Evaluation of enterotoxin gene expression and enterotoxin production capacity of the probiotic strain Bacillus toyonensis BCT-7112T

Abstract

The aim of the present study was to evaluate the safety of the probiotic strain Bacillus toyonensis BCT-7112T (active ingredient of Toyocerin) in relation to the enterotoxins haemolysin BL (Hbl) and the non-haemolytic enterotoxin (Nhe) by performing a quantitative reverse transcription (RT) real-time polymerase chain reaction (PCR) and a Western blot assay. The expression levels of the enterotoxin genes hblA, hblD, nheA, nheB and nheC, determined by means of RT real-time PCR in B. toyonensis, were lower than those in B. cereus reference strains. No expression of hblC was detected. The Western blot assays of native and 25-fold concentrated supernatants from B. toyonensis, using monoclonal antibodies directed against the Hbl component L1 and the Nhe component NheB, showed weak bands. The NheC component was not detected in the native supernatant, but weakly in the 25-fold concentrated supernatant. According to the results of the present study, the enterotoxin expression and protein levels of B. toyonensis BCT-7112T were absent or clearly lower compared to the B. cereus reference strains. Thus, their ability to form functional enterotoxins can also be considered to be lower or unlikely compared to the B. cereus reference strains. This experimental approach can be implemented when studying the health and safety as well as harmlessness of probiotic microorganisms.

Fig 1. Representative immunoblottings, membrane staining and sab controls of Hbl L1 and Hbl L2 detection.

Native and concentrated (25-fold) supernatants prepared from (1) B. cereus 1230 as positive control, (2) B. subtilis subsp. spizizenii DSM-347 as negative control and (3) B. toyonensis BCT-7112^T. Non-specific protein staining of membranes as loading control is shown as well as respective sab controls. Relevant molecular weights are indicated.

Fig 2. Representative immunoblottings, membrane staining and sab controls of NheB and NheC detection.

Native and concentrated (25-fold) supernatants prepared from (1) B. cereus 1230 as positive control, (2) B. subtilis subsp. spizizenii DSM-347 as negative control and (3) B. toyonensis BCT-7112^T. Non-specific protein staining of membranes as loading control as well as respective sab controls are shown. The sab controls of both compounds are identical because the blocking and incubation conditions were performed identically. Relevant molecular weights are indicated.

Fig 3. Sequencing of B. cereus NheC positive band.

(A) NCBI Ref. Seq. NP_831584.1 (enterotoxin C) from B. cereus ATCC-14579^T database. Appropriate peptides found by sequencing are highlighted in green. (B) NCBI GenBank No. CAB53340.2 (enterotoxin C–B. cereus NVH 1230–88) as query and Acc. No. NP_831584.1 (enterotoxin C–B. cereus ATCC-14579^T) as subject. The putative epitope of the used antibody is highlighted in grey.

Fig 4. Sequencing of B. toyonensis BCT-7112^T NheC positive band.

(A) NCBI GenBank No. AHA10308.1 (enterotoxin C) from B. toyonensis BCT-7112^T database. Appropriate peptides found by sequencing are highlighted in green. (B) NCBI GenBank No. AHA10308.1 (enterotoxin C–B. toyonensis BCT-7112^T) as query and Acc. No. NP_831584.1 (enterotoxin C–B. cereus ATCC-14579^T) as subject. The putative epitope of the NheC antibody is highlighted in grey.