Study on the Identification Methods for Effective Microorganisms in Commercially Available Organic Agriculture Materials

Abstract

The identification of microorganisms in closely related groups is challenging. The present work focused on the different molecular methodology for the accurate microbial identification in the five commercially available organic agriculture materials enriched with effective microorganisms. From the tested five organic agricultural materials, a total of seven distinct bacterial colonies (A-1, B-1, C-1, D-1, E-1, E-2, and E-3) were isolated and processed for sequential identification utilizing HiCrome™ Bacillus agar, biochemical tests with API CHB50, 16S rRNA gene analysis, random amplified polymorphic DNA (RAPD), and species-specific PCR analysis. All the isolated microorganisms were Gram-positive rods and spore former belonging to Bacillus group and appeared as a differential characteristic feature on HiCrome™ Bacillus agar. All isolates showed high-percentage similarities with the different members of Bacillus species in biochemical testing and 16S rRNA gene analysis. The collective identification results revealed isolates, A-1, B-1, and C-1, close to B. velezensis. Further RAPD-PCR and species-specific PCR discriminated and provided confirmatory evidence for D-1 as B. thuringiensis and E-1, E-2, and E-3 as B. licheniformis, respectively. In addition, presence of B. thuringiensis was also confirmed by toxin crystal protein staining. In conclusion, the species-specific primers could be used as a rapid and accurate identification tool to discriminate closely related Bacillus species such as B. subtilis, B. licheniformis, and B. thuringiensis.

Phenotypic Analysis, Molecular Characterization, and Antibiogram of Caries-Causing Bacteria Isolated from Dental Patients

“…A pure colony was suspended in saline and a loopful of this suspension was streaked on blood agar, incubated at 37 °C for 24 h [ 26 ]. Selective colonies were picked for Gram staining after observing colony morphology and were evaluated for the biochemical reaction using a commercially available rapid bacterial detection kit (API-Biomeurix France) [ 27 , 28 ].…”

Safety Analysis of Korean Cottage Industries’ Doenjang, a Traditional Fermented Soybean Product: A Special Reference to Biogenic Amines

“…Three samples (De-2, De-5, and De-19) were contaminated with B. cereus at levels above the recommended limit of 4 log CFU/g, as suggested by the national standard of Korea [26]. There is a high similarity between B. cereus and B. thuringiensis, which cannot be segregated by biochemical and molecular characterization based on 16S rRNA gene sequencing [27]. Therefore, to confirm that the isolates were B. cereus and not B. thuringiensis, crystal protein staining was performed as described by Bahuguna et al [27] (Supplementary Material M4).…”

“…There is a high similarity between B. cereus and B. thuringiensis, which cannot be segregated by biochemical and molecular characterization based on 16S rRNA gene sequencing [27]. Therefore, to confirm that the isolates were B. cereus and not B. thuringiensis, crystal protein staining was performed as described by Bahuguna et al [27] (Supplementary Material M4). None of the isolates displayed the formation of crystal proteins, a typical marker of B. thuringiensis, confirming that they were B. cereus and not B. thuringiensis.…”

In Vivo Efficacy of Bacillus velezensis Isolated from Korean Gochang Bokbunja Vinegar against Carbapenem-Resistant Klebsiella pneumoniae Infections

“…They were obtained from the Korean Collection for Type Cultures (KCTC) as reference organisms to help us to distinguish closely related species in the Bacillus genus. Extracted DNA was then diluted to run qRT-PCR using speciesspecific primers (Kwon et al 2009;Huang et al 2017a;Dunlap 2019;Bahuguna et al 2020). A primer for the macrolactin gene was used to distinguish between B. velezensis and B. amyloliquefaciens (Fan et al 2018;Li et al 2021b).…”