Incubation temperature and culture medium formulation impact the accuracy of pour-plate techniques for the enumeration of industrial Bacillus assemblages.

Aerobic plate counting assays based on the pour-plate technique are frequently used to enumerate microbial products; however, colony swarming and merging at the agar surface can reduce the accuracy of these assays. Some plating methods mitigate this risk through the inclusion of strategies including agar overlays; however, these interventions may be inadequate to mitigate swarming and merging of certain Bacillus colonies. In the present study, we assessed the accuracy of several pour-plate techniques for the enumeration of a mixed-species Bacillus assemblage. Tested modifications included a customized culture medium formulation, agar overlays, decreased incubation times and increased incubation temperature. Methods which produced countable plates were assessed for agreement with a Bacillus-specific plate counting assay and with total cell counts rendered by flow cytometry. While all tested pour-plate methods underestimated Bacillus endospore concentrations relative to flow cytometry and customized spread-plating, our results suggest that increasing incubation temperature and the inclusion of bile salts into culture medium formulations can improve the accuracy of pour-plate techniques when used to enumerate Bacillus assemblages by decreasing the incidence of spreading colonies. As Bacillus endospore preparations become more ubiquitous in the market, familiar enumeration methods such as the pour-plate technique may require methodological modifications to ensure that the cGMP compliance of Bacillus-based microbial products is assessed accurately.

Protection from Lethal Clostridioides difficile Infection via Intraspecies Competition for Cogerminant.

Clostridioides difficile, a Gram-positive, spore-forming bacterium, is the primary cause of infectious nosocomial diarrhea. Antibiotics are a major risk factor for C. difficile infection (CDI), as they disrupt the gut microbial community, enabling increased germination of spores and growth of vegetative C. difficile To date, the only single-species bacterial preparation that has demonstrated efficacy in reducing recurrent CDI in humans is nontoxigenic C. difficile Using multiple infection models, we determined that precolonization with a less virulent strain is sufficient to protect from challenge with a lethal strain of C. difficile, surprisingly even in the absence of adaptive immunity. Additionally, we showed that protection is dependent on high levels of colonization by the less virulent strain and that it is mediated by exclusion of the invading strain. Our results suggest that reduction of amino acids, specifically glycine following colonization by the first strain of C. difficile, is sufficient to decrease germination of the second strain, thereby limiting colonization by the lethal strain.IMPORTANCE Antibiotic-associated colitis is often caused by infection with the bacterium Clostridioides difficile In this study, we found that reduction of the amino acid glycine by precolonization with a less virulent strain of C. difficile is sufficient to decrease germination of a second strain. This finding demonstrates that the axis of competition for nutrients can include multiple life stages. This work is important, as it is the first to identify a possible mechanism through which precolonization with C. difficile, a current clinical therapy, provides protection from reinfection. Furthermore, our work suggests that targeting nutrients utilized by all life stages could be an improved strategy for bacterial therapeutics that aim to restore colonization resistance in the gut.

The Sporobiota of the Human Gut.

The human gut microbiome is a diverse and complex ecosystem that plays a critical role in health and disease. The composition of the gut microbiome has been well studied across all stages of life. In recent years, studies have investigated the production of endospores by specific members of the gut microbiome. An endospore is a tough, dormant structure formed by members of the Firmicutes phylum, which allows for greater resistance to otherwise inhospitable conditions. This innate resistance has consequences for human health and disease, as well as in biotechnology. In particular, the formation of endospores is strongly linked to antibiotic resistance and the spread of antibiotic resistance genes, also known as the resistome. The term sporobiota has been used to define the spore-forming cohort of a microbial community. In this review, we present an overview of the current knowledge of the sporobiota in the human gut. We discuss the development of the sporobiota in the infant gut and the perinatal factors that may have an effect on vertical transmission from mother to infant. Finally, we examine the sporobiota of critically important food sources for the developing infant, breast milk and powdered infant formula.

High counts of thermophilic spore formers in dairy powders originate from persisting strains in processing lines.

During the last decades, thermophilic spore counts became a very important quality parameter for manufacturers with regard to powdered dairy products. Low-spore count powders are highly demanded but challenging to produce when high production volume and long process times are intended. In this study a detailed monitoring of microbial levels in three skim milk powder plants was conducted. Anoxybacillus flavithermus was found to be primarily responsible for increased spore levels with increasing spore numbers being detected after 6-8 h already during initial processing steps. Simultaneously, the species composition shifted from a diverse bulk tank milk microbiota where different Bacillus species represented around 90% of the thermophilic bacteria to a dominance of A. flavithermus in the end product. The analysis of A. flavithermus isolates from different powder batches with RAPD PCR revealed recurring patterns in each of the eight German manufacturers sampled over several months. The high relatedness of isolates exhibiting identical RAPD patterns was exemplified by cgMLST based on whole genome sequences. We assume that A. flavithermus strains persisted in production plants and were not eliminated by cleaning. It is concluded that such persisting strains recurrently recontaminated subsequent powder productions. The data highlight that a targeted optimization of cleaning and disinfection procedures is the most promising measure to effectively reduce thermophilic spore counts in German dairy powders.

Initial butyrate producers during infant gut microbiota development are endospore formers.

The acquisition of the infant gut microbiota is key to establishing a host-microbiota symbiosis. Microbially produced metabolites tightly interact with the immune system, and the fermentation-derived short-chain fatty acid butyrate is considered an important mediator linked to chronic diseases later in life. The intestinal butyrate-forming bacterial population is taxonomically and functionally diverse and includes endospore formers with high transmission potential. Succession, and contribution of butyrate-producing taxa during infant gut microbiota development have been little investigated. We determined the abundance of major butyrate-forming groups and fermentation metabolites in faeces, isolated, cultivated and characterized the heat-resistant cell population, which included endospores, and compared butyrate formation efficiency of representative taxa in batch cultures. The endospore community contributed about 0.001% to total cells, and was mainly composed of the pioneer butyrate-producing Clostridium sensu stricto. We observed an increase in abundance of Faecalibacterium prausnitzii, butyrate-producing Lachnospiraceae and faecal butyrate levels with age that is likely explained by higher butyrate production capacity of contributing taxa compared with Clostridium sensu stricto. Our data suggest that a successional arrangement and an overall increase in abundance of butyrate forming populations occur during the first year of life, which is associated with an increase of intestinal butyrate formation capacity.

Hydrophobicity of abiotic surfaces governs droplets deposition and evaporation patterns.

Surface contamination with droplets containing bacteria is of concern in the food industry and other environments where hygiene control is essential. Deposition patterns after the drying of contaminated droplets is affected by numerous parameters. The present study evaluated the rate of evaporation and the shape of deposition patterns after the drying of water droplets on a panel of materials with different surface properties (topography, hydrophobicity). The influence of the particle properties (in this study 1 µm-microspheres and two bacterial spores) was also investigated. Polystyrene microspheres were hydrophobic, while Bacillus spores were hydrophilic or hydrophobic, and surrounded by different surface features. In contrast to material topography, hydrophobicity was shown to deeply affect droplet evaporation, with the formation of small, thick deposits with microspheres or hydrophilic spores. Among the particle properties, the spore morphology (size and round/ovoid shape) did not clearly affect the deposition pattern. Conversely, hydrophobic spores aggregated to form clusters, which quickly settled on the materials and either failed to migrate, or only migrated to a slight extent on the surface, resulting in a steady distribution of spores or spore clusters over the whole contaminated area. Adherent bacteria or spores are known to be highly resistant to many stressful environmental conditions. In view of all the quite different patterns obtained following drying of spore-containing droplets, it seems likely that some of these would entail enhanced resistance to hygienic processes.

Effect of copy number of the spoVA2mob operon, sourdough and reutericyclin on ropy bread spoilage caused by Bacillus spp.

Bacillus spp. cause ropy bread spoilage of bread, which is characterized by a rotten fruity odor, followed by discoloration and degradation of the crumb. Bacillus spp. are wheat grain endophytes and form heat resistant endospores, therefore, process hygiene and heating during baking do not prevent ropy spoilage. This study used 8 strains of Bacillus subtilis and Bacillus amyloliquefaciens to determine whether the presence and the copy number of spoVA2mob operon influences survival after baking; in addition, the spoilage phenotype was correlated with the presence of amylolytic enzymes in genomes of Bacillus spp.. The presence and copy number of the spoVA2mob operon had only a minor effect on survival of Bacillus endospores. Strains of B. amyloliquefaciens caused ropy spoilage faster than strains of B. subtilis, this difference correlated to the number and type of extracellular amylases encoded in the genomes of the strains of B. amyloliquefaciens and B. subtilis. The inhibitory effect of sourdough on ropy spoilage was determined by addition of 3-24% sourdough fermented with L. reuteri TMW1.656. Addition of 12% and 24% sourdough, corresponding to a bread pH of 5.93 ± 0.041 and 5.53 ± 0.040, respectively, delayed ropy spoilage for 2 and more than 5 d, respectively. The comparison of addition of 12% sourdough fermented with the reutericyclin producing L. reuteri TMW1.656 and the isogenic reutericyclin-negative strain L. reuteri TMW1.656ΔgtfAΔrtcN demonstrated that reutericyclin produced in sourdough inhibits growth of Bacillus in bread. In conclusion, sourdough inhibits germination of Bacillus spores in bread and the effect of sourdough is enhanced by reutericyclin.

High prevalence of Clostridium botulinum in vegetarian sausages.

Clostridium botulinum is a significant food safety concern due to its ability to produce highly potent neurotoxin and resistant endospores. Vegetarian sausages have become a popular source of plant protein and alternative for meat products. While vegetarian sausages have not been linked to botulism, numerous outbreaks due to preserved vegetables suggest a frequent occurrence of C. botulinum spores in the raw material. The product formulation of vegetarian sausages involves limited NaCl and preservatives, and shelf-lives may be several months. The safety of vegetarian sausages thus relies mainly on heat treatment and chilled storage. The main food safety concern is C. botulinum Group II that can grow and produce toxin at refrigeration temperatures. Here we show a high overall prevalence (32%) of C. botulinum in 74 samples of vegetarian sausages from seven producers. Both Groups I and II strains and genes for neurotoxin types A, B, E and F were detected in the products. The highest cell counts (1200 spores/kg) were observed for C. botulinum Group II in products with remaining shelf-lives of 6 months at the time of purchase. We conclude that vacuum-packaged vegetarian sausage products frequently contain C. botulinum spores and may possess a high risk of C. botulinum growth and toxin production. Chilled storage below 3°C and thorough reheating before consumption are warranted.

Sporulation and Germination in Clostridial Pathogens.

As obligate anaerobes, clostridial pathogens depend on their metabolically dormant, oxygen-tolerant spore form to transmit disease. However, the molecular mechanisms by which those spores germinate to initiate infection and then form new spores to transmit infection remain poorly understood. While sporulation and germination have been well characterized in Bacillus subtilis and Bacillus anthracis, striking differences in the regulation of these processes have been observed between the bacilli and the clostridia, with even some conserved proteins exhibiting differences in their requirements and functions. Here, we review our current understanding of how clostridial pathogens, specifically Clostridium perfringens, Clostridium botulinum, and Clostridioides difficile, induce sporulation in response to environmental cues, assemble resistant spores, and germinate metabolically dormant spores in response to environmental cues. We also discuss the direct relationship between toxin production and spore formation in these pathogens.

Identification of spore-forming bacteria isolated from contaminated Lowenstein Jensen media and effectiveness of Vancomycin to reduce Mycobacterial culture contamination in Burkina-Faso.

The type of commensal microorganisms can influence the efficiency of sputum decontamination for TB diagnosis. A basic characterization of contaminants from LJ contaminated media showed that Gram positive Spore Forming Bacteria (SFB) were the major contaminants. This study aims to identify the species of this contaminants and to evaluate the effectiveness of VCNT at 10 µg of vancomycin to reduce mycobacterial culture contamination mainly linked to SFB. Fifty-three SFB isolated between February 2016 and May 2017 were used. The effectiveness of LJ with VCNT at 10 µg of Vancomycin were evaluated with sputum collected in the same period. SFB had been stored at -20 °C and identified after subculture onto 5% sheep blood Columbia agar and incubated at 37 °C during 24 h. Bacteria cells and isolated colonies were described. API 50CH/B was performed and MALDI-TOF MS was used for external quality control. Thirty- five (66%) isolates representing 4 genera (Bacillus, Paenibacillus, Brevisbacillus and Lysinibacillus) including 10 species were identified. The most important species were Bacillus cereus (30%) and Bacillus licheniformis (21%). Eighteen (34%) isolates were non-reactive Bacillus. The overall contamination rate on LJ with VCNT at 10 µg of vancomycin was statistically lower than which without VCNT (18.7% versus 43.8%) (p = 0.01). The most important SFB identified were B. cereus and B. licheniformis. Almost all identified strains were similar to those currently isolated in fermented traditional food suggesting in part food related contaminants. VCNT containing 10 µg of vancomycin is a good alternative method to reduce mycobacterial culture contamination.

Phylogenetic diversity of aerobic spore-forming Bacillalles isolated from Brazilian soils.

The phylum Firmicutes comprises seven classes where most species are either aerobic or anaerobic endospore former. Inside Firmicutes, species allocated in the genus Bacillus and related genera are collectively named aerobic endospore-forming bacteria (AEFB), and the soil is their major reservoir. AEFB have great importance in health, agriculture, and biotechnology although the more studied species are Bacillus subtilis and the human pathogens Bacillus cereus and Bacillus anthracis. AEFB have great importance in health, agriculture, and biotechnology; although the knowledge about these organisms is based on few species, notably, Bacillus subtilis, Bacillus cereus, and Bacillus anthracis. In this work, we generated partial 16S rRNA gene sequences of both strands of 192 AEFB strains isolated from soils of Distrito Federal, Brazil (SDF strains). The resulting consensus sequences were used to obtain taxonomic assignment and establish the phylogenetic relationships among these strains. Through this approach, we could observe that classified SDF strains were distributed among genera Bacillus (169 strains; 88.02%), Paenibacillus (11; 5.73%), Lysinibacillus (6; 3.13%), Brevibacillus (4; 2.08%), Terribacillus (1; 0.52%), and Rummeliibacillus (1; 0.52%). Phylogenetic trees revealed these 192 SDF strains can be segregated into eight groups spanning families Bacillaceae and Paenibacillaceae belonging to the order Bacillales. To expand the knowledge about the diversity of these SDF strains, further studies regarding characterization with different methodologies are underway.

Spore forming Actinobacterial diversity of Cholistan Desert Pakistan: Polyphasic taxonomy, antimicrobial potential and chemical profiling.

BACKGROUND: Actinobacteria are famous for the production of unique secondary metabolites that help in controlling the continuously emerging drug resistance all over the globe. This study aimed at the investigation of an extreme environment the Cholistan desert, located in southern Punjab, Pakistan, for actinobacterial diversity and their activity against methicillin resistant Staphylococcus aureus (MRSA). The Cholistan desert is a sub-tropical and arid ecosystem with harsh environment, limited rainfall and low humidity. The 20 soil and sand samples were collected from different locations in the desert and the actinobacterial strains were selectively isolated. The isolated strains were identified using a polyphasic taxonomic approach including morphological, biochemical, physiological characterization, scanning electron microscopy (SEM) and by 16S rRNA gene sequencing. RESULTS: A total of 110 desert actinobacterial strains were recovered, which were found to be belonging to 3 different families of the order Actinomycetales, including the family Streptomycetaceae, family Pseudonocardiaceae and the family Micrococcaceae. The most frequently isolated genus was Streptomyces along with the genera Pseudonocardia and Arthrobacter. The isolated strains exhibited promising antimicrobial activity against methicillin resistant Staphylococcus aureus (MRSA) with zone of inhibition in the range of 9-32 mm in antimicrobial screening assays. The chemical profiling by thin layer chromatography, HPLC-UV/Vis and LC-MS analysis depicted the presence of different structural classes of antibiotics. CONCLUSION: The study revealed that Cholistan desert harbors immense actinobacterial diversity and most of the strains produce structurally diverse bioactive secondary metabolites, which are a promising source of novel antimicrobial drug candidates.

Ultrastructural analysis of spores from diverse Bacillales species isolated from Brazilian soil.

Many species in the order Bacillales form a specialized cell type called a spore that is resistant to a range of environmental stresses. Transmission electron microscopy (TEM) reveals that the spore is comprised of a series of concentric shells, surrounding an interior compartment harbouring the spore DNA. The outermost of these shells varies considerably in morphology among species, likely reflecting adaptations to the highly diverse niches in which spores are found. To better characterize the variation in spore ultrastructure among diverse species, we used TEM to analyse spores from a collection of 23 aerobic spore-forming bacteria from the Solo do Distrito Federal (SDF strains), spanning the genera Bacillus, Lysinibacillus, Paenibacillus and Brevibacillus, isolated from soil from central Brazil. We found that the structures of these spores varied widely, as expected. Interestingly, even though these isolates are novel strains of each species, they were structurally very similar to the known examples of each species in the literature. Because in most cases, the species we analysed are poorly characterized, our data provide important evidence regarding which structural features are likely to be constant within a taxon and which are likely to vary.

Contrasting community composition of endospores and vegetative Firmicutes in a marine sediment suggests both endogenous and exogenous sources of endospore accumulation.

Bacterial endospores are highly abundant in marine sediments, but their taxonomic identity and ecology is largely unknown. We selectively extracted DNA from endospores and vegetative cells and sequenced 16S rRNA genes to characterize the composition of the endospore and vegetative Firmicutes communities in the sediment and water column of Aarhus Bay (Denmark). The endospore community in the sediment was dominated by the families Bacillaceae, Lachnospiraceae, Clostridiaceae and Ruminoccocaceae. These families were also represented in the vegetative community in the sediment and the endospore community in the water column. OTUs of high relative abundance in the endospore community were also represented in the vegetative Firmicutes community. Other OTUs were exclusively found in the endospore communities. This suggests that endospores accumulate in marine sediments due to passive deposition from the water column and sporulation of vegetative cells in the sediment. Some OTUs were detected in the endospore community of the water column and the vegetative community the sediment indicating that endospores deposited from the water column may germinate upon burial/deposition in the sediment. We provide novel insight into the composition of endospore communities in marine sediments and highlight their role in microbial dispersal and as a seed bank in subsurface sediments.

In Planta Sporulation of Frankia spp. as a Determinant of Alder-Symbiont Interactions.

The Alnus genus forms symbiosis with the actinobacteria Frankia spp. and ectomycorrhizal fungi. Two types of Frankia lineages can be distinguished based on their ability to sporulate in planta Spore-positive (Sp+) strains are predominant on Alnus incana and Alnus viridis in highlands, while spore-negative (Sp-) strains are mainly associated with Alnus glutinosa in lowlands. Here, we investigated whether the Sp+ predominance in nodules is due to host selection of certain Frankia genotypes from soil communities or the result of the ecological history of the alder stand soil, as well as the effect of the sporulation genotype on the ectomycorrhizal (ECM) communities. Trapping experiments were conducted using A. glutinosa, A. incana, and A. viridis plantlets on 6 soils, differing in the alder species and the frequency of Sp+ nodules in the field. Higher diversity of Frankia spp. and variation in Sp+ frequencies were observed in the trapping than in the fields. Both indigenous and trapping species shape Frankia community structure in trapped nodules. Nodulation impediments were observed under several trapping conditions in Sp+ soils, supporting a narrower host range of Sp+ Frankia species. A. incana and A. viridis were able to associate equally with compatible Sp+ and Sp- strains in the greenhouse. Additionally, no host shift was observed for Alnus-specific ECM, and the sporulation genotype of Frankia spp. defined the ECM communities on the host roots. The symbiotic association is likely determined by the host range, the soil history, and the type of in plantaFrankia species. These results provide an insight into the biogeographical drivers of alder symbionts in the Holarctic region.IMPORTANCE Most Frankia-actinorhiza plant symbioses are capable of high rates of nitrogen fixation comparable to those found on legumes. Yet, our understanding of the ecology and distribution of Frankia spp. is still very limited. Several studies have focused on the distribution patterns of Frankia spp., demonstrating a combination of host and pedoclimatic parameters in their biogeography. However, very few have considered the in planta sporulation form of the strain, although it is a unique feature among all symbiotic plant-associated microbes. Compared with Sp- Frankia strains, Sp+ strains would be obligate symbionts that are highly dependent on the presence of a compatible host species and with lower efficiency in nitrogen fixation. Understanding the biogeographical drivers of Sp+ Frankia strains might help elucidate the ecological role of in planta sporulation and the extent to which this trait mediates host-partner interactions in the alder-Frankia-ECM fungal symbiosis.

The characteristics of Clostridium difficile ST81, a new PCR ribotype of toxin A- B+ strain with high-level fluoroquinolones resistance and higher sporulation ability than ST37/PCR ribotype 017.

Antibiotic exposure, Clostridium difficile toxins, and spore formation are key factors involved in the pathogenesis of Clostridium difficile infection (CDI). A high incidence of CDI due to toxin A- B+ strains, which were classified into two genotypes (ST81 and ST37) by multilocus sequence typing, was identified in Beijing Friendship Hospital in 2016-2017. ST81 was the most prevalent type, accounting for 81.25% of toxin A- B+ strains. ST81 corresponded to a novel PCR ribotype, PKI-017, with one less band than ST37/ribotype 017 in PCR ribotyping. All ST81 strains showed a high level of ciprofloxacin resistance (MICs ≥ 64 µg mL-1) and moxifloxacin resistance (MICs ≥ 128 µg mL-1) with the amino acid substitutions Thr82 to Ile in GyrA and Ser416 to Ala in GyrB. There was either no mutation or only the single amino acid mutation Thr82 to Ile in the GyrA subunit of ST37/ribotype 017 strains, which had lower MICs of ciprofloxacin (4-64 µg mL-1) and moxifloxacin (4-16 µg mL-1). In addition, ST81 strains exhibited higher spore formation ability than ST37/ribotype 017 strains. Overall, our results indicated that ST81 strains had unique characteristics distinguishable from ST37 strains and emphasized the importance of ongoing surveillance for this new genotype.

Daqu Fermentation Selects for Heat-Resistant Enterobacteriaceae and Bacilli.

Daqu is a spontaneous solid-state cereal fermentation used as saccharification and starter culture in Chinese vinegar and liquor production. The evolution of microbiota in this spontaneous fermentation is controlled by the temperature profile, which reaches temperatures from 50 to 65°C for several days. Despite these high temperatures, mesophilic Enterobacteriaceae (including Cronobacter) and bacilli are present throughout Daqu fermentation. This study aimed to determine whether Daqu spontaneous solid-state fermentation selects for heat-resistant variants of these organisms. Heat resistance in Enterobacteriaceae is mediated by the locus of heat resistance (LHR). One LHR-positive strain of Kosakonia cowanii was identified in Daqu, and it exhibited higher heat resistance than the LHR-negative K. cowanii isolated from malted oats. Heat resistance in Bacillus endospores is mediated by the spoVA2mob operon. Out of 10 Daqu isolates of the species Bacillus licheniformis, Brevibacillus parabrevis, Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus velezensis, 5 did not contain spoVA2mob, 3 contained one copy, and 2 contained two copies. The presence and copy number of the spoVA2mob operon increased the resistance of spores to treatment with 110°C. To confirm the selection of LHR- and spoVA2mob-positive strains during Daqu fermentation, the copy numbers of these genetic elements in Daqu samples were quantified by quantitative PCR (qPCR). The abundance of LHR and the spoVA2mob operon in community DNA relative to that of total bacterial 16S rRNA genes increased 3-fold and 5-fold, respectively, during processing. In conclusion, culture-dependent and culture-independent analyses suggest that Daqu fermentation selects for heat-resistant Enterobacteriaceae and bacilli.IMPORTANCE Daqu fermentations select for mobile genetic elements conferring heat resistance in Enterobacteriaceae and bacilli. The locus of heat resistance (LHR), a genomic island conferring heat resistance in Enterobacteriaceae, and the spoVA2mob operon, conferring heat resistance on bacterial endospores, were enriched 3- to 5-fold during Daqu fermentation and maturation. It is therefore remarkable that the LHR and the spoVA2mob operon are accumulated in the same food fermentation. The presence of heat-resistant Kosakonia spp. and Bacillus spp. in Daqu is not of concern for food safety; however, both genomic islands are mobile and transferable to pathogenic bacteria or toxin-producing bacteria by horizontal gene transfer. The identification of the LHR and the spoVA2mob operon as indicators of fitness of Enterobacteriaceae and bacilli in Daqu fermentation provides insights into environmental sources of heat-resistant organisms that may contaminate the food supply.

Extended Multiplicative Signal Correction Based Model Transfer for Raman Spectroscopy in Biological Applications.

The chemometric analysis of Raman spectra of biological materials is hampered by spectral variations due to the instrumental setup that overlay the subtle biological changes of interest. Thus, an established statistical model may fail when applied to Raman spectra of samples acquired with a different device. Therefore, model transfer strategies are essential. Herein we report a model transfer approach based on extended multiplicative signal correction (EMSC). As opposed to existing model transfer methods, the EMSC based approach does not require group information on the secondary data sets, thus no extra measurements are required. The proposed model-transfer approach is a preprocessing procedure and can be combined with any method for regression and classification. The performance of EMSC as a model transfer method was demonstrated with a data set of Raman spectra of three Bacillus bacteria spore species ( B. mycoides, B. subtilis, and B. thuringiensis), which were acquired on four Raman spectrometers. A three-group classification by partial least-squares discriminant analysis (PLS-DA) with leave-one-device-out external cross-validation (LODCV) was performed. The mean sensitivities of the prediction on the independent device were considerably improved by the EMSC method. Besides the mean sensitivity, the model transferability was additionally benchmarked by the newly defined numeric markers: (1) relative Pearson's correlation coefficient and (2) relative Fisher's discriminant ratio. We show that these markers have led to consistent conclusions compared to the mean sensitivity of the classification. The advantage of our defined markers is that the evaluation is more effective and objective, because it is independent of the classification models.

Bacillus safensis FO-36b and Bacillus pumilus SAFR-032: a whole genome comparison of two spacecraft assembly facility isolates.

BACKGROUND: Bacillus strains producing highly resistant spores have been isolated from cleanrooms and space craft assembly facilities. Organisms that can survive such conditions merit planetary protection concern and if that resistance can be transferred to other organisms, a health concern too. To further efforts to understand these resistances, the complete genome of Bacillus safensis strain FO-36b, which produces spores resistant to peroxide and radiation was determined. The genome was compared to the complete genome of B. pumilus SAFR-032, and the draft genomes of B. safensis JPL-MERTA-8-2 and the type strain B. pumilus ATCC7061T. Additional comparisons were made to 61 draft genomes that have been mostly identified as strains of B. pumilus or B. safensis. RESULTS: The FO-36b gene order is essentially the same as that in SAFR-032 and other B. pumilus strains. The annotated genome has 3850 open reading frames and 40 noncoding RNAs and riboswitches. Of these, 307 are not shared by SAFR-032, and 65 are also not shared by MERTA and ATCC7061T. The FO-36b genome has ten unique open reading frames and two phage-like regions, homologous to the Bacillus bacteriophage SPP1 and Brevibacillus phage Jimmer1. Differing remnants of the Jimmer1 phage are found in essentially all B. safensis / B. pumilus strains. Seven unique genes are part of these phage elements. Whole Genome Phylogenetic Analysis of the B. pumilus, B. safensis and other Firmicutes genomes, separate them into three distinct clusters. Two clusters are subgroups of B. pumilus while one houses all the B. safensis strains. The Genome-genome distance analysis and a phylogenetic analysis of gyrA sequences corroborated these results. CONCLUSIONS: It is not immediately obvious that the presence or absence of any specific gene or combination of genes is responsible for the variations in resistance seen. It is quite possible that distinctions in gene regulation can alter the expression levels of key proteins thereby changing the organism's resistance properties without gain or loss of a particular gene. What is clear is that phage elements contribute significantly to genome variability. Multiple genome comparison indicates that many strains named as B. pumilus likely belong to the B. safensis group.

Sampling and inactivation of wet disseminated spores from flooring materials, using commercially available robotic vacuum cleaners.

AIMS: Four commercially available robotic vacuum cleaners were assessed for sampling efficiency of wet disseminated Bacillus atrophaeus spores on carpet, polyvinyl chloride (PVC) and laminate flooring. Furthermore, their operability was evaluated and decontamination efficiency of one robot was assessed, using a sodium hypochlorite solution. METHODS AND RESULTS: In an environmental chamber, robots self-navigated around 4 m2 of flooring containing a single contaminated 0·25 m2 tile (c. 104 spores per cm2 ). Contamination levels at predetermined locations were assessed by macrofoam swabs (PVC and laminate) or water soluble tape (carpet), before and after sampling. Robots were dismantled postsampling and spore recoveries assessed. Aerosol contamination was also measured during sampling. Robot sampling efficiencies were variable, however, robots recovered most spores from laminate (up to 17·1%), then PVC and lastly the carpet. All robots spread contamination from the 'hotspot' (all robots spread <0·6% of the contamination to other areas) and became surface contaminated. Spores were detected at low levels during air sampling (<5·6 spores per litre). Liquid decontamination inactivated 99·1% of spores from PVC. CONCLUSIONS: Robotic vacuum cleaners show promise for both sampling and initial decontamination of indoor flooring. SIGNIFICANCE AND IMPACT OF THE STUDY: In the event of a bioterror incident, e.g. deliberate release of Bacillus anthracis spores, areas require sampling to determine the magnitude and extent of contamination, and to establish decontamination efficacy. In this study, we investigate robotic sampling methods against high concentrations of bacterial spores applied by wet deposition to different floorings, contamination spread to other areas, potential transfer of spores to the operators and assessment of a wet vacuum robot for spore inactivation. The robots' usability was evaluated and how they can be employed in real life scenarios. This will help to reduce the economic cost of sampling and the risk to sampling/decontamination teams.