Bacillus wiedmannii sp . nov . , a psychrotolerant and cytotoxic Bacillus cereus group species isolated from dairy foods and dairy environments

A facultatively anaerobic, spore-forming Bacillus strain, FSL W8-0169, collected from raw milk stored in a silo at a dairy powder processing plant in the north-eastern USA was initially identified as a Bacillus cereus group species based on a partial sequence of the rpoB gene and 16S rRNA gene sequence. Analysis of core genome single nucleotide polymorphisms clustered this strain separately from known B. cereus group species. Pairwise average nucleotide identity BLAST values obtained for FSL W8-0169 compared to the type strains of existing B. cereus group species were <95% and predicted DNA–DNA hybridization values were <70%, suggesting that this strain represents a novel B. cereus group species. We characterized 10 additional strains with the same or closely related rpoB allelic type, by whole genome sequencing and phenotypic analyses. Phenotypic characterization identified a higher content of iso-C16 : 0 fatty acid and the combined inability to ferment sucrose or to hydrolyse arginine as the key characteristics differentiating FSL W8-0169 from other B. cereus group species. FSL W80169 is psychrotolerant, produces haemolysin BL and non-haemolytic enterotoxin, and is cytotoxic in a HeLa cell model. The name Bacillus wiedmannii sp. nov. is proposed for the novel species represented by the type strain FSL W8-0169 (=DSM 102050=LMG 29269).

Strain FSL W8-0169 T was obtained from silo raw milk collected from a dairy powder processing plant in the northeastern USA in 2012 (Watterson et al., 2014;Miller et al., 2015b).The strain was initially identified as a member of B. cereus s.l.based on analysis of a partial sequence of Abbreviations: ANIb, average nucleotide identity blast; AT, allelic type; DDH, DNA-DNA hybridization; GTR, generalized time-reversible model; HBL, haemolysin BL; MLST, multilocus sequence typing; NHE, non-haemolytic enterotoxin; PI, propidium iodide; SNP, single nucleotide polymorphism; SRA, sequence read archive; WGS, whole genome sequence.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of FSL W8-0169 T is KU198626/SRX1297474.The WGS accession number for FSL W8-0169 T is LOBC00000000, for FSL H7-0353 is LXFL00000000, for FSL H8-0032 is LXFM00000000, for FSL J3-0113 is LXFN00000000, for FSL M7-0044 is LXFO00000000, for FSL M7-0938 is LXFP00000000, for FSL M7-1251 is LXFQ00000000, for FSL P2-0415 is LXFR00000000, for FSL P2-0558 is LXFS00000000, for FSL P4-0569 is LXFT00000000 and for FSL K6-0069 LOBB00000000.the rpoB gene, which encodes the b-subunit of RNA polymerase.Strain FSL W8-0169 T has rpoB allelic type (AT) 61, which was also identified for 12 other B. cereus group dairyassociated strains deposited in the Food Microbe Tracker database (http://www.foodmicrobetracker.com).Additionally, two closely related ATs (AT 410 and AT 417) were found in the database, representing 12 and two strains, respectively.Nine strains representing rpoB AT 61, one strain representing rpoB AT 417 and one strain representing rpoB AT 194 were characterized in detail in this study (Table 1).Phenotypic, phylogenetic and whole genome sequence (WGS) data failed to classify these 11 strains into existing B. cereus group species.These 11 strains are presented as representing a novel species within the B. cereus group, for which the name Bacillus wiedmannii sp.nov. is proposed.Strain FSL W8-0169 T is the type strain of B. wiedmannii sp.nov.

Phylogenetic analyses
Sequences of the 1471 bp 16S rRNA gene (Fig. 1) and a 632 bp internal fragment of the rpoB gene (Fig. S1, available in the online Supplementary Material; Miller et al., 2015a) were aligned using the MUSCLE algorithm; pairwise distance matrices were calculated in MEGA version 6.06 (Tamura et al., 2013).Maximum-likelihood phylogenetic trees were reconstructed using RaxML version 8.2.3 (Stamatakis, 2014) under the Generalized Time Reversible (GTR) model with gamma and invariant site parameters (GTRGAMMAI) and 1000 bootstrap repetitions.The 16S rRNA gene sequence of B. wiedmannii sp.nov.strain FSL W8-0169 T was checked for the presence of chimeras using DECIPHER (Wright et al., 2012) and submitted to GenBank (accession number listed in Table S1).The 16S rRNA gene sequence phylogeny (Fig. 1) supports the close relatedness of B. wiedmannii sp.nov.with existing members of the B. cereus group, as indicated by the !98.2 % sequence similarity and high bootstrap values.It is known that B. cereus group species cannot be delineated based on 16S rRNA gene sequences (Liu et al., 2015), and therefore an rpoB gene phylogeny was reconstructed to allow for a more discriminatory analysis.
Based on the rpoB gene sequence, the 11 B. wiedmannii sp.nov.strains characterized formed a monophyletic, wellsupported (bootstrap value of 97) cluster within the B. cereus group (Fig. S1).The rpoB gene sequences of all 11 B. wiedmannii sp.nov.strains were deposited in the Food Microbe Tracker database where they can be found under strain name records.
Genomes of FSL W8-0169 T and 10 other characterized B. wiedmannii sp.nov.isolates were sequenced on an Illumina MiSeq or HiSeq platform, respectively.The Nextera XT adapters were trimmed from 250 or 100 bp paired-end reads with Trimmomatic version 0.32, respectively (Bolger et al., 2014).Quality of reads was assessed using FastQC version 0.11.2.Sequences were assembled de novo using SPAdes version 3.0.0or 3.6.2,respectively (Bankevich et al., 2012).Draft genome quality was  verified by QUAST version 3.2 (Gurevich et al., 2013) and sufficient coverage was confirmed using SAMtools version 1.2 (Li et al., 2009).Short reads were submitted to the Sequence Read Archive (SRA) and draft genome assemblies to NCBI GenBank and Prokaryotic Genome Annotation Pipeline (Angiuoli et al., 2008).Assembled draft genomes ranged from 5.3 to 5.6 Mbp (Table 1).
The NCBI accession numbers for WGS and draft genome assembly statistics are listed in Table 1.
Four novel STs were identified as a result of finding a novel AT and four novel STs were identified as a result of new combinations of existing ATs (Table 1).Strains FSL J3-0113 and FSL P2-0558 carried STs that were previously observed in the PubMLST database.S2; Richter & Rosselló-Móra, 2009), with the exception of FSL K6-0069, which was classified as a borderline member of B. wiedmannii sp.nov., as it had a predicted DDH of 57.9 %, and with a 45.24 % probability of DDH being 70 % or above (Table S2).This strain was deposited with the DSMZ (DSM 102051) and BCCM (LMG 29270).
To further confirm B. wiedmannii sp.nov.as a novel taxon, the core genome single nucleotide polymorphisms (SNPs) were identified with kSNP v2 (Gardner & Hall, 2013) using a kmer size of 21 as identified by Kchooser and used to reconstruct a maximum-likelihood phylogenetic tree (Fig. 3).

Phenotypic characteristics
Phenotypic characterizations were performed for B. wiedmannii sp.nov.FSL W8-0169 T and the 10 additional B. wiedmannii sp.nov.strains (Table S3).Microscopic evaluation of FSL W8-0169 T revealed cells were rod-shaped with an average length of 2.8 µm and average width of 1.2 µm.Transmission electron microscopy (negative staining with 2 % uranyl acetate) images revealed spores in the centre of the vegetative cell (Fig. 4).Colonies grown on brain heart infusion medium (BHI, Becton Dickinson) were large, round and off-white.B. wiedmannii sp.nov.strain FSL W8-0169 T and the 10 additional strains were haemolytic on sheep's blood trypticase soy agar, were Gram-stain-positive and were positive for catalase activity, as determined according to the FDA BAM protocols (U.S. Food and Drug Administration, 2015).All strains were oxidase-negative (BBL Dry Slide Oxidase; Becton Dickinson).All strains hydrolysed starch and casein, as determined by plating on starch agar and skimmed milk agar using standard methods (Logan & De Vos, 2009).All strains were facultative anaerobes, as   64) + ( 38) determined by the BAM method (U.S. Food and Drug Administration, 2015).All tested strains were motile at 30 C, with the exception of one strain (FSL H7-0353), as determined by observing growth outside of the inoculation stab on motility agar.
The range of growth temperatures was assessed by plating overnight cultures onto BHI agar (Becton Dickinson) plates and subsequently incubating plates at 5, 10, 15, 20, 25, 30, 40, 45 and 55 C, for incubation periods as defined by Logan & De Vos (2009).The minimum growth temperature for five out of 11 B. wiedmannii sp.nov.strains was 5 C and all strains grew between 10 and 40 C. Six out of 11 strains were able to grow at 43 C. None of the strains grew at 45 C or above.Sodium tolerance was assessed by inoculating strains into tryptic soy broth (TSB) containing 2, 5, 7, 8, 9 or 10 % (w/v) NaCl, followed by incubation at 30 C for up to 14 days.Growth of seven out of 11 tested strains was observed at NaCl concentrations up to 5 % (w/v) (for four strains growth was inhibited at concentrations >2 % NaCl), but all strains were inhibited by NaCl concentrations of 7-10 % (w/v).Growth in pH-adjusted TSB incubated at 30 C for 14 days demonstrated that all strains were capable of growing at pH 5-10.
API CH 50 kits were used to characterize acid production from catabolism of carbohydrates according to the manufacturer's instructions (bioM erieux); incubation was performed at 30 C for 48 h (Table 2).Of the 11 strains tested, all were able to utilize (acid production from catabolism) D-ribose, D-glucose, D-fructose, arbutin, aesculin ferric citrate, salicin, cellobiose, maltose, trehalose, amidon and glycogen.Acid production from D-mannose (positive for one out of 11 strains), amygdalin (positive for three out of 11 strains), potassium gluconate (positive for two out of 11 strains) and N-acetylglucosamine (positive for 10 out of 11 strains) was variable.All 11 B. wiedmannii sp.nov.strains were also tested with API 20E kits, which were used as per the manufacturer's instructions; incubation was performed at 30 C for 48 h (see  containing 5 µg PI ml À1 and incubated for 30 min at 37 C with 5 % CO 2 .HeLa cells were fixed with 4 % paraformaldehyde (PFA) at room temperature for 10 min.Cells were permeabilized with 1 % Triton X-100 and incubated with 4¢,6-diamidino-2-phenylindole (DAPI; 1 µg ml À1 ) at room temperature for 1-5 min, prior to fixing coverslips and gluing onto microscope slides.Slides were imaged using a Zeiss 710 confocal microscope (Bio-imaging Facility, Cornell University).Images were processed using FIJI software (Schindelin et al., 2012).Bacterial strains were considered cytotoxic if the proportion of PI-positive cells was greater than the average proportion of PI-positive cells for the BHI-negative control.FSL W8-0169 T and all additional strains were cytotoxic (Fig. 5).
Description of Bacillus wiedmannii sp.nov.
Bacillus wiedmannii (wied.mann¢i.i;N.L. gen.masc.n. wiedmannii named in honour of Martin Wiedmann, for his contribution to the understanding of the biology of Bacillus).The type strain, FSL W8-0169 T (=DSM 102050 T =LMG 29269 T ), was isolated from a silo raw milk sample collected from a dairy powder processing plant in the north-eastern USA.The genomic DNA G+C content of the type strain is 35.3 mol%, as determined by genome sequencing.

FSLFig. 3 .Fig. 4 .
Fig. 3. Maximum-likelihood tree reconstructed in RaxML based on core SNPs using the GTR model with gamma distributed sites and 1000 bootstrap repetitions, rooted by mid-point.Bootstrap values above !70 are displayed on branches.Bar, 0.2 substitutions per site.The representative strains of B. wiedmannii sp.nov.are shown in bold type.

FSLFig. 5 .
Fig.5.Cytotoxicity of B. wiedmannii sp.nov.FSL W8-0169 T supernatants.HeLa cells grown on coverslips were co-incubated with 5 % (v/v) culture supernatants of FSL W8-0169 T and B. cereus ATCC 14579 T (included as a positive control), for 30min with 5 µg PI ml À1 (red).After fixation with 4 % PFA, HeLa cells were stained with 1 µg DAPI ml À1 (blue).All cells stained blue with DAPI, while only cells with compromised membrane integrity stained red.Incubation with BHI media was included as a negative control.The merged images demonstrate the proportion of cells with damaged membrane due to cytotoxic activity of bacterial supernatant.

Table 1 .
Characteristics of the 11 Bacillus wiedmannii sp.nov.strains characterized in this study (Richter & Rosselló-Móra, 2009)ST identities (ANIb) between the genomes for the 11 B. wiedmannii sp.nov.strains,representativestrains of the other eight validly published and three effectively published B. cereus group species were calculated (Fig.2;Richter & Rosselló-Móra, 2009).The representative strains of other B. cereus group species were type strains, except in the case of B. anthracis where the WGS was not available for the type strain; therefore, the WGS data for the widely used B. anthracis strain Ames were used.ANIb was computed using the calculate_ani.pyprogram,which is available on Github (https://github.com/widdowquinn/scripts/blob/master/bioinformatics/calculate_ani.py).A cladogram (Fig.2) was built based on the pairwise ANIb similarity matrix using the 'hclust' package in R (R Core Team, 2014).Using a 95 % cut-off for species delineation, these analyses confirmed B. wiedmannii sp.nov.as a novel species(Richter & Rosselló-Móra, 2009).The B. cereus group species closest to B. wiedmannii sp.nov.FSL W8-0169 T is B. anthracis (strain Ames with an ANIb of 93.62); the species most distant is B. cytotoxicus (strain NVH 391-98 T with an ANIb of 82.73).The minimum pairwise ANIb value between B. wiedmannii sp.nov.type strain FSL W8-0169 T and 10 other strains representative of this species was 95.92 %, confidently classifying them in the same species.
de/distcalc2.php;Meier-Kolthoffet al., 2013).Predicted pairwise DDH values between B. wiedmannii sp.nov.FSL W8-0169 T and representative strains of other B. cereus group species were substantially below the species cut-off of 70 % (Table (Guinebreti ere et al., 2010)strainFSL W8-0169 T and 10 other representatives of this species formed a monophyletic, robust clade with a bootstrap value of 99.Overall, comparative ANIb and DDH analyses, as well as WGS phylogenetic analysis provide strong evidence in favour of recognizing B. wiedmannii sp.nov.as a novel species in the B. cereus group.To examine whether strains belonging to B. wiedmannii sp.nov.werefound in previous studies, we have classified the 11 B. wiedmannii sp.nov.strainsintopreviously defined phylogenetic groups(Guinebreti ere et al., 2010).We extracted panC gene sequences from the WGS and used them for phylogenetic classification with an online tool (Guinebreti ere et al., 2010; https://www.tools.symprevius.org/bcereus/english.php).All 11 strains were affiliated to phylogenetic group II, which is known for psychrotol- (Guinebreti ere et al., 2010) 2008)and cytotoxicity(Guinebreti ere et al., 2010).The combination of these two characteristics sets phylogenetic group II apart from other phylogenetic groups within the B. cereus group.This, together with genomic evidence, supports the novel species delineation.

Table 2
Utilization of citrate as a carbon source was variable (six out of 11 strains were able to utilize citrate).All other reactions included in the API 20E kit were negative (Table2).Nitrate reduction was determined according to the FDA BAM method (U.S. Food and Drug Administration, 2015) and was variable.Strain FSL W8-0169 T and seven out of the other 10 strains were able to reduce nitrate
NR International Journal of Systematic and Evolutionary Microbiology 66 Downloaded from www.microbiologyresearch.orgby IP: 54.70.40.11On: Sun, 25 Nov 2018 01:29:44 T and seven additional strains.For three strains (FSL P4-0569, FSL K6-0069 and FSL J3-0113), HBL was not detected, but NHE was.Cytotoxicity was determined by measuring the influx of propidium iodide (PI) into HeLa cells exposed to supernatants (5 %, v/v) of B. wiedmannii sp.nov.cultures grown in BHI at 32 C for 20 h.B. wiedmannii sp.nov.supernatants were added to HeLa cells maintained in Eagle's minimal essential medium (EMEM) Cells are Gram-stain-positive rods with an average length of 2.8 µm and average width of 1.2 µm.Endospores are ellipsoidal and are present in the centre of vegetative cells; cells have a non-swollen sporangia.Colonies are positive for egg-yolk lecithinase, with the ability to hydrolyse casein and starch.Facultative anaerobe.Colonies grown on BHI agar at 37 C for 24 h appear creamcoloured, round and flat, with a rough surface.Growth temperature ranges from 5 and 43 C, with optimum growth between 20 and 40 C. Most strains are motile at 30 C. Citrate utilization and acid production from potassium gluconate and mannose are variable.Can be differentiated from other strains in the B. cereus group by the combination of an inability to produce acid from fermentation of sucrose and inability to hydrolyse arginine (arginine dihydrolase-negative).Produces toxins HBL and the non-haemolytic toxin NHE.Cells are cytotoxic in a HeLa cell culture model.Strains can be differentiated from other B. cereus group species by panC gene sequence comparisons, ANIb and WGS.