Non-typhoidal Salmonella in the Pig Production Chain: A Comprehensive Analysis of Its Impact on Human Health

Salmonellosis remains one of the most frequent foodborne zoonosis, constituting a worldwide major public health concern. The most frequent sources of human infections are food products of animal origin, being pork meat one of the most relevant. Currently, particular pig food production well-adapted and persistent Salmonella enterica serotypes (e.g., Salmonella Typhimurium, Salmonella 1,4,[5],12:i:-, Salmonella Derby and Salmonella Rissen) are frequently reported associated with human infections in diverse industrialized countries. The dissemination of those clinically-relevant Salmonella serotypes/clones has been related to the intensification of pig production chain and to an increase in the international trade of pigs and pork meat. Those changes that occurred over the years along the food chain may act as food chain drivers leading to new problems and challenges, compromising the successful control of Salmonella. Among those, the emergence of antibiotic resistance in non-typhoidal Salmonella associated with antimicrobials use in the pig production chain is of special concern for public health. The transmission of pig-related multidrug-resistant Salmonella serotypes, clones and/or genetic elements carrying clinically-relevant antibiotic resistance genes, frequently associated with metal tolerance genes, from pigs and pork meat to humans, has been reported and highlights the contribution of different drivers to the antibiotic resistance burden. Gathered data strengthen the need for global mandatory interventions and strategies for effective Salmonella control and surveillance across the pig production chain. The purpose of this review was to provide an overview of the role of pig and pork meat in human salmonellosis at a global scale, highlighting the main factors contributing to the persistence and dissemination of clinically-relevant pig-related Salmonella serotypes and clones.


Introduction
Salmonella enterica infections are a worldwide major public health concern, specifically human salmonellosis caused by non-typhoidal Salmonella (NTS) [1,2]. Salmonellosis is typically characterized by a self-limiting gastroenteritis syndrome, with diarrhea as its the main symptom; however, fever, vomiting and abdominal pain can also occur [1,2]. Despite being uncommon, more severe invasive Salmonella infections, as bacteraemia and/or other extra-intestinal infections, can occur and affect particular high-risk groups (infants, young children, older people or immunocompromised patients) [1,2]. In these cases, the use of antimicrobial agents is required, being animal samples (25%-chickens, 9%-turkeys, 22%-dairy and 8%-beef) [44]. Moreover, in developing countries (some with an expansion of food-animal industry), Salmonella was detected at high levels in pig samples (animal, carcasses and meat), ranging from 17-39% in South America [45,46], to 14-40% in Africa [47,48] and 29-100% in Asia [49,50]. Those high levels possibly reflect the different pig production practices and the absence of control measures. Overall, these data point out the need of implementing effective global measures for Salmonella control, highlighting the need for its detection at all stages of pig production chain, including in primary production [13,32,33]. This is particularly urgent in developing countries, which currently seem to present a severely underserved monitoring surveillance program [51].

Major Pig-Related Salmonella Serotypes Associated with Human Infections
In recent years, Salmonella transmission from pigs to humans through the pork food chain has been evidenced, namely through the study of serotypes prevalence in different matrices as well as food-borne outbreaks associated with consumption of pork products.
Worldwide data concerning the prevalence of Salmonella serotypes in humans, pigs and products thereof have contributed to establishing their epidemiological correlation, with particular serotypes overlapping between humans, pig and pork meat [6][7][8]14,16,17,[52][53][54]. For instance, in EU, an association between Salmonella serotypes causing human infections and those occurring in pig and pork meat was observed (Figure 1 [5][6][7][8]. These three serotypes are also among the major ones associated with human salmonellosis (second-, third-and fifth-ranked, respectively, in 2014-2016) [6][7][8]. From the 2008 EU baseline survey, S. Derby was the most frequent serotype found in both breeding (29.6%) and production holdings (28.5%) and S. Typhimurium was the second most detected (breeding holdings-25.4% and production holdings-20.1%) [32]. It is also of note the emergence of S. Rissen in pig sources (pigs: 1.5%-2014, 2.8%-2015 and 1.2%-2016; pork meat: 4.9%-2014, 5.1%-2015 and 5.9%-2016) in the EU (the fifth most common serotype since 2014 in pig sources) in spite of its low association with human infections (Figure 1) [6][7][8]. The EU baseline survey reported a high incidence of S. Rissen in breeding and production holdings, particularly in Portugal (40% and 22.4%, respectively) and Spain (25% and 29.7%, respectively), being the first or second most frequently reported serotype in those settings [32,33]. In fact, in Portugal, S. Rissen was the fourth most frequently serotype detected in human clinical isolates between 2002 and 2016 [55,56]. Although S. Enteritidis (number one in human infections) is typically associated with eggs and poultry meat, it is important to point out that in the last years, in the EU this serotype was also common in both pig and pork meat samples (varying from 1% and 3.5%) [5][6][7][8]. Moreover, Salmonella Infantis, another typically poultry-related serotype causing human infections (top 4), was detected in pigs and particularly in pork meat (varying from 3.9% to 8.8%) [5][6][7][8]. Therewithal, both S. Enteritidis and S. Infantis serotypes have been recovered from pigs/pork and products thereof in other non-EU regions and associated with human salmonellosis [57][58][59][60][61].  [6][7][8]. S. Rissen was included for being one of the five most frequent Salmonella serotypes recovered from pig meat and pig animal in EU, 2014 to 2016 [6][7][8]. The percentages were calculated based on the total number of serotyped isolates (represented by the numbers in brackets) per human salmonellosis cases, pig meat or pig animal.

Dissemination of Pig-Associated Salmonella Serotypes and Clones
Besides worldwide data concerning Salmonella serotypes prevalence in humans and pig sources, the contribution of pork meat for human salmonellosis has been also evidenced throughout the spread of certain pig-associated strains and clones. Several examples of outbreaks associated with pig-related Salmonella serotypes have been described involving diverse countries (Table 1). For instance, S. 1,4, [5],12:i:-strains causing human infections, including some particular major clones, were identified in diverse European countries and associated with the consumption of different pork products (Table 1). Moreover, since 2015, several notifications were reported by the Rapid Alert System for Food and Feed (RASFF) due to the presence of Salmonella in pork products from several European countries, including alerts of suspected multi-country foodborne outbreaks (e.g., S. Typhimurium ST19 in Denmark associated with chilled sliced salami from Spain) ( Table 1). This scenario alerts for the relevant role of pig/pork meat international trade on the dissemination of clinically-relevant pig-related Salmonella serotypes/clones, highlighting the need for global effective surveillance and detection programmes at all stages of pig production [11,13].
S. Derby and S. Rissen have been other predominant serotypes in both pig and pork meat in Europe [6][7][8], and in USA [84], despite being less implicated in human salmonellosis. Nevertheless, S. Derby has been reported at global level with identical MDR (mainly SSuT) and/or PFGE profiles in isolates from human clinical cases, pigs and products thereof, demonstrating their potential role in human infections [19,20,52,62,63,65,[85][86][87][88]. In Southern European countries, S. Rissen is considered a clinically-relevant serotype, being frequently detected the same strains in humans, pigs and products thereof [19,21,22,25,27,62,65,89,90]. Moreover, S. Rissen strains detected among humans and across pig production chain, particularly belonging to the successful MDR clone ST469, have been reported in geographic distant countries [16,19,21,22,27,62,63,65,89,90]. In particular, the circulation of a specific MDR (ASSuTTm, Trimethoprim-Tm) S. Rissen clone between the Iberian countries can be explained by the intensive trade of pigs and products thereof [21]. Additionally, some S. Rissen isolates recovered from human, pig and pork isolates in Denmark showed similar PFGE profiles with isolates from imported pigs or pork meat from Spain and Germany, as well as with isolates from human clinical cases of people who travelled to Thailand [27] (Table 1). These data enhance the contribution of live animals and international food trade to the spread of this S. Rissen clone, besides human travel to developing countries.  The enhanced ability to colonize food animals and to persist along the food chain of pig-related Salmonella serotypes and clones associated with human infections is a topic of great concern [26,64,65]. Specific adaptive features, such as colonization/virulence determinants, have been pointed out as an advantage for the maintenance and spread of these serotypes/clones in diverse environments and hosts (pig/human) [116]. Recent studies have found the presence of several virulence genes, associated with an enhanced adaptation to the food-animal host, in S. Typhimurium [116,117], in specific clones of S. 1,4, [5],12:i:-strains circulating in Europe [72,73,80,118] and in S. Rissen, including in isolates belonging to the ST469 [119,120]. Those virulence genes encode for proteins that improve colonization (e.g., clpB), adhesion (e.g., csgA, fimA/C, pefA, stbD, marT), intestinal invasion (e.g., invA, invE, spvC), survival in host tissues (e.g., sopA, avrA, sseI, mig5) and biofilm formation (e.g., bss) [83,117,119,121,122]. Interestingly, S. Typhimurium DT193 and S. 1,4, [5],12:i:-were associated with long-term survival in pig faeces comparing with other serotypes (S. Derby and S. Bredeney), due to their increased adaptation to acid fecal pH and organic acid supplementation of feed [123]. Additionally, a UK study demonstrated that SPI-23 present in S. Derby strains, which contain genes (e.g., potR) that encode Type III effector proteins, contributes for the host intestinal cells invasion in pigs [124]. More recently, the presence of this SPI-23 was also reported in French pork isolates of S. Derby ST39 and ST40, helping to explain the host pig specificity of those epidemic strains [125].
Moreover, those emergent pig-related Salmonella serotypes/clones were usually enriched with antimicrobial resistance genes, in most cases located in the mobile genetic elements that also carry virulence genes. For example, resistance plasmids of S. 1,4, [5],12:i:-isolates (from pigs and humans) circulating in Europe, carry several virulence genes, namely spvC±mig5 genes in IncA/C and IncR plasmids, associated with the Spanish and Southern European clones, respectively [83]. Furthermore, several genes associated with tolerance to metals and/or biocides (e.g., copper), widely used in food-animal production, were found in pig-related Salmonella serotypes/clones (e.g., S. Rissen MDR clone, European clone of S. 1,4, [5],12:i:-and S. Typhimurium), which might also be an additional advantage for their maintenance and spread in the food production environment and hosts (pig/human) [25,126,127].

Antimicrobial Resistance in Salmonella and the Pork Linkage
Antibiotic resistance is considered by several relevant public health entities one of the major threats to human health and a relevant concern for food safety, particularly if involves pathogenic bacteria transmitted to humans through food-chain [3,26]. The emergence and spread of Salmonella isolates presenting resistance to several antibiotics, especially to "Highest Priority Critically Important Antimicrobials" (fluoroquinolones and 3rd and higher generations cephalosporins) [3], is of concern since they are crucial to the successful treatment of NTS invasive infections [1,2]. The adverse consequences of resistance to critically important antibiotics in humans include an increase in the severity of infections and in the frequency of treatment failures, as well as the requirement of last-line antibiotics use (e.g., carbapenems, colistin) [3,26].
The common practice of antibiotic use in intensive food-animal production has been considered the main driver for the selection and transmission of antibiotic-resistant foodborne bacteria, including Salmonella, to humans [1,26,[128][129][130][131][132]. This scenario is aggravated particularly in pig production, which has been associated with a higher antimicrobial consumption, compared with other animal-food production systems, at global level [133], including in the EU [134]. In 2010, the annual average of antimicrobial consumption per kilogram of animal produced was 172 mg·kg −1 in pigs, higher than the 148 mg·kg −1 and 45 mg·kg −1 consumption in chicken and cattle, respectively [133]. Although there is still controversy about the contribution of food-animal reservoirs and food vehicles in the transmission of antibiotic-resistant bacteria with an impact in human health, there is accumulating evidence linking the pig production with antimicrobial resistance in NTS that will be discussed in the next sections.

Association between Antibiotic Use in Pig Production and Resistance in Salmonella
The first evidence of this linkage is the association between the amount and pattern of antimicrobial agents used in the pig production and the occurrence of resistant NTS in pigs, pork meat and/or humans. One illustrative case in pig production includes a study showing that the administration of tetracycline to pigs colonized with tetracycline-resistant S. Typhimurium DT104 was associated with higher pig shedding of this resistant strain compared with untreated pigs [135]. Other study reported that enrofloxacin, used for treatment of pigs, induced the selection of S. Typhimurium with decreased susceptibility to ciprofloxacin [136]. Furthermore, a Danish surveillance study performed after the ban of antibiotics as growth promoters showed higher levels of tetracycline resistance in S. Typhimurium isolates recovered from pig and human clinical cases, potentially caused by an increased usage of tetracycline in pigs [137].

Correlation of Antimicrobial Resistance Rates between Salmonella from Pigs and Humans
The correlation between antibiotic resistance rates among Salmonella from pigs, pork meat and humans obtained from systematic surveillance data evidences the impact of pig production practices on NTS antibiotic resistance. The 2016 EFSA report showed a high prevalence of MDR Salmonella in humans (29.3%), pigs (58.7%) and pork meat (40.4%), including the most used antibiotics in pig production (tetracyclines, penicillin's, sulphonamides and colistin) [26,134].  [138].
Data about MDR phenotypes are of concern due to the possible role of diverse antibiotics in the co-selection of Salmonella strains resistant to clinically-relevant ones, such as fluoroquinolones (e.g., ciprofloxacin-Cp), extended-spectrum cephalosporins (e.g., ceftazidime-Caz, cefotaxime-Ctx) and colistin (Col) [3,26,131,134]. In the last EU report, relatively low levels of Salmonella resistance to Cp (13,3%), Caz (0.9%), Ctx (0.9%), and Col (11.4%) were observed [26,139]. Moreover, the highest levels of Col resistance in humans (excluding the S. Enteritidis serotype which presents intrinsic resistance) were more common in the pig-related serotypes S. 1,4,[5],12:i:-(2.4%) and S. Typhimurium (1.5%) [26,139]. Additionally, data from 2015 in USA revealed low levels of decreased susceptibility to Cp (5.8%) among humans, being the highest levels detected in retail pork samples (5.3%) comparing with other retail meat (0% in poultry and beef) [140], which suggest an important contribution of pig production chain to the human disease burden.
In contrast, high rates of antibiotic resistance were reported among humans and pig/pork products in middle-income countries. For instance, high rates of Salmonella with resistance to Cp (15-48%), ceftriaxone-Cro (38%) and Col (36%) were observed in humans [141][142][143]. High levels of resistance to clinically-relevant antibiotics were also observed in pigs or pork meat, in particular resistance to Cp (10-49.4%) [144][145][146] and Col (7-21%) [142,147], being in most cases higher than those detected in poultry samples (Cp-0-29%, Col-3.8%) [142,144,146]. Moreover, different Chinese studies reported higher rates of acquired Col resistance mechanisms (Mobile Colistin Resistance-MCR) in pigs (10-14.8%) than in poultry samples (0.8-7.5%) [148][149][150]. Indeed, it was already suggested that pig production has the highest impact on colistin resistance in humans because of its extensive use in this system contrasting with other food-producing animals [151,152]. Overall, the high incidence of resistance to clinically-relevant antibiotics observed in Salmonella from pig production highlights the potential role of pork products in its spread and may be the consequence of the high, inappropriate or uncontrolled use of antibiotics in farming practices in middle-income countries [133,143,147,150,153,154]. In fact, it was estimated that the global consumption of antimicrobials in livestock will increase by 67% between 2010 and 2030, with an expected increase in pigs of 124%, particularly in Asia, in order to respond to the increasing demand of pork meat, one of the most consumed and traded meat products [30,133,155]. Hence, international trade of live pigs (piglets, weaner, grower or breeder pigs) and pork meat can contribute for the worldwide spread of antibiotic-resistant Salmonella, with a consequent impact on human health.

Transmission of Antimicrobial Resistant Salmonella from Pork Meat to Humans
The presence of the same mobile genetic elements carrying clinically-relevant antibiotic resistance genes and/or antibiotic-resistant Salmonella serotypes/clones in pig and human isolates is an additional evidence of transmission of antimicrobial resistant Salmonella from pork meat to humans. In the last decades, clinically-relevant antibiotic resistance genes such as those coding for extended-spectrum β-lactamases (ESBLs), acquired AmpC β-lactamases (qAmpC), plasmid-mediated quinolone resistance (PMQR) and more recently plasmid-mediated colistin resistance (MCR), have been globally reported in a wide range of Salmonella serotypes associated with pig and pork products ( Table 2). Additionally, some examples that illustrate the linkage and transmission of MDR Salmonella from pigs/pork products to humans are shown in Table 2 Table 2).
The acquisition of resistance mechanisms to antibiotics commonly used in food-animal production (e.g., ampicillin, sulphonamides, tetracyclines) is also relevant for their potential role in the co-selection of pig-related MDR Salmonella clones in pig production and further transmission to humans. Several studies have provided evidence of successful transmission of MDR Salmonella clones from diverse serotypes from pork to humans [19,20,23,24,161,162]. For example, we recently reported mcr-1 located on epidemic plasmids (IncX4 and IncHI2) in clinically-relevant MDR S. 1,4, [5],12:i:-/ST34 [159]. Additionally, in China, diverse studies reported the clonal spread of mcr-1+oqxAB+aac(6 )-Ib-cr in S. Typhimurium/ST34 in pigs presenting resistance to multiple antibiotics (e.g., A, S, Su, T) [149,150]. Moreover, some of these successful clones presented acquired metal tolerance genes, an additional feature that might be contributing for the survival and persistence of these strains in metal contaminated environments, such as the pig production setting [25,126,127,163]. For example, sil±pco genes encoding for copper/silver tolerance were often found in the emergent European clone of S. 1,4, [5],12:i:-and S. Typhimurium, as well as S. Rissen/ST469 MDR clone [25,126,127]. In fact, copper is one of the most used metal compounds in pig setting (e.g., as supplements in animal feed), suggesting that in these environments higher selective pressures could contribute for the co-selection of MDR NTS clones [25,126,127,159,[163][164][165], with consequences for food safety and human health.

Conclusions
Pork products are among the most frequent foodstuffs implicated in human salmonellosis, with pig and pork meat reported worldwide as important sources of NTS resistant to clinically-relevant antibiotics, representing a major threat to the treatment of invasive infections. Furthermore, the high incidence of resistance to clinically-relevant antibiotics reported in diverse countries together with the increasing demand for pork meat and the global trade of pig/pork products raised the current public health concern. Hence, the continuous control and monitoring of Salmonella, particularly targeting specific MDR pig-related serotypes/clones, along the food chain (from primary production to consumption) is critical to minimize their introduction in the food-animal production and further transmission to humans. Therefore, a global integrated surveillance ("One Health" approach), and the implementation of more effective measures are critically needed, including the improvement of biosecurity measures at farms (e.g., providing uncontaminated feed, isolation of new purchased animals, high standards of hygiene, regular veterinary checks, vaccination), during slaughtering/processing (e.g., prevent external sources of contamination during transport, lairage and slaughter, cross-contamination with equipment and workers and hygiene practices such as cleaning and disinfection) and retail/consumer level (e.g., avoiding cross-contamination, using safe cooking temperature). Surveillance of antibiotic resistance levels in NTS throughout the pig food chain is crucial to ensure public health, not only through the detection of new food safety risks involving foodstuffs such as pork meat but also to avoid salmonellosis treatment failures.