Diversity and antimicrobial resistance of Salmonella isolated from swines feces and carcasses

The aim of this study was to determine the most frequent Salmonella serovars in swine feces in the farm, piggery’s waiting slaughterhouse and after in their carcasses during slaughter, as well as their antimicrobial resistance profiles. Eighty six strains of Salmonella spp. were used, they were previously isolated from three different lots in different collections. The identification of serovars was done by serotyping and the resistance to antimicrobial agents was determined by disc diffusion. A variety of serovars was observed, and the 86 strains were serologically identified as: 28 (32.55%) S. Typhimurium, 20 (23.26%) S. Agona, 17 (19.77%) S. Infantis, 6 (6.98%) S. Panama, and 15 (17.44%) S. Minnesota. The diversity of serovars indicated that different factors influence the infection of finishing pigs and the persistence of microorganisms in the carcass after slaughter of these animals. More than 50% of the isolates were resistant to nine of the 11 tested antibiotics. The drugs that microorganisms had the highest percentages of sensitivity were sulphazotrim and norfloxacin, 22.1% and 14%, respectively. S. Typhimurium serovar was the most isolated, including in the carcasses, and also showed the largest difference in antimicrobial resistance when compared to other serovars. The profile of multidrug resistance observed in this study highlights to the necessity of a judicious observation of antimicrobial resistance in zoonotic foodborne bacteria. de sensibilidade foram o sulfazotrim e a norfloxacina, 22,1% e 14%, respectivamente. S. Typhimurium foi o sorovar mais isolado, inclusive nas carcaças, e também o que demonstrou maior diferença quanto à resistência aos antimicrobianos, quando comparado aos outros sorovares. O perfil de multirresistência observado neste estudo alerta para a necessidade de vigilância e monitoramento sistemático da resistência aos antimicrobianos em bactérias zoonóticas transmitidas pelos alimentos. The technique used for the antimicrobial resistance was the disk diffusion test according to the protocol described by the National Committee for Clinical Laboratory Standards (NCCLS, 2002). The antibiotics used for the test were: amoxicillin 10 μ g, norfloxacin 10 μ g, gentamicin 10 μ g, erythromycin 15 μ g, nalidixic acid 30 μ g, tetracycline 30 μ g and sulfazotrim 25 μ g (Laborclin®); ceftiofur 10 μ g, enrofloxacin 5 μ g and lincomycin 2 μ g (Sensifar®). The strain E. coli ATCC 25922 was used as a control. The plates were incubated at 37 ° C for 18 to 20 hours, and then the sensitivity halos (in millimeters) were measured using a pachymeter and subsequent classification of the microorganism as sensitive, intermediate or resistant to the tested antimicrobial. The antimicrobial resistance profile determined for the different serovars was compared using the Chi-square test with 95% of confidence. The calculations were performed using the Biostat 5.0 program (AYRES, 2007).


Introduction
Salmonellosis is one of the leading causes of foodborne gastroenteritis in humans worldwide, and is considered to be an important public health problem in both, industrialized and developing countries (EFSA, 2010;LOUREIRO et al., 2010).
The diversification of the industrial production of pork and the commercial exchange of animals and their derivatives for human consumption may be important disseminators of Salmonella spp. in the food chain. Thus, the presence of this microorganism in the production process represents an important sanitary barrier to trade food of animal origin and their by-products (SEIXAS et al., 2009).
Bacteria of the genus Salmonella are isolated worldwide, however, in areas of intensive breeding, especially of swine and birds, reports are even more frequent (OIE, 2004). The increase of the density and the indiscriminate use of antibiotics, inserted in the process of producing foods of animal origin, can act as selection pressure for some serovars and for their resistance to antimicrobials.
According to Wray;Sojka (1977) it is widely accepted that animals infected with Salmonella are the major sources of infection for other animals and humans. Thus, constant monitoring with identification of serovars and establishment of the epidemiological relationship of these along the production chain is of great importance.
Thus, we aimed to know which serovars of Salmonella spp. are infecting the pigs at the termination after transport to the slaughterhouse and in their carcasses after slaughter, as well as establish the antimicrobial resistance profile of these strains in order to contribute to a better understanding of the epidemiology of these microorganisms and provide information that may assist in the implementation of measures for the production of safe food.

Material and methods
A total of 86 strains of Salmonella spp. isolated from swine feces housed in the finishing farm, in the holding pens and from their carcasses during slaughter were used. The collection in the carcasses (after depilation, evisceration and in mesenteric lymph nodes) were performed in a non-exporter slaughterhouse under municipal inspection in the state of Minas Gerais.
The strains were previously isolated by (PACHECO, 2009)

Results and discussion
The 86  The diversity of serovars identified in feces and swine carcasses, with a higher incidence of S. Typhimuirium was also observed in another study. Bessa et al. (2004), in Rio Grande do Sul, identified 26 different serovars in 226 Salmonella isolates from 300 pigs slaughtered in three slaughterhouses.
S. Typhimurium, S. Agona and S. Infantis were the most isolated serotypes in this study. According to Loureiro et al. (2010), these serovars are associated with cases of human salmonellosis in Brazil. Serovar S.
Typhimurium was also the most isolated in swine samples in São Paulo and Rio de Janeiro by Filho (2014) and Cabral et al. (2017), respectively. In the United States, the three serotypes most frequently isolated in humans with salmonellosis are Typhimurium, Enteritidis and Newport, denoting the potential risk of these agents (CDC, 2006).
S. Infantis and S. Agona are also recognized for their pathogenic potential, which, in addition to the gastroenteric condition, may cause septicemia in young animals and humans, especially in cases of severe infections in children (TESSMANN et al., 2008).
All serovars were isolated both in the farm and in the slaughterhouse, except for S. Panama, which was not found in fecal samples collected at the termination farm, as shown in Table 1. It is possible that the source of contamination of carcasses by this serovar is equipment or instruments used at slaughter and is not related to prior infection of the animals.
Considering only the fecal samples, S. Agona was the most identified, but in the samples from animals slaughtered there was a higher incidence of  Typhimurium may possess a gene involved in the coding of a Mg 2+ carrier protein, which acts to protect the cell membrane integrity exposed to high temperatures, conferring greater thermotolerance.
The increase in the number of isolates in S. Typhimurium and S. The multiresistance of antibiotics in the Salmonella genus is a worldwide trend and is in line with the present study, since most isolates have demonstrated this profile. Of the studied isolates, more than 50% presented resistance to nine of the 11 tested antibiotics. The drugs with the highest sensitivity percentages were sulfazotrim and norfloxacin, 22.1% and 14%, respectively. However, even for these antimicrobials, the intermediate sensitivity indices observed were considered high, which may indicate the development of resistance in the medium term.
In the PREBAF report (BRASIL, 2008) on the monitoring of antimicrobial resistance of Salmonella and Enterococcus isolates from chicken carcasses, the treatment of enteropathogen infections using fluoroquinolones and third-generation cephalosporins has "a bleak outlook".
The resistance profile observed in this study confirms that this is a reality also for infection caused by Salmonella isolated from swine. No strain was sensitive to ceftiofur, which is a third generation cephalosporin, and only four (4.7%) of the isolates were sensitive to enrofloxacin (fluorquinolone).   S. Typhimurium was classified as multidrug resistant because of the resistance to three or more antimicrobial classes. All isolates of this serovar demonstrated resistance to neomycin (aminoglycoside), lincomycin (lincosamide), ceftiofur (cephalosporin) and enrofloxacin (fluorquinolone) and more than 96% showed resistance to amoxicillin (betalactam), gentamicin (aminoglycoside) and tetracycline. However, all were sensitive to sulfazotrim. In this study, the resistance of serovar Typhimurium to sulfazotrim was significantly lower than that observed for Infantis (P <0.05) and Minnesota (P <0.01) serovars.
The resistance to tetracycline in 96.4% of S. Typhimurium isolates was higher than that observed in other studies carried out in Brazil on swine samples. Resistance to this antimicrobial was also observed in 41.67% of strains isolated from fresh sausage by Spricigo (2008). Studies in Europe with porcine isolates marketed in the member countries showed lower resistance than those of this study for S. Typhimurium, with indices varying from 36.05% to 59% (EFSA, 2006;EFSA, 2008). The increase in resistance levels to ampicillin, tetracycline and chloramphenicol since 1996 in Salmonella isolated from humans, pigs and chickens characterizes the emergence of multi-resistant S. Typhimurium (BEGUM et al., 2010).
However, this author reports that isolates with these characteristics are also resistant to the sulfa and trimethoprim association (sulfazotrim), which was not observed in this study. norfloxacin in relation to serovar Agona (P <0.01). There was less resistance to sulfazotrim by the isolates when compared to those identified as S.
The resistance profile determined for the serovar Panama in this study is different from that observed by Castagna et al., (2004) in isolates of feces and mesenteric lymph nodes of pigs at slaughter in three slaughterhouses in southern Brazil.
Isolates identified as S. Infantis presented a lower percentage of resistance to enrofloxacin compared to serovar Typhimurium, but a higher number of isolates resistant to sulfazotrim compared to this same serovar (P <0.05). All isolates were resistant to lincomycin, ceftiofur, tetracycline and erythromycin. The antimicrobial resistance of S. Infantis in this study was higher than that observed by Tessmann et al. (2008). These authors determined sensitivity of 100% and 43% for nalidixic acid and tetracycline, respectively, in S. Infantis isolated from pork cuts at Pelotas, RS.
The isolates identified as S. Minnesota were less resistant to norfloxacin compared to S. Typhimurium and enrofloxacin compared to S.
In Brazil, the use of antimicrobials avilamycin, bacitracin, chlorhexidine, flavomycin, chlorohydroxyquinoline, tylosin, lincomycin and erythromycin as feed additives in finishing pigs is permitted (BRASIL, 1998). Of these, the last two were tested in this study. With the exception of 8.1% of the isolates of S. Typhimurium and 25% of S. Agona, which presented a profile classified as intermediate to erythromycin, the other isolates were 100% resistant to these drugs.
The results of this study show that, independent of the differences identified among serovars for some antimicrobials, the isolates presented a multiresistance profile, especially for those most used in clinical treatment in farms such as enrofloxacin, lincomycin, amoxicillin, tetracycline, erythromycin, norfloxacin, ceftiofur and sulfazotrim, and the observed differences are likely to be more associated to the series than to the serovar.
Resistance is worrying and demonstrates the need to reduce the use of antimicrobials in animal production as a way to reduce the selection pressure to which these microorganisms are subjected. It also warns of the difficulties that may occur in the treatment of extraintestinal infections and in groups at risk, where antibiotic therapy and the need to monitor antimicrobial resistance in foodborne zoonotic bacteria are required.
Initiatives such as the monitoring of Salmonella resistance isolated from frozen chicken carcasses throughout the country carried out by ANVISA in the PREBAF project (BRASIL, 2008) provide important data.
The information generated allows the adequate visualization of the problem to promote the implementation of effective control measures.

Conclusion
The most identified serovars were S. Typhimurium, S. Agona and S.
Infantis. The multiplicity of Salmonella serovars identified at the analyzed points of the productive process indicates that different factors influence the infection of finishing pigs and their presence and persistence in the carcass of these animals after slaughter. This reinforces the importance of a rigorous monitoring that allows to subsidize actions that promote the adequate control of these pathogens from the farm to the steps of slaughter and guarantee the health of the consumer.
The strains showed higher resistance to lincomycin and ceftiofur. The