Unique inducible filamentous motility identified in pathogenic Bacillus cereus group species

Abstract

Active migration across semi-solid surfaces is important for bacterial success by facilitating colonization of unoccupied niches and is often associated with altered virulence and antibiotic resistance profiles. We isolated an atmospheric contaminant, subsequently identified as a new strain of Bacillus mobilis, which showed a unique, robust, rapid, and inducible filamentous surface motility. This flagella-independent migration was characterized by formation of elongated cells at the expanding edge and was induced when cells were inoculated onto lawns of metabolically inactive Campylobacter jejuni cells, autoclaved bacterial biomass, adsorbed milk, and adsorbed blood atop hard agar plates. Phosphatidylcholine (PC), bacterial membrane components, and sterile human fecal extracts were also sufficient to induce filamentous expansion. Screening of eight other Bacillus spp. showed that filamentous motility was conserved amongst B. cereus group species to varying degrees. RNA-Seq of elongated expanding cells collected from adsorbed milk and PC lawns versus control rod-shaped cells revealed dysregulation of genes involved in metabolism and membrane transport, sporulation, quorum sensing, antibiotic synthesis, and virulence (e.g., hblA/B/C/D and plcR). These findings characterize the robustness and ecological significance of filamentous surface motility in B. cereus group species and lay the foundation for understanding the biological role it may play during environment and host colonization.

Fig. 1. Identification of the filamentous motile environmental isolate as Bacillus mobilis ML-A2C4.

a ML-A2C4 filamentous growth on C. jejuni lawn spots (small circles). b ML-A2C4 growth on a control 1.5% agar MH plate (left) and on a MH plate spread with a full confluent C. jejuni lawn (center) after 48 h aerobic incubation at 30 °C. The red box shows a close-up view of the filaments at the growth edge (right). c Quantification of the visible growth diameter on control MH plates (black bars) and plates with C. jejuni lawns (red bars) over time (n = 5) with error bars indicating standard deviation (SD). Statistical analysis was performed for growth diameter on C. jejuni lawn plates versus control plates using the Student’s t test with Welch’s correction, and for 48 vs. 24 h using repeated measures one-way ANOVA, with ****p < 0.0001. The limit (dotted line) represents the diameter of the MH plate. d Phylogenetic placement of ML-A2C4 (red type) based on the core genome SNP comparison with other members of the B. cereus group. Blue dots indicate additional B. cereus species selected for comparative phenotypic testing. e Cytotoxicity of B. cereus ATCC 14579, B. mobilis 0711P9-1, B. pseudomycoides DSM 12442, B. wiedmannii FSL W8-0169, and B. mobilis ML-A2C4 supernatants on HeLa (black) and Caco-2 (gray) cells relative to negative control (EMEM + FBS supplemented with BHI medium). Triton X-100 (0.05%) was included as a positive cytotoxicity control. ****p < 0.0001 indicates that there is significant difference in the cytotoxicity between the bacterial supernatants and the respective BHI negative control for each cell line as determined using the Games–Howell test.

Fig. 2. Robustness of ML-A2C4 surface motility under different conditions.

Impact of agar concentration (a), media type (b), MH richness (c), temperature (d–i), aerobic (d, g), microaerobic (e, h), and anaerobic (f, i) conditions on ML-A2C4 growth after 48 h incubation on control plates (black bars) and plates spread with C. jejuni lawns (red bars). The measurement limit represents the diameter of the plates. Testing was performed at n = 5–8 per condition. Pictures show representative plates. Red boxes show greater magnification of the indicated area. Statistical analysis was performed for growth diameter on C. jejuni lawn plates versus control plates where indicated using the Student’s t test with Welch’s correction. p values are represented as: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.

Fig. 3. Conditions that induced filamentous expansion.

a Comparison of inactive (pre-prepared plates) and live (fresh plates) C. jejuni cell lawns that induced ML-A2C4 spreading after microaerobic incubation at 38 °C (n = 5). b ML-A2C4 expansion on adsorbed C. jejuni cell lawns prepared by autoclaving cells at various densities (n = 8). The measurement limit is lower because the cell lawns were applied onto MH plates as small ~30 mm diameter spots. ML-A2C4 was inoculated at the edge of the lawn. c ML-A2C4 expansion on 15 OD₆₀₀/mL autoclaved cell lawns of multiple cell types including itself (“self”) (n = 8). Shortened species names listed in order are: B. cereus, B. thuringiensis, B. weihenstephanensis, B. cytotoxicus, B. subtilis, B. licheniformis, B. megaterium, and E. coli. ML-A2C4 expansion on multiple types of milk products (d), blood products (e), and phosphatidylcholine (PC; f, g) (n = 5). MEM minimal essential medium, RBC red blood cell, EtOH ethanol. h Representative plates from n = 3 testing of ML-A2C4 growth on 25% EtOH spots (left) and E. coli and C. jejuni inner membrane (IM) and outer membrane (OM) preparations. i Representative plates from n = 3 testing of ML-A2C4 growth on nine human fecal extracts (left: H1C, H2, and H3; center: H4, H5, and H6; right: H9, H10, and H11). Dotted lines in h and i indicate area where substrate was dried onto the plate. As a control, ML-A2C4 was spotted in the middle of plates shown in h and i where there was no substrate. All data shown were taken after 48 h incubation. Statistical analysis was performed for growth diameter on C. jejuni lawn plates versus control plates where indicated using the Student’s t test with Welch’s correction. p values are represented as: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns not significant.

Fig. 4. Growth and expansion of Bacillus spp. and E. coli.

Growth after 48 h on control 1.5% agar MH plates (left column), plates spread with C. jejuni lawns (middle left column), whole blood (middle right column), and 10% skim milk (right column) preparations. Testing was performed at n = 5, representative images are shown.

Fig. 5. Microscopy of ML-A2C4 cells on control plates and plates spread with C. jejuni lawns, whole blood, 10% skim milk, and PC.

Representative images are shown. Five distinct stages of cell and colony morphology were observed from the visible colony edge to the center of the plate. A model of the growth phenotypes was created based on these visual observations to highlight various stages of filamentous spreading.

Fig. 6. Differential expression in filamentous motility expanding cf. control colonies.

a Principle component analysis of control cells (black), filamentous cells on 10% skim milk lawns (orange) and on PC lawns (purple). b Venn diagrams of genes that showed increased (top) and decreased (bottom) transcription during filamentous expansion in comparison with control cells. c Heat map of all differently transcribed genes during filamentous growth in milk and PC (wide columns) and the gene ontology (GO) categories assigned to those genes (thin columns). d Lists of differently transcribed genes organized based on predicted function with the fold change represented as a colored box corresponding to the fold change scale in the heat map.