Most of the organisms listed in Table 17-1 are part of the normal human flora and colonize various parts of the human body, are found in the environment, or are associated with various animals. The two most notable pathogens are Listeria monocytogenes and Corynebacterium diphtheriae. However, these two species differ markedly in epidemiology. L. monocytogenes is widely distributed in nature and occasionally colonizes the human gastrointestinal tract. Many foods are contaminated with L. monocytogenes, including milk, raw vegetables, cheese, and meats. C. diphtheriae is only carried by humans, but in rare cases it is isolated from healthy individuals. Primary transmission for C. diphtheriae is through respiratory secretions or exudates from skin lesions.

TABLE 17-1

Epidemiology

Organism	Habitat (Reservoir)	Mode of Transmission
Listeria monocytogenes	Colonizer: Animals, soil, and vegetable matter; widespread in these environments Human gastrointestinal tract	Direct contact: Ingestion of contaminated food, such as meat and dairy products Endogenous strain: Colonized mothers may pass organism to fetus. Portal of entry is probably from gastrointestinal tract to blood and in some instances from blood to meninges.
Corynebacterium diphtheriae	Colonizer: Human nasopharynx but only in carrier state; not considered part of normal flora Isolation from healthy humans is not common.	Direct contact: Person to person by exposure to contaminated respiratory droplets Contact with exudate from cutaneous lesions Exposure to contaminated objects
Corynebacterium jeikeium	Colonizer: Skin flora of hospitalized patients, most commonly in the inguinal, axillary, and rectal sites	Uncertain Direct contact: May be person to person Endogenous strain: Selection during antimicrobial therapy Introduction during placement or improper care of intravenous catheters
Corynebacterium ulcerans	Normal flora: Humans and cattle	Uncertain Zoonoses: Close animal contact, especially during summer
Corynebacterium pseudotuberculosis	Normal flora: Animals such as sheep, goats, and horses	Uncertain Zoonoses: Close animal contact, but infections in humans are rare
Corynebacterium pseudodiphtheriticum	Normal flora: Human pharyngeal and occasionally skin flora	Uncertain Endogenous strain: Access to normally sterile site
Corynebacterium minutissimum	Normal flora: Human skin	Uncertain Endogenous strain: Access to normally sterile site
Corynebacterium urealyticum	Normal flora: Human skin	Uncertain Endogenous strain: Access to normally sterile site
Leifsonia aquatica (formerly Corynebacterium aquaticum)	Environment: Fresh water	Uncertain
Corynebacterium xerosis	Normal flora: Human conjunctiva Skin Nasopharynx	Uncertain Endogenous strain: Access to normally sterile site
Corynebacterium striatum	Normal flora: Skin	Uncertain Endogenous strain: Access to normally sterile site
Corynebacterium amycolatum	Normal flora: Human conjunctiva Skin Nasopharynx	Uncertain Endogenous strain: Access to normally sterile site
Corynebacterium auris	Uncertain: Probably part of normal human flora	Uncertain Rarely implicated in human infections
Kurthia spp.	Environment	Uncertain Rarely implicated in human infections
Brevibacterium spp.	Normal flora: Human Various foods	Uncertain Rarely implicated in human infections
Dermabacter hominis	Normal flora: Human skin	Uncertain Rarely implicated in human infections
Turicella otitidis	Uncertain: Probably part of normal human flora	Uncertain Rarely implicated in human infections
Arthrobacter spp., Microbacterium spp., Cellulomonas spp., and Exiguobacterium sp.	Uncertain Probably environmental	Uncertain Rarely implicated in human infections

In contrast to these two organisms, C. jeikeium is commonly encountered in clinical specimens, mostly because it tends to proliferate as skin flora of hospitalized individuals. However, C. jeikeium is not considered to be highly virulent. The penetration of the patient’s skin by intravascular devices is usually required for this organism to cause infection.

Pathogenesis and Spectrum of Disease

L. monocytogenes, by virtue of its ability to survive within phagocytes, and C. diphtheriae, by production of an extremely potent cytotoxic exotoxin, are the most virulent species listed in Table 17-2. Not all strains of C. diphtheriae are toxin-producing strains. The toxin gene is present in strains that have acquired the gene by viral transduction. The result is the incorporation of the toxin gene into the organisms’ genome. C. diphtheriae occurs in four biotypes: gravis, intermedius, belfanti, and mitis; C. gravis causes the most severe form of disease. The biotypes can be differentiated based on colonial morphology, biochemical reactions, and hemolytic patterns on blood agar.

TABLE 17-2

Pathogenesis and Spectrum of Diseases

Organism	Virulence Factors	Spectrum of Diseases and Infections
Listeria monocytogenes	Listeriolysin O: A hemolytic and cytotoxic toxin that allows for survival within phagocytes Internalin: Cell surface protein that induces phagocytosis Act A: Induces actin polymerization on the surface of host cells, producing cellular extensions and facilitating cell-to-cell spread. Siderophores: Organisms capable of scavenging iron from human transferrin and of enhanced growth of organism.*	Systemic: Bacteremia, without any other known site of infection CNS infections: Meningitis, encephalitis, bran abscess, spinal cord infections Neonatal: Early onset: Granulomatosis infantisepticum—in utero infection disseminated systemically that causes stillbirth Late onset: Bacterial meningitis Immunosuppressed patients
Corynebacterium diphtheriae	Diphtheria toxin: A potent exotoxin that destroys host cells by inhibiting protein synthesis.	Respiratory diphtheria is a pharyngitis characterized by the development of an exudative membrane that covers the tonsils, uvula, palate, and pharyngeal wall; if untreated, life-threatening cardiac toxicity, neurologic toxicity, and other complications occur. Respiratory obstruction develops and release of toxin into the blood can damage various organs, including the heart.
	Nontoxigenic strains: Uncertain	Cutaneous diphtheria is characterized by nonhealing ulcers and membrane formation. Immunocompromised patients, drug addicts, and alcoholics. Invasive endocarditis, mycotic aneurysms, osteomyelitis, and septic arthritis*
Corynebacterium jeikeium	Unknown: Multiple antibiotic resistance allows survival in hospital setting	Systemic: Septicemia Skin infections: Wounds, rashes and nodules Immunocompromised: Malignancies, neutropenia, AIDS patients. Associated with indwelling devices such as catheters, prosthetic valves, and CSF shunts*
Corynebacterium ulcerans	Unknown	Zoonoses: Bovine mastitis Has been associated with diphtheria-like sore throat, indistinguishable from C. diphtheriae Skin infections Pneumonia
Corynebacterium pseudotuberculosis	Unknown	Zoonoses: Suppurative granulomatous lymphadenitis
Corynebacterium pseudodiphtheriticum	Unknown Some stains have been identified that are resistant to macrolides*	Systemic: Septicemia Endocarditis Pneumonia and lung abscesses; primarily in immunocompromised
Corynebacterium minutissimum	Unknown Probably of low virulence	Superficial, pruritic skin infections known as erythrasma Immunocompromised: Septicemia Endocarditis Abscess formation
Corynebacterium urealyticum	Unknown Multiple antibiotic resistance allows survival in hospital setting.	Immunocompromised and elderly: Urinary tract infections Wound infections Rarely: endocarditis, septicemia, osteomyelitis, and tissue infections
Leifsonia aquatica ( formerly Corynebacterium aquaticum)	Unknown	Immunocompromised: Bacteremia Septicemia
Corynebacterium xerosis	Unknown	Immunocompromised: Endocarditis Septicemia
Corynebacterium striatum	Unknown	Immunocompromised: Bacteremia Pneumonia and lung abscesses Osteomyelitis Meningitis
Corynebacterium amycolatum	Unknown Multiple antibiotic resistance patterns	Immunocompromised: Endocarditis Septicemia Pneumonia Neonatal sepsis
Corynebacterium auris	Unknown Multiple antibiotic resistance patterns	Uncertain disease association but has been linked to otitis media
Kurthia spp., Brevibacterium and Dermabacter sp.	Unknown	Immunocompromised: Rarely causes infections in humans Bacteremia in association with indwelling catheters or penetrating injuries
Turicella otitidis	Unknown	Uncertain disease association but has been linked to otitis media
Arthrobacter spp., Microbacterium spp., Aureobacterium spp., Cellulomonas spp., and Exiguobacterium sp.	Unknown	Uncertain disease association

AIDS, Acquired immunodeficiency syndrome; CSF, cerebrospinal fluid; CNS, central nervous system.

L. monocytogenes is ingested through contaminated food. Once the organism has been phagocytized by white blood cells, it produces listeriolysin O, the major virulence factor. Listeriolysin O in combination with phospholipases enables the organism to escape from the white blood cells and spread to the bloodstream, eventually reaching the central nervous system and the placenta.

Most of the remaining organisms in Table 17-2 are opportunistic, and infections are associated with immunocompromised patients. For this reason, whenever Corynebacterium spp. or the other genera of gram-positive rods are encountered, careful consideration must be given to their role as infectious agents or contaminants. Corynebacterium urealyticum is an up-and-coming cause of cystitis in hospitalized patients, in those who have undergone urologic manipulation, and in the elderly.

Laboratory Diagnosis

Specimen Collection and Transport

No special considerations are required for specimen collection and transport of the organisms discussed in this chapter. Refer to Table 5-1 for general information on specimen collection and transport.

Specimen Processing

No special considerations are required for processing of most of the organisms discussed in this chapter. (Refer to Table 5-1 for general information on specimen processing.) One exception is the isolation of L. monocytogenes from placental and other tissue. Because isolating Listeria organisms from these sources may be difficult, cold enrichment may be used to enhance the recovery of the organism. The specimen is inoculated into a nutrient broth and incubated at 4°C for several weeks to months. The broth is subcultured at frequent intervals to enhance recovery.

Direct Detection Methods

Gram stain of clinical specimens is the only procedure used for the direct detection of these organisms. Most of the genera in this chapter (except Listeria, Rothia, and Oerskovia spp.) are classified as coryneform bacteria; that is, they are gram-positive, short or slightly curved rods with rounded ends; some have rudimentary branching. Cells are arranged singly, in “palisades” of parallel cells, or in pairs of cells connected after cell division to form V or L shapes. Groups of these morphologies seen together resemble and are often referred to as Chinese letters (Figure 17-1). The Gram stain morphologies of clinically relevant species are described in Table 17-3. L. monocytogenes is a short, gram-positive rod that may occur singly or in short chains, resembling streptococci.

TABLE 17-3

Gram Stain Morphology, Colonial Appearance, and Other Distinguishing Characteristics

Organism	Gram Stain	Appearance on 5% Sheep Blood Agar
Arthrobacter spp.	Typical coryneform gram-positive rods after 24 hr, with “jointed ends” giving L and V forms, and coccoid cells after 72 hr (i.e., rod-coccus cycle*)	Large colony; resembles Brevibacterium spp.
Brevibacterium spp.	Gram-positive rods; produce typical coryneform arrangements in young cultures (<24 hr) and coccoid-to-coccobacillary forms that decolorize easily in older cultures (i.e., rod-coccus cycle*)	Medium to large; gray to white, convex, opaque, smooth, shiny; nonhemolytic; cheeselike odor
Cellulomonas spp.	Irregular, short, thin, branching gram-positive rods	Small to medium; two colony types, one starts out white and turns yellow within 3 days and the other starts out yellow
CDC coryneform group F-1	Typical coryneform gram-positive rods	Small, gray to white
CDC coryneform group G^†	Typical coryneform gram-positive rods	Small, gray to white; nonhemolytic
Corynebacterium accolens	Resembles C. jeikeium	Resembles C. jeikeium
C. afermentans subsp. afermentans	Typical coryneform gram-positive rods	Medium; white; nonhemolytic; nonadherent
C. afermentans subsp. lipophilum	Typical coryneform gram-positive rods	Small; gray, glassy
C. amycolatum	Pleomorphic gram-positive rods with single cells, V forms, or Chinese letters	Small; white to gray, dry
C. argentoratense	Typical coryneform gram-positive rods	Medium; cream-colored; nonhemolytic
C. aurimucosum	Typical coryneform gram-positive rods	Slightly yellowish sticky colonies; some strains black-pigmented
C. auris	Typical coryneform gram-positive rods	Small to medium; dry, slightly adherent, become yellowish with time; nonhemolytic
C. coyleae	Typical coryneform gram-positive rods	Small, whitish and slightly glistening with entire edges; either creamy or sticky
C. diphtheriae group^‡	Irregularly staining, pleomorphic gram-positive rods	Various biotypes of C. diphtheriae produce colonies ranging from small, gray, and translucent (biotype intermedius) to medium, white, and opaque (biotypes mitis, belfanti, and gravis); C. diphtheriae biotype mitis may be beta-hemolytic; C. ulcerans and C. pseudotuberculosis resemble C. diphtheriae
C. falsenii	Typical coryneform gram-positive rods	Small; whitish, circular with entire edges, convex, glistening, creamy; yellow pigment after 72 hr
C. freneyi	Typical coryneform gram-positive rods	Whitish; dry; rough
C. glucuronolyticum	Typical coryneform gram-positive rods	Small; white to yellow, convex; nonhemolytic
C. jeikeium	Pleomorphic; occasionally, club-shaped gram-positive rods arranged in V forms or palisades	Small; gray to white, entire, convex; nonhemolytic
C. imitans	Typical coryneform gram-positive rods	Small, white to gray, glistening, circular, convex; creamy; entire edges
C. macginleyi	Typical coryneform gram-positive rods	Tiny colonies after 48 hr; nonhemolytic
C. matruchotii	Gram-positive rods with whip-handle shape and branching filaments	Small; opaque, adherent
C. minutissimum	Typical coryneform gram-positive rods with single cells, V forms, palisading and Chinese letters	Small; convex, circular, shiny, and moist
C. mucifaciens	Typical coryneform gram-positive rods	Small, slightly yellow and mucoid; circular, convex, glistening
C. propinquum	Typical coryneform gram-positive rods	Small to medium with matted surface; nonhemolytic
C. pseudodiphtheriticum	Typical coryneform gram-positive rods	Small to medium; slightly dry
C. pseudotuberculosis	Typical coryneform gram-positive rods	Small, yellowish white, opaque, convex; matted surface
C. riegelii	Typical coryneform gram-positive rods	Small, whitish, glistening, convex with entire edges; either creamy or sticky
C. simulans	Typical coryneform gram-positive rods	Grayish white; glistening; creamy
C. singulare	Typical coryneform gram-positive rods	Circular; slightly convex with entire margins; creamy
C. striatum	Regular medium to large gram-positive rods; can show banding	Small to medium; white, moist and smooth (resembles colonies of coagulase-negative staphylococci)
C. sundsvallense	Gram-positive rods, some with terminal bulges or knobs; some branching	Buff to slight yellow, sticky, adherent to agar
C. thomssenii	Typical coryneform gram-positive rods	Tiny after 24 hr; whitish, circular, mucoid and sticky
C. ulcerans	Typical coryneform gram-positive rods	Small, dry, waxy, gray to white
C. urealyticum	Gram-positive coccobacilli arranged in V forms and palisades	Pinpoint (after 48 hr); white, smooth, convex; nonhemolytic
C. xerosis	Regular medium to large gram-positive rods can show banding;	Small to medium; dry, yellowish, granular
Dermabacter hominis	Coccoid to short gram-positive rods	Small; gray to white, convex; distinctive pungent odor
Exiguobacterium acetylicum	Irregular, short, gram-positive rods arranged singly, in pairs, or short chains; (i.e., rod-coccus cycle*)	Golden yellow
Kurthia spp.	Regular gram-positive rods with parallel sides; coccoid cells in cultures >3 days old	Large, creamy or tan-yellow; nonhemolytic
Leifsonia aquatica	Irregular, slender, short gram-positive rods	Yellow
Listeria monocytogenes	Regular, short, gram-positive rods or coccobacilli occurring in pairs (resembles streptococci)	Small; white, smooth, translucent, moist; beta-hemolytic
Microbacterium spp.	Irregular, short, thin, gram-positive rods	Small to medium; yellow
Oerskovia spp.	Extensive branching; hyphae break up into coccoid to rod-shaped elements	Yellow-pigmented; convex; creamy colony grows into the agar; dense centers
Rothia spp.	Extremely pleomorphic; predominately coccoid and bacillary (broth, Figure 17-2, A) to branched filaments (solid media, Figure 17-2, B)	Small, smooth to rough colonies; dry; whitish; raised
Turicella otitidis	Irregular, long, gram-positive rods	Small to medium; white to cream, circular, convex

^*Rod-coccus cycle means rods are apparent in young cultures; cocci are apparent in cultures greater than 3 days old.

^†Includes strains G-1 and G-2.

^‡Includes C. diphtheriae, C. ulcerans, and C. pseudotuberculosis.

Figure 17-1 Gram stain of *Corynebacterium diphtheriae.* Note palisading and arrangements of cells in formations that resemble Chinese letters *(arrows).*

Figure 17-2 A, *Rothia dentocariosa* from broth. B, *R. dentocariosa* from solid media. (Courtesy Deanna Kiska, SUNY Upstate Medical University, Syracuse, NY.)

Cultivation

Media of Choice

Corynebacterium spp. usually grow on 5% sheep blood and chocolate agars. Some coryneform bacteria do not grow on chocolate agar, and the lipophilic (lipid loving) species (e.g., C. jeikeium, C. urealyticum, C. afermentans subsp. lipophilum, C. accolens, and C. macginleyi) produce much larger colonies when cultured on 5% sheep blood agar supplemented with 1% Tween 80 (Figure 17-3).

Figure 17-3 *Corynebacterium urealyticum* on blood agar with Tween 80 **(A)** and blood agar **(B)** at 48 hours. This organism is lipophilic and grows much better on the lipid-containing medium.

Selective and differential media for C. diphtheriae should be used if diphtheria is suspected. The two media commonly used for this purpose are cystine-tellurite blood agar and modified Tinsdale agar (TIN). Tellurite blood agar maybe used with or without cystine. Cystine enhances the growth of fastidious organisms, including C. diphtheriae. Both media contain a high concentration of potassium tellurite that is inhibitory to normal flora. Organisms capable of growing on Tinsdale agar are differentiated based on the conversion of the tellurite to tellurium. This conversion results in color variations of grey to black colonies on the two media. C. diphtheriae also produces a halo on both media. C. diphtheriae can be presumptively identified by observing brown-black colonies with a gray-brown halo on Tinsdale agar (Figure 17-4). The brown halo is produced when the organism uses tellurite to produce hydrogen sulfide. The halo produced on cystine-tellurite blood agar appears brown as a result of the organism breaking down the cystine. In addition, Loeffler medium, which contains serum and egg, stimulates the growth of C. diphtheriae and the production of metachromatic granules in the cells. C. diphtheriae grows rapidly on the highly enriched agar and produces gray to white, translucent colonies within 12 to 18 hours. Primary inoculation of throat swabs to Loeffler serum slants is no longer recommended because of the inevitable overgrowth of normal oral flora.

Figure 17-4 Colony of *Corynebacterium diphtheriae* on Tinsdale agar. Note black colonies with brown halo.

Corynebacterium spp. are unable grow on MacConkey agar. They all are capable of growth in routine blood culture broth and nutrient broths, such as thioglycollate or brain-heart infusion. Lipophilic coryneform bacteria demonstrate better growth in broths supplemented with rabbit serum.

Incubation Conditions and Duration

Detectable growth of corynebacterium on 5% sheep blood and chocolate agars, incubated at 35°C in either ambient air or in 5% to 10% carbon dioxide, should occur within 48 to 72 hours after inoculation. The lipophilic organisms grow more slowly; it takes 3 days or longer to identify visible growth on routine media. For growth of C. diphtheriae, cystine-tellurite blood agar and modified Tinsdale agar should be incubated for at least 48 hours in ambient air. Five percent to 10% carbon dioxide (CO₂) retards the formation of halos on TIN agar.

Colonial Appearance

Table 17-3 describes the colonial appearance and other distinguishing characteristics (e.g., hemolysis and odor) of each clinically relevant genus or species of corynebacteria on blood agar. Colonies of C. diphtheriae on cystine-tellurite blood agar appear black or gray, whereas those on modified Tinsdale agar are black with dark brown halos (see Figure 17-4). C. diphtheriae colonies may be recognized by one of four varieties of colony morphologies. These colony types are referred to as gravis, intermedius, belfanti, and mitis, based on the phenotypic characteristics of size, texture, color, hemolysis, and the presence of metachromatic granules.

Approach to Identification

Except for L. monocytogenes and a few Corynebacterium spp., identification of the organisms in this chapter generally is complex and problematic. A multiphasic approach is required for definitive identification. This often requires biochemical testing, whole-cell fatty acid analysis, cell wall diamino acid analysis, or 16S rRNA gene sequencing. The last three methods are usually not available in routine clinical laboratories, so identification of isolates requires expertise available in reference laboratories. Further complicating the situation is the fact that coryneforms are present as normal flora throughout the body. Thus, only clinically relevant isolates should be identified fully. Indicators of clinical relevance include (1) isolation from normally sterile sites or multiple blood culture bottles; (2) isolation in pure culture or as the predominant organism from symptomatic patients who have not yielded any other known etiologic agent; and (3) isolation from urine if present as a pure culture at greater than 10,000 colony-forming units per milliliter (CFU/mL) or the predominant organism at greater than 100,000 CFU/mL. Coryneforms are more likely to be the cause of a urinary tract infection if the pH of the urine is alkaline or if struvite crystals composed of phosphate, magnesium, and ammonia are present in the sediment.

The API Coryne strip (bioMérieux, St. Louis, Missouri) and the RapID CB Plus (Remel, Lenexa, Kansas) are commercial products available for rapid identification of this group of organisms; however, the databases may not be current with recent taxonomic changes. Therefore, misidentifications can occur if the code generated using these kits is the exclusive criterion used for identification.

Molecular methods for the identification of C. diphtheriae, including ribotyping, pulsed- field gel electrophoresis, and multilocus sequence typing, have been demonstrated to be more sensitive and effective for identification during an outbreak. Various polymerase chain reaction (PCR) techniques have been developed for the quantitative detection of L. monocytogenes in food products. L. monocytogenes DNA in cerebrospinal fluid (CSF) and tissue (fresh or paraffin blocks) can be detected by molecular assays, although these are not available in most clinical laboratories.

Table 17-4 shows the key tests needed to separate the genera discussed in this chapter. In addition to the features shown, the Gram stain and colonial morphology should be carefully noted.

TABLE 17-4

Catalase-Positive, Non–Acid-Fast, Gram-Positive Rods ^a

Organism	Metabolism^b	Motility	Pigment^c	Nitrate Reduction	Esculin	Glucose Fermentation	CAMP^d	Mycolic Acid^e	Cell Wall Diamino Acids^f	Other Comments
Corynebacterium	F/O	–	n, w, y, bl	v	–^g	v	v	+^h	meso-DAP
Arthrobacter	O	vⁱ	w, g	v	v	–^j	–	–	L-lys	Gelatin-positive
Brevibacterium	O	–	w, g, sl y, t	v	–	–^j	–	–	meso-DAP	Gelatin- and casein-positive; cheese odor
Microbacterium^k	F^l/O^m	vⁿ	y, o, y-o	v	v^o	v	–^p	–	L-lys, D-orn	Gelatin and casein variable
Turicella otitidis	O	–	w	–	–	–	+	–	meso-DAP	Isolated from ears
Dermabacter hominis	F	–	n, w	–	+	+	–	–	meso-DAP	Pungent odor; decarboxylates lysine and ornithine; gelatin positive
Cellulomonas	F^l	v	sl y, y	+	+	+	–	–	L-orn	Gelatin-positive; casein-negative
Leifsonia aquatica	O	+	y	v	v	–^q	–	–	DAB	Gelatin- and casein-negative
Rhodococcus equi	O	–	p	v	–	–	+	+	meso-DAP	Usually mucoid; can be acid-fast; urease-positive
Cellulosimicrobium cellulans (formerly Oerskovia xanthineolytica)	F	–	y	+	+	+	NT	–	L-lys	Hydrolyzes xanthine; colonies pit agar
Oerskovia turbata	F	v	y	+	+	+	NT	–	L-lys	Does not hydrolyze xanthine
Listeria monocytogenes	F	+^r	w	–	+	+	+	–	meso-DAP	Narrow zone of beta hemolysis on sheep blood agar; hippurate-positive
Kurthia	O	+^r	n, c	–	–	–	NT	–	L-lys	Large, “Medusa-head” colony with rhizoid growth on yeast nutrient agar; may be H₂S-positive in TSI butt; gelatin-negative
Exiguobacterium acetylicum	F	+	Golden	v	+	+	NT	–	L-lys	Most are oxidase positive; casein and gelatin positive
Rothia dentocariosa	F	–	w	+	+	+	–	–	L-lys	If sticky, probably R. mucilaginosa; some strains are black pigmented
Actinomyces neuii	F	–	n	v	–	+	+	–	NT	Nonhemolytic
Actinomyces viscosus	F	–	n	+	–	+	–	–	NT
Propionibacterium avidum/granulosum	F	–	w, n	–	v	v	+	–	NT	Beta-hemolytic, branching

NT, Not tested; TSI, triple sugar iron agar; v, variable reactions; +, ≥90% of species or strains positive; –, ≥90% of species or strains negative.

^a The aerotolerant catalase-positive Propionibacterium spp. and Actinomyces spp. are also included in Table 23-4.

^b F, Fermentative; O, oxidative.

^c c, Cream; g, gray; n, nonpigmented; o, orange; sl, slightly; t, tan; w, white; y, yellow; y-o, yellowish-orange; p, pink; bl, black.

^d CAMP test using a beta-lysin–producing strain of Staphylococcus aureus.

^e Mycolic acids of various lengths are also present in the partially acid-fast Nocardia, Gordona, Rhodococcus, and Tsukamurella and the completely acid-fast Mycobacterium genera.

^f DAB, diaminobutyric acid; D-orn, d-ornithine; L-lys, L-lysine; L-orn, L-ornithine; meso-DAP, meso-diaminopimelic acid.

^g Of the significant clinical Corynebacterium isolates, only C. matruchotii and C. glucuronolyticum are esculin-positive.

^h Of the significant Corynebacterium isolates, Corynebacterium amycolatum does not have mycolic acid as a lipid in the cell wall, as determined by high-performance liquid chromatography (HPLC) profiling methods.

ⁱ Rod forms of some species are motile.

^j Glucose may be variably oxidized, but it is not fermented.

^k Microbacterium spp. now include the former Aureobacterium spp.

^l Some grow poorly anaerobically.

^m Slow and weak oxidative production of acid from some carbohydrates.

ⁿ Only the orange-pigmented species M. imperiale and M. arborescens are motile at 28°C.

^o Positive reaction may be delayed.

^p Some strains of M. arborescens are CAMP-positive.

^q Glucose is usually oxidized, but it is not fermented.

^r Motile at 20° to 25°C.

Comments on Specific Organisms

Two tests (halo on Tinsdale agar and urea hydrolysis) can be used to separate C. diphtheriae from other corynebacteria. Definitive identification of a C. diphtheriae as a true pathogen requires demonstration of toxin production by the isolate in question. A patient may be infected with several strains at once, so testing is performed using a pooled inoculum of at least 10 colonies. Several toxin detection methods are available:

• Guinea pig lethality test to ascertain whether diphtheria antitoxin neutralizes the lethal effect of a cell-free suspension of the suspect organism

• Immunodiffusion test originally described by Elek (Figure 17-5)

Figure 17-5 Diagram of an Elek plate for demonstration of toxin production by *Corynebacterium diphtheriae.* A filter paper strip impregnated with diphtheria antitoxin is buried just beneath the surface of a special agar plate before the agar hardens. Strains to be tested and known positive and negative toxigenic strains are streaked on the agar’s surface in a line across the plate and at a right angle to the antitoxin paper strip. After 24 hours of incubation at 37°C, the plates are examined with transmitted light for the presence of fine precipitin lines at a 45-degree angle to the streaks. The presence of precipitin lines indicates that the strain produced toxin that reacted with the homologous antitoxin. Line 1 is the negative control. Line 2 is the positive control. Line 3 is an unknown organism that is a nontoxigenic strain. Line 4 is an unknown organism that is a toxigenic strain.

• Tissue culture cell test to demonstrate toxicity of a cell-free suspension of the suspect organism in tissue culture cells and the neutralization of the cytopathic effect by diphtheria antitoxin

• PCR to detect the toxin gene

Because the incidence of diphtheria in the United States is so low (fewer than 5 cases/year), it is not practical to perform these tests in routine clinical laboratories. Toxin testing is usually performed in reference laboratories.

Identification criteria for Corynebacterium spp. (including C. diphtheriae) are shown in Tables 17-5 through 17-9. Most clinically relevant strains are catalase positive, nonmotile, nonpigmented, and esculin and gelatin negative. Therefore, isolation of an organism failing to demonstrate any of these characteristics provides a significant clue that another genus shown in Table 17-4 should be considered. In addition, an irregular, gram-positive rod isolate that is strictly aerobic, nonlipophilic and oxidizes or does not utilize glucose, will likely be Leifsonia aquatica, or Arthrobacter, Brevibacterium, or Microbacterium spp.

TABLE 17-5

Fermentative, Nonlipophilic, Tinsdale-Positive Corynebacterium spp.*

Organism	Urease^†	Nitrate Reduction^†	Esculin Hydrolysis^†	Fermentation of Glycogen^†	Lipophilic
C. diphtheriae subsp. gravis	–	+	–	+	–
C. diphtheriae subsp. mitis	–	+	–	–	–
C. diphtheriae subsp. belfanti	–	–	–	–	–
C. diphtheriae subsp. intermedius	–	+	–	–	+
C. ulcerans^‡^§	+	–	–	+	–
C. pseudotuberculosis^‡^§	+	v	–	–	–

+, ≥90% of species or strains positive; –, ≥90% of species or strains are negative; v, variable reactions.

^*Separation of lipophilic and nonlipophilic species can be determined by comparing growth on sheep blood agar and sheep blood agar with 1% Tween 80 or growth in brain-heart infusion broth with and without 1 drop of Tween 80 or rabbit serum.

^†Reactions from API Coryne.

^‡Propionic acid produced as a product of glucose metabolism.

^§Reverse CAMP positive.

Data compiled from Coyle MB, Lipsky BA: Coryneform bacteria in infectious diseases: clinical and laboratory aspects, Clin Microbiol Rev 3:227, 1990; Funke G, Carlotti A: Differentiation of Brevibacterium spp. encountered in clinical specimens, J Clin Microbiol 32:1729, 1994; and Gruner E, Steigerwalt AG, Hollis DG et al: Human infections caused by Brevibacterium casei, formerly CDC groups B-1 and B-3, J Clin Microbiol 32:1511, 1994.

TABLE 17-6

Fermentative, Nonlipophilic, Tinsdale-Negative Clinically Relevant Corynebacterium spp.*^†

Organism	Urea^‡	Nitrate Reduction^‡	Propionic Acid^§	Motility	Esculin Hydrolysis^‡	Fermentation of Glucose^‡	Maltose^‡	Sucrose^‡	Xylose^‡	CAMP^¶
C. amycolatum^a	v	v	+	–	–	+	v	v	–	–
C. argentoratense	–	–	+	–	–	+	–	–	–	–
C. aurimucosum^b,c	–	–	–	–	–	+	+	+	–	–
C. coyleae	–	–	ND	–	–	(+)	–	–	–	+
C. falsenii^d	(+)	v	ND	–	v	(+)	v	–	–	–
C. freneyi^e	–	v	–	–	–	+	+	+	–	ND
C. glucuronolyticum	v	v	+	–	v	+	v	+	v	+
C. imitans	–	v	ND	–	–	+	+	(+)	–	+
C. matruchotii	–	+	+	–	v +	+	+	+	–	–
C. minutissimum^f	–	–	–	–	–	+	+	v	–	–
C. riegelii	+	–	ND	–	–	–	(+)	–	–	–
C. simulans^g	–	+	–	–	–	+	–	+	–	–
C. singulare	+	–	–	–	–	+	+	+	–	–
C. striatum	–	+	–	–	–	+	–	v	–	v
C. sundsvallense^b	+	–	ND	–	–	(+)	+	+	–	–
C. thomssenii^b	+	–	ND	–	–	(+)	+	+	–	–
C. xerosis	–	v	–	–	–	+	+	+	–	–

ND, No data; v, variable reactions; +, ≥90% of species or strains are positive; +^W, (+), delayed positive reaction; –, ≥90% of species or strains are negative.

^*Consider also Dermabacter, Cellulomonas, Exiguobacterium, and Microbacterium spp. if the isolate is pigmented, motile, or esculin or gelatin positive (see Table 17-4). The aerotolerant catalase-positive Propionibacterium spp. and Actinomyces spp. (see Table 18-4) should also be considered in the differential with the organisms in this table.

^†Separation of lipophilic and nonlipophilic species can be determined by comparing growth on sheep blood agar and sheep blood agar with 1% Tween 80 or growth in brain-heart infusion broth with and without 1 drop of Tween 80 or rabbit serum.

^‡Reactions from API Coryne.

^§Propionic acid as an end-product of glucose metabolism.

^¶CAMP data using a beta-lysin-producing strain of Staphylococcus aureus.

^a Most frequently encountered species in human clinical material; frequently misidentified as C. xerosis.

^b Sticky colonies.

^c Yellow or black-pigmented; black-pigmented strains have been previously listed as C. nigricans; may be pathogenic from female genital tract.

^d Yellow after 72 hours.

^e Grows at 42°C; frequently misidentified as C. xerosis.

^f DNase positive.

^g Nitrite reduced.

TABLE 17-7

Strictly Aerobic, Nonlipophilic, Nonfermentative, Clinically Relevant Corynebacterium spp.a,b

Organism	Oxidation of Glucose	Nitrate Reduction^c	Urease^c	Esculin Hydrolysis^c,d	Gelatin^c,d	Camp^e	Other Comments
C. afermentans subsp. afermentans	–	–	–	–	–	v	Isolated from blood; nonadherent colony
C. auris^f	–	–	–	–	–	+	Isolated from ears; dry, usually adherent colony
C. mucifaciens	+	–	–	–	NT	–	Slightly yellow, mucoid colonies
C. pseudodiphtheriticum	–	+	+	–	–	–
C. propinquum	–	+	–	–	–	–

NT, Not tested; v, variable reactions; +, ≥90% of species or strains are positive; –, ≥90% of species or strains are negative.

^a Kurthia sp. is also a strictly aerobic, nonlipophilic, nonfermentative organism. However, as described in Table 17-3, the colonial and cellular morphology of Kurthia organisms should easily distinguish it from the organisms in this table.

^b Separation of lipophilic and nonlipophilic species can be determined by comparing growth on sheep blood agar and sheep blood agar with 1% Tween 80 or growth in brain-heart infusion broth with and without 1 drop of Tween 80 or rabbit serum.

^c Reactions from API Coryne.

^d Consider also Brevibacterium and Microbacterium spp., Leifsonia aquatica, and Arthrobacter sp. in the differential if the isolate is gelatin or esculin positive (see Table 17-4).

^e CAMP test using a beta-lysin–producing strain of Staphylococcus aureus.

^f For isolates from the ear, also consider Turicella otitidis, which is nitrate and urease positive, in the differential (see Table 17-4).

TABLE 17-8

Strictly Aerobic, Lipophilic, Nonfermentative, Clinically Relevant Corynebacterium spp.*

OXIDATION OF
Organism	Nitrate Reduction^†	Urease^†	Esculin Hydrolysis^†	Glucose	Maltose
C. lipophiloflavum^‡	–	–	–	–	–
C. jeikeium^§	–	–	–	+	v
C. afermentens subsp. lipophilum	–	–	–	–	–
C. urealyticum^§	–	+	–	–	–

+, ≥90% of species or strains positive; –, ≥90% of species or strains negative; v, variable reactions.

^†Reactions from API Coryne.

^‡Yellow.

^§Isolates are usually multiply antimicrobial resistant.

Data compiled from Coyle MB, Lipsky BA: Coryneform bacteria in infectious diseases: clinical and laboratory aspects, Clin Microbiol Rev 3:227, 1990; Funke G, Carlotti A: Differentiation of Brevibacterium spp. encountered in clinical specimens, J Clin Microbiol 32:1729, 1994; Mandell GL, Bennett JE, Dolin R: Principles and practices of infectious diseases, 2010, Churchill Stone and Livingston, Elsevier; and Riegel P, de Briel D, Prévost G et al: Genomic diversity among Corynebacterium jeikeium strains and comparison with biochemical characteristics, J Clin Microbiol 32:1860, 1994.

TABLE 17-9

Lipophilic, Fermentative, Clinically Relevant Corynebacterium spp.*

Organism	Urease^†	Esculin Hydrolysis^†	Alkaline Phosphatase^†	Pyrazinamidase^†
C. kroppenstedtii^‡	–	+	–	+
C. bovis	–	–	+	–
C. accolens^§	–	–	–	v
C. macginleyi^§	–	–	+	–
CDC coryneform group F-1	+	–	–	+
CDC coryneform group G	–	–	+	+

+, ≥90% of species or strains positive; –, ≥90% of species or strains negative; v, variable reactions.

^†Reactions from API Coryne.

^‡Propionic acid produced as a product of glucose metabolism.

^§Nitrate reduced.

Data compiled from Coyle MB, Lipsky BA: Coryneform bacteria in infectious diseases: clinical and laboratory aspects, Clin Microbiol Rev 3:227, 1990; Funke G, Carlotti A: Differentiation of Brevibacterium spp. encountered in clinical specimens, J Clin Microbiol 32:1729, 1994; Mandell GL, Bennett JE, Dolin R: Principles and practices of infectious diseases, 2010, Churchill Stone and Livingston, Elsevier; and Riegel P, Ruimy R, de Briel D et al: Genomic diversity and phylogenetic relationships among lipid-requiring diphtheroids from humans and characterization of Corynebacterium macginleyi sp nov, Int J Syst Bacteriol 45:128, 1995.

The enhancement of growth by lipids (e.g., Tween 80 or serum) of certain coryneform bacteria (e.g., C. jeikeium and C. urealyticum) is useful for preliminary identification. These two species are also resistant to several antibiotics commonly tested against gram-positive bacteria.

L. monocytogenes can be presumptively identified by observation of motility by direct wet mount. The organism exhibits characteristic end-over-end tumbling motility when incubated in nutrient broth at room temperature for 1 to 2 hours. Alternatively, characteristic motility can be seen by an umbrella-shaped pattern (Figure 17-6) that develops after overnight incubation at room temperature of a culture stabbed into a tube of semisolid agar. L. monocytogenes ferments glucose and is Voges-Proskauer positive and esculin positive. Isolation of a small, gram-positive, catalase-positive rod with a narrow zone of beta-hemolysis from blood or CSF should be considered strong presumptive evidence for listeriosis. L. monocytogenes can be differentiated from other Listeria spp. by a positive result on the Christie, Atkins, Munch-Petersen (CAMP) test, as described in Chapter 15 for the identification of Streptococcus agalactiae. A reverse CAMP reaction (i.e., an arrow of no hemolysis formed at the junction of the test organism with the staphylococci) is used to identify C. pseudotuberculosis and C. ulcerans. C. urealyticum is rapidly urea positive.

Figure 17-6 Umbrella motility of *Listeria monocytogenes* grown at room temperature.

Serodiagnosis

Serodiagnostic techniques are not generally used for the laboratory diagnosis of infections caused by the organisms discussed in this chapter. Anti–listeriolysin O antibodies (IgG) can be detected in cases of listeriosis, although IgM antibodies are undetectable. However, these tests are not commonly used for the clinical diagnosis.

Antimicrobial Susceptibility Testing and Therapy

Definitive guidelines have been established for antimicrobial therapy for L. monocytogenes against certain antimicrobial agents. Because there is no resistance to the therapeutic agents of choice, antimicrobial susceptibility testing is not routinely necessary (Table 17-10).

TABLE 17-10

Antimicrobial Therapy and Susceptibility Testing

Organism	Therapeutic Options	Resistance to Therapeutic Options	Validated Testing Methods*
Listeria monocytogenes	Ampicillin, or penicillin (MIC ≤2 µg/mL), with or without an aminoglycoside	Occasional resistance to tetracyclines	Yes, but testing is rarely needed to guide therapy; typically treated empirically.
Corynebacterium diphtheriae	Antitoxin to neutralize diphtheria toxin plus penicillin or erythromycin to eradicate organism	Not to recommended agents; rare instances of penicillin or macrolide resistance	See CLSI document M45-A: Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria.
Other Corynebacterium spp.	No definitive guidelines. All are susceptible to vancomycin and teicoplanin.	Multiple resistance to penicillins, macrolides, aminoglycosides, fluoroquinolones, tetracyclines, clindamycin and cephalosporins	See CLSI document M45-A: Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria.
Kurthia spp., Brevibacterium spp., Dermabacter sp., Arthrobacter spp., Microbacterium spp., Cellulomonas spp., and Exiguobacterium sp.	No definitive guidelines	Unknown	Not available

CLISI, Clinical and Laboratory Standards Institute; MIC, minimum inhibitory concentration.

^*Validated testing methods include the standard methods recommended by CLSI and commercial methods approved by the U.S. Food and Drug Administration (FDA).

As shown in Table 17-10, Clinical and Laboratory Standards Institute (CLSI) document M45 provides some guidelines for testing of Corynebacterium spp. Chapter 12 should be reviewed for strategies that can be used to provide susceptibility information and data when warranted. It is important to note that some strains of Corynebacterium spp. may require 48 hours of incubation for growth. If growth is insufficient or if the isolate appears susceptible to β-lactams at 24 hours, the medium should be incubated for a total of 48 hours before the result is reported.