Phylogenetic Analysis of Bacillus cereus sensu lato Isolates from Commercial Bee Pollen Using tRNACys-PCR

Abstract

Endospore-forming bacteria related to the Bacillus cereus group produce toxins that cause illnesses in organisms from invertebrates to mammals, including foodborne illnesses in humans. As commercial bee pollen can be contaminated with these bacteria, a comprehensive microbiological risk assessment of commercial bee pollen must be incorporated into the relevant regulatory requirements, including those that apply in Mexico. To facilitate detection of members of this group of bacteria, we have developed a PCR strategy that is based on the amplification of the single-copy tRNACys gene and specific genes associated with tRNACys to detect Bacillus cereus sensu lato (B. cereus s.l.). This tRNACys-PCR-based approach was used to examine commercial bee pollen for endospore-forming bacteria. Our analysis revealed that 3% of the endospore-forming colonies isolated from a commercial source of bee pollen were related to B. cereus s.l., and this result was corroborated by phylogenetic analysis, bacterial identification via MALDI-TOF MS, and detection of enterotoxin genes encoding the HBL and NHE complexes. The results show that the isolated colonies are closely related phylogenetically to B. cereus, B. thuringiensis, and B. bombysepticus. Our results indicate that the tRNACys-PCR, combined with other molecular tools, will be a useful approach for identifying B. cereus s.l. and will assist in controlling the spread of potential pathogens.

Diversity of aerobic spore-forming bacteria isolated from fresh bee pollen intended for human consumption in Argentina

“…The microbiota of spore-forming bacteria isolated from bee pollen in Argentina was similar to those reported by other authors in different countries (Dinkov, 2018;Gilliam, 1979;Gilliam et al, 1990;Hern andez Flores et al, 2020;Heydenreich et al, 2012). Nevertheless, to our knowledge, the present study is the first report of the presence of viable spores of B. clausii, B. coagulans, B. simplex, and R. stabekisii in bee pollen.…”

“…Another study, using a tRNA Cys-PCR-based approach, examined commercial bee pollen samples from Mexico revealing that 3% of the endospore-forming colonies isolated from commercial bee pollen were related to B. cereus s.l. (Hern andez Flores et al, 2020). That analysis identified members of the genus Bacillus as the most common species found in association with commercial bee pollen, followed by Paenibacillus species.…”

“…That analysis identified members of the genus Bacillus as the most common species found in association with commercial bee pollen, followed by Paenibacillus species. Besides B. cereus, other identified species were B. licheniformis, B. pumilus, B. subtilis, B. sonorensis B. subtilis, B. mojavensis, B. megaterium, B. altitudinis, B. endophyticus, P. odorifer, P. chitinolyticus, P. peoriae, P. polymyxa and, P. rhizosphaeriae (Hern andez Flores et al, 2020). In Germany, B. subtilis, B. cereus L. sphaericus, Brevibacillus brevis, and P. alvei were isolated from grass pollen (Heydenreich et al, 2012).…”

“…Also, due to its structure and nutritional composition, bee pollen provides a unique microhabitat for yeasts and bacterial communities, mainly Proteobacteria, Actinobacteria, and Firmicutes (Ambika Manirajan et al, 2016;Moreno Andrade et al, 2018). Within Firmicutes, spore-forming bacteria can survive in pollen grains for several months, and some representatives of this group are pathogens, i.e., Paenibacillus larvae, the causal agent of American foulbrood disease of honey bee larvae (AFB) (de Sousa Pereira et al, 2019;Genersch, 2010;Gochnauer & Corner, 1974;Moreno Andrade et al, 2019;Sekulja et al, 2014); Bacillus cereus, the etiological agent of gastrointestinal diseases in humans Fern andez et al, 2020;Hern andez Flores et al, 2020;L opez et al, 2020;Stenfors Arnesen et al, 2008), Bacillus megaterium ( Alvarez Gilliam, 1979;Gilliam et al, 1990;Hern andez Flores et al, 2020;Hosny et al, 2018;L opez et al, 2013;L opez & Alippi, 2010); and Clostridium botulinum that produces several toxins to humans (G€ uc€ uko glu et al, 2020;Johnson & Bradshaw, 2001). Gonz alez et al (2005) studying the mycobiota of fresh bee pollen in Spain and Argentina concluded that the most critical stage is pollen collection from traps, mainly if the collection is delayed for a long time, favoring fungal growth and mycotoxin production.…”

Detection of Bacillus cereus sensu lato Isolates Posing Potential Health Risks in Mexican Chili Powder

“…Unfortunately, knowledge about the presence of the pathogen B. cereus in Mexican chili powder is limited and of equal or greater concern is the lack of phylogenetic information on the microbes that may be present as contaminants in the chili powder. To address these deficiencies in our knowledge, we used standard and molecular microbial procedures on Mexican chili powder samples elaborated from Capsicum annuum L., and extended our tRNA Cys -PCR method previously reported [24] to detect the presence of antibiotic-resistant B. cereus s.l. isolates.…”