Isolation, Partial Characterization and Application of Bacteriophages in Eradicating Biofilm Formation by Bacillus cereus on Stainless Steel Surfaces in Food Processing Facilities

Abstract

The Bacillus cereus (B. cereus) group is a widespread foodborne pathogen with a persistent ability to form biofilm, and with inherent resistance to traditional treatment in the food industry. Bacteriophages are a promising biocontrol agent that could be applied to prevent or eliminate biofilms formation. We have described, in this study, the isolation from sewage samples and preliminary characterization of bacteriophages that are active against the B. cereus group. The effectiveness of phage treatment for reducing B. cereus attachment and biofilms on stainless steel surfaces has been also assessed using three incubation periods at different titrations of each phage. Out of 62 phages isolated, seven showed broad-spectrum lytic action against 174 B. cereus isolates. All selected phages appeared to be of the Siphoviridae family. SDS-PAGE proved that two phages have a similar profile, while the remainder are distinct. All isolated phages have the same restriction pattern, with an estimated genome size of around 37 kb. The isolated bacteriophages have been shown to be effective in preventing biofilm formation. Reductions of up to 1.5 log10 UFC/cm2 have been achieved, compared to the untreated biofilms. Curative treatment reduced the bacterial density by 0.5 log10 UFC/cm2. These results support the prospect of using these phages as a potential alternative strategy for controlling biofilms in food systems.

Caracterización fenotípica y molecular de colifagos de granjas de pollos de engorde con Colibacilosis y plantas beneficiadoras de aves en Azuay, Ecuador

“…Para la caracterización de bacteriófagos específicos de biopelículas por Bacillus cereus en superficies de acero inoxidable, en instalaciones de procesamiento de alimentos, se utilizó con éxito la técnica RFLP; los ADN de bacteriófagos aislados extraídos se digirieron con cinco endonucleasas de restricción (BamHI, EcoRI, Hind III, Hin6I y TaqI); ninguno de los ADN de fagos extraídos fue susceptible a BamHI y Hind III, mientras que las enzimas EcoRI, Hin6I y TaqI dieron como resultado perfiles de restricción muy similares para los fagos seleccionados; indicando que los fagos seleccionados tenían tamaños de genoma similares y posiblemente estaban muy relacionados [14].…”

The Use of Natural Methods to Control Foodborne Biofilms

“…Whereas E. coli and Salmonella tend to be foodborne pathogens that get the most attention with the common public, B. cereus is another highly resistant widespread foodborne pathogen known to produce biofilms and to plague the food industry [ 25 ]. As already mentioned, phages can be a uniquely ideal means of naturally treating or preventing the most resistant of biofilm bacteria.…”

“…As already mentioned, phages can be a uniquely ideal means of naturally treating or preventing the most resistant of biofilm bacteria. In one recent study, phages proved to be highly versatile against B. cereus , wherein 62 phages were isolated from sewage samples, and seven were found to have broad-spectrum lytic antimicrobial activity in the prevention and reduction of 174 B. cereus isolates [ 25 ]. Transmission electron microscopy demonstrated the phages to be Siphoviridae , and SDS-PAGE analysis of structural proteins, along with restriction analysis of genomes, showed strong relatedness but great diversity amongst the phages [ 25 ].…”

“…In one recent study, phages proved to be highly versatile against B. cereus , wherein 62 phages were isolated from sewage samples, and seven were found to have broad-spectrum lytic antimicrobial activity in the prevention and reduction of 174 B. cereus isolates [ 25 ]. Transmission electron microscopy demonstrated the phages to be Siphoviridae , and SDS-PAGE analysis of structural proteins, along with restriction analysis of genomes, showed strong relatedness but great diversity amongst the phages [ 25 ]. Most significant was the low-percentage-of-lysis characterization of these phages, as that was selected to reduce the chances of resistance development and minimize the potential harm to important normal flora [ 25 ].…”

“…Transmission electron microscopy demonstrated the phages to be Siphoviridae , and SDS-PAGE analysis of structural proteins, along with restriction analysis of genomes, showed strong relatedness but great diversity amongst the phages [ 25 ]. Most significant was the low-percentage-of-lysis characterization of these phages, as that was selected to reduce the chances of resistance development and minimize the potential harm to important normal flora [ 25 ]. Overall, however, bacteriophage studies point out that the success of bacteriophage treatment of biofilms is highly variable depending on factors, such as strains, temperature, multiplicity of infection (MOI), biofilm content or age, and the surfaces or foods involved [ 26 ].…”