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Bacterial endospores are highly specialized cellular forms that allow Endospore forming Firmicutes (EFF) to tolerate harsh environmental conditions. EFF are considered ubiquitous in natural environments in particular those subjected to stress conditions. In addition to natural habitats EFF are often the cause of contamination problems in anthropogenic environments such as industrial production plants or hospitals. It is therefore desirable to assess their prevalence in environmental and industrial fields. To this end a high sensitivity detection method is still needed. The aim of this study was to develop and evaluate an approach based on quantitative PCR. For this the suitability of functional genes specific for and common to all EFF were evaluated. Seven genes were considered but only spo0A was retained to identify conserved regions for qPCR primer design. An approach based on multivariate analysis was developed for primer design. Two primer sets were obtained and evaluated with 16 pure cultures including representatives of the genera Bacillus Paenibacillus Brevibacillus Geobacillus Alicyclobacillus Sulfobacillus Clostridium and Desulfotomaculum as well as with environmental samples. The primer sets developed gave a reliable quantification when tested on laboratory strains with the exception of Sulfobacillus and Desulfotomaculum. A test using sediment samples with a diverse EFF community also gave a reliable quantification compared to 16S rRNA gene pyrosequencing. A detection limit of about 104 cells (or spores) per gram of initial material was calculated given this method a promising potential for the detection of EFF in a wide range of applications.

In this study, we developed and validated a cultureindependent method for diversity surveys to specifically detect endospore-forming Firmicutes. The global transcription regulator of sporulation (spo0A) was identified as a gene marker for endosporeforming Firmicutes. To enable phylogenetic classification, we designed a set of primers amplifying a 602 bp fragment of spo0A that we evaluated in pure cultures and environmental samples. The amplification was positive for 35 strains from 11 genera, yet negative for strains from Alicyclobacillus and Sulfobacillus. We also evaluated various DNA extraction methods because endospores often result in reduced yields. Our results demonstrate that procedures utilizing increased physical force improve DNA extraction. An optimized DNA extraction method on biomass pre-extracted from the environmental sample source (indirect DNA extraction) followed by amplification with the aforementioned primers for spo0A was then tested in sediments from two different sources. Specifically, we validated our cultureindependent diversity survey methodology on a set of 8338 environmental spo0A sequences obtained from the sediments of Lakes Geneva (Switzerland) and Baikal (Russia). The phylogenetic affiliation of the environmental sequences revealed a substantial number of new clades within endospore-formers. This novel culture-independent approach provides a significant experimental improvement that enables exploration of the diversity of endospore-forming Firmicutes.

Bacterial endospores are highly specialized cellular forms that allow endospore-forming Firmicutes (EFF) to tolerate harsh environmental conditions. EFF are considered ubiquitous in natural environments, in particular, those subjected to stress conditions. In addition to natural habitats, EFF are often the cause of contamination problems in anthropogenic environments, such as industrial production plants or hospitals. It is therefore desirable to assess their prevalence in environmental and industrial fields. To this end, a high-sensitivity detection method is still needed. The aim of this study was to develop and evaluate an approach based on quantitative PCR (qPCR). For this, the suitability of functional genes specific for and common to all EFF were evaluated. Seven genes were considered, but only spo0A was retained to identify conserved regions for qPCR primer design. An approach based on multivariate analysis was developed for primer design. Two primer sets were obtained and evaluated with 16 pure cultures, including representatives of the genera Bacillus, Paenibacillus, Brevibacillus, Geobacillus, Alicyclobacillus, Sulfobacillus, Clostridium, and Desulfotomaculum, as well as with environmental samples. The primer sets developed gave a reliable quantification when tested on laboratory strains, with the exception of Sulfobacillus and Desulfotomaculum. A test using sediment samples with a diverse EFF community also gave a reliable quantification compared to 16S rRNA gene pyrosequencing. A detection limit of about 104 cells (or spores) per gram of initial material was calculated, indicating this method has a promising potential for the detection of EFF over a wide range of applications.

A novel endospore forming bacterium designated strain GSsed3T was isolated from deposits clogging aboveground filters from the geothermal power plant of Gross Schoenebeck in Northern Germany. The novel isolate was Gram staining positive facultative anaerobe catalase positive and oxidase positive. Optimum growth occurred at 60 °C 0.5 (w/v) NaCl and pH 7 8. Analysis of the 16S rRNA sequence similarity indicated that strain GSsed3T belongs to the genus Anoxybacillus and showed 99.8 sequence similarity to Anoxybacillus rupiensis R270T 98.2 similarity to Anoxybacillus tepidamans GS5 97T 97.9 similarity to Anoxybacillus voinovskiensis TH13T 97.7 similarity to Anoxybacillus caldiproteolyticus DSM 15730T and 97.6 similarity to Anoxybacillus amylolyticus MR3CT. DNA DNA hybridization (DDH) indicated only 16 relatedness to A. rupiensis DSM 17127T. Furthermore DDH estimation based on genomes analysis indicated only 19.9 overall nucleotide similarity to A. amylolyticus DSM 15939T. The major respiratory menaquinone was MK 8. The polar lipid profile consisted of phosphatidylethanolamine phosphatidylglycerol diphosphatidylglycerol one unknown phosphoglycolipid and one unknown phospholipid. The predominant cellular fatty acids were iso C15:0 iso C17:0 C16:0 iso C16:0 and anteiso C17:0. The peptidoglycan type was A1? meso Dpm direct. The genomic DNA G+C content of the strain was 46.9 mol. The phenotypic genotypic and chemotaxonomic characterization indicated that strain GSsed3T differs from related species of the genus. Therefore strain GSsed3T is considered to be a novel species of the genus Anoxybacillus for which the name Anoxybacillus geothermalis sp. nov. is proposed. The type strain of Anoxybacillus geothermalis is GSsed3T ( = CCOS808T = ATCC BAA2555T).