SciELO - Scientific Electronic Library Online

 
vol.38 issue1Hallmarks of liver lesions in pigs naturally infected by hepatitis E virus genotype 3Profile of dairy farmers of the Joanópolis/SP region, Brazil: how they deal with Rhipicephalus microplus control and other diseases of veterinary interest author indexsubject indexarticles search
Home Pagealphabetic serial listing  

Services on Demand

Journal

Article

Indicators

Related links

Share


Pesquisa Veterinária Brasileira

Print version ISSN 0100-736XOn-line version ISSN 1678-5150

Pesq. Vet. Bras. vol.38 no.1 Rio de Janeiro Jan. 2018

http://dx.doi.org/10.1590/1678-5150-pvb-4928 

LIVESTOCK DISEASES

Biofilm formation capacity of Salmonella serotypes at different temperature conditions

Capacidade de produção de biofilme por cepas de diferentes sorovares de Salmonella em quatro temperaturas de incubação

Karen A. Borges2  * 

Thales Q. Furian2 

Sara N. Souza2 

Rafaela Menezes2 

Eduardo C. Tondo3 

Carlos T.P. Salle2 

Hamilton L.S. Moraes2 

Vladimir P. Nascimento2 

2 Centro de Diagnóstico e Pesquisa em Patologia Aviária, Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 8824, Porto Alegre, RS 91540-000, Brazil.

3 Instituto de Ciência e Tecnologia dos Alimentos (ICTA), UFRGS, Agronomia, Av. Bento Gonçalves 9500, Porto Alegre, RS 91501-970, Brazil.

ABSTRACT:

Salmonella spp. are one of the most important agents of foodborne disease in several countries, including Brazil. Poultry-derived products are the most common food products, including meat and eggs, involved in outbreaks of human salmonellosis. Salmonella has the capacity to form biofilms on both biotic and abiotic surfaces. The biofilm formation process depends on an interaction among bacterial cells, the attachment surface and environmental conditions. These structures favor bacterial survival in hostile environments, such as slaughterhouses and food processing plants. Biofilms are also a major problem for public health because breakage of these structures can cause the release of pathogenic microorganisms and, consequently, product contamination. The aim of this study was to determine the biofilm production capacity of Salmonella serotypes at four different temperatures of incubation. Salmonella strains belonging to 11 different serotypes, isolated from poultry or from food involved in salmonellosis outbreaks, were selected for this study. Biofilm formation was investigated under different temperature conditions (37°, 28°, 12° and 3°C) using a microtiter plate assay. The tested temperatures are important for the Salmonella life cycle and to the poultry-products process. A total of 92.2% of the analyzed strains were able to produce biofilm on at least one of the tested temperatures. In the testing, 71.6% of the strains produced biofilm at 37°C, 63% at 28°C, 52.3% at 12°C and 39.5% at 3°C, regardless of the serotype. The results indicate that there is a strong influence of temperature on biofilm production, especially for some serotypes, such as S. Enteritidis, S. Hadar and S. Heidelberg. The production of these structures is partially associated with serotype. There were also significant differences within strains of the same serotype, indicating that biofilm production capacity may be strain-dependent.

INDEX TERMS: Salmonella serotype; biofilm; temperature; poultry process; bacterioses

RESUMO:

Salmonella spp. são um dos mais importantes agentes causadores de doenças transmitidas por alimentos em vários países, inclusive no Brasil. Produtos avícolas e ovos são os principais alimentos envolvidos na transmissão dos sorovares de Salmonella que são responsáveis por surtos de salmonelose em humanos. Salmonella possui a capacidade de formar biofilmes em diversas superfícies. O processo de formação de biofilme depende da interação entre as células bacterianas, a superfície de adesão e as condições do ambiente onde a bactéria se encontra. Estas estruturas favorecem a sobrevivência bacteriana em ambientes hostis, como em matadouros-frigoríficos e em indústrias processadoras de alimentos. Biofilmes são um grande problema em saúde pública, pois a ruptura destas estruturas pode provocar a liberação de microrganismos patogênicos e, consequentemente, a contaminação dos produtos. O objetivo deste estudo foi avaliar a capacidade de produção de biofilme por diferentes sorovares de Salmonella submetidos a quatro temperaturas de incubação. Cepas de Salmonella de 11 sorovares foram selecionadas. A produção de biofilme foi avaliada através do método de incubação em microplacas de poliestireno incubadas a 37°, 28°, 12° e 3°C. Estas temperaturas são importantes durante o ciclo de vida de Salmonella e para o processamento de produtos avícolas. Do total de cepas avaliadas, 92,2% foram capazes de produzir biofilme em pelo menos uma das quatro temperaturas testadas. Neste estudo, 71,6% das cepas produziram biofilme a 37°C, 63% a 28°C, 52,3% a 12°C e 39,5% a 3°C, independentemente do sorovar. Os resultados indicam uma forte influência da temperatura na produção de biofilme, especialmente para os sorovares S. Enteritidis, S. Hadar e S. Heidelberg. A produção de biofilme está parcialmente associada com o sorovar da cepa. Também foi observado que existe variação quanto à produção destas estruturas dentro de um mesmo sorovar, indicando que possivelmente a produção de biofilme é cepa-dependente.

TERMOS DE INDEXAÇÃO: sorovares de Salmonella; biofilme; temperatura; cadeia avícola

Introduction

According to the World Health Organization (WHO), despite the growing concern regarding emerging pathogens in recent years, Salmonella spp. remain one of the most common agents of foodborne diseases in several countries, including Brazil (Brasil 2015, WHO 2016). The Centers for Disease Control and Prevention (CDC) estimate that approximately 48 million people get sick and 20,000 need to be hospitalized. Each year, almost 3,000 persons die because of foodborne illness in the United States. Of this total, approximately 1 million cases are caused by Salmonella (CDC 2011a). Poultry-derived products are the most common food products, including meat and eggs, involved in outbreaks of human salmonellosis and account for up to 47 % of all infections (CDC 2011b).

During the Salmonella life cycle, host colonization is alternated with periods of survival outside of the host (Winfield & Groisman 2003). To survive in this environment, Salmonella spp. have developed several mechanisms, including surface adherence and biofilm formation (Steenackers et al. 2012). Biofilms are complex communities composed of cellular organisms of the same or different species that are attached to both each other and an abiotic or biotic surface (Costerton et al. 1995, Donlan & Costerton 2002). This multicellular complex is surrounded by an extracellular matrix mainly composed of exopolysaccharides (Donlan & Costerton 2002, Hall-Stoodley & Stoodley 2009). The major components of the extracellular matrix are curli and cellulose. Curli fimbriae are involved in surface adhesion, cell aggregation, persistence in the environment, biofilm production, and host cell adhesion and invasion (Barnhart & Chapman 2006). Cellulose production confers adhesion to abiotic surfaces and bacterial cell-cell interactions (Römling 2007). The advantages of biofilm production include greater resistance to physical forces and the host immune system and toxic molecules like antimicrobials and disinfectants, metabolic cooperation and more efficient DNA sharing (Stewart & Costerton 2001, Hall-Stoodley & Stoodley 2009, Hung & Henderson 2009, Steenackers et al. 2012, Satpathy et al. 2016).

The biofilm formation process depends on interactions among bacterial cells (microbial shape, structure, molecular composition, presence of flagella, pili, capsules or exopolymeric substances), the attachment surface (chemistry, topography, and physicochemistry) and the environmental conditions (pH, nutrient availability, temperature, host proteins, and fluid dynamics) (Van Houdt & Michiels 2010, Campoccia et al. 2013, Cappitelli et al. 2014, Whitehead & Verran 2015). Temperature is one of the most important factors influencing the capacity to form biofilm (Stepanović et al. 2003, De Oliveira et al. 2014, Cabarkapa et al. 2015, Piras et al. 2015).

Biofilms favor bacterial survival in hostile environments, such as slaughterhouses and food processing plants (Steenackers et al. 2012). Animal food production systems are favorable places for biofilm formation because they offer ideal conditions for the development of these structures (Chmielewski & Frank 2003). Biofilm formation in the food industry can lead to economic losses due to facility and equipment impairments and food spoilage (Gram et al. 2007, Hung & Henderson 2009). Biofilms are also a major problem in public health because breakage of these structures can cause the release of pathogenic microorganisms and, consequently, product contamination (Costerton et al. 1995, Hung & Henderson 2009). The National Institutes of Health of the United States estimate that over 80 % of human bacterial infections could be related to biofilm formation (Hung & Henderson 2009).

Salmonella spp. have demonstrated the capacity to form biofilms on several surfaces including different types of stainless steel (AISI 304 or 316), polyethylene, polystyrene, acrylic, glass, and metal inert gas (MIG) and tungsten inert gas (TIG) melts (Casarin et al. 2014, Nguyen et al. 2014, Silva et al. 2014, Cabarkapa et al. 2015, Tondo et al. 2015, Casarin et al. 2016). Vestby et al. (2009) suggest that biofilm formation capacity may be an important factor for the persistence of Salmonella on food contact surfaces in the factory environment.

The influence of Salmonella serotypes on biofilm production capacity has not yet been completely elucidated, and there are few studies that compare the differences among the serotypes. Characteristics inherent to a specific serotype can influence its capacity for biofilm production (Díez-García et al. 2012, Schonewille et al. 2012, Castelijn et al. 2013). However, due to the variability within strains of the same serotype, it might not be possible to demonstrate this correlation (Agarwal et al. 2011, Lianou & Koutsoumanis 2012).

In this context, the aim of this study was to evaluate and compare the biofilm-forming capacities of Salmonella serotypes at four different temperatures of incubation.

Materials and Methods

Bacterial strains and inoculum preparation. A total of 243 strains of Salmonella enterica belonging to 11 different serotypes (Table 1) were used in this study. These strains were isolated between 1996 and 2013 from either poultry or food involved in salmonellosis outbreaks in southern Brazil. A complete antigenic characterization and serovar identification were performed by the Enteric Pathogens Laboratory in the Oswaldo Cruz Institute Foundation (Fiocruz, Rio de Janeiro, Brazil).

Table 1. Biofilm production of Salmonella enterica strains according to serotype at 37°C, 28°C, 12°C and 3°C 

Serotype Total number
of samples
Temperature
(°C)
Production of Biofilm Total number of
producers (%)
NP (%) WP (%) MP (%) SP (%)
S. Enteritidis 150 37°C 42 (28) 66 (44) 31 (20.7) 11 (7.3) 108 (72)
28°C 51 (34) 68 (45.3) 30 (20) 1 (0.7) 99 (66)
12°C 62 (41.3) 80 (53.3) 7 (4.7) 1 (0.7) 88 (58.7)
3°C 97 (64.7) 51 (34) 2 (1.3) 0 53 (35.3)
S. Heidelberg 49 37°C 21 (42.9) 25 (51) 3 (6.1) 0 28 (57.1)
28°C 23 (46.9) 21 (42.9) 5 (10.2) 0 26 (53.1)
12°C 35 (71.4) 13 (26.5) 1 (2.1) 0 14 (28.6)
3°C 32 (65.3) 16 (32.7) 1 (2) 0 17 (34.7)
S. Hadar 14 37°C 2 (14.3) 10 (71.5) 1 (7.1) 1 (7.1) 12 (85.7)
28°C 8 (57.1) 5 (35.8) 1 (7.1) 0 6 (42.9)
12°C 5 (35.8) 7 (50.1) 1 (7.1) 1 (7.1) 9 (64.3)
3°C 4 (28.6) 9 (64.3) 0 1 (7.1) 10 (71.4)
S. Typhimurium 8 37°C 0 7 (87.5) 1 (12.5) 0 8 (100)
28°C 2 (25) 6 (75) 0 0 6 (75)
12°C 4 (50) 4 (50) 0 0 4 (50)
3°C 3 (37.5) 5 (62.5) 0 0 5 (62.5)
S. Anatum 5 37°C 1 (20) 2 (40) 1 (20) 1 (20) 4 (80)
28°C 1 (20) 2 (40) 2 (40) 0 4 (80)
12°C 2 (40) 2 (40) 1 (20) 0 3 (60)
3°C 2 (40) 3 (60) 0 0 3 (60)
S. Bredeney 5 37°C 1 (20) 3 (60) 1 (20) 0 4 (80)
28°C 1 (20) 1 (20) 2 (40) 1 (20) 4 (80)
12°C 2 (40) 0 1 (20) 2 (40) 3 (60)
3°C 2 (40) 3 (60) 0 0 3 (60)
S. Agona 4 37°C 1 (25) 3 (75) 0 0 3 (75)
28°C 2 (50) 2 (50) 0 0 2 (50)
12°C 3 (75) 1 (25) 0 0 1 (25)
3°C 4 (100) 0 0 0 0
S. Tennessee 3 37°C 1 (33.3) 2 (66.7) 0 0 2 (66.7)
28°C 2 (66.7) 1 (33.3) 0 0 1 (33.3)
12°C 3 (100) 0 0 0 0
3°C 2 (66.7) 1 (33.3) 0 0 1 (33.3)
S. Infantis 2 37°C 0 2 (100) 0 0 2 (100)
28°C 0 1 (50) 1 (50) 0 2 (100)
12°C 0 1 (50) 1 (50) 0 2 (100)
3°C 1 (50) 1 (50) 0 0 1 (50)
S. Brandenburg 2 37°C 0 0 2 (100) 0 2 (100)
28°C 0 2 (100) 0 0 2 (100)
12°C 0 2 (100) 0 0 2 (100)
3°C 0 2 (100) 0 0 2 (100)
S. Schwarzengrund 1 37°C 0 1 (100) 0 0 1 (100)
28°C 0 0 0 1 (100) 1 (100)
12°C 0 0 1 (100) 0 1 (100)
3°C 0 1 (100) 0 0 1 (100)

NP = no biofilm producer, WP = weak biofilm producer, MP = moderate biofilm producer, SP = strong biofilm producer.

The bacterial isolates were kept frozen at -80°C in brain heart infusion broth (BHI) (Oxoid, Basingstoke, Hampshire, United Kingdom) and were supplemented with 15 % glycerin (Synth, Diadema, São Paulo, Brazil). The bacteria were retrieved from frozen culture stocks and were cultured overnight at 37°C in trypticase soya agar (TSA) (Merck, Darmstadt, Germany). All strains were subsequently transferred to trypticase soya broth without glucose (TSB) (Difco Laboratories, Detroit, Michigan, United States) and were incubated again at 37°C for 24 hours. To prepare the inoculum, McFarland standard No. 1 (Probac do Brasil, São Paulo, Brazil) was used as a reference to adjust the turbidity of the bacterial suspension in TSB without glucose to a concentration of 3 × 108 CFU/mL.

Quantification of biofilm formation. The biofilm formation capacities of Salmonella serotypes were investigated under different temperature conditions (37°, 28°, 12° and 3°C) using a micro-titer plate method. These temperatures were chosen due to their importance in the poultry production process: 37°C is the optimum temperature for Salmonella growth (Gast 2008), 28 °C is the optimum temperature for the expression of extracellular matrix components in some Salmonella serotypes (Barnhart & Chapman 2006, Römling 2007, Steenackers et al. 2012), 12°C is required by the Brazilian sanitary service in cutting rooms of broiler processing plants (Brasil 1998), and 3°C is the ideal average temperature of domestic refrigerators (Silva et al. 2008).

To evaluate biofilm formation, 200 μL of each bacterial suspension were inoculated in three wells of four sterile 96-well flat-bottomed polystyrene plates. Negative control wells containing only TSB without glucose were inoculated in triplicate. For biofilm evaluation, we used biological triplicates. The plates were covered and incubated aerobically for 24 h at 37°C, 28°C, 12°C or 3°C. After incubation, the contents of the microtiter plate were poured off, and the wells were washed three times with 250 μL of sterile saline solution (0.9 %). The attached bacteria were then fixed with 200 μL of methanol (Nuclear, Diadema, São Paulo, Brazil) per well for 15 minutes, after which the plates were emptied and dried at room temperature. Then, the plates were stained with 200μL per well of 2% Hucker crystal violet for 5 min. The stain was removed, and the plate was washed under running tap water. The plates were air-dried. The biofilm was resuspended in 200μL per well of 33% glacial acetic acid (Nuclear). Salmonella Enteritidis ATCC 13076 was used as a reference strain for all temperatures of incubation. In addition, for each temperature, we included a non-producer and a producer strain. These strains belonged to the serotype S. Enteritidis and were selected from our stock collection. The optical density (OD) of each well was measured at 550 nm in an ELx800 Absorbance Reader (Biotek, Winooski, Vermont, United States). The optical density of each strain (ODS) was obtained from the arithmetic mean value of the respective three wells. The strains were classified as previously described (Stepanović et al. 2004). The cut-off OD (ODC) for the microtiter-plate test was defined as three standard deviations above the mean OD of the negative control. The strains were classified into four categories: no biofilm producer (ODS ≤ ODC), weak biofilm producer (ODC <ODS ≤2×ODC), moderate biofilm producer (2×ODC <ODS ≤4×ODC) or strong biofilm producer (4×ODC <ODS).

Statistics. The data obtained were subjected to statistical analysis using PASW Statistic software (IBM, Hong Kong). Descriptive statistical analysis was used to determine the grouping of the samples according the biofilm formation capacity. The non-parametric chi-square (χ2) was performed to analyze the distribution of the strains according to the biofilm capacity production and to compare the evaluated temperatures. One-Way ANOVA and the post hoc LSD test were carried out to compare serotypes. The significance was defined as p <0.05.

Results

Among the 243 analyzed strains, 224 (92.2 %) were able to produce biofilm in a minimum of one of the tested temperature conditions, but to various extents. A total of 174 (71.6 %) strains produced biofilm at 37°C, 153 (63%) at 28°C, 127 (52.3%) at 12°C and 96 (39.5%) at 3°C, regardless of the serotype. Among the producer strains, most of them were classified as weak, and just a small number were strong biofilm producers. Strong and moderate producer strains were more common when the incubations were performed at 37°C and 28°C, respectively. Biofilm production was independent of the sources of the analyzed samples (data not shown).

The results per serotype according to the incubation temperature are shown in Table 1. All serotypes produced biofilm at 37°C and 28°C, and all produced these structures at 12°C and 3°C, except Salmonella Tennessee and S. Agona, respectively. No strains of S. Heidelberg, S. Typhimurium, S. Agona, S. Infantis, S. Brandenburg and S. Tennessee were classified as strong producers, regardless of the incubation temperature.

There were no significant differences (p>0.05) in biofilm production for S. Typhimurium, S. Agona, S. Anatum, S. Bredeney, S. Tennessee, S. Infantis and S. Schwarzengrund at the four evaluated temperatures. For S. Enteritidis and S. Hadar, biofilm production varied significantly (p<0.05) among all temperatures. For S. Heidelberg, the differences in biofilm production were significant (p<0.05) at 37°C and 28°C compared with biofilm production at 3°C.

To compare biofilm production among serotypes, only those with five or more strains were included in the statistical analysis. There were significant differences (p<0.05) among the serotypes at all tested temperatures. At 37°C, S. Enteritidis differed from S. Heidelberg, whereas S. Heidelberg differed from S. Enteritidis and S. Anatum. At 28°C, S. Enteritidis differed from S. Heidelberg and S. Bredeney, whereas S. Heidelberg differed from S. Enteritidis and S. Bredeney, and S. Hadar and S. Typhimurium differed from S. Bredeney. At 12°C, S. Enteritidis differed from S. Heidelberg and S. Bredeney, whereas S. Hadar differed from S. Heidelberg and S. Bredeney, and S. Typhimurium and S. Anatum differed from S. Bredeney. At 3°C, S. Enteritidis and S. Heidelberg differed from S. Hadar.

The results for S. Enteritidis and S. Heidelberg at 37°C were selected to compare biofilm production within strains of the same serotype at the same temperature. In both cases, there were significant differences (p<0.05) within the two serotypes.

Discussion

Laboratory conditions are favorable for Salmonella growth and may not be encountered in food processing facilities (Wang et al. 2013). However, these conditions represent a reproducible method to evaluate the biofilm formation capacity among Salmonella (Stepanović et al. 2000, Stepanović et al. 2004, Naves et al. 2008, Agarwal et al. 2011, Díez-García et al. 2012, Lianou & Koutsoumanis 2012). The applied technique permitted the detection of biofilm production by Salmonella strains. Incubation in TSB without glucose, a low nutrient availability broth, favored biofilm formation (Rodrigues et al. 2009, Agarwal et al. 2011).

The microtiter plates used for this in vitro experiment mimic some of the plastics used in poultry production and kitchens (Díez-García et al. 2012, Piras et al. 2015). Our findings indicate great capability in Salmonella biofilm formation, possibly related to the increased capacity of these strains to adhere to hydrophobic surfaces, such as polystyrene, compared to hydrophilic ones, such as glass and stainless steel, as previously demonstrated by other authors (Rodrigues et al. 2009, Tondo et al. 2010, Steenackers et al. 2012). These results are of particular concern because plastic materials are widely used on breeding farms, in poultry slaughterhouses, in the food processing industry and in kitchens (Díez-García et al. 2012).

The incubation temperatures evaluated in this study are important for the Salmonella life cycle and to the poultry-products process (Brasil 1998, Barnhart & Chapman 2006, Römling 2007, Gast 2008, Silva et al. 2008, Steenackers et al. 2012). A decrease in the incubation temperature can reduce bacterial growth but can also favor biofilm production because the strains are able to express components that were not produced under other conditions (Stepanović et al. 2003, Agarwal et al. 2011, Lianou & Koutsoumanis 2012, Schonewille et al. 2012). For example, curli and cellulose production occurs only at temperatures below 30°C, especially between 25°C and 28°C (Gerstel & Römling 2003, Stepanović et al. 2003, De Oliveira et al. 2014, Cabarkapa et al. 2015, Piras et al. 2015). Biofilm phenotypes are correlated with the production of extracellular matrix components (Cabarkapa et al. 2015). This characteristic could explain the fact that some strains did not produce biofilm at 37°C but acquired this capacity at 28°C. Stepanović et al. (2003) observed increased biofilm production at 30°C compared to incubation at 37°C. Piras et al. (2015) reported a higher biofilm production at 22°C than at 35°C. These findings indicate that the factors involved in biofilm production have different responses, according to the temperature of incubation (Cabarkapa et al. 2015).

More than 50% of the Salmonella strains were able to produce biofilms at 12°C. This result should serve as a warning to the food processing industry, especially poultry slaughterhouses, because it is the temperature required by the Brazilian sanitary service in the cutting rooms of broiler processing plants (Brasil 1998). It is also important to emphasize Salmonella’s capacity to produce biofilms at 3°C, which is the average temperature of a kitchen’s refrigerator (Silva et al. 2008); this finding is important because most of the foodborne diseases in Brazil occur in the consumer’s residence and mainly due to maintenance of food at incorrect temperatures (Brasil 2015).

The influence of serotypes in biofilm production capacity by Salmonella strains has not yet been completely elucidated, and few studies have compared their differences. The specific characteristics of each serovar could increase or decrease their capacity to produce biofilm (Vestby et al. 2009, Díez-García et al. 2012, Schonewille et al. 2012, Castelijn et al. 2013, Wang et al. 2013). Vestby et al. (2009) suggested that the biofilm-forming capacity might be an important factor for the persistence of some Salmonella serotypes in the food processing industry. Serotypes S. Agona and S. Montevideo are supposed to be “good biofilm producers” and have been considered persistent in Norwegian feed-factories for a number of years. In contrast, S. Typhimurium is rarely isolated from factories, and the tested strains produced little biofilm. However, in our study, both S. Typhimurium and S. Agona seem to be mostly weak biofilm producers.

Only 14.4% of the S. Enteritidis strains were not able to produce biofilm at any of the tested temperatures. Other authors reported similar results, and this serotype has been described as the strongest biofilm producer (Schonewille et al. 2012, Puffal 2013). According to Cabarkapa et al. (2015), the frequent involvement of S. Enteritidis in salmonellosis outbreaks may be a consequence of the strong capacity of some strains to produce biofilms. It has been previously demonstrated that S. Enteritidis has higher adhesion capabilities on several surfaces that are usually used in the food industry, including different types of glass, stainless steel, polyethylene, polystyrene, polypropylene, granite, and MIG and TIG melts (Oliveira et al. 2006, Mafu et al. 2011, Casarin et al. 2014, Cabarkapa et al. 2015, Tondo et al. 2015, Casarin et al. 2016).

Most strains of S. Heidelberg, S. Hadar and S. Typhimurium were weak biofilm producers at all temperatures. These results are similar to previous findings (Marin et al. 2009, Vestby et al. 2009, Castelijn et al. 2013). It has been previously demonstrated that strains of S. Typhimurium produce more biofilms on stainless steel than on acrylic surfaces, which could explain the low adhesion to polystyrene (Nguyen et al. 2014). Our results indicate that S. Agona is a weak biofilm producer, which is different from what has been observed in previous studies (Vestby et al. 2009, Díez-García et al. 2012, Lianou & Koutsoumanis 2012, Corcoran et al. 2013). Few studies have evaluated the biofilm-forming capacity of Salmonella strains belonging to serotypes S. Tennessee, S. Brandenburg, S. Bredeney, S. Anatum, S. Infantis and S. Schwarzengrund. It was previously observed that most of these strains showed a low capacity to produce biofilms. These data were similar to those found by Agarwal et al. (2011) and Castelijn et al. (2013). However, these authors also studied a few examples of these serotypes, and the small differences observed could be due to the reduced sample size.

The significant differences (p<0.05) within serotypes indicates that even within a serovar, there are important variations in the capacity to produce biofilms, as previously described (Castelijn et al. 2013). It is possible that variability within a single serotype makes it difficult to compare the strains’ biofilm production capacities (Agarwal et al. 2011, Lianou & Koutsoumanis 2012). According to Agarwal et al. (2011), the intrinsic characteristics of each strain, such as the presence of fimbriae, flagella, membrane proteins and other cellular appendages, could be more important than the environmental conditions for biofilm production.

Conclusions

Most Salmonella strains had the capacity to produce biofilms in microtiter plates at all tested temperatures.

The strains showed different behaviors at different incubation temperatures, demonstrating that there is a strong influence of temperature on biofilm production.

The production of these structures is partially associated with serotype.

The data obtained indicate that biofilm production is strain-dependent and that their intrinsic characteristics determine their potential to form these structures. However, the regulation of biofilm formation by Salmonella spp. strains are very complex and further studies are necessary

Acknowledgements

This study was supported by the Brazilian National Council of Technological and Scientific Development (CNPq, grant number 476092/2013-2)

References

Agarwal R.K., Singh S., Bhilegaonkar K.N. & Singh V.P. 2011. Optimization of microtitre plate assay for the testing of biofilm formation ability in different Salmonella serotypes. Int. Food Res. J. 18:1493-1498. [ Links ]

Barnhart M.M. & Chapman M.R. 2006. Curli biogenesis and function. Annu. Rev. Microbiol. 60:131-147. [ Links ]

Brasil 1998. Ministério da Agricultura, Pecuária e Abastecimento. Portaria n° 210. Regulamento Técnico da Inspeção Tecnológica e Higiênico-Sanitária de carne de Aves. Diário Oficial [Da] União, Poder Executivo, Brasília, DF, 10 November 1998. [ Links ]

Brasil 2015. Doenças Transmitidas por Alimentos. Ministério da Saúde e Secretaria de Vigilância em Saúde, Brasília. Available at <Available at http://u.saude.gov.br/images/pdf/2015/novembro/09/Apresenta----o-dados-gerais-DTA-2015.pdf > Accessed on Nov. 15, 2005. [ Links ]

Cabarkapa I., Skrinjar M., Levic J., Kokic B., Blagojev N., Milanov D. & Suvajdzic L. 2015. Biofilm forming ability of Salmonella Enteritidis in vitro. Acta Vet., Beogr. 65:371-389. [ Links ]

Campoccia D., Montanaro L. & Arciola C.R. 2013. A review of the biomaterials technologies for infection-resistant surfaces. Biomaterials 34:8533-8554. [ Links ]

Cappitelli F., Polo A. & Villa F. 2014. Biofilm formation in food processing environments is still poorly understood and controlled. Food Eng. Rev. 6:29-42. [ Links ]

Casarin L.S., Brandelli A., De Oliveira Casarin F., Soave P.A., Wanke C.H. & Tondo E.C. 2014. Adhesion of Salmonella Enteritidis and Listeria monocytogenes on stainless steel welds. Int. J. Food Microbiol. 191:103-108. [ Links ]

Casarin L.S., Casarin F.dO., Brandelli A., Novello J., Ferreira S.O. & Tondo E.C. 2016. Influence of free energy on the attachment of Salmonella Enteritidis and Listeria monocytogenes on stainless steels AISI 304 and AISI 316. LWT Food Sci. Technol. 69:131-138. [ Links ]

Castelijn G.A.A., Parabirsing J.A., Zwietering M.H., Moezelaar R. & Abee T. 2013. Surface behaviour of S. Typhimurium, S. Derby, S. Brandenburg and S. Infantis. Vet. Microbiol. 161:305-314. [ Links ]

CDC 2011a. CDC 2011 estimates: findings. Center for Disease Control. Available at <Available at http://www.cdc.gov/foodborneburden/2011-foodborne-estimates.html > Accessed on Jan. 16, 2005. [ Links ]

CDC 2011b. Making food safer to eat: reducing contamination from the farm to the table. Center for Disease Control. Available at <Available at http://www.cdc.gov/vitalsigns/foodsafety/ > Accessed on Mar. 16, 2003. [ Links ]

Chmielewski R.A.N. & Frank J.F. 2003. Biofilm formation and control in food processing facilities. Comp. Rev. Food Sci. Food Saf. 2:23-32. [ Links ]

Corcoran M., Morris D., De Lappe N., O’Connor J., Lalor P., Dockery P. & Cormican M. 2013. Salmonella enterica biofilm formation and density in the Centers for Disease Control and Prevention’s biofilm reactor model is related to serovar and substratum. J. Food Prot. 76:662-667. [ Links ]

Costerton J.W., Lewandowski Z., Caldwell D.E., Korber D.R. & Lappin-Scott H.M. 1995. Microbial biofilms. Annu. Rev. Microbiol. 49:711-745. [ Links ]

De Oliveira D.C., Fernandes Júnior A., Kaneno R., Silva M.G., Araújo Júnior J.P., Silva N.C. & Rall V.L. 2014. Ability of Salmonella spp. to produce biofilm is dependent on temperature and surface material. Foodborne Pathog. Dis. 11:478-483. [ Links ]

Díez-García M., Capita R. & Alonso-Calleja C. 2012. Influence of serotype on the growth kinetics and the ability to form biofilms of Salmonella isolates from poultry. Food Microbiol. 31:173-180. [ Links ]

Donlan R.M. & Costerton J.W. 2002. Biofilms: survival mechanisms of clinically relevant microorganisms. Clin. Microbiol. Rev. 15:167-193. [ Links ]

Gast R.K. 2008. Salmonella infections - paratyphoid infections, p. 636-665. In: Saif Y.M. (Ed.), Disease of Poultry. 12th ed. Blackwell Publishing, Ames, IA. [ Links ]

Gerstel U. & Römling U. 2003. The csgD promoter, a control unit for biofilm formation in Salmonella Typhimurium. Res. Microbiol. 154:659-667. [ Links ]

Gram L., Bagge-Ravn D., Ng Y.Y., Gymoese P. & Vogel B.F. 2007. Influence of food soiling matrix on cleaning and disinfection efficiency on surface attached Listeria monocytogenes. Food Control 18:1165-1171. [ Links ]

Hall-Stoodley L. & Stoodley P. 2009. Evolving concepts in biofilm infections. Cell. Microbiol. 11:1034-1043. [ Links ]

Hung C.S. & Henderson J.P. 2009. Emerging concepts of biofilms in infectious diseases. Mo. Med. 106:292-296. [ Links ]

Lianou A. & Koutsoumanis K.P. 2012. Strain variability of the biofilm-forming ability of Salmonella enterica under various environmental conditions. Int. J. Food Microbiol. 160:171-178. [ Links ]

Mafu A.A., Plumety C., Deschênes L. & Goulet J. 2011. Adhesion of pathogenic bacteria to food contact surfaces: influence of pH of culture. Int. J. Microbiol. 2011:972494. [ Links ]

Marin C., Hernandiz A. & Lainez M. 2009. Biofilm development capacity of Salmonella strains isolated in poultry risk factors and their resistance against disinfectants. Poult. Sci. 88:424-431. [ Links ]

Naves P., Del Prado G., Huelves L., Gracia M., Ruiz V., Blanco J., Dahbi G., Blanco M., Ponte Mdel C. & Soriano F. 2008. Correlation between virulence factors and in vitro biofilm formation by Escherichia coli strains. Microb. Pathog. 45:86-91. [ Links ]

Nguyen H.D.N., Yang Y.S. & Yuk H.G. 2014. Biofilm formation of Salmonella Typhimurium on stainless steel and acrylic surfaces as affected by temperature and pH level. LWT Food Sci. Technol. 55:383-388. [ Links ]

Oliveira K., Oliveira T., Teixeira P., Azeredo J., Henriques M. & Oliveira R. 2006. Comparison of the adhesion ability of different Salmonella Enteritidis serotypes to materials used in kitchens. J. Food Prot. 69:2352-2356. [ Links ]

Piras F., Fois F., Consolati S.G., Mazza R. & Mazzette R. 2015. Influence of temperature, source, and serotype on biofilm formation of Salmonella enterica isolates from pig slaughterhouses. J. Food Prot. 78:1875-1878. [ Links ]

Puffal J. 2013. Investigação de genes de resistência a antimicrobianos e da capacidade de formação de biofilme em isolados de Salmonella Enteritidis. Dissertação de Mestrado em Biologia Celular e Molecular, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre. 52p. [ Links ]

Rodrigues L.B., Santos L.R., Rizzo N.N., Tagliari V.Z., Oliveira A.P., Trenhago G., Rodegheri S.C., Taglieti R.M., Dickel E.L. & Nascimento V.P. 2009. Avaliação da hidrofobicidade e da formação de biofilmes em poliestireno por Salmonella Heidelberg isoladas de abatedouro avícola. Acta Scient. Vet. 37:225-230. [ Links ]

Römling U. 2007. Cellulose biosynthesis in Enterobacteriaceae, p.107-122. In: Brown Jr R.M. & Saxena I.M. (Eds), Cellulose: Molecular and Structural Biology. Springer Verlag, Dordrecht. [ Links ]

Satpathy S., Sen S.K., Pattanaik S. & Raut S. 2016. Review on bacterial biofilm: an universal cause of contamination. Biocatal. Agric. Biotechnol. 7:56-66. [ Links ]

Schonewille E., Nesse L.L., Hauck R., Windhorst D., Hafez H.M. & Vestby L.K. 2012. Biofilm building capacity of Salmonella enterica strains from the poultry farm environment. FEMS Immunol. Med. Microbiol. 65:360-365. [ Links ]

Silva C.F., Gehlen S.S., Webber B., Diedrich L.N., Pilotto F., Santos L.R., Tondo E.C., Nascimento V.P. & Rodrigues L.B. 2014. Salmonella Enteritidis formadoras de biofilmes são multirresistentes a antimicrobianos. Acta Scient. Vet. 42:1-8. [ Links ]

Silva D.L., Celidonio F.A. & Oliveira K.M.P. 2008. Verificação da temperatura de refrigeradores domésticos para minimizar a deterioração e possíveis doenças veiculadas por alimentos. Revta Hig. Aliment. 22:42-45. [ Links ]

Steenackers H., Hermans K., Vanderleyden J. & De Keersmaecker S.C.J. 2012. Salmonella biofilms: an overview on occurrence, structure, regulation and eradication. Food Res. Int. 45:502-531. [ Links ]

Stepanović S., Vukovic D., Dakic I., Savic B. & Svabic-Vlahovic M. 2000. A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J. Microbiol. Methods 40:175-179. [ Links ]

Stepanović S., Ćirković I., Mijač V. & Švabić-Vlahović M. 2003. Influence of the incubation temperature, atmosphere and dynamic conditions on biofilm formation by Salmonella spp. Food Microbiol. 20:339-343. [ Links ]

Stepanović S., Cirković I., Ranin L. & Svabić-Vlahović M. 2004. Biofilm formation by Salmonella spp. and Listeria monocytogenes on plastic surface. Lett. Appl. Microbiol. 38:428-432. [ Links ]

Stewart P.S. , Costerton J. 2001. Antibiotic resistance of bacteria in biofilms. Lancet 358:135-138. [ Links ]

Tondo E.C., Machado T.R., Malheiros P. da S., Padrão D.K., de Carvalho A.L. & Brandelli A. 2010. Adhesion and biocides inactivation of salmonella on stainless steel and polyethylene. Braz. J. Microbiol. 41:1027-1037. [ Links ]

Tondo E.C., Ritter A.C. & Casarin L.S. 2015. Involvement in foodborne outbreaks, risk factors and options to control salmonella Enteritidis SE86: an important food pathogen in Southern Brazil, p.65-77. In: Hackett C.B. (Ed.), Salmonella. Nova Science Publishers, New York. [ Links ]

Van Houdt R. & Michiels C.W. 2010. Biofilm formation and the food industry, a focus on the bacterial outer surface. J. Appl. Microbiol. 109:1117-1131. [ Links ]

Vestby L.K., Møretrø T., Langsrud S., Heir E. & Nesse L.L. 2009. Biofilm forming abilities of Salmonella are correlated with persistence in fish meal- and feed factories. BMC Vet. Res. 5:20. [ Links ]

Wang H., Ye K., Wei X., Cao J., Xu X. & Zhou G. 2013. Occurrence, antimicrobial resistance and biofilm formation of Salmonella isolates from a chicken slaughter plant in China. Food Control 33:378-384. [ Links ]

Whitehead K.A. & Verran J. 2015. Formation, architecture and functionality of microbial biofilms in the food industry. Curr. Opin. Food Sci. 2:84-91. [ Links ]

WHO 2016. Salmonella. World Health Organization, Geneva. < http://www.who.int/topics/salmonella/en/ > Accessed on Apr. 16, 2005. [ Links ]

Winfield M.D. , Groisman E.A. 2003. Role of nonhost environments in the lifestyles of Salmonella and Escherichia coli. Appl. Environ. Microbiol. 69:3687-3694 [ Links ]

Received: February 17, 2017; Accepted: May 15, 2017

*Corresponding author: karen.borges@ufrgs.br

Conflict of interest statement.- The authors have no competing interests.

Creative Commons License This is an open-access article distributed under the terms of the Creative Commons Attribution License