Bacteria display two modes of growth: free-living planktonic or the sessile and surface-attached within biofilms (Rumbaugh and Sauer 2020). The bacteria colonize by adhering to surfaces, growing, and forming a self-produced polymeric matrix in which microbial species may grow together as a biofilm (González-Rivas et al. 2018). Biofilm growth is observed in many industrial and indigenous areas such as dairy, water systems, maritime, dentistry, food, paper, oil, optics, and healthcare fields (Garrett et al. 2008). One of the most prevalent biofilm-forming microorganisms in dairy manufacturing is the thermophilic bacilli (Burgess et al. 2013). The presence of the bacteria is an indicator of poor plant hygiene (Burgess et al. 2013).
Biofilms have become problematic in a wide range of food industries (González-Rivas et al. 2018). Examples of biofilms’ harmful effects are product spoilage, reduced production capacity, corrosion, equipment failure, pipe blockages, and infection (Garrett et al. 2008). Spoilage bacteria are responsible for nearly one-third of losses in the food chain supply (González-Rivas et al. 2018). Biofilm formation of the bacteria in food manufacturing concerns the dairy industry (Lindsay and Flint 2009). The reason behind is that thermophilic bacilli are hard to remove due to their broad temperature range of growth; they have a fast growth rate; their spores show high resistance to heat and chemicals; and they can form biofilms (Scott et al. 2007; Burgess et al. 2009; Eijlander et al. 2019). Eijlander et al. (2019) reported that
In our previous studies, we carried out the preliminary biofilm experiments, including pellicle formation, complex exopolysaccharide production, biofilm morphotypes, and viable biofilm cell counting on stainless steel of
Treatment of biofilms with sanitation agents.
Agent (Concentration) | Temperature – Time | References |
---|---|---|
AP (0.16 U/g) | 37°C - 60 min | Parkar et al. 2004 |
Protease 0.16 U/g) | 37°C - 60 min | Parkar et al. 2004 |
Subtilisin (1%) | 37°C - 30 min | Parkar et al. 2004 |
Trypsin (3%) | 37°C - 3 h | Parkar et al. 2003 |
SDS (3%) | 100°C - 10 min | Parkar et al. 2003 |
a-Amylase (1%) | 37°C - 30 min | Parkar et al. 2004 |
Cellulase (1.66%) | 37°C - 30 min | Parkar et al. 2004 |
SM (100 mM) | 22°C - 60 min | Parkar et al. 2003 |
Lysozyme (2%) | 37°C - 60 min | Parkar et al. 2003 |
TCA (10%) | 100°C - 15 min | Parkar et al. 2003 |
Nisin (2 mg/ml) | 37°C - 24 h | Parkar et al. 2003 |
PM (2 mg/ml) | 22°C - 30 min | Parkar et al. 2003 |
ST (10 mg/ml) | 22°C - 5 min | Parkar et al. 2003 |
2(5H)-Furanone (1 mg/ml) | 22°C - 60 min | Ponnusamy et al. 2010 |
Triclosan (2 mg/ml) | 22°C - 60 min | Tabak et al. 2007 |
Agarose gel electrophoresis photographs displaying differences between the gDNA and eDNA of D413 and E134. M, Marker (Fermentas Gene Ruler 1 kb Plus DNA Ladder, 75–20.000 bp) (A) DNase I (1.45 mg/ml), RNase A (0.90 mg/ml) and proteinase K (0.85 mg/ml) treatment of both the gDNA and eDNA; (B) Different DNase I concentrations on eDNA (1.7, 2.5 and 3.0 mg/ml).
Treatment of mature biofilms with DNase I.
The viable cell counts of D413 biofilms formed on surfaces (Tukey test;
The viable cell counts of E134 biofilms formed on surfaces (Tukey test;
The effects of sanitation agents on the biofilm of D413 and E134 isolates (Dunnett’s test;
The temperature range of growth for
Exopolysaccharide, proteinaceous polymers, lipids, and eDNA may be important biofilm matrix components (Allesen-Holm et al. 2006; Soler-Arango et al. 2019). Many microorganisms release eDNA within their biofilm matrix. Moreover, eDNA was reported as a component of the EPS matrix of numerous Gram-negative and Gram-positive bacteria (Ibáñez de Aldecoa et al. 2017; Ramirez et al. 2019). However, as far as it is known, there is no information about the eDNA of
Potential target sites of Gram-positive bacteria to antimicrobials are the cell wall, the cytoplasmic membrane, functional and structural proteins, DNA, RNA, and other cytosolic components (Bridier et al. 2011). In this study, eDNA in the biofilm matrix was not affected by DNase I, RNase A, and proteinase K enzymes. However, the gDNA was degraded only by DNase I. It seemed that the eDNA of the D413 and E134 had resistance to DNase I when purified (Fig. 1). Böckelmann et al. (2006) reported that agarose gel electrophoresis of purified microfilaments of strain F8 resulted in a distinct band of large size (more than 29 kb) and the band of the eDNA of the strain exactly disappeared after treatment with DNase I but remained stable after treatment with RNase A and proteinase K. Dengler et al. (2015) indicated that
Qin et al. (2007) showed that DNase I severely decreased the biofilm formation of
Thermophilic bacteria can attach to stainless steel coupons and support biofilms’ development (Jindal et al. 2016; Gupta and Anand, 2018). In this paper, six abiotic surfaces were compared in terms of viable cell counts within biofilms. D413 and E134 were observed to adhere to all surfaces. The viable cell numbers ranged from 3.91 to 5.12 log cfu/cm2 and 2.25 to 4.70 log cfu/cm2, respectively. Polystyrene surface (4.70 log cfu/cm2) and glass surface (5.12 log cfu/cm2) were determined to be the most effective surface for biofilm formation of D413 and E134 isolates, respectively (
Biofilm dispersal can be provided by the disruption of the polysaccharide matrix, proteins, and eDNA. To remove irreversibly attached cells, the implementation of a powerful shear force as scrubbing, scraping or chemical breaking of the adherence forces through the applications of enzymes, sanitizers, and heat is required (Elhariry et al. 2012). It was determined that various sanitation agents could help to reduce the number of D413 and E134 cells on the polystyrene surface (Fig. 5). The best results were provided by nisin for D413 (80%) and α-amylase for E134 (98%) (
In conclusion, this paper showed that DNase I degraded the eDNA of