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. 2019 Jul 10;56(10):4595–4604. doi: 10.1007/s13197-019-03924-1

Characterization of biofilm production by Pseudomonas fluorescens isolated from refrigerated raw buffalo milk

Karine Lauer Cruz 1, Amanda de Souza da Motta 1,✉
PMCID: PMC6801270  PMID: 31686691

Abstract

Pseudomonas fluorescens can often be isolated from refrigerated raw milk. Two strains of P. fluorescens PL5.4 and PL7.1, isolated from raw buffalo milk, were evaluated for their proteolytic capacity, exopolysaccharide production and biofilm production. Proteolytic activity was observed in both strains. The P. fluorescens PL5.4 strain presented fluorescence in the presence of calcofluor, indicating exopolysaccharide production. Both strains were able to produce biofilm at 7 °C for 72 h. For the biofilm production test on stainless steel, adherent cell counts of up to 7.1, 7.3 and 8.8 log CFU/cm2 at 7, 23 and 30 °C were obtained. Through scanning electron microscopy, it was possible to observe the biofilm produced by the P. fluorescens PL5.4 strain. Proper cleaning and disinfection practices in order are important to reduce bacterial contamination and extend the useful life of raw material and its derivatives.

Keywords: Biofilm, Pseudomonas fluorescens, Psychrotrophic bacteria, Raw buffalo milk

Introduction

The production of buffalo milk has been expanding in the Brazilian market, mainly for the supply of dairy products. At around 12.8%, its international yield parallels that of other types of milk, making it the second most produced type of milk in the world (Bernardes 2014). However, the availability of nutrients in the milk, along with a lack of good hygienic practices during the milking process, make this product a favorable medium for microbial growth (Martins et al. 2015).

The cooling of raw milk as an alternative strategy to control the growth of mesophilic microorganisms led to the creation of selective conditions for psychrotrophic bacteria (Puga et al. 2016). Psychrotrophic bacteria are organisms that have the ability to grow at low temperatures, but their optimal growth temperatures range between 15 and 20 °C (Oliveira et al. 2015). This group is more expressive in products that are preserved or stored under refrigerated conditions for relatively long periods. For this reason, in order to reduce initial contamination it is extremely important to collect and receive raw milk under adequate hygienic/sanitary conditions (De Jonghe et al. 2011). While psychrotrophic bacteria are not resistant to the pasteurization process, during the production process, they are able to produce thermoresistant enzymes that can subsequently degrade proteins and fats present in the milk (Oliveira et al. 2015). Milk with high microbiological counts, which is subjected to a long storage period, will suffer major alterations in its final product (pasteurized milk, ultra-pasteurized milk and cheeses) and consequently have a shortened shelf life (Arslan et al. 2011).

Pseudomonas fluorescens is a psychrotolerant bacterium, which can frequently be isolated from refrigerated raw milk and are commonly associated with food spoilage (Martins et al. 2014; Oliveira et al. 2015; Puga et al. 2016). They are important because they have proteolytic and lipolytic enzymes, which are able to withstand thermal treatments (Boari et al. 2009; Martins et al. 2014). Two other notable characteristics of P. fluorescens are their ability to adhere to the surface and to produce biofilms (Rossi et al. 2016). This biofilm production on surfaces, such as those used for food production, has been increasingly studied because these act as contamination points, triggering the release of pathogenic or spoilage microorganisms. It also compromises the quality of these raw materials and their derivative products (Cleto et al. 2012; Marchand et al. 2012).

At present, there has been little research conducted on biofilms produced by P. fluorescens in milk, despite the fact that this microorganism is most often isolated from the raw material (Scatamburlo et al. 2015; Lin et al. 2016; Rossi et al. 2016). For these reasons the objective of the present study was to evaluate and characterize the biofilm production process by two P. fluorescens isolated from raw buffalo milk refrigeration tanks.

Materials and methods

Bacterial strains and culture conditions

Two strains of P. fluorescens were used in this study. Both were obtained from raw milk refrigeration tank, during the period 2012–2013 and identified in previous work as P. fluorescens PL5.4 and P. fluorescens PL7.1, under codes MH046417 and MH046418, respectively, in the Standard Nucleotide BLAST (retrieved from http://www.ncbi.nlm.nih.gov). These two strains showed at least 98% similarity to the available sequences in the database. The same strains were isolated from samples of raw buffalo milk taken from a cooling tank of a dairy farm located in the municipality of Glorinha–RS. The strains were frozen in skimmed milk and kept at − 20 °C. After thawing, the bacteria were cultured in Tryptic Soy Broth (TSB) (Himedia, India) and incubated at 30 °C for 48 h. After growth, they were striated by depletion on Trypticase Soy Agar (TSA) (Himedia, India) plates and incubated at 30 °C for 48 h.

Evaluation of proteolytic activity in milk agar

The proteolytic activity of the isolates was evaluated according to Ruaro et al. (2013), with some modifications. The P. fluorescens PL5.4 and PL7.1 strains were inoculated on milk agar plates (5 g/L meat peptone, 3 g/L yeast extract, 12 g/L agar, 10% skimmed ultra-high temperature-UHT milk) by stab technique and incubated at 30 °C for 48 h. The presence of a proteolysis halo around the colony was measured and expressed in millimeters (mm). Pseudomonas aeruginosa ATCC 27853 was used as a positive control.

Investigation of the production of exopolysaccharides (EPS) on solid medium containing calcofluor white

Strains of P. fluorescens PL5.4 and PL7.1 were grown in Petri dishes using a synthetic medium (K2HPO4 0.6 g/L, KH2PO4 1.8 g/L, MgSO4·7H2O 0.2 g/L; NaCl 0.1 g/L, CaCl2·2H2O 0.2 g/L, Na2MoO4·2H2O 2 mg/L, MnSO4H2O 2.4 mg/L, H3BO3 2.8 mg/L, CuSO4·5H2O 0.08 mg/L, ZnSO4·7H2O 0.24 mg/L, supplemented with 4 mL of FeEDTA 1.64%, 1 mL of biotin 100 μg/mL, mannitol 5.0 g/L, monosodium l-glutamate monohydrate 1.87 g/L) plus the epi-fluorescent dye calcofluor white (final concentration 200 μg/mL). One plaque was divided into five parts and the lines of P. fluorescens PL5.4 and PL7.1, together with the controls Staphylococcus epidermidis ATCC 35984, P. aeruginosa ATCC 27853 and P. fluorescens NCTC 10038, were seeded. After 24 h of growth in an incubator at 30 °C, the EPS production of each strain was observed by illuminating the plates with ultraviolet (UV) light at a wavelength of 365 nm (Serrato et al. 2006).

Crystal violet assay for the evaluation of biofilm production

For biofilm production, the method described by Stepanović et al. (2000) was used. The colonies of P. fluorescens PL5.4 and PL7.1 strains, from TSA plates, were picked up and resuspended, individually, in 3 mL of a 0.85% saline solution and the turbidity was standardized according to the McFarland scale of 0.5. The assay was performed on 96 well microtiter plates of flat bottom polystyrene material (NEST, China). The strains were individually inoculated into octuplicates. One hundred eighty (180) μl of TSB (Himedia, India) were added to the wells, plus 0.25% glucose and 20 μl of bacterial suspension from each of the strains individually. The wells with the negative control were treated under the same conditions, but without the addition of the strains. For positive control, a strong biofilm-producing culture of S. epidermidis ATCC 35984 (Moura et al. 2015) was used. Plates were incubated under different conditions: at 7 °C for 72 h, 23 °C for 24 h and 37 °C for 24 h. After incubation, the wells were aspirated and the samples were washed three times with 200 μL of a 0.85% saline solution. Bacterial fixation was performed using 200 μL of 99.8% methanol (PA) for 20 min. The methanol was then aspirated and the microplates were inverted and allowed to dry overnight at room temperature.

The staining procedure was performed with 200 μl of a 0.5% crystal violet solution for 15 min, followed by a washing of the plates with sterile distilled water. After drying the plates, the bacterial cells fixed and stained at the bottom of the wells were resuspended in 200 μl of 95% ethanol for 30 min before the biofilm quantification was done. The optical density (OD) of the bacterial biofilm was quantified with the aid of a spectrophotometer microplate reader, at a wavelength of 570 nm (Anthos 2010 Type 17 550 S. No. 17 550 4894). All microorganisms were separated into categories using the OD of the bacterial biofilms. The samples were classified as: non-biofilm forming producer (ODbiofilm < ODc−), weak biofilm producer (ODc− < ODbiofilm ≤ 2 × ODc−), moderate biofilm producer (2 × ODc− < ODbiofilm ≤ 4 × ODc−) and strong biofilm producer (ODbiofilm > 4 × ODc−), ODc− (optical density negative control).

Effects of the co-cultivation of Listeria monocytogenes ATCC 7644 on the biofilm of P. fluorescens PL5.4 in the crystal violet assay

To evaluate the effect of the co-culture of L. monocytogenes ATCC 7644 on the biofilm production of the P. fluorescens PL5.4 strain, colonies of both strains, previously cultured in TSA, were resuspended individually in a 0.85% saline solution and turbidity was standardized according to the McFarland scale of 0.5. 10 μl of each bacterial suspension were added to the same wells, followed by the same protocol described by Stepanović et al. (2000). Three replicates were used to evaluate the production of biofilms under different conditions: at 7 °C for 72 h, 23 °C for 24 h and 37 °C for 24 h.

Evaluation of biofilm production by a strain of P. fluorescens PL5.4 on stainless steel surfaces

The biofilm formation capacity of the P. fluorescens PL5.4 strain was evaluated on American Iron and Steel Institute (AISI 304) stainless steel test specimens (1 cm × 1 cm, negligible thickness), according to the methodology described by Nörnberg et al. (2011) with some modifications. To clean the stainless steel specimens, they were immersed in acetone for 30 min to remove grease or fingerprints, followed by a rinsing in sterile, distilled water (dH2O) and sterilization by autoclaving. For the experiment, bacterial cultures were grown in TSB medium and incubated for 24 h at 30 °C. A 2 mL aliquot at a concentration of 108 CFU/mL (adjusted using a spectrophotometer with an OD of 600 nm), was transferred to Erlenmeyer flask containing 18 mL of TSB plus 0.25% glucose. For each Erlenmeyer, three stainless steel specimens were added. They were subsequently incubated without shaking at 7 °C, 23 °C and 30 °C for 24, 24, 48 e 72 h. Every 24 h, a test specimen from each Erlenmeyer was aseptically removed using sterile forceps and washed three times in sterile dH2O to remove poorly adherent cells. The specimens containing biofilm cells were immersed in 10 mL of a 0.85% saline solution and subjected to a Unique Model USC700 ultrasound bath, at a frequency of 40 kHz for a period of 10 min, in order to release cells from the biofilm. Decimal dilutions were made from each solution containing the specimen. They were then plated and incubated at 30 °C for 24 h. Counts were performed to determine the number of sessile cells and planktonic cells, both being expressed in log CFU/cm2. All counts were performed in duplicate and each assay was repeated twice.

Scanning electron microscopy of biofilm produced by a strain of P. fluorescens PL5.4

The biofilm production by the P. fluorescens PL5.4 strain on a stainless steel specimen (1 cm × 1 cm, negligible thickness) and its planktonic cells were examined by Scanning Electron Microscopy (SEM) at the Microscopy Center and Microanalysis of the Federal University of Rio Grande do Sul (UFRGS). For this analysis, an Erlenmeyer flask was used with 18 mL of culture medium TSB broth plus 0.25% glucose. To this Erlenmeyer flask, 2 mL of a pre-inoculum containing the P. fluorescens PL5.4 strain was added, at a concentration of around 108 CFU/mL.

To hygienization of the specimens, they were immersed in acetone for 30 min to remove grease or fingerprints, followed by a rinsing in sterile, distilled water (dH2O) and sterilization by autoclaving. After, it was immersed in the broth containing the P. fluorescens PL5.4 strain and incubated at 30 °C for 24 h. Next, for the assay of sessile cells, the surface with the adherent cells was fixed with 3% (v/v) glutaraldehyde in 0.2 M phosphate buffer (pH 7.2) for 3 days. To check for free (planktonic) cells, 1 mL of the inoculated broth was removed and added to a microcentrifuge tube, which was centrifuged at 10,000 rpm for 10 min to obtain a pellet with the cells. From this, the supernatant was removed and glutaraldehyde added, according to the same specifications stated above, for fixing. After this process, the materials were washed three times with the same buffer and dehydrated in an acetone gradient of a 30% to 100% concentration (v/v). The material was then processed in a critical-point dryer (Balzers CPD030; Bal-Tec, Balzers, Liechtenstein). Subsequently, gold and platinum particles were deposited on the samples. The viewing was conducted via SEM JSM6060 (JEOL, Tokyo, Japan) (Goldbeck et al. 2014).

Statistical analysis

The count values were converted to decimal logarithms (log CFU/cm2) to correspond to a normal distribution. The results were submitted to analysis of variance (ANOVA), using generalized linear models of the SPSS program (version 18), followed by the Bonferroni test to evaluate for significant differences, with p < 0.05 as the accepted value for significant difference.

Results

Evaluation of proteolytic activity in milk agar

The isolates were tested in milk agar culture medium, an area of degradation around the colony growth of the two strains was observed, indicating proteolytic activity in the culture medium. The halos measured from the P. fluorescens PL7.1 strain ranged from 10 to 16 mm, whereas, the halos measured from the P. fluorescens PL5.4 strain ranged from 11 to 15 mm. Both expressed larger halos than the ones produced by the positive control, which ranged from 5 to 8 mm.

Investigation of the production of exopolysaccharides (EPS) by solid medium containing calcofluor white

In the evaluation of EPS production, the P. fluorescens PL5.4, P. fluorescens NCTC 10038 and P. aeruginosa ATCC 27853 bacteria showed fluorescence when exposed to UV light at a wavelength of 365 nm. The P. fluorescens PL7.1 strain did not produce fluorescence, and differed from the other tested bacteria. The S. epidermidis ATCC 35984 strain showed no growth in this culture medium (Table 1). Fluorescence on calcofluor-containing media is indicative of EPS production by bacteria (Fig. 1).

Table 1.

Fluorescence by strains exposed to UV light in culture medium containing calcofluor white

Strain Observation
P. aeruginosa ATCC 27853 Fluorescent
P. fluorescens NCTC 10038 Fluorescent
S. epidermidis ATCC 35984 Non-fluorescent
P. fluorescens PL5.4 Fluorescent
P. fluorescens PL7.1 Non-fluorescent
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Fig. 1.

Fig. 1

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Fluorescence in culture medium containing calcofluor white at 365 nm. S. epidermidis ATCC 35984, P. fluorescens NCTC 10038, P. aeruginosa ATCC 27853, PL5.4 (P. fluorescens PL5.4), PL7.1 (P. fluorescens PL7.1)

Crystal violet assay for the evaluation of biofilm production

In the evaluations of biofilm formation, bacterial strains were found to present similar behavior when submitted to different temperatures, differing only at a temperature of 37 °C. For both strains, the best conditions for the production of biofilm was noted at a temperature of 7 °C after 72 h, thus qualifying them as strong biofilm producers (OD > 4 × negative control) under these circumstances. Under conditions of 23 °C after 24 h, both presented as moderate biofilm producers. At 37 °C, the P. fluorescens PL5.4 strain presented as a moderate producer whereas the P. fluorescens PL7.1 strain presented as a non-biofilm producing agent. S. epidermidis ATCC 35984, a positive control, presented as a strong producer at 37 °C and 23 °C, and as a non-biofilm producer at a temperature of 7 °C.

Effects of the co-cultivation of L. monocytogenes ATCC 7644 on the production of biofilm by P. fluorescens PL5.4

In the presence of the bacterium L. monocytogenes ATCC 7644, the P. fluorescens PL5.4 strain maintained its ability to produce biofilm. It presented as a strong biofilm producer at a temperature of 7 °C, however, at 37 °C and at 23 °C, it presented as a weak producer. Listeria monocytogenes ATCC 7644, when evaluated alone, presented as a poor biofilm at 37 °C and as a non-biofilm producer at temperatures of 7 °C and 23 °C. The positive control S. epidermidis ATCC 35984 presented as a strong producer at 37 °C and at 23 °C and as a weak producer at a temperature of 7 °C.

Evaluation of the biofilm production of the P. fluorescens PL5.4 strain on stainless steel surfaces

Regarding temperature variation and the P. fluorescens PL5.4 strain, there was no significant difference (p > 0.05) in the number of adherent cells between temperatures of 7 °C (ranging from 6.0 to 7.0 log UFC/cm2) and 23 °C (ranging from 5.0 to 7.0 log CFU/cm2). However, a significantly larger number of adherent cells (p < 0.05) was observed at a temperature of 30 °C (ranging from 5.0 to 8.0 log CFU/cm2).

With reference to time variation, during a time interval of 24–48 h at a temperature of 7 °C, P. fluorescens PL5.4 showed a significant decrease (p < 0.05) in the number of adherent cells (ranging from 7.0 to 6.0 log CFU/cm2). In the time interval of 48–72 h there was a significant increase (p < 0.05) of adherent cells to stainless steel (ranging from 6.0 to 7.0 log CFU/cm2). At a temperature of 23 °C, during a time interval of 24–48 h there was a significant drop (p < 0.05) in the number of adherent cells to the stainless steel (ranging from 7.0 to 5.0 log CFU/cm2) and during a time interval of 48–72 h this count continued to decline (ranging from 5.0 to 4.0 log UFC/cm2). At 30 °C, depending on the time variation, the behavior of P. fluorescens PL5.4 was the same as described above at 23 °C (Fig. 2).

Fig. 2.

Fig. 2

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Counting of sessile P. fluorescens PL5.4 cells on stainless steel surface in TSB culture medium at temperatures of 7, 23 and 30 °C after 24, 48 and 72 h

For the three temperatures tested, the planktonic cell count of the P. fluorescens PL5.4 strain in the initial inoculum (Time 0) resulted in a mean value of 8.5 log CFU/cm3. The mean number of planktonic cells after 24 h was 9.1 log CFU/cm3 at 7 °C, 9.7 log CFU/cm3 at 23 °C and 9.6 log CFU/cm3 at 30 °C. The mean number of planktonic cells in 48 h was 9.4 log CFU/cm2 at 7 °C, 9.7 log CFU/cm3 at 23 °C and 9.3 log CFU/cm3 at 30 °C. The mean number of planktonic cells after 72 h was 9.5 log CFU/cm3 at 7 °C, 9.1 log CFU/cm3 at 23 °C and 8.8 log CFU/cm3 at 30 °C (Fig. 3).

Fig. 3.

Fig. 3

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Pseudomonas fluorescens PL5.4 plankton cells on a stainless steel surface in TSB culture medium at temperatures of 7, 23 and 30 °C after 24, 48 and 72 h

Scanning electron microscopy (SEM) of the biofilm produced by the P. fluorescens PL5.4 strain

SEM showed the P. fluorescens PL5.4 strain in its two forms: sessile and planktonic. Figure 4a–c show the cell architecture adherent to the American Iron and Steel Institute’s AISI 304 stainless steel coupon after 24 h at a temperature of 30 °C in 0.25% TSB broth. EPS and locomotion structures were not observed, although these microorganisms have the capacity to produce them. The length of the bacterial cell was approximately 1 μm. The cell surface has a rough appearance. Figure 4d–f show the cells in their planktonic form, grown under the same conditions. The images were captured at the same respective magnifications mentioned previously. The length of the bacteria in this planktonic form is approximately 2 μm. The cell surface is smoother when compared to its form in biofilm.

Fig. 4.

Fig. 4

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Scanning electron microscopy (SEM) of the P. fluorescens PL5.4. Images a–c scanning electron micrographs of the P. fluorescens PL5.4 strain in sessile form, after growth in TSB at 30 °C for 24 h. Images d–f scanning electron micrograph of the P. fluorescens PL5.4 strain in planktonic form, after growth in TSB at 30 °C for 24 h. In b, c and f the arrows indicate structures that resemble fibers, being structures that are related to the formation and establishment of the structures of biofilms

Discussion

The genus Pseudomonas spp. is already known to be responsible for the deterioration of milk and many dairy products (Arslan et al. 2011; Oliveira et al. 2015). Although several studies have reported the isolation of Pseudomonas spp. from samples of raw milk, P. fluorescens is actually the group most often involved in the alteration of derived products (Arslan et al. 2011; De Jonghe et al. 2011; Martins et al. 2014; Puga et al. 2016; Rossi et al. 2016). There is still a shortage of information on biofilms produced by P. fluorescens and isolated from milk (Rossi et al. 2016).

Both P. fluorescens PL5.4 and PL7.1 strains showed high proteolytic capacity when exposed to the culture medium plus milk. The production of heat-resistant exoenzymes by these microorganisms, such as lipases and proteases, may remain stable after the pasteurization process and ultimately deteriorate milk and its future derivatives (Boari et al. 2009). Pseudomonas spp. are able to produce and secrete proteolytic enzymes (Cleto et al. 2012; Martins et al. 2015). The results found in this study are in agreement with Martins et al. (2015), who described the production of proteolytic activity by P. fluorescens. These findings underscore the point that refrigeration processes adopted by the dairy industry during milk processing and storage do not completely inhibit enzymatic activity (Boari et al. 2009). This fact reinforces the importance of the precepts of good manufacturing practices during milk production and processing, in order to limit contamination and the alteration of its organoleptic properties by contaminating bacteria (Martins et al. 2014, 2015; Rossi et al. 2016).

To evaluate the production capacity of exopolysaccharides (EPS), a plaque growth experiment was carried out with the addition of the calcofluor white dye. Calcofluor white is a specific fluorescence dye for polysaccharides containing β − 1 → 4 or β − 1 → 3 bonds (Serrato et al. 2006). The colonies that produced the highest amount of EPS had a higher fluorescence intensity when the plates were exposed to UV light (365 nm). The P. fluorescens PL5.4 strain is known for its high fluorescent intensity, caused by the large amount of EPS produced under these conditions. In comparison, the P. fluorescens PL7.1 strain presented low fluorescent intensity. Formed biofilm demands stricter sanitation procedures since some sanitizers cannot penetrate satisfactorily into EPS, which act as a protective layer for the bacterial cell (Castro et al. 2017).

Biofilms stand out as a serious problem for the food industry. Well-established biofilms are difficult to remove from surfaces, thus becoming a constant source of contamination by bacteria (Koo et al. 2013). The strains evaluated in this study demonstrated that the time and temperatures under which the raw material is exposed to the after to surfaces directly influence the production of biofilms. It was observed that these bacteria, were better able to produce biofilms when exposed to a temperature of 7 °C for a period of 72 h. For the P. fluorescens PL5.4 and PL7.1 strains, the longer the exposure time, the higher the concentration of cells present in the resulting biofilm. Data gathered by Souza et al. (2014) and Castro et al. (2017) showed most intense biofilm formation at the ideal temperature tested (35–37 °C) for S. aureus and Enterococcus spp. growth. This finding can be explained by the fact that P. fluorescens has a psychrotrophic characteristic (Puga et al. 2016). Even though they prefer an optimal temperature of about 28 °C, they are capable of developing at temperatures between 7 and 10 °C (De Jonghe et al. 2011; Oliveira et al. 2015). As such, the refrigeration of raw milk, while providing control of undesirable mesophilic microorganisms, can end up favoring the growth of psychrotrophs such as P. fluorescens and L. monocytogenes (Boari et al. 2009). In this work we used autochthonous Pseudomonas cultures of buffalo milk and observed that there are no papers showing the maintenance of these isolates in the form of biofilms in this raw material. Also buffalo milk is a raw material with different properties of bovine milk, being the milk with higher solids content (Bernardes 2014). There is no legislation for this milk in the State of Rio Grande do Sul, hence work is needed to evaluate this raw material and its characteristics.

Another aspect to be considered in the milk processing environment is the development of polymicrobial biofilms. Different species may coexist in a microbial biofilm, like P. fluorescens and L. monocytogenes (Puga et al. 2016). Listeria monocytogenes is known to be a human foodborne pathogen (Piercey et al. 2016). In the experiment to evaluate the co-cultivation of the P. fluorescens PL5.4 strain with L. monocytogenes ATCC 7644, the performance of the P. fluorescens PL5.4 strain was found to be superior to that of the L. monocytogenes strain in biofilm production. The result of the interaction between species on surfaces depended on the conditions (time, temperature, nutrients) influencing the strains involved and the respective sizes of their populations (Kives et al. 2005; Boari et al. 2009). Studies report that species of Pseudomonas spp. are more prevalent in food industries than strains of L. monocytogenes (Martins et al. 2014; Oliveira et al. 2015; Puga et al. 2016; Rossi et al. 2016). As reported by Puga et al. (2016), a commensal relationship was observed in the biofilm shared between the two species, where the primary colonizer (P. fluorescens) provides shelter for poor or non-biofilm producers. Apparently, when this dual-species consortium produces biofilms on a solid surface, the interaction between them, especially at low temperatures, contributes to a more compact structure than the one constructed by L. monocytogenes in monoculture biofilm (Puga et al. 2016). Multispecies biofilms have attracted attention primarily because their partners may withstand antimicrobials better than biofilms of individual species (Kives et al. 2005; Boari et al. 2009). At the same time, in the dairy industry the polypropylene material is less used than stainless steel, the latter of which cooling tanks, pipelines, transport tanks, surfaces and utensils used in milk processing are generally composed (Picoli et al. 2017).

In the present study, it was noted that the P. fluorescens PL5.4 strain, on stainless steel surface, had a higher number of adherent cells at 7 °C, showing no significant difference (p > 0.05) in the time between 24 and 72 h. This demonstrates its preference for a lower temperature and its ability to maintain structure during extended storage. Since this bacterium was isolated from refrigerated raw buffalo milk, this study shows that it could produce biofilm on stainless steel composite equipment, at a temperature of 7 °C–as early as within the first 24 h–and remain structured for up to 72 h of storage. Biofilm formation was also observed at temperatures of 30 °C. This shows the importance of hygiene processes adequately, in the sense that when the cooling tanks have been emptied and switched off, they must be cleaned immediately and properly. At 30 °C, in hotter periods of the year, the maintenance of residues of organic matter in these equipment may suggest the establishment of bacteria in the form of biofilm (Castro et al. 2017).

It is known that raw milk can be kept for several hours in refrigerated tanks until the beginning of its processing of its derivatives (Bernardes 2014). This period may be sufficient for the growth of microorganisms, with the possible production of biofilms and the subsequent impairment of raw material (Castro et al. 2017). The final quality of dairy products can be highly influenced by the contamination of the raw milk which, in turn, can cause degradation and reduction of its useful life (Rossi et al. 2016; Pinto et al. 2017). The number of adherent microorganisms on the surface is also influenced by hydrophobicity and surface charge, since hydrophobic surfaces such as metal have a high number of these adherent microorganisms (Souza et al. 2014). The presence of biofilm on stainless steel is likewise associated with the production of EPS by these microorganisms as well as the time the cell is exposed to a determined surface (Castro et al. 2017). It is important to note that, considering the raw material milk, especially buffalo milk, investigations are necessary in order to evaluate the real conditions in which biofilms are established, mainly in relation to temperature and time factor (Zhu et al. 2018).

Through SEM, it was possible to view the P. fluorescens PL5.4 strain and to compare its planktonic and sessile forms. Each cell state presented distinct architecture. Its sessile form shows large, massive clusters of cells composed of many layers. However, it was not possible to verify the presence of motile structures or the production of EPS. Simões et al. (2013) reported that biofilms produced under a turbulent flow were more metabolically active and had a greater ability to produce matrix structures and proteins when compared to biofilms produced under laminar flow. This detailed characterization of biofilm in response to stress may explain the possible lack of these structures. The absence of these structures in biofilms can also be caused by the chemical and physical processes that the sample undergoes during preparation for observation in the SEM (Al-Shabib et al. 2018; Hrubanova et al. 2018; Zhu et al. 2018).

In the visualization of the planktonic and sessile forms, one can observe a greater grouping and density between the cells, by field visualized, in images a, b and c (Fig. 4). This data demonstrates the capacity of formation of these structures called biofilm, considering the experimental conditions established in the previous experiments. Still, according to other works that indicate in more detail this structure, our images are compatible with the biofilms described by other researchers, where we also identify structures in the form of fibers, which have been related to the establishment of biofilms in Pseudomonas. Cell densification and overlapping were also observed in a multilayer structure (Fig. 4a–c) (Baum et al. 2009; Alhede et al. 2012).

In the food industry, it is necessary for surfaces to be well sanitized so that they do not harbor pathogenic microorganisms, and reducing the load of degrading microbes to safe levels (Pinto et al. 2017). Milk farms and dairy processing industries may perform poor hygiene practices or have limited access to training in food safety for workers, which is a biological hazard to the handling environment. The principles of food safety systems based on Hazard Analysis and Critical Control Points (HACCP) are necessary and recommended for the prevention of raw milk contamination (Pinto et al. 2017).

Conclusion

Biofilm is produced by microorganisms as a way of survival in hostile environments, which makes them more resistant to antimicrobial substances and sanitizing agents. For this reason, they are a serious concern for the food industry. They are constituted by harmful pathogens or spoilage microorganisms, which can cause diseases and economic damage due to the depreciation of the final product through physicochemical and sensorial alterations. The present study showed that the P. fluorescens PL5.4 strain is capable of producing biofilm and maintaining this structure for up to 72 h at refrigeration temperatures. This bacterium was able to produce biofilm in co-culture with L. monocytogenes ATCC 7644 under the same conditions. These findings highlight the importance of carrying out appropriate cleaning procedures in order to reduce contamination by these bacteria.

Acknowledgements

The Centro de Microscopia e Microánalise (CMM)–UFRGS, Cooperbúfalo and CAPES–Coordenação de Aperfeiçoamento de Pessoal de Nível Superior.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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