Microbial contamination, caused by biofilm-forming bacteria, is one of the main threats to the quality, safety, stability and nutritional value of dairy products [1
]. Moreover, biofilms are not only a potential source of contamination; they can also increase the corrosion rate of equipment used in the milk industry, impair heat transfer, and increase fluid frictional resistance [3
]. Therefore, controlling biofilm formation is of major importance to the dairy industry [4
Members of the Bacillus
genus are among the most commonly found biofilm-formers in dairy farms and processing plants [7
]. In addition to aggressive biofilm, these bacteria are able to form heat-resistant endospores [10
]. To this end, the biofilm matrix can serve as an epicenter for the ripening of spores, which can be released from it and cause continuous contamination of the production environment [12
]. Spores, as well as biofilm cells, are highly resistant to antimicrobial agents, which makes it rather difficult to eliminate them [11
]. Moreover, biofilm matrix offers additional protection for embedded endospores, allowing their survival and colonization in the surrounding environment, when conditions are favorable [15
]. In B. subtilis
, the matrix has two main components, an exopolysaccharide (EPS) and amyloid-like fibers. Another extracellular polymer, γ-poly-dl
-glutamic acid (PGA), is produced in copious amounts by some B. subtilis
The main strategy to prevent biofilm formation, applied in the dairy industry, is to clean and disinfect regularly before bacteria attach firmly to surfaces [19
]. Cleaning and disinfection in dairy processing plants have been incorporated into the cleaning-in-place (CIP) regimes, which include regular cleaning of processing equipment with alkaline and acidic liquids at high temperatures and flow velocities [4
]. However, a weak point of CIP processes, evident in both industrial- and laboratory-scale systems, is their variable efficiency in eliminating established biofilms [4
]. It is conceivable that biofilm formation can facilitate bacterial adaptation and survival in certain environmental niches. We therefore hypothesized that aggressive biofilm formation by dairy-associated bacteria might increase their resistance to industrial cleaning procedures.
In the present study, we evaluated the susceptibility of strong biofilm-forming dairy Bacillus isolates to cleaning-in-place procedures using two different model systems, which resemble industrial cleaning conditions. Our results show that the dairy-associated Bacillus isolates demonstrate enhanced resistance to different aspects of the CIP procedures, including mechanical, chemo-biological and disinfecting effects. Such reduced susceptibility can be attributed to robust biofilm formation by the tested dairy Bacillus.
2. Materials and Methods
2.1. Bacterial Strains and Growth Conditions
The following bacterial strains were used in this study: (i) dairy-associated isolates, such as B. paralicheniformis
], B. licheniformis
MS310, B. subtilis
MS302, B. paralicheniformis
]; (ii) non-dairy isolate B. subtilis
NCIB3610 (descendant of B. subtilis
Marburg); (iii) poly-γ-glutamic acid (PGA)-overproducing mutant derivatives of B. subtilis
3610, B. subtilis
) and B. subtilis
) (a gift of Y. Chai [18
]). B. licheniformis
MS310, B. subtilis
MS302 and B. paralicheniformis
MS303 whole-genome shotgun projects are deposited at DDBJ/EMBL/GenBank, under accession numbers MIPQ00000000, MIZD00000000, MIZE00000000 respectively.
For routine growth, the strains were propagated in Lysogeny broth (LB; 10 g tryptone, 5 g yeast extract, 5 g NaCl per liter, pH 7) or on a solidified LB medium, supplemented with 1.5% agar at 37 °C.
2.2. Generation of Biofilm-Derived Spores
Biofilm colonies were generated at 30 °C in a biofilm-promoting medium (LBGM = LB + 1% v
glycerol + 0.1 mM MnSO4
]. Biofilm-derived spores were obtained from colonies, as described previously [21
]. Briefly, the grown (three-day-old) colonies, harvested and suspended in phosphate buffered saline (PBS; 0.01 M phosphate buffer, 0.0027 M KCl, 0.137 M NaCl per 200 mL, Sigma Aldrich, St. Louis, MO, USA), were disrupted by mild sonication (Vibra Cell, Sonics, Newtown, CT, USA; amplitude 60%, pulse 10 s, pause 10 s, duration 2 min, instrument power: 7.2 Joules per second). During sonication, the samples were kept on ice. Then, heat killing was performed at 80 °C for 20 min. Cell numbers after heat killing were quantified by the spread plating method.
2.3. Staining Extracellular Matrix of Biofilm-Derived Spores
Biofilm-derived spores were stained using the FilmTracer™ SYPRO® Ruby Biofilm Matrix Stain (Molecular Probes, Eugene, OR, USA), according to the manufacturer’s protocol. Stained samples were visualized by confocal laser scanning microscopy (CLSM; Olympus IX81, Tokyo, Japan) at a 10 μm scale.
2.4. Preparation for Cleaning Tests and Enumeration of Biofilm-Derived Spores
The preparation of biofilm-derived spores for cleaning tests was performed, as described in the previous study [21
]. Briefly, 200-µL aliquots of the spore suspension (containing approximately two million spores) were applied in the sampling area of stainless-steel sampling plates and dried in a biological laminar hood for 1 h. Two sampling plates were not exposed to the cleaning procedures (control). Following each cleaning test, the sampling plates were immediately subjected to abundant rinsing with tap water at RT (similar to the CIP procedures at Israeli dairy farms, where the rinsing with water stage is introduced after applying a cleaning agent). For the enumeration of the spores, the sampling area on each plate was carefully swabbed with cotton swabs, moistened in PBS buffer. Swabs from each plate were then agitated in PBS in separate test tubes. Serial dilutions from each sample were prepared, followed by spread plating on LB agar for CFU analysis. Plates were incubated for 24 h at 37 °C, before the colonies were counted. The efficiency of a cleaning procedure was evaluated by comparing the number of viable spores (attached to sampling plates), before and after cleaning.
2.5. Cleaning Solutions
The following cleaning solutions were used in this study: Caustic soda (NaOH), sodium hypochlorite (NaOCl) and six different commercial alkaline detergents, defined as solutions I (10–15% NaOH, 3–5% NaOCl), A (polycarboxylate, phosphates, 3.6% NaOCl), M (>5% polycarboxylate, 5–15% phosphates, 3.6% NaOCl), F (5% phosphonates, polycarboxylates), D (active chlorine, alkaline-based) and H (active chlorine, phosphates, additives, alkaline-based), which are commonly used in the Israeli dairy farms. The pH value of the tested solutions varied between 11–12; the pH of NaOH was 13; and the pH of NaOCl was 4. In accordance with the manufacturer’s recommendations, the agents were used at the following concentrations: (i) 0.5% (v/v) for solutions A, M, F, D, H; (ii) 0.6% (v/v) for solution I; (iii) 0.5% (m/v) for caustic soda and detergent H; (iv) 0.018% (v/v) for sodium hypochlorite (similar to the NaOCl concentration in working solutions of the examined cleaning agents, such as A, M and I). As a control, tap water was used (pH value around 7.7), with a standard level of hardness (50 mg/L Ca2+, 50 mg/L Mg2+), without the addition of any detergent.
2.6. Cleaning Test Installations
The cleaning tests were carried out either using the cleaning-in-place (CIP) model system (closely resembling the typical conditions for milking systems) [21
] or using the simplified laboratory procedure, developed in this study.
2.6.1. CIP Model System
The main components of the CIP model system were described in the previous study [21
]. In brief, the system consists of a 5-m stainless-steel milk line (fitted with a test unit) for pumping the cleaning agents from the basin, milk releaser, and a stainless-steel return line to the basin. The test unit has T-junctions, protruding 35, 125 or 275 mm from the main loop, reflecting different degrees of cleaning difficulty. Sampling plates with the spores were mounted on the T-junctions and cleaned in the installation. The temperature of the cleaning solution during the cleaning tests was 50 °C. To generate flushing pulsation of the circulating liquid, air was introduced into the system every 8 s. The duration of each cleaning cycle was 10 min.
2.6.2. Laboratory System
For cleaning tests in the laboratory system, sampling plates with the spores were placed into 100 mL plastic vessels (Yoel Naim, Rehovot, Israel), containing 50 mL of cleaning solution (preliminarily warmed to 50 °C). The samples were incubated in closed vessels at conditions simulating those in the CIP-model system (50 °C, 250 rpm) for 10 min.
2.7. Evaluation of the Effect of the Cleaning Agents on the Viability of Bacillus Spores
The tested solutions were added to spore suspension within tap water containing around 1 × 107 CFU/mL spores. The spore suspension without the addition of detergents was used as a control. The samples were incubated in closed tubes under the conditions of the laboratory system (50 °C, 250 rpm) for 10 min. The CFU measurements of the number of viable spores were made immediately after the addition of the tested cleaning agents and following 10 min of incubation.
2.8. Statistical Analysis
The results of the study are the means and standard deviation (SD) of at least two independent biological experiments, performed in triplicate. The Student’s t test was used to calculate the significance of the difference between the mean expression of a given experimental sample and the control sample. A p value of <0.05 was considered significant.
It becomes increasingly clear that biofilm formation by Bacillus
species can facilitate their survival in the dairy environment [11
]. Our current study investigated the effect of CIP procedures on strong biofilm-forming dairy Bacillus
, compared to the non-dairy B. subtilis
3610, using differently designed model systems. As in our previous study [21
], we used biofilm-derived spores to simulate the type of hygiene problem common in practice. Thus, similarly to actual dairy biofilm, biofilm-derived spores combine the presence of biofilm matrix [21
] and a high content of spores [29
]. Moreover, the resistance of vegetative cells/spores to cleaning and disinfection can be greatly enhanced by the presence of EPS [21
]. At the same time, the presence of spores within the Bacillus
biofilm may also modify biofilm properties, e.g., interaction forces [12
In the current study, two model systems were used to ensure that the enhanced resistance of the dairy isolates to cleaning procedures is observed under different experimental conditions, which are relevant to the industrial CIP systems. Moreover, the design of the CIP system, employed in our previous study does not allow for the evaluation of the disinfecting effect of the cleaning agents on Bacillus
spores directly in this system [21
]. The laboratory system, developed in this study, provides sufficient conditions both for determining the mechanical, chemo-biological and disinfecting effects of the cleaning agents.
A first notable finding of the study was the enhanced resistance of the dairy Bacillus
to the mechanical effect of liquid circulation. Thus, the most expressed difference in cleaning susceptibility between the dairy-associated strains and B. subtilis
3610 was observed at high levels of turbulence (35- and 125-mm T-junctions, CIP model system; Figure 2
). In the case of a lower turbulence (275-mm T-junction), the difference between the dairy Bacillus
isolates and the non-dairy strain is markedly decreased, and for some strains, it was insignificant (Figure 2
). These results suggest that the protective effect of Bacillus
biofilm matrix is most strongly expressed under a high turbulence of liquid flow. Previous studies demonstrate that a high turbulence may facilitate the removal of surface-attached bacteria [21
], but may also increase the rate of attachment by bringing the microbial cells and the substrate in close proximity [35
]. Thus, biofilm formation by the dairy-associated Bacillus
can be detrimental not only in so-called “dead legs” (equipment details, in which the flow of liquid is significantly less turbulent), but also in main pipelines.
Furthermore, we showed that the biofilm-derived spores of the dairy Bacillus
isolates are much more resistant to commercial cleaning agents, compared to B. subtilis
3610. Presumably, the causes of this resistance differ between the tested strains. Thus, the biofilm-derived spores of MS310 are, apparently, less susceptible both to the mechanical and chemo-biological effects of the employed solutions (Figures S2 and S3
). At the same time, B. paralicheniformis
S127 has the highest resistance to the mechanical removal of spores but shows a variable susceptibility to the chemo-biological effect of the tested agents.
As shown in our previous study [21
], the chemo-biological effect of cleaning agents comprises a disinfecting effect (inactivating bacteria) and/or removal of them from the surfaces of dairy equipment (cleaning effect). According to our results, the dairy Bacillus
isolates are significantly less susceptible to the disinfecting effect of the tested agents, compared to the non-dairy strain (except solution I in the case of S127; Figure 4
; Figure 5
). The observed differences in the mechanical and chemo-biological effects between the tested strains might be explained by the dissimilarities in the biofilm structure. For instance, a correlation between colony biofilm phenotype of the tested strains, and their resistance to the cleaning procedures, was observed (Figure 1
). Thus, the dairy-associated Bacillus
, characterized by a mucoid biofilm phenotype, were less susceptible to mechanical and chemo-biological effects during the CIP procedures. Since biofilm matrix components can be responsible for binding and/or neutralizing detergents and antimicrobial agents [36
], differences in the matrix structure/composition can lead to differences in cleaning and/or disinfection susceptibility. Thereby, the biofilm matrix composition was shown to affect the susceptibility of food-associated staphylococci to cleaning and disinfection agents, with polysaccharide matrix-producing strains being more resistant to the lethal effect of benzalkonium chloride [38
]. Likewise, the efficiency of monochloramine disinfection was dependent on the quantity and composition of EPS in Pseudomonas
biofilms. Protein-based EPS-producing P. putida
was less sensitive to monochloramine than polysaccharide-based EPS-producing P. aeruginosa
, since monochloramine had a selective reactivity with proteins over polysaccharides [39
]. According to Bridier et al. (2011) [40
], the biofilm of the P. aeruginosa
clinical isolate, in which a high delay of benzalkonium chloride penetration is recorded, was characterized by a large quantity of proteinacious matrix. Moreover, the authors report that, in P. aeruginosa
, resistance to antimicrobial agents is intimately related to the inherent three-dimensional organization of cells into the exopolymeric matrix. Therefore, the low sensitivity of the dairy Bacillus
isolates to the CIP procedures (compared to B. subtilis
3610) may be connected to differences in the structure/composition of the biofilm matrix.
Importantly, mucoid colony formation, observed for the dairy Bacillus
isolates, was viewed as a hallmark of poly-γ-glutamic acid (PGA) production in multiple previous studies [17
]. Significant production of PGA could result in a stronger attachment to surfaces due to its adhesive properties [41
]. To this end, PGA-overproducing derivatives of B. subtilis
3610 (B. subtilis
YC295 and B. subtilis
YY54) were significantly more resistant to the mechanical effect of water circulation, compared to the wild type (Figure 6
C). Notably, biofilm colonies of these mutant strains were more mucoid, compared to the WT (Figure 6
A). Moreover, the biofilm-derived spores of PGA-overproducing B. subtilis
were surrounded by higher amounts of proteinaceous extracellular matrix, which resembles the tested dairy Bacillus
isolates (Figure 6
B). Therefore, the presence of PGA in the biofilm matrix of the examined bacterial strains may be one of the factors enhancing resistance to the CIP procedures. We believe that the role of PGA and other presumptive EPS components of the dairy-associated Bacillus
in relation to cleaning and disinfecting agents is an important subject for further investigation.
Relatively low cleaning and, especially, disinfecting effects of the tested solutions (Figure 5
) might lead to undesirable implications regarding the hygiene level in dairy environments. For instance, the rapid recovery of biofilms after inappropriate disinfectant treatment is often observed. This may be due to the re-growth of surviving cells, residual biofilm, providing a conditioning layer for further cell attachment, or the selection of resistant microorganisms that survive and thrive after antimicrobial treatment [5
]. In addition, biofilm cells exposure to low (sub-lethal) concentrations of disinfecting compounds, including chlorine-based detergents, can stimulate further biofilm development [10
]. Therefore, we speculate that the composition of commercial CIP agents should be revised and evaluated under the experimental conditions suggested in this study.