Pseudomonas spp. and Brevundimonas spp.

The genera Pseudomonas and Brevundimonas comprise several environmental species that rarely inhabit human skin or mucosal surfaces. In the clinical setting, P. aeruginosa is the most commonly encountered gram-negative species that is not a member of the family Enterobacteriaceae and is an uncommon member of the normal human flora. The organism survives in various environments in nature and in homes and hospitals (see Table 22-1). Brevundimonas spp. are environmental and are encountered primarily in nature in water, soil, and on plants, including fruits and vegetables. Because of the ubiquitous nature of P. aeruginosa and Brevundimonas spp., the transmission of to humans can occur in a variety of ways.

P. fluorescens, P. putida, and P. stutzeri are environmental inhabitants, but they are much less commonly found in clinical specimens than is P. aeruginosa. The other pseudomonads and Brevundimonas spp. listed in Table 22-1 are also environmental organisms. Because they are rarely encountered in patient specimens, the mode of transmission to humans remains uncertain.

Pseudomonas spp. and Brevundimonas spp.

Of the species in the Pseudomonas and Brevundimonas genera, P. aeruginosa is the most thoroughly studied with regard to infections in humans. Brevundimonas spp. are rarely associated with human infection. B. vesicularis has been isolated in clinical cases of bacteremia and from cervical specimens. B. diminuta has been recovered from cancer patients in blood, urine and pleural fluid. Although P. aeruginosa is an environmental inhabitant, it is also a very successful opportunistic pathogen. Factors that contribute to the organism’s pathogenicity include production of exotoxin A, which kills host cells by inhibiting protein synthesis, and production of several proteolytic enzymes and hemolysins capable of destroying cells and tissue. On the bacterial cell surface, pili mediate attachment to host cells. Some strains produce alginate, a polysaccharide polymer that inhibits phagocytosis and contributes to the infection potential in patients with cystic fibrosis. Pyocyanin, the blue phenazine pigment that contributes to the characteristic green color of P. aeruginosa, damages cells by producing reactive oxygen species. The reactive oxygen species are also bacteriocidal to the organism. In order to protect itself from destruction, the organism must produce catalase enzymes.

P. aeruginosa also contains several genes involved in quorum sensing, a mechanism for detecting bacterial products in the immediate environment. When the growth of the organism or neighboring bacteria reaches a critical mass, the concentration of these “inducing” molecules reaches a level that activates transcription of virulence factors, including genes related to metabolic processes, enzyme production, and the formation of biofilm. Although many in vitro studies have examined biofilm formation, no clear evidence exists that demonstrates a clear role for biofilm in the organism’s pathogenesis. Although biofilm studies have been examined in the laboratory, it is evident that P. aeruginosa does not form the same type of biofilm in vivo as is seen on artificial surfaces. Biofilm production related to the overproduction of alginate and the mucoid phenotype isolated from patients with cystic fibrosis is associated with serious infections. P. aeruginosa forms microcolonies in tissue that are associated with quorum-sensing, biofilm-producing strains, which indicates that the quorum sensing is also linked to the formation of microcolonies. These microcolonies contain DNA, mucus, actin, and other products from dying bacterial and host cells. Additionally, P. aeruginosa can survive harsh environmental conditions and displays intrinsic resistance to a wide variety of antimicrobial agents, two factors that facilitate the organism’s ability to survive in the hospital setting (see Table 22-2).

Even with the variety of potential virulence factors discussed, P. aeruginosa remains an opportunistic pathogen that requires compromised host defenses to establish infection. In normal, healthy hosts, infection is usually associated with events that disrupt or bypass protection provided by the epidermis (e.g., burns, puncture wounds, use of contaminated needles by intravenous drug abusers, eye trauma with contaminated contact lenses). The result is infections of the skin, bone, heart, or eye (see Table 22-2).

In patients with cystic fibrosis, P. aeruginosa has a predilection for infecting the respiratory tract. Although organisms rarely invade through respiratory tissue and into the bloodstream of these patients, the consequences of respiratory involvement alone are serious and life-threatening. In other patients, P. aeruginosa is a notable cause of nosocomial infections of the respiratory and urinary tracts, wounds, bloodstream, and even the central nervous system. For immunocompromised patients, such infections are often severe and frequently life-threatening. In some cases of bacteremia, the organism may invade and destroy the walls of subcutaneous blood vessels, resulting in the formation of cutaneous papules that become black and necrotic. This condition is known as ecthyma gangrenosum. Similarly, patients with diabetes may suffer a severe infection of the external ear canal (malignant otitis externa), which can progress to involve the underlying nerves and bones of the skull.

No known virulence factors have been associated with P. fluorescens, P. putida, or P. stutzeri. When infections caused by these organisms occur, they usually involve a compromised patient exposed to contaminated medical materials. Such exposure has been known to result in infections of the respiratory and urinary tracts, wounds, and bacteremia (see Table 22-2). However, because of their low virulence, whenever these species are encountered in clinical specimens, their significance should be highly suspect. Similar caution should be applied whenever the other Pseudomonas spp. or Brevundimonas spp. listed in Table 22-2 are encountered.

Specimen Processing

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

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 pseudomonads, 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 multilocus sequence typing (MLST). Discriminatory techniques are typically limited to specialized reference laboratories and are not considered routine laboratory testing.

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 22-1). 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 22-3). 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 distinguishable from other bacteria.

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 22-4 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 22-2 and 22-3)

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 22-4. 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.

Serodiagnosis

Serodiagnostic techniques are not generally used for laboratory diagnosis of infections caused by the organisms 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.