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. 2016 May 23:7:718.
doi: 10.3389/fmicb.2016.00718. eCollection 2016.

Probiotic Bacillus cereus Strains, a Potential Risk for Public Health in China

Kui Zhu  1 , Christina S Hölzel  2 , Yifang Cui  3 , Ricarda Mayer  2 , Yang Wang  3 , Richard Dietrich  2 , Andrea Didier  2 , Rupert Bassitta  2 , Erwin Märtlbauer  2 , Shuangyang Ding  3
Affiliations

Probiotic Bacillus cereus Strains, a Potential Risk for Public Health in China

Kui Zhu et al. Front Microbiol. .

Abstract

Bacillus cereus is an important cause of foodborne infectious disease and food poisoning. However, B. cereus has also been used as a probiotic in human medicine and livestock production, with low standards of safety assessment. In this study, we evaluated the safety of 15 commercial probiotic B. cereus preparations from China in terms of mislabeling, toxin production, and transferable antimicrobial resistance. Most preparations were incorrectly labeled, as they contained additional bacterial species; one product did not contain viable B. cereus at all. In total, 18 B. cereus group strains-specifically B. cereus and Bacillus thuringiensis-were isolated. Enterotoxin genes nhe, hbl, and cytK1, as well as the ces-gene were assessed by PCR. Enterotoxin production and cytotoxicity were confirmed by ELISA and cell culture assays, respectively. All isolated B. cereus group strains produced the enterotoxin Nhe; 15 strains additionally produced Hbl. Antimicrobial resistance was assessed by microdilution; resistance genes were detected by PCR and further characterized by sequencing, transformation and conjugation assays. Nearly half of the strains harbored the antimicrobial resistance gene tet(45). In one strain, tet(45) was situated on a mobile genetic element-encoding a site-specific recombination mechanism-and was transferable to Staphylococcus aureus and Bacillus subtilis by electro-transformation. In view of the wide and uncontrolled use of these products, stricter regulations for safety assessment, including determination of virulence factors and transferable antimicrobial resistance genes, are urgently needed.

Keywords: Bacillus cereus; China; antimicrobial resistance; dif site; enterotoxin; probiotic; site-specific recombination; tetracycline resistance gene tet(45).

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Figures

Figure 1
Figure 1
Partial amino acid sequence of Tet(45). Amino acid sequence alignment of tet(45)-amplicons (a, strain No. 6f, 7d, 8h, 11, 13d, 14e and 15d) and the mobile genetic element- tet(45)-amplicon (b, strain No. 9i) with Tet(45) from Bhargavaea spp. (Ref, You et al., ; blastx, https://npsa-prabi.ibcp.fr).
Figure 2
Figure 2
Agarose gel electrophoresis after plasmid extraction of B. cereus 9i. 1–3: three independent replicates. S supercoiled ladder, L linear ladder. Note: two putative plasmid bands were visible in a second agarose gel electrophoresis of extract 9i-3, at the same height as in extract 9i-1.
Figure 3
Figure 3
Genetic environment of tet(45) in B. cereus 9i. Surrounding sequences of tet(45) in B. cereus 9i (this study) and B. cereus MSX-D12 (Timmery et al., 2011) are shown relatively to B. cereus NC7401 (AP007209.1). Note: most Genbank entries for Bacillus cereus show the same 5'-3'-orientation of the YdcF-like-/ S-layer protein encoding sequences and the Pas domain S-box-/Cof-like protein encoding sequences as 9i and MSX-D12, respectively, not as NC7401. NC7401 was chosen as reference sequence since the palindromic sequence between the Pas domain S-box-/Cof-like protein encoding sequences is conserved here, but not in the Bacillus cereus reference genomes.
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