High Prevalence of Multidrug-Resistant Nontyphoidal Salmonella Recovered from Broiler Chickens and Chicken Carcasses in Brazil

21/December/2019 Approved: 13/January/2020 ABSTRACT The extensive use of antimicrobial agents has contributed to the emergence of antimicrobial resistance and multidrug resistance (MDR) in Salmonella, an important zoonotic pathogen that causes outbreaks and sporadic cases of gastroenteritis in humans. The study aimed to investigate the antimicrobial resistance profile of Salmonella strains isolated from poultry in Brazil. A total of 230 Salmonella strains, isolated from cloacal swabs (n=56) and broiler carcasses swabs (n=174) before and after chilling from slaughterhouses under Federal Inspection Service within the period 2012-2017, were analyzed. Serotyping and antibiotic susceptibility testing were performed on all the isolates. Serotyping results showed that 41% of the strains were Salmonella Heidelberg, 29% S . Minnesota, 12% S . Saintpaul, 6.5% S . Enteritidis, 3.9% S . Anatum, 2.2% S . Cerro, 2.2% S . Senftenberg, 1.7% S . Newport, 0.4% S . Ealing, 0.4% S . O:4,5 and 0.4% S . O:9,12. MDR rates of the isolates were 67.4%. S . Heidelberg 89.5%, S . Minnesota 51.5%, S . Saintpaul 82.1%, S. Anatum 66.7%, S . Cerro 60%, S . Senftenberg 40%. Out of the 230 strains, 41.3% presented resistance to Penicillins + betalactamase inhibitor, Penicillin, 1 st and 2 nd Generation Cephalosporin, 3 rd and 4 th Generation Cephalosporin, Tetracycline and Sulfonamide. Salmonella Heidelberg, S . Saintpaul, S . Anatum, S . Cerro, S . Senftenberg and S . Minnesota were isolated after chilling tank highlighting a food safety concern for the industry of poultry and poultry products indicating a risk to collective health. The high prevalence of MDR nontyphoidal Salmonella obtained in this study limit the options available to treat infectious disease in


INTRODUCTION
Surveillance of antimicrobial resistance (AMR) can be very valuable for orienting treatment choices, understanding AMR trends, identifying priority zones for interventions and monitor the impact of interventions in order to contain resistance spread. The lack of adequate surveillance in various parts of the world leaves large gaps in existing knowledge of the distribution and dimension of this phenomenon (Prestinaci et al., 2015).
Furthermore, surveillance data in nontyphoidal Salmonella enterica is essential to monitor the transmission of resistance from the food chain to humans, and to establishing effective treatment protocols (Neuert et al., 2018).
Due to the overuse of antibiotics in animals and humans, the number of foodborne multidrug resistant Salmonella isolates has increased rapidly in the last years. These multidrug resistant strains cause a heavy burden on clinical diagnosis and treatment of salmonellosis and have become a major public health issue in Brazil and in several other countries (Zishiri et al., 2016;Xu et al., 2019).

eRBCA-2019-1206
Salmonella is an important zoonotic pathogen that causes outbreaks and sporadic cases of gastroenteritis in humans all around the world (Zishiri et al., 2016). According to "The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2017", 91,662 human salmonellosis cases were reported in the European Union and it was identified in 1.241 (24.4%) foodborne outbreaks (FBO), affecting 9.600 people (EFSA; ECDC, 2018).
Most people recover from illness without antibiotic treatment. In rare cases, when the infection spreads from the gut to the bloodstream it may lead to hospitalization, antibiotic treatment and even to death, (CDC, 2016). A rapid increase of resistance to extended-spectrum cephalosporins, including the third and fourth generation cephalosporins, poses a significant threat to public health worldwide (Nguyen et al., 2016).
The aim of this study was to characterize the phenotypic profile of antimicrobial resistance and establish multidrug resistance (MDR) patterns of Salmonella strains isolated from broiler chickens and carcasses obtained from slaughterhouses in Brazil.

Sample collection, Isolation and Serotyping
A total of 230 Salmonella strains previously isolated, recovered from cloacal swabs (n=56) and broiler carcasses swabs (n=174) before and after chilling, from slaughterhouses under Federal Inspection Service (West-Center, Southeast and South regions of Brazil) between the year of 2012 and 2017 were received by the Poultry Health Laboratory/Universidade Federal Fluminense and stored in Nutrient Agar (Merck). After reactivation, the isolates were sent to serotyping at the Enterobacteria Laboratory, Oswaldo Cruz Foundation, in Rio de Janeiro, Brazil.

Multidrug Resistance
Antimicrobial susceptibility patterns were evaluated by the disk diffusion method and the strains classified as resistant to at least one antimicrobial in three or more categories was considered multidrug resistant, as defined by the European Centre for Disease Prevention and Control (ECDC) and the Centers for Disease Control and Prevention (CDC) (Gieraltowski et al., 2016;Magiorakos et al., 2012).
The isolates presented variable resistance levels to 16 out of the 19 tested antibiotics; only Ertapenem, Imipenem, Meropenem totally inhibited the growth of tested strains. All S. Newport, S. Ealing and S. Enteritidis strains were fully susceptible to the antimicrobials tested. The results in table 2 shows the highest resistances observed among the evaluated categories.

DISCUSSION
Several studies have found high prevalence of Salmonella Heidelberg in Brazil Neves et al., 2016;Webber et al., 2019), corroborating with this study, where S. Heidelberg accounted for 41% of isolated serotypes. The high prevalence of this serotype has a great zoonotic importance, since S. Heidelberg has been shown to be more invasive compared to Typhimurium and Enteritidis in a study about Salmonella surveillance data from FoodNet collected during 1996-2006 in the United States (Jones et al., 2008). In the present study, we observed serotypes that have recently been reported worldwide, as S. Enteritidis (6.5%) and S. Newport (1.7%) (CDC, 2018; EFSA; ECDC, 2018), besides others not so frequently reported, as S. Senftenberg (2.2%) and S. Cerro (2.2%) (Baptista et al., 2018). Knowledge of serotypes distribution constitutes an important epidemiological tool for the country, especially when it comes to nontyphoidal serotypes, important zoonotic pathogens that can cause gastroenteritis in humans worldwide (Prestinaci et al., 2015).
The poultry industry has been frequently implicated in Salmonella outbreaks, with reports of human pathogenic S. enterica serotypes in poultry products representing a major food safety concern for the

High Prevalence of Multidrug-Resistant Nontyphoidal Salmonella Recovered from Broiler Chickens and Chicken Carcasses in Brazil
eRBCA-2019-1206 industry (Neves et al., 2016;Rothrock Jr et al., 2015). Each official poultry slaughter establishment must ensure that all poultry carcasses, parts, and giblets will be chilled immediately after slaughter operations so that there is no outgrowth of pathogens (USDA-FSIS, 2014). Nevertheless, in the current study, most of the strains (59.6%, n=137/230), were recovered after being in the chilling tank, where contact between the carcasses processed during the day occur, being S. Heidelberg the most frequent followed by S. Saintpaul, S. Anatum, S. Cerro, S. Senftenberg and S. Minnesota. These findings are worrisome, since cold water used for chilling the carcasses can act as a cross-contamination vehicle among them (Demirok et al., 2013). The most observed MDR pattern in S. Heidelberg and S. Minnesota was Penicillin + betalactamase inhibitor, Penicillin, 1 st and 2 nd Generation Cephalosporin, 3 rd and 4 th Generation Cephalosporin, Tetracycline and Sulfonamide, 54.1% (n=46/85) and 50% (n=17/34) respectively. The threat of gramnegative bacteria resistant to multiple antimicrobials including to cephalosporins (WHO, 2017) is a growing global concern. These results show that Salmonella spp. is a challenge regarding AMR because of the frequent use of the 3rd generation cephalosporins (3GC) in salmonellosis treatment in human medicine, and because of the frequent use of ceftiofur in food animals (Neves et al., 2016;Nguyen et al., 2016). Cephalosporin use, specifically ceftiofur, the only 3GC available for animal food, is considered a major driver of selection and development of 3CG resistance in animal food (Carson et al., 2019). Microorganism resistant to ceftiofur are cross-resistant to ceftriaxone, important antimicrobial agent used to treat children with severe salmonellosis (Neves et al., 2016). Among S. Heidelberg, the dissemination of AMR determinants is likely due to plasmid-mediated conjugative transfer and by transposons (Deblais et al., 2018). S. Heidelberg has a propensity to acquire and disseminate multiple plasmids encoding for MDR (Foley et al., 2011). Besides that, it was observed in the present study an increased resistance to tetracyclines and sulfonamides, the oldest agents used in the treatment of bacterial infections, both in human and in veterinary medicine. Until 1998, these antimicrobial agents were used as additives in animal feeds in Brazil, when their use was restricted to therapeutic purposes. Nevertheless, these drugs still exert selection pressure on the microorganisms. In Brazil, manufacturing, handling, fractionation, marketing, import and the use of chloramphenicol for  veterinary use is also prohibited (Brasil, 2003), even so, we observed a high susceptibility to this agent in the current study. The concern about MDR was also reported by Borges et al., 2019, who studied Samonella enterica serotypes isolated from poultry sources in Brazil and 18% were classified as multidrug resistant strains. Neves et al., 2016 found MDR strains in 22.8% of Samonella enterica serotypes (including Heidelberg) from poultry origin in Brazil, being the most common resistance pattern gentamicin, nalidixic acid and tetracycline for 14 Salmonella Heidelberg isolates, followed by ceftiofur, nalidixic acid and tetracycline for 12 Salmonella Heidelberg isolates. Jeon et al., 2018 analyzed Salmonella enterica serotypes from chicken meat in Korea and 50.9% were multidrug resistant strains, being the most common resistance profiles ampicillin, chloramphenicol, nalidixic acid, trimethoprim-sulfamethoxazole, tetracycline and ampicillin, chloramphenicol, nalidixic acid, trimethoprim-sulfamethoxazole for 5 isolates each. These authors found a lower frequency of MDR isolates compared to this study, where 67.4% of the strains were classified as multidrug resistant. Moreover, their resistance patterns were also different, as Table 3 shows that 27.8% (n=64/230) of Salmonella strains presented resistance to Penicillin, Penicillin + betalactamase inhibitors, 1 st and 2 nd Generation Cephalosporin, 3 rd and 4 th Generation Cephalosporin, Tetracycline, Sulfonamide and 7.8% (n=18/230) to Penicillin, 1 st and 2 nd Generation Cephalosporin, 3 rd and 4 th Generation Cephalosporin, Aminoglycoside, Sulfonamide.
A study to evaluate the population dynamics and AMR pattern of the most prevalent poultry-associated Salmonella serotypes from the United States in the years 2002 to 2012, suggested that serotypes such as Heidelberg, Typhimurium, Kentucky, and Sentfenberg are more likely to be multidrug resistant whereas Enteritidis, Montevideo, Schwarzengrund, Hadar, Infantis, Thompson, and Mbandaka are generally pansusceptible or display resistance to fewer antimicrobials (Shah et al., 2017). These results corroborate with our findings, where 89.5% of S. Heidelberg and 40% of S. Senftenberg were multidrug resistant, and all strains of S. Enteritidis were susceptible to all antimicrobials. However, it is unclear why S. Enteritidis isolated from the United States poultry have remained recalcitrant to acquisition of MDR. A recent emergence MDR S. Enteritidis from other parts of the world raised a possibility that if clonal expansion and international spread of such MDR clones occurs this may pose a significant public health concern worldwide (Shah et al., 2017).
Considering the widespread degree of resistance observed in this and other studies related to nontyphoidal Salmonella, caution should be exercised when using antimicrobials that are still efficient to human salmonellosis treatment, in order to avoid or at least delay the resistance and, consequently, the appearance of new super-resistant bacterial strains.
The data from this study reinforce the importance of epidemiological surveillance and the need for improved communication between veterinarians and producers.