BACKGROUND
acute gastroenteritis caused by salmonella spp.
represents a public health concern because of its high welfare and socio-economical impact in developed countries
<cit> .
in the usa, salmonellosis is the main cause of foodborne illness with  <dig> , <dig> human cases of non-typhoidal salmonellosis in  <dig>  of which a total of  <dig>   required hospitalization and  <dig>  had a fatal outcome
<cit> .
in the european union , salmonellosis is, after campylobacteriosis, the most common zoonosis, registering a total of  <dig>  human cases in 2011
<cit> .
besides laying hens and poultry, asymptomatically salmonella-infected pigs are a major source of human salmonellosis
<cit> , by intermittently shedding the pathogen in their faeces and thus contaminating pork and products thereof.
however, faecal excretion is not neccesarily indicative of a true infection of the animal.
in fact, after being ingested, salmonella may be present in faecal samples and pass through the pig gut lumen without invading the enterocytes.
to cause active infection, salmonellae should invade the enterocyte barrier and reach the local lymphoid system
<cit> .
accordingly, the proper diagnosis of this infection in pigs requires the identification of this pathogen in the mesenteric lymph nodes .
thus, eu reference studies in finishing pigs have been based on the detection of salmonella spp.
in mln at slaughter.
pigs are considered susceptible to most of salmonella serotypes and, although typhimurium is the most common, a large variety of other serotypes are also reported in surveillance studies at farm level
<cit> .
however, the presence of multiple infections in mln of a single animal, although suggested, has never been confirmed.
an additional challenge for human health is the emergence of multi-antimicrobial resistant  salmonella strains and the subsequent spread of the ar clones
<cit> .
pigs and other domestic species are recognized as a primary reservoir of multi-ar bacteria, usually associated with the selective pressure exerted by antimicrobial treatments
<cit> .
the emergence and spread of multi-ar salmonella are often related to both the acquisition and the fixation of bacterial mobile genetic elements such as plasmids, transposons or integrons
<cit> .
five classes of integrons carrying antibiotic resistance gene cassettes have been reported so far
<cit> .
class  <dig> integrons  are the most prevalent in the enterobacteriaceae family, containing different ar gene cassettes  that can be located either extrachromosomally or integrated in the salmonella genomic island  <dig>
<cit> .
coexistence in the same animal of salmonella strains showing different ar genes has been postulated to support the horizontal ar genetic exchange.
the aim of the present study was to ascertain whether different salmonella serotypes and/or strains can be simultaneously isolated from the same animal.
methods
experimental design, salmonella spp.
isolation and serotyping
a total of  <dig> fattening pig farms identified previously
<cit>  with high herd salmonella-prevalence and showing multiple circulating strain types  were selected for this study.
one pig from each farm was randomly selected at the slaughter line in the abattoir.
animal handling and slaughtering procedures were performed according to the current national legislation .
the whole intestinal package was removed from the selected carcasses at the evisceration point of the slaughter line, and mln samples  were collected in a sterile plastic bag , transported at 4°c to the laboratory and immediately processed for salmonella isolation.
isolation procedures were performed according to iso 6579:2002/amd 1: <dig> rules
<cit> , as described previously
<cit> .
after selective growth  on xylose lysine deoxycholate agar  and brilliant green agar  plates,  <dig> presumptive salmonella spp.
colonies from each mln sample were tranferred from selective plates to agar, then tested biochemically  and further confirmed by serotyping at the national reference laboratory centre for animal salmonellosis , following the kauffmann-white scheme
<cit> .
antimicrobial resistance
a total of  <dig> salmonella colonies were tested by the kirby-bauer disk diffusion method
<cit>  using the antimicrobials and concentrations recommended by the current eu legislation for harmonized monitoring of antimicrobial resistance of salmonella in poultry and pigs
<cit> , namely, ampicillin and amoxicillin plus clavulanic acid , chloramphenicol , streptomycin , gentamicin, sulfisoxazole and trimethoprim plus sulfamethoxazole , tetracycline , nalidixic acid , enrofloxacin, and cefotaxime .
e. coli strain atcc  <dig>  and serovar typhimurium strains atcc  <dig> and dt <dig> were used as controls.
antimicrobial susceptibility was determined by measuring the inhibition halo generated after incubation .
strains were classified as resistant or susceptible, according to the clinical and laboratory standard institute  recommendations
<cit> .
the presence of ic <dig> was analysed by pcr using the primers 5′cs-3′cs described previously
<cit> , and the resulting amplicons were purified with a commercial kit , cloned in pgem®-t , and then sequenced .
dna sequences were analysed by expasy protein translation  followed by protein basic local alignment search tool  analysis.
the presence of sgi <dig> was also determined by pcr using u7-l <dig> and lj-r <dig> primers specific for sgi <dig> left junction amplification
<cit> .
pulsed-field gel electrophoresis
to identify simultaneous infections by different salmonella strains in a pig, the strains isolated from each animal showing identical phenotypic  and ar genotypic  characteristics, were analysed by pfge, following the pulse-net protocol described by the centre for disease control and prevention
<cit> .
briefly, the agarose plugs containing dna were digested with  <dig> u xbai .
dna fragments were separated  in 1% agarose gels with  <dig> x tris borate edta buffer, using a cheff-dr ii system , and dna was stained with 5% aqueous ethidium bromide solution.
lambda ladder  was used as molecular weight marker, and the dna obtained from serotype braenderup was used as control.
salmonella strains showing less than 95% pfge profile similarity were considered as different.
RESULTS
eight different serotypes were identified among the  <dig> salmonella strains obtained.
as shown in table
<dig>  serotypes typhimurium, rissen, derby, and kapemba were the most frequently isolated  and widely distributed .
fifty per cent of the pigs analysed  were found infected simultaneously by different salmonella strains, since  <dig> or  <dig> different serotypes were identified in each pig.
typhimurium was the serotype most frequently isolated  in these  <dig> pigs.
interestingly, serovar kapemba was always found simultaneously with typhimurium.
the remaining  <dig> pigs were found infected with a single serotype.
salmonella
strains isolated from fattening pigs mesenteric lymph nodes
a
b
c
aa total of  <dig> cfu  isolated in selective bga or xld media were purified in agar and characterized; bantimicrobial agents showing ar strains:  ampicillin and/or amoxicillin+clavulanic acid;  chloramphenicol;  streptomycin and/or gentamicin;  sulfonamides and/or trimetoprim+sulphometoxazole;  tetracycline and/or doxicycline;  nalidixic acid; cgenes identified by ic <dig> amplicons sequencing; dflagellar antigen phase  <dig> was not detected; esgi <dig> was detected; na: not applicable.
regarding phenotypic ar characteristics, pigs were infected with at least one multi-ar strain, and a total of  <dig> different ar profiles were identified .
the ar profile most frequently identified  was acssut, with or without additional resistance to nx .
genotypically, strains showing  <dig> types of ic <dig> were identified in  <dig> pigs , showing amplicons of either 1000 bp , 2000 bp , or a double band of 1000 bp and 1200 bp each  .
these ic <dig> were absent in the  <dig> strains found susceptible to all antimicrobials as well as in the other  <dig> strains showing ar to one  or several  agents.
amplicon sequencing allowed the identification of ic <dig> carrying  <dig> different ar gene cassettes:  blaoxa30-aada <dig> contained in 2000 bp amplicons of typhimurium strains;  drfa12-aada <dig> contained in 2000 bp amplicons of goldcoast, rissen and bredeney;  aada <dig> contained in 1000 bp amplicons of kapemba and derby; and  aada2-pse <dig> contained in  <dig> plus 1200 bp amplicons .
this latter ic <dig> was found only in typhimurium strains, and associated with both the acssut penta-ar profile and the presence of sgi <dig>  accordingly, these strains showed the characteristics of the dt <dig> phage-type.
the remaining  <dig> typhimurium strains  also showed the acssut penta-ar profile but only the 2000 bp blaoxa30-aada <dig> ic <dig> amplicon not associated with sgi <dig> was amplified .
similarly, the  <dig> kapemba strains  were resistant to cssut and carried a single 1000 bp ic <dig> containing the aada <dig> ar gene .
interestingly, in  <dig> out of the  <dig> pigs infected with a unique salmonella serotype ,  <dig> or  <dig> different ar profiles were identified, regardless of the presence/absence and size/sequence of ic <dig> amplicons.
this clearly indicates also the presence of different salmonella strains infecting the same animal.
the remaining  <dig> pigs  were infected by a unique and homogeneous salmonella strain, as confirmed by pfge.
overall,  <dig> out of the  <dig> pigs studied were infected simultaneously by at least  <dig> different salmonella strains.
discussion
to the best of our knowledge, this is the first report in pigs demonstrating that the same animal may be naturally infected by multiple salmonella strains.
for this, a thorough microbiological analysis of mln was carried out in a limited number of animals belonging to farms with high salmonellosis prevalence and where multiple circulating salmonella strain types were previously identified.
although it was not the objective of this study, our results suggest that salmonella co-infections may be quite common in pig herds with multiple salmonella circulating strains.
the existence of multiple infections in the same animal suggests that pigs can be either infected simultaneously during a brief period either through one or multiple sources  or re-infected along the different stages of their productive life .
the possibility of re-infection has been previously proposed in sows from which different salmonella serotypes were isolated from faecal samples collected at different time points
<cit> .
nevertheless, the presence of the pathogen in faeces does not necessarily mean an active infection, as salmonella can circulate passively through the animal’s gut lumen.
faecal culture results should interpreted with caution since these samples can also be easily cross-contaminated during collection.
in our study, however, the presence of salmonella in mln would reflect a true infection.
in the present study, the possibility of mln cross-contamination was very limited because  sampling was performed at different dates;  we used single-use gloves and clothes, liquid disinfectant  and sterilized instruments each time;  mln samples were individually collected in sterile plastic bags; and  once in the laboratory, mln samples were defatted and externally decontaminated through alcohol immersion and flaming, as recommended by the iso method
<cit> .
thus, our results demonstrate the presence of active multiple infections as different salmonella strains were isolated from mln tissue, which could be colonised only after active enterocyte invasion
<cit> .
typhimurium and rissen were the most prevalent salmonella serotypes identified, which is in agreement with the findings of a large study performed previously in the same pig population
<cit> .
it is worth to note that kapemba was also found in a relative high frequency  but always accompanied by typhimurium.
in contrast, kapemba was rarely isolated at both individual  and herd  levels in the previous large study
<cit> , and also in the baseline study carried out in the eu
<cit> .
such differences could be due to the different identification strategy used in these studies, since serotyping was performed exclusively on one colony from each animal in these large-scale studies.
for epidemiological purposes, the international standards recommend confirming the presence of salmonella by typing one  colony per sample
<cit> .
although this microbiological approach may be useful to confirm infection, it could easily overlook the presence of the less predominant strains, since the more prevalent ones appear to be always present in mln co-infections .
therefore, epidemiological studies based on the serotyping of a single bacterial colony, such as those focused on the eradication of specific serotypes  may be overrepresenting the prevalent strains and understimating other potentially pathogenic but less predominant serotypes.
likewise, outbreak investigations would require the analysis of several colonies from the same animal to identify the main source of infection.
systematic screening of multiple colonies from individual pig samples could contribute to the trace back of many salmonella outbreaks origin in humans
<cit> .
the coexistence of salmonella strains with different multi-ar profiles within the same pig as primary reservoir may have important epidemiological consequences.
this can promote exchange and propagation of mobile genetic elements between bacterial strains that share the same biological niche in vivo.
in this study, co-infections by salmonella strains showing different ar profiles were relatively frequent, regardless of the serotype.
in fact, most of animals studied  were simultaneously infected by strains showing  <dig> or  <dig> different ar profiles.
the finding that pigs with co-infections showed different ar profiles against common antimicrobial agents suggested that genetic exchange could be taking place within the same animal, generating a genetic variability in salmonella.
horizontal transfer of ar genes or ic <dig> was not observed in three animals  harbouring both susceptible and multi-ar strains, but genetic exchanges could not be excluded in these animals
<cit> .
sgi <dig> was detected only in typhimurium strains from two animals  containing also the characteristic ic <dig> 1000-1200 bp double band with the double aada2-pse <dig> gene cassette, and the typical penta-ar  of dt <dig> phagetype
<cit> .
the widespread dissemination of typhimurium dt <dig> clone was particularly relevant since it was first isolated in the early 80´s in uk cattle and subsequently reported worldwide in a wide variety of animal species including pigs, animal foodstuff, and humans
<cit> .
similarly, other emergent variants, such as the monophasic variant of typhimurium dt <dig> phagetype carrying the multi-ar assut
<cit>  have been detected, and epidemiological surveillance is therefore recommended
<cit> .
ic <dig> genotypes are the most frequent carriers of ar genes in salmonellae, but these genes could also be present in other integrons
<cit> .
in fact, ic <dig> was not detected in some strains resistant to one  or more  antimicrobial agents.
however, a quick detection of ar strains is critical for a successful treatment in human beings.
thus, the ic <dig> pcr analysis of several salmonella colonies from a salmonella-positive sample should be considered as a suitable  screening approach for detecting multi-ar genetic mobile elements.
the presence of simultaneous infections by salmonella strains of different serotype, serogroup and ar profiles could also have immunological implications on the host-pathogen interaction.
thus, if infections occur over time, our results may suggest a limited genus-, serogroup- and species- specific protection of pigs after a primary salmonella infection, but further studies are needed for a better understanding of the host-pathogen interactions.
the existence of co-infections in a single animal and within the same herd may assist in the development of effective vaccines, therapeutics and control programmes against pig salmonellosis.
CONCLUSIONS
this study demonstrates the presence of simultaneous infections by different salmonella strains in asymptomatic pigs.
systematic screening for multiple strains from individual mln samples is a time-consuming strategy not routinely applied in laboratory protocols but essential to understanding both the pathogenesis and epidemiology of salmonella infections in pigs.
it may also be useful to trace back the origin of salmonellosis outbreaks in humans.
further studies in larger pig populations should be carried out to confirm that salmonella co-infections are a common event in swine.
competing interest
the authors declare that they have no competing interests.
authors’ contributions
all authors participated in the draft of the manuscript.
moreover, vg, bsr, ss and azb carried out the sampling collection in slaughterhouses, microbiological isolation, biochemical identification, antimicrobial resistance characterization, and pfge studies; ydt and cdf carried out the serotyping; rcmj participated in the design of the study and discussion of results; and mjg conceived, designed, and coordinated the study, and wrote the final manuscript.
all authors read and approved the final manuscript.

 acute gastroenteritis caused by salmonella spp. represents a public health concern because of its high welfare and socio-economical impact in developed countries. in the usa, salmonellosis is the main cause of foodborne illness with, human cases of non-typhoidal salmonellosis in of which a total of required hospitalization and had a fatal outcome. in the european union, salmonellosis is, after campylobacteriosis, the most common zoonosis, registering a total of human cases in 2011. besides laying hens and poultry, asymptomatically salmonella-infected pigs are a major source of human salmonellosis, by intermittently shedding the pathogen in their faeces and thus contaminating pork and products thereof. however, faecal excretion is not neccesarily indicative of a true infection of the animal. in fact, after being ingested, salmonella may be present in faecal samples and pass through the pig gut lumen without invading the enterocytes. to cause active infection, salmonellae should invade the enterocyte barrier and reach the local lymphoid system. accordingly, the proper diagnosis of this infection in pigs requires the identification of this pathogen in the mesenteric lymph nodes. thus, eu reference studies in finishing pigs have been based on the detection of salmonella spp. in mln at slaughter. pigs are considered susceptible to most of salmonella serotypes and, although typhimurium is the most common, a large variety of other serotypes are also reported in surveillance studies at farm level. however, the presence of multiple infections in mln of a single animal, although suggested, has never been confirmed. an additional challenge for human health is the emergence of multi-antimicrobial resistant salmonella strains and the subsequent spread of the ar clones. pigs and other domestic species are recognized as a primary reservoir of multi-ar bacteria, usually associated with the selective pressure exerted by antimicrobial treatments. the emergence and spread of multi-ar salmonella are often related to both the acquisition and the fixation of bacterial mobile genetic elements such as plasmids, transposons or integrons. five classes of integrons carrying antibiotic resistance gene cassettes have been reported so far. class integrons are the most prevalent in the enterobacteriaceae family, containing different ar gene cassettes that can be located either extrachromosomally or integrated in the salmonella genomic island. coexistence in the same animal of salmonella strains showing different ar genes has been postulated to support the horizontal ar genetic exchange. the aim of the present study was to ascertain whether different salmonella serotypes and/or strains can be simultaneously isolated from the same animal. methods experimental design, salmonella spp. isolation and serotyping a total of fattening pig farms identified previously with high herd salmonella-prevalence and showing multiple circulating strain types were selected for this study. one pig from each farm was randomly selected at the slaughter line in the abattoir. animal handling and slaughtering procedures were performed according to the current national legislation. the whole intestinal package was removed from the selected carcasses at the evisceration point of the slaughter line, and mln samples were collected in a sterile plastic bag, transported at 4°c to the laboratory and immediately processed for salmonella isolation. isolation procedures were performed according to iso 6579:2002/amd 1: rules, as described previously. after selective growth on xylose lysine deoxycholate agar and brilliant green agar plates, presumptive salmonella spp. colonies from each mln sample were tranferred from selective plates to agar, then tested biochemically and further confirmed by serotyping at the national reference laboratory centre for animal salmonellosis, following the kauffmann-white scheme. antimicrobial resistance a total of salmonella colonies were tested by the kirby-bauer disk diffusion method using the antimicrobials and concentrations recommended by the current eu legislation for harmonized monitoring of antimicrobial resistance of salmonella in poultry and pigs, namely, ampicillin and amoxicillin plus clavulanic acid, chloramphenicol, streptomycin, gentamicin, sulfisoxazole and trimethoprim plus sulfamethoxazole, tetracycline, nalidixic acid, enrofloxacin, and cefotaxime. e. coli strain atcc and serovar typhimurium strains atcc and dt were used as controls. antimicrobial susceptibility was determined by measuring the inhibition halo generated after incubation. strains were classified as resistant or susceptible, according to the clinical and laboratory standard institute recommendations. the presence of ic was analysed by pcr using the primers 5′cs-3′cs described previously, and the resulting amplicons were purified with a commercial kit, cloned in pgem®-t, and then sequenced. dna sequences were analysed by expasy protein translation followed by protein basic local alignment search tool analysis. the presence of sgi was also determined by pcr using u7-l and lj-r primers specific for sgi left junction amplification. pulsed-field gel electrophoresis to identify simultaneous infections by different salmonella strains in a pig, the strains isolated from each animal showing identical phenotypic and ar genotypic characteristics, were analysed by pfge, following the pulse-net protocol described by the centre for disease control and prevention. briefly, the agarose plugs containing dna were digested with u xbai. dna fragments were separated in 1% agarose gels with x tris borate edta buffer, using a cheff-dr ii system, and dna was stained with 5% aqueous ethidium bromide solution. lambda ladder was used as molecular weight marker, and the dna obtained from serotype braenderup was used as control. salmonella strains showing less than 95% pfge profile similarity were considered as different.  eight different serotypes were identified among the salmonella strains obtained. as shown in table  serotypes typhimurium, rissen, derby, and kapemba were the most frequently isolated and widely distributed. fifty per cent of the pigs analysed were found infected simultaneously by different salmonella strains, since or different serotypes were identified in each pig. typhimurium was the serotype most frequently isolated in these pigs. interestingly, serovar kapemba was always found simultaneously with typhimurium. the remaining pigs were found infected with a single serotype. salmonella strains isolated from fattening pigs mesenteric lymph nodes a b c aa total of cfu isolated in selective bga or xld media were purified in agar and characterized; bantimicrobial agents showing ar strains: ampicillin and/or amoxicillin+clavulanic acid; chloramphenicol; streptomycin and/or gentamicin; sulfonamides and/or trimetoprim+sulphometoxazole; tetracycline and/or doxicycline; nalidixic acid; cgenes identified by ic amplicons sequencing; dflagellar antigen phase was not detected; esgi was detected; na: not applicable. regarding phenotypic ar characteristics, pigs were infected with at least one multi-ar strain, and a total of different ar profiles were identified. the ar profile most frequently identified was acssut, with or without additional resistance to nx. genotypically, strains showing types of ic were identified in pigs, showing amplicons of either 1000 bp, 2000 bp, or a double band of 1000 bp and 1200 bp each. these ic were absent in the strains found susceptible to all antimicrobials as well as in the other strains showing ar to one or several agents. amplicon sequencing allowed the identification of ic carrying different ar gene cassettes: blaoxa30-aada contained in 2000 bp amplicons of typhimurium strains; drfa12-aada contained in 2000 bp amplicons of goldcoast, rissen and bredeney; aada contained in 1000 bp amplicons of kapemba and derby; and aada2-pse contained in plus 1200 bp amplicons. this latter ic was found only in typhimurium strains, and associated with both the acssut penta-ar profile and the presence of sgi accordingly, these strains showed the characteristics of the dt phage-type. the remaining typhimurium strains also showed the acssut penta-ar profile but only the 2000 bp blaoxa30-aada ic amplicon not associated with sgi was amplified. similarly, the kapemba strains were resistant to cssut and carried a single 1000 bp ic containing the aada ar gene. interestingly, in out of the pigs infected with a unique salmonella serotype, or different ar profiles were identified, regardless of the presence/absence and size/sequence of ic amplicons. this clearly indicates also the presence of different salmonella strains infecting the same animal. the remaining pigs were infected by a unique and homogeneous salmonella strain, as confirmed by pfge. overall, out of the pigs studied were infected simultaneously by at least different salmonella strains. discussion to the best of our knowledge, this is the first report in pigs demonstrating that the same animal may be naturally infected by multiple salmonella strains. for this, a thorough microbiological analysis of mln was carried out in a limited number of animals belonging to farms with high salmonellosis prevalence and where multiple circulating salmonella strain types were previously identified. although it was not the objective of this study, our results suggest that salmonella co-infections may be quite common in pig herds with multiple salmonella circulating strains. the existence of multiple infections in the same animal suggests that pigs can be either infected simultaneously during a brief period either through one or multiple sources or re-infected along the different stages of their productive life. the possibility of re-infection has been previously proposed in sows from which different salmonella serotypes were isolated from faecal samples collected at different time points. nevertheless, the presence of the pathogen in faeces does not necessarily mean an active infection, as salmonella can circulate passively through the animal’s gut lumen. faecal culture results should interpreted with caution since these samples can also be easily cross-contaminated during collection. in our study, however, the presence of salmonella in mln would reflect a true infection. in the present study, the possibility of mln cross-contamination was very limited because sampling was performed at different dates; we used single-use gloves and clothes, liquid disinfectant and sterilized instruments each time; mln samples were individually collected in sterile plastic bags; and once in the laboratory, mln samples were defatted and externally decontaminated through alcohol immersion and flaming, as recommended by the iso method. thus, our results demonstrate the presence of active multiple infections as different salmonella strains were isolated from mln tissue, which could be colonised only after active enterocyte invasion. typhimurium and rissen were the most prevalent salmonella serotypes identified, which is in agreement with the findings of a large study performed previously in the same pig population. it is worth to note that kapemba was also found in a relative high frequency but always accompanied by typhimurium. in contrast, kapemba was rarely isolated at both individual and herd levels in the previous large study, and also in the baseline study carried out in the eu. such differences could be due to the different identification strategy used in these studies, since serotyping was performed exclusively on one colony from each animal in these large-scale studies. for epidemiological purposes, the international standards recommend confirming the presence of salmonella by typing one colony per sample. although this microbiological approach may be useful to confirm infection, it could easily overlook the presence of the less predominant strains, since the more prevalent ones appear to be always present in mln co-infections. therefore, epidemiological studies based on the serotyping of a single bacterial colony, such as those focused on the eradication of specific serotypes may be overrepresenting the prevalent strains and understimating other potentially pathogenic but less predominant serotypes. likewise, outbreak investigations would require the analysis of several colonies from the same animal to identify the main source of infection. systematic screening of multiple colonies from individual pig samples could contribute to the trace back of many salmonella outbreaks origin in humans. the coexistence of salmonella strains with different multi-ar profiles within the same pig as primary reservoir may have important epidemiological consequences. this can promote exchange and propagation of mobile genetic elements between bacterial strains that share the same biological niche in vivo. in this study, co-infections by salmonella strains showing different ar profiles were relatively frequent, regardless of the serotype. in fact, most of animals studied were simultaneously infected by strains showing or different ar profiles. the finding that pigs with co-infections showed different ar profiles against common antimicrobial agents suggested that genetic exchange could be taking place within the same animal, generating a genetic variability in salmonella. horizontal transfer of ar genes or ic was not observed in three animals harbouring both susceptible and multi-ar strains, but genetic exchanges could not be excluded in these animals. sgi was detected only in typhimurium strains from two animals containing also the characteristic ic 1000-1200 bp double band with the double aada2-pse gene cassette, and the typical penta-ar of dt phagetype. the widespread dissemination of typhimurium dt clone was particularly relevant since it was first isolated in the early 80´s in uk cattle and subsequently reported worldwide in a wide variety of animal species including pigs, animal foodstuff, and humans. similarly, other emergent variants, such as the monophasic variant of typhimurium dt phagetype carrying the multi-ar assut have been detected, and epidemiological surveillance is therefore recommended. ic genotypes are the most frequent carriers of ar genes in salmonellae, but these genes could also be present in other integrons. in fact, ic was not detected in some strains resistant to one or more antimicrobial agents. however, a quick detection of ar strains is critical for a successful treatment in human beings. thus, the ic pcr analysis of several salmonella colonies from a salmonella-positive sample should be considered as a suitable screening approach for detecting multi-ar genetic mobile elements. the presence of simultaneous infections by salmonella strains of different serotype, serogroup and ar profiles could also have immunological implications on the host-pathogen interaction. thus, if infections occur over time, our results may suggest a limited genus-, serogroup- and species- specific protection of pigs after a primary salmonella infection, but further studies are needed for a better understanding of the host-pathogen interactions. the existence of co-infections in a single animal and within the same herd may assist in the development of effective vaccines, therapeutics and control programmes against pig salmonellosis.  this study demonstrates the presence of simultaneous infections by different salmonella strains in asymptomatic pigs. systematic screening for multiple strains from individual mln samples is a time-consuming strategy not routinely applied in laboratory protocols but essential to understanding both the pathogenesis and epidemiology of salmonella infections in pigs. it may also be useful to trace back the origin of salmonellosis outbreaks in humans. further studies in larger pig populations should be carried out to confirm that salmonella co-infections are a common event in swine. competing interest the authors declare that they have no competing interests. authors’ contributions all authors participated in the draft of the manuscript. moreover, vg, bsr, ss and azb carried out the sampling collection in slaughterhouses, microbiological isolation, biochemical identification, antimicrobial resistance characterization, and pfge studies; ydt and cdf carried out the serotyping; rcmj participated in the design of the study and discussion of results; and mjg conceived, designed, and coordinated the study, and wrote the final manuscript. all authors read and approved the final manuscript. 
the presence of simultaneous infections by different salmonella strains in the same animal has never been reported and could have important epidemiological implications. this may have implications in the interpretation of results from baseline studies, and also help to better understand human salmonellosis outbreaks and the horizontal transmission of antimicrobial resistance genes. baseline studies on salmonellosis prevalence in fattening pigs in europe are based on direct pathogen isolation from mesenteric lymph nodes. fourteen finishing pigs belonging to farms that showed high salmonellosis prevalence and a variety of circulating salmonella strains, were found infected by salmonella spp, and of them were simultaneously infected with strains of or different serotypes. this is the first report clearly demonstrating that pigs naturally infected by salmonella may harbour different salmonella strains simultaneously. four animals were found infected by salmonella spp.
 salmonellosis is a major worldwide zoonosis, and salmonella-infected finishing pigs are considered one of the major sources of human infections in developed countries. baseline studies on salmonellosis prevalence in fattening pigs in europe are based on direct pathogen isolation from mesenteric lymph nodes. this procedure is considered the most reliable for diagnosing salmonellosis in apparently healthy pigs. the presence of simultaneous infections by different salmonella strains in the same animal has never been reported and could have important epidemiological implications.  fourteen finishing pigs belonging to farms that showed high salmonellosis prevalence and a variety of circulating salmonella strains, were found infected by salmonella spp, and of them were simultaneously infected with strains of or different serotypes. typhimurium isolates showing resistance to several antimicrobials and carrying mobile integrons were the most frequently identified in the colonized mln. four animals were found infected by salmonella spp. of a single serotype but showing or different antimicrobial resistance profiles, without evidence of mobile genetic element exchange in vivo.  this is the first report clearly demonstrating that pigs naturally infected by salmonella may harbour different salmonella strains simultaneously. this may have implications in the interpretation of results from baseline studies, and also help to better understand human salmonellosis outbreaks and the horizontal transmission of antimicrobial resistance genes. salmonellamultiple infectionspigsserotypesantimicrobial resistance 