Pseudomonas, Burkholderia, and Similar Organisms

Chapter 22


Pseudomonas, Burkholderia, and Similar Organisms



Objectives



1. Describe the normal sources (habitat) for Pseudomonas aeruginosa, Burkholderia cepacia, Burkholderia pseudomallei, and Burkholderia mallei, including the routes of transmission.


2. Identify the factors that contribute to the pathogenicity of P. aeruginosa and explain the physiologic mechanism for each.


3. List the various disease states associated with P. aeruginosa and Burkholderia spp.


4. Compare and contrast the Gram stain appearance of the gram-negative bacilli discussed in this chapter.


5. List the appropriate scheme for identifying P. aeruginosa.


6. Describe the media and chemical principle of each used, including differential and selective agars that aid the cultivation of Pseudomonas, Brevundimonas, and Ralstonia spp.


7. Describe the potential therapies for B. cepacia and B. pseudomallei and the concerns about optimal therapy.


8. Describe and identify the patterns of antibiotic resistance in P. aeruginosa.



Genera and Species to Be Considered








































































Current Name Previous Name
Acidovorax delafieldii Pseudomonas delafieldii
Acidovorax facilis  
Acidovorax temperans  
Brevundimonas diminuta Pseudomonas diminuta
Brevundimonas vesicularis Pseudomonas vesicularis
Burkholderia cepacia complex Pseudomonas cepacia
Burkholderia pseudomallei Pseudomonas pseudomallei
Burkholderia mallei Pseudomonas mallei
Pandoraea spp. CDC group WO-2 (five distinct species)
Pseudomonas aeruginosa  
Pseudomonas fluorescens  
Pseudomonas mendocina  
Pseudomonas monteilii  
Pseudomonas putida  
Pseudomonas stutzeri (includes CDC group Vb-3) CDC group IVd
Pseudomonas veronii  
Pseudomonas-like group 2  
CDC group Ic
Ralstonia mannitolilytica “Pseudomonas thomasii,” Ralstonia pickettii biovar 3
Ralstonia insidiosa CDC group IVc-2
Ralstonia pickettii Pseudomonas pickettii, Burkholderia pickettii, Va-1, Va-2


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General Characteristics


At one time, most of the species belonging to the genera Brevundimonas, Burkholderia, Ralstonia, and Acidovorax were members of the genus Pseudomonas. Organisms in these genera have many similar characteristics. They are aerobic, non–spore-forming, straight, slender, gram-negative bacilli with cells that range from 1 to 5 µm long and 0.5 to 1 µm wide. All species except B. mallei are motile, having one or several polar flagella. Members of these genera use a variety of carbohydrate, alcohol, and amino acid substrates as carbon and energy sources. Although they are able to survive and possibly grow at relatively low temperatures (i.e., as low as 4°C), the optimum temperature range for growth of most species is 30° to 37°C; that is, they are mesophilic. Burkholderia gladioli, Pseudomonas luteola, and Pseudomonas oryzihabitans are oxidase negative and are discussed in Chapter 21. Pseudomonas alcaligenes, Pseudomonas pseudoalcaligenes, Ralstonia paucula, Ralstonia gilardii, Comamonas spp. (including the former Pseudomonas testosteroni), and Delftia acidovorans (formerly Pseudomonas acidovorans) are not able to utilize glucose and are discussed in Chapter 25. Acidovorax facilis is MacConkey negative. Pseudomonas spp. are catalase positive. The organisms in this chapter are all oxidase-positive, grow on MacConkey agar, and oxidize glucose.



Epidemiology


Burkholderia spp. and Ralstonia Pickettii


Burkholderia spp. and Ralstonia pickettii are inhabitants of the environment and are not considered part of the normal human flora (Table 22-1). As such, their transmission usually involves human contact with heavily contaminated medical devices or substances encountered in the hospital setting.



TABLE 22-1


Epidemiology












































Species Habitat (Reservoir) Mode of Transmission
Burkholderia cepacia Environmental (soil, water, plants); survives well in hospital environment; not part of normal human flora; may colonize respiratory tract of patients with cystic fibrosis Exposure of medical devices and solutions contaminated from the environment; person-to-person transmission also documented
B. pseudomallei Environmental (soil, streams, surface water, such as rice paddies); limited to tropical and subtropical areas, notably Southeast Asia; not part of human flora Inhalation or direct inoculation from environment through disrupted epithelial or mucosal surfaces
B. mallei Causative agent of glanders in horses, mules, and donkeys; not part of human flora Transmission to humans is extremely rare; associated with close animal contact and introduced through mucous membranes or broken skin.
Ralstonia pickettii Environmental (multiple sources); found in variety of clinical specimens; not part of human flora Mode of transmission is not known; likely involves exposure to contaminated medical devices and solutions
Pseudomonas aeruginosa Environmental (soil, water, plants); survives well in domestic environments (e.g., hot tubs, whirlpools, contact lens solutions) and hospital environments (e.g., sinks, showers, respiratory equipment); rarely part of normal flora of healthy humans Ingestion of contaminated food or water; exposure to contaminated medical devices and solutions; introduction by penetrating wounds; person-to-person transmission is assumed to occur
P. alcaligenes, P. pseudoalcaligenes, Pseudomonas sp. CDC group 1, “P. denitrificans,” Pseudomonas-like group 2, and CDC group Ic Environmental; not part of normal human flora Uncertain. Rarely encountered in clinical specimens
P. fluorescens, P. putida, P. stutzeri, (including Vb-3), P. luteola, and P. mendocina Environmental (soil and water); not part of normal human flora Exposure to contaminated medical devices and solutions
Brevundimonas vesicularis and B. diminuta Environmental; not part of normal human flora Uncertain. Rarely encountered in clinical specimens
Acidovorax spp. Environmental, soil; not part of human flora Unknown. Rarely found in humans

B. cepacia, which is among the Burkholderia spp. found in the United States, is a complex of 10 distinct genomic species (genomovars) isolated from clinical specimens. Plants, soil, and water serve as reservoirs. These organisms are able to survive on or in medical devices and disinfectants. Intrinsic resistance to multiple antimicrobial agents contributes to the organism’s survival in hospitals. Human acquisition of B. cepacia that results in colonization or infection usually involves direct contact with contaminated foods, devices such as respiratory equipment, or medical solutions, including disinfectants. Person-to-person transmission also has been documented.


B. pseudomallei is another environmental inhabitant of niches similar to those described for B. cepacia; however, it is geographically restricted to tropical and subtropical areas of Australia and Southeast Asia. The organism is widely disseminated in soil, streams, ponds, and rice paddies. Human acquisition occurs through inhalation of contaminated debris or by direct inoculation through damaged skin or mucous membranes.


Although B. mallei causes severe infections in horses and related animals, it has been identified in rare human localized suppurative or acute pulmonary infections. When transmission has occurred, it has been associated with close animal contact. B. gladioli is a plant pathogen that is only rarely found in the sputa of patients with cystic fibrosis or associated with chronic granulomatous disease; the mode of transmission to humans and its clinical significance are unknown.


R. pickettii is another environmental organism that is occasionally found in a variety of clinical specimens, such as blood, the sputa of patients with cystic fibrosis, and urine. The mode of transmission is uncertain, but isolates have been found in contaminated sterile hospital fluids.



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.



Pathogenesis and Spectrum of Disease


Burkholderia spp. and Ralstonia Pickettii


Because Burkholderia spp. and R. pickettii are uncommon causes of infection in humans, very little is known about what, if any, virulence factors they exhibit. Except for B. pseudomallei, the species listed in Table 22-2 generally are nonpathogenic for healthy human hosts.



TABLE 22-2


Pathogenesis and Spectrum of Disease












































Species Virulence Factors Spectrum of Disease and Infections
Burkholderia cepacia Unknown. Binding of mucin from patients with cystic fibrosis may be involved. Intrinsic resistance to multiple antibiotics complicates therapy and may promote organism survival in hospital Nonpathogenic to healthy human hosts; able to colonize and cause life-threatening infections in patients with cystic fibrosis or chronic granulomatous disease; other patients may suffer nonfatal infections of the urinary tract, respiratory tract, and other sterile body sites
B. pseudomallei Unknown. Bacilli can survive within phagocytes Wide spectrum from asymptomatic infection to melioidosis, of which there are several forms, including infections of the skin and respiratory tract, multisystem abscess formation, and bacteremia with septic shock
B. mallei Unknown for human infections Human disease is extremely rare. Infections range from localized acute or chronic suppurative infections of skin at site of inoculation to acute pulmonary infections and septicemia
Ralstonia pickettii Unknown Rarely encountered as cause of disease; nonpathogenic to healthy human host, but may be isolated from a variety of clinical specimens, including blood, sputum, and urine; when encountered environmental contamination should be suspected
Pseudomonas aeruginosa Exotoxin A, endotoxins, proteolytic enzymes, alginate, and pili; intrinsic resistance to many antimicrobial agents Opportunistic pathogen that can cause community- or hospital-acquired infections
Community-acquired infections: skin (folliculitis); external ear canal (otitis externa); eye, following trauma; bone (osteomyelitis), following trauma; heart (endocarditis) in IV drug abusers; and respiratory tract (patients with cystic fibrosis)
Hospital acquired infections: respiratory tract, urinary tract, wounds, bloodstream (bacteremia), and central nervous system
Key pathogen that infects lungs of cystic fibrosis patients
P. fluorescens, P. putida, and P. stutzeri (includes Vb-3) Unknown. Infection usually requires patient with underlying disease to be exposed to contaminated medical devices or solutions Uncommon cause of infection; have been associated with bacteremia, urinary tract infections, wound infections, and respiratory tract infections; when found in clinical specimen, significance should always be questioned
P. mendocina, P. alcaligenes, P. pseudoalcaligenes, Pseudomonas sp. CDC group 1, “P. denitrificans,” Pseudomonas-like group 2, and CDC group Ic Unknown Not typically known to cause human infections. P. mendocina has been isolated from a patient with endocarditis (R)
Brevundimonas vesicularis and B. diminuta Unknown Rarely associated with human infections. B. vesicularis is rare cause and of bacteremia in patients suffering underlying disease
Acidovorax spp. Unknown Rarely isolated from clinical specimens. Not implicated in human infections

The capacity of B. cepacia to survive in the hospital environment, which may be linked to the organism’s intrinsic resistance to many antibiotics, provides the opportunity for this species to occasionally colonize and infect hospitalized patients. In patients with cystic fibrosis or chronic granulomatous disease, the organism can cause fulminant lung infections and bacteremia, resulting in death. In other types of patients, infections of the blood, urinary tract, and respiratory tract usually result from exposure to contaminated medical solutions or devices but are rarely fatal.


Infections caused by B. pseudomallei (capable of survival in human macrophages) can range from asymptomatic to severe. The disease is referred to as melioidosis; it has several forms, including the formation of skin abscesses, sepsis and septic shock, abscess formation in several internal organs, and acute pulmonary disease.


The remaining species listed in Table 22-2 are rarely encountered in human disease, and their clinical significance should be questioned when they are found in clinical specimens.



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.



Laboratory Diagnosis


Specimen Collection and Transport


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





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.



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 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.



TABLE 22-3


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







































































































































































Organism Medium Appearance
Acidovorax delafieldii BA No distinctive appearance
Mac NLF
Acidovorax facilis BA No distinctive appearance
Mac Unable to grow
A. temperans BA No distinctive appearance
Mac NLF
Brevundimonas diminuta BA Chalk white
Mac NLF
B. vesicularis BA Orange pigment
Mac NLF, but only 66% grow
Burkholderia cepacia complex BA Smooth and slightly raised; dirtlike odor
Mac NLF; colonies become dark pink to red due to oxidation of lactose after 4-7 days
PC or OFPBL Smooth
B. pseudomallei BA Smooth and mucoid to dry and wrinkled (may resemble P. stutzeri)
Mac  
Ashdown NLF
Dry, wrinkled, violet-purple
B. mallei BA No distinctive appearance
Mac NLF
Pandoraea spp. BA No distinctive appearance
Mac NLF
Pseudomonas aeruginosa BA Spreading and flat, serrated edges; confluent growth; often shows metallic sheen; bluish green, red, or brown pigmentation; colonies often beta–hemolytic; grapelike or corn tortilla–like odor; mucoid colonies commonly seen in patients with cystic fibrosis
Mac NFL
P. fluorescens BA No distinctive appearance
Mac NLF
P. mendocina BA Smooth, nonwrinkled, flat, brownish–yellow pigment
Mac NLF
P. monteilii BA No distinctive appearance
Mac NLF
P. mosselii BA No distinctive appearance
Mac NLF
No acid production from xylose
P. putida BA No distinctive appearance
Mac NLF
P. stutzeri and CDC group Vb–3 BA Dry, wrinkled, adherent, buff to brown
Mac NLF
P. veronii BA No distinctive appearance
Mac NLF
Pseudomonas–like group 2 BAP No distinctive appearance but colonies tend to stick to agar
Mac
NLF
CDC group Ic BAP No distinctive appearance
Mac NLF
Ralstonia mannitolilytica BAP No distinctive appearance
Mac NLF
R. pickettii BAP No distinctive appearance but may take 72 hr to produce visible colonies
Mac NLF

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Aug 25, 2016 | Posted by in MICROBIOLOGY | Comments Off on Pseudomonas, Burkholderia, and Similar Organisms

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