Specimen Collection and Transport

No special considerations are required for specimen collection and transport of organisms discussed in this chapter.

 Specimen Processing

No special considerations are required for processing of the organisms discussed in this chapter.

Direct Detection Methods

 Other than Gram staining, no specific procedures have been established for the direct detection within clinical samples of the organisms discussed in this chapter. These organisms usually appear as medium-size, straight rods on Gram staining. Exceptions are B. diminuta, which is a long, straight rod; B. mallei, which is a coccobacillus; P. pseudomallei, which is a small, gram-negative rod with bipolar staining; and CDC group Ic, which is a thin, pleomorphic rod.

 Nucleic Acid Detection

Culture remains the standard approach for organism identification. However, rapid screening may be useful when evaluating a large outbreak or during environmental epidemiologic studies. Polymerase chain reaction (PCR) assays have been developed for various genes, including 16s rRNA, heat shock protein, and exotoxin A. Undoubtedly, with further development and expansion in molecular diagnostics, useful clinical assays related to rapid diagnosis for respiratory infections and other serious infections will continue to emerge.

Several genotyping methods have been developed to examine the heterogeneity and diversity of the pseudo monads, including restriction fragment length polymorphism (RFLP); pulsed-field gel electrophoresis (PFGE); additional PCR-based typing methods, such as rapid amplification of polymorphic DNA (RAPD); and multi locus sequence typing (MLST). Discriminatory techniques are typically limited to specialized reference laboratories and are not considered routine laboratory testing.

Cultivation

Media of Choice

 Pseudomonas spp., Brevundimonas spp., Burkholderia spp., R. pickettii, and CDC group Ic grow well on routine laboratory media, such as 5% sheep blood agar and chocolate agar (Figure 1). Except for B. vesicularis, all usually grow on MacConkey agar. All four genera also grow well in broth-blood culture systems and common nutrient broths, such as thioglycollate and brain-heart infusion. Specific selective media, such as Pseudomonas cepacia (PC) agar or oxidative–fermentative base–polymyxin B–bacitracin–lactose (OFPBL) agar may be used to isolate B. cepacia from the respiratory secretions of patients with cystic fibrosis (Table 1). PC agar contains crystal violet, bile salts, polymyxin B, and ticarcillin to inhibit gram positive and rapid-growing, gram-negative organisms. Inorganic and organic components, including pyruvate and phenol red, also are added. B. cepacia breaks down the pyruvate, creating an alkaline pH and resulting in a color change of the pH indicator (phenol red) from yellow to pink. OFPBL incorporates bacitracin as an added selective agent and uses lactose fermentation to differentiate isolates. B. cepacia ferments lactose and appears yellow, whereas nonfermenters appear green. Ashdown medium is used to isolate B. pseudomallei when an infection caused by this species is suspected. The medium contains crystal violet and gentamicin as selective agents to suppress the growth of contaminating organisms. Neutral red is incorporated into the medium and is taken up by the organism, making it distinguish able from other bacteria.

Fig1. Burkholderia cepacia on chocolate agar. Note green  pigment.

Table1. Colonial Appearance and Other Characteristics of Pseudomonas, Brevundimonas, Burkholderia, Ralstonia,  and Other Organisms

Table1. Colonial Appearance and Other Characteristics of Pseudomonas, Brevundimonas, Burkholderia, Ralstonia,  and Other Organisms—cont’d

Incubation Conditions and Duration

 Detectable growth on 5% sheep blood and chocolate agars, incubated at 35°C in carbon dioxide or ambient air, generally occurs in 24 to 48 hours after inoculation. Growth on MacConkey agar incubated in ambient air at 35°C is detectable within this same time frame. Selective media used for patients with cystic fibrosis (e.g., PC or OFPBL) may require incubation at 35°C in ambient air for up to 72 hours before growth is detected.

 Colonial Appearance

 Table 1 describes the colonial appearance and other distinguishing characteristics (e.g., hemolysis and odor) of each genus on common laboratory media.

Approach to Identification

 Most of the commercial systems available for identification of these organisms reliably identify Pseudomonas aeruginosa and Burkholderia cepacia complex, but their reliability for identification of other species is less certain.

Table 1 provides the key phenotypic characteristics for identifying the species discussed in this chapter. These tests provide useful information for presumptive organism identification, but definitive identification often requires the use of a more extensive battery of tests performed by reference laboratories.

Comments Regarding Specific Organisms

A convenient and reliable identification scheme for P. aeruginosa involves the following conventional tests and characteristics:

 • Oxidase-positive

 • Triple sugar iron slant with an alkaline/no change (K/NC) reaction

 • Production of bright bluish (pyocyanin) green (pyoverdin), red (pyorubin), or brown (pyomelanin) diffusible pigment on Mueller-Hinton agar or trypticase soy agar (Figures 2 and 3)

Fig2. Pseudomonas aeruginosa on tryptic soy agar (B).  Note bluish-green color. Uninoculated tube (A) is shown for  comparison. 

Fig3. Pseudomonas aeruginosa on MacConkey agar. 

 P. aeruginosa, P. fluorescens, P. putida, P. veronii, and P. monteilii comprise the group known as the fluorescent pseudomonads. P. aeruginosa can be distinguished from the others in this group by its ability to grow at 42°C. Mucoid strains of P. aeruginosa from patients with cystic fibrosis may not exhibit the characteristic pigment and may react more slowly in biochemical tests than nonmucoid strains. The organisms may undergo several phenotypic changes, including slow growth, changes in pigment production, and altered biochemical activity. Therefore, standard biochemicals should be held for the complete 7 days before being recorded as negative. This slow biochemical activity is often what prevents the identification of mucoid P. aeruginosa by commercial systems. P. monteilii can be distinguished from P. putida by its inability to oxidize xylose. Both can be distinguished from P. fluorescens by their inability to liquefy gelatin.

B. cepacia should be suspected whenever a nonfermentative organism that decarboxylates lysine is encountered. Lysine decarboxylation is positive in 80% of strains. Correct identification of the occasional lysine-negative (20%), or oxidase-negative (14%) strains requires full biochemical profiling. Pandoraea spp. may be differentiated from B. cepacia by their failure to decarboxylate lysine and their inability to liquefy gelatin. Unlike R. paucula, they do not hydrolyze Tween 80,

The presumptive identification of other species in this chapter is fairly straightforward using the key characteristics given in Table 2. However, a few notable exceptions exist. First, when B. cepacia complex is identified by a commercial system in a patient with cystic fibrosis, species confirmation should be completed by a combination of phenotypic and genotypic methods. This is also true if a rapid system identifies an organism as B. gladioli or R. pickettii. The B. cepacia complex has 10 genomovars, and appropriate speciation is crucial.

Table2. Biochemical and Physiologic Characteristics

Serodiagnostic

Serodiagnostic techniques are not generally used for laboratory diagnosis of infections caused by the organ isms discussed in this chapter. An indirect hemagglutination assay is available in endemic areas in the Far East to diagnose infections caused by P. pseudomallei; acute and convalescent sera are required. Cross-reactions with other organisms (e.g., B. cepacia complex) occur, and interpretation of any serology must include compatible clinical symptoms.

An indirect hemagglutination assay has been used in the diagnosis of B. pseudomallei infections. The serologic test is not available commercially and has limited value in endemic areas. No current validated method exists; therefore, results should be interpreted carefully.

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