Biological Relevance of Colony Morphology and Phenotypic Switching by Burkholderia pseudomallei

Abstract

Melioidosis is a notoriously protracted illness and is difficult to cure. We hypothesize that the causative organism, Burkholderia pseudomallei, undergoes a process of adaptation involving altered expression of surface determinants which facilitates persistence in vivo and that this is reflected by changes in colony morphology. A colony morphotyping scheme and typing algorithm were developed using clinical B. pseudomallei isolates. Morphotypes were divided into seven types (denoted I to VII). Type I gave rise to other morphotypes (most commonly type II or III) by a process of switching in response to environmental stress, including starvation, iron limitation, and growth at 42°C. Switching was associated with complex shifts in phenotype, one of which (type I to type II) was associated with a marked increase in production of factors putatively associated with in vivo concealment. Isogenic types II and III, derived from type I, were examined using several experimental models. Switching between isogenic morphotypes occurred in a mouse model, where type II appeared to become adapted for persistence in a low-virulence state. Isogenic type II demonstrated a significant increase in intracellular replication fitness compared with parental type I after uptake by epithelial cells in vitro. Isogenic type III demonstrated a higher replication fitness following uptake by macrophages in vitro, which was associated with a switch to type II. Mixed B. pseudomallei morphologies were common in individual clinical specimens and were significantly more frequent in samples of blood, pus, and respiratory secretions than in urine and surface swabs. These findings have major implications for therapeutics and vaccine development.Burkholderia pseudomallei is a biothreat agent and the cause of melioidosis (29). This gram-negative motile bacillus is present in soil and water over a wide area of the Far East, where infection is acquired by inoculation or inhalation (29). B. pseudomallei causes 20% of community-acquired septicemias in northeast Thailand (7) and is the most common cause of fatal community-acquired pneumonia in Darwin, Australia (10, 14). Overall, mortality is around 50% in northeast Thailand (35% in children) and 20% in Australia (10, 11, 29).A major feature of melioidosis is that bacterial eradication is difficult to achieve. The clinical response to intravenous antibiotics is slow (median fever clearance time, 8 days), and recurrent disease is common (6% in the first year in Thailand), despite appropriate antibiotic therapy for 12 to 20 weeks (6, 9). A prolonged period of dormancy may also occur between exposure to B. pseudomallei and clinical manifestations of infection, with the maximum recorded time being 62 years (8,20,21). It is clear that B. pseudomallei can become adapted for survival in vivo, but the mechanisms by which this occurs in humans have not been demonstrated.In the 1930s, it was observed that colony morphology could change in vitro between rough and smooth colonies (22). We have observed over a period of 20...

Simultaneous Infection with More than One Strain of Burkholderia pseudomallei Is Uncommon in Human Melioidosis

“…This included the characteristic colony morphology (purple, flat, dry, and wrinkled) together with six additional colony morphotypes, as described previously (2). Colonies suspected to be B. pseudomallei were tested using an oxidase test and then confirmed using a highly specific latex agglutination test (positive for B. pseudomallei but negative for Burkholderia thailandensis) (1,12).…”

“…Genotyping of 2,058 bacterial colonies isolated from 215 samples taken from 133 patients demonstrated that mixed infection is uncommon (2/133 cases [1.5%; 95% confidence interval, 0. 2 …”

Variations in Ceftazidime and Amoxicillin-Clavulanate Susceptibilities within a Clonal Infection of Burkholderia pseudomallei

“…An alternative explanation is that resistant colonies 76161w and 76161y were present in low numbers in the initial blood culture and were undetected. B. pseudomallei appears to exhibit multiple phenotypes within a clonal population and can switch between them in response to the environment, resulting in variations in morphological appearance, intracellular replication, and persistence (2). Highly resistant small-colony variants of B. pseudomallei can revert to the parental (larger) size phenotype and antibiotic susceptibility (7).…”

Colony Morphology Variation of Burkholderia pseudomallei Is Associated with Antigenic Variation and O-Polysaccharide Modification

“…The type I morphotype represents the predominant type (88% of 241 clinical isolates), but this can switch to other types in vivo and in vitro. The different colonies that were observed on Ashdown selective agar are related to phenotypic variation, including the ability to produce extracellular enzymes, flagella, motility, and biofilm formation, but the relationship between colony morphotype and LPS variation is unclear (19). We have subsequently noted the presence of two distinct colony types in a proportion of clinical samples plated onto nonselective agar, such as Trypticase soy agar (TSA) and blood agar (BA), which are nonselective media commonly used in routine hospital laboratories and in our research laboratory.…”