Salmonella Minnesota Emergence in Poultry Production: Danger to Public Health?

Purpose: The aim of this study was to analyze in S. Minnesota the presence of virulence genes, phylogenetic relationship, bio�lm formation index (BFI) and its ultrastructure at temperatures of 4 °C, 25 °C and 36 °C and also the ability of biocidal agents to promote inhibition of the sessile structure formed by 20 strains isolated in the years 2009, 2010 and 2014 in broiler slaughter plants of two Brazilian companies. Methods: The presence of speci�c genes was evaluated by PCR and the phylogeny between the strains by PFGE, while BFI was performed by the classical method using TSB broth supplemented with 5% of chiken juice and its structure observed by SEM Chlorhexidine 1%, sodium hypochlorite 1% and peracetic acid 0.8% were tested for bio�lm inhibition. Results: The presence of speci�c genes indicates that S. Minnesota has the potential to cause disease in humans and to adapt to adverse conditions. In general, temperatures of 25 °C and 36 °C favored more bio�lm formation, although at 4 °C there is still biomass with potential contaminant from the �nal product. Tolerant strains (12/20–60%) to all biocides pose a real risk for the development of adaptation mechanisms, especially for resistance to sodium hypochlorite. Phylogenetic analysis indicated the existence of cross contamination problems and the spread among companies, probably related to the formation of bio�lms. Conclusion: The results show the necessity of attention to this serovar considering its resistance to sodium hypochlorite in addition to the need for rigorous monitoring and control measures based on the adoption of low temperatures in order to prevent the formation of bio�lms in the poultry industry.


Introduction
The genus Salmonella comprises a group of pathogenic and zoonotic microorganisms frequently associated with foodborne diseases, having as main source of human infection the ingestion of chicken meat and its derivatives [13,21].
The control of Salmonella in food of animal origin, speci cally the chicken meat, is performed by searching the agent in 25 g of the sample.The presence of the genus Salmonella spp. is already a reason for sanitary embargoes or international warnings due to the compromised meat quality [22,19,16,43,12].In particular, the S. Minnesota species has been gaining prominence among serovars due to the increasing number of isolations in the poultry production chain, prompting research to know its danger as an agent of human salmonellosis.In Brazil, this serovar was the second highest incident between 2007 and 2011 in an investigation of 12,582 strains obtained from chicken carcasses and poultry products [19].
The participation of S. Minnesota in human infection and the strategies that this agent uses for its maintenance in the poultry chain is still little known, so there is a need of more studies focused on the epidemiological characterization through phylogenetic analysis to understand the spread of the agent.
Pulsed Field Gel Electrophoresis (PFGE) subtyping provides information that favors genotypic characterization and molecular grouping, and subsequently the creation of measures for its control [42].
Considering the complexity of Salmonella and host/environment interaction, the investigation of virulence factors and their persistence in the environment via genotypic and phenotypic analysis allows a better understanding of the pathogen.Identify genes associated with apoptosis (avrA), oxidative stress (sodC), invasion (invA), adhesion and bio lm (agfA, sefA and lpfA) and quorum sensing (luxS) [15,39,6] helps to characterize the pathogenic potential and to understand the strategies for perpetuation in the environment.At the same time, among the hypotheses that may explain S. Minnesota persistence in the environment, its ability to form bio lms stands out, which hinders its control in the food industry [46].In this condition microorganisms have different responses to heat treatments, biocides and antimicrobials, and are therefore a constant source of contamination [18,27,40].
Knowing the emergence of S. Minnesota, the aim of this work was to evaluate the presence of virulence and environmental adaptation genes, the ability of bio lm formation under different conditions and to establish the phylogenetic relationship of 20 strains of S. Minnesota in order to elucidate the spread, the potential danger that they may represent for human health and possible control measures of the sessile structure.

Bio lm formation x temperature
From the 20 strains evaluated, it was observed that all (100%) were unable to form bio lm at 4 °C, according to the classi cation by Naves in 2008 [30] (Table 2).2).No strong BFI was identi ed at any of the temperatures tested.
Six BFI pro les were identi ed in the strains (A-F), the most prevalent being those classi ed as nonexistent at the three temperatures (9/20-45,0%) (Table 3).Despite the non-existent bio lm classi cation for all strains when kept at 4 o C, when we considered the statistical analysis compared to the negative control, we found that 9/20 (45.0%) strains presented biomass formation at 4 °C, 19/20 (95.0%) at 25 °C and 100% at 36 °C (Table 2).The evaluation of the formed biomass showed that the temperatures of 25ºC and 36 °C favor the formation of S. Minnesota bio lms in the same intensity, compared to the temperature of 4 °C (Fig. 1).However, in Fig. 2 we can observe three examples of S. Minnesota strains that exhibited distinct behaviors and therefore different BFI pro les.

Performance of chemical agents in S. Minnesota sessile
All chemical agents tested, peracetic acid, sodium hypochlorite and chlorhexidine, reduced S. Minnesota sessile counts after exposure for 15 minutes.However, signi cant difference was only observed for peracetic acid and chlorhexidine.For both agents it was observed that, besides reducing the counts, in 8/20 (40.0%) strains no growth was observed after the test.Figure 3 illustrates the quantitative results obtained from untreated bio lms, which obtained a mean value of 6.87 ± 0.38 Log CFU.mL − 1 (p > 0.05), and from bio lms treated with different products, which showed growth after 15 minutes of contact.
All strains (20/20-100.0%)showed resistance to sodium hypochlorite 1%, so that the mean count (6.57± 0.55 Log CFU.mL − 1 ) did not differ from that obtained in the control group (p > 0.05).The use of peracetic acid and chlorhexidine demonstrated the same e ciency in signi cantly reducing counts for resistant strains.The mean count after treatments was equivalent to 3.63 ± 2.84 and 2.96 ± 2.55 Log CFU.mL − 1 , respectively.This result indicated an average decrease of 3.24 and 3.91 log cycles, respectively, compared with the control group.
For both agents, the reduction in the number of CFU varied signi cantly (p < 0.001) between tolerant strains.Six chemical resistance pro les were identi ed, and pro le VI 12/20 (60%) with resistance to all agents (Table 4).

Phylogenetic Analysis
The dendrogram, constructed from the results of the PFGE, was compared considering the isolation site, the date of collection and the genotypic and phenotypic characteristics evaluated in our study.The similarity analysis of the 20 strains of S. Minnesota showed the presence of 14 pulsotypes (A to N) (Fig. 4), in which three groups, presented genotypic similarities above 80%: "C", "K" and "M".

Ultrastructure of treated bio lms
In the SEM test it became evident that the mature bio lm was not characterized when the bacteria are kept at a temperature of 4 ° C (Fig. 5a).As we found in the microbiological test, the bio lm formed under this temperature is of low intensity, demonstrated by being at the beginning of its development process or the occurrence of a possible stagnation in the initial stages, of which we detected only the presence of punctual microcolonies in the eld.The temperatures of 25 and 36 ° C allowed the development of dense clusters of bacteria with evident matrix production between the bacteria (Fig. 5b) and in the outermost region (Fig. 5c) providing the necessary protection to the bacterial community.
The group of bacteria was maintained after treatment with the three agents tested, which is consistent with the expression of the phenotype identi ed in the quantitative assay.The biomass was maintained for the three strains and had similar characteristics between them, the difference being the type of chemical agent used (Fig. 6). Figure 6a illustrates the maintenance of the integral structure of the bio lm without altering the three-dimensional conformation of the bio lm, highlighted by the bacterial agglomeration.This pattern was identi ed in water-treated bio lms for the three strains.The negative control allowed the characterization of the bio lm formed by this serovar, which has a more dense and stable architecture and the presence of a compact coverage along the surface associated with the presence of exchange channels.
The external cover of extracellular matrix was a less evident parameter in bacteria after treatment with sodium hypochlorite (Fig. 6b), but the maintenance of macrocolonies shows the presence of matrix connecting bacteria and the contact surface, showing the characteristic of mature bio lm (Fig. 6c).The treatment with peracetic acid (Fig. 6d) and chlorhexidine (Fig. 6e) promoted not only the reduction of the external EPS matrix, but also the weakening of the connection between the bacteria and the greater access of the microorganism to the chemical agent consistent with the reduction logarithmic identi ed in the counting analyzes.The existence of dispersed groups of bacterial aggregates evidenced the loss of bio lm conformation and cellular viability or bio lms still immature with primary production of extracellular matrix.Part of the bacterial cells showed altered morphology and impaired membrane integrity (Fig. 6f).

Discussion
Presence of virulence genes Our study showed variations in the frequency of virulence genes in S. Minnesota strains.However, the detection of avrA, sodC and invA genes was unanimous.For the invA gene, this pattern was expected by Rowlands in 2014 [38], when analyzing 237 strains of Salmonella spp.isolated from food in Brazil also reported the presence of the invA gene in all strains.Zou in 2012 [47] in a study conducted in North Carolina (USA), from June 2009 to September 2010, found that 99.3% of S. Enteritidis strains isolated from human outbreaks of salmonellosis had the gene.According to Whang in 2009 [44], the invA gene seems to be very conserved in Salmonella spp., which justi es its high occurrence.
In addition to invasiveness, all strains demonstrated survival potential under oxidative stress (sodC) and apoptosis induction in infected cells (avrA).The existence of these virulence mechanisms reinforces the pathogenic potential of strains and, despite the absence of reported cases of salmonellosis by this serovar, demonstrates the possibility of causing disease in humans.Therefore, its isolation in environmental samples, chicken meat and its derivatives may indicate a potential source of transmission of this agent to humans.
Borges in 2013 [6] evaluated 84 strains of S. Enteritidis isolated from 1996 to 2010 in the state of Rio Grande do Sul, Brazil, from different avian sources, and observed a similar result to our study, with the presence of the invA and avrA genes in 100% of the isolates and the IpfA and agfA genes were identi ed in 99% (83/84) and 96% (81/84) of the isolates, respectively.In 2016, Ahmed [2] also observed similar results in Egypt in 20 isolates of S. Typhimurium from chicken, and in 10 isolates of human origin, with frequency of 100% of invA and avrA genes (30/30).They concluded that a high frequency of these genes is observed in serovars potentially causing salmonellosis in humans.
The absence of the sefA gene in the studied strains may be justi ed according to the study by Amini in 2010 [3], in which they observed that this speci c gene is not present in all serovars, being restricted to group D Salmonella, such as Enteritidis, Dublin, Moscow and Blegdon serotypes.
Although not present in all strains, the genetic potential to form bio lms, associated with the presence of agfA, lpfA and luxS genes, was observed in 75% (15/20) of the strains.The presence of these genes demonstrate that the studied strains are fully capable of adapting to adverse environmental conditions through the acquisition of sessile life form.But the expression of only one of them already allows the initiation of the process of acquiring sessile form.Similar studies with different Salmonella serotypes have shown the presence of the agfA gene in over 91,4%, the lfpA gene in 80.63% and the three genes together in more than 73.34% of strains isolated from birds [7,6,45,28].
According to Borges in 2013 [6], the frequency of virulence determinants and the establishment of genetic pro les of isolates can be used to determine more effective control protocols and prevention measures in industries.
In uence of temperature on S. Minnesota bio lms In addition to genotypic characteristics, it is known that extrinsic factors also directly affect the sessile lifestyle.Among them, the presence of moist, nutrient-poor surfaces and temperature variations, which in poultry production offer the ideal conditions for Salmonella to settle and form bio lms [4].
The absence of bio lms at 4 °C was de ned according to the BFI determination described by Naves in 2008 [30].These data are in agreement with the work done by Dhakal in 2019 [20], who found optical density values equal to those of the negative control for six Salmonella serovars tested when kept at refrigeration temperature.However, a study conducted with a standard S. Minnesota strain showed that this serovar has the capacity to form medium intensity bio lms under in vitro conditions using the traditional methodology [40].
In addition, our study indicates that the BFI classi cation for this serovar is strain-dependent, and there are probably other factors that determine this variability, such as the presence and expression of genes linked to bio lm formation (agfA, lpfA and luxS), which encodes aggregative mbriae and the quorum sensing system whose functions are linked to the process of surface xation and bacterial communication, respectively, facilitating bio lm formation [28].In our study, all strains had at least one of these genes, indicating the potential for sessile life that was expressed differently and according to external factors and the expression of this potential.
The increase in bio lm intensity was directly related to the increase in temperature.This is consistent with the study by Borges in 2013 [6] in bio lms in S. Heidelberg, S. Typhimurium, S. Agona, S. Infantis, S.
Brandenburg and S. Tennessee at temperatures of 3 °C, 12 °C, 28 °C and 37 °C, whose BFIs ranged from nonexistent to moderate.
Considering the presence of biomass identi ed as statistically different from the negative control, we observed a higher number of strains capable of forming bio lms at different temperatures.Thus, even under thermal stress conditions under low temperatures, S. Minnesota is still capable of forming a biomass that allows its viability, presenting potential contaminant.
The results found in our study indicate that S. Minnesota can become a di cult problem to control during broiler processing in industry.Considering the high prevalence of the microorganism in Brazilian broiler poultry and slaughterhouses [43], it is possible to suggest its permanence in the industrial environment, even under stress conditions, through the production of bio lm.This information serves as a warning to the poultry industry as an incentive to develop more rigorous and effective control measures for this agent.
The same behavior in relation to the bio lm formation identi ed at temperatures of 25ºC and 36 °C contradicts the results found in the literature, which states that temperatures below the optimal growth temperature and near the environment intensify the biomass production in Salmonella bio lms [41,35].
According to Cabarkapa in 2015 [11], the factors involved in bio lm production have different responses depending on the bacterial strain and according to the incubation temperature.
Signi cant differences (p < 0.05) in relation to BFI within the same serotype indicate that there is probably in uence of intrinsic characteristics, such as the presence of mbriae, agella, membrane proteins and others [1], which even present at the molecular level vary in their expression.Thus, environmental conditions alone are not decisive and limiting in the formation of bio lms.Genotypic diversity and the way in which these factors are expressed and determine the phenotype which, in association, in uence the production of sessile biomass by S. Minnesota should also be considered.
The image analysis allowed to con rm the differences identi ed in the bio lm according to the temperatures (Fig. 5).The ultrastructure of the bio lm formed by S. Minnesota at temperatures of 25 and 36 ° C showed a more stable and mature conformational characteristic compared to the temperature of 4 ° C consistent with that found in different Salmonella serovars [40].
Chemical agents reduced S. Minnesota bio lm Peracetic acid, sodium hypochlorite and chlorhexidine are chemical agents widely used in the cleaning of slaughterhouses, including the industry where strains have been isolated.These biocides promoted a reduction in S. Minnesota bio lm counts after exposure for 15 minutes.However, the use of peracetic acid and chlorhexidine demonstrated the same e ciency in signi cantly reducing counts for resistant strains, differently of what was observed for sodium hypochlorite, which in all strains showed resistance.This is alarming because sodium hypochlorite is one of the most widely used cleaning and disinfecting agents in the industry.
The presence of strains tolerant to different sanitizers suggests that the inappropriate use of these agents in the routine of the industrial environment may result from sublethal exposure to these biocides, representing a real risk to the resistance and adaptation of these bacteria, besides favoring the production of bio lms [24], since under these conditions the adaptive response mechanisms of stress bacteria are activated, promoting their survival even in harsh environments [33].
Regarding the high resistance observed to sodium hypochlorite, it is also possible to associate the involvement of molecular factors, such as RpoS, Dps and sodC genes, linked to oxidative stress, being the last one identi ed in all strains of our study.These genes are actively expressed in S. Enteritidis SE86, resistant to the presence of sodium hypochlorite at 200 ppm [37].It is also possible that for S. Minnesota there are similar mechanisms at work in this process.In addition, the properties of this sanitizer can be altered according to the pH variation and the presence of organic matter that alternate the availability of hypochlorous acid, reducing its e ciency [34].
The variation in the counts for the different strains identi ed after contact with peracetic acid and chlorhexidine demonstrated that the persistence of the presence of these microorganisms may be a strain-dependent characteristic.
The existence of strains with resistance pro le to all agents (pro le IV) (Table 4) (12/20-60%) indicates that there are probably intrinsic or extrinsic adaptive mechanisms that allow their survival.This fact may characterize a potential risk in industry due to the di culty in eliminating these microorganisms and the risk of spreading this characteristic to other bacterial strains.
Although the use of chemical compounds brings bene ts in the disinfection stage, these agents usually have the limitation of not destroying the residual structures of the bacterial bio lm matrix, which can facilitate the resurgence or even the maintenance of these structures on the surfaces [32].In our study, we observed that the external matrix was maintained only when the strains were treated with sodium hypochlorite.Therefore, bacterial resistance seems to be related to the limitation of the presence of organic matter and, for this case, special efforts are required for the complete removal of S. Minnesota bio lms adapted to this biocide.Probably, the effectiveness in controlling these microorganisms will be achieved through sanitation plans that combine cleaning measures focused on the elimination of the extrapolymeric matrix combined with the use of different agents and on the periodic rotation of disinfectants, respecting the periods between disinfections.
In a different way, peracetic acid and chlorhexidine proved to be effective in eliminating the external matrix and in disrupting the conformation of mature bio lm.This pro le may be associated with the mechanism of action aimed at the denaturation of proteins, cellular enzymes, increased permeability of the bacterial cell and cell lysis [5].

Similarity between strains
Phylogenetic analysis identi ed strains with distinct molecular pro les.Only pulsotypes C, K and M grouped strains that allowed an epidemiological evaluation.Pulsotype "C" grouped two strains, M17 and M14, with similarity of 82.8%, coming from the industry B broiler poultry in different periods, 2009 and 2010, with the presence of avrA, sodC, invA, agfA and luxS genes in common.Although they belong to the same industry (B), they are strains of distinct ocks; one from the state of São Paulo and one from Mato Grosso do Sul, indicative of a possible dissemination of this pro le, possibly in uenced by transport.In addition, the strains behavior in bio lm tests was identical with resistance to the three chemical agents evaluated in sessile form.
The pulsotype "K" grouped four strains, two (M04 and M03) clones isolated from the slaughterhouse of industry A, located in the state of Minas Gerais.Cross-contamination may be the reason for the occurrence of this pro le, since they were isolated in very close periods, both dated in November 2014.
The presence of avrA, sodC, invA, agfA and lpfA genes was common in both.
Other strains of the K (M20 and M16) pulsotypes were isolated in 2010; both in the aviary and in the industry B, cutting room in Mato Grosso do Sul.The avrA, sodC, invA and luxS genes and resistance to sodium hypochlorite and peracetic acid in bio lms were common among these strains.These data indicates that the maintenance of these microorganisms in sessile form and contamination of the nal product may be caused by cross contamination between utensils and the slaughtering environment itself [8].
In addition to cross contamination, there are also signs of neglect of biosecurity standards due to the presence of the agent in clean and dirty areas of processing.According to Moura in 2014 [29], this oversight is decisive for the maintenance of the microorganism in the environment.At the same time, the pulsotype K indicates that there was a spread of this pro le, since there are similar strains in both Mato Grosso do Sul and Minas Gerais, in different slaughter units.
The "M" pulsotype grouped three strains (M08; M07 and M05) with similarity equal to 83,6% of 2014, coming from industry A slaughterhouse in the state of Minas Gerais.The strains presented the avrA, sodC, invA and agfA genes in common.
In addition to cross contamination, it is possible to suggest that strains with homology greater than 80% persist in both the poultry and slaughterhouse environment, probably due to the presence of bio lms, which, even if identi ed at low intensities in our assays, demonstrated the viability of the strains sessile bacteria at high counts, even in the presence of biocidal agents.
The sessile life form probably caused this serovar to be isolated at different times, as well as facilitating its spread among the different industry environments.

Conclusions
The presence of virulence, bio lm formation and oxidative stress survival genes in S. Minnesota strains indicates that this serovar has the potential to cause disease in humans and adapt to adverse conditions.
In general, temperatures of 25 °C and 36 °C favored the formation of bio lms with higher intensity than those found at 4 °C, where S. Minnesota inhibits bio lm formation, but biomass formation is still present potential contaminant of the nal product.
The existence of tested biocide tolerant strains suggests a possible exaggerated expression of genes related to oxidative stress and/or the existence of exposure to sublethal concentrations in the environment, posing a real risk for the development of adaptation mechanisms, especially for hypochlorite resistance sodium.
Phylogenetic analysis has shown that strains have high genetic diversity and that the presence of similar strains is probably associated with cross-contamination problems and spread among companies, both related to bio lm formation.
The emergence of serovar combined with the identi ed genotypic and phenotypic characteristics undoubtedly indicate the need for special attention to serovar in broiler production, aiming at preventing its presence, especially through stricter measures to prevent installation and ensure removal bio lms, allied to monitoring protocols.

Samples and Sampling
Twenty S. Minnesota strains isolated from 2009, 2010 and 2014 were used, originated from broiler slaughtering plants of two Brazilian companies (A and B), with complete production cycle and integration system inspected by the Federal Inspection Service (SIF), quali ed for internal and external trade.
From company A, located in the state of Minas Gerais, nine isolates were used and from industry B, with slaughtering plants in the states of São Paulo and Mato Grosso do Sul, seven and four isolates were used, respectively.
Isolations were performed in industrial laboratories, with samples collected at points previously established in internal control programs and/or required by the PRP-Pathogen Reduction Program [9].The strains were assigned to the study after biochemical identi cation, as recommended by Normative Instruction 62 [9] and serological (Oswaldo Cruz Institute Foundation in the State of Rio de Janeiro (IOC/FIOCRUZ, Rio de Janeiro, Brazil), and were stored in the library from the Applied Animal Biotechnology Laboratory of the Federal University of Uberlândia (LABIO/UFU, Minas Gerais, Brazil).

Reactivation of strains and extraction of genomic DNA
The strains were obtained from pure cultures maintained on NA (nutrient agar -OXOID®, Roskilde, Denmark) and reactivated in BHI broth (brain heart infusion -OXOID®) followed by subculturing on TSA Agar (tryptone soybean agar -OXOID®), respecting the conditions of incubation at 37 °C for 24 hours.
Bacterial suspension obtained in TSB broth (tryptone soyl broth -OXOID®) overnight was used for the extraction of genomic DNA using the Wizard® Genomic DNA Puri cation commercial kit (Promega®, Wisconsin, United States), following a protocol provided by the manufacturer.

Identi cation of speci c genes
Conventional PCR was to identify genes linked to the apoptosis process (avrA), potential survival under oxidative stress (sodC), invasion (invA), adhesion and bio lm formation (agfA, sefA and lpfA) and quorum-sensing (luxS) as described in Table 5.

Pulsed-Field Gel Electrophoresis (PFGE)
The Pulse Net protocol, recommended by the CDC in 2013 [14] was used.Bacteria grown at 37 °C overnight on TSA agar (OXOID®)were suspended in tubes containing 2 mL of phosphate buffered saline (PBS: 0.01 M phosphate buffer; pH 7.2; 0.85% NaCl).After agarose blocking, genomic DNA digestion was performed with 30U of Xba I enzyme (Invitrogen®) for two hours at 25 °C.
The DNA fragments were separated on 1% agarose gel (SeaKem Gold®) in 0.5X TBE buffer in CHEF DRIII (Bio-Rad®, California, United States) for a period of 18 hours with the following parameters: 200v, 120°a ngle, 6v / cm gradient and 14 °C buffer temperature.The gels were stained with ethidium bromide, photographed under UV light in a transilluminator (Loccus Biotechnology®) and evaluated using the GelCompar II program.

Bio lm Formation Index
The of bio lm formation index (BFI) was performed according to Kudirkienein 2012 [25] and Naves in 2008 [30], with modi cations.The strains were cultivated in TSB broth (OXOID®) supplemented with 5% of chicken juice to simulate nutritional stress in a slaughterhouse environment.Incubation was performed for 24 hours at 37 °C under constant agitation (175 rpm) until reaching OD 600 close to 0,25.The suspension was centrifuged at 5000 rpm/10 min/4 °C, and the pellet obtained was washed with 0.9% NaCl solution in three successive centrifugations.The last wash suspension was added to the TSB broth (OXOID®) supplemented with 5% of chicken juice at a 1:100 concentration, which was then added to a polystyrene microplate (Kasvi®, Paraná, Brazil) and incubated for 24 hours at the three pre-set temperatures determined at 4ºC, 25ºC and 36ºC.As control the strain of Salmonella Enteritidis ATCC13076 was used.Three repetitions were performed with eight replicates for each of the temperatures.
The bio lm formed at the bottom of the wells was washed twice with 0.9% NaCl solution and dried at 55 °C for 50 minutes.After drying, 200 µL of 1% crystal violet solution (Synth®, São Paulo, Brazil) was added to each well for 5 minutes.The plates were then washed three times with ultrapure water and dried again at 55 °C for 15 minutes, then eluting the dye with methyl alcohol solution (Synth®) and reading the absorbance at OD 600 .Results from suspended cells and adhered cells were tabulated to determine Bio lm Formation Index (BFI) using the formula: BFI = AB-PC/SB.
BFI is the result of the detected index, AB the reading of adherent bacteria, PC the absorbance reading of the control wells without microorganisms and SB the absorbance reading of the suspended bacteria.After incubation, the membranes were removed and transferred back to a new TSA agar plate (OXOID®), incubated under the same conditions and again repeated after 24 hours.On the third day the membranes were added to the sterile vial treatments, the rst being the TSB broth control, the second with 1% chlorhexidine (Vicpharma®, São Paulo, Brazil), the third with sodium hypochlorite 1% (Sanikoll®, São Paulo, Brazil) and the fourth with 0,8% peracetic acid (Synth®).Each treatment was incubated for 15 minutes at room temperature.The membranes were washed with saline solution and after washing 1% trypsin solution (ThermoFisher®, Massachusetts, United States) was added and maintained for 15 minutes.Serial dilutions of the detached content were made to dilution 10 − 6 , which was then added to TSA plates incubated at 36 °C for 24 hours to perform counts.

Scanning electron microscopy
Con rmation of the phenotype obtained in the bio lm formation assays, three selected strains that showed resistance to the three chemical agents tested were evaluated for the ultrastructure formed at temperatures of 4, 25 and 36 ° C and, separately, in treatments with sodium hypochlorite at 1%, 1% chlorhexidine and 1% peracetic acid after the production of the sessile structure at 36 ° C. The assays were performed by Scanning Electron Microscopy.
The preparation of the material for SEM analysis was done according to Brown et al. (2014) [10], with modi cations.Bio lms were formed in 5 mm diameter glass beads, respecting the growth conditions described above.After biomass formation, the bio lms bio lms were treated for 15 minutes as mentioned in the previous topic.The samples were xed with 72.5% of glutaraldehyde and 2.5% of paraformaldehyde in 0.1 M PBS buffer (pH 7.4) overnight at 4 °C.The xative was removed, and the samples washed three times with PBS buffer.The beads were post xed with 1% of osmium tethoxide for two hours and washed three times with PBS buffer.The beads were dehydrated in a series of ethanol solutions (30,40,50,60,70, 80 and 90% and then three times at 100%) for 15 minutes for each step.
Samples were dried on CPD (Critical Drying Point) (CPD 030, Baltec®, Liechtenstein, Deutsch) using liquid carbon dioxide as the transition uid, then coated with a 20 nm of gold layer (SCD 050, Baltec®) and displayed on SEM Zeiss Supra 55 FEG SEM operating at 20 kV.

Analysis of Results
The virulence gene results were analyzed using descriptive statistics, with calculation of percentages.The analysis for dendrogram construction was performed using GelCompar II software.The comparison of the band patterns was performed by the UPGMA analysis method, using the Dice similarity coe cient, with a tolerance of 1.5% in the comparison of the position of the bands.
The interpretation of data related to bio lms was done using the GraphPadPrism software®, version 7.0.
Simple analysis of variance test (ANOVA) was applied for qualitative and quantitative bio lm formation tests and for comparison between treatments.For the bio lm inhibition test, a nonparametric Kruskal-Wallis test was used.For all tests a 95% of con dence interval was used.

Declarations
Ethics approval and consent participate Not applicable

Consent for publication
Comparative dendrogram of 20 S. Minnesota strains, constructed from PFGE results considering the isolation site, date of collection, and the presence or absence of avrA, sodC, invA, agfA, lpfA and luxS genes using the Dice similarity coe cient with 1.5% tolerance and UPGMA method with 0.80% optimization.Presence of 14 pulsotypes (A to N) -three showed genotypic similarities above 80% "C", "K" and "M".

Figure 3 S
Figure 3

Table 1 S
. Minnesota virulence pro les isolated from slaughter plants located in three brazilian states during 2009, 2010 and 2014.

Table 2
Classi cation of S. Minnesota strains according to Bio lm Formation Index (BFI) at different temperatures.

Table 5
Primers used to identify speci c genes in S. Minnesota strains.The PCR reaction preparation consisted of 12.5 µL of GoTaq® Green Master Mix (Promega®), 1 µL of DNA at 10 ng / µL, 1 µL of the gene-speci c primer pairs (Table5) and 10.5 µL of Milli-Q® Water (Merck®, Darmstadt, Germany).The microtubes were transferred to the thermal cycler (Eppendorf®, Hamburg, Germany) for ampli cation according to the cycles: an initial denaturation cycle at 94 °C for 5 minutes, 35 cycles of denaturation at 94 °C for 45 seconds, annealing for 30 seconds at 58 °C (invA); at 50 °C (sefA and 1pfA), at 66 °C (agfA) and 62 °C (avrA, sodC and luxS); extension at 72 °C for 90 seconds, with a nal extension at 72 °C for 10 minutes.The positive reaction control used was the strain S. Enteritidis ATCC 13076 and, as negative control, sterile ultrapure water.
They were classi ed as strong if ≥ 1.10, medium: 0.70-1.10,weak:0.36-0.69ornonexistentif≤0.35.Bio lm Formation Inhibitionassay was performed according to the protocol described by Lu in 2012[26].The bio lm formation was performed from a 100 µL of inoculum containing 10 7 CFU/mL of the bacteria on four cellulose membranes placed in TSA (OXOID®) agar plates incubated at 36 °C for 24 hours.