A total of 61 isolates of B. cereus collected between 2007 and 2015 were analysed in the study. Twenty-six of them originated from patients hospitalised in different clinical wards in Ljubljana hospitals. Isolates were obtained from various specimens such as, wounds, burns, faeces, other excreta, ear ducts, nose mucosa swabs, etc. Another thirty were isolated from food samples. Their sources were raw milk from individual farms, pasteurised milk, cream, ice-cream, skim milk powder, ultra-high temperature-treated milk produced by a Slovenian dairy (21 strains), and salad, rice meal, pudding, infant food, selected sauces, dumplings, spices, beefsteaks, etc. prepared in public catering plants (9 strains). Five isolates were obtained from drinking and underground water. Identification of the strains was carried out by conventional methods including colony morphology, cell morphological and physiological characteristics and haemolytic activity (ISO 7932 2004).
The isolates were biochemically identified with the API 50CHB and API10 S test systems using the API WEB identification programme Vitek 2.1 (bioMerieux, Marcy–I’Etoile, France) and by multiplex PCR according to Park et al. (2007) and Leski et al. (2009). Total DNA was extracted using the SDS method, followed by purification using the phenol-chloroform-isoamyl alcohol protocol, as previously described (Mäntynen and Lindström 1998; Moore et al. 2004; Sambrook et al. 1989).
Reference strains used for the quality control: B. cereus ATCC 14579T, B. mycoides IAM 1190, B. thuringiensis ATCC 10792 (CCM, Brno, Czech Republic), B. cereus ATCC 11778 (Oxoid, Cambridge, UK) and B. subtilis BGA (Merck, Darmstadt, Germany).
DNA extraction and PFGE for detection of genetic diversity of B. cereus strains
PFGE analysis of the B. cereus isolates was performed according to Liu et al. (1997) and Sjölund et al. (2005) with some modifications.
All strains were cultured on blood agar (BD BBLTM ) with the ampicillin discs (AM-10 mg, BD BBL ™) placed on the surface of each plate. After incubation at 37 °C for 24 h, the colonies were transferred into 2 ml of the BHI broth (Merck, Germany) and incubated with shaking at 37 °C for 4 h. Cells were harvested by centrifugation and the cell pellet was resuspended in 0.5 ml of SE buffer (75 mM NaCl, 25 mM EDTA [pH 7.5], both Sigma-Aldrich, Germany) and the centrifugation was repeated. The supernatants were removed, and the cell lysis took place in 500 mL of SE buffer containing 105 mg of lysozyme (Sigma-Aldrich, Germany) and 10 U of lysostaphin (Sigma-Aldrich, Germany) at 37 °C for 1 h.
The bacterial suspension was then mixed with 500 mL of 1% Low-Melting agarose (Invitrogen, USA), dispensed in a plug mould (Bio-Rad Laboratories, USA), and allowed to solidify at 4 °C.
For lysis, the resulting plugs were then placed in a mixture of 6 mM/L Tris base (Sigma-Aldrich, Germany), 100 mM/L EDTA [pH 7.5], Sigma-Aldrich, Germany), NaCl 58,4 g (Merck, Germany), 0.5% Brij®58 (Merck, Germany), 0.5% sodium lauryl sarcosine (Sigma-Aldrich, Germany), 0.2% sodium deoxy-cholate (Merck, Germany), 1.5 mg of lysozyme (our modification instead 1 mg) per mL, and 5 U of lysostaphin per mL. Following 72 h incubation at 37 °C, the plugs were transferred to a solution which contained 1% sodium lauryl sarcosine, 0.5 M EDTA [pH 9.5], and 200 mg of proteinase K per mL, and the mixture was incubated for 24 h at 50 °C under gentle shaking. The plugs were washed in TE buffer (10 mM Tris-HCl [pH 7.5], 10 mM EDTA) six times for 30 min at room temperature.
A slice of each plug (2.5 mm) was cut out and incubated 1 h at 37 °C with 25 U of SmaI restriction endonuclease (Roche, Switzerland) in the relevant buffer. The enzyme solution was removed, and a fresh one was added afterwards for further 24 h incubation at 37 °C. The slices were then loaded into the wells of 1% Pulsed Field Certified Agarose (Bio-Rad Laboratories, USA) in 0.5 × Tris borate EDTA (TBE) buffer.
Electrophoresis was done in a contour-clamped homogeneous electric field apparatus (CHEF-DR®III, Bio-Rad Technologies, USA) for 30 h at 11 °C, with an electric field of 6 V/cm at an angle of 120 °; the pulse time was increased from 5.3 to 34.9 s. After electrophoresis the gel was stained with ethidium bromide (0.5 µg/mL) for 30 min and destained in distilled water for 1 h, afterwards, DNA was visualised under UV light. A low range Lambda Ladder PFGE Marker 50-1000 kb (BioLabs, New England) was used as the molecular weight marker (Liu et al. 1997; Sjölund et al. 2005).
Isolation of large circular and linear plasmids by PFGE
A method for detecting and estimating the sizes of large bacterial plasmids in the presence of genomic DNA by pulsed-field gel electrophoresis (PFGE) was used according to Barton et al. (1995) with a few modifications. The agarose plugs with DNA, prepared in the same way as for studying the genetic diversity between the isolates, were cut into two slices with a sterile glass coverslip. The first one was soaked in the buffer S1 for 1 h at 37 °C, while to the second one appr. 450 U (0.3 mL) of endonuclease S1 in 1 × S1 buffer (100 mL) (Thermo Fisher Scientific, ZDA) was added and incubated for 30 min at room temperature. Digested and undigested slices were applied to wells in 1% agarose gel, prepared in 0.5 × TBE buffer (45 mM Tris-OH [pH 8.0], 45 mM boric acid, 1 mM EDTA), and run in a CHEF-DR®III apparatus for 24 h at 11 °C, with an electric field of 6 V/cm at an angle of 120 °, the pulse time was increased from 1 to 12 s. The presence of linear plasmids was demonstrated in undigested slices, while the circular plasmids were detected in digested slices.
Macrorestriction profile analysis was made in BioNumerics 7.1 (Applied Maths, Saint-Martens, Belgium) using the Dice coefficient, and represented by unweighted pair grouping by mathematical averaging (UPGMA) with 0.5% band tolerance and 0.5% optimisation settings. Images of B. cereus ATCC 14579T were used as a marker to calibrate images’ position, with manual correction if necessary.
The SPSS software (version 25.0; IBM, USA) was used for statistical analyses. The statistical difference of the PFGE clonal complexes with the source of the strains and the presence of plasmids was calculated using the Kullbach 2Ȋ (Likelihood Ratio) and Pearson chi-square tests. A p-value lower than 0.05 was considered statistically significant.