been reported. A Gram stain of the isolated colony quickly eliminates the possibility of group A streptococci, because A. haemolyticum is a gram-positive rod.
A. haemolyticum is both lipase and lecithinase positive. It exhibits a reverse CAMP reaction (CAMP inhibition reaction) because the hemolysis produced by a B-Iysin-producing Staphylococcus aureus is inhibited by a phospholipase D excreted by A. haemolyticum.
Erythromycin is the drug of choice for treatment; A. haemolyticum is penicillin resistant.
Rhodococcus equi (formerly Corynebacterium eqUl) is found in soil and causes respiratory tract infections in animals. Human infection is rare, although an increased incidence in immunosuppressed patients, particularly AIDS patients, is being reported. On Gram stain, R. equi may demonstrate filaments, some with branching. R. equi may be partially acid fast. On SBA, the colonies resemble Klebsiella and can form a salmon-pink pigment upon prolonged incubation, especially at room temperature. Biochemical identification is difficult because
it does not ferment carbohydrates and shows a variable reaction to a number of characteristics (e.g., nitrate reduction and urease). Key features for the identification of Rhodococcus is the salmon-pink pigment and a Gram stain showing characteristic diphtheroid gram-positive rods with traces of branching.
A member of the normal human oropharyngeal flora, Rothia dentocariosa may be found in saliva and supragingival plaque. It has been isolated from patients with endocarditis. Microscopically, this organism resembles the coryneform bacilli, forming short gram-positive rods but also branching filaments that resemble those of facultative actinomycetes. When placed in broth, however,
the species produces coccoid cells, a characteristic differentiating it from the Actinomyces. Rothia is nitrate positive, nonmotile, and urease negative. Approximately two thirds of the isolates are catalase positive.
Undesignated CDC Coryneform Groups
Several coryneform bacilli are unnamed and remain designated with CDCgroup numbers and letters while awaiting proper species designation. These organisms
have been isolated from a wide range of clinical samples, and they should be regarded as potential nosocomial pathogens or opportunistic pathogens in the immunocompromised patient.