Direct Detection Methods

 Other than Gram staining of patient specimens, there are no specific procedures for the direct detection of these organisms in clinical material. B. bronchiseptica is a medium-sized straight rod, whereas O. urethralis, Psychrobacter spp., Roseomonas spp., and Moraxella spp. are all coccobacilli, although Psychrobacter phenylpyruvicus may appear as a broad rod, and some Roseomonas spp. may appear as short, straight rods. O. ureolytica is a short, straight rod; Myroides spp. are pleomorphic rods and are either short or long and straight to slightly curved.

Alcaligenes and Achromobacter spp. are medium to long straight rods, as are CDC Alcaligenes-like group 1, Cupriavidus pauculus, Delftia acidovorans, P. alcaligenes, and P. pseudoalcaligenes. The Comamonas spp. are pleomorphic and may appear as long, paired, curved rods or filaments. The cells of CDC group IIg appear as small, coccoid-to rod forms or occasionally as rods with long filaments.

Cultivation

Media of Choice

 B. bronchiseptica grows on 5% sheep blood, chocolate, and MacConkey agars, usually within 1 to 2 days after inoculation. It should also grow in thioglycollate broth. Psychrobacter spp., Myroides spp., Oligella spp., Achromobacter spp., D. acidovorans, Alcaligenes spp., CDC Alcaligenes-like group 1, Comamonas spp., Roseomonas spp., P. alcaligenes, P. pseu doalcaligenes, C. pauculus, and CDC group IIg all grow well on 5% sheep blood, chocolate, and MacConkey agars. Most of these genera should also grow well in the broth of blood culture systems, as well as in common nutrient broths such as thioglycollate and brain-heart infusion.

Incubation Conditions and Duration

 Most of the organisms produce detectable growth on media incubated at 35°C in ambient air or 5% carbon dioxide (CO2). Psychrobacter spp. usually grow better at 25°C than at 35°C.

Colonial Appearance

Table 1 describes the colonial appearance and other distinguishing characteristics (e.g., pigment and odor) of each genus on 5% sheep blood and MacConkey agars.

Table1. Colonial Appearance and Characteristics

Approach to Identification

The ability of most commercial identification systems to accurately identify the organisms discussed in this chapter is limited or uncertain. Strategies for identification of these genera therefore are based on the use of conventional biochemical tests and special staining for flagella. Although most clinical microbiology laboratories do not routinely perform flagella stains, motility and flagella placement are the easiest ways to differentiate among these organisms.

Many microbiologists groan at the mere mention of having to perform a flagella stain. At the very least, a simple wet mount to observe cells for motility helps distinguish between the motile and nonmotile genera. The pseudomonads and Brevundimonas, Burkholderia, and Ralstonia species described in Chapter 22 are motile by means of single or multiple polar flagella; the motile organisms described in this chapter have peritrichous flagella (e.g., B. bronchiseptica, Alcaligenes spp., and Achromobacter spp.), or polar flagella (e.g., Delftia, Comamonas spp.).

Organisms are first categorized on the basis of Gram stain morphology (i.e., coccoid [Table 2] or rod shaped [Tables 3 through 5]). They are then further characterized based on whether the organisms are non motile (see Table 3), peritrichously flagellated (see Table 4), or flagellated by polar tufts (see Table 5).

Table2. Key Biochemical and Physiologic Characteristics for Coccoid Species

Table3. Key Biochemical and Physiologic Characteristics  for Rod-Shaped Nonmotile Species

Table4. Key Biochemical and Physiologic Characteristics for Rod-Shaped Motile Species with Polar Flagella

Table5. Key Biochemical and Physiologic Characteristics for Rod-Shaped Motile Species with Peritrichous Flagella

Comments Regarding Specific Organisms

 B. bronchiseptica is oxidase-positive, motile, and rapidly urease positive, sometimes in as little as 4 hours. This organism must be differentiated from C. pauculus and O. ureolytica.

Urea hydrolysis is a key test for Myroides spp., which is also distinguished by production of a characteristic fruity odor. CDC group IIg is the only indole-positive, nonmotile species included in this chapter.

The genus Oligella includes one nonmotile species (O. urethralis) and one motile species (O. ureolytica). Urease hydrolysis is a key test for differentiating between these species; O. ureolytica often turns positive within minutes. O. urethralis is urease and nitrate reductase negative. P. phenylpyruvicus is nonmotile and urease positive, arginine dihydrolase positive and phenylalanine deaminase positive.

Achromobacter denitrificans and Alcaligenes piechaudii reduce nitrate to nitrite, but only the former reduces nitrite to gas. Achromobacter species are oxidase and catalase positive and negative for urease, DNase, lysine decarboxylase, ornithine decarboxylase, arginine dihydrolase, and gelatinase. A. faecalis has a fruity odor and also reduces nitrite to gas. CDC Alcaligenes-like group 1 is similar to Achromobacter denitrificans but is usually urea positive.

Delftia acidovorans is unique in producing an orange color when Kovac’s reagent is added to tryptone broth (indole test).

Roseomonas spp. must be separated from other pink pigmented, gram-negative (e.g., Methylobacterium spp.) and gram-positive (e.g., certain Rhodococcus spp. or Bacillus spp.) organisms. Roseomonas spp. differ from Rhodococcus and Bacillus spp. by being resistant to vancomycin, as determined by using a 30-µg vancomycin disk on an inoculated 5% blood agar plate. Unlike Methylobacterium spp., Roseomonas spp. grow on MacConkey agar and at 42°C. All Roseomonas species strongly hydrolyze urea but not esculin and are β-galactosidase negative.

P. alcaligenes is differentiated from P. pseudoalcaligenes by its inability to oxidize fructose. These two species are often referred to as “Pseudomonas spp., not aeruginosa” in clinical situations.

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