Quantification of Endospore-Forming Firmicutes by Quantitative PCR with the Functional Genespo0A

Abstract

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 10 4 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 Combination of Extreme Environmental Conditions Favor the Prevalence of Endospore-Forming Firmicutes

“…Relative GCN ( spo0A GCN/16S rRNA gene GCN) ranged from <0.0001 to 100 %, with an average GCN of 6.79% ( Supplementary Table 3 ). Considering that the 16S rRNA gene is found in multiple copies per bacterial genome (Farrelly et al, 1995), while spo0A gene is a single-copy gene (Galperin et al, 2012; Bueche et al, 2013), in addition to the comparison without normalization, a second calculation was made using for normalization the average number of rRNA gene operons found in Bacteria and in Firmicutes (Ratio % rrnDB; Supplementary Table 3 ). The normalization did not change the patterns of relative abundance obtained and thus further analyses were conducted using the non-normalized ratio.…”

“…First, for quantifying gene copy numbers (GCN) of total bacteria, qPCR amplification of the V3 hyper-variable region from the 16S rRNA gene was carried out. The primers used were 338f (5′-ACTCCTACGGGAGGCAGCAG-3′) and 520r (5′-ATTACCGCGGCTGCTGG-3′) (Muyzer et al, 1995; Bakke et al, 2011) and amplification was carried out under conditions previously described (Bueche et al, 2013). For the quantification of GCN for EFF, a primer pair targeting the spo0A gene was used as previously described (Bueche et al, 2013).…”

“…The primers used were 338f (5′-ACTCCTACGGGAGGCAGCAG-3′) and 520r (5′-ATTACCGCGGCTGCTGG-3′) (Muyzer et al, 1995; Bakke et al, 2011) and amplification was carried out under conditions previously described (Bueche et al, 2013). For the quantification of GCN for EFF, a primer pair targeting the spo0A gene was used as previously described (Bueche et al, 2013). Reactions were carried out in a final volume of 10 with 5 μl Rotor-Gene SYBR green PCR master mix (Qiagen, Germany), 1 and 0.45 μM of primers spo0A655f and spo0A834r, respectively, and 4 ng of DNA template.…”

“…Reactions were carried out in a final volume of 10 with 5 μl Rotor-Gene SYBR green PCR master mix (Qiagen, Germany), 1 and 0.45 μM of primers spo0A655f and spo0A834r, respectively, and 4 ng of DNA template. Amplification conditions were previously described (Bueche et al, 2013). All reactions were performed in triplicates.…”

“…Abundance was estimated by measuring the gene count numbers (GCN) for either a spore-specific gene marker ( spo0A gene), or a general bacterial gene marker (16S rRNA gene). The selection of spo0A as molecular marker for sporulation is a compromise as this gene has been in some cases found outside EFF (Galperin et al, 2012), but has been identified as the most prevalent gene among the genomes of known EFF (Bueche et al, 2013). For the first time, we present experimental evidence for the ubiquity of this group and we propose an ecological explanation for the under-detection of EFF in microbial community studies.…”