Environmental persistence and virulence of Salmonella spp. Isolated from a poultry slaughterhouse

Journal Pre-proofs Environmental persistence and virulence of Salmonella spp. isolated from a poultry slaughterhouse Stéfani T.A. Dantas, Carlos H. Camargo, Monique R. Tiba-Casas, Ricardo C. Vivian, José P.A.N. Pinto, José C.F. Pantoja, Rodrigo T. Hernandes, Ary Fernandes Júnior, Vera L.M. Rall PII: DOI: Reference:

S0963-9969(19)30721-5 https://doi.org/10.1016/j.foodres.2019.108835 FRIN 108835

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Food Research International

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23 August 2019 12 November 2019 18 November 2019

Please cite this article as: Dantas, S.T.A., Camargo, C.H., Tiba-Casas, M.R., Vivian, R.C., Pinto, J.P.A., Pantoja, J.C.F., Hernandes, R.T., Fernandes Júnior, A., Rall, V.L.M., Environmental persistence and virulence of Salmonella spp. isolated from a poultry slaughterhouse, Food Research International (2019), doi: https://doi.org/ 10.1016/j.foodres.2019.108835

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Environmental persistence and virulence of Salmonella spp. isolated from a poultry slaughterhouse

Stéfani T. A. Dantasa, Carlos H. Camargob, Monique R. Tiba-Casasb, Ricardo C. Vivianc, José P. A. N. Pintoc, José C. F. Pantojac, Rodrigo T. Hernandesa, Ary Fernandes Júniora, Vera L. M. Ralla*

a

Department of Microbiology and Immunology, Institute of Biosciences, São Paulo State University,

Botucatu, SP, Brazil b

c

Adolfo Lutz Institute Bacteriology Division, São Paulo, SP, Brazil

Departament of Veterinary Hygiene and Public Health, Faculty of Veterinary Medicine, São Paulo State

University, Botucatu, SP, Brazil

*Corresponding author: [email protected] Department of Microbiology and Immunology, Institute of Biosciences, Sao Paulo State University - UNESP. Post Office Box 510. 18618-970, Botucatu, Sao Paulo, Brazil.

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ABSTRACT Salmonella spp. is responsible for severe foodborne disease, and is one of the main agents involved in foodborne outbreaks worldwide. Contamination occurs mainly as a result of poultry and egg consumption since they can carry some serotypes pathogenic to humans. The aim of the study was to evaluate the persistence and pathogenic potential of Salmonella spp. (n = 40) isolated from poultry slaughterhouse mats, using adhesion and invasion assays, antimicrobial susceptibility by disc diffusion, and biofilm production as phenotypic tests and genotypic analyses. Polystyrene mats presented 3.2 times greater chance of isolating Salmonella than canvas mats. Besides, we observed resistance to tetracycline

(17.5%),

ampicillin

(10%),

cefotaxime

(7.5%),

trimethoprim-

sulfamethoxazole (5%), and chloramphenicol (2.5%). All strains possessed the invA, sipB, sipD, ssaR, sifA, sitC, iroN, tolC, flgK, fljB, and flgL genes. The genes sopB and sipA were both present in 92.5% of the isolates, while sopD and spvB were observed in 90% and 32.5% of strains, respectively. All strains adhered to and invaded HeLa cells. Regarding biofilm production, 31 (77.5%) strains were able to produce biofilm on polystyrene microplates. Using PFGE, we detected the persistence of clones in the environment for up to 18 fromthe 20 weeks. The ability of these strains to produce a biofilm and thus persist in the environment and disperse through contact surfaces in the processing plant favors the contamination of food, aggravated by the pathogenic potential of these isolates demonstrated by their adhesion capacity, invasion and resistance to various antibiotic agents.

Keywords: antibiotic resistance; biofilm; invasion; PFGE; virulence genes

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1. INTRODUCTION Salmonellosis is one of the major frequent foodborne disease in the world (Lamas et al., 2018; Tack et al, 2019). Depending on the serovar involved, the concentration of the inoculum, the virulence factors expressed by the agent, and the host’s immune status, Salmonella spp. may result in a mild gastrointestinal infection, septicemia, or death (Coburn, Grassl & Finlay, 2007). The strongest virulence-related genes are located in the Salmonella pathogenicity islands (SPIs) and in a virulence-associated plasmid (pSTV) (Fardsanei, Dallal, Douraghi, Salehi, Mahmoodi et al., 2017). Both SPI-1 and SPI-2 encode a type III secretion system (T3SS) that forms a channel in the host cell membrane, allowing bacterial effector proteins to be internalized within these cells (Coburn et al., 2007). SPI-1 encodes for genes such as invA, sipA, sipB, sipD, sopB, sopD involved in the invasion of epithelial cells, whereas SPI-2 encodes the genes sifA and ssaR related to the survival and replication of Salmonella within phagocytic cells, in addition to playing an important role in systemic infection (Fardsanei et al., 2017; Chakroun et al., 2018). Salmonella can form biofilm on different surfaces, such as stainless steel (Wang et al., 2013), glass (Oliveira et al, 2014), wood, and plastic (Dantas et al., 2018). Biofilm production can be also considered a survival strategy for the pathogen, since a biofilm protects the microorganisms embedded in the matrix, increasing resistance to the action of antibiotics and sanitizers and allowing their propagation in the environment (Zhao, Zhao, Wang & Zhong, 2017; Chuah, Syuhada, Suhaimi, Hanim & Rusul, 2018). In addition to the virulence factors expressed by Salmonella, the emergence of multidrug-resistant (MDR) isolates has been a public health problem. The improper use of antibiotics in therapeutic treatment, for prophylaxis, and as growth promoters in

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poultry farming, may have contributed to the increase in MDR Salmonella strains (Chuah et al., 2018). Considering the importance of Salmonella in the poultry production chain, its pathogenic potential for infection in humans, and its capacity for cross-transmission, knowing the profile of virulence, sensitivity and diversity of these pathogens can provide information about their dissemination and propose measures for control. Therefore, the aim of this work was to evaluate the pathogenic potential of Salmonella spp., previously isolated from mats in a poultry slaughterhouse, by phenotypic tests assessing adhesion, invasion, and biofilm production, genotypic analysis of the presence of several genes related to virulence factors, and antimicrobial susceptibility testing.

2. MATERIAL AND METHODS 2.1. Samples A total of 240 samples from the surfaces of two types of mats (120 canvas and 120 polystyrene) were collected in a poultry slaughterhouse under federal inspection in São Paulo State, Brazil, during 20 weeks, between March and July, in 2017. The collection interval was established according to the production schedule of the slaughterhouse, but always in different days of the week. The first collection was carried out before the start of production (I), the second was during morning activities (II), the third was after the lunch break (III), the fourth was at the beginning of afternoon activities (IV), the fifth was during afternoon production (V) and the sixth and last one was after the end of activities (VI). During the lunch break (between collections III and IV), the room was cleaned with a hose and high-pressure jets of hot water (temperature below

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60°C). A complete hygienization procedure with detergents and sanitizers was performed after the end of activities. The presence of Salmonella spp. was assessed according to the American Public Health Association (Cox et al., 2015). After rubbing the swab moistened with saline solution (0.9%) against the mat, the swab was transported to the laboratory, under refrigeration (4°C), in a sterile bag containing Dey-Engley broth (All culture media, except wherever specified, were from Difco, Detroit, MI, USA), and the analysis began on the same day of collection. Each swab was incubated in 225 mL of lactose broth (pH 6.8 ± 0.2,), maintained for 1 h at room temperature, and then, incubated at 35°C for 24h. After this incubation period, 0.1 mL and 1 mL was transferred to 10 mL of RappaportVassiliadis broth (42°C) and to 10 mL of Tetrathionate Brilliant Green broth (35°C), respectively; and both broths were incubated for 24 h. For Salmonella isolation, a loopful of each broth was seeded onto Xylose Lysine Desoxycholate Agar and Bismuth Sulfite Agar, and the plates were incubated for 24h/35°C. Characteristic colonies were identified using API 20 E (bioMerieux, l’Étoile, France) and polyvalent somatic and flagellar antisera (Probac, São Paulo, Brazil). The genera were confirmed by polymerase chain reaction (PCR), based on the presence of the invA gene (Arnold, Scholz, Marg & Hensel, 2004). The isolates were serotyped by the Oswaldo Cruz Institute (FioCruz) in Rio de Janeiro.

2.2. PCR for Virulence Genes For the extraction of DNA, Salmonella strains were inoculated into brain heart infusion broth (BHI, Oxoid, Basingstoke, UK) and incubated at 35°C/24 h and 1 mL of each inoculum was centrifuged at 10,000 g for 10 min. The cell pellet was analyzed according to Arnold et al. (2004) with modification. The supernatant was discarded, and

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theellet resuspended in 200 μL ultrapure water and incubated in a 95°C/10 min water bath. A new centrifugation at 13,000 g for 5 min was performed and the supernatant was frozen at -20°C until use in the PCR. For the PCR reactions, samples were prepared in a final volume of 25 μL with 2X GoTaq Green Master Mix (Promega, Madison, WI) according to manufacturer recommendations. Amplification was performed on a Pro Flex PCR System thermocycler (Applied Biosystem, Wellesley, MA) using the following cycling program: initial denaturation at 94°C for 5 min; 35 cycles of denaturation at 94°C for 30 s, annealing at different temperatures (Table 1) for 30 s and primer extension at 72°C for 30 s; a final extension period was performed at 72°C for 4 min. In all reactions, a negative control was prepared by replacing nucleic acid with ultrapure water. A standard strain of Salmonella ser. Typhimurium ATCC 14028 was used as a positive control (Arnold et al., 2004). The PCR products were electrophoresed (Electrophoresis Power Supply Model EPD 600; AmershamPharmacia Biotech, Inc.) in a 1.5% agarose gel (Sigma-Aldrich, St. Louis, MO) in Tris-borate-EDTA (TBE) buffer, and the bands were stained with SYBR Safe (Invitrogen, Grand Island, NY).

2.3. Molecular Characterization by PFGE PulseNet's standard protocol (CDC, 2017a) with XbaI (Thermo Scientific, Waltham MA) (50 U for 2 h at 37°C) was used as the restriction endonuclease to digest Salmonella genomic DNA. Digested DNA was separated by electrophoresis using a CHEF (clamped homogeneous electric field) mapper (Biorad, Hercules, California), with an initial switch time of 2.2 s, final switch time of 63.8 s, voltage of 6 V and an included angle of 120°, through a run time of 18 hours. In addition, for the PFGE run, the electrophoresis buffer was supplemented with 50 μM of thiourea.

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The images were analyzed using Bionumerics software v.7.6 and the PFGE fingerprints were analyzed using a band position tolerance of 1.5%. Clustering was carried out by the unweighted pair-group method with arithmetic mean (UPGMA), using the Dice coefficient, and a similarity index of 80% according to Kitchel et al. (2009). The DNA fragments of ATCC BAA-664 (S. ser. Braenderup H9812) were used as molecular weight markers for the XbaI digestion standards. 2.4. Adhesion and Invasion Assay The adhesion assay was performed according to Mellor, Gouter, Raymond Chia & Dikes (2009) with modifications. Each isolate was grown in BHI at 37°C for 24 h and diluted in Dulbecco’s Modified Eagle’s Medium (DMEM) to obtain a suspension of approximately 1.5 x 106 CFU/mL. An established HeLa cell line was grown in 24-well plates. Before the adherence assay, each well was washed four times with PBS+ buffer (0.01 M PBS, supplemented with 0.1g/L CaCl2 and 0.2g/L MgCl2; Sigma-Aldrich), and 1 mL of the bacterial suspension was added to each well. After incubation at 37°C for 2 h in 5% CO2, the monolayers were washed four times with PBS+ buffer to remove nonadherent bacteria. Next, the cells were resuspended in 500 µL of 0.1% trypsin + 0.04% EDTA (Fermentas, St. Leon Rot, Germany) in each well. The plates were incubated at 37°C for 15 min in 5% CO2. After resuspension, trypsinization was stopped by adding 500 µL of DMEM without antimicrobial agents. Serial 10-fold dilutions of the cells were performed and inoculated into trypticase soy agar plates (TSA; Oxoid) and incubated at 35°C for 24 h. After, thecolonies were counted, representing the number of bacteria that were adhered to the cells. For the invasion assays, the isolates were diluted as described for the adhesion test. Cells were washed four times with PBS+ buffer, and 1 mL of the bacterial suspension was added to each well and incubated at 37°C for 2 h in 5% CO2. Then, the cell

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monolayers were washed four times with PBS+ buffer to remove extracellular bacteria; 1 mL of DMEM containing 5% FBS and 300 µL/mL gentamicin was added to each well, and the plate was reincubated at 37°C for 2 h in 5% CO2. Next, the wells were washed four times with 1 mL PBS+, and 10 µL from the last wash was plated onto TSA agar to check for the absence of bacteria in the extracellular medium. Afterward, 1 mL of Triton X-100 (Sigma-Aldrich) at a final concentration of 0.1% (v/v) was added to the wells to lyse the cells. Next, we prepared a serial 10-fold dilutions from the lysed cells, inoculated 10 µL onto TSA plates, and incubated at 35°C for 24 h. So, we counted the colonies, calculated the relative percentage, and compared it with the values determined in the adhesion assay (adhered bacteria/internalized bacteria × 100) (Gagnon et al., 2013).

2.5. Biofilm formation Salmonella isolates were incubated in BHI broth at 35°C for 24 h. Next, the culture was diluted to approximately 1.5 x 108 CFU/mL (0.5 on the MacFarland scale) with the aid of Densicheck (Biomeriéux), using the same broth supplemented with 0.5% glucose. An aliquot of 300 μL was seeded in quadruplicate into a 96-well microplate. After 96 hours at 28°C (Oliveira et al, 2014), the wells were washed three times with PBS (pH 7.4) and added of 250 ml of methanol per well for 15min to fix the biofilm. After its removal, the microplate was dried at room temperature and stained with 1% crystal violet for 5 minutes. After three washes with distilled water to remove excess dye, the biofilm was resuspended in 200 µL of 33% (v/v) glacial acetic acid for 10 minutes and the optical density (OD) was measured on an ELISA plate reader (Babsystems, MultiSkan EX) at 570 nm. The tests were performed in triplicate. From the average of the replicates and according to the relationship between OD and ODc (negative control), the samples were classified according to Stepanović, Ćirković, Mijac & Svabić-Vlahović (2003) as non-

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biofilm producer (DO ≤ DOc), weak [DOc
2.7. Statistical analysis A generalized ordered logistic regression model was used to estimate the chances of isolation of Salmonella between the two types of mats studied. An autoregressive covariance matrix was used to consider repeated measurements within the same sampling day. Spearman's correlation coefficient was applied to estimate the correlation between adhesion and invasion in HeLa cells. The analysis was performed using the PROC GLIMMIX procedure (SAS Institute, Cary, NC, USA), adopting a statistical significance level of 5%.

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3. RESULTS 3.1. Salmonella spp. Isolation and their Virulence Genes Among the 240 samples randomly collected from the two types of mats (canvas and polystyrene) over a period of 20 weeks. Forty samples werecontaminated with Salmonella spp. We observed that the chance of isolating Salmonella from polystyrene was 3.2 times greater (95% confidence interval: 1.5-6.8) than from canvas (p = 0.0048). Forty Salmonella strains belonging to eleven different serovars were observed, including S. ser. Heidelberg (27.5%), S. ser. Enteritidis (17.5%), S. ser. Ohio (12.5%), S. ser. Mbandaka (10%), S. ser. Agona, S. ser. Typhimurium (7.5% each), S. ser. Kentucky (5%), S. ser. Abony (2.5%), S. ser. Bredeney (2.5%), S. ser. Saintpaul (2.5%), and S. ser. Worthington (2.5%). One (2.5%) non-typeable S. enterica subsp. enterica (rough) was observed. All isolated strains were positive for the invA, sipB, sipD, ssaR, sifA, sitC, tolC, flgK, fljB, and flgL genes. The sopB and sipA genes were found in 37 strains (92.5%) and sopD in 36 strains (90%). The least frequent gene was spvB, present in only 13 isolates (32.5%).

3.2. Molecular Characterization by PFGE Molecular typing by the PFGE technique was performed among isolates belonging to the same serovar (Figure 1). Figure 1A shows seven different restriction profiles (H1- H7) in 11 S. ser. Heidelberg strains, with similarity ranging from 57.6% to 100%. Four of them showed the same profile (H1); this indicates that the same clone had been isolated between week 2 and week 17 of collection.

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Among the seven strains belonging to serovar S. ser Enteritidis (Figure 1B), it was possible to observe the existence of four restriction profiles (E1-E4), sharing at least 89.2% similarity. Regarding serovar S. ser Ohio (Figure 1C), all five strains evaluated were classified with the same profile (O1), while two different profiles (A1 and A2) of S. ser Agona were present in three strains (Figure 1D). We observed that the isolates obtained in weeks 3 and 20 are the same clone. On the other hand, the four isolates of S. ser Mbandaka generated four restriction profiles without any related clones among them (up to 58.3% similarity). Furthermore, the three isolates of serovar S. ser Typhimurium presented three different profiles, which shared up to 75.9% similarity. Additionally, the two evaluated strains of S. Kentucky generated two profiles with 96.6% similarity (data not shown).

3.3. Adhesion and Invasion of Strains in HeLa Cells All strains were able to adhere to and invade HeLa cells, with invasion index ranging from 1.4% to 73.8%, and there was a moderate correlation between adhesion and invasion (r = 0.44; p = 0.0041). We observed considerable variation in the rate of adhesion and invasion among strains belonging to the same serovar and possessing the same virulence genes. The cellular recovery values ranged from 0.2 x 106 to 3.0 x 106 CFU/mL. S. ser. Heidelberg was the serovar that presented the highest adhesion values. Recovery values in the invasion test ranged from 5 x 103 to 9 x 105 CFU/mL. More details are available in supplementar data.

3.4. Biofilm Formation Among the 40 strains analyzed, 31 (77.5%) were found to be biofilm producers (Table 2); 15 (37.5%) were classified as poor biofilm producers, 12 (30%) as moderate

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and 4 (10%) as strong producers. The strongest producers belonged to serovars S. Enteritidis (2), S. Ohio (1), and S. Mbandaka (1) (Figure 2)

3.5. Antibiotic Susceptibility Testing Seven antibiotics were tested and 13 (32.5%) isolates were resistant to at least one of them. One S. ser. Heidelberg strain was resistant to ampicillin, cotrimoxazole, and cefotaxime; another strain was also resistant to ampicillin and cotrimoxazole and presented intermediate resistance to chloramphenicol and tetracycline. Table 3 presents general data about the resistence profile and detailed results can be accessed in supplementary material

4. DISCUSSION Poultry are considered reservoirs of Salmonella spp., and poultry products are the main source of human infection. A total of 40 isolates were obtained during the 20 weeks of collection from canvas and polystyrene mats used in a poultry slaughterhouse. S. ser. Heidelberg, S. ser. Enteritidis, and S. ser. Ohio were the most prevalent serovars. Human gastroenteritis can be caused by different S. enterica serovars, including S. ser. Enteritidis and S. ser. Typhimurium as the most commonly involved serovars (LaRock, Chaudhary & Miller, 2015; Antunes, Mourão, Campos & Peixe, 2016). In 2017, in the United States, 841 foodborne disease outbreaks were reported and Salmonella spp. was responsible for 29% of them, with S. ser. Enteritidis, S. ser. Typhimurium, S. ser. Newport, S. ser. Heidelberg, S. Braenderup and S. ser. Javiana as the main serovars identified (CDC, 2017b). For years, S. ser. Enteritidis has been highlighted as the main causal agent of gastroenteritis, but with the implementation of control measures such as vaccination, its frequency has decreased substantially in Europe and USA (Antunes et al, 2016, EFSA,

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2017) while other commonly multidrug resistant serovars such as S. ser. Infantis, S. ser. Heidelberg and S. ser. Kentucky are increasing (Antunes et al, 2016; Campos et al., 2018). This trend was also observed in the results of the present study, which showed S. ser. Heidelberg as the predominant serovar. Although cases of human salmonellosis are self-limiting and do not require the use of antibacterials, antibiotic therapy can be applied in more severe cases, such as in extra-intestinal disease or in immunocompromised patients. In this context, multiresistant Salmonella serovars are considered to be a concern since they can compromise the effective treatment of the disease, thereby prolonging the hospitalization period or leading to death (Sing & Mustapha, 2013), and in recent decades, an increase has been reported in the incidence of antimicrobial resistance in foodborne Salmonella isolates (Iglesias, Kroning, Decol, Franco & Silva, 2017; ).. Chuah et al. (2018) investigating the MDR of Salmonella strains isolated from poultry and environmental samples collected from markets and smale-scale processing plant from northern Malaysia observed high resistance

rates

to

ampicillin,

tetracycline,

chloramphenicol

and

sulfametazole/trimethoprim. Similar observations have also been made by other researchers (Shang, Wei, Jang, & Kang, 2019; Vinueza-Burgos, Baquero, Medina, & De Zutter, 2019; Yang et al., 2019). Different results were observed by Procura, Bueno, Bruno & Rogé (2019) in which all Salmonella strains isolated from chicken liver obtained in markets in Argentina were sensitive to all antibiotics tested except for erythromycin and streptomycin. In our study, we observed higher resistance rates for tetracycline and ampicillin, but with lower percentages than those found in the studies cited. This difference may be due to the fact that antibiotic resistance may vary by region, source of isolation, different antibiotics and other factors (Khan et al., 2019).

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.Similarly, we also observed prevalent resistance to tetracycline and ampicillin. The results presented here provide useful data on the antimicrobial susceptibility of different Salmonella serovars and highlight the diversity of multiresistance to the tested antibiotics. This is an important aspect related to foodborne diseases which, together with different virulence factors and mechanisms of bacterial pathogenicity, make it difficult to control these diseases. The virulence genes invA, sipA, sipB, sipD, sopB, sopD, spvB, ssaR, sifA, tolC, flgK, fljB, and flgL encode for proteins linked to the processes of adhesion, invasion, survival, and replication within cells, while the sitC and iroN genes are involved in the acquisition of iron. Considering these 15 genes, 11 were found in all isolates, demonstrating the high pathogenic potential of these strains. SPI-1-associated genes such as sipA, sipD and sopD were observed in most isolates and encode proteins involved in Salmonella adhesion and invasion, causing the formation of “membrane rufing” and actin structure disruption (Karacan Sever & Akan, 2019). Similar results were obtained by Shang et al. (2019), who studied several virulence genes(invA, sipA, sopB, sopD, and ssaR), in Salmonella sp. from chicken samples obtained in retail markets in South Korea and they found that all isolates carried most of the genes searched (except ssaR, with 94.2%). It’s important to highlight these results, since the pathogenicity of Salmonella is mediated by different virulence factors and an insufficient number of virulence genes may interfere in the establishment of the disease (Sharma et al., 2019)

For successful colonization and persistence in a host, bacteria often invade eukaryotic cells to avoid competing with commensal bacteria and/or to evade the host’s immune system (Hauck, Agerer, Muenzner & Schmitter 2006). By evaluating the invasion rates of each serovar, a variation in this rate was observed among isolates of the

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same serovar. Shah et al. (2011) tested 53 isolates of S. ser. Enteritidis from poultry and observed that all were able to invade intestinal epithelial cells (Caco-2) at different intensities, with no correlation between the invasion rate and the presence of the spvB gene, which has been associated with virulence in Salmonella. Similar results were observed in the present work. In addition to invasion, biofilms can also protect bacteria from antimicrobials and the immune system and Salmonella is well-known for its ability to produce a biofilm on different surfaces, a fact that contributes to its survival in these hostile environments and favors its transmission to new hosts and surfaces. In the environment, a biofilm can protect bacteria, making them less sensitive to sanitizers when compared to planktonic cells and complicating their elimination even under unfavorable environmental conditions (Zhao et al., 2017; Zadernowska & Chajęcka-Wierzchowska, 2017). A study conducted by Zadernowska & Chajęcka-Wierzchowska (2017) verified that 87.5% of the 16 isolates of Salmonella spp. were classified as biofilm producers. Borges et al. (2018) evaluated biofilm production by S. Enteritidis and S. Typhimurium and found 66% of biofilm producing strains when incubated at 37°C and 54.8% at 28°C. In both studies, strong biofilm producers strains were not observed, but we found 4 isolates classified in this way. Using PFGE, Cao et al. (2018) compared 168 isolates of Salmonella collected from baseline surveys in chicken and pork production chains and ill chickens collected between 2012 and 201, and this analysis identified high similarity between strains from different sources and environments. Similar results were obtained by Shang et al. (2019) who evaluated the clonal relationship of 72 strains of S. ser. Albany, 37 S. ser. Montevideo, 22 S. ser. Virchow and 4 S. ser. Typhimurium, isolated from a single poultry slaughterhouse and its related retail markets, and observed routes of Salmonella cross-

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contamination within the slaughterhouse and between the slaughterhouse and the retail markets. These data added to our results can indicate that the persistence of these clones in the environment may be associated with the production of biofilm, since most of our strains were classified as biofilm producers. This finding demonstrates possible failures in hygiene, raising the risk of contamination of the carcasses and compromising the entire production line. Importantly, the same S. ser. Agona clone remained in the environment for 18 weeks, and all three strains of this clone were biofilm producers. Conversely, the four isolates of S. ser. Mbandaka presented four different clonal profiles and two of them (50%) did not produce a biofilm. Fardsanei et al. (2017) compared the genetic diversity of 34 strains of S. ser. Enteritidis isolated from meat (poultry, lamb, and beef) and eggs, and they observed indistinguishable patterns among most strains of S. ser. Enteritidis, despite the origin and also showed that there was no association between virulence profile and antibacterial susceptibilityAll our S. ser. Enteritidis strains also presented indistinguishable patterns (Figure 1B). This could happen due to the low genetic diversity in this serovar, so we cannot claim that these isolates share the same origin.

5. CONCLUSION Although all Salmonella isolates were able to adhere to and invade HeLa cells in this sudy, it is not possible to establish a direct relationship between a low or high adhesion rate and invasion with the absence or presence of genes coding for effector proteins involved in these processes, indicating the possible presence of other genes or mechanisms. Salmonella ser. Heidelberg, S. ser. Enteritidis, S. ser. Ohio, S. ser. Agona, and S. ser. Kentucky clones can be detected for up to 18 weeks, demonstrating the persistence and propagation of these pathogens on contact surfaces in the processing

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plant, potentially causing cross-contamination in good quality carcasses. This persistence could occur mainly due to the production of a biofilm by most strains. Food contamination is problematic as these isolates can invade the cell and be resistance to some antibiotics.

ACKNOWLEDGMENT This study was financed in part by the Coordenação de Aperfeiçoamento Pessoal de Nível Superior – Brasil (CAPES)- Finance Code 001. The authors have no conflicts of interest in conducting this work.

REFERENCES Antunes, P., Mourão, J., Campos, J., & Peixe, L. (2016). Salmonellosis: the role of poultry meat.

Clinical

Microbiology

and

Infection,

22,

110–121.

https://doi.org/10.1016/j.cmi.2015.12.004 Arnold, T., Scholz, H. C., Marg, H., & Hensel, A. (2004). Impact of invA-PCR and culture detection methods on occurrence and survival of salmonella in the flesh, internal organs and lymphoid tissues of experimentally infected pigs. Journal of Veterinary Medicine series B, 51(10), 459-463. https://doi.org/10.1111/j.14390450.2004.00808.x Borges, K. A., Furian, T. Q., de Souza, S. N., Menezes, R., de Lima, D. A., Fortes, F. B. B., Salle, 17

C. T. P., Moraes, H. L. S., & Nascimento, V. P. (2018). Biofilm formation by Salmonella Enteritidis and Salmonella Typhimurium isolated from avian sources is partially related with their

in

vivo

pathogenicity.

Microbial

Pathogenesis,

118,

238–241.

https://doi.org/10.1016/j.micpath.2018.03.039.

Campioni, F., Bergamini, A. M. M., & Falcão, J. P. (2012). Genetic diversity, virulence genes and antimicrobial resistance of Salmonella Enteritidis isolated from food and humans over a 24-year period in Brazil. Food Microbiology, 32, 254-264. https://doi.org/10.1016/j.fm.2012.06.008 Campos, J., Mourão, J., Silevira, L., Saraiva, M., Correia, C. B., Maçãs, A. P., Peixe, L., & Antunes, P. (2018). Imported poultry meat as a source of extended-spectrum cephalosporinresistant CMY-2-producing Salmonella Heidelberg and S. minnesota in European Union, 2014-2015.

International

Journal

of

Antimicrobial

Agents,

51,

151-154.

https://doi.org/10.1016/j.ijantimicag.2017.09.006 Cao, Y., Shen, Y., Cheng, L., Zhang, X., Wang, C., Wang, Y., Zhou, X., Chao, G., & Wu, Y. (2018). Combination of multilocus sequence typing and pulsed-field gel electrophoresis reveals an association of molecular clonality with the emergence of extensive-drug resistance (XDR) in Salmonella. Microbiological Research, 207, 170–176. https://doi.org/10.1016/j.micres.2017.12.001.

CDC. (2017a). Standard Operating Procedure for PulseNet PFGE of Escherichia coli O157:H7, Escherichia coli non-O157 (STEC), Salmonella serotypes, Shigella sonnei and Shigella flexneri. (2017). https://www.cdc.gov/pulsenet/pdf/ecoli-shigellasalmonella-pfge-protocol-508c.pdf. Accessed 15 May 2017. CDC. (2017b). Surveillance for Foodborne Disease Outbreaks United States, 2017: Annual

Report.

Centers

for

Disease

Control

and

Prevention,

USA.

https://www.cdc.gov/fdoss/pdf/2017_FoodBorneOutbreaks_508.pdf. Accessed 01 November 2019.

18

Chakroun, I., Mahdhi, A., Morcillo, P., Cordero, H., Cuesta, A., Bakhrouf, A., Mahdouani, K., & Esteban, M. A. (2018). Motility, biofilm formation, apoptotic effect and virulence gene expression of atypical Salmonella Typhimurium outside and

inside

Caco-2

cells.

Microbial

Pathogenesis,

114,

153-162.

https://doi.org/10.1016/j.micpath.2017.11.010 Chuah, L. O., Syuhada, A. K. S., Suhaimi, I. M., Hanim, T. F., & Rusul, G. (2018). Genetic relatedness, antimicrobial resistance and biofilm formation of Salmonella isolated from naturally contaminated poultry and their processing environment in northern

Malaysia.

Food

Research

International,

105,

743-751.

https://doi.org/10.1016/j.foodres.2017.11.066 CLSI. (2018). Performance Standards for Antimicrobial Susceptibility Testing; TwentySeven Informational Supplement. CLSI document M100-S28. Clinical and Laboratory Standards Institute, Wayne, PA. Coburn, B., Grassl, G. A., & Finlay, B. B. (2007). Salmonella, the host and disease: a brief

review.

Immunology

and

Cell

Biology,

85,

112-118.

https://doi.org/10.1038/sj.icb.7100007 Cox, N. A., Frye, J. G., McMahon, W., Jackson, C. R., Richardson, J., Cosby, D.E., Mead, G., & Doyle, M.P. (2015). Salmonella. In: Salfinger, Y., & Tortorello, M.L. (Eds). Compendium of Methods for the Microbiological Examination of Foods. 5th

Ed

.

Washington: Apha, 445-463. Dantas, S. T. A., Rossi, B. F., Bonsaglia, E. C. R., Castilho, I. G., Hernandes, R. T., Fernandes Júnior, A., & Rall, V. L. M. (2018). Cross-contamination and biofilm formation by Salmonella enterica serovar Enteritidis on various cutting boards. Foodborne

Pathogens

and

Disease,

15(2),

81-85.

https://doi.org/10.1089/fpd.2017.2341

19

European Food Safety Authority (EFSA), European Centre for Disease Prevention and Control (ECDC). The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2015. EFSA J 2017.EN-1353. Available

from:

https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/sp.efsa.2017.EN-1353. [Accessed 23 November 2018]. Fardsanei, F., Dallal, M. M. S., Douraghi, M., Salehi, T. Z., Mahmoodi, M., Memariani, H., & Nikkhahi, F. (2017). Genetic diversity and virulence genes of Salmonella enterica subspecies enterica serotype Enteritidis isolated from meats and eggs. Microbial Pathogenesis, 107, 451-456. https://doi.org/10.1016/j.micpath.2017.04.026 Gagnon, M., Berner, A. Z., Chervet, N., Chassard, C., & Lacroix, C. (2013). Comparison of the Caco-2, HT-29 and the mucus-secreting HT29-MTX intestinal cell models to investigate Salmonella adhesion and invasion. Journal of Microbiological Methods, 94, 274-279. https://doi.org/10.1016/j.mimet.2013.06.027 Hauck, C. R., Agerer, F., Muenzner, P., & Schmitter T. (2006). Cellular adhesion molecules as targets for bacterial infection. European Journal of Cell Biology, 85(34), 235-242. https://doi.org/10.1016/j.ejcb.2005.08.002 Iglesias, M. A., Kroning, I. S., Decol, L. T., Franco, B. D. G. M., & Silva, W. P. (2017). Occurrence

and

phenotypic

and

molecular

characterization

of

Listeria

monocytogenes and Salmonella spp. in slaughterhouses in southern Brazil. Food Research International, 100, 96-101. https://doi.org/10.1016/j.foodres.2017.06.023 Karacan Sever, N., & Akan, M. (2019). Molecular analysis of virulence genes of Salmonella Infantis isolated from chickens and turkeys. Microbial Pathogenesis, 126, 199–204. https://doi.org/10.1016/j.micpath.2018.11.006. 20

Khan, S. B., Khan, M. A., Ahmad, I., ur Rehman, T., Ullah, S., Dad, R., Sultan, A., & Memon, A. M. (2019). Phentotypic, gentotypic antimicrobial resistance and pathogenicity of Salmonella enterica serovars Typimurium and Enteriditis in poultry and poultry products. Microbial Pathogenesis, 129, 118–124. https://doi.org/10.1016/j.micpath.2019.01.046.

Kitchel, B., Rasheed, J. K., Patel, J.B., Srinivasan, A., Navon-Venezia, S., Carmeli, Y., Brolund, A., & Giske, G. G. (2009). Molecular Epidemiology of KPC-Producing Klebsiella pneumoniae isolates in the United States: Clonal Expansion of Multilocus Sequence Type 258. Antimicrobial Agents and Chemotherapy, 53(8), 3365–3370. https://dx.doi.org/10.1128%2FAAC.00126-09 Lamas, A., Paz-Mendez, A.M., Regal, P., Vazquez, B., Miranda, J.M., Cepeda, A., & Franco, C.M. (2018). Food preservatives influence biofilm formation, gene expression and small RNAs in Salmonella enterica. LWT - Food Science and Technology, 97, 1–8. https://doi.org/10.1016/j.lwt.2018.06.042 LaRock, D. L., Chaudhary, A., & Miller, S. I. (2015). Salmonellae interactions with host processes.

Nature

Reviews

Microbiology,

13,

191-205.

https://doi.org/10.1038/nrmicro3420 Mellor, G. E., Goulter, R. M., Raymond Chia, T. W., & Dikes, G. A. (2009). Comparative analysis of attachment of Shiga-Toxigenic Escherichia coli and Salmonella strains to cultured HT-29 and Caco-2 cell lines. Applied and Environmental Microbiology, 75(6), 1796-1799. https://doi.org/10.1128/AEM.02232-08 Oliveira, D. C. V., Fernandes Jr, A., Kaneno, R., Silva, M. G., Silva, N. C. C., & Rall, V. L. M. (2014). Ability of Salmonella spp. to produce biofilm is dependent on temperature and surface material. Foodborne Pathogens and Disease, 11, 478-483. https://doi.org/10.1089/fpd.2013.1710

21

Procura, F., Bueno, D. J., Bruno, S. B., & Rogé, A. D. (2019). Prevalence, antimicrobial resistance profile and comparison of methods for the isolation of Salmonella in chicken liver from Argentina. Food Research International, 119, 541-546. http://dx.doi.org/10.1016/j.foodres.2017.08.008 Sharma, J., Kumar, D., Hussain, S., Pathak, A., Shukla, M., Prasanna Kumar, V., Anisha, P. N., Richa Rautela, Upadhyay, A. K., & Singh, S. P. (2019). Prevalence, antimicrobial resistance and virulence genes characterization of nontyphoidal Salmonella isolated from retail chicken meat

shops

in

Northern

India.

Food

Control,

102,

104–111.

https://doi.org/10.1016/j.foodcont.2019.01.021. Shang, K., Wei, B., Jang, H.-K., & Kang, M. (2019). Phenotypic characteristics and genotypic correlation of antimicrobial resistant (AMR) Salmonella isolates from a poultry slaughterhouse and its downstream retail markets. Food Control, 100, 35–45. https://doi.org/10.1016/j.foodcont.2018.12.046.

Shah, D.H., Zhou, X., Addwebi, T., Davis, M. A., Orfe, L., Call, D. R., Guard, J., & Besser, T. E. (2011). Cell invasion of poultry-associated Salmonella enterica serovar Enteritidis isolates is associated with pathogenicity, motility and proteins secreted by the

type

III

secretion

system.

Microbiology,

157,

1428-1445.

https://doi.org/10.1099/mic.0.044461-0 Sing, P. & Mustapha, A. (2013). Multiplex TaqMan® detection of pathogenic and multidrug resistant Salmonella. Intenational Journal of Food Microbiology, 166, 213-128. https://doi.org/10.1016/j.ijfoodmicro.2013.07.023 Skyberg, J.A., Logue, C.M., & Nolan, L.K. (2006). Virulence genotyping of Salmonella spp. with multiplex PCR. Avian Diseases, 50, 77-81. https://doi.org/10.1637/7417.1 Stepanović, S., Ćirković, I., Mijac, V., & Svabić-Vlahović, M. (2003). Influence of the incubation temperature, atmosphere and dynamic conditionson biofilm formation by

22

Salmonella spp. Food Microbiology, 20, 339-343. https://doi.org/10.1016/S07400020(02)00123-5 Tack, D.M., Marder, E.P., Griffin, P.M., Cieslak, P.R., Dunn, J., Hurd, S., Scallan, E., Lathrop, S., Muse, A., Ryan, P., Smith, K., Tobin-D’Angelo, M., Vugia, D.J., Holt, K.G., Wolpert, B.J., Tauxe, R., & Geissler, A.L. (2019). Preliminary incidence and trends of infections with pathogens transmitted commonly through food — Foodborne Diseases Active Surveillance Network, 10 U.S. Sites, 2015–2018. Morbidity

and

Mortality

Weekly

Report,

68

(16),

369-373.

https://dx.doi.org/10.15585%2Fmmwr.mm6816a2Turki, Y., Ouzari H., Mehri, I., Aissa, R. B., & Hassen, A. (2012). Biofilm formation, virulence gene and multi-drug resistance in Salmonella Kentucky isolated in Tunisia. Food Research International, 45, 940–946. https://doi.org/10.1016/j.foodres.2011.05.031 Vinueza-Burgos, C., Baquero, M., Medina, J., & De Zutterb, L. (2019). Occurrence, genotypes and antimicrobial susceptibility of Salmonella collected from the broiler production chain within an integrated poultry. Company. International Journal of Food Microbiology, 16, 17. https://doi.org/10.1016/j.ijfoodmicro.2019.03.014 Yang, J., Zhang, Z., Zhou, X., Cui, Y., Shi, C., & Shi, X. (2019). Prevalence and Characterization of Antimicrobial Resistance in Salmonella enterica Isolates from Retail Foods in Shanghai, China.

Foodborne

Pathogens

and

Disease,

XX,

XX,

fpd.2019.2671.

https://doi.org/10.1089/fpd.2019.2671. IN PRESS

Zadernowska, A., & Chajęcka-Wierzchowska, W. (2017). Prevalence, biofilm formation and virulence markers of Salmonella sp. and Yersinia enterocolitica in food of animal origin in Poland. LWT – Food Science and Technology, 75, 552-556. https://doi.org/10.1016/j.lwt.2016.10.007

23

Zhao, X., Zhao, F., Wang, J., & Zhong, N. (2017). Biofilm formation and control strategies of foodborne pathogens: food safety perspectives. Royal Society of Chemistry Advances, 7, 36670-36683. https://doi.org/10.1039/C7RA02497E Wang, H., Ding, S., Dong, Y., Ye, K., Xu, X., & Zhou, G. (2013). Biofilm formation of Salmonella serotypes in simulated meat processing environments and its relationship to cell characteristics. Journal of Food Protection, 76(10), 1784-1789. http://dx.doi.org/10.4315/0362-028X.JFP-13-093

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25

Highlights - Clones of Salmonella remained in the environment for a long time - All Salmonella isolates presented cell invasion capacity. - Salmonella isolates showed resistance to different antimicrobials. - There was no correlation between phenotypic tests and the presence of virulence genes

26

A

B

C

D

27

Figure 1. Dendrogram of pulsed-field gel electrophoresis (PFGE) patterns of different strains from the same Salmonella serovar, obtained from mats of a poultry slaughterhouse. A) Salmonella ser. Heidelberg, B) Salmonella ser. Enteritidis, C) Salmonella ser. Ohio, D) Salmonella ser. Agona. C: canvas; P: polystyrene

28

Figure 2: Classification of biofilm production, according to the Salmonella serovars tested.

29

Table 1: Primers and their properties used in PCR reactions, for detection of virulence genes in Salmonella spp. Gene

Sequence

Amplicon

Annealing

(5' - 3')

size (bp)

temperature

284

60°C

Arnold et al., 2004

220

66.5°C

Skyberg et al., 2006

1291

56.5°C

Hur et al., 2011

550

66.5°C

Skyberg et al., 2006

875

66.5°C

Skyberg et al., 2006

1029

53°C

Shah et al., 2011

1628

50°C

Hur et al., 2011

449

66.5°C

Skyberg et al., 2006

717

66.5°C

Skyberg et al., 2006

768

66,5°C

Skyberg et al., 2006

161

66.5°C

Skyberg et al., 2006

1205

66.5°C

Skyberg et al., 2006

1659

53°C

Shah et al., 2011

1515

53°C

Shah et al., 2011

951

53°C

Shah et al., 2011

invA F

TCATCGCACCGTCAAAGGAAC

invA R

CACGATATTGATATTAGCCCG

sopB F

CGGACCGGCCAGCAACAAAACAAGAAGAAG

sopB R

TAGTGATGCCCGTTATGCGTGAGTGTATT

sopD F

ACGACCATT TGCGGCG

sopD R

GAGACACGCTTCTTCG

sipA F

CCAGGGGTCGTTAGTGTATTGCGTGAGATG

sipA R

CGCGTAACAAAGAACCCGTAGTGATGGATT

sipB F

GGACGCCGCCCGGGAAAAACTCTC

ksipB R

ACACTCCCGTCGCCGCCTTCACAA

sipD F

ATGCTCCTTGCAGGAAGCTTTTG

sipD R

TTAATATTCAAAATTATTCCG

ssaR F

GTTCGGATTCATTGCTTCGG

ssaR R

TCTCCAGTGACTAACCCTAACCAA

sifA F

TTTGCCGAACGCGCCCCCACACG

sifA R

GTTGCCTTTTCTTGCGCTTTCCACCCATCT

spvB F

CTATCAGCCCCGCACGGAGAGCAGTTTTTA

spvB R

GGAGGAGGCGGTGGCGGTGGCATCATA

sitC F

CAGTATATGCTCAACGCGATGTGGGTCTCC

sitC R

CGGGGCGAAAATAAAGGCTGTGATGAAC

tolC F

TACCCAGGCGCAAAAAGAGGCTATC

tolC R

CCGCGTTATCCAGGTTGTTGC

iroN F

ACTGGCACGGCTCGCTGTCGCTCTAT

iroN R

CGCTTTACCGCCGTTCTGCCACTGC

flgK F

ATGTCCAGCTTGATTAATCAC

flgK R

GCGAATATTCAATAACGCATC

fljB F

ATGGCACAAGTCATTAATACAAAC

fljB R

ACGCAGTAAAGAGAGGAC

flgL F

ATGCGTATCAGTACCCAGATG

flgL R

CCGGTTCAACTGGAAAAGC

Reference

Bp: base pair

30

Table 2. Genotypic pic characterization of some virulence factors in Salmonella spp., isolated from mats in a poultry slaughterhouse. Genotype Serovar (N)

fljB

flgK

flgL

invA

sipA

sipB

sipD

tolC

sopB

sopD

ssaR

sifA

spvB

sitC

iroN

S. Heidelberg (11)

100

100

100

100

100

100

100

100

72.7

91

100

100

18.2

100

100

S. Enteritidis (7)

100

100

100

100

100

100

100

100

100

100

100

100

85.7

100

100

S. Ohio (5)

100

100

100

100

80

100

100

100

100

100

100

100

0

100

100

S. Mbandaka (4)

100

100

100

100

100

100

100

100

100

75

100

100

25

100

100

S. Agona (3)

100

100

100

100

100

100

100

100

100

100

100

100

66.7

100

100

S. Typhimurium (3) S. Kentucky (2)

100

100

100

100

100

100

100

100

100

100

100

100

33.3

100

100

100

100

100

100

100

100

100

100

100

100

100

100

0

100

100

S. Abony (1)

100

100

100

100

0

100

100

100

100

100

100

100

100

100

100

S. Bredeney (1)

100

100

100

100

100

100

100

100

100

100

100

100

0

100

100

S. Saintpaul (1)

100

100

100

100

100

100

100

100

100

100

100

100

0

100

100

S. Worthington (1) S. enterica subs. enterica (1)

100

100

100

100

0

100

100

100

100

100

100

100

0

100

100

100

100

100

100

100

100

100

100

100

0

100

100

0

100

100

fljB, flgK e flgL: flagella-encoding chromosomal genes; invA, sipA, sipB, sipD, sopB, sopD, tolC: encoding invasion associated proteins; sitC, iroN: siderophores; ssaR, sifA, spvB: survival and bacterial replication within host cells

31

Table 3. Antimicrobial susceptibility of Salmonella spp. isolated from mats in a poultry slaughterhouse. Salmonella spp.(n=40) Antimicrobial

Sensitive

Intermediate

Resistant

N (%)

N (%)

N (%)

Ampicillin

36 (90)

0

4 (10)

Cotrimoxazole

38 (95)

0

2 (5)

Chloramphenicol

35 (87,5)

4 (10)

1(2.5)

Ciprofloxacin

40 (100)

0

0

Cefotaxime

37 (92.2)

0

3 (7.5)

Gentamicin

40 (100)

0

0

Tetracycline

32 (80)

1 (2.5)

7 (17.5)

32

The authors have no conflicts of interest in conducting this work.

33

Environmental persistence and virulence of Salmonella spp. isolated from a poultry slaughterhouse

Stéfani T. A. Dantas: Conceptualization, investigation and writing original draft preparation Carlos H. Camargo: Investigation Monique R. Tiba-Casas: Investigation Ricardo C. Vivian: Investigation José P. A. N. Pinto: Supervision José C. F. Pantoja: Formal analysis Rodrigo T. Hernandes: Writing, reviewing and editing Ary Fernandes Júnior: Investigation and reviewing Vera L. M. Rall: Conceptualization and management and coordination responsibility for the research activity planning and execution

34