Chromatin accessibility profiling reveals that human fibroblasts respond to mechanical stimulation in a cell-specific manner.

Fibroblasts in the skin are highly heterogeneous, both in vivo and in vitro. One difference between follicular (dermal papilla fibroblasts [DP]) and interfollicular fibroblasts (papillary fibroblasts [PFi]) in vitro is their ability to differentiate in response to osteogenic media (OM), or mechanical stimulation. Here, we asked whether differences in the ability of DP and PFi to respond to differentiation stimuli are due to differences in chromatin accessibility. We performed chromatin accessibility and transcriptional profiling of DP and PFi in human skin, which arise from a common progenitor during development, yet display distinct characteristics in adult tissue and in vitro. We found that cells cultured in growth media had unique chromatin accessibility profiles; however, these profiles control similar functional networks. Upon introduction of a chemical perturbation (OM) to promote differentiation, we observed a divergence not only in the accessible chromatin signatures but also in the functional networks controlled by these signatures. The biggest divergence between DP and PFi was observed when we applied 2 perturbations to cells: growth in OM and mechanical stimulation (a shock wave [OMSW]). DP readily differentiate into bone in OMSW conditions, while PFi lack differentiation capability in vitro. In the DP we found a number of uniquely accessible promoters that controlled osteogenic interaction networks associated with bone and differentiation functions. Using ATAC-seq and RNA-seq we found that the combination of 2 stimuli (OMSW) could result in significant changes in chromatin accessibility associated with osteogenic differentiation, but only within the DP (capable of osteogenic differentiation). De novo motif analysis identified enrichment of motifs bound by the TEA domain (TEAD) family of transcription factors, and inter-cell comparisons (UpSet analysis) displayed large groups of genes to be unique to single cell types and conditions. Our results suggest that these 2 stimuli (OMSW) elicit cell-specific responses by modifying chromatin accessibility of osteogenic-related gene promoters.

Intraspecific Diversity and Pathogenicity of Bacillus thuringiensis Isolates from an Emetic Illness.

This study describes an emetic food-borne intoxication associated with a Bacillus cereus group species and the characterization of the bacterial isolates from the incident in aspects of molecular tying, genetic factors, cytotoxicity, and pathogenic mechanisms relating to emetic illness. Through the polyphasic identification approach, all seven isolates obtained from food and clinical samples were identified as Bacillus thuringiensis. According to multilocus sequence typing (MLST) analysis, intraspecific diversity was found within the B. thuringiensis isolates. Four allelic profiles were found, including two previously known STs (ST8 and ST15) and two new STs (ST2804 and ST2805). All isolates harbored gene fragments located in the cereulide synthetase (ces) gene cluster. The heat-treated culture supernatants of three emetic B. thuringiensis isolates, FC2, FC7, and FC8, caused vacuolation and exhibited toxicity to Caco-2 cells, with CC50 values of 56.57, 72.17, and 79.94 µg/mL, respectively. The flow cytometry with the Annexin V/PI assay revealed both apoptosis and necrosis mechanisms, but necrosis was the prominent mechanism that caused Caco-2 cell destruction by FC2, the most toxic isolate.

Demethylation of ITGAV accelerates osteogenic differentiation in a blast-induced heterotopic ossification in vitro cell culture model.

Trauma-induced heterotopic ossification is an intriguing phenomenon involving the inappropriate ossification of soft tissues within the body such as the muscle and ligaments. This inappropriate formation of bone is highly prevalent in those affected by blast injuries. Here, we developed a simplified cell culture model to evaluate the molecular events involved in heterotopic ossification onset that arise from the shock wave component of the disease. We exposed three subtypes of human mesenchymal cells in vitro to a single, high-energy shock wave and observed increased transcription in the osteogenic master regulators, Runx2 and Dlx5, and significantly accelerated cell mineralisation. Reduced representation bisulfite sequencing revealed that the shock wave altered methylation of gene promoters, leading to opposing changes in gene expression. Using a drug to target ITGAV, whose expression was perturbed by the shock wave, we found that we could abrogate the deposition of mineral in our model. These findings show how new therapeutics for the treatment of heterotopic ossification can be identified using cell culture models.

Evaluating Primary Blast Effects In Vitro.

Exposure to blast events can cause severe trauma to vital organs such as the lungs, ears, and brain. Understanding the mechanisms behind such blast-induced injuries is of great importance considering the recent trend towards the use of explosives in modern warfare and terrorist-related incidents. To fully understand blast-induced injury, we must first be able to replicate such blast events in a controlled environment using a reproducible method. In this technique using shock tube equipment, shock waves at a range of pressures can be propagated over live cells grown in 2D, and markers of cell viability can be immediately analyzed using a redox indicator assay and the fluorescent imaging of live and dead cells. This method demonstrated that increasing the peak blast overpressure to 127 kPa can stimulate a significant drop in cell viability when compared to untreated controls. Test samples are not limited to adherent cells, but can include cell suspensions, whole-body and tissue samples, through minor modifications to the shock tube setup. Replicating the exact conditions that tissues and cells experience when exposed to a genuine blast event is difficult. Techniques such as the one presented in this article can help to define damage thresholds and identify the transcriptional and epigenetic changes within cells that arise from shock wave exposure.

Methods for the isolation and 3D culture of dermal papilla cells from human hair follicles.

The dermal papilla is a cluster of mesenchymal cells located at the base of the hair follicle which have a number of important roles in the regulation of hair growth. As a consequence, in vitro models of these cells are widely used to study the molecular mechanisms which underlie hair follicle induction, growth and maintenance. While dermal papilla from rodent hair follicles can be digested prior to cell isolation, the unique extracellular matrix composition found in human dermal papilla renders enzymes such as trypsin and collagenase insufficient for digestion of the dermal papilla into a single cell suspension. As such, to grow human dermal papilla cells in vitro, the papilla has to first be isolated via a micro-dissection approach from the follicle. In this article we describe the micro-dissection and culture methods, which we use within our laboratory, for the study of human dermal papilla cells.

Osteogenic stem cell selection for repair and regeneration.

The first osteogenic cells to attach to a titanium (Ti) implant after placement are the multipotent stromal cells (MSCs) that circulate in the bloodstream and are recruited to the site of tissue damage. The reservoirs of these cells are heterogeneous in nature, consisting of a mixture of cells with varying differentiation abilities. In order to utilise these cells and to reduce the chance of unwanted events during regenerative therapies, the selection of a subset of cells that is truly multipotent is required. The behaviour of these cells has been shown to be altered by modifications to Ti implant surfaces, most notably rough, hydrophilic Ti. These changes in behaviour underpin the differences seen in clinical performance of these surfaces. In this study Human bone marrow derived stromal cells (hBMSCs) have been cultured on modified Ti surfaces in order to analyse these changes in cell behaviour. The results demonstrate the different effects of the surfaces and suggest that one surface selectively enriches the population with osteogenic adult 'stem cells' by inducing the cell death of the more differentiated cells. Combined with subsequent expansion in bioreactors before implantation, this may lead to a new source of cells for regenerative therapies.

Mesenchymal stem cell response to topographically modified CoCrMo.

Surface roughness on implant materials has been shown to be highly influential on the behavior of osteogenic cells. Four surface topographies were engineered on cobalt chromium molybdenum (CoCrMo) in order to examine this influence on human mesenchymal stem cells (MSC). These treatments were smooth polished (SMO), acid etched (AE) using HCl 7.4% and H2SO4 76% followed by HNO3 30%, sand blasted, and acid etched using either 50 µm Al2O3 (SLA50) or 250 µm Al2 O3 grit (SLA250). Characterization of the surfaces included energy dispersive X-ray analysis (EDX), contact angle, and surface roughness analysis. Human MSCs were cultured onto the four CoCrMo substrates and markers of cell attachment, retention, proliferation, cytotoxicity, and osteogenic differentiation were studied. Residual aluminum was observed on both SLA surfaces although this appeared to be more widely spread on SLA50, whilst SLA250 was shown to have the roughest topography with an Ra value greater than 1 µm. All substrates were shown to be largely non-cytotoxic although both SLA surfaces were shown to reduce cell attachment, whilst SLA50 also delayed cell proliferation. In contrast, SLA250 stimulated a good rate of proliferation resulting in the largest cell population by day 21. In addition, SLA250 stimulated enhanced cell retention, calcium deposition, and hydroxyapatite formation compared to SMO (p < 0.05). The enhanced response stimulated by SLA250 surface modification may prove advantageous for increasing the bioactivity of implants formed of CoCrMo.

TiO2-coated CoCrMo: improving the osteogenic differentiation and adhesion of mesenchymal stem cells in vitro.

The current gold standard material for orthopedic applications is titanium (Ti), however, other materials such as cobalt-chromium-molybdenum (CoCrMo) are often preferred due to their wear resistance and mechanical strength. This study investigates if the bioactivity of CoCrMo can be enhanced by coating the surface with titanium oxide (TiO2 ) by atmospheric pressure chemical vapor deposition (CVD), thereby replicating the surface oxide layer found on Ti. CoCrMo, TiO2-coated CoCrMo (CCMT) and Ti substrates were used for this study. Cellular f-actin distribution was shown to be noticeably different between cells on CCMT and CoCrMo after 24 h in osteogenic culture, with cells on CCMT exhibiting greater spread with developed protrusions. Osteogenic differentiation was shown to be enhanced on CCMT compared to CoCrMo, with increased calcium ion content per cell (p < 0.05), greater hydroxyapatite nodule formation (p < 0.05) and reduced type I collagen deposition per cell (p < 0.05). The expression of the focal adhesion protein vinculin was shown to be marginally greater on CCMT compared to CoCrMo, whereas AFM results indicated that CCMT required more force to remove a single cell from the substrate surface compared to CoCrMo (p < 0.0001). These data suggest that CVD TiO2 coatings may have the potential to increase the biocompatibility of CoCrMo implantable devices.

The Control of Mesenchymal Stromal Cell Osteogenic Differentiation through Modified Surfaces.

Stem cells continue to receive widespread attention due to their potential to revolutionise treatments in the fields of both tissue engineering and regenerative medicine. Adult stem cells, specifically mesenchymal stromal cells (MSCs), play a vital role in the natural events surrounding bone healing and osseointegration through being stimulated to differentiate along their osteogenic lineage and in doing so, they form new cortical and trabecular bone tissue. Understanding how to control, manipulate, and enhance the intrinsic healing events modulated through osteogenic differentiation of MSCs by the use of modified surfaces and biomaterials could potentially advance the fields of both orthopaedics and dentistry. This could be by either using surface modification to generate greater implant stability and more rapid healing following implantation or the stimulation of MSCs ex vivo for reimplantation. This review aims to gather publications targeted at promoting, enhancing, and controlling the osteogenic differentiation of MSCs through biomaterials, nanotopographies, and modified surfaces for use in implant procedures.

Taxonomic revision of the genus Geobacillus: emendation of Geobacillus, G. stearothermophilus, G. jurassicus, G. toebii, G. thermodenitrificans and G. thermoglucosidans (nom. corrig., formerly 'thermoglucosidasius'); transfer of Bacillus thermantarcticus to the genus as G. thermantarcticus comb. nov.; proposal of Caldibacillus debilis gen. nov., comb. nov.; transfer of G. tepidamans to Anoxybacillus as A. tepidamans comb. nov.; and proposal of Anoxybacillus caldiproteolyticus sp. nov.

Sixty-two strains of thermophilic aerobic endospore-forming bacteria were subjected to polyphasic taxonomic study including 16S rRNA gene sequence analysis, polar lipid and fatty acid analysis, phenotypic characterization, and DNA-DNA hybridization experiments. Distinct clusters of the species Geobacillus stearothermophilus, Geobacillus thermodenitrificans, Geobacillus toebii and Geobacillus thermoglucosidasius were formed, allowing their descriptions to be emended, and the distinctiveness of the poorly represented species Geobacillus jurassicus, Geobacillus subterraneus and Geobacillus caldoxylosilyticus was confirmed. It is proposed that the name Geobacillus thermoglucosidasius be corrected to Geobacillus thermoglucosidans nom. corrig. Bacillus thermantarcticus clustered between Geobacillus species on the basis of 16S rRNA gene sequence analysis, and its transfer to the genus Geobacillus as Geobacillus thermantarcticus comb. nov. (type strain LMG 23032(T)=DSM 9572(T)=strain M1(T)=R-35644(T)) is proposed. The above-mentioned species, together with Geobacillus thermoleovorans and Geobacillus thermocatenulatus, form a monophyletic cluster representing the genus Geobacillus. The distinctiveness of 'Geobacillus caldoproteolyticus' was confirmed and it is proposed that it be accommodated, along with Geobacillus tepidamans, in the genus Anoxybacillus as Anoxybacillus caldiproteolyticus sp. nov. (type strain DSM 15730(T)=ATCC BAA-818(T)=LMG 26209(T)=R-35652(T)) and Anoxybacillus tepidamans comb. nov. (type strain LMG 26208(T)=ATCC BAA-942(T)=DSM 16325(T)=R-35643(T)), respectively. The type strain of Geobacillus debilis was not closely related to any members of the genera Anoxybacillus and Geobacillus, and it is proposed that this species be placed in the new genus Caldibacillus as Caldibacillus debilis gen. nov. comb. nov. The type strain of the type species, Caldibacillus debilis, is LMG 23386(T) (=DSM 16016(T)=NCIMB 13995(T)=Tf(T)=R-35653(T)).

Emended descriptions of Bacillus sporothermodurans and Bacillus oleronius with the inclusion of dairy farm isolates of both species.

Bacillus sporothermodurans is an industrially important micro-organism because of its ability to produce endospores which resist ultra-high temperature (UHT) and industrial sterilization processes. It was described by Pettersson et al. (1996) [Pettersson, B., Lembke, F., Hammer, P., Stackebrandt, E. & Priest, F. G. (1996). Int J Syst Bacteriol 46, 759-764] based on seven genetically homogeneous isolates all from UHT milk. Bacillus oleronius, the closest phylogenetic neighbour of B. sporothermodurans, was described by Kuhnigk et al. (1995) [Kuhnigk, T., Borst, E.-M., Breunig, A., König, H., Collins, M. D., Hutson, R. A. & Kämpfer, P. (1995). Can J Microbiol 41, 699-706] based on a single strain, isolated from the hindgut of the termite Reticulitermes santonensis. A polyphasic study of a heterogeneous collection of B. sporothermodurans and B. oleronius strains isolated from various sources and geographical origins led to an emended description of both species. Additional data presented are the results of fatty acid, quinone and/or cell wall (polar lipid) analyses. DNA-DNA hybridization confirmed 3 subgroups of strains obtained after SDS-PAGE analysis of cellular proteins as B. sporothermodurans. One named B. sporothermodurans strain (R-7489) was reclassified as a Bacillus fordii strain. The phenotypic profiles of both species were rather heterogeneous, sometimes different from the original descriptions and did not differ in a large number of characteristics, although B. oleronius generally gave stronger reactions in its positive tests than did B. sporothermodurans; the variable and weak reactions for both organisms with some substrates blurred the distinction between the two. However, differences in polar lipid, SDS-PAGE and menaquinone profiles clearly allow distinction between the two species.

Lysinibacillus macroides sp. nov., nom. rev.

'Bacillus macroides' ATCC 12905(T) ( = DSM 54(T) = LMG 18474(T)), isolated in 1947 from cow dung, was not included in the Approved Lists of Bacterial Names and so it lost standing in bacteriological nomenclature. Reinvestigation of the strain, including DNA-DNA relatedness experiments, revealed that 'Bacillus macroides' is genomically distinct from its closest relatives Lysinibacillus xylanilyticus, Lysinibacillus boronitolerans and Lysinibacillus fusiformis (as determined by 16S rRNA gene sequence analysis, with pairwise similarity values of 99.2, 98.8 and 98.5 %, respectively, with the type strains of these species). Further analysis showed that 'Bacillus macroides' shares the A4α L-Lys-D-Asp peptidoglycan type with other members of the genus Lysinibacillus and can thus be attributed to this genus. These results, combined with additional phenotypic data, justify the description of strain LMG 18474(T) ( = DSM 54(T) = ATCC 12905(T)) as Lysinibacillus macroides sp. nov., nom. rev.

Recognition of greater diversity of Bacillus species and related bacteria in human faeces.

In a study looking at culturable aerobic Actinobacteria associated with the human gastrointestinal tract, the vast majority of isolates obtained from dried human faeces belonged to the genus Bacillus and related bacteria. A total of 124 isolates were recovered from the faeces of 10 healthy adult donors. 16S rRNA gene sequence analyses showed the majority belonged to the families Bacillaceae (n=81) and Paenibacillaceae (n=3), with Bacillus species isolated from all donors. Isolates tentatively identified as Bacillus clausii (n=32) and Bacillus licheniformis (n=28) were recovered most frequently, with the genera Lysinibacillus, Ureibacillus, Oceanobacillus, Ornithinibacillus and Virgibacillus represented in some donors. Phenotypic data confirmed the identities of isolates belonging to well-characterized species. Representatives of the phylum Actinobacteria were recovered in much lower numbers (n=11). Many of the bacilli exhibited antimicrobial activity against one or more strains of Clostridium difficile, Clostridium perfringens, Listeria monocytogenes and Staphylococcus aureus, with some (n=12) found to have no detectable cytopathic effect on HEp-2 cells. This study has revealed greater diversity within gut-associated aerobic spore-formers than previous studies, and suggests that bacilli with potential as probiotics could be isolated from the human gut.

Emended descriptions of Geobacillus thermoleovorans and Geobacillus thermocatenulatus.

Nineteen thermophilic, aerobic, endospore-forming bacterial strains were subjected to 16S rRNA gene sequence analysis. Eight of these strains had been received as cultures of Geobacillus kaustophilus, G. lituanicus, G. stearothermophilus, 'G. thermoleovorans subsp. stromboliensis', G. vulcani, 'Bacillus caldolyticus', 'B. caldotenax' and 'B. caldovelox', but they showed close relationships with the type strain of G. thermoleovorans, as did two other strains received as G. thermoleovorans. All strains underwent further taxonomic analysis by API and other phenotypic tests and fatty acid methyl ester analysis, and selected strains were analysed for their polar lipids and for DNA relatedness. The 11 strains that formed the G. thermoleovorans 16S rRNA cluster also showed some phenotypic similarities, and DNA relatedness data support the reassignment of the strains received as G. kaustophilus, G. lituanicus, 'G. thermoleovorans subsp. stromboliensis', G. vulcani, 'B. caldolyticus', 'B. caldotenax' and 'B. caldovelox', and one of the G. stearothermophilus strains, as members of the species G. thermoleovorans. Four other strains received as G. kaustophilus were misnamed; two were identified as G. stearothermophilus and two appeared to be closely related to Anoxybacillus rupiensis. One strain received as G. stearothermophilus remained unidentified. On the basis of a single strain, Geobacillus thermocatenulatus was shown to represent a distinct species, but study of the type strain of Geobacillus gargensis showed this species to be a later heterotypic synonym of Geobacillus thermocatenulatus. Emended descriptions of Geobacillus thermoleovorans and Geobacillus thermocatenulatus are therefore presented.

Bacillus thermolactis sp. nov., isolated from dairy farms, and emended description of Bacillus thermoamylovorans.

A polyphasic taxonomic study was performed on 22 thermotolerant, aerobic, endospore-forming bacteria from dairy environments. Seventeen isolates were retrieved from raw milk, one from a filter cloth and four from grass, straw or milking equipment. These latter four isolates (R-6546, R-7499, R-7764 and R-7440) were identified as Bacillus thermoamylovorans based on DNA-DNA hybridizations (values above 70 % with Bacillus thermoamylovorans LMG 18084(T)) but showed discrepancies in characteristics with the original species description, so an emended description of this species is given. According to 16S rRNA gene sequence analysis and DNA-DNA hybridization experiments, the remaining 18 isolates (R-6488(T), R-28193, R-6491, R-6492, R-7336, R-33367, R-6486, R-6770, R-31288, R-28160, R-26358, R-7632, R-26955, R-26950, R-33520, R-6484, R-26954 and R-7165) represented one single species, most closely related to Bacillus thermoamylovorans (93.9 % 16S rRNA gene sequence similarity), for which the name Bacillus thermolactis is proposed. Cells were Gram-stain-positive, facultatively anaerobic, endospore-forming rods that grew optimally at 40-50 °C. The cell wall peptidoglycan type of strain R-6488(T), the proposed type strain, was A1γ based on meso-diaminopimelic acid. Major fatty acids of the strains were C(16 : 0) (28.0 %), iso-C(16 : 0) (12.1 %) and iso-C(15 : 0) (12.0 %). MK-7 was the predominant menaquinone, and major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and some unidentified phospholipids. DNA G+C content was 35.0 mol%. Phenotypic properties allowed discrimination from other thermotolerant species of the genus Bacillus and supported the description of the novel species Bacillus thermolactis, with strain R-6488(T) ( = LMG 25569(T)  = DSM 23332(T)) as the proposed type strain.

Bacillus ruris sp. nov., from dairy farms.

Four novel ellipsoidal spore-forming Bacillus isolates with swollen sporangia, isolated from raw milk and feed concentrate, showed a high level of similarity in SDS-PAGE, fatty acid methyl esters and routine phenotypic tests. However, 16S rRNA gene sequence comparisons showed that this taxon was different from other related Bacillus species, and only a low level of DNA relatedness was found with the closest phylogenetic and phenotypic relative, Bacillus galactosidilyticus. This taxon could be differentiated from B. galactosidilyticus on the basis of morphological differences, stronger acid reactions with a wide range of substrates after 48 h incubation, and qualitative and quantitative differences in fatty acid content. On the basis of these data, a novel species, Bacillus ruris sp. nov., is proposed, with LMG 22866T (=DSM 17057T) as the type strain.

Paenibacillus wynnii sp. nov., a novel species harbouring the nifH gene, isolated from Alexander Island, Antarctica.

Soil taken from 12 different locations at Mars Oasis on Alexander Island, Antarctica, yielded unidentified isolates of endospore-forming bacteria. Soil from four of the locations contained Gram-negative, facultatively anaerobic, motile rods that were able to grow at 4 degrees C and which formed ellipsoidal spores that lay paracentrally or subterminally in swollen or slightly swollen sporangia. All of the strains harboured the nitrogenase gene nifH. Phenotypic tests, amplified rDNA restriction analysis (ARDRA), fatty acid analysis and SDS-PAGE analysis suggested that the isolates represented a novel taxon of Paenibacillus. 16S rRNA gene sequence comparison supported the proposal of a novel species, Paenibacillus wynnii sp. nov. (type strain, LMG 22176(T)=CIP 108306(T)).

Cases of emesis associated with bacterial contamination of an infant breakfast cereal product.

A commercial product for infants containing cereal mixed with dried infant formula was diagnosed as producing rapid projectile vomiting in two infants. Analysis of multiple samples of the cereal product revealed significant contamination with two spore-forming species, Bacillus subtilis and a strain of Bacillus cereus. The latter is the most likely cause of the emetic food poisoning, but we were unable to detect B. cereus emetic toxin. This raises the possibility of the cause being either a new cereulide-type toxin, or the bacterial load, in which case the presence of B. subtilis could have been a contributing factor.

Bacillus arenosi sp. nov., Bacillus arvi sp. nov. and Bacillus humi sp. nov., isolated from soil.

A group of nine Gram-positive endospore-forming bacteria was isolated from soil of the Drentse A agricultural research area in The Netherlands. Using (GTG)5-PCR genomic fingerprinting and fatty acid analysis, the nine isolates were divided into three consistent groups. On the basis of 16S rRNA gene sequence similarity of representative strains, the nine isolates were shown to belong to the genus Bacillus. The first group of four isolates was most closely related to Bacillus carboniphilus (95.5 %) and Bacillus sporothermodurans (95.5 %). The second and third groups of three and two isolates, respectively, showed highest sequence similarity to Bacillus neidei (97.0 and 97.1 %, respectively) and Bacillus pycnus (both 96.7 %). A DNA-DNA relatedness study confirmed the consistency of the three groups delineated by (GTG)5-PCR and fatty acid analysis. A small number of phenotypic characters allowed differentiation of the three groups of isolates. The three groups therefore represent novel species, for which the names Bacillus humi, Bacillus arenosi and Bacillus arvi are proposed, with LMG 22167T (=DSM 16318T), LMG 22166T (=DSM 16319T) and LMG 22165T (=DSM 16317T) as the respective type strains.

Study of mural painting isolates, leading to the transfer of 'Bacillus maroccanus' and 'Bacillus carotarum' to Bacillus simplex, emended description of Bacillus simplex, re-examination of the strains previously attributed to 'Bacillus macroides' and description of Bacillus muralis sp. nov.

A group of 24 strains was isolated from deteriorated mural paintings situated in Spain (necropolis of Carmona) and Germany (church of Greene-Kreiensen). (GTG)5-PCR genomic fingerprinting was performed on these strains to assess their genomic variability and the strains were delineated into four groups. Representatives were studied by 16S rRNA gene sequencing and were found to be closely related to Bacillus simplex and the species 'Bacillus macroides' (strain NCIMB 8796) and 'Bacillus maroccanus' (names not validly published) according to a fasta search. The close similarity between B. simplex, 'B. macroides' NCIMB 8796, 'B. maroccanus' and the mural painting isolates was confirmed by additional (GTG)5-PCR, ARDRA, FAME and SDS-PAGE analyses. Furthermore, these techniques revealed that strains of 'Bacillus carotarum', another name that has not been validly published, also showed high similarity to this group of organisms. On the other hand, it was shown that the strains labelled 'B. macroides' in different collections do not all belong to the same species. Strain NCIMB 8796 can be allocated to B. simplex, while strain DSM 54 (=ATCC 12905) shares the highest 16S rRNA gene sequence similarity with Bacillus sphaericus and Bacillus fusiformis (both around 98.6 %). On the basis of further DNA-DNA hybridization data and the study of phenotypic characteristics, one group of five mural painting strains was attributed to a novel species in the genus Bacillus, for which the name Bacillus muralis sp. nov. is proposed. Finally, the remaining mural painting strains, one (LMG 18508=NCIMB 8796) of two strains belonging to 'B. macroides' and strains belonging to 'B. maroccanus' and 'B. carotarum' are allocated to the species B. simplex and an emended description of B. simplex is given.