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Historical species definitions for many prokaryotes, including pathogens, have relied on phenotypic characteristics that are inconsistent with genome evolution. This scenario forces microbiologists and clinicians to face a tradeoff between taxonomic rigor and clinical interpretability. Using the Bacillus cereus group as a model, a conceptual framework for the taxonomic delineation of prokaryotes which reconciles genomic definitions of species with clinically and industrially relevant phenotypes is presented. The nomenclatural framework outlined here serves as a model for genomics-based bacterial taxonomy that moves beyond arbitrarily set genomospecies thresholds while maintaining congruence with phenotypes and historically important species names. The Bacillus cereus group comprises numerous closely related species, including bioterrorism agent B. anthracis , foodborne pathogen B. cereus , and biopesticide B. thuringiensis . Differentiating organisms capable of causing illness or death from those used in industry is essential for risk assessment and outbreak preparedness. However, current species definitions facilitate species-phenotype incongruences, particularly when horizontally acquired genes are responsible for a phenotype. Using all publicly available B. cereus group genomes ( n  = 2,231), we show that current species definitions lead to overlapping genomospecies clusters, in which 66.2% of genomes belong to multiple genomospecies at a conventional 95 average nucleotide identity (ANI) genomospecies threshold. A genomospecies threshold of ≈92.5 ANI is shown to reflect a natural gap in genome similarity for the B. cereus group, and medoid genomes identified at this threshold are shown to yield resolvable genomospecies clusters with minimal overlap (six of 2,231 genomes assigned to multiple genomospecies; 0.269%). We thus propose a nomenclatural framework for the B. cereus group which accounts for (i) genomospecies using resolvable genomospecies clusters obtained at ≈92.5 ANI, (ii) established lineages of medical importance using a formal collection of subspecies names, and (iii) heterogeneity of clinically and industrially important phenotypes using a formalized and extended collection of biovar terms. We anticipate that the proposed nomenclature will remain interpretable to clinicians, without sacrificing genomic species definitions, which can in turn aid in pathogen surveillance; early detection of emerging, high-risk genotypes; and outbreak preparedness. IMPORTANCE Historical species definitions for many prokaryotes, including pathogens, have relied on phenotypic characteristics that are inconsistent with genome evolution. This scenario forces microbiologists and clinicians to face a tradeoff between taxonomic rigor and clinical interpretability. Using the Bacillus cereus group as a model, a conceptual framework for the taxonomic delineation of prokaryotes which reconciles genomic definitions of species with clinically and industrially relevant phenotypes is presented. The nomenclatural framework outlined here serves as a model for genomics-based bacterial taxonomy that moves beyond arbitrarily set genomospecies thresholds while maintaining congruence with phenotypes and historically important species names.

Bacillus strains produce non-ribosomal lipopeptides that can be grouped into three families: surfactins or lichenysins, iturins and fengycins or plispastatins. These biosurfactants show a broad spectrum of biological activities. To detect strains able to produce these lipopeptides, a new polymerase chain reaction screening approach was developed using degenerated primers based on the intraoperon alignment of adenylation and thiolation nucleic acid domains of all enzymes implicated in the biosynthesis of each lipopeptide family. The comparative bioinformatics analyses of each operon led to the design of four primer pairs for the three families taking into account the differences between open reading frames of each synthetase gene. Tested on different Bacillus sp. strains, this technique was used successfully to detect not only the expected genes in the lipopeptide producing strains but also the presence of a plispastatin gene in Bacillus subtilis ATCC 21332 and a gene showing a high similarity with the polyketide synthase type I gene in the B. subtilis ATCC 6633 genome. It also led to the discovery of the presence of non-ribosomal peptide synthetase genes in Bacillus thuringiensis serovar berliner 1915 and in Bacillus cereus LMG 2098. In addition, this work highlighted the differences between the fengycin and plipastatin operon at DNA level.

The soil-related Bacillus and Paenibacillus species have increasingly been implicated in various human diseases. Nevertheless, their identification still poses problems in the clinical microbiology laboratory and, with the exception of Bacillus anthracis and Bacillus cereus, little is known on their pathogenicity for humans. In this study, we evaluated the use of matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS) in the identification of clinical isolates of these genera and conducted genotypic and phenotypic analyses to highlight specific virulence properties. Seventy-five clinical isolates were subjected to biochemical and MALDI-TOF MS identification. 16S rDNA sequencing and supplemental tests were used to solve any discrepancies or failures in the identification results. MALDI-TOF MS significantly outperformed classical biochemical testing for correct species identification and no misidentification was obtained. One third of the collected strains belonged to the B. cereus species, but also Bacillus pumilus and Bacillus subtilis were isolated at high rate. Antimicrobial susceptibility testing showed that all the B. cereus, B. licheniformis, B. simplex, B. mycoides, Paenibacillus glucanolyticus and Paenibacillus lautus isolates are resistant to penicillin. The evaluation of toxin/enzyme secretion, toxin-encoding genes, motility, and biofilm formation revealed that B. cereus displays the highest virulence potential. However, although generally considered nonpathogenic, most of the other species were shown to swim, swarm, produce biofilms, and secrete proteases that can have a role in bacterial virulence. In conclusion, MALDI-TOF MS appears useful for fast and accurate identification of Bacillus and Paenibacillus strains whose virulence properties make them of increasing clinical relevance.

Emerging B. cereus strains that cause anthrax-like disease have been isolated in Cameroon (CA strain) and Côte d'Ivoire (CI strain). These strains are unusual, because their genomic characterisation shows that they belong to the B. cereus species, although they harbour two plasmids, pBCXO1 and pBCXO2, that are highly similar to the pXO1 and pXO2 plasmids of B. anthracis that encode the toxins and the polyglutamate capsule respectively. The virulence factors implicated in the pathogenicity of these B. cereus bv anthracis strains remain to be characterised. We tested their virulence by cutaneous and intranasal delivery in mice and guinea pigs; they were as virulent as wild-type B. anthracis. Unlike as described for pXO2-cured B. anthracis, the CA strain cured of the pBCXO2 plasmid was still highly virulent, showing the existence of other virulence factors. Indeed, these strains concomitantly expressed a hyaluronic acid (HA) capsule and the B. anthracis polyglutamate (PDGA) capsule. The HA capsule was encoded by the hasACB operon on pBCXO1, and its expression was regulated by the global transcription regulator AtxA, which controls anthrax toxins and PDGA capsule in B. anthracis. Thus, the HA and PDGA capsules and toxins were co-regulated by AtxA. We explored the respective effect of the virulence factors on colonisation and dissemination of CA within its host by constructing bioluminescent mutants. Expression of the HA capsule by itself led to local multiplication and, during intranasal infection, to local dissemination to the adjacent brain tissue. Co-expression of either toxins or PDGA capsule with HA capsule enabled systemic dissemination, thus providing a clear evolutionary advantage. Protection against infection by B. cereus bv anthracis required the same vaccination formulation as that used against B. anthracis. Thus, these strains, at the frontier between B. anthracis and B. cereus, provide insight into how the monomorphic B. anthracis may have emerged.

Some members of the Bacillus velezensis ( Bv ) group (e.g., Bv FZB42T and AS3.43) were previously assigned grouping with B. subtilis and B . amyloliquefaciens , based on the fact that they shared a 99% DNA–DNA percentage phylogenetic similarity. However, hinging on current assessments of the pan-genomic reassignments, the differing phylogenomic characteristics of Bv from B . subtilis and B. amyloliquefaciens are now better understood. Within this re-grouping/reassignment, the various strains within the Bv share a close phylogenomic resemblance, and a number of these strains have received a lot of attention in recent years, due to their genomic robustness, and the growing evidence for their possible utilization in the agricultural industry for managing plant diseases. Only a few applications for their use medicinally/pharmaceutically, environmentally, and in the food industry have been reported, and this may be due to the fact that the majority of those strains investigated are those typically occurring in soil. Although the intracellular unique biomolecules of Bv strains have been revealed via in silico genome modeling and investigated using transcriptomics and proteomics, a further inquisition into the Bv metabolome using newer technologies such as metabolomics could elucidate additional applications of this economically relevant Bacillus species, beyond that of primarily the agricultural sector.

Bacteria of the Bacillus cereus group colonize several ecological niches and infect different hosts. Bacillus cereus , a ubiquitous species causing food poisoning, Bacillus thuringiensis , an entomopathogen, and Bacillus anthracis , which is highly pathogenic to mammals, are the most important species of this group. These species are closely related genetically, and their specific toxins are encoded by plasmids. The infectious cycle of B. thuringiensis in its insect host is regulated by quorum-sensing systems from the RNPP family. Among them, the Rap–Phr systems, which are well-described in Bacillus subtilis , regulate essential processes, such as sporulation. Given the importance of these systems, we performed a global in silico analysis to investigate their prevalence, distribution, diversity and their role in sporulation in B. cereus group species. The rap – phr genes were identified in all selected strains with 30% located on plasmids, predominantly in B. thuringiensis . Despite a high variability in their sequences, there is a remarkable association between closely related strains and their Rap–Phr profile. Based on the key residues involved in RapH phosphatase activity, we predicted that 32% of the Rap proteins could regulate sporulation by preventing the phosphorylation of Spo0F. These Rap are preferentially located on plasmids and mostly related to B. thuringiensis . The predictions were partially validated by in vivo sporulation experiments suggesting that the residues linked to the phosphatase function are necessary but not sufficient to predict this activity. The wide distribution and diversity of Rap–Phr systems could strictly control the commitment to sporulation and then improve the adaptation capacities of the bacteria to environmental changes.

We evaluated the minimum inhibitory concentrations of clindamycin and erythromycin toward 98 Bacillus licheniformis strains isolated from several types of fermented soybean foods manufactured in several districts of Korea. First, based on recent taxonomic standards for bacteria, the 98 strains were separated into 74 B. licheniformis strains and 24 B. paralicheniformis strains. Both species exhibited profiles of erythromycin resistance as an acquired characteristic. B. licheniformis strains exhibited acquired clindamycin resistance, while B. paralicheniformis strains showed unimodal clindamycin resistance, indicating an intrinsic characteristic. Comparative genomic analysis of five strains showing three different patterns of clindamycin and erythromycin resistance identified 23S rRNA (adenine 2058-N6)-dimethyltransferase gene ermC and spermidine acetyltransferase gene speG as candidates potentially involved in clindamycin resistance. Functional analysis of these genes using B. subtilis as a host showed that ermC contributes to cross-resistance to clindamycin and erythromycin, and speG confers resistance to clindamycin. ermC is located in the chromosomes of strains showing clindamycin and erythromycin resistance and no transposable element was identified in its flanking regions. The acquisition of ermC might be attributable to a homologous recombination. speG was identified in not only the five genome-analyzed strains but also eight strains randomly selected from the 98 test strains, and deletions in the structural gene or putative promoter region caused clindamycin sensitivity, which supports the finding that the clindamycin resistance of Bacillus species is an intrinsic property.

Background Plastic pollution is a significant environmental problem caused by its high resistance to degradation. One potential solution is polyhydroxybutyrate (PHB), a microbial biodegradable polymer. Mexico has great uncovered microbial diversity with high potential for biotechnological applications. The best polymer producers tend to be isolated from environments that require survival adaptations from microorganisms, the high-producing Bacillus cereus strain saba.zh comes from refinery wastewater, the costs of production have been a limiting factor for biopolymer production, and one of the focuses of interest has been finding novel strains with better production or singular traits that help in industrial processes. Results The isolates were taxonomically classified as Bacillus cereus MSF4 and Bacillus inaquosorum MSD1 from Mina, Nuevo Leon; B. cereus S07C; and Paenibacillis dendritiformis from the active volcano “El Chichonal” on Chiapas. The strains had growth temperatures ranging from 35 to 50 °C and pH tolerance values ranging from 3 to 9. The best PHB-producing strain, B. cereus MSF4 , produced 0.43 g/kg PHB on orange peels, followed by B. inaquosorum MSD1 at 0.40 g/kg, B. cereus S07C at 0.23 g/kg and P. dendritiformis at 0.26 g/kg. Conclusions The findings of this study affirm the potential of the Mexican isolated strains as PHB-producing organisms, enabling further studies to test their viability as industrial producers. The ability of P. dendritiformis and B. inaquosorum to synthetize PHB was also confirmed by the observations made providing novel evidence to consider these species as potential producers.

Whole-genome sequencing and phenotypic testing of 104 strains of Bacillus licheniformis and Bacillus paralicheniformis from a variety of sources and time periods was used to characterize the genetic background and evolution of (putative) antimicrobial resistance mechanisms. Core proteins were identified in draft genomes and a phylogenetic analysis based on single amino acid polymorphisms allowed the species to be separated into two phylogenetically distinct clades with one outlier. Putative antimicrobial resistance genes were identified and mapped. A chromosomal ermD gene was found at the same location in all B. paralichenformis and in 27% of B. licheniformis genomes. Erythromycin resistance correlated very well with the presence of ermD. The putative streptomycin resistance genes, aph and aadK, were found in the chromosome of all strains as adjacent loci. Variations in amino acid sequence did not correlate with streptomycin susceptibility although the species were less susceptible than other Bacillus species. A putative chloramphenicol resistance gene (cat), encoding a novel chloramphenicol acetyltransferase protein was also found in the chromosome of all strains. Strains encoding a truncated CAT protein were sensitive to chloramphenicol. For all four resistance genes, the diversity and genetic context followed the overall phylogenetic relationship. No potentially mobile genetic elements were detected in their vicinity. Moreover, the genes were only distantly related to previously-described cat, aph, aad and erm genes present on mobile genetic elements or in other species. Thus, these genes are suggested to be intrinsic to B. licheniformis and B. paralicheniformis and part of their ancient resistomes. Since there is no evidence supporting horizontal transmission, these genes are not expected to add to the pool of antibiotic resistance elements considered to pose a risk to human or animal health. Whole-genome based phylogenetic and sequence analysis, combined with phenotypic testing, is proposed to be suitable for determining intrinsic resistance and evolutionary relationships.

Phosphorus (P) use efficiency in alkaline/calcareous soils is only 20% due to precipitation of P 2 O 5 with calcium and magnesium. However, coating Diammonium Phosphate (DAP) with phosphorus solubilizing bacteria (PSB) is more appropriate to increase fertilizer use efficiency. Therefore, with the aim to use inorganic fertilizers more effectively present study was conducted to investigate comparative effect of coated DAP with PSB strains Bacillus subtilis ZE15 (MN003400), Bacillus subtilis ZR3 (MN007185), Bacillus megaterium ZE32 (MN003401) and Bacillus megaterium ZR19 (MN007186) and their extracted metabolites with uncoated DAP under axenic conditions. Gene sequencing was done against various sources of phosphorus to analyze genes responsible for phosphatase activity. Alkaline phosphatase (ALP) gene amplicon of 380bp from all tested strains was showed in 1% w/v gel. Release pattern of P was also improved with coated fertilizer. The results showed that coated phosphatic fertilizer enhanced shoot dry weight by 43 and 46% under bacterial and metabolites coating respectively. Shoot and root length up to 44 and 42% with metabolites coated DAP and 41% with bacterial coated DAP. Physiological attributes also showed significant improvement with coated DAP over conventional. The results supported the application of coated DAP as a useful medium to raise crop yield even at lower application rates i.e., 50 and 75% DAP than non-coated 100% DAP application which advocated this coating technique a promising approach for advancing circular economy and sustainable development in modern agriculture.