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...

Burkholderia pseudomallei is a soil-dwelling saprophyte and the causative agent of melioidosis, a lifethreatening human infection. Most cases are reported from northeast Thailand and northern Australia. Using multilocus sequence typing (MLST), we have compared (i) soil and invasive isolates from northeast Thailand and (ii) invasive isolates from Thailand and Australia. A total of 266 Thai B. pseudomallei isolates were characterized (83 soil and 183 invasive). These corresponded to 123 sequence types (STs), the most abundant being ST70 (n ‫؍‬ 21), ST167 (n ‫؍‬ 15), ST54 (n ‫؍‬ 12), and ST58 (n ‫؍‬ 11). Two clusters of related STs (clonal complexes) were identified; the larger clonal complex (CC48) did not conform to a simple pattern of radial expansion from an assumed ancestor, while a second (CC70) corresponded to a simple radial expansion from ST70. Despite the large number of STs, overall nucleotide diversity was low. Of the Thai isolates, those isolated from patients with melioidosis were overrepresented in the 10 largest clones (P < 0.0001). There was a significant difference in the classification index between environmental and disease isolates (P < 0.001), confirming that genotypes were not distributed randomly between the two samples. MLST profiles for 158 isolates from Australia (mainly disease associated) contained a number of STs (96) similar to that seen with the Thai invasive isolates, but no ST was found in both populations. There were also differences in diversity and allele frequency distribution between the two populations. This analysis reveals strong genetic differentiation on the basis of geographical isolation and a significant differentiation on the basis of virulence potential.

Human melioidosis is associated with a high rate of recurrent disease, despite adequate antimicrobial treatment. Here, we define the rate of relapse versus the rate of reinfection in 116 patients with 123 episodes of recurrent melioidosis who were treated at Sappasithiprasong Hospital in Northeast Thailand between 1986 and 2005. Pulsed-field gel electrophoresis was performed on all isolates; isolates from primary and recurrent disease for a given patient different by one or more bands were examined by a sequence-based approach based on multilocus sequence typing. Overall, 92 episodes (75%) of recurrent disease were caused by the same strain (relapse) and 31 episodes (25%) were due to infection with a new strain (reinfection). The interval to recurrence differed between patients with relapse and reinfection; those with relapses had a median time to relapse of 228 days (range, 15 to 3,757 days; interquartile range [IQR], 99.5 to 608 days), while those with reinfection had a median time to reinfection of 823 days (range, 17 to 2,931 days; IQR, 453 to 1,211 days) (P ‫؍‬ 0.0001). A total of 64 episodes (52%) occurred within 12 months of the primary infection. Relapse was responsible for 57 of 64 (89%) episodes of recurrent infection within the first year after primary disease, whereas relapse was responsible for 35 of 59 (59%) episodes after 1 year (P < 0.0001). Our data indicate that in this setting of endemicity, reinfection is responsible for one-quarter of recurrent cases. This finding has important implications for the clinical management of melioidosis patients and for antibiotic treatment studies that use recurrent disease as a marker for treatment failure.Melioidosis is a severe infection caused by the gram-negative bacillus Burkholderia pseudomallei, an environmental saprophyte present in Southeast Asia and northern Australia (17). A major feature of human disease is that bacterial eradication is difficult to achieve. The clinical response to intravenous antibiotics is slow, with a median fever clearance time in our patient population of 8.5 days. Although prolonged courses of antibiotic treatment (at least 10 days of intravenous antibiotics, followed by 12 to 20 weeks of oral antibiotics) are recommended, recurrent disease is common (at a rate of Ն6% in the first year) (2, 5). A prolonged period of dormancy of up to 62 years may also occur between a presumed exposure to B. pseudomallei and clinical manifestations of infection (9).Bacterial mechanisms and host susceptibility for recurrent melioidosis are poorly understood. However, the ability of B. pseudomallei to survive in the human host supports the idea that recurrence is more likely to be associated with a relapse caused by bacteria persisting within a sequestered focus than to reinfection with a different strain. This is consistent with previous small studies in which isolates from the first and second episodes of infection in a given patient usually had the same genotype (5,6,14). Here, we report the results of a large genotyping study of isolates fr...