Burkholderia pseudomallei

• Domain: Bacteria
•     Phylum: Proteobacteria
•         Class: Betaproteobacteria
•             Order: Burkholderiales
•                 Family: Burkholderiaceae
•                     Genus: Burkholderia
•                         Species: Burkholderia pseudomallei

B. pseudomallei is a small aerobic Gram-negative, motile rod-shaped bacterium, approximately 3-5 µm by 0.5-1 µm. Gram-stained smears of 18-24 h cultures on nutrient agars incubated at 37°C usually show bipolar staining. They are present singly, in pairs or sometimes in chains. After 48 h, the cells become oval, only staining at the periphery and may be mistaken for spores. Bipolar or uneven staining may be apparent in the bacteria in smears of clinical specimens, but the organism is not always seen in clinical specimens and definitive diagnosis depends on isolation and identification (Dance, 1998). Although an aerobe, B. pseudomallei can also grow anaerobically in complex media, requiring nitrate as the oxygen source (Groves, 1979).

The bacterium grows readily on or in simple nutrient media and can be recovered on most aerobically incubated primary isolation media used in clinical microbiology for enteric Gram negatives; notable exceptions to this are salmonella-shigella (SS) agar and deoxycholate-citrate agar (DCA) on which it is apparently reluctant to grow. On nutrient agar after 24 h at 37°C, colonies are small (0.5 to 1 mm in diameter), round, translucent, moist and slightly raised. By 48 h they become opaque, cream to yellow, 2-3 mm in diameter. With continued incubation, rough, heaped-up, 'corrugated' colonies may appear. On blood agar at 24 h, the colonies are transparent, shiny and convex; the confluent areas are grey to white with a sheen, and may be surrounded by a slight greening of the agar. On MacConkey’s agar, colonies are usually pink, but may be pink to colourless. Broths become uniformly turbid after 12 to 18 h at 37°C with a surface pellicle forming after continued incubation which becomes wrinkled and rough. A characteristic earthy, musty odour is frequently associated with these cultures.

The resistance of the Burkholderia pathogens to various dyes has allowed the formulation of media for selective separation from Pseudomonas aeruginosa (Pitt, 1990). Ashdown’s medium (Ashdown, 1979), containing glycerol (which induces the characteristic wrinkled colony morphology), crystal violet and gentamicin at concentrations inhibitory to various other bacteria, including Proteus, and neutral red (selectively absorbed by B. pseudomallei), is especially recommended for B. pseudomallei. On this, only pale pink pinpoint colonies are visible at 18-24 h; by 48 h, they are 1-2 mm, pinkish-purple, flat, slightly dry with a sheen. At 72 h, they are 2-3 mm, drier and wrinkled, deeper purple, though there may be mucoid variants or different coloured variants (Walsh and Wuthiekanun, 1996). Dry wrinkled, violet-purple colonies with the Gram-stain characteristics already described, permit a presumptive identification of B. pseudomallei (Gilligan and Whittier, 1991). An enrichment broth consisting of Ashdown medium supplemented with 50 mg/l of colistin enhances selective recovery of B. pseudomallei (Walsh et al., 1995).

Primary distinguishing characteristics when differentiating B. pseudomallei from its nearest pathogenic relatives, in particular, B. mallei, B. cepacia, P. aeruginosa and P. stutzeri, are its distinctive colonial morphology (P. stutzeri has a similar morphology), characteristic odour (B. cepacia has a similar odour) and motility (B. mallei is non-motile). A full bank of confirmatory tests is given in Table 1. Walsh and Wuthiekanun (1996) consider that complete accuracy in the identification of B. pseudomallei can be achieved with the simplified schema outlined in Table 2. A diagnostic phage was included in the earlier literature (Howe et al., 1971), but is not included in more recent texts. [CAUTION: It should be pointed out that, because of the potential seriousness of melioidosis in humans and inhalation is a method of acquisition, odour should only be a first-time observation and should not be used as a follow-up test (Ashdown 1992a). Laboratory infections have occurred (Sewell, 1995), and once it is suspected that B. pseudomallei is present, further manipulations should be carried out in a biological safety cabinet under Biohazard Level 3 conditions.]

Commercial identification systems recorded as good for identifying B. pseudomallei are the Microbact 24E (Thomas, 1983), API 20NE (BioMerieux-Vitek, Hazlewood, MO, USA) (Dance et al., 1989; Walsh and Wuthiekanun, 1996; Dance, 1998) and Titertek NF with the Minitek system (BBL Microbiology Systems, Cockeysville, MD, USA) good for final confirmation where other systems have given a presumptive identification of either B. pseudomallei or B. cepacia (Ashdown, 1992b).

Table 1. Distinguishing characteristics for B. pseudomallei and close relatives*

*From Cowan (1974) and Gilligan and Whittier (1991).
+ = >85%; ± = 10-85%; - = <10%
^§Results are method dependent

Table 2. Simplified identification scheme of Walsh and Wuthiekanun (1996)

The pathogenesis of melioidosis is not well understood. Putative virulence factors include a thermolabile exotoxin, thought to suppress cellular immune functions (Yahya and Chui Lik, 1995), several hydrolytic enzymes, including proteases and phospholipases, at least one haemolysin and lipopolysaccharides/endotoxin (Rapaport et al., 1961; Ashdown and Koehler, 1990; Kanai and Kondo, 1994; Perry et al., 1995). Evidence suggests that B. pseudomallei can colonize mucosal tissue and that pili may be the means of adherence enabling this (Woods et al., 1999). Siderophores may be utilized (Yang et al., 1993). There is increasing evidence that a capsular polysaccharide is an important virulence determinant (Reckseidler et al., 2001). Resistance to complement-mediated bacteriolysis and ability to survive and possibly multiply in macrophages and neutrophils are believed to be important contributors to virulence (Egan and Gordon, 1996; Woods et al., 1999). At least in humans, it appears that infection can remain latent with reactivation occurring up to many years later, and this has been attributed to the ability of B. pseudomallei to persist intracellularly in a dormant state (Woods et al., 1999). This can provoke a state of delayed type hypersensitivity (Howe et al., 1971).

Interest in B. pseudomallei as a potential biological warfare or bioterrorism agent is promoting further research into better understanding of the pathogenesis of this bacterium.