Two stable variants of <i>Burkholderia pseudomallei</i> strain MSHR5848 express broadly divergent <i>in vitro</i> phenotypes associated with their virulence differences

<div><p><i>Burkholderia pseudomallei</i> (<i>Bp</i>), the agent of melioidosis, causes disease ranging from acute and rapidly fatal to protracted and chronic. <i>Bp</i> is highly infectious by aerosol, can cause severe disease with nonspecific symptoms, and is naturally resistant to multiple antibiotics. However, no vaccine exists. Unlike many <i>Bp</i> strains, which exhibit random variability in traits such as colony morphology, <i>Bp</i> strain MSHR5848 exhibited two distinct and relatively stable colony morphologies on sheep blood agar plates: a smooth, glossy, pale yellow colony and a flat, rough, white colony. Passage of the two variants, designated “Smooth” and “Rough”, under standard laboratory conditions produced cultures composed of > 99.9% of the single corresponding type; however, both could switch to the other type at different frequencies when incubated in certain nutritionally stringent or stressful growth conditions. These MSHR5848 derivatives were extensively characterized to identify variant-associated differences. Microscopic and colony morphology differences on six differential media were observed and only the Rough variant metabolized sugars in selective agar. Antimicrobial susceptibilities and lipopolysaccharide (LPS) features were characterized and phenotype microarray profiles revealed distinct metabolic and susceptibility disparities between the variants. Results using the phenotype microarray system narrowed the 1,920 substrates to a subset which differentiated the two variants. Smooth grew more rapidly <i>in vitro</i> than Rough, yet the latter exhibited a nearly 10-fold lower lethal dose for mice than Smooth. Finally, the Smooth variant was phagocytosed and replicated to a greater extent and was more cytotoxic than Rough in macrophages. In contrast, multiple locus sequence type (MLST) analysis, ribotyping, and whole genome sequence analysis demonstrated the variants’ genetic conservation; only a single consistent genetic difference between the two was identified for further study. These distinct differences shown by two variants of a <i>Bp</i> strain will be leveraged to better understand the mechanism of <i>Bp</i> phenotypic variability and to possibly identify <i>in vitro</i> markers of infection.</p></div

Western blot of LPS from <i>Bp</i> strains.

<p>Purified LPS from MSHR5848 Smooth and Rough, and <i>Bp</i> strain 1106a, were separated by SDS-PAGE and a western blot was done using monoclonal antibody (mAb) 11G3-1, specific for <i>Bp</i> LPS O polysaccharide (OPS). Unlike the typical Type A banding pattern of most <i>Bp</i> strains (1106a), the variants displayed identical higher molecular weight patterns.</p

Infection of J774.A1 macrophage cultures with the Smooth and Rough variants of <i>Bp</i> MSHR5848.

<p>The MOIs were 19.4 and 19.1, respectively. The MSHR5848 Smooth was phagocytosed to an almost fivefold greater extent than the MSHR5848 Rough strain, as shown by the 3 h viable counts. The counts recovered from Smooth-infected cells at all three time point were greater than those from Rough-infected macrophages (p < 0.0001).</p

Smooth and Rough variant colony and microscopic morphologies.

<p>A: Sheep blood agar plates. The smooth, glossy, pale yellow colonies are the Smooth morphotype (arrow) and the flat dry grey-white colonies are Rough (arrowhead). B: Ashdown’s agar plates. The raised shiny, smooth deep pink colonies are Smooth (arrow) and the wrinkled dry pink and purple colonies are Rough (arrowhead). C and D: BSCA plates with Rough (lactose-positive, yellow dye) and Smooth (lactose-negative, red dye) growth, respectively. E and F: Gram stains of Smooth and Rough, respectively (100x objective). The Rough cells exhibit a”safety pin” appearance with stained poles and unstained center whereas the Smooth bacteria have a typical gram-negative rod morphology. G–J: The BURK production stock was grown on SBA plates at 37°C for three days. Smooth or Rough colonies were suspended in PBS and the suspensions were dried on microscope slides and stained with the fluorescent DNA binding dye PI. The slides were viewed on an Olympus BX51 microscope with phase contrast (100x, oil immersion objective) or fluorescence (ex. 535 nm/em. 617 nm) microscopy. G: Smooth colony bacteria viewed by phase contrast. H: Smooth colony bacteria stained with PI and viewed with fluorescence. I: Rough colony bacteria viewed by phase contrast. J: Rough colony bacteria stained with PI and viewed with fluorescence.</p

Growth conditions used to produce colony morphotype switching in MSHR5848 variant colony stocks^a.

<p>Growth conditions used to produce colony morphotype switching in MSHR5848 variant colony stocks<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171363#t002fn001" target="_blank">^a</a>.</p

Summary of phenotypic differences of <i>Bp</i> MSHR5848 (BURK178) variants.

<p>Summary of phenotypic differences of <i>Bp</i> MSHR5848 (BURK178) variants.</p

Phenotypic microarray data.

<p>The 20 PM plates are used to identify strain differences in ability to metabolize different sources of carbon, nitrogen, phosphorus or sulfur; or differences in sensitivity to antimicrobial conditions. Kinetic data were collected every 15 min for 48 h and the variant curves were compared, as shown. <b>Gray</b>: overlapping activity; <b>blue</b> area: greater Smooth response; and <b>red</b> area: greater Rough activity.</p

Comparison of relative virulence for mice of strains derived from <i>Bp</i> MSHR5848.

<p>Comparison of relative virulence for mice of strains derived from <i>Bp</i> MSHR5848.</p

Comparison of the virulence of variants Smooth and Rough in a mouse infection model.

<p>BALB/c mice were challenged IP with five ten-fold dilutions of Smooth (panel A) or Rough (panel B), as described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171363#pone.0171363.ref011" target="_blank">11</a>]. The decline in numbers of mice surviving with time for each dose group (10 mice/group) is shown. Infection with Rough was associated with more lethality/morbidity and a faster loss of survival than infection with Smooth.</p

Cytotoxicity of the Smooth and Rough variants of <i>Bp</i> MSHR5848 in J774.A1 macrophage cultures.

<p>Cytotoxicity of the Smooth and Rough variants of <i>Bp</i> MSHR5848 in J774.A1 macrophage cultures.</p