Approach To Identification

If Gram-stain morphology or colonial morphology is suggestive of a possible actinomycetes (see Table 21-7), a Ziehl-Neelsen acid-fast stain should first be performed to rule out rapidly growing mycobacteria (see Chapter 45), followed by a modified add-fast stain (Procedure 21-1), If the modified acid-fast stain results are positive, the isolate is a probable partially acid-fast aerobic acti-nomycete, that is, Nocardia, Rhodococcus, Tbukamurella, or Gordonia. (If acid-fast stain-negative, these organisms are still not completely ruled out because of the variability of acid-fastness among isolates belonging to this group.) Aerobic actinomycetes can be initially placed into major groupings by taking into consideration the following:

  • Gram-stain morphology (see Figures 21-1 and Figure 21-2)
  • Modified add-fast stain results
  • Presence or absence of aerial hyphae when grown on tap water agar
Table 21-7 Typical Gram-Stain Morphology and Colonial Appearance

Organism

Gram Stain*

Colonial Appearance on Routine Agar

Nocardia spp.

Branching, fine, delicate filaments with fragmentation

Extremely variable; adherent; some isolates are beta-hemolytic on sheep blood agar; wrinkled; often dry, chalky-white appearance to orange-tan pigment; crumbly

Rhodococcus

Diphtherold-like with minimal branching or coccobacillary; colonial growth appears as coccobacilli In zigzag configuration

Nonhemolytic; round; often mucoid with orange to red, salmon-pink pigment developing within 4 to 7 days (pigment may vary widely)

Gordonia

Non-motile, short rods

Somewhat pigmented; fi. spun smooth, mucoid and adherent to media; G. bronchialis dry anil raised

Tsukamureila

Mostly long rods that fragment, no spores or aerial hyphae

May have rhizoid edges, dry, white to creamy to orange

Streptomyces spp.

Extensive branching with chains and spores; does not fragment easily

Glabrous or waxy heaped colonies; variable morphology

Actinomadura spp.

Moderate, fine, intertwining branching with short chains of spores, fragmentation

White-to-pink pigment mucoid, moiar tooth appearance after 2 weeks' incubation

Dermatophilus sp.

Branched filaments divided in transverse and longitudinal planes; fine, tapered filaments

Round, adherent, gray-white colonies that later develop orange pigments; often beta-hemolytic

Nocardiopsis sp.

Branching with internal spores

Coarsely wrinkled and folded with well-developed aerial mycelium

Data compiled from Brown JH, Mcneil MM: In Murray PR, Baron EJ, Pfaller MA et al, editors: Manual of clinical microbiology, ed 8, Washington, DC, American Society for Microbiology,

McNeil MM, Brown JM: CtinMmbio!Rev7:351,1994.

>lc actnomycetes are gram-positive organisms that are often beaded in appearance.

Data compiled from Brown JH, Mcneil MM: In Murray PR, Baron EJ, Pfaller MA et al, editors: Manual of clinical microbiology, ed 8, Washington, DC, American Society for Microbiology,

McNeil MM, Brown JM: CtinMmbio!Rev7:351,1994.

>lc actnomycetes are gram-positive organisms that are often beaded in appearance.

Figure 21-2 Gram stains of different aerobic actinomycetes. A, Nocardia asteroides grown on Lôwenstein-Jensen media. The arrows indicate branching rods. B, Rhodococcus equi from broth. C, R. equi grown on chocolate agar. D, Streptomyces spp. on Sabouraud dextrose agar.
Nocardia Beta Hemolytic

Figure 21-4 Lysozyme (A) and glycerol broths (B). The lysozyme broth is growing better, which is typical of Nocardia asteroides.

Figure 21-3 Aerobic actinomycetes grown on solid media. A, Nocardia asteroides grown on L&wenstein-Jensen media. B, Rhodococais equi grown on chocolate agar.

  • Growth or no growth in nutrient broth containing lysozyme (250 |ig/mL; Figure 21-4, Procedure 21-2)
  • Other tests: urea hydrolysis, nitrate reduction, and ability to grow anaerobically

Table 21-8 summarizes these key characteristics for aerobic actinomycetes.

Accurate identification of Nocardia to the species level is important, in that differences among the species have emerged in terms of virulence, antibiotic susceptibility, and epidemiology. However, identification of the pathogenic Nocardia to the species level can be problematic because no single method can identify all Nocardia isolates and because the methods employed are time consuming, often requiring 2 weeks. Useful pheno-typic tests include the use of casein, xanthine, and tyrosine hydrolysis; growth at 45° C; acid production from rhamnose; gelatin hydrolysis; opacification of Middlebrook agar; and antimicrobial susceptibility patterns.2-4'7,11'18 Some of these reactions with the nocardial pathogens are summarized in Table 21-9. A number of molecular approaches such as polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis and PCR of a sequence of 16S rRNA gene with subsequent sequencing (see Chapter 8) have been used to rapidly and accurately identify these organisms.8,17 Of note, using the MicroSeq System for identification (see Chapter 8), almost 15% of isolates were identified as Nocardia but no definitive species was given.8 Therefore, identification approaches employing phenotypic and molecular methods will continue to evolve and be refined for the aerobic actinomycetes.

To confirm the identification of the other actinomycetes and speciate them, many tests are needed; these are beyond the capabilities of most routine clinical microbiology laboratories and should therefore be referred to a reference laboratory.

Figure 21-4 Lysozyme (A) and glycerol broths (B). The lysozyme broth is growing better, which is typical of Nocardia asteroides.

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Bacterial Vaginosis Facts

Bacterial Vaginosis Facts

This fact sheet is designed to provide you with information on Bacterial Vaginosis. Bacterial vaginosis is an abnormal vaginal condition that is characterized by vaginal discharge and results from an overgrowth of atypical bacteria in the vagina.

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