Incidence and characterisation of Bacillus cereus bacteriophages from Thua Nao, a Thai fermented soybean product

Samples collection

Twenty-six Thua Nao samples (nine fresh and 17 dried samples) were purchased from different local markets in Chiang Rai (19 samples), Chiang Mai (five samples), Lampang (one sample), and Mae Hong Son (one sample). In general, approximately 100 g of each sample was collected in sampling bags, transferred to the laboratory, and stored at 4°C until required.

Isolation of phages

Bacterial strains used for bacteriophage isolation were three B. cereus reference strains (ATCC 11778, TISTR 687, and TISTR 1527), B. cereus TN69, and B. thuringiensis S2–3. These bacteria were grown in either tryptic soy broth (TSB) or agar (Difco Laboratory, Detroit, MI, USA) at 37°C overnight. To isolate the bacteriophages, 20 g of the sample was mixed with 80 ml of TSB and 1 ml of each overnight-grown bacterial host culture. The mixture was incubated at 37°C with shaking (180 rpm) for 72 h. Then, the culture was centrifuged at 8000 × g, 10 min, and the supernatant was filtered through a 0.2 μm membrane filter (Whatman, Little Chalfont, UK). The filtrate was used in a plaque assay in order to detect bacteriophages, that were then propagated and purified following the method previously described by Sambrook and Russel [27].

Host range determination

Isolated bacteriophages were tested for host range against B. cereus strains (ATCC 11778, TISTR 687, TISTR 1527, and TN69); B. subtilis strains (ATCC 6633, ASA, S1–13, S4–5, TN3, and TN51); B. thuringiensis S2–3; B. licheniformis TISTR 1109; and five non-Bacillus species (Enterobacter aerogenes TISTR 1540, Escherichia coli TISTR 527, Micrococcus luteus TISTR 884, Pseudomonas aeruginosa TISTR 781, and Staphylococcus aureus TISTR 746). The spot test was then performed to determine the host range of the isolated phages. For this, the soft agar overlayer containing the indicator strain was prepared, on which 20 μl of the phage stock (10⁴ – 10⁶ PFU/ml) was spotted and incubated overnight for lysis zone formation.

Determination of multiplicity of infection (MOI)

MOI was typically defined as the ratio of virus particles to potential host cells. In order to determine the optimal MOI, the early exponential phase bacterial host strain cultured in TSB broth at 37°C were inoculated with dilutions of the phage samples to give the MOI of 0.1, 1, 10 and 100. Bacteria-free suspensions and phage-free suspensions were also prepared as the controls for all experiments. After incubation at 37°C for 2–3 h, the samples in triplicate were taken from each MOI experiment, and the viable bacterial cell counts were determined by plating samples on TSB agar and then incubating overnight at 37°C. To evaluate the optimal MOI, a reduction rate (log Nt/Nc) was calculated by comparing the number of the host cells in the control (Nc) with the number of the host cells treated with the bacteriophage (Nt) after 2–3 h incubation.

Phage morphology examination

A drop of purified phage stock (approximately 10⁸ PFU/ml) was spotted onto a carbon-coated copper grid and left at room temperature for 6 min. After removal of excess fluid, negative staining with 2% (w/v) uranyl acetate for 10 min was then applied on the sample. Excess fluid was removed by absorbent paper. After drying, the phage morphology was observed with a JEOL transmission electron microscope (JEM-2010, Japan) at an accelerating voltage of 160kV. The phages were classified based on the information of the International Committee on Taxonomy of Viruses [28]. The phage size was determined from the average of at least three independent measurements.

Phage stability under different conditions

The stability of the isolated phages was then evaluated under different conditions, including pH, temperature, UV, and chloroform. Phage samples were incubated in TSB broths with different pH values in the range of 3 – 13 at 37°C for 2 h. Similarly, phage stocks were incubated at 4, 37, 50, 55 and 60°C for 2 h. For UV stability assay, the phage samples were placed in sterile Petri dishes and irradiated for 0, 15, 30, 45, 60 and 120 min with a UV lamp (λ = 254 nm) in a laminar flow cabinet. Resistance to chloroform was also determined by adding 5% chloroform to the phage samples. In addition, to investigate the presence of lipid in the viral capsid, an equal volume of the phage sample was mixed with chloroform and incubated for 30 min at room temperature [29]. After all these experiments, the bacteriophage titre was determined using the double-layer agar technique.

Ethical approval: The conducted research is not related to either human or animals use.

Incidence of B. cereus bacteriophages in Thua Nao

Twenty-six Thai fermented soybean samples (Thua Nao), including 17 dried samples and nine fresh samples, were used to screen and isolate B. cereus bacteriophages using five B. cereus sensu lato strains as indicators. Based on this experiment, only two Thua Nao samples were shown to contain bacteriophages (to form plaques), and two bacteriophages were thus isolated. These included BaceFT01 and BaceCM02 obtained from Chiang Rai and Chiang Mai sources, respectively. Both bacteriophages produced clear plaques with different sizes (Figure 1) on B. cereus TISTR687 bacterial lawn, and thus this bacterial strain was used for all subsequent experiments. For further characterization, the two phages were propagated and purified using the liquid culture technique. A high-titre stock was also prepared from the plate elution method.

Figure 1

Plaque formation by double layer nutrient agar plates of B. cereus bacteriophages isolated from Thua Nao: BaceFT01 (A) and BaceCM02 (B).

The isolation rate of B. cereus bacteriophages obtained from our study was quite low (7.7%), when compared to the data reported by Shin et al. [20], Bandara et al. [30], and Oh et al. [21]. These previous works showed that the isolation rate of B. cereus phages from Cheonggukjang products (Korean fermented soybean samples) was high ranging from 37.8 to 100%. Thua Nao and Cheonggukjang are similar in terms of the manufacturing process and product type. Other similar fermented soybean products include Japanese Natto and Indian Kinema [22]. B. cereus, an opportunistic foodborne pathogenic bacterium, can be found in various food products including cereals, dairy products, and dried foods. The presence of B. cereus in the food products can be either from the original food substrates, production process, or environmental contamination of the food products. Interestingly, there might be a relationship between the B. cereus host and the B. cereus bacteriophages. Based on our study, B. cereus was not found in all Thua Nao samples when initially screened by mannitol egg yolk polymyxin (MYP) agar plates (data not shown). The low incidence (8.3%) of B. cereus in Thua Nao samples was also found as reported by Suriyanoi et al. [31]. It should be noted, however that the high incidence of B. cereus was also detected in Thua Nao (78%) as described by Inatsu et al. [32].

Host range of B. cereus bacteriophages

The host range of the two bacteriophages BaceFT01 and BaceCM02 was determined against both Bacillus (twelve strains) and non-Bacillus bacterial species (five strains). Our data revealed that the BaceFT01 and BaceCM02 bacteriophages could infect only two B. cereus strains (ATCC 11778 and TISTR 687). Both bacteriophages did not infect other bacterial hosts used (including the remaining three B. cereus sensu lato strains and other Bacillus species). This finding was consistent with the previous work demonstrating that B. cereus bacteriophages isolated from the fermented soybean products were only specific to B. cereus bacterial host [20, 21, 30]. It is well known that most bacteriophages are specific for a bacterial species and many of them can only infect the specific strains within a species. There are advantages and disadvantages of this limited host range. For example, these two B. cereus bacteriophages may be of great importance if they could be used to prevent contamination of B. cereus during a soybean fermentation. Such a high specificity will not cause a harm to other microbes especially for the beneficial microbes (e.g., B. subtilis, a bacterial starter culture in the soybean fermentation process). In contrast, the bacteriophages with a broad host infectivity (i.e., to many B. cereus strains) would be of great significance for controlling the disease outbreaks.

Optimal multiplicity of infection (MOI)

The lytic activity of the two bacteriophages against B. cereus was then assessed at different MOIs ranging from 0.1 to 100, and our data showed that the optimal MOI value of both bacteriophages was 100 (Figure 2). In general, the bacterial growth inhibition seemed to be MOI-dependent. This was evident especially for the bacterial growth inhibition derived from the treatment of the bacteriophage BaceFT01. Although the addition of the phage BaceCM02 with MOI values of 1 and 10 seemed not to affect the bacterial growth, it could be clearly seen that the phage BaceCM02 with an MOI of 100 showed the highest reduction in bacterial growth (more than 2 log reduction). The knowledge on the phage characteristics (e.g., growth and stability) is crucial considering from the application viewpoint. For example, the level of the bacteriophage may be a limiting factor, considering that during the initial infection process, the ratio between the phage and the host affects the attachment and adsorption, leading to specificity in the phage-host interactions [33]. In addition, the optimal MOI is suggested to be the recommended mixing ratio for large-scale proliferation and enrichment of the bacteriophage [34].

Figure 2

Log reduction of B. cereus TISTR 687 with different multiplicities of infection (MOIs) of bacteriophage BaceFT01 (Δ) and BaceCM02 (×).

Morphology of B. cereus bacteriophages

The morphological characteristics of B. cereus bacteriophages were determined using transmission electron microscopy (Figure 3). Both isolates consisted of an icosahedral head (90 and 62 nm in size for the phages BaceFT01 and BaceCM02, respectively), and a long contractile tail (166 and 188 nm, respectively) (Figure 3). Both bacteriophages were classified as Myoviridae phages based on their morphology. To date, most known bacteriophages (more than 96%) belong to the order Caudovirales (or tailed bacteriophage group) [35]. Based on this distinct tailed structure, the phages in the order Caudovirales can be classified into three families: Myoviridae (long contractile tails), Siphoviridae (long non-contractile tails), and Podoviridae (short non-contractile tails). At present, it has been reported that the bacteriophages infecting B. cereus are tailed bacteriophages being the members of the three families, although it should be noted that the two families of Myoviridae and Siphoviridae are the most abundant in the B. cereus group phage [36]. This finding coincided with previous studies, especially when considering that the B. cereus bacteriophages isolated from the fermented soybean products, were members of the Myoviridae and Siphoviridae families [26].

Figure 3

Transmission electron micrographs of B. cereus bacteriophages: BaceFT01 (A) and BaceCM02 (B). The scale bars shown are 100 nm.

Phage stability

To evaluate the phage suitability for potential applications (e.g., as a biocontrol agent in the food industry), we undertook the phage stability assays under different conditions including pH, temperature, UV, and chloroform. We found that the activity of the two phages (BaceFT01 and BaceCM02) was similar as both phages were stable between 4 and 37°C (Figure 4). The temperature is one of the key factors for bacteriophage survival [37]. It is known that the temperature involves the mechanism of attachment, penetration, multiplication, and the length of the latent period (in the case of lysogenic phages). For example, at lower than optimal temperatures, fewer phage genetic materials could penetrate into the bacterial hosts leading to a slow (or delayed) rate in the multiplication phase. Moreover, bacteriophages could potentially lose the ability to attach to the host cells or might simply disintegrate in the presence of high temperature [38]. For pH stability, both bacteriophages were stable (>60% of the phage titre remained) at pH values from 6 to 10 (Figure 5). The pH value is another important factor affecting the phage occurrence, stability, and survivability. Under an acidic condition, this also causes phage coagulation and precipitation [38]. The phage stability when treated with UV (λ = 254 nm) was also investigated. Figure 6 showed that the two phages (BaceFT01 and BaceCM02) remained active after 15 min of UV irradiation. Both phages were completely inactive after 2 h-UV treatment. The ultraviolet light is known to cause DNA damage, and besides, it can also cause a peptide bond alteration. The previous work has shown that UV is a major cause of phage mortality especially in marine habitats [39]. In the chloroform test, our results showed that the viability of both phages was unaffected (>90% of the phage titre remained) in the presence of 5% chloroform. Additionally, the treatment of chloroform (using an equal volume of chloroform and phage sample and incubating for 30 min) had no effect on the activity of both phages, suggesting an absence of lipids in the phage capsid.

Figure 4

Influence of temperature on stability of the B. cereus bacteriophages BaceFT01 (Δ) and BaceCM02 (×).

Figure 5

Survival of the B. cereus bacteriophages BaceFT01 (Δ) and BaceCM02 (×) after 2 h at 37°C in TSB broths with different pH values in the range of 3 – 13.

Figure 6

Effect of ultraviolet irradiation on viability of the B. cereus bacteriophages BaceFT01 (Δ) and BaceCM02 (×).