Specific oligonucleotide primers for detection of endoglucanase positive Bacillus subtilis by PCR

Ashe, Stephanie; Maji, U. J.; Sen, R.; Mohanty, Sujata; Maiti, N. K.

doi:10.1007/s13205-013-0177-6

Cited by 23 publications

(17 citation statements)

References 22 publications

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“…subtilis , B. licheniformis , B. velezensis , B. atrophaeus , B. mojavensis , B. vallismortis , and B. subtilis subsp. spizizenii 27 . In particular, B. subtilis subsp.…”

Section: Discussionmentioning

confidence: 99%

Understanding the ontogeny and succession of Bacillus velezensis and B. subtilis subsp. subtilis by focusing on kimchi fermentation

Cho¹,

Jin²,

Kang³

et al. 2018

Sci Rep

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Bacillus subtilis and B. velezensis are frequently isolated from various niches, including fermented foods, water, and soil. Within the Bacillus subtilis group, B. velezensis and B. subtilis subsp. subtilis have received significant attention as biological resources for biotechnology-associated industries. Nevertheless, radical solutions are urgently needed to identify microbes during their ecological succession to accurately confirm their action at the species or subspecies level in diverse environments, such as fermented materials. Thus, in this study, previously published genome data of the B. subtilis group were compared to exploit species- or subspecies-specific genes for use as improved qPCR targets to detect B. velezensis and B. subtilis subsp. subtilis in kimchi samples. In silico analyses of the selected genes and designed primer sequences, in conjunction with SYBR Green real-time PCR, confirmed the robustness of this newly developed assay. Consequently, this study will allow for new insights into the ontogeny and succession of B. velezensis and B. subtilis subsp. subtilis in various niches. Interestingly, in white kimchi without red pepper powder, neither B. subtilis subsp. subtilis nor B. velezensis was detected.

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“…subtilis , B. licheniformis , B. velezensis , B. atrophaeus , B. mojavensis , B. vallismortis , and B. subtilis subsp. spizizenii 27 . In particular, B. subtilis subsp.…”

Section: Discussionmentioning

confidence: 99%

Understanding the ontogeny and succession of Bacillus velezensis and B. subtilis subsp. subtilis by focusing on kimchi fermentation

Cho¹,

Jin²,

Kang³

et al. 2018

Sci Rep

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“…However, the microbiological identity of probiotic preparation genus/species can be investigated by the unique microbial protein spectrum (proteome) mass spectroscopy analysis in MALDI-TOF assays (Azarko & Wendt, 2011;Kosikowska et al, 2015), as presented for closely related Bacillus species: Bacillus subtilis, Bacillus mojavensis, Bacillus vallismortis, Bacillus pumilus residing in the same lyophilized preparation sample . Alternatively, a specific determination can be achieved with qualitative PCR reactions, amplifying specific genome regions (Table 5) characteristic for either a group of probiotic bacteria (Wattiau et al, 2001) or a single Bacillus species (Ashe et al, 2014). Many species from the Bacillus genus are known to display high similarity in the conserved regions of 16S rRNA genes, like Bacillus subtilis and Bacillus pumilus (Berkeley et al, 2008).…”

Section: Characteristics Of Bacillus Sp Probiotic Preparationsmentioning

confidence: 99%

“…An even more specific determination is possible when the unique marker of a strain is determined, like endo-β-1,4-glucanase of B. subtilis (excluding B. pumilus, licheniformis, amyloliquefaciens, thuringiensis, megaterium). In this case, combining the results of two PCR primer sets leads to an accurate determination of group (genus) and species (Ashe et al, 2014). The PCR reaction products can, of course, also be further cloned and/or sequenced or mapped by the RFLP technique (Restriction Fragments Length Polymorphism), providing even more accurate and precise data.…”

Section: Characteristics Of Bacillus Sp Probiotic Preparationsmentioning

confidence: 99%

The promises and risks of probiotic Bacillus species

et al. 2018

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Supplementing the human microbiome with probiotic microorganisms is a proposed solution for civilization syndromes such as dysbiosis and gastrointestinal tract (GIT) disorders. Bimodal probiotic strains of the Bacillus genus constitute the microbiota of the human environment, and are typically found in soil, water, a number of non-dairy fermented foods, as well as in human and animal GIT. Probiotic Bacillus sp. are Gram positive rods, with the ability of sporulation to survive environmental stress and preparation conditions. In vitro models of the human stomach and human studies with probiotic Bacillus reveal the mechanisms of its life cycle and sporulation. The Bacillus sp. probiotic biofilm introduces biochemical effects such as antimicrobial and enzymatic activity, thus contributing to protection from GIT and other infections. Despite the beneficial activity of Bacillus strains belonging to the safety group 1, a number of strains can pose a substantial health risk, carrying genes for various toxins or antibiotic resistance. Commercially available Bacillus probiotic preparations include strains from the subtilis and other closely related phylogenetic clades. Those intended for oral administration in humans, often encapsulated with appropriate supporting materials, still tend to be mislabeled or poorly characterized. Bacillus sp. MALDI-TOF analysis, combined with sequencing of characteristic 16S rRNA or enzyme coding genes, may provide accurate identification. A promising future application of the probiotic Bacillus sp. might be the microflora biocontrol in the human body and the closest human environment. Environmental probiotic Bacillus species display the potential to support human microflora, however controversies regarding the safety of certain strains is a key factor in their still limited application.

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“…The results presented in Table 1 indicate that this method still needs refinement and it would be beneficial to complement it with other techniques. However, in general, a reliable identification of Bacillus species is a challenge due to their very high genome, proteome, and metabolic similarities, and thus a more specific determination requires DNA analysis techniques targeting unique markers of a given strain, such as the presence of endo-ß-1,4-glucanase (Ashe et al, 2014). Table 1 also includes all potentially identified species, not belonging to the Bacillus genus, even with weak species discrimination and showing low ID number after MALDI-TOF evaluation.…”

Section: Preparation 14: All Purpose Cleaner With Probioticsmentioning

confidence: 99%

Identification of bacterial species in probiotic consortiums in selected commercial cleaning preparations

et al. 2019

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The role of environmentally coexisting microflora that often comprises human commensal microbiome is still underestimated. Modern lifestyle changes include hygienic practices, food preparation and eradication of many contagious diseases. In this context, probiotic microorganisms are biocontrol remedies still under development, solving a number of gastrointestinal and immunological issues, while fighting hazardous microbiological biofilms on different surfaces. Probiotics are mainly associated with Lactic Acid Bacteria, however environmental, non-dairy sources are promising ecological niches of probiotic spore-forming Bacillus species. Industrial applications of these “unconventional” probiotics take an advantage of their sporulating activity which greatly enhances their compatibility with chemical formulations used in the household, cosmetic or pharmaceutical chemistry. We have analysed 14 commercially available chemical products, labelled or described to contain a probiotic or biologically active component. It was determined that in the most part they relay on consortiums of spore-forming, very closely related Bacillus species, exhibiting bimodal existence in the environment and the gastrointestinal tract (GIT). In addition, we have found a number of non-sporulating species. Overall, the microorganisms found included: Bacillus licheniformis, Bacillus subtilis, Bacillus pumilus, Citrobacter freundii, Klebsiella oxytoca, Stenotrophomonas malthophila, Serratia liquefaciens, Bacillus altitudinis, Lactobacillus gastricus, Bacillus megaterium, Lactobacillus nagelii, Aromatoleum buckelii, Trichosporon mucoides, Clostridium novyi, Bacteroides uniformis. As some of the listed species may become opportunistic pathogens, this raises an important question concerning general safety of probiotics, as apparently the manufacturing procedures do not always lead to microbiologically defined or sufficiently controlled microorganism consortiums.

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Specific oligonucleotide primers for detection of endoglucanase positive Bacillus subtilis by PCR

Cited by 23 publications

References 22 publications

Understanding the ontogeny and succession of Bacillus velezensis and B. subtilis subsp. subtilis by focusing on kimchi fermentation

Understanding the ontogeny and succession of Bacillus velezensis and B. subtilis subsp. subtilis by focusing on kimchi fermentation

The promises and risks of probiotic Bacillus species

Identification of bacterial species in probiotic consortiums in selected commercial cleaning preparations

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