Introduction

Pork is the most produced and consumed animal protein in the world [1]. In Brazil, swine production has been showing relevant growth rates since the 1980s, mainly due to the intensive breeding of animals, associated with genetic, nutritional, and health improvement [2]. However, an intensive farming system can enhance the spread of important pathogens in the swine production, such as the use of collective drinking and feeders, high stool density, direct contact between animals, and number of animals per barn [3, 4].

Different foodborne pathogens can be associated to the swine chain. Yersinia enterocolitica is considered an emerging pathogen and pigs are described as reservoirs, once they can carry this pathogen asymptomatically into their lymph nodes, tonsils, and intestines [5, 6]. As consequence, poor hygienic conditions and inadequate procedures during pigs slaughtering and processing can facilitate the contamination and spread of Y. enterocolitica to carcasses, environment, utensils, and end products [7,8,9].

The pathogenic potential of Y. enterocolitica to humans is determined by virulence features encoded by genes located at the bacterial chromosome or in a plasmid: pYV [10, 11]. Virulence activities of chromosomal genes are mainly related to adhesion and invasion (ail, inv, mvf, hreP), toxin production (yst), enterotoxic activity (tccC), iron production (fes, fep), and virulence modulation (ymoA) [10, 12,13,14,15]. Among the pYV genes, virF is described as a transcriptional regulator of genes associated with important proteins, such as YadA (adhesin A) and Yops (other Yersinia proteins), related to adherence and activity against host immune cells [16, 17].

Besides the pathogenic potential of foodborne pathogens, there is a current worldwide concern related to antibiotic resistance [18]. Y. enterocolitica is usually susceptible to different antibiotics, such as aminoglycosides, tetracycline, chloramphenicol, extended spectrum cephalosporins, and trimethoprim-sulfamethoxazole [19]. However, as Y. enterocolitica can be hidden in lymphatic tissues since early ages of pigs, it is consequently subjected to all antibiotic treatments during the different stages of swine production and it leads to a natural development of resistance against these substances [20, 21]. In addition, the contact with other bacteria allows transference of genetic material, leading to modifications and acquisition of resistance-related genes and plasmids, resulting in increase of the antibiotic resistance characteristics by Y. enterocolitica strains [22, 23].

There are no official data or report of human yersiniosis associated to pork consumption in Brazil, but some studies have demonstrated the relevance of the pork production chain in harboring Y. enterocolitica [24,25,26,27]. Thus, studies that characterize the distribution and the virulence of Y. enterocolitica in Brazil are necessary to support the epidemiological characterization of this foodborne pathogen in the Brazilian pork production chain. Here, we characterized the distribution, virulence, and antibiotic resistance of Y. enterocolitica in Western Paraná, a Southern Brazilian region known by its relevance in the pork production.

Material and methods

Sampling

A pork production chain from Western Paraná, a Southern Brazilian state, was selected for this study. Ten pig lots from different farms were selected and samples of water (n = 10, 25 mL), feed (n = 10, 25 g), and barn floors (n = 10, footprint, as described by Botteldoorn et al. [28]) were obtained. During the slaughtering of the selected pig lots, carcasses were surface sampled at different stages: after bleeding (n = 100), after singeing (n = 100), after evisceration (n = 100), and after final rinse (n = 100): four sterile molds of 100 cm2 were placed in different carcass sites and swabbed with pre-moistened sponges (3 M Microbiology, St. Paul, MN, USA), as described by ISO 17,604 [29]. Prior to slaughtering, the lairage floors of pig lots were sampled by footprint (n = 10). Mesenteric lymph nodes (n = 100, 12.5 g) and palatine tonsils (n = 100, 12.5 g) were also samples from the selected carcasses. During the processing of the selected lots, surface samples of end cuts (n = 40), knives (n = 40), steel gloves (n = 40), cutting boards (n = 20), and conveyor belts (n = 20) were also obtained, as described above. All samples were transferred to sterile bags and kept refrigerated until laboratory analysis.

Detection of Yersinia enterocolitica

The samples were subjected to Y. enterocolitica detection based on ISO 10,273 [30], with modifications. Aliquots of 25 mL of water and 25 g of feed were diluted in 225 mL peptone-sorbitol-bile (PSB, Sigma-Aldrich, St. Louis, MO, USA), homogenized in Stomacher Seward 400® for 1 min (230 rpm). Samples from surface, lymph nodes, and tonsil were diluted in 160 mL and 112.5 mL of 0.1% peptone saline, respectively, and homogenized. Forty milliliters of aliquots of these samples were transferred to falcon tubes and centrifuged at 2,000 × g for 15 min. The obtained pellet was suspended in 10 mL of PSB broth and incubated at 25° C for 72 h. After incubation, 0.5 mL aliquots of the PSB cultures were transferred to 4.5 mL 0.5% potassium hydroxide (KOH) solution for 20 s and then streaked on cefsulodin-irgasan-novobiocin agar (CIN, Oxoid, Basingstoke, England). The plates were incubated at 30 °C for 18 to 48 h, when typical colonies of Y. enterocolitica were observed (small colonies with “red bull’s eyes red” center). Up to three suspected colonies from each plate were selected, purified, and subjected to biochemical tests of urease, indole, citrate, glucose fermentation, glucose gas production, lactose fermentation, H2S production, mobility, and liquid disposal of lysine to confirm the results [30]. Y. enterocolitica subsp. enterocolitica ATCC 9610 was used as positive control.

Isolates that presented biochemical results coherent with Y. enterocolitica were subjected to DNA extraction by boiling [31] and PCR assays targeting inv and a specific region of 16 s rRNA, for Y. enterocolitica identification [32]. Also, PCR assays were performed targeting per, wbbU, wbcA, and wzt for characterization of serotypes O:9, O:3, O:8, and O:5,27, respectively [32]. Amplification reactions were conducted using Gotaq Green Master Mix (Promega Corp., Madison, WI, USA), 200 nMol of each primer, 40 ng of extracted DNA, and nuclease free water with 25 μL final volume. PCR products were visualized after 1.5% agarose gel electrophoresis in GelRed ™ Tris–borate-EDTA (TBE) buffer (Biotium, Inc., Fremont, CA, USA). Primer sequences, conditions used for PCR amplifications, and expected product sizes are specified in the Supplementary Table. Y. enterocolitica subsp. enterocolitica ATCC 9610 was considered as the positive control for PCR assays.

Isolates identified as Y. enterocolitica were subject to DNA macro-restriction with XbaI (Promega) and pulsed field gel electrophoresis (PFGE), as indicated by PulseNet (Centers for Disease Control and Prevention, Atlanta, GA, USA), following the protocol described by Ribot et al. [33]. The obtained band profiles were compared using the software Bionumerics 6.6 (Applied Maths, Ghent, Belgium), considering 5% optimization and 5% Dice coefficient. Band profiles from Y. enterocolitica isolates (n = 8) were included for a comparative analysis. These isolates were obtained from different steps of the pork production chain (tonsils, mesenteric lymph nodes, pork carcasses) in Minas Gerais state, Brazil, using the same isolation protocol adopted in this study and identified as bioserotype 4/O:3 [25].

Based on the obtained band profiles, one isolate was selected and biotyped in the Yersinia Research Reference Laboratory of the College of Pharmaceutical Sciences at the University of São Paulo (USP; Ribeirão Preto, SP, Brazil) using the protocol described by Petersen et al. [34].

Virulence-related genes

Isolates identified as Y. enterocolitica were subjected to PCR assays for detection of virulence-related genes, as described by Martins et al. [25]. virF, myfA, ystA, ystC, fepA, fepD, fes, tccC, ymoA, and hreP genes were screened by individual PCR assays using primers described by Bhagat and Virdi [35]. Primer sequences, PCR conditions, and expected product sizes are specified in the Supplementary Table.

Antibiotic resistance

Y. enterocolitica isolates were characterized according their antibiotic resistance based on disk diffusion assay, following the recommendations of the Clinical and Laboratory Standards Institute [36]. Seventeen antibiotics from eleven classes were considered: (1) aminoglycosides: gentamicin (10 μg) and amikacin (30 μg); (2) fluoroquinolones: ciprofloxacin (5 μg) and norfloxacin (10 μg); (3) tetracyclines: doxycycline (30 μg) and tetracycline (30 μg); (4) phenicols: chloramphenicol (30 μg); (5) third-generation cephalosporins: ceftriaxone (30 μg); (6) folate pathway inhibitors: trimethoprim (5 μg) and sulfonamide (300 μg); (7) carbapenem: meropenem (10 μg) and imipenem (10 μg); (8) quinolone: nalidixic acid (30 μg); (9) penicillins: ampicillin (10 μg) and amoxicillin (10 μg); (10) macrolides: azithromycin (15 μg); (11) lipopeptides: polymyxin B (300 IU). All antibiotics were purchased from Sigma-Aldrich. Escherichia coli ATCC 25,922 was considered as a pan-susceptible quality control. Results were interpreted according to enterobacterial susceptibility standards [36].

Also, the extracted DNA of the Y. enterocolitica isolates was subjected to PCR assays for detection of the antibiotic resistance–related genes yfhD, emrD, marC (multidrug resistance), and sat (streptogramins), as described previously [25, 35]. Primer sequences, PCR conditions, and expected product sizes are specified in the Supplementary Table.

Results

Considering all samples (n = 800), 108 (13.5%) presented characteristic colonies on CIN agar, allowing the selection of 307 typical isolates. After biochemical assays, 91 isolates from 48 samples presented results coherent with Y. enterocolitica, but only three, from a single sample (tonsil), were confirmed as such after PCR targeting 16 s rRNA and inv (Table 1). Based on these results, the frequency of Y. enterocolitica was 0.12% in the pork production chain, and 1.0% among tonsil samples. In addition, the three Y. enterocolitica isolates presented positive results only for wbbu, what characterize them as belonging to serotype O:3. The macrorestriction profiles of the three isolates are presented in Fig. 1. The isolates presented identical band profiles, also identical to 6 out of the 8 isolates obtained in Minas Gerais state [25]. These obtained isolates were grouped into two clusters with more than 86% genetic similarity between them. Once the obtained isolates presented identical macrorestriction profiles, one was selected and identified as belonging to biotype 4.

Table 1 Frequencies of Y. enterocolitica positive samples (isolates) obtained from a pork production chain in Western Paraná, Southern Brazil, based on the presence of suspect and confirmed isolates
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Fig. 1
figure 1

Schematic representation of macrorestriction profile (XbaI) by PFGE (Dice coefficient with 5% tolerance) and phenotypic profiles of antimicrobial resistance in disk diffusion test (CLSI, 2017) of Y. enterocolitica isolates obtained from state of Paraná and Minas Gerais. AMP, ampicillin; AMX, amoxicillin; TRI, trimethoprim; NAL, nalidixic acid; POL, polymyxin B; SUL, sulfonamides; DOX, doxycycline; NOR, norfloxacin

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All isolates presented PCR amplification production for inv, ail, ystA, myfA, hrep, ymoA, tccC, and virF. However, fepA, fepD, and fes were not found in these isolates. Based on antibiotic resistance assays, the isolates presented PCR amplification products for yfhD, emrD, and marC. The three isolates were characterized as resistant to amoxicillin, ampicillin, and trimethoprim.

Discussion

The identification of the steps that have major influence on Y. enterocolitica contamination, from the farms to the animals’ slaughtering, is important for specific control measures and to monitor this pathogen along the production chain, resulting in safety of the end products [37]. Despite the low frequency of isolation, the identification of Y. enterocolitica in a palatine tonsil sample is consistent with the literature data, which indicates this site as the main location of this pathogen in pigs [8, 20, 25, 38, 39]. The presence of Y. enterocolitica in swine tonsils is considered as one of the main risk factors for carcass contamination in the slaughtering process and consequently in the end products, as these tissues are incised during inspection and slaughtering, increasing the chances of cross contamination [40, 41].

Low frequencies of Y. enterocolitica in pig slaughtering, especially in tonsils, have been reported in other studies conducted in Brazil. However, in São Paulo state, the occurrence of Y. enterocolitica positive samples was 8.0% in tonsils, tongues, submandibular, and mesenteric lymph nodes and knives [42]. Another study in São Paulo isolated 442 strains of Y. enterocolitica from a total of 792 samples collected from slaughtered pigs, slaughterhouse environment, and retail market [27]. From 400 samples of swine tonsils from Western Santa Catarina, 101 (25.25%) were positive for Y. enterocolitica [43]. In a similar study in Minas Gerais state, approximately 5% of swine tonsil samples were positive for Y. enterocolitica [25]. In other countries, especially in Europe, the presence of Y. enterocolitica in pig tonsils can be considered as high when compared to Brazilian studies, ranging from 13 to 90% [12, 38, 39, 44,45,46], while in the USA this prevalence was reported as 10% [47] and in China as 19.5% [48]. It is important to highlight that these differences can occur due to different isolation and detection approaches [17]. Conventional PCR and real-time PCR assays directly from samples and pre-enrichment broths have shown higher detection capacity of Y. enterocolitica than conventional procedures, and some studies already described that although the pathogen is present in samples, it is not always able to form colonies, which undermines the results obtained exclusively by culture plating [20, 21, 49]. Pork products usually contain a rich and diverse background microbiota, what can jeopardize the proper isolation of Y. enterocolitica by culture dependent methods [50]. Also, the low occurrence of Y. enterocolitica can be associated with the competing microbiota in the tested samples. It was already demonstrated that the serological prevalence of Y. enterocolitica is inversely proportional to the serological prevalence of Salmonella spp. in swine herds [51, 52]. In a parallel study with the same samples, Viana et al. [53] observed Salmonella occurrence of 45% in palatine tonsils, supporting this hypothesis.

Y. enterocolitica is described as an emerging pathogen in Europe due to the high number of reported yersiniosis cases in recent decades [5]. Fosse et al. [54] estimate that 77.3% of worldwide cases of yersiniosis may be associated with pork consumption. In Brazil, only a few studies describe the isolation and characterization of Y. enterocolitica from food, and the lack of epidemiological data from foodborne disease cases and outbreaks does not allow a proper characterization of the described yersiniosis, neither their potential link with contaminated pork products [26]. Y. enterocolitica isolates belonging to bioserotype 4/O:3 are described as the main pathogenic agents of yersiniosis in humans and animals worldwide, including in Brazil [10, 24, 32, 48, 55]. Martins et al. [25] identified Y. enterocolitica isolates from this bioserotype in a pork production chain in Minas Gerais, Brazil, and Paixão et al. [27] described that among 442 Y. enterocolitica isolates, all obtained from swine tonsils were characterized as serotype O:3.

The presence of the main virulence plasmid, pYV, is indicated by positive results for virF, responsible for coding one of the major pathogenicity factors of Y. enterocolitica, the type III secretion system [14, 17]. Despite presenting pYV, other chromosomal virulence genes are required for full pathogenicity capacity of Y. enterocolitica, such as inv, ail, ystA, myfA, hrep, ymoA, and tccC [14, 56]. These genes encode proteins that act in synergy for adhesion, internalization, and production of molecules necessary for enterotoxic activity to occur and to escape the host immune system [10, 12, 14, 24, 56, 57]. However, the absence of fepA, fepD, and fes indicates that the isolates obtained in this study have limited capacity for iron utilization, an essential factor for the development of most microorganisms [58].

Resistance to amoxicillin and ampicillin was already expected, once Y. enterocolitica is intrinsically resistant to these drugs [36]. All antibiotics usually recommended for yersiniosis treatment were effective against the obtained isolates: tetracycline, chloramphenicol, gentamicin, and third-generation cephalosporins [13]. Some studies have demonstrated increasing frequencies of multidrug-resistant Y. enterocolitica, as we observed based on phenotypical and molecular assays [25, 28, 31, 59]. Besides presenting amplification for yfhD, emrD, and marC, related to multidrug resistance, the three isolates also presented positive results for sat, related to resistance to streptogramins (virginiamycin), an antibiotic used as a feed additive [35, 60]. The use of antibiotics in animal production as growth promoters and prophylaxis is considered one of the main causes for the development of resistance, and the pork production is known by the wide use of drugs with these purposes when compared to other livestock systems [61]. Considering the strong epidemiological link between Y. enterocolitica and the pork production chain, monitoring the antibiotic resistance profiles of pork-related bacteria can be considered critical with regard to food safety and the performance of yersiniosis treatments in humans [19].

The results from the Minas Gerais’ strains are different probably because of the variances in pork production between these Brazilians states, and distinct drugs are probably being considered during production and resulting in these different profiles. Identification of Y. enterocolitica from Paraná and Minas Gerais states from a same serotype (O:3) and sharing identical band patterns suggests the low variability of this pathogen circulating in these two Brazilian states. Rusak et al. [26] reported a high similarity based on PFGE after XbaI macro-restriction among Y. enterocolitica from different bioserotypes and samples (swine, food, and clinical patients) in Brazil: all isolates identified as O:3 were grouped in a single cluster, with high similarity index.

Here we reported a low occurrence of Y. enterocolitica in a pork production chain, specifically in Western Paraná, a relevant pork production region in Southern Brazil. Despite the low occurrence, the Y. enterocolitica 4/O:3 isolates obtained presented high pathogenic potential and resistance to three antibiotics, but identical XbaI macro-restriction patterns with isolates obtained from other Brazilian state, Minas Gerais. These results suggest that strains descending from a common subtype may be circulating in the pork production chain of two states from different regions of Brazil, leading to further studies to elucidate their genomic profiles and potential clonality.