Evaluation of the biofilm-forming ability and molecular characterization of dairy Bacillus spp. isolates

Abstract

Food processing lines represents a suitable environment for bacterial biofilm formation. One of the most common biofilm-forming genera in dairy processing plants is Bacillus, which includes species that may have a negative impact on safety and/or quality of dairy products. In the current study, we evaluated the biofilm forming ability and molecular characteristics of dairy Bacillus spp. isolates (B. cereus and B. subtilis). Reference strains (B. cereus ATCC 14579 and B. subtilis NCTC 3610) were also included in the experiment. All isolates were screened by micro-titer plate (96 wells) to assess their ability to form biofilm. Then, they were tested on two common food contact surfaces (polystyrene and stainless steel) by using 6-well plates and AISI 316 stainless steel coupons. Biofilm formation, expressed as biofilm production index (BPI), was higher on polystyrene than stainless steel (except for B. cereus ATCC 14579). These observations were further confirmed by scanning electron microscopy, which allowed the microscopy observation of biofilm structure. Moreover, a possible correlation among total viable cell counts (CFU) and BPI was examined, as well as a connection among biofilm formation and bacterial cell hydrophobicity. Finally, whole genome sequencing was performed highlighting a genetic similarity among the strains belonging to the same species. The presence of selected genes involved in biofilm formation was also examined showing that strains with a greater presence of these genes were able to produce more biofilm in the tested materials. Additionally, for B. cereus strains enterotoxin genes were detected.

Keywords: Bacillus; SEM; biofilm; polystyrene; stainless-steel; whole genome sequencing.

Figure 1

Biofilm forming ability of (A) Bacillus cereus (BC) dairy isolates and ATCC 14579 reference strain and (B) Bacillus subtilis (BS) dairy isolates and NCTC 3610 reference strain in different growth media (BHI and TSB) and incubation time (24h and 48h). The threshold of biofilm formation (dotted line) is equal to OD values ≤ 0.11. Error bars indicate standard deviation. Different letters indicated significant difference among the isolates, p< 0.05.

Figure 2

Biofilm formation of reference strains ATCC 14579 and NCTC 3610, Bacillus cereus (BC) and Bacillus subtilis (BS) food isolates, on polystyrene and stainless-steel coupons. Biofilms were formed in BHI at 30°C for 24 h and biofilm production index (BPI) was measured by the crystal violet assay. Each bar represents the result of the average of three biological experiments. Error bars indicate standard deviation. Letters on the top of the bars indicate significant statistically differences (p< 0.05).

Figure 3

Total CFU counts in the biofilms of reference strains, B. cereus (BC) and B. subtilis (BS). Biofilms were grown on polystyrene wells and stainless-steel coupons in BHI at 30°C for 24 h. Each bar represents the result of the average of three biological experiments and standard deviation for each strain. To compare the number of sessile cells yielded among strains, one-way ANOVA and Tukey’s post hoc test were performed. Groups with different alphabets indicate significant difference (p< 0.05).

Figure 4

Scanning electron microscope (SEM) images of biofilms formed by reference B. subtilis strain NCTC 3610 in (A) polystyrene and (B) stainless-steel and dairy B. subtilis isolate BS_42 in (C) polystyrene and (D) stainless-steel. On the left the representation of 2000x, on the right 5000x magnification. The white arrows show the EPS matrix. Biofilms were formed in BHI broth at 30°C for 24h. a,b,c,d: structure details and biofilm matrix visualized by SEM.

Figure 5

Scanning electron microscope (SEM) images of biofilms formed by reference B. cereus strain ATCC 14579 in (A) polystyrene and (B) stainless-steel and dairy B. cereus isolate BC_14 in (C) polystyrene and (D) stainless-steel. On the left the representation of 2000x, on the right 10000x magnification for (a, c) and 5000x magnification for (b, d). The white arrows show the EPS matrix. Biofilms were formed in BHI broth at 30°C for 24h.

Figure 6

Venn diagrams depicting the overlap between genes common or unique between (A) B. cereus or (B) B. subtilis genomes. Phylogenetic tree built on concatenated (C) B. cereus or (D) B. subtilis genes.