Biofilm Formation by Staphylococcus aureus Isolated from Food Contact Surfaces in the Dairy Industry of Jalisco, Mexico

Staphylococcus aureus is an important food-borne pathogen able to form biolms. is pathogen is responsible for outbreaks of food-borne illnesses associated with the consumption of milk and dairy products. e aim of this study was to evaluate the biolm-forming ability of S. aureus isolates, recovered from food contact surfaces in the dairy industry of Jalisco, Mexico. A total of 84 S. aureus strains were evaluated. e isolates were characterized phenotypically by culture on Congo red agar plates. e ability of the strains to form biolms was investigated in 96-well at-bottomed microtiter polystyrene plates. Stainless-steel coupons were used as an experimental surface. Biolm formation was observed, using epiuorescence microscopy and scanning electron microscopy. Detection of the icaADBC genes in S. aureus was performed by the PCR technique. A total of 52.3% (44/84) of the S. aureus strains contained the icaADBC gene that synthesizes polysaccharide intercellular adhesion (PIA) molecules. On Congo red agar, 75% (63/84) of the S. aureus isolates were biolm producers, 16.6% (14/84) were non-biolm formers, and 8.3% (7/84) showed a noncharacteristic phenotype. e biolm production of the S. aureus strains SA-4E, SA-9, SA-13, and SA-19 on stainless-steel coupons was investigated at 25°C for 8 days, and the detected cell population density was approximately 7.15–7.82 log CFU cm. In addition to the ability of biolm production, it is important to highlight that these strains are potential enterotoxin producers as se genes have been previously detected in their genomes. A part of the ability of biolm production and the determination of the presence of virulence determinants in the genome of S. aureus can contribute to the pathogenicity of strains. erefore, vigilant food safety practices need to be implemented in the dairy industries regarding FCS to prevent foodborne infections and intoxications due to S. aureus contamination.


Introduction
In the food industry, bio lms increase bacterial resistance to environmental stresses including cleaning, disinfection, and inhibition, enabling these microorganisms to persist on surfaces and processing equipment, compared to planktonic cells [1][2][3].Formation of bio lms can occur on all types of surfaces of technological systems in the dairy industry.e detection of bio lms in the food industry can be related to the presence of pathogenic microorganisms in the industrial settings.
Staphylococcus aureus is a food-borne pathogen that can cause staphylococcal food poisoning.In the USA, staphylococcal food poisoning is estimated to account for 241,188 illnesses, 1,064 hospitalizations, and six deaths, annually [4].S. aureus can adhere to and develop bio lms on food contact surfaces, thereby a ecting the quality and safety of food products [5,6].e extracellular matrix of S. aureus bio lms is usually composed of exopolysaccharide (PIA/PNAG), but the proteinaceous and extracellular DNA matrix can also be present in staphylococcal bio lms [7].Depending on the environment in which the bio lm was developed, the bio lm matrix can also contain blood components or noncellular materials such as mineral crystals, corrosion particles, and clay or silt particles [8].PIA is linked to the irreversible attachment phase [9].e formation of biofilm of Staphylococcus aureus is not only mediated by the PIA-dependent biofilm formation, but it can exist in PIA-independent biofilm.In the PIAindependent biofilm, despite the importance of the ica gene locus in biofilm development, biofilms can occur in an ica-independent fashion where biofilm-associated protein (Bap) and Bap-related proteins of S. aureus can confer biofilm development independently or PIA production through cell-to-cell aggregation and are characterized by their high molecular weight, presence of the bacterial surface, role as a virulence factor, and occasional containment in mobile elements [10,11].
e aim of this study was to evaluate the biofilm-forming ability of S. aureus isolates, recovered from food contact surfaces in the dairy industry of Jalisco, Mexico.

S. aureus Isolates. S. aureus (SA1-SA84
) strains were isolated from food contact surfaces (FCS) of six dairy industries in the Mexican state of Jalisco [13].
e S. aureus strains were identified by the methods described in the Bacteriological Analytical Manual (Gram staining, the coagulase and Voges-Proskauer tests, tests for catalase and thermostable nuclease, and glucose and mannitol utilization test), and finally, PCR was used for confirmation (PCR amplification of genes encoding for 23S ribosomal RNA (rRNA) and thermonuclease (nuc)) [13].
e strains were cultivated in tryptic soy broth (TSB; Becton Dickinson Diagnostic Systems) for 24 h at 37 °C.All strains were subcultured in TSB with 0.25% glucose (w/v) for 24 h at 37 °C for the quantification of biofilm formation and in TSB with 0.5% glucose (w/v) for 8 d at 25 °C for biofilm formation on stainless steel.e S. aureus strain ATCC 25923, a strong biofilm former, was used as a positive control.

Phenotype Analysis of Biofilm Production.
e isolates were characterized phenotypically by culture on Congo red agar (CRA) plates, as described by Arciola et al. [14].Briefly, agar plates were prepared by adding 0.8 g Congo red (Sigma-Aldrich) and 36 g saccharose (Sigma-Aldrich) to 1 L blood agar (Becton Dickinson Diagnostic Systems), followed by incubation at 37 °C for 24 and 48 h.e macroscopic characteristics of the S. aureus isolates in the CRA were observed.Crusty black colonies, with a dry filamentous appearance, were recorded as biofilm producers, smooth pink colonies as nonproducers, and intermediate colony morphology (pink with dark centers resembling bull's eyes), as potential biofilm producers [15].

Quantification of Biofilm Formation.
e ability of the strains to form biofilms was investigated in 96-well flatbottomed microtiter polystyrene plates [16].For each strain, three wells of the microtiter plate were filled with 200 μL bacterial suspension in TSB with 0.25% glucose (w/v) (TSB + 0.25% G). en, the plates were incubated at 37 °C for 24 h.Wells filled with the broth medium (TSB + 0.25% G) were used as negative controls, and S. aureus ATCC 25923 was used as the positive control.Next, the content of each well was aspirated and washed three times with phosphatebuffered saline (PBS; 7 mM Na 2 HPO 4 , 3 mM NaH 2 PO 4 , and 130 mM NaCl, pH 7.4) to remove the planktonic bacteria.e attached bacteria were fixed with 95% ethanol for 5 min; then, the plates were emptied and left to dry. e plates were stained with 100 µL of 1% (w/v) crystal violet solution per well for 5 min.e excess stain was rinsed off with sterile distilled water, and the microtiter plates were airdried.

Detection of icaADBC Genes.
Genomic DNA was extracted, using the protocol described by Pu et al. [17].Detection of the icaADBC genes in S. aureus was performed, as stated by Diemond-Hernández et al. [18].e amplifications were performed using the ermal Cycler (TechNet; Bibby Scientific Ltd., UK). e initial step (94 °C for 5 min) was followed by 30 cycles with annealing at 60 °C for 1 min (icaA), 59 °C for 1 min (icaB), 42 °C for 1 min (icaC), or 59 °C for 1 min (icaD) and a final step at 72 °C for 7 min (Table 1).After amplification, the products were electrophoresed on a 2% agarose gel (ultrapure agarose; Invitrogen), containing 0.5 µg/mL ethidium bromide (Sigma-Aldrich), and visualized by transillumination under ultraviolet light.S. aureus ATCC 25923 was used as the positive control.

Conditions for Biofilm Formation.
Stainless-steel (SS) coupons (AISI 316, 0.8 × 2.0 × 0.1 cm) were used as an experimental surface.e coupons were consecutively cleaned, according to the method described by Marques et al. [5].For the biofilm formation, each SS coupon was individually introduced into glass test tubes (20 × 150 mm) containing 10 mL of TSB with 0.5% glucose (TSB + 0.5% G). e monospecies biofilms were inoculated with 100 μL of cultures incubated at 37 °C/24 h (containing approximately 10 8 CFU/mL of the corresponding strains) (Table 2); after that, the biofilms were incubated at 25 °C for 8 d.Afterwards, cell viability was determined by the standard plate count technique on standard agar (Becton Dickinson Diagnostic Systems) with incubation at 37 °C for 24 h.Biofilm formation was observed, using epifluorescence microscopy and scanning electron microscopy (SEM).ree replicates were performed for each strain.S. aureus ATCC 25923 was used as the positive control.As a negative control, an SS coupon without inoculum was included in all assays.

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Journal of Food Quality

Epifluorescence Microscopy.
After the incubation at 25 °C for 8 d, the SS coupons were removed from the glass test tubes containing 10 mL of TSB with 0.5% glucose using sterile forces.Each coupon was washed with 1 mL PBS for 10 s to eliminate nonadhered cells.e coupons were stained with 5(6)-carboxyfluorescein diacetate (CFDA; 10 μg/mL), rinsed with sterile distilled water, dried in a level II cabinet, and observed under a Nikon Eclipse E400 epifluorescent microscope, using 100× oil immersion lens and the filter BA 515 B2a at 450-900 nm.At least 18 fields were observed.Once inside the cell, the diacetate is hydrolyzed by intracellular nonspecific esterases, producing carboxyfluorescein (CF), which is retained by live cells with an intact plasma membrane [19,20].

SEM.
After the incubation at 25 °C for 8 d, the SS coupons were treated as indicated in Section 2.3.1.ey were further dried and transferred to 2% glutaraldehyde at 4 °C for 2 h to fix the sample [21,22].Next, the samples were dehydrated in serial dilutions of ethanol at 30, 50, 60, 70, 90, and 95% at 4 °C for 10 min each.Furthermore, three transfers were performed in 100% ethanol for 10 min each.e samples were vacuum-dried and gold-coated for 30 s. Biofilms were observed, using a TESCAN Mira3 LMU scanning electron microscope.

Genomic Fingerprinting of S. aureus Isolates.
e differentiation of the S. aureus isolates with genotypic and phenotypic characteristics associated with biofilm formation (presence of icaADBC genes, in addition to the presence of virulence determinants in their genome) was performed by the RAPD-PCR method.Primers used for this purpose were OPL5, RAPD5, P1, and P2 (Table 2) according to the method previously described [23,24].Strains of S. aureus ATCC 25923 and 51811 and Lactobacillus delbrueckii subsp.bulgaricus ATCC 11778 were included to enable the comparison of genetic variability.RAPD-PCR band patterns from each primer were scanned, and profile grouping (dendrogram) was performed with the PAST (PAleontological Statistics) version 3.20 software (University of Oslo, Noruega), using Jaccard's coefficient and the unweighted pair-group method with arithmetic averages (UPGMA) cluster analysis [25].

Statistical Analysis.
Pearson's chi-squared test was employed at the p < 0.05 significance level to compare differences between groups.Statistical analysis was performed with SPSS for the Windows software, version 11.0.

Results
A total of 84 S. aureus strains (SA1−SA84) were studied to estimate their potential to adhere to, and, subsequently, form biofilms on food contact surfaces.Biofilms were quantified regarding biomass accumulation, using the crystal violet staining method.
Among 84 S. aureus strains, four S. aureus (SA-4E, SA-9, SA-13, and SA-19) were examined by epifluorescence and SEM. e four S. aureus strains were considered, according to the genotypic and phenotypic characteristics associated with biofilm formation (Table 4).In addition to the ability of biofilm production, it is important to highlight that these strains are potential enterotoxin producers as se genes have been previously detected in their genomes [13].e genetic variability of these strains of S. aureus isolates was determined by RAPD-PCR genotyping using four different primers (Table 2).Strains were grouped into five main clusters (I-V) (Figure 2).Cluster I is composed of strains SA-13 and SA19.Cluster II includes the strain SA-9.Cluster III includes the strain SA-4E.Group IV comprises the strains of Staphylococcus aureus ATCC 25923 and 51811.And finally, cluster V comprises the strain of Lactobacillus delbrueckii subsp.bulgaricus ATCC 11778.
e genetic variability of the strains of S. aureus was demonstrated by RAPD-PCR analysis.e four S. aureus strains showed the ability to form single-species biofilms on SS coupons at 25 °C; cell adhesion was visualized during biofilm maturation by epifluorescence microscopy (Figure 3).With this technique, it is possible to observe the presence of metabolically active living cells, and the diacetate is hydrolyzed by intracellular nonspecific esterases, producing carboxyfluorescein (CF) that indicates the integrity of the plasma membrane and esterase activity.Moreover, in the SEM microphotographs, the surface of microcolonies of the biofilm of the four S. aureus strains was visualized as well as probably the presence of the EPS (Figure 4).All isolates evaluated in this study had a concentration ranging from 7.15 ± 0.15 to 7.82 ± 0.25 log CFU cm −2 on the SS coupons, and no significant differences (p > 0.05) were observed among them.

Discussion
Biofilms formed on food contact surfaces can lead to significant health problems.Biofilms reduce the effectiveness of sanitizers, cause economic losses to industries, and contaminate food and can increase the level of antimicrobial resistance [26].Our results indicated that most of the examined S. aureus strains had at least one intercellular adhesion gene involved in the formation of PIA.Of note, 44 strains harbor the 4 genes of the icaADBC locus, which support their ability to produce biofilms.Most of the S. aureus strains formed the biofilm in an ica-dependent mechanism.is finding is consistent with results reported by Tang et al. [12], who detected icaAD and icaBC in 87.5% (n � 57) of S. aureus strains isolated from several sources (chicken, food samples, and goats).Gutiérrez et al. [27] also showed that 100% of S. aureus (n � 63) strains collected from various food contact surfaces in the dairy, meat, and seafood industries were positive for the icaA and icaD genes.
In the current study, most of the evaluated strains were S. aureus biofilm producers.Similar results were obtained by Szczuka et al. [9], who reported that, of 74 biofilm-positive strains, 56 carried the icaA (76%) gene and produced slime on CRA.However, the variation between phenotypic and genotypic methods for detection of the biofilm produced by S. aureus has been reported, regarding CRA [28].Congo red can directly interact with certain polysaccharides, forming   colored pigments [29], and some metabolic reactions that form secondary products with the dye can influence the formation of dark colonies [14].Nevertheless, Kim et al. [30] determined slime production under various environmental conditions (1% dextrose, 5% NaCl, and their combination) in four S. aureus strains (ATCC 12600, D8, D29, and C52), but the results did not indicate any influence of the tested conditions on slime production.Consequently, the CRA technique could be used as the presumptive test for the formation of a biofilm.However, Arciola et al. [14] suggest that the phenotypic change may be caused by a deletion of the ica operon rather than an insertion event which inactivates the ica genes.e type of the food contact surface and diverse environmental factors, such as osmolarity, nutrient content, and temperature, and genetics, such as the presence of sarA, ica, and agr genes [31], may influence the development of a biofilm by S. aureus and, consequently, its persistence on contact surfaces within the food industry [32].Moreover, the ica operon expression is strongly influenced by environmental factors, such as glucose, temperature, osmolarity, and growth under anaerobic conditions [33].Li et al. [34] reported that, besides icaAD and icaBC, other virulence regulators including bap, sigB, and sar might be crucial biofilm-associated genes because these genes are expressed more often in biofilm-positive strains than in biofilm-negative strains.
Rode et al. [32] demonstrated that temperatures suboptimal for growth increased the biofilm formation in eleven S. aureus strains and the highest biofilm production occurred at 25, 30, and 46 °C, whereas, in general, biofilm formation was low at 42 °C.Da Silva Meira et al. [22] evaluated the biofilm formation of three food industry-associated S. aureus isolates on SS and polypropylene surfaces, incubated in a vegetablebased medium at two temperatures (7 and 28 °C/15 d), deducing that the biofilm development was favored at 28 °C, without significant differences between the type of surface.Journal of Food Quality  6 Journal of Food Quality Bae et al. [35] found that populations of five food-borne pathogens including S. aureus formed biofilms with 8.8-9.3 and 9.4-10.3log CFU/coupon on SS and polypropylene surfaces, respectively.Consequently, these isolates of S. aureus (SA-4E, SA-9, SA-13, and SA-19) have the ability to form biofilms on food contact surfaces.

Conclusion
In conclusion, this study showed the biofilm-forming ability of S. aureus, isolated from food contact surfaces in the dairy industry.Biofilm formation can cause public health problems and economic losses, associated with food contamination by the pathogen and equipment damage, by favoring equipment corrosion or resistance to hygiene treatments of food contact surfaces.

Table 2 :
Primers used for the RAPD-PCR method.

Table 3 :
icaADBC genes in Staphylococcus aureus isolates from food contact surfaces.

Table 4 :
Association between the biofilm phenotype on Congo red agar, slime production, adherence assay, and the presence of icaADBC genes in Staphylococcus aureus.