Salmonellosis in swine results in tremendous economic losses in the pork industry [1
]. Salmonella enterica
serovar Choleraesuis (S.
Choleraesuis) causes clinical salmonellosis in pigs and wild boar (WB) [2
], and the identification of epidemiologic groups strongly suggests an exchange of this serovar between WB and domestic pigs [3
]. Nowadays, S. Choleraesuis
is still very common in North America and Asia and, although it is not considered a dominant serovar in pigs from Europe [4
], different outbreaks have occasionally been reported in recent years [6
] including in WB [2
The Iberian pig (IP) is an autochthonous breed that originated in the Iberian Peninsula, for which the production system is mainly associated with extensive management deeply linked to the Mediterranean ecosystem and traditional agroforestry in the southwest of the Iberian Peninsula [12
]. This means that WB and IP share the same habitats, leading to subsequent interactions among them [13
]. WB, as an omnivorous species, is prone to multiple pathogen exposure. They have been shown to carry resistant bacteria [15
] and could be a gateway for spread of this resistance from domestic animals or humans to wildlife [16
]. Besides, several studies have shown WB as a possible asymptomatic persistent reservoir of S.
Choleraesuis is swine-specific and rarely infects other hosts, it is the second most predominant serovar among human isolates in Taiwan and exhibits the highest degree of invasiveness [19
], which may result in severe disease and death [21
]. Most S.
Choleraesuis isolates from humans and swine exhibit closely related DNA fingerprints, indicating that human infections were acquired from pigs [22
], reinforcing the importance of controlling this serotype in Suidae.
Choleraesuis strains that have caused infections in humans, mainly in Asian countries, are multidrug resistant (MDR) [19
], which has been associated with classical mobile genetic elements (i.e., transposons and plasmids) and integrative elements that can spread antimicrobial resistance genes within the bacterial host genome through gene cassettes by site-specific recombination [24
]. In addition, plasmids can carry other gene functions such as those involved in virulence by pSCV50 in S. Choleraesuis
]. This 50 kb plasmid does not carry antimicrobial resistance genes, although it can recombine with larger sized plasmids detected in S. Choleraesuis
, and extended-spectrum beta-lactamase genes are located [27
In contrast to the limited administration of colistin (polymyxin E) to humans as a last resort antibiotic, it has historically been used for prophylaxis in animal production [30
]. Consequently, a dramatic increase of colistin resistance has arisen in naturally sensitive Gram-negative bacteria, with the spread of plasmid carrying mcr-1
among other resistance determinants [31
]. Among different reservoirs, livestock is considered the main source of mcr
genes worldwide [32
], and a global concern exists due to their high mobilization potential by plasmids carrying other resistance determinants [33
, one of the most clinically relevant enterobacteria, carries colistin resistance genes in many serovars via different plasmids, including IncHI2 mega-plasmids larger than 200 kb with multiple resistance determinants [34
]. In S.
Choleraesuis, this has been described very recently in one MDR isolate from a human blood infection in Brazil, linked to a 40 kb IncX4 plasmid [35
The aim of the present investigation was to study the genetic relationship between strains of S. Choleraesuis from IP and WB raised in the southwest of the Iberian Peninsula and to address the mechanism of spread of its antimicrobial resistance determinants, including through screening for low-susceptible isolates to colistin in this bacterial pathogen.
In this study, isolates of S. Choleraesuis
from IP and WB have been analyzed in order to trace the spread potential of antimicrobial resistance determinants carried by this serotype in the “dehesa”, a traditional agrosystem consisting of grassland with Holm’s oaks found in the Iberian Peninsula. The XbaI-PFGE profile of S.
Choleraesuis isolates revealed different PT, but most of the strains (16/20) belonged to the same cluster with a degree of similarity above 75% (Cluster A), among which PT1, PT3, and PT5 might represent clones with high potential spread both in space and time, in agreement with previous studies in WB [2
] and domestic pigs [5
]. With regard to phylogeographic analysis, a recent study demonstrated cross-border transmission of S. Choleraesuis
from pigs between countries that was concordant with the trading network [18
]. In our study, genetic relationships were detected not only among bacteria from the same species, but also with the wild ancestor of pigs, the WB, which share the “dehesa” environment with IP [14
]. In our study, the geographical link between animals is maximal for WB from estates E4 and E1, the closest to IP farms E6 and E11 (Figure 3
) from which S. Choleraesuis
isolates share PT1, PT2, or PT3 in closely related backgrounds (>95%, Figure 1
). On the other hand, it should be noted that there are large distances between these estates; approximately 70 km between E6 and E1 and all of them (E1, E4, E6, and E11) in a radius of 90 km (Figure 3
). Apart from the distance, the estates are also separated by several highways (E11 and E4) and a large river (E4). Moreover, one WB isolate from a faraway estate, E12, also shares PT1. When considered together, all these facts suggest that proximity itself is not the main reason for the bacterial relationship and that other factors may be responsible, i.e., human carriers or animal trading, although evidence is lacking. Together with studies showing the spread of S.
Choleraesuis between WB and domestic pigs [3
], including asymptomatic WB in Europe [17
], our results show a wildlife reservoir that may spill over to farmed pigs or vice versa.
MDR was detected in 83.3% (10/12) of isolates from IP in this study, higher than the 37.5% (3/8) observed in WB. Similar prevalences of antimicrobial resistance have been reported in S. Choleraesuis
from domestic pigs in Asia [26
] but these are higher than previous reports in Europe [5
]. The data from WB are similar to those previously described by our group [2
]. Likewise, the antibiotic groups with higher resistances differ between S. Choleraesuis
from the analyzed Suidae, showing resistance to ampicillin and sulfonamide for bacteria from IP, and sulfonamide, tetracycline, and streptomycin from WB, although streptomycin resistance had the same ratio in bacteria from both hosts, similarly to previous reports [2
]. The lack of resistance found against quinolones and cephalosporins is in accordance with most of the studies from Europe [18
], although outbreaks or sporadic cases of infections caused by Salmonella
spp. with resistance to these antibiotics are being increasingly reported [39
Isolates of S. Choleraesuis
from the two hosts screened in this study, IP and WB, presented quantitative differences in antibiotic resistance found against ampicillin and trimethoprim/sulfamethoxazole, which are higher in the autochthonous pig breed. In contrast, resistance to chloramphenicol, gentamicin or colistin was only detected in IP. This could be due to the fact that many of these antibiotics have been extensively used as growth promoters (beta-lactams) or as prophylactic agents for common diseases such as colibacillosis (colistin) or coccidiosis (sulfonamides) in pig farms for a long time [44
], which has been associated with increases in resistant bacteria [45
]. Although the IP production system is linked to the dehesa in the last period of fattening, the first stages of breeding mostly take place on farms with semi intensive management systems. It was in these stages where antibiotic abuse has taken place in the past. Considering that frequent use has a stronger association with resistance than sporadic use [46
], it could explain the lower number of resistances found in WB, as the treatments applied to them, when applied, are scarce and limited to certain short periods of time, which is different to the IP, especially in the early stages of breeding. However, even on estates that did not apply any antibiotic treatment, antibiotic resistances were found in S. Choleraesuis
from WB. This could be due to the omnivorous behavior of WB, which means they visit communal refuse sites as well as the proximity of farmed animals like IP in free range production systems, where horizontal transmission of bacteria might occur [48
Resistance genes have been previously detected in S.
Choleraesuis from pigs and humans [18
], but information is scarce in WB [2
]. In this study, we described Class 1 integrons in 42% of the S. Choleraesuis
isolates from IP and none in WB. Around 41% of these integrons carried a resistance gene cassette. These genetic elements play an important role in the development of antibiotic resistance and have a worldwide distribution in Gram-negative bacteria, colonizing both humans and animals [50
]. In S.
Choleraesuis from pigs, finding these elements in a large number of isolates is very common [39
]. Interestingly, our study shows that the sul3
gene occurs in 3 out of 5 Salmonella isolates carrying class 1 integrons, although the presence of this integron is more frequently related to the spread of the sul1
Our study reveals that, with exceptions, S.
Choleraesuis strains from IP or WB carry plasmids which are around 50 kb in size (Figure 1
), that isolates lacking antimicrobial resistance did not present additional plasmids, and that bacteria expressing multiple antimicrobial resistance share mega-plasmids, alone or in addition to the 50 kb bands (Table 1
). The fact that only closely related isolates share plasmid bands and/or antimicrobial resistance patterns might suggest that clonal spread prevails over horizontal transfer as the common mechanism for dispersion of antimicrobial resistance determinants in the analyzed environment. This study also shows the presence of the colistin-resistant gene mcr-1
in one of the isolates studied from IP. In this strain, mcr-1
is carried by a high-molecular weight plasmid (>240 kb), possibly conferring MDR and most probably belonging to the IncHI2-type replicon (different to the recent finding in a human isolate) [35
], which could represent a risk for accumulation and/or spread antimicrobial resistance determinants through food chain environments, as for Iberian pigs, and their processed products and humans. Although more studies are needed to determine its prevalence, due to its clinical importance in human health, the presence of these colistin-resistant Salmonella isolates should be monitored in order to control their evolution.