Unique inducible filamentous motility identified in pathogenic Bacillus cereus group species

Isolation of an environmental contaminant with preferential expansion on C. jejuni cell lawns

We observed a contaminant colony that paradoxically grew preferentially on small, spot-plated lawns of C. jejuni cells on Mueller Hinton (MH) agar (1.5% w/v). The MH plate had previously been inoculated with C. jejuni cells spotted and incubated microaerobically at 38 °C overnight before being stored for several days aerobically at room temperature. Transfer of contaminant cells onto new, similarly prepared spot-plated lawns of C. jejuni resulted in the contaminant again growing preferentially atop the C. jejuni lawns, with minimal growth on the rich agar in between spots of C. jejuni lawns (Fig. 1a). The contaminant was isolated for further study and the strain named ML-A2C4.

A two day spread plating technique was developed to achieve full, dense, and confluent lawns of C. jejuni (“C. jejuni lawn” plates; refer to Supplementary Methods S1), which were stored aerobically at room temperature for up to 5 days. Control conditions were comparable plates without C. jejuni. ML-A2C4 was inoculated at the center of each plate, and expanded rapidly on those spread with C. jejuni lawns but not on control plates (Fig. 1b). The expanding ML-A2C4 colony was flat and often showed a dense white inner ring with a less dense outer ring. The outer ring had fine tendrils that exhibited a distinctive filamentous pattern radiating outward in random directions at the edges (Fig. 1b). When incubated aerobically at 30 °C, ML-A2C4 expanded rapidly on C. jejuni cell lawns at ~32 mm/day, filling the entire plate (83 mm diameter) between 48 and 72 h post inoculation (Fig. 1c). In contrast, colonies on control plates grew modestly over time (~4 mm/day) and did not exhibit filamentous edges (Fig. 1b, c). Further investigation into this strain and the filamentous surface migration phenomenon was undertaken as bacterial motility on hard agar is relatively rare, surface motility dependence on the presence of other cell lawns had not yet been reported, and the distinctive filamentous expansion of the colony appeared unique.

The contaminant was identified as a new B. mobilis strain

We hypothesized that ML-A2C4 originated from the air since the plate on which it was originally isolated had been dried with the lid off prior to use. Light microscopy of cells from control plates showed that they were rod-shaped spore formers (data not shown). Initial 16S rRNA sequencing revealed that ML-A2C4 belonged to the B. cereus group, and panC sequencing using the 7-clade panC classification scheme demonstrated that it belonged in clade II [13]. Therefore, we performed whole-genome sequencing of ML-A2C4 using both PacBio and Illumina MiSeq and compared it with the other members of the B. cereus s.l. species. The genome of ML-A2C4 was 5.47 Mb with an average GC content of 35.6%. Core genome comparative analyses with 18 valid and three effective B. cereus s.l. species type strains revealed a close relatedness to the B. mobilis type strain 0711P9-1 based on 1142 core SNPs identified using kSNP3 [27] (Fig. 1d and Supplementary Table S2). The genome of B. mobilis ML-A2C4 was uploaded into Genbank as NCBI reference sequence NZ_CP031443.1 and represents the first complete annotated genome for B. mobilis.

ML-A2C4 possesses 23 known or putative virulence genes and five antimicrobial resistance genes (Supplementary Table S3), including the hblABCD and nheABC genes encoding the well-characterized diarrheal enterotoxin hemolysin BL and nonhemolytic enterotoxin Nhe [28,29,30,31]. To further characterize the pathogenic potential of this strain, we evaluated HeLa and Caco-2 cell intoxication using bacterial supernatants (Fig. 1e). Survival was determined based on the metabolic conversion of WST-1 into a colored dye. ML-A2C4 supernatant reduced HeLa and Caco-2 cell viability to 23% and 22% of untreated, respectively. This was comparable with the cytotoxic-positive control B. cereus ATCC 14579 supernatant, which reduced HeLa and Caco-2 cell viability to 32% and 22% of untreated, respectively, and B. pseudomycoides DSM 12442 (19% viability for HeLa and 21% for Caco-2 cells). The B. mobilis reference strain 0711P9-1 and B. wiedmannii FSL W8-0169 did not demonstrate cytotoxicity in either HeLa or Caco-2 cells. The pronounced cytotoxicity of ML-A2C4 may have implications for pathogenesis of certain B. mobilis strains.

Filamentous motility was robust under a wide range of environmental conditions

B. mobilis is phylogenetically closely related to B. cereus s.s. (Fig. 1d), which swarms by elongation and hyper-flagellation of individual cells when inoculated on the surface of semi-soft (0.4–1.0%) agar [4, 32]. ML-A2C4 inoculated onto similar swarm plates (LB and TrB with 0.5 and 0.7% agar) failed to swarm (data not shown). A plcR deletion mutant of B. cereus s.s. also demonstrates dendritic colony morphology, which is thought to occur as a result of a sliding action of the filamentous cells after 96 h incubation on 0.7% agar made with low-nutrient defined media [33]. In contrast, the filamentous growth of ML-A2C4 on C. jejuni lawns occurred much earlier (before 24 h), was morphologically distinct, was robust even on rich MH agar plates at a much higher agar concentration, had a faster expansion rate, and was inducible. Therefore, we concluded that the filamentous growth of ML-A2C4 was distinct from the previously reported swarming and dendritic sliding phenotypes of B. cereus s.s.

We assessed ML-A2C4 filamentous growth under a wide range of conditions using C. jejuni lawn plates prepared with different agar concentrations, nutrient types, nutrient richness, and incubated at different temperatures and oxygen concentrations. No notable variability was observed in the C. jejuni lawns on these plates prior to ML-A2C4 inoculation. Control conditions were the corresponding plates without C. jejuni. ML-A2C4 filamentous motility occurred on C. jejuni lawn plates at all agar concentrations tested (0.5–5% w/v); however, it was fastest when plate agar concentrations were less than 2% (Fig. 2a). Motility of ML-A2C4 on C. jejuni lawn plates containing up to 5% agar was unexpected, since even the robust swarmer P. mirabilis exhibits minimal motility on 3% agar and does not swarm at all on 4% agar [22, 34], while other swarming cells like P. aeruginosa are inhibited at 0.6% agar [35]. ML-A2C4 showed filamentous expansion on C. jejuni lawns atop 1.5% agar plates made with different nutrient types including TrB, MH, LB, and BHI; however, the expansion rates appeared inversely correlated with nutrient richness and were faster on plates with less nutrient rich media (e.g., TrB, 15 g/L solids) versus highly rich media (e.g., BHI, 37 g/L solids) (Fig. 2b). To test different nutrient richness levels using the same medium, we made plates containing 0.5× and up to 2× the amount of MH powder normally used prior to inoculation of C. jejuni lawns. ML-A2C4 exhibited filamentous motility under all MH conditions tested; however, expansion was fastest when the lawn plates contained lower levels of MH (Fig. 2c). ML-A2C4 growth on control plates without C. jejuni lawns was comparable for all agar concentrations, media types, and MH concentrations (Fig. 2a–c). Inoculation of ML-A2C4 onto C. jejuni lawn plates and subsequent incubation at multiple temperatures (22–42 °C) and oxygen levels (aerobic, microaerobic, and anaerobic conditions) demonstrated that cell growth and filamentous motility occurred over a wide range of temperature and oxygen conditions but was most rapid under microaerobic incubation at 30 °C (Fig. 2d–i). Bacillus spp. are facultative anaerobes, but fermentative growth is much less effective than aerobic growth [36]. As expected, ML-A2C4 growth on both control and C. jejuni lawn plates was inhibited when incubated under anaerobic conditions (Fig. 2f, i). However, filamentous expansion was still evident, especially at 37 °C, despite lack of accumulated cell mass (Fig. 2i). For all the experiments described below, ML-A2C4 filamentous surface motility was evaluated using 1.5% MH agar plates incubated aerobically at 30 °C unless otherwise stated.

Filamentous motility was induced by other metabolically inactive bacterial cells, blood, and milk

As noted above, C. jejuni lawn plates were prepared and stored aerobically at room temperature for up to 5 days before each experiment. C. jejuni is microaerophilic and thermophilic and does not grow aerobically or at room temperature, thus the cells on these plates are likely metabolically inactive and/or dead [37]. To determine if ML-A2C4 could expand on C. jejuni lawns containing actively growing cells, we inoculated freshly grown C. jejuni plates with ML-A2C4 and incubated them microaerobically at 38 °C, a condition under which most C. jejuni cells are expected to remain viable. Filamentous expansion on live C. jejuni cell lawns was reduced versus on plates containing the inactive lawns (Fig. 3a). ML-A2C4 filamentous growth was also inhibited when inoculated next to or on top of live lawns of other bacteria such as E. coli (data not shown), suggesting that either the compound(s) triggering filamentous motility were not accessible in living bacteria, or that living bacteria were capable of inhibiting ML-A2C4. Lawns of autoclaved C. jejuni spotted and dried onto agar plates induced filamentous growth in a concentration-dependent manner (Fig. 3b), and ML-A2C4 showed filamentous motility on heat-killed lawns of all bacterial cell types tested, including itself (Fig. 3c).

A variety of additional compounds, selected to represent different nutrients, cellular products, stressors, host factors, and other environmental factors that Bacillus might encounter, were screened to determine if there were additional triggers. Filamentous motility was not induced when plates were inoculated with protein derivatives, nucleotides, sugars, high levels of nutrients, or various other chemicals (see Supplementary Table S1 for a full list of tested substrates and concentrations). However, filamentous motility was very robust on plates containing adsorbed lawns of milk and blood (Fig. 3d, e). These findings were particularly notable since Bacillus spp. are commonly isolated from contaminated milk and have been shown to cause bacteremia in humans [15]. Filamentous expansion was robustly induced on lawns of commercial cow-milk products ranging from skim to half-and-half, and was modestly induced with soy milk but not coconut or almond milk (Fig. 3d). We separated whole blood into red blood cells [RBCs; re-suspended in minimal essential medium (MEM)] and plasma by centrifugation. RBCs induced filamentous growth comparable with whole blood, but filamentous expansion was reduced and inconsistent in plasma (Fig. 3e), while fetal bovine serum likewise did not induce filamentous growth (data not shown). Direct contact also appeared to be required for initiation of filamentous growth, since ML-A2C4 did not move in the direction of tested compounds when inoculated near (but not touching) spots of C. jejuni cell lawns, blood, or milk, and did not expand when inoculated on top of 5% or 15% sheep blood agar where the RBCs were embedded within the agar plate (data not shown).

Filamentous motility was induced by phospholipids and human fecal extracts

Phospholipids are present in membranes of bacterial cells and RBCs, and soluble phospholipids are found in cow milk and soy milk [38,39,40,41,42]; therefore, we suspected that phospholipids might be an inducer. PC was utilized as a representative phospholipid for initial testing. Filamentous growth readily occurred on plates spread with PC (0.5 mL of 2% w/v in 50% EtOH) (Fig. 3f, g); EtOH did not impact ML-A2C4 growth (Fig. 3f). Filamentous growth also occurred readily on extracted inner and outer membrane fractions from E. coli and C. jejuni, which are composed of primarily of phosphatidylglycerol and phosphatidylethanolamine [40, 43], suggesting that filamentous growth could be induced by multiple phospholipid types (Fig. 3h).

Since filamentous expansion was optimally induced under low nutrient, moist, microaerobic conditions between 30 and 37 °C while in contact with dead bacterial cells and/or phospholipids, and as B. cereus s.l. organisms are often gut colonizers, we hypothesized that this filamentous motility might occur in the intestinal milieu. While the lumen of the gut can be largely anaerobic, the oxygen content near the intestinal epithelia is better described as microaerobic, and aerotolerant microbes are well suited to thrive in this ecological niche [44]. To determine if human gut contents could induce filamentous motility, nine sterile human fecal extracts collected and purified from a separate study were spotted onto agar plates [45]. ML-A2C4 exhibited filamentous expansion when inoculated on five out of nine human fecal extracts (Fig. 3i). This indicated that human gut contents could selectively induce filamentous motility. As filamentous expansion was not induced with all human fecal extract samples, we hypothesize that other factors impacting extract composition such as person to person differences, diet, stool frequency, and variability during extract preparation also impacted ML-A2C4 motility [45, 46].

Filamentous motility was conserved to varying degrees amongst members of the B. cereus s.l. group

To determine if filamentous motility was conserved amongst other Bacillus spp., reference strains of eight different species distributed across the Bacillus genus were screened. These included five from the B. cereus s.l. group (B. cereus s.s. ATCC 14579, B. thuringiensis DSM 2046, B. wiedmannii FSL W8-0169, B. weihenstephanensis DSM 11821, and B. cytotoxicus DSM 22905), and three others including B. subtilis DSM 23778, B. licheniformis DSM 13, and B. megaterium DSM 32. B. subtilis DSM 23778 does not produce surfactin, but B. licheniformis DSM 13 does [47, 48]. E. coli DH5α was used as a negative control. Filamentous motility on C. jejuni cell lawns, 10% skim milk, and whole blood adsorbed onto 1.5% MH agar plates was observed for four out of five B. cereus species (Fig. 4). B. cytotoxicus displayed expansion on blood and milk but not on C. jejuni cell lawns (Fig. 4). B. cytotoxicus is the most phylogenetically distant member of the B. cereus s.l. group tested (Fig. 1d), so while filamentous growth appeared generally conserved, different members of the group could have different growth preferences. Filamentous expansion was not observed with B. subtilis, B. megaterium, B. licheniformis, or E. coli when incubated on lawns of C. jejuni cells, blood or milk (Fig. 4), indicating that filamentous surface motility is partially conserved in the B. cereus s.l. organisms but not in other environmental Bacillus species.

Microscopy revealed chains of elongated cells at expanding edge

Under light microscopy, filamentously expanding colonies of ML-A2C4 on plates with C. jejuni cell lawns, milk, blood, and PC showed that edges of the expanding circle were populated by very long chains of cells with minimal visible septation and/or cell division (Fig. 5 “Expansion front” column), whereas the edges of colonies on control plates were mostly populated by a single layer of rod-shaped individual cells. The elongated filamentous chains appeared oriented in all directions and undulated through multiple focal planes of the C. jejuni or RBC cell lawns versus resting on top of the cell layers or between the cells and the agar, suggesting that they might be burrowing through the cell layers. The tips of the elongated cells also appeared to extend over time with a smooth continuous forward motion, which we hypothesize was driven by growth and extension of the long filament. At the visible edge of the filamentous colony, the cell population was more dense and appeared as bundles of elongated cell chains (Fig. 5 “Ring edge” column), and in the first few millimeters of the growth ring the cells had distinct septa (“Outer ring” column). The denser, inner circle of cell growth was populated with a high proportion of sporulating cells (Fig. 5 “Dense edge” column), and the population near the center of the colony largely consisted of spores (Fig. 5 “Near center” column).

Comparable with ML-A2C4, the expanding fronts of the five other B. cereus s.l. species displaying the above-described filamentous motility also exhibited elongated cells when grown on lawns of C. jejuni, milk, and blood (Supplementary Fig. S1), with the exception of B. cytotoxicus which, as noted above, did not expand on C. jejuni cell lawns. We also examined the morphology of B. subtilis, B. licheniformis, and B. megaterium that did not exhibit filamentous motility. Unexpectedly, B. subtilis cells grown on C. jejuni and blood lawns showed elongated cells comparable with those observed for the B. cereus s.l. organisms, even though there was no characteristic colony expansion observed on those plates (Supplementary Fig. S1). B. licheniformis, B. megaterium, and E. coli cell morphology remained comparable with the control situation under all growth conditions.

Motile cells exhibited significant gene transcription differences compared with control cells

We performed RNA-Seq transcriptomic analysis using the Illumina HiSeq 2500 system on the highly motile cells from milk and PC plates to better understand putative genetic mechanisms associated with and driving filamentous growth. ML-A2C4 cells were collected from the edges (outermost 1–2 mm) of filamentous colonies on milk and PC lawn plates incubated aerobically at 30 °C for 24 h to isolate actively growing cells, and gene expression profiles were compared with those of cells collected from the edges of colonies on control MH agar plates without adsorbed milk or PC incubated under the same conditions. Principle component analysis showed a clear distinction between gene expression patterns of filamentous motility cells when compared with control cells (Fig. 6a). Filamentous cells harbored 456 genes with higher and 792 genes with lower transcription during growth on milk and/or PC, with the majority of the differentially transcribed genes common to both milk- and PC-induced growth conditions (Fig. 6b, c, Supplementary Table S4). Functional classification based on gene ontology (GO) categorization showed that filamentous motility cells differentially modulated transcription of genes involved in amino acid biosynthesis, de-novo inosine monophosphate (IMP) biosynthesis, aromatic acid biosynthesis, ion and transmembrane transport and oxidation–reduction processes (Fig. 6c, Supplementary Table S5). However, ~60% of genes could not be assigned a functional GO classification in part because this strain provided the first fully circular annotated genome for B. mobilis.

Genes exhibiting the highest dysregulation in filamentously motile versus control cells included several involved in IMP biosynthesis (including all 12 genes of the purine biosynthesis operon purEKBCSQLFMNHD) and virulence [including hblA/B/C/D, hlyR, inhA2, galU (homolog of bpsE, an exopolysaccharide gene), and plcR], which showed some of the largest increases in transcription, while genes involved in sporulation and antibiotic synthesis showed the greatest downregulation of transcription in filamentous cells (Fig. 6d). Reduced transcription of sporulation related genes, some having up to 75-fold lower transcription than in control cells, was consistent with microscopic observations that cells near the edge did not have spores (Fig. 5). At least 15 regulators were more highly expressed, and 12 had lower expression during growth on milk and PC lawns (Supplementary Table S6). Many of the regulators appeared related to sporulation (e.g., spoIIID, spo0F), virulence (e.g., plcR), antibiotic response (e.g., tipA), and metabolism (e.g., spxA, yeiL, 10045) (Fig. 6d). Genes likely involved in quorum sensing that were differentially transcribed in filamentous colonies included plcR–papR (1.7- to 2.2-fold higher), two out of three clusters of the opp oligopeptide transport system genes (oppBCDF; 2.0- to 5.2-fold higher), and autoinducer-2 (AI-2) genes (22- to 182-fold lower) (Fig. 6d, see Supplementary Table S6 for a full list) [49,50,51]. The reduction in genes responsible for AI-2 intracellular processing suggested that it might be involved in filamentous growth, but further assessment is required.

To determine if gene regulation changes we observed during filamentous motility on milk and PC were similar to differential gene expression during B. cereus s.s. swarming in semi-soft agar, we compared the microarray transcription profile of B. cereus ATCC 14579 swarming versus non-swarming conditions (published in Salvetti et al. [4]) to the RNA-Seq results obtained for ML-A2C4. ML-A2C4 possesses 86 of the reported 119 B. cereus genes showing more than a twofold difference in transcription during swarming (including hemolysin genes) (Supplementary Table S7). 21 of 86 genes (24%) showed expression patterns similar to that observed during B. cereus swarming [including hemolysin genes (hblABC), fermentation and oxidative phosphorylation genes (pflAB and alcohol dehydrogenases), and membrane transporters and stress proteins], 25 (29%) had the opposite pattern of expression [including purine, histidine, and lysine biosynthesis genes (purEBSLN, xpt, hisAFHL, and lysA), and many hypothetical proteins], and 15 (17%) did not show differences in expression during filamentous growth. While swarming B. cereus cells showed a 4.0-fold higher expression of flagellin [4], no such increase was observed in filamentously growing ML-A2C4 on either milk or PC lawns. To further assess whether filamentous motility requires flagella, we specifically looked at the ML-A2C4 44-gene flagellar locus 08515 to 08735 involved in biosynthesis and control of the flagellar apparatus. Only the cheY response regulator showed >2-fold difference in transcription during filamentous growth in milk and PC (2.7- and 2.3-fold, respectively). To confirm flagella were not required for filamentous motility, we tested B. cereus 407 cells with deletions in motA and the fla locus for surface motility. The Δfla strain does not produce flagella, and both the Δfla and the ΔmotA strains are nonmotile in a semi-soft agar swim plates [52]. The wild-type, ΔmotA, and Δfla strains showed comparable filamentous motility on C. jejuni lawns, blood, and milk, which verified that filamentous motility likely does not require the flagellar apparatus (Supplementary Fig. S2). Thus, while many similar genes appear to be controlled in both swarming and our above-described filamentous growth, the overall regulation of these metabolic and motility systems, as well as the mechanism of motility, are unique.

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