Bacillus Cereus Group Research Articles

Biocontrol potential of phage Deep-Blue against psychrotolerant Bacillus weihenstephanensis

Some strains of the Bacillus cereus group can be implicated in two types of foodborne intoxication syndromes, namely diarrheal and emetic. The emetic syndrome results from the ingestion of the cereulide toxin produced by particular strains of B. cereus and Bacillus weihenstephanensis. It represents a major concern since it can lead, in severe cases, to the death of the intoxicated patients. This study aimed to characterize the virulent phage Deep-Blue and investigate its biocontrol potential against psychrotolerant B. weihenstephanensis. This myovirus was isolated from an agricultural soil and is stable for at least 6 months at 4 °C. It is specific to members of the B. cereus group and shows a lytic activity against five of twelve B. weihenstephanensis. Its lytic cycle lasts less than 1 h with a final phage progeny of ca. 300 virions. Regarding its bactericidal potential, Deep-Blue was able to reduce B. weihenstephanensis contamination under laboratory conditions (LB broth) and in milk, and to control bacterial growth in food matrices such as milk rice. Finally, the application of Deep-Blue as preventive treatment was shown to efficiently control the development of B. weihenstephanensis biofilm.

Rapid and reasonable molecular identification of bacteria and fungi in microbiological diagnostics using rapid real-time PCR and Sanger sequencing

Analyses of short subunit gene sequences have been established for taxonomic classification and identification of bacteria and fungi. To produce partial bacterial ribosomal 16S rRNA and rpoB and fungal ribosomal ITS/LSU gene sequences for DNA sequencing, real-time PCR assays supplemented with the nucleic acid stain SYBR Green were created. Generation of PCR products was monitored based on amplification and melting curves. The PCR products were subsequently subjected to Sanger sequencing on demand for identification of bacteria and fungi in routine microbiological diagnostics within a period of two days.From a total of 78 bacterial isolates 40 (51%) or 67 (86%) could be identified at species level using only partial 16S rRNA or additionally rpoB gene sequences based on BLASTN (NCBI) database queries, respectively. Using partial 16S rRNA and rpoB gene sequencing unambiguous assignment was not possible for the closely related species of the Bacillus (B.) cereus group, Bordetella (B.) pertussis/ B. parapertussis/ B. bronchiseptica, Brucella spp., Enterobacter cloacae complex, Escherichia/ Shigella spp., Staphylococcus (S.) hyicus/ S. agnetis and Yersinia (Y.) pseudotuberculosis/ Y. pestis. However, partial rpoB gene sequencing succeeded in identifying 27 bacterial isolates at species level in addition to 16S rRNA gene sequencing.Regarding ITS/LSU gene sequencing, best results could be achieved by ITS gene sequencing followed by LSU gene sequencing, resulting in 32 (63%) and 21 (43%) of a total of 51 fungal isolates that could be identified at species level, respectively. Insufficient identification at species level was observed for the genera Apiotrichum, Aspergillus, Cladosporium, Cryptococcus, Microsporum, Nannizziopsis, Penicillium, Trichosporon, and Tolypocladium included in this study.The concept of this procedure is suitable for rapid and reasonable molecular identification of bacteria and fungi within two days and is therefore applicable in routine microbiological diagnostic laboratories.

PXO16, the large conjugative plasmid from Bacillus thuringiensis serovar israelensis displays an extended host spectrum

pXO16, the large conjugative plasmid from Bacillus thuringiensis serovar israelensis is able to efficient self-transfer, to mobilize and retro-mobilize non-conjugative plasmids, including “non-mobilizable” plasmids, and to transfer chromosomal loci. It also displays a remarkable aggregation phenotype associated with conjugation under liquid conditions. However, it was recently shown that aggregation boosts pXO16 transfer but is not mandatory. In this paper, we have further explored pXO16 transfers under various mating conditions and with different members of the Bacillus cereus group. The results indicated that colony or filter mating largely compensate the transfer deficit observed when using a pXO16 aggregation-minus mutant. Using filter mating, pXO16 transfer efficiency and host range were both improved. For instance, pXO16 was shown to transfer itself, and to mobilize the small pUB110 plasmid, from B. thuringiensis serovar israelensis to the thermotolerant Bacillus cytotoxicus at frequencies of 3.3 × 10−3 and 5.2 × 10−4 transconjugants per donor (T/D), respectively. All together, these results indicate that pXO16 can potentially “circulate” among members of the Bacillus cereus group. Yet, this is contrasting with pXO16's known natural distribution, which is apparently limited to the israelensis serovar of B. thuringiensis.

Whole-genome-based phylogeny of Bacillus cytotoxicus reveals different clades within the species and provides clues on ecology and evolution

Bacillus cytotoxicus is a member of the Bacillus cereus group linked to fatal cases of diarrheal disease. Information on B. cytotoxicus is very limited; in particular comprehensive genomic data is lacking. Thus, we applied a genomic approach to characterize B. cytotoxicus and decipher its population structure. To this end, complete genomes of ten B. cytotoxicus were sequenced and compared to the four publicly available full B. cytotoxicus genomes and genomes of other B. cereus group members. Average nucleotide identity, core genome, and pan genome clustering resulted in clear distinction of B. cytotoxicus strains from other strains of the B. cereus group. Genomic content analyses showed that a hydroxyphenylalanine operon is present in B. cytotoxicus, but absent in all other members of the B. cereus group. It enables degradation of aromatic compounds to succinate and pyruvate and was likely acquired from another Bacillus species. It allows for utilization of tyrosine and might have given a B. cytotoxicus ancestor an evolutionary advantage resulting in species differentiation. Plasmid content showed that B. cytotoxicus is flexible in exchanging genes, allowing for quick adaptation to the environment. Genome-based phylogenetic analyses divided the B. cytotoxicus strains into four clades that also differed in virulence gene content.

Characterization of Emetic and Diarrheal Bacillus cereus Strains From a 2016 Foodborne Outbreak Using Whole-Genome Sequencing: Addressing the Microbiological, Epidemiological, and Bioinformatic Challenges.

The Bacillus cereus group comprises multiple species capable of causing emetic or diarrheal foodborne illness. Despite being responsible for tens of thousands of illnesses each year in the U.S. alone, whole-genome sequencing (WGS) is not yet routinely employed to characterize B. cereus group isolates from foodborne outbreaks. Here, we describe the first WGS-based characterization of isolates linked to an outbreak caused by members of the B. cereus group. In conjunction with a 2016 outbreak traced to a supplier of refried beans served by a fast food restaurant chain in upstate New York, a total of 33 B. cereus group isolates were obtained from human cases (n = 7) and food samples (n = 26). Emetic (n = 30) and diarrheal (n = 3) isolates were most closely related to B. paranthracis (group III) and B. cereus sensu stricto (group IV), respectively. WGS indicated that the 30 emetic isolates (24 and 6 from food and humans, respectively) were closely related and formed a well-supported clade distinct from publicly available emetic group III genomes with an identical sequence type (ST 26). The 30 emetic group III isolates from this outbreak differed from each other by a mean of 8.3 to 11.9 core single nucleotide polymorphisms (SNPs), while differing from publicly available emetic group III ST 26 B. cereus group genomes by a mean of 301.7–528.0 core SNPs, depending on the SNP calling methodology used. Using a WST-1 cell proliferation assay, the strains isolated from this outbreak had only mild detrimental effects on HeLa cell metabolic activity compared to reference diarrheal strain B. cereus ATCC 14579. We hypothesize that the outbreak was a single source outbreak caused by emetic group III B. cereus belonging to the B. paranthracis species, although food samples were not tested for presence of the emetic toxin cereulide. In addition to showcasing how WGS can be used to characterize B. cereus group strains linked to a foodborne outbreak, we also discuss potential microbiological and epidemiological challenges presented by B. cereus group outbreaks, and we offer recommendations for analyzing WGS data from the isolates associated with them.

Whole genome sequence of Bacillus thuringiensis ATCC 10792 and improved discrimination of Bacillus thuringiensis from Bacillus cereus group based on novel biomarkers

BackgroundAmong the Bacillus cereus group, B. thuringiensis, is one of the most extensively used biological control agent. The present study reports the complete genome and four novel plasmid analysis of the type strain B. thuringiensis ATCC 10792. MethodsComplete genome sequencing of Bacillus thuringiensis ATCC 10792, assembled using de-novo (v.3.2.0, assembly name MIRA3), Pac-Bio sequencers and Hierarchical Genome Assembly Process software (version 4.1) and real-time polymerase chain reaction (qPCR) is a consistent technique for quantifying gene expression based on specific biomarkers, in addition the efficiency of the primers were analysed based on artificially spiked food samples on lettuce, kimbab and spinach with B. thuringiensis ATCC 10792. ResultsComplete genome annotation was performed, and a total of 6269 proteins with 5427594 bps were identified and four novel plasmid (poh2, poh3, poh4, poh5) a total of 134, 131, 96, 21 proteins with 113294; 92,949; 86488; 11332 bps were identified. Six selective genes (lipoprotein-lipo, methyltransferase-MT, S-layer homology domain protein-BC, flagellar motor protein-motB, transcriptional regulator-XRE, crystal protein-cry2) and associated four novel plasmids were investigated along with the characteristics and expression profiles of two housekeeping genes (chaperonin protein-GroEL and topoisomerase enzyme-gyrB). Although from the assessment of 120 strains, both GroEL and gyrB showed 100% specificity towards detection of both B. thuringiensis in artificially spiked vegetable samples. All the eight genes revealed no specificity towards any of the 9 non- Bacillus strains. ConclusionIn our study based on the complete genome and plasmid sequence of B. thuringiensis ATCC 10792, among the six discriminating genes, specifically GroEL, gyrB and XRE showed promising results in identifying B. thuringiensis ATCC 10792, and there detection limit was 3.0–9.6 log CFU/g in the food samples respectfully. The key role in control of the predatory biological agent.

Bacillus cereus biofilm formation on central venous catheters of hospitalised cardiac patients

Formation of bacterial biofilms is a risk with many in situ medical devices. Biofilm-forming Bacillus species are associated with potentially life-threatening catheter-related blood stream infections in immunocompromised patients. Here, bacteria were isolated from biofilm-like structures within the lumen of central venous catheters (CVCs) from two patients admitted to cardiac hospital wards. Isolates belonged to the Bacillus cereus group, exhibited strong biofilm formation propensity, and mapped phylogenetically close to the B. cereus emetic cluster. Together, whole genome sequencing and quantitative PCR confirmed that the isolates constituted the same strain and possessed a range of genes important for and up-regulated during biofilm formation. Antimicrobial susceptibility testing demonstrated resistance to trimethoprim-sulphamethoxazole, clindamycin, penicillin and ampicillin. Inspection of the genome revealed several chromosomal β-lactamase genes and a sulphonamide resistant variant of folP. This study clearly shows that B. cereus persisting in hospital ward environments may constitute a risk factor from repeated contamination of CVCs.

Identification of cereulide producing Bacillus cereus by MALDI-TOF MS

The Bacillus (B.) cereus group consists of nine recognized species which are present worldwide. B. cereus play an important role in food-borne diseases by producing different toxins. Yet, only a small percentage of B. cereus strains are able to produce the heat stable cereulide, the causative agent of emetic food poisoning. To minimize the entry of emetic B. cereus into the food chain, food business operators are dependent on efficient and reliable methods enabling the differentiation between emetic and non-emetic strains.Currently, only time-consuming cell bioassays, molecular methods and tandem mass spectrometry are available for this purpose. Thus, the aim of the present study was to establish a fast and reliable method for the differentiation between emetic/non-emetic strains by MALDI-TOF MS. Selected strains/isolates of the B. cereus group as well as other Bacillus spp. (total n = 121) were cultured on sheep blood agar for 48 h before analysis.Subsequently, the cultures were directly analyzed by MALDI-TOF MS without prior extraction steps. The samples were measured in the mass range of m/z 800–1800 Da. Using ClinProTools 3.0 statistical software and Flex analysis software (Bruker Daltonics GmbH, Bremen, Germany), a differentiation between emetic/non-emetic isolates was possible with a rate of correct identification of 99.1% by means of the evaluation of two specific biomarkers (m/z 1171 and 1187 Da).

Characteristics of Potential Gamma-Aminobutyric Acid-Producing Bacteria Isolated from Korean and Vietnamese Fermented Fish Products.

Gamma-aminobutyric acid (GABA) is a neurotransmitter that exerts several physiological functions and positive effects on human health. The aim of this study was to isolate and characterize the strains that had GABA-producing abilities from various fermented fish products. A total of 91 acid-producing strains were isolated from 41 samples of fermented fish products, and 27 strains showing GABA-producing abilities were identified by the 16S rDNA sequences. Among the strains, 31% strains tolerated at high-salt environment of 10-20% throughout the fermentation of fish sauces. The 27 isolates that produced GABA at various concentrations did so in the range of 5 to 454 mM. These GABA-producing isolates were identified as lactic acid bacteria of 14 strains, which included twelve Lactococcus lactis, one Enterococcus faecium, and one Lactococcus pentosus; eight Bacillus cereus group, which included seven B. thuringiensis and one B. cereus; and five Staphylococcus spp. Interestingly, with Vietnamese fish sauces, we mostly identified species of B. thuringiensis and Staphylococcus spp., while with Korean fermented fish products, the majority of the strains identified belonged to L. lactis. Among the strains, B. thuringiensis LH2134 produced the highest levels of GABA at 366 mM among the strains identified from Vietnamese fish sauces, whereas L. lactis LA43, a new strain isolated from Korean jeotgal (salted shrimp paste), produced the highest amount of GABA at 454 mM and the glutamate concentration in the medium was essential for GABA accumulation. Therefore, such the isolates might serve as good starters for development of more GABA-reinforced foods among fermented fish products.

Comprehensive proteomic analysis of exoproteins expressed by ERIC I, II, III and IV Paenibacillus larvae genotypes reveals a wide range of virulence factors

ABSTRACTAmerican foulbrood is a quarantine disease of the honeybee Apis mellifera L. in many countries and contributes greatly to colony losses. We performed a label-free proteomics study of exoprotein fractions produced in vitro by Paenibacillus larvae reference strains of the ERIC I–IV genotypes. A quantitative comparison was performed of previous studied protein-based virulence factors and many newly identified putative virulence factors. Among the multiple proteases identified, key virulence factors included the microbial collagenase ColA and immune inhibitor A (InhA, an analog of the Bacillus thuringiensis protein InhA). Both of these virulence factors were detected in ERICs II–IV but were absent from ERIC I. Furthermore, the different S-layer proteins and polysaccharide deacetylases prevailed in ERICs II–IV. Thus, the expression patterns of these virulence factors corresponded with the different speeds at which honeybee larvae are known to be killed by ERICs II–IV compared to ERIC I. In addition, putative novel toxin-like proteins were identified, including vegetative insecticidal protein Vip1, a mosquitocidal toxin, and epsilon-toxin type B, which exhibit similarity to homologs present in Bacillus thuringiensis or Lysinibacillus sphaericus. Furthermore, a putative bacteriocin similar to Lactococcin 972 was identified in all assayed genotypes. It appears that P. larvae shares virulence factors similar to those of the Bacillus cereus group. Overall, the results provide novel information regarding P. larvae virulence potential, and a comprehensive exoprotein comparison of all four ERICs was performed for the first time. The identification of novel virulence factors can explain differences in the virulence of isolates.

Isolation and Characterization of A Bacillus Phage from Equine Carcass Disposal Site

Bacteriophages play an important role in bacterial control in natural niche however a little is known about Bacillus sp. phages prevailing in cadaver affected soils. In the current study, the Bacillus sp. phage was isolated from the equine cadaver disposal site and characterised to gain an insight into the issue of role of phages in biological dynamics of manure thus formed over years. Firstly, the host bacterium was isolated and identified as Bacillus cereus group member as assessed by phylogenetic analysis and secondly it’s corresponding phage from same soil sample was also enriched and characterised. The phage (VTCCBPA38) was found to belong to family Myoviridae and was active within the temperature range of 4°C - 45°C. As assessed by biological sensitivity by spot test, the phage was active against 6/19 (31.6 %) Bacilli tested including Bacillus cereus from goat mastitis. Thus the phagemay find potential use in biocontrol of diseases caused by Bacillus sp. Furthermore, this report is valuable as the first study for investigation of Bacillus sp. phage in carcass burial sites.

Bacillus species at the Canberra Airport: A comparison of real-time polymerase chain reaction and massively parallel sequencing for identification

Anthrax, caused by the Gram-positive, spore forming bacterium Bacillus anthracis, is a disease with naturally occurring outbreaks in many parts of the world, primarily in domestic and wild herbivores. Due to the movement of people and stock, B. anthracis could, however, be at transportation hubs including airports. The continuous threat to national and international security from a biological agent release, or hoax attack, is a very real concern. Sensitive, robust and rapid (hours-day) methods to identify biological agents, including B. anthracis, and distinguish pathogenic from non-pathogenic species, is an essential cornerstone to national security. The aim of this project was to determine the presence of Bacillus species at the Canberra Airport using two massively parallel sequencing (MPS) approaches and compare with previous results using real-time polymerase chain reaction (qPCR).Samples were collected daily for seven days each month from August 2011–July 2012 targeting movement of people, luggage and freight into and out of the Canberra Airport. Extracted DNA was analysed using qPCR specific for B. anthracis. A subset of samples was analysed using two MPS approaches. Approach one, using the Ion PGM™ (Thermo Fisher Scientific; TFS) and an in-house assay, targeted the two B. anthracis virulence plasmids (cya and capB genes) and a single conserved region of the 16S rRNA gene. Approach two, using the Ion S5™ (TFS) and the commercial Ion 16S™ Metagenomics Kit (TFS), targeted multiple regions within the bacterial 16S rRNA gene.Overall there was consistency between the two MPS approaches and between MPS and qPCR, however, MPS was more sensitive, particularly for plasmid detection. Whilst the broad-range 16S genomic target(s) used in both MPS approaches in this study was able to generate a metagenomic fingerprint of the bacterial community at the Canberra Airport, it could not resolve Bacillus species beyond the level of the Bacillus cereus group. The inclusion of B. anthracis virulence plasmid targets in the in-house assay did allow for the potential presumptive identifications of pathogenic species. No plasmid targets were in the Ion 16S™ Metagenomics Kit.This study shows the choice of target(s) is key in MPS assay development and should be carefully considered to ensure the assay is fit for purpose, whether as an initial screening (presumptive) or a more specific (but not entirely confirmatory) test. Identification approaches may also benefit from a combination of MPS and qPCR as each has benefits and limitations.

Quality control of batches of bacteriological examination kits «Selective differential-diagnostic medium for recovery of Bacillus anthracis and closely related bacilli of Bacillus cereus group»

Possibilities of quality control of experimental series of a set of reagents for bacteriological studies «Selective differential diagnostic environment for the isolation of the causative agent of anthrax and closely related bacilli of the Bacillus cereus group» is justified. Findings of the investigation of growth properties of the selective medium showed typical colonial morphology of virulent strains of Bacillus anthracis (B. anthracis 81/1, B. anthracis 1(CО), B. anthracis 1193, B. anthracis И-363, B. anthracis 1283) and its vaccine strains B. anthracis CTI-1, B. anthracis Ihtiman, B. anthracis Sterne 34 F2, B. anthracis 55, B. anthracis 71/12 - II Tsenkovsky’s vaccine). The colour of the batches of selective medium and Hottinger’s agar around the Bacillus anthracis colonies was unchanged. Whereas, the colour of selective medium around colonies of closely related saprophytes of genus Bacillus discoloured to bright-yellow, allowing to differentiate colonies of anthracic micro germs and closely related microorganisms. Presence of inhibiting properties of the selective medium was confirmed using heterologous germ cultures (Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Proteus vulgaris HX-19) which showed no growth in all series of the tested medium. Meanwhile, the typical colonial morphology of strains of these microorganisms was observed on Hottinger’s agar.

Efficiency of Different Disinfectants on Bacillus cereus Sensu Stricto Biofilms on Stainless-Steel Surfaces in Contact With Milk.

The species of the Bacillus cereus group have the ability to adhere to and form biofilms on solid surfaces, including stainless steel, a material widely used in food industries. Biofilms allow for recontamination during food processing, and the “clean-in-place” (CIP) system is largely used by industries to control them. This study thus proposes to evaluate the efficacy of peracetic acid and sodium hypochlorite against biofilms induced on stainless-steel surfaces. The SAMN07414939 isolate (BioProject PRJNA390851), a recognized biofilm producer, was selected for biofilm induction on AISI 304 stainless steel. Biofilm induction was performed and classified into three categories: TCP (Tindalized, Contaminated, and Pasteurized milk), TCS (Tindalized milk Contaminated with Spores), and TCV (Tindalized milk Contaminated with Vegetative cells). Subsequently, the coupons were sanitized simulating a CIP procedure, on a pilot scale, using alkaline and acid solutions followed by disinfectants (peracetic acid and sodium hypochlorite). Microorganism adhesion on the surfaces reached 6.3 × 105 to 3.1 × 107 CFU/cm-2. Results did not show significant differences (p > 0.05) for surface adhesion between the three tested categories (TCP, TCS, and TCV) or (p > 0.05) between the two disinfectants (peracetic acid and sodium hypochlorite). Microbial populations adhered to the stainless-steel coupons are equally reduced after treatment with peracetic acid and sodium hypochlorite, with no differences in the control of B. cereus s.s. biofilms on AISI 304 stainless-steel surfaces.

Isolation of A Novel Bacillus thuringiensis Phage Representing A New Phage Lineage and Characterization of Its Endolysin.

Phages, the parasites of bacteria, are considered as a new kind of antimicrobial agent due to their ability to lyse pathogenic bacteria. Due to the increase of available phage isolates, the newly isolated phage showed increasing genomic similarities with previously isolated phages. In this study, the novel phage vB_BthS_BMBphi, infecting the Bacillus thuringiensis strain BMB171, is isolated and characterized together with its endolysin. This phage is the first tadpole-like phage infecting the Bacillus strains. Genomic analysis shows that the phage genome is dissimilar to all those of previously characterized phages, only exhibiting low similarities with partial regions of the B. thuringiensis prophages. Phylogenetic analysis revealed that the phage was distant from the other Bacillus phages in terms of evolution. The novel genome sequence, the distant evolutionary relationship, and the special virion morphology together suggest that the phage vB_BthS_BMBphi could be classified as a new phage lineage. The genome of the phage is found to contain a restriction modification system, which might endow the phage with immunity to the restriction modification system of the host bacterium. The function of the endolysin PlyBMB encoded by the phage vB_BthS_BMBphi was analyzed, and the endolysin could lyse all the tested Bacillus cereus group strains, suggesting that the endolysin might be used in controlling pathogenic B. cereus group strains. The findings of this study enrich the understanding of phage diversity and provide a resource for controlling the B. cereus group pathogenic bacteria.

Bacillus cereus group: genetic aspects related to food safety and dairy processing

ABSTRACT: Bacillus cereus group includes not pathogenic and high pathogenic species. They are considered as a risk to public health due to foodborne diseases and as an important cause of economic losses to industries due to production of spoilage enzymes. Some researches have been performed in order to assess the possible factors that contribute to put public health into risk because of consumption of food contaminated with viable cells or toxins which have complex mechanisms of production. The control of these bacteria in food is difficult because they are resistant to several processes used in industries. Thus, in this way, this review focused on highlighting the risk due to toxins production by bacteria from B. cereus group in food and the consequences for food safety and dairy industries.

Yeasts and bacteria associated with kocho, an Ethiopian fermented food produced from enset (Ensete ventricosum)

Enset (Ensete ventricosum) is the basis of the staple food consumed by about 20% of the Ethiopian population. Kocho is one of the food products generated from enset by spontaneous fermentation of decorticated and pulverized pseudostem and corm sections. We isolated culturable microbes associated with kocho from different stages of fermentation. Twelve yeast species, six lactic acid bacteria (LABs) species and eleven species of aerobic bacteria were identified by sequencing ITS/D1D2 regions of 26S rDNA of yeasts and 16S rDNA of bacteria, respectively. More yeast species were identified in fresh (fermented for 2–5 days) kocho, compared to long-term (7–12 months) fermented kocho, while we observed an opposite trend for LABs. In fresh kocho, the most frequently isolated yeast species were Pichia exigua, Galactomyces geotrichum, and Pichia fermentans. From mid-term (3–4 months) kocho most frequently Candida cabralensis, G. geotrichum, and Candida ethanolica were isolated. In the long-term fermentations, the most frequently isolated yeast was Saturnispora silva. Lactobacillus plantarum was the most frequently isolated LAB in both fresh and mid-term kocho. In long-term fermented kocho, Acetobacter pasteurianus and L. plantarum were most frequently isolated. L. plantarum was consistently isolated from all the three stages of fermentation. Aerobic bacteria in fresh kocho were mostly gram-negative, with Raoultella planticola and Pantoea agglomerans being the most frequently isolated species. In long-term fermented kocho, mainly gram-positive, spore-forming bacteria of the genus Bacillus were found, among them also species of the Bacillus cereus group, Bacillus anthracis and Bacillus thurigiensis.

Genomic characterization of three novel Basilisk-like phages infecting Bacillus anthracis

BackgroundIn the present study, we sequenced the complete genomes of three novel bacteriophages v_B-Bak1, v_B-Bak6, v_B-Bak10 previously isolated from historical anthrax burial sites in the South Caucasus country of Georgia. We report here major trends in the molecular evolution of these phages, which we designate as “Basilisk-Like-Phages” (BLPs), and illustrate patterns in their evolution, genomic plasticity and core genome architecture.ResultsComparative whole genome sequence analysis revealed a close evolutionary relationship between our phages and two unclassified Bacillus cereus group phages, phage Basilisk, a broad host range phage (Grose JH et al., J Vir. 2014;88(20):11846-11860) and phage PBC4, a highly host-restricted phage and close relative of Basilisk (Na H. et al. FEMS Microbiol. letters. 2016;363(12)). Genome comparisons of phages v_B-Bak1, v_B-Bak6, and v_B-Bak10 revealed significant similarity in sequence, gene content, and synteny with both Basilisk and PBC4. Transmission electron microscopy (TEM) confirmed the three phages belong to the Siphoviridae family. In contrast to the broad host range of phage Basilisk and the single-strain specificity of PBC4, our three phages displayed host specificity for Bacillus anthracis. Bacillus species including Bacillus cereus, Bacillus subtilis, Bacillus anthracoides, and Bacillus megaterium were refractory to infection.ConclusionsData reported here provide further insight into the shared genomic architecture, host range specificity, and molecular evolution of these rare B. cereus group phages. To date, the three phages represent the only known close relatives of the Basilisk and PBC4 phages and their shared genetic attributes and unique host specificity for B. anthracis provides additional insight into candidate host range determinants.

Fine-scale differentiation between Bacillus anthracis and Bacillus cereus group signatures in metagenome shotgun data.

BackgroundIt is possible to detect bacterial species in shotgun metagenome datasets through the presence of only a few sequence reads. However, false positive results can arise, as was the case in the initial findings of a recent New York City subway metagenome project. False positives are especially likely when two closely related are present in the same sample. Bacillus anthracis, the etiologic agent of anthrax, is a high-consequence pathogen that shares >99% average nucleotide identity with Bacillus cereus group (BCerG) genomes. Our goal was to create an analysis tool that used k-mers to detect B. anthracis, incorporating information about the coverage of BCerG in the metagenome sample.MethodsUsing public complete genome sequence datasets, we identified a set of 31-mer signatures that differentiated B. anthracis from other members of the B. cereus group (BCerG), and another set which differentiated BCerG genomes (including B. anthracis) from other Bacillus strains. We also created a set of 31-mers for detecting the lethal factor gene, the key genetic diagnostic of the presence of anthrax-causing bacteria. We created synthetic sequence datasets based on existing genomes to test the accuracy of a k-mer based detection model.ResultsWe found 239,503 B. anthracis-specific 31-mers (the Ba31 set), 10,183 BCerG 31-mers (the BCerG31 set), and 2,617 lethal factor k-mers (the lef31 set). We showed that false positive B. anthracis k-mers—which arise from random sequencing errors—are observable at high genome coverages of B. cereus. We also showed that there is a “gray zone” below 0.184× coverage of the B. anthracis genome sequence, in which we cannot expect with high probability to identify lethal factor k-mers. We created a linear regression model to differentiate the presence of B. anthracis-like chromosomes from sequencing errors given the BCerG background coverage. We showed that while shotgun datasets from the New York City subway metagenome project had no matches to lef31 k-mers and hence were negative for B. anthracis, some samples showed evidence of strains very closely related to the pathogen.DiscussionThis work shows how extensive libraries of complete genomes can be used to create organism-specific signatures to help interpret metagenomes. We contrast “specialist” approaches to metagenome analysis such as this work to “generalist” software that seeks to classify all organisms present in the sample and note the more general utility of a k-mer filter approach when taxonomic boundaries lack clarity or high levels of precision are required.

Corrigendum: Proposal of nine novel species of the Bacillus cereus group.

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