Robust Biofilm-Forming Bacillus Isolates from the Dairy Environment Demonstrate an Enhanced Resistance to Cleaning-in-Place Procedures

Abstract

One of the main strategies for maintaining the optimal hygiene level in dairy processing facilities is regular cleaning and disinfection, which is incorporated in the cleaning-in-place (CIP) regimes. However, a frail point of the CIP procedures is their variable efficiency in eliminating biofilm bacteria. In the present study, we evaluated the susceptibility of strong biofilm-forming dairy Bacillus isolates to industrial cleaning procedures using two differently designed model systems. According to our results, the dairy-associated Bacillus isolates demonstrate a higher resistance to CIP procedures, compared to the non-dairy strain of B. subtilis. Notably, the tested dairy isolates are highly persistent to different parameters of the CIP operations, including the turbulent flow of liquid (up to 1 log), as well as the cleaning and disinfecting effects of commercial detergents (up to 2.3 log). Moreover, our observations indicate an enhanced resistance of poly-γ-glutamic acid (PGA)-overproducing B. subtilis, which produces high amounts of proteinaceous extracellular matrix, to the CIP procedures (about 0.7 log, compared to the wild-type non-dairy strain of B. subtilis). We therefore suggest that the enhanced resistance to the CIP procedures by the dairy Bacillus isolates can be attributed to robust biofilm formation. In addition, this study underlines the importance of evaluating the efficiency of commercial cleaning agents in relation to strong biofilm-forming bacteria, which are relevant to industrial conditions. Consequently, we believe that the findings of this study can facilitate the assessment and refining of the industrial CIP procedures.

Keywords: Bacillus species; biofilm; biofilm derived spores; cleaning-in-place; dairy industry; disinfecting effect.

Figure 1

Dairy-associated Bacillus isolates exhibit robust biofilm formation. (A) Colony type biofilm formation by the tested Bacillus strains in the biofilm-promoting medium, LBGM. The images were taken using a stereoscopic microscope (Zeiss Stemi 2000-C; Carl Zeiss, Gottingen, Germany). (B) Biofilm-derived spores of the dairy Bacillus strains are surrounded by high amounts of the extracellular matrix. Protein components of the biofilm matrix were stained red. The samples were analyzed using a confocal laser scanning microscope (CSLM, Olympus, Japan). Scale: 10 µm.

Figure 2

Effect of the cleaning procedure with tap water on the removal of biofilm-derived spores of the dairy-associated Bacillus in the CIP model system. Sampling plates, each containing approximately 2 million spores of B. subtilis 3610 or dairy Bacillus isolates, were mounted on T-junctions, protruding 35, 125, and 275 mm from the main loop of the CIP model system, and cleaned in the installation. Tap water, without the addition of any detergent, was used as the cleaning agent. A basic assumption was the similar adhesion efficiency of the spores of each tested strain in different experimental repeats (since the spores were obtained using previously validated experimental procedures [21]). The cleaning effect was evaluated by comparing the number of viable spores (attached to the sampling plates), before and after cleaning. The results represent the means and standard deviations (SD) of two independent biological experiments, performed in triplicate. * Statistically significant difference (p < 0.05) between the reduction in the viable spore counts of a given sample and the reduction in the spore counts for B. subtilis 3610 (control).

Figure 3

Effect of commercial cleaning agents on the removal of the biofilm-derived spores of the dairy-associated Bacillus in the simplified laboratory system. Sampling plates, each maintaining approximately 2 million spores of the tested Bacillus strains, were cleaned in the laboratory system. Caustic soda, sodium hypochlorite and the following cleaning solutions—I, A, M, F, D and H (compositions and dosages are described in Methods)—were used as the cleaning agents. The cleaning effect was evaluated by comparing the numbers of viable spores (attached to sampling plates), before and after cleaning. The results represent the means and standard deviation (SD) of two independent biological experiments, performed in triplicate. * Statistically significant difference (p < 0.05) between the reduction in the viable spore counts in a given sample and the reduction in the spore counts for B. subtilis 3610 (control). ** Statistically significant difference (p < 0.05) between the reduction in the viable spore counts, after treatment with a given cleaning agent, and the reduction in the spore counts for the same strain, after incubation with tap water.

Figure 4

Effect of the examined cleaning agents on the viability of the biofilm-derived spores of the tested Bacillus strains. Caustic soda, sodium hypochlorite, and different cleaning solutions—I, A, M, F, D, and H (compositions are described in Methods)—were added to the tubes, with spore suspension of the tested Bacillus isolates. Spore suspension, without any detergent, was used as the control. The effect on spore viability was evaluated by comparing the numbers of viable spores in the control and after the treatment with the tested agents (following 10 min of incubation at 50 °C, 250 rpm). The results represent the means and standard deviation (SD) of two independent biological experiments, performed in duplicate. * Statistically significant difference (p < 0.05) between the viable spore counts in a given sample versus the spore counts after cleaning with water (control).

Figure 5

Correlation between the cleaning and disinfecting effects of the examined agents for each tested strain. Caustic soda and different cleaning solutions—I, A, M, F, D, and H (compositions are described in Methods)—were added to the tubes, with spore suspension, of the tested Bacillus isolates and incubated for 10 min at 50 °C, 250 rpm. The ability of a cleaning agent to reduce the number of viable spores was defined as the disinfecting effect. The percentage of the disinfecting effect was compared to the total chemical/biological effect of a cleaning agent (taken as 100%). The difference between the total chemical/biological effect of a cleaning agent and the disinfecting effect was defined as the cleaning effect. The results represent the means and standard deviation (SD) of two independent biological experiments, performed in duplicate. * Statistically significant difference (p < 0.05) between the reduction in the spore counts due to the cleaning or disinfecting effects versus the total chemo-biological effect of a tested agent. ** Statistically significant difference (p < 0.05) between the reduction in the viable spore counts in a given sample and the reduction in the spore counts for B. subtilis 3610 (control).

Figure 6

PGA-overproducing derivatives of B. subtilis 3610 exhibit increased resistance to the CIP procedures due to enhanced biofilm formation. (A) Colony biofilm formation by the tested Bacillus strains in the biofilm-promoting medium, LBGM. The images were taken using a stereoscopic microscope (Zeiss Stemi 2000-C; Carl Zeiss, Gottingen, Germany). (B) Biofilm-derived spores of the PGA-overproducing B. subtilis strains are surrounded by high amounts of extracellular matrix. Protein components of the biofilm matrix were stained red. The samples were analyzed using a confocal laser scanning microscope (CSLM, Olympus, Japan). Scale: 10 µm. (C) The effect of water circulation on the removal of biofilm-derived spores of the PGA-overproducing derivatives of B. subtilis 3610 in the laboratory CIP system. * Statistically significant difference (p < 0.05) between the reduction in the viable spore counts in a given sample and the reduction in the spore counts for B. subtilis 3610 (control).