Geographic map of Poland with locations of soil sampling.

<p>Soil samples were collected in Białowieża National Park (Białowieża NP; N 52°42′, E 23°54′), the natural forest with no human activity, in marshes of Biebrza National Park (Biebrza NP; N 53°36′, E 22°56′) with limited human activity, and a farmland in Jasienowka (N 52°30′, E 22°58′) in northeastern Poland.</p

Genetic diversity in the seven loci within three <i>B. cereus sensu lato</i> populations from northeastern Poland.

a<p><i>B.c.</i>, <i>Bacillus cereus s.s.</i>; <i>B.t.</i>, <i>Bacillus thuringiensis</i>; <i>B.m.</i>, <i>Bacillus mycoides</i>.</p>b<p>ST, sequencing type; number of new STs described in this study is given in the parentheses.</p>c<p>Number of new alleles described in this study are given in the parenthesis.</p>d<p>Ratio of nonsynonymous (<i>d</i>_N) to synonymous (<i>d</i>_S) substitutions per nucleotide site where <i>d</i>N/<i>d</i>S<1 indicates that the loci is subjected to purifying selection.</p

Genetic diversity in housekeeping genes within <i>B. cereus s.l.</i> originated from three locations in northeastern Poland.

<p>The percentage of the polymorphic sites was much higher in <i>B. cereus</i> and <i>B. thuringiensis</i> than in <i>B. mycoides</i> originating from Białowieża National Park (A) and Biebrza National Park (B). Contradictory for bacteria isolated from the farm soil (C), the percentage of the polymorphic sites was similar among bacilli classified to the three species.</p

geoBURST analysis showing the clonal assignment of STs present in <i>B. cereus s.l.</i>

<p>Bacteria originated from Białowieża National Park, Biebrza National Park, and the Jasienowka farm in northeastern Poland. The CCs are named based on the ST assigned as a founder genotype (marked with a star) of the complex. The relative size of the circles indicates their prevalence among the <i>B. cereus s.l.</i> isolates. New STs characterized in this study are accentuated by a green halo, while STs present in the MLST database are accentuated by a blue halo.</p

NJ phylogenic trees of the <i>B. cereus s.l.</i> environmental isolates originated from three locations in northeastern Poland and 28 reference strains.

<p>The trees prepared separately for each population were constructed as given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080175#pone-0080175-g005" target="_blank">Figure 5</a>. The isolates originated from Białowieża and Biebrza National Parks pertained to three clades, while those isolated from the farm samples clustered to four clades, with only two isolates in clade IV containing <i>B. pseudomycoides</i> reference strains. Detailed information on the isolates and the reference strains used in phylogenetic trees are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080175#pone.0080175.s003" target="_blank">Table S3</a>. Yellow box indicates the <i>cytK</i> positive isolates. Red asterisk indicates the isolates that express the <i>cytK</i> gene in Real-Time PCR analysis. Blue asterisk indicates isolates able to grow at 7°C. <i>B.c.</i>, <i>B. cereus s.s.</i>; <i>B.t.</i>, <i>B. thuringiensis</i>; <i>B.m.</i>, <i>B. mycoides</i>, <i>B.a.</i>, <i>B. anthracis</i>, <i>B.w.</i>, <i>B. weihenstephanensis</i>.</p

geoBURST analysis showing the clonal assignment of the STs present in soil isolates of <i>B. cereus</i>, <i>B. thuringiensis</i>, and <i>B. mycoides</i> originating from northeastern Poland.

<p>The CCs are named based on the ST assigned as a founder genotype (marked with a star) of the complex. The relative size of the circles indicates their prevalence among the <i>B. cereus s.l.</i> isolates. New STs characterized in this study are accentuated by a green halo, while STs present in the MLST database are accentuated by a blue halo.</p

Soil types, chemical properties and density of <i>B. cereus sensu lato</i> in the samples.

a<p>M, mineral soil; O, organic soil;</p>b<p>soil organic matter;</p>c<p>humic substances;</p>d<p>given as an average CFU per gram of soil.</p

Phylogenetic tree of 273 <i>B. cereus s.l.</i> environmental isolates from northeastern Poland.

<p>The tree was constructed based on seven concatenated housekeeping loci (<i>glpF</i>, <i>gmk</i>, <i>ilvD</i>, <i>pta</i>, <i>pur</i>, <i>pycA</i>, and <i>tpi</i>) representing a total of 2,829 nucleotides, using the Neighbor-Joining (NJ) method implemented in MEGA4 software. Branch quality was evaluated using 1,000 replicates bootstraps <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080175#pone.0080175-Tamura1" target="_blank">[44]</a>. The <i>B. cereus s.l.</i> isolates originated from northeastern Poland were separated into four major clades containing 10.6% (Clade I), 14.7% (Clade II), 74.0% (Clade III), and 0.7% (Clade IV) of environmental isolates. The pathogenic <i>B. cereus s.l.</i> references were clustered mostly with the minority of the environmental isolates of the clades I and II, whereas the environmental references were grouped together with the bacilli isolated in this study classified as the clade III.</p

Eco-Genetic Structure of <i>Bacillus cereus sensu lato</i> Populations from Different Environments in Northeastern Poland

<div><p>The <i>Bacillus cereus</i> group, which includes entomopathogens and etiologic agents of foodborne illness or anthrax, persists in various environments. The basis of their ecological diversification remains largely undescribed. Here we present the genetic structure and phylogeny of 273 soil <i>B. cereus s.l.</i> isolates from diverse habitats in northeastern Poland, with samplings acquired from the last European natural forest (Białowieża National Park), the largest marshes in Europe (Biebrza National Park), and a farm. In multi-locus sequence typing (MLST), despite negative selection in seven housekeeping loci, the isolates exhibited high genetic diversity (325 alleles), mostly resulting from mutation events, and represented 148 sequencing types (131 STs new and 17 STs already described) grouped into 19 complexes corresponding with bacterial clones, and 80 singletons. Phylogenetic analyses showed that 74% of the isolates clustered with <i>B. cereus s.l.</i> environmental references (clade III), while only 11 and 15%, respectively, grouped with isolates of clinical origin (clade I), and <i>B. cereus</i> ATCC 14579 and reference <i>B. thuringiensis</i> (clade II). Predominantly within clade III, we found lineages adapted to low temperature (thermal ecotypes), while putative toxigenic isolates (<i>cytK</i>-positive) were scattered in all clades of the marsh and farm samplings. The occurrence of 92% of STs in bacilli originating from one habitat, and the description of new STs for 78% of the isolates, strongly indicate the existence of specific genotypes within the natural <i>B. cereus s.l.</i> populations. In contrast to the human-associated <i>B. cereus s.l.</i> that exhibit a significant level of similarity, the environmental isolates appear more complex. Thus we propose dividing <i>B. cereus s.l.</i> into two groups, the first including environmental isolates, and the second covering those that are of clinical relevance.</p></div

Melanin-like pigment production in Luria-Bertoni broth by <i>B</i>. <i>weihenstephanensis</i>.

<p><i>B</i>. <i>cereus</i> ATCC 10987 was used as the negative control.</p