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 1 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.

Table1. Catalase-Positive, Non–Acid-Fast, Gram-Positive Rods^a

Table1. Catalase-Positive, Non–Acid-Fast, Gram-Positive Rods—cont’d

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 1)

• 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

Fig1. 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. 

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 2 through 6. 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 1 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.

Table2. Fermentative, Nonlipophilic, Tinsdale-Positive Corynebacterium spp.*

Table3. Fermentative, Nonlipophilic, Tinsdale-Negative Clinically Relevant Corynebacterium spp.*

Table4. Strictly Aerobic, Nonlipophilic, Nonfermentative, Clinically Relevant Corynebacterium spp.a,b

Table5. Strictly Aerobic, Lipophilic, Nonfermentative, Clinically Relevant Corynebacterium spp.*

Table6. Lipophilic, Fermentative, Clinically Relevant Corynebacterium spp.*

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 organ ism 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 2) 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.

Fig2. Umbrella motility of Listeria monocytogenes grown at  room temperature

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مواضيع ذات صلة

Direct Detection Methods for Erysipelothrix, Lactobacillus, and Similar Organisms

Pathogenesis and Spectrum of Disease Erysipelothrix, Lactobacillus, and Similar Organisms

Antimicrobial Susceptibility Testing and Therapy for Listeria, Corynebacterium, and Similar Organisms

GROWTH AND METABOLISM

SPORES AND SPORULATION

Susceptibility Testing

Immunologic Detection of Microorganisms

Identification of Bacteria

Growing Bacteria in Culture

Cultivation of Listeria, Corynebacterium, and Similar Organisms

Pathogenesis and Spectrum of Disease for Listeria, Corynebacterium, and Similar Organisms

The Growth Curve in Batch Culture