Tolerance to benzalkonium chloride and antimicrobial activity of Butia odorata Barb. Rodr. extract in Salmonella spp. isolates from food and food environments

Accepted Manuscript Tolerance to benzalkonium chloride and antimicrobial activity of Butia odorata Barb. Rodr. extract in Salmonella spp. isolates from food and food environments

Louise Haubert, Maiara Lindemann Zehetmeyr, Ytacyana Maria Nascimento Pereira, Isabela Schneid Kroning, Darla Silveira Volcan Maia, Carla Pohl Sehn, Graciela Völz Lopes, Andreia Saldanha de Lima, Wladimir Padilha da Silva PII: DOI: Reference:

S0963-9969(18)30682-3 doi:10.1016/j.foodres.2018.08.092 FRIN 7900

To appear in:

Food Research International

Received date: Revised date: Accepted date:

2 May 2018 21 July 2018 19 August 2018

Please cite this article as: Louise Haubert, Maiara Lindemann Zehetmeyr, Ytacyana Maria Nascimento Pereira, Isabela Schneid Kroning, Darla Silveira Volcan Maia, Carla Pohl Sehn, Graciela Völz Lopes, Andreia Saldanha de Lima, Wladimir Padilha da Silva , Tolerance to benzalkonium chloride and antimicrobial activity of Butia odorata Barb. Rodr. extract in Salmonella spp. isolates from food and food environments. Frin (2018), doi:10.1016/j.foodres.2018.08.092

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ACCEPTED MANUSCRIPT Tolerance to benzalkonium chloride and antimicrobial activity of Butia odorata Barb. Rodr. extract in Salmonella spp. isolates from food and food environments

Louise Haubert1, Maiara Lindemann Zehetmeyr2, Ytacyana Maria Nascimento

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Pereira1, Isabela Schneid Kroning1, Darla Silveira Volcan Maia1, Carla Pohl

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Sehn3, Graciela Völz Lopes1, Andreia Saldanha de Lima1, Wladimir Padilha da

de Ciência e Tecnologia Agroindustrial, Faculdade de Agronomia

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1Departamento

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Silva1,2*

Eliseu Maciel, Universidade Federal de Pelotas (UFPel), Pelotas, RS, Brazil. de

Biotecnologia,

Centro

de

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2Núcleo

Desenvolvimento

Tecnológico,

Universidade Federal de Pelotas (UFPel), Pelotas, RS, Brazil. de Nutrição, Universidade Federal do Pampa (UNIPAMPA), Itaqui, RS,

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3Curso

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Brazil.

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*Corresponding author: Campus Capão do Leão s/nº, Universidade Federal de Pelotas (UFPel), Capão do Leão, RS, Brazil, Caixa Postal 354, 96160-000. E-mail: [email protected]

ACCEPTED MANUSCRIPT

Abstract Salmonellosis, caused by the consumption of contaminated foods, is a major

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health problem worldwide. The aims of this study were to assess the susceptibility of Salmonella spp. isolates to benzalkonium chloride (BC) disinfectant and the

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antimicrobial activity of Butia odorata Barb. Rodr. extract against the same

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isolates from food and food environments. Moreover, phenotypic and genotypic resistance profiles, the presence of virulence genes and biofilm forming ability

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were determined. The minimum inhibitory concentration (MIC) of B. odorata

ampicillin,

streptomycin,

nalidixic

trimethoprim,

acid,

sulfonamide,

tetracycline,

and/or

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trimethoprim/sulfamethoxazole,

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extract against Salmonella spp. ranged from 10 to >19 mg.mL-1. Resistance to

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chloramphenicol was observed. In addition, multidrug resistance was observed in seven isolates (26.92%). The MIC of BC ranged from 32 to 64 mg.L -1, higher concentrations in comparison with wild-type MICs, and therefore were considered

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tolerant. Several resistance genes were detected, of which the most common

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were aadA, qacEΔ1, blaTEM, int1, sul1, and tetA. All isolates carried at least one virulence gene and produced biofilms on stainless steel surfaces at 10 and 22 °C. On the other hand, the B. odorata extract showed activity against Salmonella spp., and it has the potential to be used as a natural antimicrobial to control this important foodborne pathogen, despite the latter’s virulence potential and antimicrobial resistance profile.

ACCEPTED MANUSCRIPT Keywords: Foodborne isolates; Virulence genes; Biofilm; Resistance genes;

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Plant extract

ACCEPTED MANUSCRIPT 1 Introduction Salmonella spp. are Gram-negative, facultative anaerobe and rod-shaped bacteria belonging to the Enterobacteriaceae family (Agbaje et al., 2011). Although the Salmonella genus contains only two species, Salmonella enterica and Salmonella bongori, the former is divided into six subspecies consisting of

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more than 2500 serovars (Barco et al., 2013). Salmonella enterica serovars

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Typhimurium and Enteritidis are most frequently detected worldwide, and the

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prevalence of other serovars appears to depend on the region (Hendriksen et al., 2011; Lopes et al., 2014).

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The contamination of food and food supplements by Salmonella spp. poses a significant health concern, since this pathogen is responsible for causing

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foodborne diseases throughout the world (EFSA - European Food Safety Authority, 2015). This microorganism can cause salmonellosis, a disease with

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high rates of morbidity. Its pathogenicity involves several virulence factors that

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are located in the bacterial chromosome, frequently as part of pathogenicity

al., 2001).

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islands (SPI-1 and SPI-2), or in plasmids (Campioni et al., 2012; McClelland et

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The capacity of Salmonella spp. to form complex surface-associated communities, called biofilms, is described as an important virulence factor. Several reports have demonstrated the ability of Salmonella strains to form biofilms on abiotic surfaces outside the host, especially on surfaces commonly encountered in the food processing environment. As Salmonella cells in biofilms are more resistant to several environmental stress factors such as desiccation, antimicrobial agents and disinfectants, their ability to form biofilms in a food

ACCEPTED MANUSCRIPT industry environment must be investigated (Karaca et al., 2013; Steenackers et al., 2012; Wang et al., 2013). It is now accepted that the widespread use of antimicrobials in both animal and human populations has led to the emergence of antimicrobial resistant Salmonella isolates. In recent years multi-resistant Salmonella spp. isolates have

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emerged and spread rapidly, constituting a severe and rising concern for public

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health (Anjum et al., 2011). Furthermore, the common use of disinfectants in food

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processing chains may facilitate the selection of strains that exhibit acquired disinfectant resistance and can carry genes encoding cross-resistance to

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antibiotics, thereby creating a new threat to public health (Long et al., 2016). For instance, benzalkonium chloride (BC) is a disinfectant belonging to the class of

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quaternary ammonium compounds (QACs), widely used for sanitizing surfaces, utensils and instruments in the food industry, which contributes to the emergence

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Wu et al., 2015).

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of resistant bacteria like Salmonella spp. (Garrido et al., 2015; Long et al., 2016;

Several studies have reported the use of natural antimicrobials, notable

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among them plant extracts (Côté et al., 2011; Fernandes et al., 2014a; Medina et al., 2011; Shen et al., 2014). Butia odorata Barb. Rodr., belonging to the botanical

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family Arecaceae (Hoffmann et al., 2014; Lorenzi et al., 2010), is a subtropical palm that occurs in open areas and in Araucaria forests, distributed in some countries in South America. Extracts from this palm can be used as an inhibition strategy for microorganisms, including Salmonella isolates (Hoffmann et al., 2014; Maia et al., 2017). The aims of this study were to assess the susceptibility of Salmonella spp. isolates from food and food environments to BC, evaluate their antimicrobial

ACCEPTED MANUSCRIPT resistance profile as well as biofilm forming ability. Additionally, we intend to test an alternative natural inhibitor from B. odorata extract against Salmonella spp. foodborne isolates.

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2 Material and methods

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2.1 Bacterial strains

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A total of 26 Salmonella isolates from food and food environments randomly selected, were evaluated in this study (Table 1). These isolates belong

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to the culture collection of the Laboratório de Microbiologia de Alimentos (DCTAFAEM-UFPel) located in southern Rio Grande do Sul state, Pelotas, Brazil and

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were isolated according to International Organization for Standardization (ISO-

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2.2 Serotyping

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6579) method.

Serological tests were performed at the National Reference Center,

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Fundação Instituto Oswaldo Cruz, Rio de Janeiro, Brazil, by standard slide

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agglutination using commercially available antisera.

2.3 Molecular identification of Salmonella isolates and detection of virulence genes The confirmation of Salmonella isolates by the detection of hilA gene and the detection of virulence genes (invA, spvC, sefA, and pefA) was performed by PCR assays. The oligonucleotides used are shown in Table 2. The reaction mixtures contained 12.5 L of GoTaq® Green Master Mix 2x (Promega, USA), 1

ACCEPTED MANUSCRIPT L of each primer at a concentration of 10 mol, 2 L of DNA (10 ng) and 8.5 L of ultra-pure water (Promega, USA) to a total volume of 25 L. The mixtures were subjected to a MJ Research® PTC 100 thermocycler (Bio-Rad, USA). Next, the PCR products were subjected to electrophoresis at 80 V for 70 min on a 1.5% (w/v) agarose gel (Invitrogen, USA) in a 0.5 Tris/Acetate/EDTA buffer (TAE) using

2.4 Antimicrobial susceptibility testing

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visualized in an UV transilluminator (Loccus, Brazil).

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1 Kb molecular weight marker (Invitrogen, USA). The amplified products were

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The antimicrobial susceptibility of the Salmonella isolates was evaluated by the agar disk diffusion method, according to the Clinical and Laboratory

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Standards Institute for the Enterobacteriaceae group (CLSI, 2018). The following antimicrobial disks were used: ampicillin 10 μg (AMP), amoxicillin/clavulanic acid

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30 μg (AMC), cefotaxime 30 μg (CTX), cephalothin 30 μg (CFL), gentamicin 10

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μg (GEN), tobramycin 10 μg (TOB), streptomycin 10 µg (STR), imipenem 10 µg (IMP), nalidixic acid 30 μg (NAL), ciprofloxacin 5 μg (CIP), sulfonamide 300 μg

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(SUL), trimethoprim/sulfamethoxazole 1.25/23.75 μg (SUT), trimethoprim 5 μg (TRI), tetracycline 30 μg (TET), and chloramphenicol 30 μg (CHL) acquired from

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Laborclin (Laborclin Produtos para Laboratórios Ltda, Brazil).

2.5 Detection of antimicrobial resistance genes The isolates that presented antimicrobial resistance in the agar disk diffusion method were evaluated for the presence of resistance genes. The coding resistance genes to β-lactams (blaZ and blaTEM), aminoglycosides (aadA, aadB, aac(6’)-Ib, strA, and strB), class 1 integrase (int1), folate pathway inhibitors

ACCEPTED MANUSCRIPT (sul1, sul2, sul3, dfrA, dfrD, and dfrG), tetracyclines (tetA and tetB), phenicols (catA1 and floR), and QACs (qacEΔ1) were investigated by PCR assays using the oligonucleotides listed in Table 2. The cycling conditions of the PCR assays followed the recommendations of the referenced authors’ studies.

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2.6 Biofilm forming ability

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Biofilm formation was evaluated using AISI 304 stainless steel coupons

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(0.366 μm roughness, 10 mm x 10 mm x 1 mm). Before use, the stainless steel coupons were sanitized according to the protocol proposed by Parizzi et al.,

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(2004) with adaptations. After that, the coupons were placed in tubes containing Tryptone Soy broth (TSB) (Acumedia, Brazil). Twenty-six Salmonella isolates and

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S. Typhimurium ATCC® 14028 (ST) were first inoculated on Petri dishes with Tryptone Soy agar (TSA) (Oxoid, UK) and were incubated at 37 °C for 24 h. The

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bacterial growth was adjusted at a concentration of 0.5 on the McFarland scale

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(108 CFU.mL-1), and 1 mL was inoculated in tubes containing 9 mL of TSB plus coupons (final concentration 107 CFU.mL-1) followed by incubation at 10 and 22

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°C for 48 h. Subsequently, the stainless steel coupons were transferred to 5 mL of 0.1% (w/v) peptone water (PW) (Oxoid, UK) and were immersed for 1 minute

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to remove planktonic cells. The coupons were transferred to 10 mL of PW and vortexed for 2 minutes to remove the sessile cells (Andrade et al., 1998; Fernandes et al., 2014b). Ten-fold dilutions were made in duplicate, and plated on TSA, followed by incubation for 24 h at 37 °C. The test was performed in triplicate.

2.7 Determination of minimum inhibitory concentration (MIC) of BC

ACCEPTED MANUSCRIPT The MIC of BC (Sigma-Aldrich, UK) was determined according to Wu et al., (2015). Firstly, the 26 Salmonella isolates were incubated on TSA at 37 ºC for 24 h. After incubation, the isolates were diluted on 0.5 McFarland standard in 0.9% (w/v) saline solution (SS) (Synth, Brazil) followed by a 1:10 dilution, and 1 µL (~104 CFU.mL-1) was spotted on Mueller-Hinton agar (MH) (Oxoid, UK) using

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variable concentrations of BC (0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, 128, 256,

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and 500 mg.L-1) with incubation of the plates at 37 ºC for 24 h. The MIC of BC

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was defined as the lowest concentration that completely inhibited the bacterial

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growth.

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2.8 Antimicrobial activity of B. odorata extract

2.8.1 B. odorata extract preparation

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A methanolic extract from B. odorata was prepared according to the

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method proposed by Shen et al., (2014). A 500 mL Erlenmeyer flask was filled with 30 g of lyophilized B. odorata fruit plus 300 mL of methanol (Synth, Brazil).

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The flasks were placed in a shaker for 2 h (150 rpm) and then in an ultrasonic bath (48 A / 15 min). After filtering the extract through filter paper and centrifuging

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for 20 min (7500 rpm), the supernatant was rotary-evaporated at 40 °C to constant weight.

2.8.2 Agar disk diffusion testing The antimicrobial activity of B. odorata extract against Salmonella spp. isolates was determined firstly by agar disk diffusion method. The inoculum was standardized at a concentration of 108 CFU.mL-1 and plated on Petri dishes

ACCEPTED MANUSCRIPT containing MH. Thereafter, sterile paper filter disks (6 mm) (Laborclin, Produtos para Laboratórios Ltda, Brazil) impregnated with 20 µL of B. odorata extract were placed on the agar, and the plates were incubated at 37 °C for 24 h. The tests were performed in all 26 isolates and in the strain ST; water was used in place of the B. odorata extracts as negative control and streptomycin 10 µg (STR) as

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positive control.

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2.8.3 MIC and minimum bactericidal concentration (MBC) of B. odorata extract

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The MIC was performed according to Weerakkody et al. (2010) with adaptations. Butia odorata extracts were tested at concentrations of 19, 16, 13,

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10, 6, and 2 mg.mL-1 (Maia et al., 2017). In tubes containing 1 mL of MH broth, 105 CFU.mL-1 of the target bacterium was inoculated with B. odorata extract at

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the predetermined concentration, and incubated at 37 °C for 24 h under shaking

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(150 rpm). MH broth containing streptomycin inoculated with 10 5 CFU.mL-1 was used as positive control. MH broth inoculated with 105 CFU.mL-1 without

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streptomycin was used as negative control. MIC was defined as the lowest extract concentration at which no microbial growth in the culture broth was visually

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detectable.

After incubation, 100 µL samples from tubes with no visible microbial growth were seeded on Petri dishes containing TSA and incubated at 37 °C for 24 h. After this step, colonies were counted. The MBC was defined as the lowest extract concentration at which 99.9% of the initially inoculated cells were killed.

2.8.4 Chemical characterization of B. odorata extract by GC-MS

ACCEPTED MANUSCRIPT For chemical characterization of the B. odorata extract, the sample was diluted in ethyl acetate and 1 µL was injected by Splitless in a RTx 5-MS column (Restek, USA), at a temperature of 35 °C and injection temperature of 250 °C. The analysis conditions used were: 35 °C/2 min, 5 °C/min until 80 °C, 2 °C/min until 120 °C, 120 °C/2 min, 2 °C/min until 230 °C, 230 °C/2 min, 10 °C/min until

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300 °C, remaining at 300 °C for 10 min. The temperature of the ion source was

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200 °C, and the interface temperature was 240 °C. For identification of the

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compounds, the library NIST-05 was used.

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2.9 Statistical analysis

Biofilm formation data on stainless steel coupons were submitted to

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statistical analysis of variance (ANOVA) using Tukey's test (p < 0.05) with

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3 Results and discussion

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STATISTIX 8.0 software.

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3.1 Salmonella serotyping

This study investigated Salmonella isolates from food and food processing

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environments. Among these isolates, only one (S15), which came from a food processing plant, was identified as Salmonella enterica subsp. diarizonae. Among the other isolates, all from food, Salmonella enterica subsp. enterica was identified in 25 isolates that included 12 different serovars (Table 1). Salmonella Derby and Salmonella Typhimurium were the most frequent serovars (n=5, 19.23%), followed by Salmonella Rissen (n=3, 11.53%).

ACCEPTED MANUSCRIPT In Europe, Salmonella ser. Derby is one of the most prevalent serovars in slaughter pigs, and it also ranks among the 10 most frequently isolated serovars in humans (EFSA - European Food Safety Authority, 2015). In Southern Brazil it has also been found in samples from swine finishing herds, slaughterhouses and pork sausage (Kich et al., 2011; Lopes et al., 2014; Murmann et al., 2009) as well

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as in our work, where three of the five isolates came from sausages containing

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pork meat.

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Also in Brazil, Salmonella Typhimurium was the most isolated serovar until mid-1990s, and after this period, it became the second most frequently isolated

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serovar surpassed by Salmonella Enteritidis (Fernandes et al., 2006; Tavechio et

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al., 2002), differently from that found in this study.

3.2 Molecular identification and detection of virulence genes

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All the isolates were confirmed as belonging to the genus Salmonella,

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since there was amplification of a 413 bp fragment related to the hilA gene, specific to the Salmonella genus. Moreover, all the isolates harbored the

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Salmonella invasion gene invA, which is also widely used to confirm Salmonella at the genus level (Favier et al., 2013; Tafida et al., 2013). The other virulence

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genes, pefA and spvC, were detected in five (19.23%) and four (15.38%) isolates, respectively, while the sefA gene was detected in just one isolate (3.84%) (Table 1). Many of virulence factors of Salmonella isolates are clustered within SPI1 and SPI-2. For example, the HilA protein regulates the transcription of the SPI1 genes (Boddicker et al., 2003). The invA gene is involved in the internalization of Salmonella in epithelial cells (Swamy et al., 1996). The chromosomal sefA

ACCEPTED MANUSCRIPT gene encoding structural subunits of fimbriae SEF14 (Amini et al., 2010; Crâciunaş et al., 2012), that contribute to Salmonella spp. virulence. On the other hand, the pefA and spvC genes are plasmid virulence genes of Salmonella spp., where pefA codifies Pef fimbriae involved in the intestinal epithelium adhesion and the spvC gene is the central effector gene of the operon

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spvRABCD (Salmonella plasmid virulence), which contributes to the systemic

3.3 Antimicrobial susceptibility testing

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phase of Salmonella infection (Swamy et al., 1996).

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All the 26 isolates (100%) were susceptible to amoxicillin/clavulanic acid, cefotaxime, cephalothin, tobramycin, imipenem, and ciprofloxacin. Furthermore,

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resistance to streptomycin and sulfonamide was observed in six isolates (23.07%), tetracycline in five isolates (19.23%), ampicillin, nalidixic acid,

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trimethoprim/sulfamethoxazole and trimethoprim in four isolates (15.38%), and

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chloramphenicol in three isolates (11.53%). Intermediate resistance was observed for gentamicin and streptomycin in two isolates (7.69%). While 16

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isolates (61.53%) were susceptible to all the antimicrobials evaluated, 10 isolates (38.46%) presented a phenotypic resistance profile, and multi-resistance was

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observed in seven isolates (26.92%). Resistance to streptomycin and sulfonamides was the most commonly observed, followed by tetracycline. Similar results were observed by SanchezMaldonado et al. (2017) that found 28.3, 29.1 and 26.8% of the resistance to streptomycin, sulfonamide and tetracycline, respectively in isolates from pork processing plants.

ACCEPTED MANUSCRIPT The great resistance to these antimicrobial agents is commonly associated with the use of antimicrobials in animal feed and/or at therapeutic or subtherapeutic levels to prevent some infections in cattle, poultry and swine production (Brown et al., 2016; Haubert et al., 2016; Zhang et al., 2015). The findings of our study are significant and highlight the need for more planning

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regarding the use of antimicrobials in animal production in Brazil, to avoid the

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appearance of multi-resistant Salmonella spp. isolates in foods and to protect

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animal and human health.

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3.4 Detection of antimicrobial resistance genes

Salmonella isolates with phenotypic resistance profile were evaluated for

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antimicrobial resistance genes (blaZ, blaTEM, aadA, aadB, aac(6’)-Ib, strA, strB, int1, sul1, sul2, sul3, dfrA, dfrD, dfrG, tetA, tetB, catA1, and floR,) and for qacEΔ1

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gene, which promotes resistance to QACs. Eight isolates presented antimicrobial

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resistance genes, and those detected were aadA and qacEΔ1; blaTEM, int1, sul1 and tetA; strA, strB and floR; sul2, tetB and catA1 in five, four, two and one isolate,

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respectively. All isolates with resistance profile for ampicillin (n=4) and tetracycline (n=5) harbored resistance genes for the respective class (β-lactams

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and tetracyclines). Of eight isolates resistant to the class of aminoglycosides, five showed resistance genes for this class. For the class of folate pathway inhibitors (n=7), five isolates harbored resistance genes. Of the three isolates resistant to chloramphenicol (phenicols), two harbored resistance genes evaluated in this study. The QAC resistance gene stood out, with just five of the 26 isolates harboring the qacEΔ1 gene. Table 1 shows the antimicrobial resistance phenotypes and genotypes of the 26 isolates evaluated in this study.

ACCEPTED MANUSCRIPT The most frequent resistance genes were aadA and qacEΔ1, followed by blaTEM, int1, sul1, and tetA genes. These resistance genes are frequently associated with class 1 integrons (Lopes et al., 2016). Class 1 integrons typically show the 5’ conserved segment (5’-CS) and 3’ conserved segment (3’-CS) flanking a central variable region, which may harbor gene cassettes, including

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aadA and blaTEM genes. The 5’-CS includes the integrase gene (int1), while the

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3’-CS consists of a qacEΔ1, which encodes a semi-functional derivate of the

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QACs resistance gene qacE and the sulfonamide resistance gene sul1 (Lopes et al., 2015; Michael & Schwarz, 2016; Sanchez-Maldonado et al., 2017). Integrons

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are commonly inserted into transposons, like the Tn21 transposon, and play an important role in the dissemination of antimicrobial resistance genes (Miriagou,

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Carattoli, & Fanning, 2006).

The tet genes which code efflux pumps have been identified in Salmonella

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isolates (tetA, tetB, tetC, tetD, and tetG), although tetA and tetB have been

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detected most frequently (Michael & Schwarz, 2016). The presence of these genes is a challenger problem due to they are generally encoded by mobile

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genetic elements such as conjugative plasmids and/or conjugative transposons, highlighting a possible reservoir and potential transfer of resistance genes to

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other bacteria.

3.5 Biofilm forming ability of Salmonella isolates All the Salmonella isolates produced biofilms on stainless steel surfaces at 10 and 22 ºC after 48 h of incubation, although the highest values were at 22 ºC, according to Figure 1. Through the statistical analysis, it was observed that there was a

ACCEPTED MANUSCRIPT significant difference (p < 0.05) between the isolates at the temperature of 10 ºC. The highest mean bacterial concentration that adhered to the coupon was for isolate S9 (6.52 log CFU.cm-2). Although it did not differ significantly from most other isolates, it differed from isolates S16 and S20 (5.30 and 5.54 log CFU.cm2,

respectively). The lowest mean was observed for S25 and S17 (5.18 log

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CFU.cm-2), which differed significantly from the other isolates.

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At 22 ºC, a significant difference (p < 0.05) was observed between the

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isolates. The highest mean was for isolate S9 (7.61 log CFU.cm-2), which differed from isolates S10, S13, S14, S16, S17, S20 and S23 (values between 6.30 and

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6.81 log CFU.cm-2). The lowest ability for biofilm formation was observed for isolates S18 and S25 (6.18 log CFU.cm-2), with a significant difference (p < 0.05)

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from the others.

The value of 5 log CFU.cm-2 was used as the threshold, according to

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Kroning et al. (2016) and Ronner & Wong (1993). In this study, the average of

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bacterial counts varied between 5.18 and 7.61 log CFU.cm -2 at 10 and 22 °C, respectively. These results indicate that the isolates evaluated in this study were

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able to adhere on stainless steel at both temperatures, resulting in the formation of biofilms. According to Steenackers et al., (2012), the extracellular matrix

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components of Salmonella spp. biofilms vary considerably with the environmental conditions. The incubation temperature of the coupons in the culture media, for example, influences the adhesion and biofilm formation of Salmonella isolates. A study with Salmonella Typhimurium from different sources of isolation showed bacterial counts of 6.06 log CFU.cm-2 on stainless surfaces at 20 °C (Gkana et al., 2016). In another study, Salmonella spp. isolates from meat products and chicken showed the highest bacterial adherence on stainless steel surfaces at 20

ACCEPTED MANUSCRIPT °C (Karaca et al., 2013) when compared with 37 °C and 5 °C. In our study, we also detected the highest bacterial concentration adhered to stainless steel at 22 °C when compared with 10 ºC.

3.6 MIC of BC

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Twenty-one isolates (80.77%) presented MIC 32 mg.L-1 and the other five

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isolates (19.23%) showed MIC 64 mg.L-1 for BC (Table 1).

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QACs such as BC are disinfectants frequently used for sanitization in food processing lines and surfaces in food industries (Bore et al., 2007). Salmonella

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strains with higher tolerance to disinfectants would be expected to also have a greater chance of surviving disinfection processes when biocide solutions

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become diluted below their effective concentrations (Karatzas et al., 2007) or if biocide tolerant strains are attached to surfaces in the form of biofilms (Chylkova

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et al., 2017). In the current study, all Salmonella spp. isolates proved to be

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tolerant to BC, with MIC values ranged from 32 to 64 mg.L -1. These biocide tolerant isolates also have the ability to form biofilm on stainless steel, which may

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contribute to their persistence in the food industry. The increased use of disinfectants has raised concerns about a possible in

selection

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role

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antimicrobial

resistant

bacteria.

The

molecular

characterization of QAC resistance is poorly elucidated; however, some QAC resistance genes, including qacEΔ1, have been identified on mobile genetic elements in Gram-negative microorganisms, encoding an antimicrobial resistance profile (Wu et al., 2015). The qacE gene is located in the 3’-CS of class 1 integrons, whereas qacEΔ1 is a defective version of qacE that has been associated with an increased MIC for BC. In this study, all Salmonella spp.

ACCEPTED MANUSCRIPT isolates were evaluated for the presence of qacEΔ1 gene, and it was possible to amplify this gene in just five isolates. This fact suggests that other mechanisms, such as multidrug efflux pumps, are involved in the tolerance phenotype to BC in the other 21 isolates.

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3.7 Antimicrobial activity of B. odorata extract

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In general, the B. odorata extract showed activity against Salmonella

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isolates evaluated by the agar disk diffusion method, with the exception of isolate S10. The inhibition zones varied between 8 and 14 mm, as shown in Table 3.

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Little is known about the antimicrobial activity of B. odorata extract towards foodborne bacteria (Maia et al., 2017), but the antimicrobial activity of a variety of

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fruit extracts against Salmonella spp. have been studied. The activity of Moroccan date fruit (Phoenix dactylifera L.) extracts against Salmonella Abony was

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evaluated by Bouhlali et al. (2016). The authors found inhibition zones induced

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by Moroccan date fruit extracts varying from 10.0 to 14.6 mm, similar to our results.

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The MIC of B. odorata extract ranged from 10 to 19 mg.mL-1 for most isolates, except for S16, S17, S19, S20 and S21, which were not inhibited using

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the highest tested concentration (MIC >19 mg.mL-1). The MBC was 19, 16, and 13 mg.mL-1 for 7.69% (n=2), 3.84% (n=1), and 3.84% (n=1) of the isolates, respectively, but the MBC was >19 mg.mL-1 for 84.61% of the isolates (n=22) (Table 3). The comparison of our results to those in the literature is difficult, because there are no standardized methodologies to evaluate the MIC of the extracts. In addition, different parts of the fruits and different solvent systems are used to

ACCEPTED MANUSCRIPT extract the bioactive compounds. Salmonella Enteritidis and Salmonella Kentucky isolates were inhibited by Nana variety pomegranate peel extracts (Punica granatum L.), and the MIC values ranged from 10.75 to 12.50 mg.mL -1 (Wafa et al., 2017). In turn, blueberry extracts (Vaccinium corymbosum L.) were

according to the cultivar analyzed (Shen et al., 2014).

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1,

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tested against S. Enteritidis and the MIC values ranged from 450 to 1200 mg.mL -

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3.7.1 Chemical characterization

GC-MS analysis identified 17 compounds in the B. odorata extract,

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representing 97.46% of the total composition. The major component present was 5-(Hydroxymethyl)-2-furfural (65.17%), followed by 2,3-Dihydro-3,5-dihydroxy-

MA

6-methyl-4H-pyran-4-one (pyranone) (8.49%) (Table 4). The major compound present in the B. odorata extract was 5-

D

(Hydroxymethyl)-2-furfural. This compound is formed by the Maillard reaction or

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during dehydration of reducing saccharides, for example fructose and glucose (Polovková and Šimko, 2017). Considering that high temperature was not used,

was

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the second hypothesis is more likely. The second most common compound found

2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-onen(pyranone)

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(8.49%). Bioactive properties of pyranones have been extensively studied, regarding antifungal activity (Schiller et al., 2010) as well as antioxidant activity (Li et al., 2018). The pyranone derivative exhibited antimicrobial properties against Gram-negative and Gram-positive bacteria, including S. Typhi and S. Paratyphi (Shiva Krishna et al., 2015). Due to the development of adverse effects and antimicrobial resistance to chemically synthesized drugs, particular interest in natural products, such as fruit

ACCEPTED MANUSCRIPT extract, has grown throughout the world. The obtained results indicate that B. odorata extract could be an effective means to inhibit the growth of multi-resistant Salmonella spp. isolates from food and food environments. Besides that, B. odorata extract could be used as an alternative to disinfectants such as BC, since it inhibited Salmonella spp. isolates even with tolerance profile (MIC 32 and 64

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mg.L-1).

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4 Conclusions

Salmonella spp. isolates evaluated in this study presented virulence as

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well as biofilm forming ability on stainless steel surfaces, which are widely used in food processing environments. Moreover, the isolates presented resistance

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and multi-resistance profiles, and considering that the antimicrobial resistance is a public health concern, new strategies should be developed to solve this

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problem, such as natural inhibitors. In this way, Butia odorata extract showed

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activity against Salmonella spp. isolates, and show potential to be used as a

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natural antimicrobial to control this important foodborne pathogen.

Acknowledgements

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The authors thank the Coordination for the Improvement of Higher Level Education Personnel (CAPES) and the National Council for Scientific and Technological Development (CNPq) for the scholarships of Louise Haubert, Maiara Lindemann Zehetmeyr, Isabela Schneid Kroning, Darla Volcan Maia, and Graciela Völz Lopes. We would like to thank the Laboratório de Enterobactérias, Fundação Instituto Oswaldo Cruz (FIOCRUZ) for serotyping Salmonella spp. isolates. Also, we thank the Laboratório de Medicina Veterinária Preventiva at the

ACCEPTED MANUSCRIPT Universidade Federal do Rio Grande do Sul, especially Professor Dra. Marisa

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Ribeiro de Itapema Cardoso for her collaboration with this study.

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ACCEPTED MANUSCRIPT Tables

Table 1 Characteristics of Salmonella spp. isolates from food and food environments in Southern Brazil Table 2 Oligonucleotides used in this study

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Table 3 Antimicrobial activity of Butia odorata extract against Salmonella isolates

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Table 4 List of compounds tentatively identified by GC-MS in methanol Butia

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odorata extract

ACCEPTED MANUSCRIPT Figure legends

Fig. 1 Biofilm formation (CFU.cm-2) of Salmonella isolates cultured on TSB at 10 and 22 °C for 48 h. Error bars indicate the standard deviation. Numbers 1 to 26 correspond to isolates S1 to S26, and number 27 to the strain S. Typhimurium

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ATCC® 14028.

ACCEPTED MANUSCRIPT Table 1 Characteristics of Salmonella spp. isolates from food and food environments in Southern Brazil Resistanc Virulenc Isolat

Yea Source

e

MIC Resistanc BCb

e Serovar

e

e

r

Phenotype genes

(mg.L Genotype

a

-1

PT

)

aadA, int1,

sausage

0

S1

STR-SULDerby

invA

RI

201

sul1, tetA,

32

TET

SC

Fresh

qacEΔ1

AMP-CHL-

Colonial S2

mixed

Typhimurium

pefA

0

201

pudding

0

Meat and

201

bone flour

0

Meat and bone flour

SUT-TRI

Susceptible

-

32

invA

Susceptible

-

32

Adelaide

invA

Susceptible

-

64

invA,

AMP-NALblaTEM

64

pefA

SUT-TRI

Muenster

invA

Susceptible

-

32

Muenster

invA

Susceptible

-

32

201 0

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Fresh

32

invA

Senftenberg

S5

blaTEM

STR-SUL-

Infantis

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S4

GENc-NAL-

D

White

MA

sausage

S3

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invA,

201

mixed

AC

homemad

S6

e

201

Typhimurium 0

seasoned sausage Meat and

201

bone flour

0

Meat and

201

bone flour

0

S7

S8

ACCEPTED MANUSCRIPT AMP-CHL-

blaTEM,

Pork

201

Schwarzengru

invA,

STR-SUL-

aadA, strA,

sausage

1

d

spvC

SUT-TET-

strB, sul2,

TRI

tetB, floR

Susceptible

-

64

-

32

S9

32

Mixed

201

sausage

1

S10

Rissen

invA

PT

invA,

Frozen 201 S11

food

Typhimurium

pefA,

Susceptible

spvC

ingredient

invA, 201

sausage

1

S12

Derby

pefA,

201

sausage

1

qacEΔ1

32

invA

Susceptible

-

32

invA

STRc

-

32

(O:6,7)

invA

Susceptible

-

32

Livingstone

invA

Susceptible

-

32

(O:6,7)

invA

Susceptible

-

32

Give 1

manipulato

PT E

Hand

D

sausage

invA

MA

201 calabrese

qacEΔ1

NAL

Derby

Fresh S14

32

NU

Poultry

sul1, tetA,

TET

spvC

S13

aadA, int1,

STR-SUL-

SC

Pork

RI

1

S. enterica

201

S15

r during

subsp.

CE

0

execution

diarizonae

S16

AC

of cuts

Carcass

201

53-1

1

Carcass

201

53-1

1

Carcass

201

58-1

1

S17

S18

ACCEPTED MANUSCRIPT aadA, int1, Carcass

201

43-1

0

S19

STR-SULDerby

invA

sul1, tetA,

32

TET qacEΔ1

Carcass

201

S20 58-1

Ohio

invA

STRc

Rissen

invA

Susceptible

Rissen

invA

-

32

-

32

-

32

1

sausage

201

with

1

S21

RI

cheese

sausage

201

with

1

Skinless

201

bacon

1

Typhimurium

CE

AC

invA

PT E

S23

D

MA

cheese

Beef

Susceptible

NU

S22

SC

Pork

S24

PT

Poultry

2012

blaTEM, AMP-CHL-

aadA, int1,

GENc-NAL-

strA, strB,

STR-SUL-

sul1, tetA,

SUT-TET-

catA1,

TRI

floR,

64

qacEΔ1 invA,

Typhimurium

pefA,

Susceptible

-

32

Susceptible

-

64

invA, sefA Susceptible

-

32

spvC

Mixed S25

2012

Give

2012

Derby

invA

sausage Pork S26

sausage with

ACCEPTED MANUSCRIPT Parmesan cheese aAMP:

ampicillin, AMC: amoxicillin/clavulanic acid, CTX: cefotaxime, CFL: cephalothin, GEN:

gentamicin, TOB: tobramycin, STR: streptomycin, TET: tetracycline, IMP: imipenem, CHL: chloramphenicol, NAL: nalidixic acid, CIP: ciprofloxacin, SUL: sulfonamide, SUT: trimethoprim/sulfamethoxazole, TRI: trimethoprim inhibitory concentration of benzalkonium chloride resistance

AC

CE

PT E

D

MA

NU

SC

RI

cIntermediate

PT

bMinimum

ACCEPTED MANUSCRIPT Table 2 Oligonucleotides used in this study Target

Amplicon Sequence (5’ – 3’)

Reference

genes

size (bp) Fw: GCGAGATTGTGAGTAAAAACACC

413

Crâciunaş et al.

invA

Rv: CTGCCCGGAGATATAATAATCG Fw: TTGTTACGGCTATTTTGACCA

521

(2012) Swamy et al. (1996)

sefA

Rv: CTGACTGCTACCTTGCTGATG Fw: GCAGCGGTTACTATTGCAGC

330

pefA

Rv: TGTGACAGGGACATTTAGCG Fw: TTCCATTATTGCACTGGGTG

spvC

Rv: AAGCCACTGCGAAAGATGCC Fw: CGGAAATACCATCTACAA ATA

blaZ

Rv: CCCAAACCCATACTTACTCTG Fw: ACTTCAACACCTGCTGCTTTC

blaTEM

Rv: TGACCACTTTTATCAGCAACC Fw: ATGAGTATTCAACATTTCCG

aadA

Rv: TTAATCAGTGAGGCACCTAT Fw: GTGGATGGCGGCCTGAAGCC

aadB

Rv: ATTGCCCAGTCGGCAGCG Fw: GGGCGCGTCATGGAGGAGTT

RI

PT

hilA

SC

497

Woodward and

Kirwan (1996) Haneda et al. (2001)

Swamy et al. (1996)

172

Martineau et al.

851

(2000) Grimm et al., (2004)

526

Sandvang and

328

Aarestrup (2000) Sandvang and

aac(6’)-Ib

Rv: TATCGCGACCTGAAAGCGGC Fw: TTGCGATGCTCTATGAGTGGCTA

482

Aarestrup (2000) Park et al. (2006)

strA

Rv: CTCGAATGCCTGGCGTGTTT Fw: TGACTGGTTGCCTGTCAGAGG

645

Kehrenberg and

510

Schwarz (2001) Kikuvi et al. (2007)

871

Sandvang et al.

int1

MA

D

PT E

CE

Rv: CCAGTTGTCTTCGGCGTTAGCA Fw: ATCGTCAAGGGATTGAAACC

AC

strB

NU

669

Rv: GGATCGTAGAACATATTGGC Fw: CGGAATGGCCGAGCAGATC

Rv: CAAGGTTCTGGACCAGTTGCG Fw: ATGGTGACGGTGTTCGGCATTCTG

sul1

(2002) 840

Grape et al. (2003)

Rv: CTAGGCATGATCTAACCCTCGGTT

sul2

Fw: GCGCTCAAGGCAGATGGCATT

293

Kerrn et al. (2002)

sul3

Rv: GCGTTTGATACCGGCACCCGT Fw: GGGAGCCGCTTCCAGTAAT

500

Chuanchuen et al.

Rv: TCCGTGACACTGCAATCATTA Rv:CTAGGCATGATCTAACCCTCGGTCT

(2008)

ACCEPTED MANUSCRIPT dfrA

Fw: CCTTGGCACTTACCAAATG

350

Perreten et al.

dfrD

Rv: CTGAAGATTCGACTTCCC Fw: GGGCAGATTTGTTTAGTAAGG

785

(2005) Bertsch et al.

dfrG

Rv: GTATCTCCTTCGAATTCATGATG Fw: TTTCTTTGATTGCTGCGATG

422

(2013a) Bertsch et al.

tetA

Rv: CCCTTTTTGGGCAAATACCT Fw: GTAATTCTGAGCACTGT

953

(2013b) Frech and Schwarz

tetB

Rv: CCTGGACAACATTGCTT Fw: ACGTTACTCGATGCCAT

catA1

Rv: AGCACTTGTCTCCTGTT Fw: GGCATTTCAGTCAGTTG

floR

Rv: CATTAAGCATTCTGCCG Fw: AGGGTTGATTCGTCATGACCA

AC

CE

PT E

D

MA

Rv: CAAGCTTTTGCCCATGAAGC Fw: forward primer; Rv: reverse primer

PT

RI

1169

SC

551

NU

qacEΔ1

Rv: CGGTTAGACGACTGGCGACT Fw: ATCGCAATAGTTGGCGAAGT

(2000) Frech and Schwarz

1291

225

(2000) Kikuvi et al. (2007)

Kadlec et al. (2007)

Paulsen et al. (1993)

ACCEPTED MANUSCRIPT Table 3 Antimicrobial activity of Butia odorata extract against Salmonella isolates Inhibition halo

MICa BEb (mg.mL-

MBCc BE (mg.mL-

(mm)

1)

1)

S1

10

/16

>19

S2

12

13

>19

S3

9

16

S4

12

10

S5

12

13

S6

8

S7

11

S8

9

S9

12

19 >19 >19

13

>19

13

16

16

>19

16

>19

NId

19

>19

14

19

>19

11

13

13

S13

10

13

>19

S14

14

13

>19

S15

11

10

19

S16

11

>19

>19

MA D

PT E

AC

S12

CE

S10 S11

NU

SC

RI

PT

Isolate

ACCEPTED MANUSCRIPT 12

>19

>19

S18

11

19

>19

S19

9

>19

>19

S20

10

>19

>19

S21

10

>19

>19

S22

12

19

S23

10

13

S24

13

16

S25

10

S26

9

ST

9

odorata extract

cMinimum

inhibited

RI

SC

>19 >19

19

>19

19

>19

>19

>19

NU MA D

bactericidal concentration

AC

dNot

inhibitory concentration

CE

bButia

PT E

aMinimum

PT

S17

>19

ACCEPTED MANUSCRIPT

PT

Table 4 List of compounds tentatively identified by GC-MS in methanol Butia

Compound

retention

Percentage (%)

SC

Time

RI

odorata extract

Furfural

8.923

2,4-Dihydroxy-2,5-dimethyl-3(2H)-furanone

1.37

12.124

2,2-Dimethylpropyl furan-2-carboxylate

1.01

12.442

Cyclopentane, 1-acetyl-1,2-epoxy-

2.32

14.080

Levulinic acid

0.95

Pyranone

8.49

5-(Hydroxymethyl)-2-furfural

65.17

6-Acetyl-β-D-mannose

0.59

3,4-Anhydro-d-galactosan

0.69

26.633

(3E)-5-Hydroxy-2-methyl-3-hexenoic acid

1.60

32.610

1,6-Anhydro-β-D-glucopyranose

4.93

55.913

Palmitic acid

0.37

58.094

Pentadecanoic acid

3.11

63.534

9,12-Octadecadienoic acid, methyl ester

0.52

65.187

Salicylidene aniline

2.20

D

MA

NU

5.129

PT E

14.371

19.585

AC

22.068

CE

19.011

2.21

ACCEPTED MANUSCRIPT 65.602

9,12-Octadecadienoic acid (Z,Z)-

0.90

66.510

9,12-Octadecadienoic acid (Z,Z)-, ethyl

1.03

AC

CE

PT E

D

MA

NU

SC

RI

PT

ester

ACCEPTED MANUSCRIPT Anderson Sant’Ana, Editor-in-Chief Food Research International

Highlights to accompany the manuscript submitted to Food Research

PT

International for application as an original research paper. The manuscript is

RI

entitled:

SC

“Tolerance to benzalkonium chloride and antimicrobial activity of Butia

NU

odorata Barb. Rodr. extract in Salmonella spp. isolates with biofilm

MA

forming ability”

PT E

D

Highlights

Virulence genes in Salmonella spp. from food and food environment



Salmonella spp. with biofilm forming ability on stainless steel surface



Multidrug resistant Salmonella harboring several resistance genes



Tolerance to benzalkonium chloride in foodborne Salmonella isolates



Butia odorata extract presents antimicrobial activity against Salmonella

AC

CE



isolates

Figure 1