High Genetic Similarity Among Salmonella Heidelberg Isolated from Poultry Farms, Wild Animals, Beef, Poultry and Pork Meat, and Humans in Brazil

ABSTRACT Salmonellosis is an important gastrointestinal infection in humans and cause of foodborne outbreaks in the world. In this context, molecular characterization is essential to understand how the strains circulate. The aim of this study was to evaluate the genotypic distribution of S. Heidelberg according to the source of isolation. The genetic relatedness of the S. Heidelberg isolates was determined by pulsed-field gel electrophoresis (PFGE). The most prevalent pulsotypes of cluster A were BRJF6X01.006 (27/95 = 28,42%) related between 1995 and 2011 in broilers, poultry meat and poultry farms, meat product and human, and BRJF6X01.001 (21/95 = 22,10%) related between 2011 and 2017 in wild animals, broilers, poultry meat, poultry farms, meat product, animal feed, and pork meat. The pulsotype BRJF6X01.001 shows a high distribution in the environmental and productive chain. The degree of similarity between pulsotypes BRJF6X01.006 and BRJF6X01.001 is 88%. To ensure the safety of human and animal health, holistic approaches, including surveillance of Salmonella throughout the environment and in the production chain, together with control measures, are critical. As transmission of Salmonella from food producing animals to wildlife and to the environment is considered potential public health problem, information on the survival and persistence of Salmonella in the environment and in potential reservoirs is of considerable importance.


INTRODUCTION
Salmonellosis is the second most reported gastrointestinal infection in humans after Campylobacteriosis, and an important cause of foodborne outbreaks in the world (CDC, 2019b;European Food Safety et al., 2021).Salmonella can be classified into typhoid and non-typhoid regarding their ability to develop specific pathologies in humans and animals.Typhoid serovars are a subcategory of serovars capable of infecting and colonizing only a very narrow range of hosts and highly adapted to humans, presenting only higher primates and humans as reservoirs; in contrast, the non-typhoid serovars are capable of triggering infections in both humans and animals (Knodler & Elfenbein, 2019).Symptoms caused by non-typhoid serovars are usually limited to diarrhea, and there is no need for the use of antibiotics, but severe illness can occur.Such cases may include extra-intestinal infections such as septicemia and myocarditis, and can result in death, especially in children, pregnant women, and the elderly, as infection can be complicated by resistance to third generation cephalosporins -one of the antimicrobial classes for treating severe or invasive Salmonellosis (Parisi et al., 2018;Collineau et al., 2020).
The most common non-typhoid Salmonella reservoir is the intestinal tract of a wide range of domestic and wild animals and a eRBCA-2022-1628 eRBCA-2022-1628 variety of food matrices that can serve as a vehicle for transmission of Salmonella spp. to humans through fecal contamination (European Food Safety, 2021).Salmonella Heidelberg is a serotype widely distributed and frequently associated with human diseases, being more frequent in North America than in other parts of the world (Ferrari et al., 2019;Collineau et al., 2020;Melo et al., 2021).Since 2013, Salmonella Heidelberg joined the group of 10 serovars with the highest incidence in Brazil, in which food sources were the largest contributor with 65%, followed by environmental samples, animals, raw materials, and human beings (Santos et al., 2022).
Despite the lower costs of genome sequencing techniques, they are still expensive for laboratories in developing countries, (Voss-Rech et al., 2019) and PFGE still can be considered the golden standard for genotyping of Salmonella due to the stability of the generated profiles, the discriminatory power and reproducibility of the results (Ferrari et al., 2017).
Considering the relevant frequency of isolation Heidelberg in the last 25 years in Brazil, the aim of this study was to evaluate the genotypic distribution of S. Heidelberg according to the source of isolation and the relevance to public health.

MATERIAL AND METHODS
Isolates were received in The National Reference Laboratory of Cholera and Enteric Diseases of the Oswaldo Cruz Institute Foundation -FIOCRUZ, Rio de Janeiro, Brazil (NRL) inoculated in nutrient agar and on arrival, confirmed the identification as S. enterica by serotyping according to the Kauffman-White scheme (Issenhuth-Jeanjean et al., 2014).NRL receives Salmonella strains from Brazilian meat industries, health services laboratories and universities for serotyping and genotyping.
Genotyping protocol was applied according to the Center of Disease Control of the PulseNet Network (CDC, 2017).The isolates were genotyped by DNA macrorestriction analysis, using 40 U of the enzyme XbaI (New England Biolabs, Beverly, MA, USA) followed by PFGE, as previously described (Ribot et al., 2006).Salmonella Braendrup H9812 was used as size standard.Restriction fragments were electrophoresed in certificated 1.2% PFGE agarose gels (Bio-Rad, Hercules, CA, USA) in tris-borate buffer (TBE; tris-borate 0.045 M, EDTA 0.001M) at 14ºC, using the CHEF DR III system (Bio-Rad), with an initial switch time of 2.2s and a final switch of 63.8s at 6V/s for 18 h.Gels were stained in ethidium bromide (1µlg/mL) and visualized under UV light.Images were captured using a digital camera, and macrorestriction patterns were compared using BioNumerics 7.6 software (AppliedMaths, Sint-Martens-Latem, Belgium).Similarity was calculated by the Dice coefficient with 1.7% tolerance.A dendrogram was generated by cluster analysis using the unweighted pair of group method with arithmetic mean (UPGMA).Strains sharing the same number and position of DNA macrorestriction fragments were considered to belong to the same genotype.
The interpretation of PFGE profile was evaluated according to Tenover et al. (1995) and Barrett et al. (2006), considering the difference from one another.The pulsotypes should be considered closely related when the difference is up to three bands, and profiles that differ by up to six bands should be considered possibly related.The rationale behind this recommendation is that a single genetic event (a point mutation in a local restriction, a deletion or insertion) would result in up to three bands of difference.Thus, a three-band difference would be the result of one genetic event and a six-band difference the result of two genetic events.
PFGE is a method based on genomic DNA restriction well established for Salmonella genotyping (Cosby et al., 2015), and can be used to trace routes of strains dispersion and elucidate outbreaks.Voss-Rech et al. (2019) detected undifferentiated Salmonella Heidelberg genotypes by PFGE in poultry environment of two companies supporting the clonal dispersing of this serovar.S. Heidelberg emerged in commercial poultry farms and derived products from Brazil (Voss-Rech et al., 2019;Rodrigues et al., 2020), making this serovar a concern to public health.The persistence in poultry chain is probably due to the capacity of S. Heidelberg to resist to different litter treatments (Voss-Rech et al., 2017) and to form biofilms under different temperatures and the tolerance to biocidal agents (Melo et al., 2021).A study using multi-locus sequence typing supported the clonality of this serovar in poultry chain (Campos et al., 2018), and in another study, using whole genome sequence, the strains were clustered together with isolates from poultry and swine in Brazil and poultry products from other countries (Kipper et al., 2021).The high level of genomic similarity in S. Heidelberg indicated a probable clonal origin.The detection of this cluster could be explained by the dissemination among livestock industry worldwide as reported in other bacteria (Peirano & Pitout, 2010); or because Brazil is one of the largest chicken meat exporters in the world, our products are available all over the globe for researchers (Campos et al., 2018;Van Den Berg et al., 2019).
The   related to human cases (Santos et al., 2022).This fact reinforces the importance of these programs to the control of Salmonella serovars all over the food chain, considering that the strategy of control should be based on good practices, hazards and risks analysis to support a farm to fork strategy.The degree of similarity between pulsotypes BRJF6X01.006and BRJF6X01.001was 88% and the timeline shows that the pulsotype BRJF6X01.006was mostly present in poultry chain sources between 1995 to 2011 and was detected in human cases between 2009 to 2011; the pulsotype BRJF6X01.001appears since 2011, showing a high distribution in the environmental and food chain, but was not more isolated from human cases.Due to the high degree of similarity between the BRJF6X01.006and BRJF6X01.001pulsotypes and the fact that BRJF6X01.006was no longer detected after the appearance of BRJF6X01.001 in 2011, we believe that the BRJF6X01.006pulsotype would be the precursor of BRJF6X01.001.
Humans can be asymptomatic or can develop enteric disease, which can include bloody diarrhea and fever, and in some cases trigger severe systemic disease (Knodler & Elfenbein, 2019).S. Heidelberg has been associated with invasive human infections and mortality rates (Folster et al., 2012;Gieraltowski et al., 2016;Palmeira et al., 2016).In most cases, salmonellosis does not require antibiotic therapy, but in patients with comorbidities or invasive infection the antibiotic treatment can be performed (CDC, 2019b).In 2019, 87,923 cases of human salmonellosis were reported in European Union with a rate of 20.0 cases per 100,000 population (European Food Safety, 2021).In the United States, CDC estimates that Salmonella causes around 1.35 million infections, 26500 hospitalizations, and 420 deaths in the United States every year and food is the main source of illness (CDC, 2019a).The annual cost associated with the major foodborne pathogens in the US has been estimated to be approximately $14 billion, and non-typhoid Salmonella alone accounts for $3.3 billion of the total (Hoffmann et al., 2012).Regarding Brazilian status about salmonellosis, as in other developing countries, outbreak information is frequently incomplete because health authorities lack the capabilities or resources for detection, or because diarrheal diseases are widespread and outbreaks may be less common or clear than in developed countries (Panzenhagen et al., 2015).
The phylogenetic comparison between the strains from food, poultry, humans, and wild animals suggest a probable common source since they presented, approximately, 90% of genetic similarity.The most common non-typhoid Salmonella reservoir is the intestinal tract of a wide range of domestic and wild animals and a variety of food matrices which can serve as a vehicle for transmission of Salmonella spp. to humans through fecal contamination.Failure in biosecurity measures in animal production can allow free-living animals to invade the livestock environment, allowing for the exchange of strains between farm and wild animals.The transfer frequently occurs when theses microorganisms are introduced into food preparation areas, with subsequent proliferation in food items through improper storage temperature, inadequate cooking, and/or cross contamination, as well as through direct contact with infected animals and humans (European Food Safety, 2021).
The analyzed S. Heildelberg isolates confirm to us an example of connection between humans, poultry farms, beef, poultry, pork and wild animals.Strains of Salmonella were also frequently found in farm animals, which could be due to contamination from different sources such as wildlife or the environment.
There are several limitations in this study that must be considered in the prevalence values.Furthermore, when flour made from dairy, bovine and pork products are mixed with poultry, they appeared more likely to have Salmonella strains of public health importance, such as S. Heidelberg.This finding suggests that foraging animals not normally associated with these important human pathogens can be exposed, colonized, and become exterminators in mixed avian operations.Although our data are insufficient to assess whether certain host species are important for human infection, the distribution of different pulsotypes may arise from a combination of factors, including variation in host range and sampling structure for different species, which suggest that companion animals and wild animals deserve more attention.

CONCLUSION
The results from this study lend support to the hypothesis that S. Heidelberg circulates between several sources and some cases could be infections carried by wild animals.As transmission of Salmonella from food producing animals to wildlife and to the environment is considered a potential public health problem, information on the survival and persistence of Salmonella in the environment and in potential reservoirs is of considerable importance.Genetic surveillance of Salmonella from different sources is necessary to understand the complex epidemiology National Program of Poultry Health (PNSA) andThe National Program of Pathogens Control performs regular monitoring in the poultry production chain to check the prevention and control Salmonella programs of industries, which could explain the higher frequency of isolates from poultry and poultry products.Since the beginning of these programs in 1994 and 2003, there has been a decrease in the frequency of Salmonella Enteritidis and Typhimurium in poultry products and by-products, especially after the introduction of vaccine in the primary production.These serovars have been replaced by other serovars such as Heidelberg and Minnesota(Voss-Rech et al., 2017;Voss-Rech et al., 2019;Rodrigues et al., 2020), however S. Enteritidis and S. Typhimurium remain the most common serovars

Figure 1 -
Figure 1 -Dendrogram of pulsotypes detected in Salmonella Heidelberg from wild animals, broilers, poultry meat, poultry farms, beef, pork meat, humans, meat products and animal feed between 1995 to 2017, Brazil.
Farms, Wild Animals, Beef, Poultry and Pork Meat, and Humans in Brazilof this bacterium, due to its relevance for human and animal health.

Table 1 -
Cluster, pulsotype, source and year of isolation of 124 Salmonella Heidelberg strains.