Biofilm Formation on Stainless Steel by Streptococcus thermophilus UC8547 in Milk Environments Is Mediated by the Proteinase PrtS

Abstract

In Streptococcus thermophilus, gene transfer events and loss of ancestral traits over the years contribute to its high level of adaptation to milk environments. Biofilm formation capacity, a phenotype that is lost in the majority of strains, plays a role in persistence in dairy environments, such as milk pasteurization and cheese manufacturing plants. To investigate this property, we have studied S. thermophilus UC8547, a fast-acidifying dairy starter culture selected for its high capacity to form biofilm on stainless steel under environmental conditions resembling the dairy environment. Using a dynamic flow cell apparatus, it was shown that S. thermophilus UC8547 biofilm formation on stainless steel depends on the presence of milk proteins. From this strain, which harbors the prtS gene for the cell wall protease and shows an aggregative phenotype, spontaneous mutants with impaired biofilm capacity can be isolated at high frequency. These mutants lack the PrtS expendable island, as confirmed by comparison of the genome sequence of UC8547Δ3 with that of the parent strain. The prtS island excision occurs between two 26-bp direct repeats located in the two copies of the ISSth1 flanking this genomic island. The central role of PrtS was confirmed by analyzing the derivative strain UC8547Δ16, whose prtS gene was interrupted by an insertional mutation, thereby making it incapable of biofilm formation. PrtS, acting as a binding substance between the milk proteins adhered to stainless steel and S. thermophilus cell envelopes, mediates biofilm formation in dairy environments. This feature provides S. thermophilus with an ecological benefit for its survival and persistence in this environment.IMPORTANCE The increased persistence of S. thermophilus biofilm has consequences in the dairy environment: if, on the one hand, the release of this microorganism from biofilm can promote the fermentation of artisanal cheeses, under industrial conditions it may lead to undesirable contamination of dairy products. The study of the molecular mechanism driving S. thermophilus biofilm formation provides increased knowledge on how an ancestral trait affects relevant phenotypes, such as persistence in the environment and efficiency of growth in milk. This study provides insight into the genetic factors affecting biofilm formation at dairy plants.

Role of Temperate Bacteriophage ϕ20617 on Streptococcus thermophilus DSM 20617T Autolysis and Biology

“…The results obtained clearly showed that the strain carrying the prophage had a significantly higher adhesion phenotype, compared to the A33 phage-cured strain (Table 2 ), thus highlighting that in S. thermophilus , likewise in L. lactis , the cell wall integrity is correlated with biofilm formation. More recently, S. thermophilus biofilm formation was correlated to the presence of the cell-wall protease PrtS which was demonstrated to trigger an aggregative phenotype ( Bassi et al, 2017 ), but this cannot be the case of strain DSM20617 T because it is protease-negative ( Arioli et al, 2007 ). In addition, to investigate the role of phage lysin in the S. thermophilus chain length, we evaluated the differential sedimentation rates of bacteria according to their chain-forming ability and/or cell size in semi-liquid medium (i.e., liquid M17 – lactose medium containing 0.15% wt/vol agarose).…”

Effect of recycled manure solids as bedding on bulk tank milk and implications for cheese microbiological quality

“…Indeed, some strains can form biofilms on stainless steel. These can cause many problems in cheese manufacturing plants such as undesirable contamination of dairy products and uncontrolled milk acidification during food production (Bassi et al, 2017).…”

Phenotypic and Genotypic Investigation of Two Representative Strains of Microbacterium Species Isolated From Micro-Filtered Milk: Growth Capacity and Spoilage-Potential Assessment

“…The remaining attached cells were marked with 0.3% crystal violet. We removed the excess crystal violet solution by washing with bi-distilled water; the attached cells were resuspended with a 10% acetic acid solution and quantified by a spectrophotometric measurement at 590 nm, as described by Bassi et al (2017) with some modifications. To assess whether these strains showed significant differences in their ability to form biofilms (crystal violet staining method), 12 inoculation replicates were made, and an ANOVA was carried out with Tukey's honestly significant difference test.…”