Salmonella isolated from chicken carcasses from a slaughterhouse in the state of Mato Grosso, Brazil: antibiotic resistance profile, serotyping, and characterization by repetitive sequence-based PCR system

Salmonella isolated from chicken carcasses from a slaughterhouse in the state of Mato Grosso, Brazil: antibiotic resistance profile, serotyping, and characterization by repetitive sequence-based PCR system Adelino da Cunha-Neto,∗,† Larrayane Albuˆes Carvalho,† Ricardo C´esar Tavares Carvalho,† D´ alia dos aquio de Souza Figueiredo,† Prazeres Rodrigues,‡ Sergio Borges Mano,∗ Eduardo Eust´ and Carlos Adam Conte-Junior∗,1 ∗

†

Department of Food Technology, Faculty of Veterinary, Universidade Federal Fluminense, Niter´ oi, Brazil; Department of Food and Nutrition, Faculty of Nutrition, Universidade Federal do Mato Grosso, Cuiab´ a, Brazil; and ‡ National Reference Laboratory Diagnosis of Enteric Bacteria, Oswaldo Cruz Institute, Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro, Brazil The predominant serovars were Salmonella Infantis (35.4%, 11/31), and S. Abony (25.8%, 8/31), followed by serovars S. Agona (12.9%, 4/31), S. Schwarzengrund (9.7%, 3/31), S. Anatum and Salmonella enterica O:4,5 (6.5%, 2/31), and only one Salmonella enterica O:6,7 strain (3.2%, 1/31). All isolates were resistant to one to 5 classes of antibiotics in decreasing order: folate pathway inhibitors, β -lactams (cephalosporins, penicillin, monobactams), tetracyclines, chloramphenicol, and gentamicin. However, strains sensitive to florfenicol, streptomycin, nalidixic acid, ciprofloxacin, enrofloxacin, and nitrofurantoin were also found in this study. Genotyping revealed 98 to 99% homology between 3 Salmonella strains, which displayed high phenotypic resistance similarity to β -lactams and folate pathway inhibitors. Detection of MDR non-typhoid Salmonella in chicken slaughterhouses with quality assurance systems such as Hazard Analysis and Critical Points and Implemented Good Manufacturing Practices is a concern, reinforcing the need for constant monitoring of these pathogens, with the purpose of safeguarding the safety of their products.

ABSTRACT Salmonella is one of the major causative agents of foodborne infections. Salmonellosis becomes more dangerous when strains resistant to several antibiotics are found in food, especially in chicken, one of the primary transmission vehicles of this pathogen for humans. The present study aimed to estimate the occurrence of Salmonella in chicken carcasses from the state of Mato Grosso, Brazil, as well as determine the antibiotic resistance profile and genotypic characteristic of multi-drug resistant (MDR) isolates. During a 15-month period, from 01/2014 to 05/2015, 850 samples of chilled fresh chicken carcasses were sampled from a slaughterhouse and submitted to Salmonella determinations according to the ISO6579/2002 method, serotyping and multiplex PCR. The disc diffusion test was applied for 17 antibiotics, according to CLSI (2014). Five isolates were genotyped by repetitive sequence-based PCR using the semiR automated DiversiLab (bioM´erieux ) system. The occurrence of Salmonella in chicken carcasses was of 3.7% (31/850), with only 4 strains (12.9%) presenting as MDR, and 6 strains (19.35%) displaying ESBL.

Key words: multidrug resistance, chicken meat, genotyping, PCR multiplex, pathogen 2018 Poultry Science 0:1–9 http://dx.doi.org/10.3382/ps/pex406

INTRODUCTION

6 subspecies: enterica, salamae, arizonae, diarizonae, houtenae, and indica. Enteric subspecies serotypes cause 99% of infections in animals and humans (Grimont and Weill, 2007; Issenhuth-Jeanjean et al., 2014). In humans, enteric infection symptoms include nausea, vomiting, and non-bloody diarrhea. Symptoms may include fever, cold, abdominal pain, myalgia, joint, and headaches, and can progress to bacteremia and endocarditis (S´ anchez-Vargas et al., 2011; Thiennimitr et al., 2012). Prevalence studies and epidemiological investigations have identified chicken products and raw or undercooked eggs as potential transmission vehicles

Salmonella is a gram-negative bacillus belonging to the Enterobacteriaceae family, classified into approximately 2,600 serotypes by the Kauffmann-White scheme (Grimont and Weill, 2007; Issenhuth-Jeanjean et al., 2014). This genus comprises Salmonella bongori and Salmonella enterica, the latter divided into

 C 2018 Poultry Science Association Inc. Received August 1, 2017. Accepted November 24, 2017. 1 Corresponding author: [email protected]

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for Salmonella in humans (Fallah et al., 2013; Cabral et al., 2014). Serotypes that frequently occur in different countries and are commonly isolated from chicken carcasses, other cuts, and by-products include Salmonella Enteritidis, S. Typhimurium, S. Newport, S. Infantis, S. Montevideo, S. Heidelberg, S. Senftenberg, and S. Schwarzengrund (Foley et al., 2011). The most common serotypes are known to cause enteritis and are causative agents of foodborne infections (Antunes et al., 2016). These serotypes detected in chicken carcass studies developed in Asia and Africa. In these studies, the presence of serotypes Salmonella Indiana and S. Derby in China were also reported (Li et al., 2013; Lai et al., 2014; Bai et al., 2015; Hu et al., 2017); in Korea, S. Bellevue and S. London are among the most frequently isolated serotypes (Bae et al., 2013; Chon et al., 2015). In Senegal and Morocco, in addition to the most frequent serotypes, S. Kentucky, S. Muenster, S. Bancaster, and S. Hadar were among the most isolated serotypes (Bada-Alambedji et al., 2006; Abdellah et al., 2009). Commonly occurring serotypes in broiler chickens in the previously mentioned regions of the world have been found in Brazil. Other serotypes such as Salmonella Agona, S. Mbandaka, S. Orion, S. Albany, S. Tennessee, S. Cerro, S. Livingstone, S. Muenster, and S. Corvallis presented high prevalence in chicken carcasses in this country (Linhares et al., 2005; Ribeiro et al., 2007; Boni et al., 2011; Medeiros et al. 2011; Moraes et al., 2014; Cardoso et al., 2015; Minharro et al., 2015; Fitch et al., 2016). The emergence of multidrug-resistant (MDR) Salmonella is a worldwide concern (Abdellah et al., 2009; Alvarez-Fern´ andez et al., 2012; Chon et al., 2015), since the occurrence of MDR Salmonella in food is a risk condition, representing increasing foodborne disease severity, leading to increased hospitalization rates and the possibility of death (Crump et al., 2015). This phenomenon is a consequence of the extensive use of antibiotics in the treatment of humans and animals. Strains with this profile represent a worldwide problem for both the hospital and the public health sectors (Bada-Alambedji et al., 2006; Van Boeckel et al., 2015). The increase in the prevalence of Salmonella strains resistant to multiple antibiotics in products of animal origin, particularly poultry, has been observed (Bai et al., 2015; Chon et al., 2015; Dan et al., 2015; Ziech et al., 2016). Studies evaluating this aspect in Salmonella strains are scarce. Therefore, investigations that verify the prevalence of MDR strains in poultry are important to produce information that subsidizes control programs regarding the indiscriminate use of antibiotics in animal production. Molecular techniques, such as pulsed-field electrophoresis (PFGE), multiple-locus variable number tandem repeats analysis (MLVA), multilocus sequence typing (MLST) and, recently, whole genome sequencing (WGS), have been used in Brazil for the identification, typing, and sub-typing of Salmonella strains

(Almeida et al., 2013; Medeiros et al., 2015; Almeida R et al., 2016; Panzenhagen et al., 2017). The Diversilab system is a repetitive extragenic palindromicpolymerase chain reaction (PCR) that targets the highly conserved, interspersed, repetitive elements found in several sites within the bacterial genome (Tang and Stratton, 2006). The advantage of this technique is that it is faster, efficient, and presents higher reproducibility (Anderson et al., 2010). Through this method, it is also possible to enhance the discrimination power between Salmonella strains, to verify their genetic diversity (Anderson et al., 2010; Luo et al., 2015). In addition, there is also a lack of scientific information regarding the presence, antibacterial susceptibility behavior, and genotypic profile of Salmonella in chicken meat, taking into account that Brazil is the leading poultry exporter worldwide. In this context, the present study aimed to isolate Salmonella from chicken carcasses processed at a slaughterhouse in the state of Mato Grosso, Brazil, to determine the antibiotic resistance profile, serotype, and genotype characteristics of MDR isolates.

MATERIAL AND METHODS Sampling A total of 850 chilled chicken carcasses were sampled from a slaughterhouse that slaughters 90,000 birds daily. Twelve and 15 samples were taken alternately per wk for 63 wk, from 01/2014 to 05/2015. The establishment is a medium-sized enterprise located in the state of Mato Grosso, Brazil. This state comprises 3 biomes, the Pantanal, Cerrado, and the Amazon rainforest, presenting a hot, humid and rainy tropical climate, in which 59,733,435 chickens were slaughtered in 2014 and 59,681,284 in 2015 (IBGE, 2017).

Isolation and Identification of Salmonella Species The isolation method was based on the protocol recommended by the International Organization for Standardization (2002). Briefly, 25 g of each sample were inoculated in Buffered Peptone Water R (Code-M614, Himedia , Mumbai, India), incubated at ◦ 37 C/24 h and then enriched in Rappaport-Vassiliadis R , United Kingdom), inBroth (Code-M614, Oxoid ◦ cubated at 42 C/24 h in Muller Kauffmann NovoR , biocin Tetrathionate Broth (Code-M14961, Himedia ◦ Mumbai, India), at 37 C/24 h, with subsequent plating on Xylose Lysine Deoxycholate Agar (Code-M031, R Himedia , Mumbai, India) and Rambach Agar (Code107,500, Merck, Darmstadt, Germany), incubated at 37◦ C/24 h. Typical colonies were selected, purified on R , Mumbai, India) Nutrient Agar (Code-M001, Himedia and subsequently inoculated on API 20E (Code-Ref.20 R 100/20 160, BioM´erieux , Lyon, France). The isolates

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SALMONELLA MDR FROM A POULTRY SLAUGHTERHOUSE

with a typical Salmonella reaction were subjected to serum agglutination by anti-salmonella O polyvalent serum.

DNA Extraction Strains identified as Salmonella were inoculated into 10 mL BHI Broth-Brain Heart Infusion (Code-M211, R Himedia , Mumbai, India), and incubated at 35◦ C for 24 h. Subsequently, a 1.5 mL aliquot was centrifuged at 14,000 × g for 5 min; the pellets were dissolved in 500 μL Milli-Q water and heated at 100◦ C for 10 min on a heating plate (BioGPRO, Brazil), and subsequently cooled at 4◦ C for 10 min. After lysate centrifugation at 14,000 × g for 5 min, 200 μL of the supernatant were separated, stored in a freezer and subsequently subjected to multiplex PCR.

Multiplex-PCR The PCR was performed in a total volume of 25 μL R containing 1 U Taq Polymerase (Invitrogen ), 1× Taq buffer (5 mM KCl Tris-HCl, pH 8.5) 1.5 mM MgCl 2, R ), 0.9 MM primer Inv-A, 0.1 mM dNTPs (Promega R and 0.4 μM of IE-1 and Flic-C primers (Invitrogen ). Conditions were based on the study performed by Pai˜ ao et al. (2013). The m-PCR assay was performed with an initial denaturation for 5 min at 95◦ C, followed by 30 cycles for 1 min at 95◦ C, 1 min at 58◦ C, and 30 s at 75◦ C, with a final extension step at 72◦ C for 7 min. The PCR products were analyzed by electrophoresis on 1.5% agarose gels with TBE buffer (45 mmol L−1 Tris pH 8.3, 45 mmol L−1 borate, and 2 mmol L−1 EDTA) as the running buffer. The gels were then stained with Gel R ) and photodocumented (MiniBis-Pro Red (Invitrogen R DNT, Bio-Imaging Systems ).

Salmonella Serotyping Complete antigen characterization and serotype identification were carried out at the National Reference Laboratory for the Diagnosis of Enteric Bacteria, at the Oswaldo Cruz Institute, Oswaldo Cruz Foundation (FIOCRUZ, Rio de Janeiro, Brazil) by the rapid slide agglutination test using somatic and flagellar antisera produced by the laboratory (Voss-Rech et al., 2015).

Antibiotic Susceptibility Test The isolates were subjected to antibiotic susceptibility tests by the disk diffusion method employing 17 antimicrobials: ampicillin 10 μg, aztreonam 30 μg, cephalothin 30 μg, cefoxitin 30 μg, ceftiofur 30 μg, chloramphenicol 30 μg, florfenicol 30 μg, streptomycin 300 μg, gentamicin 10 μg, nalidixic acid 30 μg, ciprofloxacin 5 μg, enrofloxacin 5 μg, tetracycline 300 μg, sulfamethoxazole-trimethoprim 25 μg, sulfonamide 300 μg, trimethoprim 5 μg, and nitrofurantoin

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R , S˜ ao Paulo, Brazil). The disks were 300 μg (Cecon distributed equidistantly in plates, which were then incubated at 35◦ C for 16 to 20 h (CLSI, 2014). Inhibition halos were measured, and the results were compared to standards contained in the Clinical and Laboratory Standards Institute (Patel et al., 2016), and classified as sensitive, intermediate or resistant. Screening of strains suggestive of extended-spectrum β -lactamase (ESBL) producers were also carried out, using the β -lactams breakpoint criterion for Enterobacteriaceae, that detects all resistance mechanisms, including ESBL and plasmid-mediated AmpC. According to Cavalieri et al. (2005), strains that present halos ≤22 mm for ceftazidime 30 μg, and ≤27 mm for aztreonam 30 μg are potential β -lactamase-producing strains suggestive of ESBL.

MDR Isolate Genotyping Genotyping of 5 MDR isolates was performed by repetitive sequence-based PCR using the semiR automated DiversiLab (bioM´erieux) system. The R bacterial DNA was amplified with the DiversiLab Salmonella kit. The amplified fragments were separated, and fluorescence intensities were detected with a microfluidic chip on a 2100 Agilent bioanalyzer (Agilent Technologies, Santa Clara, CA). The results were R illustrated with the web-based DiversiLab software, version v.r.3.3.40. The Pearson correlation coefficient and the unweighted pair group method with arithmetic means were used to determine the genetic similarity of the tested samples. A similarity cut-off of 95% was considered indicative of clonal relatedness, as described previously.

RESULTS AND DISCUSSION The prevalence of Salmonella in chicken carcasses produced in the state of Mato Grosso, Brazil was of 3.6% (31/850). Seven serotypes were identified among the 31 Salmonella samples. The complete antigen characterization of the 31 strains revealed that Salmonella Infantis was the most frequently isolated (35.4%, 11/31), with the second most frequent being S. Abony (25.8%, 8/31), followed by S. Agona (12.9%, 4/31), S. Schwarzengrund (9.7%, 3/31), S. Anatum and Salmonella enterica subspecies enterica O:4,5 (6.5%, 2/31), with the least frequent detection of Salmonella enterica subspecies enterica O:6,7 (3.2%, 1/31). The multiplex PCR identified the 31 isolates as Salmonella and did not characterize any strain as S. Enteritidis or S. Typhimurium. Comparing the results obtained in this slaughterhouse study with other Salmonella investigations performed in Brazil in chicken carcasses by the conventional isolation method and PCR, the results described herein were slightly lower when compared to those reported by Carvalho and Cortez (2005), that observed

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20%, Linhares et al. (2005), that detected 19.8%, Minharro et al. (2015), that verified the presence of 18.33%, while prevalences of 14.6% and 11.28%, were observed by Cardoso et al. (2015) and Boni et al. (2011), respectively. They were, however, similar to the prevalence observed by Moraes et al. (2014), of 4.7%, Panzenhagen et al. (2016), of 6.67%, and Matias et al. (2010), of 8.3%. A high variation in contamination levels in different chicken slaughter systems is observed in Brazil, as well as in developing countries as in Transylvania, 4.17% (Dan et al., 2015), Thailand, 7.3% (Chotinun et al., 2014), and North Korea, 9.2% (Chon et al., 2015). In southwest China, higher frequencies (21.7%) are reported in broiler chickens (Li et al., 2013). It is known that worker training and the implementation of programs such as Good Manufacturing Practices (GMP) and Hazard Analysis and Critical Control Points (HACCP) influence contamination levels during the slaughter process. The implementation or not of these programs may be a partial reason for variations in contamination levels of different chicken slaughter systems in Brazil and developing countries (Matias et al., 2010; Panzenhagen et al., 2016). The visited slaughterhouse in the present study had implemented Good Manufacturing Practices (GMP) and Hazard Analysis and Critical Control Points (HACCP), which could explain the low prevalence described herein. The prevalence of Salmonella cross-contamination in chicken carcasses may be related to activities related to the production of these animals, including the breeding, management, transport and processing of poultry and their by-products. This hypothesis has been proven by Chotinun et al. (2014) in Thailand, who detected Salmonella contamination in the environment (water and slaughterhouse floor), equipment, utensils, and carcasses, with prevalence ranging from 2.4 to 29.3%, with the only Salmonella-free area being the manipulator’s hands. These data demonstrate the difficulty of controlling Salmonella in slaughterhouses, especially at critical control points, since contamination can be caused by several slaughter operations, such as scalding, plucking and evisceration (Von R¨ uckert et al., 2009). Another critical point of contamination can originate from feed of animal origin, such as bone meal and feathers, fish, and offal. This has been a concern for the Brazilian Ministry of Agriculture, Livestock and Food Supply (MAPA), which has established constant monitoring of contamination levels by this pathogen in poultry slaughterhouses in Brazil, in the form of a program aimed at the reduction of pathogens—microbiological monitoring and control of Salmonella in chicken and turkey carcasses, also known as IN 70 (Normative Instruction) (BRASIL, 2003). According to the IN 70 program, the findings of this study are within the tolerable limits of acceptance, of a maximum of 12 positive samples (c = 12) in 51 samples, corresponding to the number of plots sampled in a year (one cycle) (BRASIL, 2003).

Serological characterization of the isolated strains revealed S. Infantis (35.4%), S. Abony (25.8%), S. Agona (12.9%), S. Anatum (6.5%), S. Schwarzengrund (9.7%), Salmonella (O:4,5) (6.5%), and Salmonella (O:6,7) (3.2%) in chicken carcasses from Mato Grosso. However, serotypes Salmonella Enteritidis, S. Typhimurium, S. Newport, S. Montevideo, S. Heidelberg, and S. Senftenberg, frequently found in broilers, were not detected, and only the presence of serotypes S. Infantis and S. Schwarzengrund as representatives of serotypes commonly detected in chicken in Brazil and worldwide were observed. The serotypes detected in this study were found in a comprehensive study of refrigerated chicken carcasses carried out in 15 Brazilian states, in which serotypes S. Infantis (7.6%), S. Agona (3.6%) and S. Schwarzengrund (1.2%) were detected (Medeiros et al., 2011). In regional studies, other researchers have also observed a prevalence of 7.70% for S. Agona and S. Infantis; and 3.84% for S. Anatum and S. Schwarzengrund among those detected in chicken carcasses and by-products in Tocantins (Minharro et al., 2015). The following prevalences were observed in chicken carcasses slaughtered in S˜ao Paulo between 2000 and 2010: 33.3% for Salmonella Infantis, 20.8% for S. Agona, and 6.7% for Salmonella enterica subspecies enterica O:4,5 (Cardoso et al., 2015). In the states of Mato Grosso do Sul, Paran´ a, and Santa Catarina, on farms that supply chickens to regional slaughterhouses, a prevalence of 14.63% of serotype S. Infantis was observed in the environment and chickens, making it the second most frequent. However, the prevalence of S. Schwarzengrund was of 4.87%, S. Anatum, of 2.44% and Salmonella enterica subspecies enterica O:4,5 and S. Agona of 1.22% (Voss-Rech et al., 2015). The low prevalence of serotypes S. Anatum and Salmonella enterica subspecies enterica O:4,5 (6.5%, 2/31) and Salmonella enterica subspecies enterica O:6,7 (3,2%, 1/31), found in the present study are similar to previously mentioned prevalences. Serotypes rarely reported in chicken carcasses in Brazil, like S. Abony and Salmonella enterica subspecies enterica O:6,7 cited only in the 1990s by Hofer et al. (1997), were detected. Recent citations of the S. Abony serotype in chicken carcasses have been carried out in countries such as Albania and China (Beli et al., 2001; Yang et al., 2014). According to the National Agency of Sanitary Surveillance (ANVISA), serotypes Salmonella Enteritidis, S. Infantis, S. Typhimurium, S. Heidelberg, and S. Mbandaka are the most prevalent in refrigerated chicken carcasses in Brazil (BRASIL, 2008). Regarding the antibiotic susceptibility evaluation, all serotypes were resistant to antibiotics belonging to one or more classes, and among the 31 Salmonella isolates, 100% showed resistance to sulfonamide, 87.5% were resistant to trimethoprim, and 75% showed resistance to trimethoprim/sulfamethoxazole. All strains (100%) were sensitive to florfenicol, streptomycin, nalidixic acid, ciprofloxacin, enrofloxacin, and nitrofurantoin

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Figure 1. Antimicrobial resistance profile of Salmonella spp. isolated from chilled chicken carcasses. AMP, ampicillin; ATM, aztreonam; CFL, cephalothin; CFO, cefoxitin; CTF, ceftiofur; CLO, chloramphenicol; FLF, florfenicol; ET, streptomycin; GEN, gentamicin; NAL, nalidixic acid; CIP, ciprofloxacin; ENO, enrofloxacin; TET, tetracycline; SUT, trimethoprim+sulfamethoxazole; SUL, sulfonamide; TRI, trimethoprim; NIT, nitrofurantoin.

(Figure 1). The resistance prevalence of β -lactams ranged from 6.25% to ceftiofur to 25% to ampicillin and cephalothin. One hundred percent of the strains were resistant to antibiotics belonging to the folate pathway inhibitor class, sulfonamides, and trimethoprim, or associated trimethoprim/sulfamethoxazole. Resistance to older-generation antibiotics, such as ampicillin, cephalothin, and trimethoprim/sulfamethoxazole, has increased in recent years (Antunes et al., 2016). Combinations of trimethoprim/sulfonamides have been used in veterinary practice due to low cost and their broad spectrum and clinically effective characteristics (Cosby et al., 2015). Studies on Salmonella from chicken in 15 different regions in Brazil indicate strains resistant to ampicillin (38%), ceftiofur (28%), aztreonam (19.2%), cefoxitin (13.2%), and cephalothin (12%), similar to the prevalence detected in the present study. However, strains resistant to streptomycin (78%), florfenicol (62%), nalidixic acid (40%), and enrofloxacin (19.2%) were also observed (Medeiros et al., 2011). Another comprehensive study conducted in Brazil with 1939 Salmonella isolates from 5 Brazilian regions identified strains resistant to tetracycline (20.9%), ampicillin (10%), trimethoprim/sulfamethoxazole (4.9%), and chloramphenicol (4.7%), as well as enrofloxacin (0.6%) (Fitch et al., 2016). In addition, Salmonella isolated from farms in the states of Santa Catarina, Paran´ a, and Mato Grosso do Sul expressed resistance to tetracycline (52.44%), streptomycin (24.39%), trimethoprim/sulfamethoxazole (17.07%), ceftiofur (12.19%), and gentamicin (6.09%) (Voss-Rech et al., 2015). The behavior of the 31 Salmonella strains found herein was represented by resistance to 11 different antibiotics with 11 sets of behavior patterns (Table 1). These strains were resistant to one to 5 classes of an-

tibiotics in decreasing order: folate pathway inhibitors, β -lactams (cephalosporins, penicillin, monobactams), tetracyclines, chloramphenicol, and gentamicin. Strains resistant to 3 or more classes of antibiotics were classified as MDR, a characteristic expressed by 4 of 31 strains (12.9%). One S. Abony serotype was resistant to folate, chloramphenicol, and tetracycline inhibitors, while 2 Salmonella enterica O:4,5 serotypes were resistant to antifolates, β -lactams, gentamicin and tetracycline and Salmonella enterica O:6,7 was resistant to antifolates, β -lactams, and tetracycline (Table 1). Strains were resistant to antibiotics belonging to the β -lactams classes, namely first-, second-, and third- generation cephalosporin, penicillin, and monobactam, with 75% resistance being most frequent among MDR strains, in addition to 100% resistance to the folate pathway inhibitor class. Most serotypes were resistant to first- and second-generation cephalosporins, cephalothin, and cefoxitin, although the S. Abony and Salmonella (O:4,5) strains were resistant to ceftiofur (third-generation cephalosporin). The emergence of Salmonella resistant to gentamicin and third-generation cephalosporin, extended spectrum antibiotics, is a concern for human health, since this increases the severity, morbidity, and mortality of the diseases caused by these strains (Antunes et al., 2016). In this study, the prevalence of Salmonella MDR strains was of 12.9%, in agreement with the 11.5% detected by Minharro et al. (2015) in Tocantins. However, it is lower than the prevalence of 33.3% verified by Ribeiro et al. (2007) in Rio Grande do Sul, and 53.2% verified in 15 cities in several states of Brazil (Medeiros et al., 2011). Furthermore, the prevalence of Salmonella MDR found in studies conducted on chicken carcasses in Brazil is lower than those found in China, that ranged from 36.1 to 99.1% (Lai et al., 2014; Lu

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2 1 1

11 10

Salmonella (O:6,7)

4 1 2

Salmonella (O:4,5)

S. Infantis

8 1 2 1 1

S. Shwarzengrund

S. Anatum

Antibiotic resistance profile SUL, TRI SUL, SUT, TRI AMP, CFL, CFO, SUL CLO, SUL, SUT, TRI, TET AMP, SUL, SUT, TRI, TET AMP, ATM, CFL, CFO, SUL, TRI, AMP, ATM, CFL, CFO, SUT, SUL CFL, CFO, SUL, SUT, TET, TRI, AMP, ATM, CFL, CFO, CTF, SUL, TRI, AMP, ATM, CFL, CFO, SUL, SUT, TRI AMP, ATM, CFL, CTF, GEN, SUL, SUT, TRI,

S. Agona

Salmonella serovar number from resistant strains

S. Abony

Table 1. Cluster of antibiotic profile of resistance in each Salmonella serovar, from 31 strains isolated in a poultry slaughterhouse in Mato Grosso, Brazil.

3 1 2

2

1

1 1 1

1 1

1 1 1

1 AMP, ampicillin; ATM, aztreonam; CFL, cephalothin; CFO, cefoxitin; CTF, ceftiofur (β -lactams); CLO, chloramphenicol; GEN, gentamicin; TET, tetracycline; TRI, trimethoprim; SUL, sulfonamide; and SUT, trimethoprim+sulfamethoxazole (folate pathway inhibitors).

et al., 2014; Hu et al., 2017; Li et al., 2017), and in Korea, that ranged from 87.2 to 100% (Kim et al., 2012; Chon et al., 2015). A 100% prevalence of strains of Salmonella MDR in chicken meat was observed in ´ Spain (Alvarez-Fern´ andez et al., 2012) and Iran (Fallah et al., 2013). The low prevalence of Salmonella MDR strains in Brazil can be attributed to regulations, control programs, surveillance, and monitoring of the use and residues of veterinary drugs in products of animal origin (Gouvˆea et al., 2015), regulated by the Ministry of Agriculture, Livestock and Food Supply, and Ministry of Health, based on regulations published between 1986 and 2013 (Gouvˆea et al., 2015). Probably, the use of these antibiotics in the chickens in frequent doses as zootechnical strategies has created a selective pressure that allowed for the growth of these MDR isolates. Among the 11 different behavior sets pertinent to antibiotic resistance, 6 of 31 Salmonella strains (19.35%) were classified as potential ESBL producers, using the breakpoint criterion described by Cavalieri et al. (2005). A common feature presented by the 6 strains was a resistance halo ≤27 mm for the antibiotic aztreonam. This group of strains is represented by 3 S. Abony serotype strains and one S. Infantis, S. Agona and Salmonella enterica subspecies enterica O:4,5 (ceftiofur resistant) strain, respectively. All strains were resistant to antibiotics belonging to the β -lactams class, in increasing order ampicillin, cephalothin, and cefoxitin (Table 1), to antibiotics belonging to the folate pathway inhibitor class (sulfonamides, trimethoprim and trimethoprim/sulfamethoxazole), and also to gentamicin, expressed by the serotype Salmonella enterica O:4,5. This resistance profile to antibiotics belonging to other classes is characteristic of ESBL strains, since plasmids that transfer resistance to cephalosporins are frequently found to be resistant to other antibiotics (Ziech et al., 2016).

According to the National Program for Monitoring Prevalence and Bacterial Resistance in Chicken (PREBAF) report (BRASIL, 2008), the major antibiotics to which Brazilian isolates have shown resistance, in decreasing order from 2004 to 2008, are streptomycin, sulfonamide, florfenicol, ampicillin, nalidixic acid, ceftiofur, aztreonam, enrofloxacin, cephalothin and cefoxitin. On the other hand, Salmonella isolated from chickens from Mato Grosso was sensitive to florfenicol, streptomycin, and nalidixic acid. This fact may reflect the search for an appropriate use of antibiotics or distribution of a distinct pattern for this region/timeframe. There is a consensus that the resistance pattern of Enterobacteriaceae depends on the season, region, serotype, farm, hens and chickens and antibiotic agent (Gyles, 2008). Genotyping was carried out in strains resistant to 4 or more antibiotics and suspected of producing ESBL through repetitive extragenic palindromic PCR (RepPCR). Five strains were evaluated, among which 3 were found to be similar, with 98 to 99% genetic homology (3,4,5), in order, Salmonella Infantis (3), S. Abony (4) and S. Agona (5). Strains with genetic homology (3,4,5) presented common characteristics, with resistance to β -lactams, namely first- and second-generation penicillin, monobactams, and cephalosporins, in addition to folate pathway inhibitors. The other 2 strains (1,2) are dissimilar, respectively, from Salmonella enterica subspecies enterica O:4,5 and S. Abony (Figure 2), which are distinguished from the other 3 strains by being resistant to ceftiofur (third-generation cephalosporin), and differing from each other by virtue of resistance to gentamicin presented by Salmonella enterica O:4,5. Genetically closely related strains might have been exposed to the same selective pressure and can transmit their antibiotic resistance genes both vertically and

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SALMONELLA MDR FROM A POULTRY SLAUGHTERHOUSE

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Figure 2. Dendrogram, sample ID, gel-like image, serotypes, and AMR profile of the Salmonella spp. serovars isolated from chilled chicken carcasses produced in Mato Grosso, Brasil. AMP, ampicillin; ATM, aztreonam; CFL, cephalothin; CFO, cefoxitin; CTF, ceftiofur; CLO, chloramphenicol; FLF, florfenicol; ET, streptomycin; GEN, gentamicin; NAL, nalidixic acid; CIP, ciprofloxacin; ENO, enrofloxacin; TET, tetracycline; SUT, trimethoprim+sulfamethoxazole; SUL, sulfonamide; TRI, trimethoprim; NIT, nitrofurantoin.

horizontally to their descendants (Fallah et al., 2013; Chon et al., 2015). This corroborates with the genotyping of the MDR strains, since all strains that showed genotype similarities were resistant to the same β lactam and folate pathway inhibitor classes, which suggest a horizontal transmission of resistance genes. Circulation of resistance genes between Enterobacteriaceae genera and species can be mediated by mobile genetic elements, such as plasmids, transposons, and integrons (Corrˆea et al., 2014; Bai et al., 2015; Fitch et al., 2016). Scientific information regarding the presence, antibacterial susceptibility profile and genotypic characteristic of Salmonella circulating in the chicken production chain and their products, as well as monitoring Salmonella MDR serotypes and clones, is of paramount importance to public health, mainly due to the globalized poultry meat trade (Antunes et al., 2016) and the fact that Brazil is a leading country in this scenario (IBGE, 2017).

CONCLUSIONS Despite the fact that occurrence of Salmonella in chicken carcasses produced in Mato Grosso is relatively low (3.65%), the observed strains present a wide diversity of serotypes. Serovars Salmonella Infantis and S. Abony were prevalent, although other, less frequent, serovars were also isolated, such as Salmonella Agona, S. Schwarzengrund, S. Anatum, Salmonella enterica subspecies enterica O:4,5 and Salmonella enterica subspecies enterica O:6,7. Of a total of 31 Salmonella isolates, 4 were MDR, being resistant to 3 or more classes of antibiotics, and sensitive to chloramphenicol, florfenicol, streptomycin, nalidixic acid, ciprofloxacin, enrofloxacin, and nitrofurantoin. In addition, some of the isolates were suggestive of ESBL. Salmonella Abony, S. Infantis, and S. Agona serovars showed high genetic and phenotypic similarity and may be transferring antibiotic

resistance genes vertically or horizontally among the population.

ACKNOWLEDGMENTS The authors would like to thank the Funda¸c˜ao de Amparo a` Pesquisa do Estado do Rio de Janeiro (process no. E-26/201.185/2014, FAPERJ, Brazil), Funda¸c˜ao de Amparo a` Pesquisa do Estado de Mato Grosso (process no.222388/2015, FAPEMAT, Brazil), and the Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnol´ogico (process no. 311422/2016–0, CNPq, Brazil), for financial support.

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