Biochemical Testing and the Critical Identification of Burkholderia pseudomallei
Burkholderia pseudomallei is a Gram-negative, rod-shaped bacterium and the causative agent of melioidosis, a serious and often fatal disease. The organism is classified as a high-priority biological agent due making its accurate and timely laboratory identification a critical public health and safety concern. Due to its ubiquitous presence in soil and water in endemic regions, and the risk of laboratory-acquired melioidosis, established protocols are necessary for all clinical laboratories.
Accurate identification is complicated by the organism’s pleomorphic nature—it may demonstrate bipolar staining, often referred to as a “safety pin” appearance—and its highly variable colonial morphology. Furthermore, *B. pseudomallei* is morphologically and biochemically similar to other members of the *Burkholderia* genus, particularly the *Burkholderia cepacia* complex, leading to frequent misidentification by automated systems and conventional methods. Therefore, a definitive identification relies on a combination of classical biochemical tests, growth characteristics, antimicrobial susceptibility profiling, and, ideally, molecular confirmation.
Essential Conventional Biochemical Reactions
The core set of biochemical reactions are used to rule in or rule out a suspected isolate. The traditional, manual tests remain essential, particularly since identification systems not specific for non-fermenting organisms can produce erroneous results. The characteristic reactions for *B. pseudomallei* are summarized by several key tests:
Oxidase Test: *B. pseudomallei* is typically oxidase-positive. This reaction, catalyzed by the enzyme cytochrome *c* oxidase, is a crucial first step in differentiating the organism from many other enterics and non-fermenting bacilli, although some strains may exhibit a negative or variable result.
Indole Test: *B. pseudomallei* is indole-negative. This differentiates it from many other organisms that metabolize tryptophan to indole, a key step in its presumptive identification flowchart.
Catalase Test: The organism is catalase-positive, demonstrating its ability to break down hydrogen peroxide, which is a common characteristic among many aerobic bacteria, including other *Burkholderia* species.
Motility Test: *B. pseudomallei* is motile, a significant differentiator from its close relative, *Burkholderia mallei*, which is non-motile. This test is typically performed using Motility Test Medium, sometimes supplemented with 2,3,5-triphenyltetrazolium chloride (TTC) to visualize growth.
Arginine Dihydrolase Test: *B. pseudomallei* is arginine dihydrolase-positive. This enzymatic reaction, which breaks down arginine, is a very reliable differential test for *B. pseudomallei* and is commonly included in identification protocols for glucose non-fermenting organisms. The organism is generally lysine and ornithine decarboxylase-negative, further defining its metabolic profile.
OF Test (Glucose): The organism is a glucose oxidizer, producing acid (A) in the oxidative tube of the oxidative/fermentative (OF) test, which is characteristic of non-fermenting Gram-negative rods.
Growth and Colony Morphology on Differential Media
Observation of growth patterns on various media at specific time intervals and temperatures provides additional crucial identification points:
Sheep Blood Agar (BAP): Initial growth at 24 hours often presents as pinpoint colonies, which develop into smooth, creamy-white colonies by 48 hours. Importantly, these colonies are non-hemolytic. Over time (48 to 72 hours or more), the colonies may develop a characteristic dry or mucoid and wrinkled morphology, a feature that can lead to confusion if staff mistakenly believe the culture is mixed.
MacConkey Agar (MAC): *B. pseudomallei* typically grows on MAC within 48 hours, yielding colonies that are pink or colorless. The pink color is due to the oxidation of lactose, though some may remain colorless or turn colorless over time. Growth on MAC is a standard screening test for this Gram-negative rod.
Temperature Tolerance: The ability to grow at 42°C is another key characteristic that helps distinguish *B. pseudomallei* from some other environmental bacteria.
Musty Odor: While not a definitive biochemical test, some isolates may produce a distinctive musty or earthy odor. However, this olfactory characteristic is not present in all strains and cannot be relied upon to rule out the organism.
Antimicrobial Profile as a Presumptive Tool
The antimicrobial susceptibility profile serves as a strong presumptive identification characteristic, especially when dealing with oxidase-positive, Gram-negative bacilli.
Resistance to Colistin and Polymyxin B: *B. pseudomallei* is characteristically resistant to both colistin and polymyxin B. This resistance allows for the use of selective media, such as Ashdown’s agar or Threonine Basal Salt Solution (TBSS) containing colistin, which is often necessary to isolate the organism from polymicrobial clinical or environmental samples.
Susceptibility to Amoxicillin-clavulanate: Approximately 99% of *B. pseudomallei* strains are susceptible to amoxicillin-clavulanate. This susceptibility, in combination with the resistance to colistin, forms a highly specific presumptive identification signature that aids frontline sentinel laboratories in the immediate decision to refer an isolate for confirmatory testing.
Challenges and Biosafety Considerations
The complexity of *B. pseudomallei* identification is compounded by the high risk associated with handling the organism. It is an agent that requires Biosafety Level 3 (BSL-3) precautions for handling high-titer cultures, and all aerosol-producing procedures must be conducted within a Biological Safety Cabinet (BSC) to prevent laboratory-acquired infection.
A significant challenge in clinical microbiology remains the misidentification by widely used automated systems. Devices such as Vitek 2 or the API 20NE manual strip frequently misidentify *B. pseudomallei* strains as members of the *B. cepacia* complex or even *Chromobacterium violaceum*. Therefore, any identification that is consistent with *B. pseudomallei* using the conventional biochemical profile—oxidase positive, indole negative, motile, arginine positive, and resistant to colistin—must be treated as presumptive and referred to a reference laboratory for definitive confirmation via methods like real-time PCR or DNA sequencing.
In summary, the conventional biochemical tests are not obsolete but are essential for an initial, accurate rule-out or presumptive identification of *Burkholderia pseudomallei*. These tests, particularly the reactions for oxidase, indole, motility, and arginine dihydrolase, coupled with the unique antimicrobial resistance to polymyxin B/colistin, serve as the backbone for the laboratory workflow required to manage this medically important and hazardous organism.