Prevalence and Tetracycline Susceptibility of Chlamydia suis in Different Intestinal Sections of Pigs from Commercial Farms

TL;DR

This study examines where Chlamydia suis is most common in pigs' intestines and how it responds to tetracycline.

Contribution

The study reveals segment-specific prevalence and tetracycline susceptibility patterns of C. suis in pigs.

Findings

C. suis was detected most frequently in the colon (40%) compared to the jejunum and ileum (4.5%).

Viable C. suis was most commonly isolated from the ileum, suggesting active replication there.

Tetracycline resistance was observed in 12.5% of isolates, with resistant strains found in the ileum and colon.

Abstract

Chlamydia suis, a close relative of the human pathogen C. trachomatis, can be detected in the porcine gut, yet its prevalence and viability across intestinal segments remain poorly defined. This study aimed to assess the segment-specific prevalence, isolation success, and tetracycline susceptibility of C. suis in grower-finisher pigs. Jejunal, ileal, and colonic samples (n = 200 per intestinal segment) were collected from 600 pigs at slaughter and analyzed using C. suis-specific real-time PCR and culture. PCR revealed significantly higher detection rates in the colon (40%) than in the jejunum or ileum (both 4.5%), accompanied by significantly higher calculated bacterial loads in colonic samples. In contrast, viable C. suis was most frequently isolated from ileal material, indicating that the ileum may provide a more favorable condition for active bacterial replication. Among 24 culture-confirmed isolates, 75% were susceptible to tetracycline (MIC/MBC < 2 µg/mL), 12.5% exhibited an intermediate phenotype (2 µg/mL < MIC/MBC < 4 µg/mL) and another 12.5% were resistant (MIC/MBC > 4 µg/mL). Intermediate phenotypes were recovered from the jejunum and ileum, whereas resistant isolates were found in the ileum and colon. These findings suggest that the porcine colon may serve as an intestinal reservoir for C. suis, while the ileum supports more robust bacterial replication. Overall, these data contribute to our understanding of the intestinal ecology of C. suis under field conditions and its tetracycline susceptible patterns.

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1. Introduction

Chlamydia suis is a Gram-negative, obligate intracellular bacterium that is phylogenetically closely related (>95% genome sequence identity) to the human pathogen C. trachomatis [1], which causes ocular and genital infections in humans. Chlamydia suis is endemic in many pig populations worldwide, and some studies found C. suis in humans. Dean et al. [2] detected C. suis mRNA in the eyes of trachoma patients in Nepal who lived in close contact with pigs. Similarly, De Puysseleyr et al. [3] isolated C. suis and detected its DNA in ocular samples from slaughterhouse employees. Thus, while C. suis can be detected in human samples, it is not considered a confirmed zoonotic pathogen.

Chlamydia suis in pigs is mainly associated with conjunctival and genital infections, and it can also be detected in the intestinal tract. Despite its high prevalence, C. suis is often isolated from healthy animals and is generally considered an opportunistic or secondary pathogen rather than a primary cause of disease [4]. Subclinical intestinal infections are common, suggesting that C. suis typically coexists with the host without causing overt disease. Detection in the gastrointestinal (GI) tract of clinically healthy pigs indicates that it may serve as a natural reservoir, consistent with frequent isolation from fecal swabs of pigs without intestinal symptoms [5]. Fecal shedding from subclinical infected pigs facilitate transmission via the fecal–oral route, as well as through aerosols or direct contact. The concept of the GI tract as a reservoir is well established in C. muridarum mouse models [6,7,8,9] and is increasingly recognized in humans, where persistent, subclinical intestinal C. trachomatis infections may contribute to recurrent genital infections and facilitate transmission, as indicated by rectal detection in asymptomatic individuals [10].

There are limited data comparing the prevalence of C. suis in different intestinal regions, and our understanding of C. suis host–cell interactions and the presumed balanced relationship in the porcine gut remains poor. However, the role of intestinal colonization by Chlamydia suis in bacterial persistence, reinfection dynamics, and potential treatment failure remains poorly understood. Most existing studies relay on porcine intestinal cell cultures [11], archived histological intestinal slides [12,13], or pig models following intragastric or oral inoculation [14,15,16]. Consequently, we still lack systematic, segment-specific data on C. suis distribution along the porcine intestinal tract under field conditions.

On farms, C. suis infection is considered a multifactorial disease, often triggered by immunosuppressive factors [17]. There are no vaccines, and the infection is most often treated with tetracyclines, to which the bacterium is generally highly sensitive. However, tetracycline-resistant C. suis strains (carrying the tetracycline resistance class C gene, tet(C)) are emerging worldwide, and have been reported in Austria, Belgium, China, Cyprus, Estonia, Germany, Israel, Italy, Switzerland, and the U.S. [18,19,20,21]. In cases of phenotypically confirmed tetracycline-resistant C. suis infections, the use of fluoroquinolones, currently listed on EMA’s category B list of veterinary drugs [22,23], and therefore restricted in swine production, is permitted. Tetracycline resistance therefore not only threatens food security but also increases the risk of untreatable C. suis infections and facilitates the spread of these potentially zoonotic strains in pigs. Furthermore, the tet(C) gene, which can be horizontally transferred to C. trachomatis in vitro [24], could potentially be transferred to C. trachomatis in humans. This is concerning, as human C. trachomatis infections are sometimes treated with doxycycline [25,26].

This study aimed to assess the segment-specific prevalence, isolation success, and tetracycline susceptibility of C. suis in pigs under field conditions. Jejunal, ileal, and colonic samples (n = 200 per intestinal segment) were collected from 600 animals at slaughter and analyzed using C. suis-specific real-time PCR and culture. The tetracycline susceptibility of intestinal isolates was assessed phenotypically by their minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC).

2. Materials and Methods

2.1. Sample Collection and Study Design

Samples were collected between February 2025 and September 2025 at a slaughterhouse in Flanders, Belgium. At three independent time points, intestinal samples were obtained from different pig populations to collect distinct gut segments. Specifically, jejunal swabs were collected from 20 pigs originating from 10 farms (farms 1 to 10), ileal swabs from 20 pigs from 10 other farms (farms 11 to 20), and colon descendens swabs from 20 pigs from a further 10 farms (farms 21 to 30). Each intestinal region thus represents an independent dataset derived from separate animals and farms. This design aimed to explore the occurrence of C. suis across intestinal compartments at a population level, rather than to compare tissue-specific differences within individual pigs. Consequently, comparisons among tissues should be interpreted descriptively, not as paired intra-animal differences.

Two swabs (FlocQSwab^®^, Copan, Brescia, Italy) were collected from each intestinal sample by firmly swabbing the mucosal surface: one was placed in DNA/RNA stabilization buffer (Merck, Life Science BV, Overijse, Belgium), and the other in Chlamydia transport medium [27], resulting in two swabs per pig. All swabs were transported on ice. Upon arrival in the lab, swabs were shaken for 1 h at 4 °C and subsequently centrifuged for 10 min at 2700× g. The resulting supernatants were collected and stored at −80 °C until further processing.

All swabs in DNA/RNA stabilization buffer (n = 600) were examined using a C. suis-specific real-time PCR [28]. The samples that were PCR-positive, as well as the samples that contained DNA-polymerase inhibitors, were cultured and analyzed by immunofluorescence staining using a genus-specific anti-lipopolysaccharide (LPS) antibody. The presence of C. suis in cell cultures was further confirmed using the same C. suis-specific real-time PCR. Isolates confirmed as C. suis were phenotypically tested for tetracycline susceptibility via MIC and MBC determination. An overview of the analyses can be found in Figure 1.

2.2. DNA Extraction from Intestinal Swabs

Genomic DNA from swabs was extracted using the QIAamp^®^ DNA Mini Kit (Qiagen, Antwerp, Belgium). The manufacturer’s protocol for buccal swabs was followed.

2.3. Chlamydia suis Real-Time PCR

The species-specific TaqMan probe-based C. suis real-time PCR was performed on a Rotor-Gene Q (Qiagen, Hilden, Germany) instrument, as previously described [1], with one modification: the inhibition control plasmid was replaced by a Universal Exogenous Internal Positive Control for TaqMan assays (Eurogentec, Seraing, Belgium). The analytical sensitivity of the PCR is 10 copies of 23S rDNA per reaction. All samples (n = 600) were tested in duplicate, and the cycle threshold was set at 1.0 for each run. Samples with a mean cycle threshold (Ct value) < 35 were considered positive for C. suis DNA, and were used to calculate the corresponding bacterial load. Positive samples, as well as samples for which the amplification of the internal control DNA was inhibited (n = 109), were further used for culture.

The bacterial load (IFU/mL) of positive intestinal swabs was calculated from the obtained Ct values, which were converted using a previously established standard curve describing the correlation between IFU/mL and Ct. This curve was generated by performing a real-time PCR on a ten-fold serial dilution of genomic DNA from C. suis S45 with a known inclusion-forming unit (IFU) quantity per reaction.

2.4. Isolation in McCoy Cells

McCoy cells were seeded onto glass slides (VWR, Leuven, Belgium) in 24-well cell culture plates (VWR, Leuven, Belgium) using standard procedures [29]. The swab sample in transport medium was inoculated on a 24 h old McCoy monolayer, pre-treated with PBS-DEAE dextran and incubated at 37 °C and 5% CO_2_ for 6 days after centrifugation (1900× g) for 1 h at 37 °C [29].

For a blind passage, monolayers were harvested using a sterile cell scraper, followed by sonication for 1 min and centrifugation at 1100× g for 10 min, after which the supernatant was inoculated on fresh McCoy monolayers. Following incubation at 37 °C and 5% CO_2_ for 6 days, the monolayers were fixed with methanol, and Chlamydia LPS staining was performed as detailed below.

2.5. Chlamydia LPS Staining

The inoculated McCoy monolayers were fixed with methanol for 10 min before detection of C. suis with the IMAGEN™ Chlamydia Reagent (Oxoid™, Geel, Belgium), which contains a genus-specific FITC-conjugated monoclonal antibody to Chlamydia LPS [27]. The 24 well plates were incubated in a humid chamber at 37 °C for 45 min. The cells were washed twice with 2 mL of PBS (Gibco, Paisley, Scotland) and twice with 2 mL of Aqua BiDest. The glass inserts were removed from the wells, air dried, and placed with cells facing downwards onto a droplet of Mounting Fluid (ThermoFisher Scientific, Hampshire, UK) on microscope slides (VWR, Leuven, Belgium). All slides were examined by immunofluorescence microscopy (630×, Leica Thunder, Diegem, Belgium).

2.6. Chlamydia suis Real-Time PCR on Cell Cultures

To confirm the presence of C. suis in cell cultures, monolayers were harvested using a sterile cell scraper, followed by centrifugation at 18,000× g for 30 min. The resulting pellet was resuspended in 200 µL and stored at −80 °C until DNA extraction using the QIAamp^®^ DNA Mini Kit, following the manufacturer’s protocol for blood or body fluids. C. suis real-time PCR was performed as described above.

2.7. MIC and MBC Determination

The tetracycline susceptibility of all C. suis isolates (n = 24) was determined as described by Wanninger et al. [4], using 1 × 10^7^ IFU/mL per isolate. Prior to inoculation, McCoy monolayers were treated with PBS-DEAE dextran, and the inoculum (1 × 10^7^ IFU/mL) was added to the cells. After centrifugation (1900× g) for 1 h at 37 °C, chlamydia culture medium containing a serial two-fold dilution of tetracycline (Sigma-Aldrich, Hoeilaart, Belgium) at concentrations ranging from 0.06 µg/mL to 4 µg/mL was added. Each tetracycline concentration was added in duplicate. Following incubation at 37 °C for 48 h, one infected monolayer per tetracycline concentration was fixed with methanol and stained with the IMAGEN Chlamydia Reagent, as described above. The minimum inhibitory concentration (MIC) of tetracycline was defined as the lowest drug concentration that reduced the number of detectable chlamydial inclusions by more than 90% compared with untreated control cultures [30,31]. In parallel, the remaining infected monolayers were washed twice with phosphate-buffered saline (PBS) to remove tetracycline and then incubated for an additional 48 h in tetracycline-free chlamydia culture medium. After this recovery period, the monolayers were fixed and stained using the IMAGEN Chlamydia Reagent. The minimum bactericidal concentration (MBC) was determined using the same inclusion-counting criterion as for the MIC. Because tetracycline acts as a bacteriostatic agent against Chlamydia, the MBC does not represent true bactericidal activity. Rather, it indicates the lowest tetracycline concentration at which Chlamydia fails to resume growth after the antibiotic has been removed [32]. Images were taken with a Zeiss Imager M2 microscope (630×). As described by Wanninger et al. [4], C. suis isolates with a MIC/MBC of ≥ 4 µg/mL were considered resistant, those with 2 µg/mL≤ MIC/MBC < 4 µg/mL were defined as intermediate, and isolates with a MIC/MBC of < 2 µg/mL were considered sensitive.

2.8. Statistical Analysis

The number of real-time PCR-positive swabs and C. suis isolates, both at the farm level and across intestinal segments, was summarized in contingency tables. Comparisons were performed using a chi-squared test. Additionally, Ct values, bacterial load, and MIC/MBC values across different intestinal segments were compared using a one-way Welch ANOVA, as the data were heteroscedastic. p-values below 0.05 were considered statistically significant. Full statistical details are provided in Table A4. All statistical analyses were conducted using GraphPad Prism, version 8.0.1.

3. Results

3.1. C. suis Real-Time PCR on Intestinal Swabs

All swabs collected in DNA/RNA stabilization buffer (n = 600) were first examined using a C. suis-specific real-time PCR, and 98 (16.33%) were positive (Ct-value < 35), corresponding to 21 of 30 (70%) PCR-positive farms. Chlamydia suis DNA was detected in the jejunum in six of 10 (60%) farms, in the ileum in five of 10 (50%) farms, and in the colon of all 10 farms examined (100%) (Table 1). Accordingly, significantly more farms tested positive in the colon than in the jejunum (p = 0.0253) or ileum (p = 0.0098). No significant difference was observed between the jejunum and ileum.

Regarding the intestinal segments, C. suis DNA was identified in nine of 200 (4.5%) jejunal swabs, in nine of 200 (4.5%) ileal swabs, and in 80 of 200 (40%) colonic swabs (Table 1). The number of positive pigs per farm and intestinal segment ranged from 1 of 20 (5%) to 3 of 20 (15%) for the jejunum, from 1 of 20 (5%) to 3 of 20 (15%) for the ileum, and from 2 of 20 (10%) to 13 of 20 (65%) for the colon (Table 1). Accordingly, C. suis DNA was detected more frequently in colonic swabs than in jejunal or ileal swabs (p < 0.001), whereas no significant difference in detection frequency was observed between the jejunum and ileum.

The Ct-values of positive swabs and the corresponding calculated bacterial loads are presented in Table A1. The calculated bacterial load in colonic swabs was significantly higher than in jejunal swabs (p = 0.0453) and ileal swabs (p = 0.0096). No significant differences were observed between jejunal and ileal swabs.

A total of 68 of 600 (11.3%) swabs showed DNA-polymerase inhibition, resulting in inconclusive results. Inhibition was observed in 55 of 200 (27.5%) jejunal swabs and in 13 of 200 (6.5%) ileal swabs, whereas DNA polymerase inhibitors were absent in all 200 colonic swabs (0%). Swabs with DNA polymerase inhibitors were subjected to culture, as described below.

3.2. Isolation and Confirmation of C. suis

As mentioned above, 68 swabs contained DNA polymerase inhibitors. Eleven of these (16.2%) were positive for Chlamydia LPS following culture, including eight of 55 (14.5%) jejunal swabs and three of 13 (23%) ileal swabs. These 11 swabs, together with the PCR-positive swabs (n = 98), were used for isolation and subsequent PCR identification of C. suis in McCoy cell culture harvests, (Figure 1). Thus, Chlamydia isolation was performed on 109 of 600 (17.5%) swabs, comprising 17 of 200 (8.5%) jejunal swabs, 12 of 200 (6%) ileal swabs, and 80 of 200 (40%) colonic swabs.

Viable C. suis was detected in the jejunum on four of 10 farms (40%), in the ileum on five of 10 farms (50%), and in the colon on six of 10 farms (60%) (Table 1), as illustrated by a reproductive infection in McCoy cell cultures (Figure 2). At the farm level (n = 30), no significant differences were observed in the number of farms harboring viable C. suis among the intestinal sites.

C. suis was isolated from six of 17 jejunal swabs (35.3%), nine of 12 ileal swabs (75.0%), and nine of 80 colon swabs (11.25%) (Table 2), resulting in a total of 24 C. suis isolates. At the farm level, the number of isolation-positive pigs per intestinal segment ranged from one to two pigs per 20 sampled pigs (5–10%) for the jejunum, ileum, and colon (Table 2). Overall, viable C. suis was detected significantly more frequently in the ileum than in the jejunum (p = 0.0351) and the colon (p < 0.001). In addition, viable Chlamydia was detected significantly more often in the jejunum than in the colon (p = 0.0128).

The Ct values were significantly lower for ileal isolates compared with the jejunal and the colonic isolates (p = 0.0009 and p = 0.0042, respectively). No significant differences in Ct values were observed between jejunal and colonic isolates. The corresponding bacterial loads in ileal isolates were significantly higher than in jejunal and colonic isolates (p = 0.0066 and p = 0.0126, respectively), whereas no significant differences were observed between jejunal and colonic isolates.

Next, we evaluated whether the bacterial load of intestinal swabs influenced isolation success. Neither higher nor lower bacterial loads were associated with successful isolation, indicating that the amount of C. suis DNA present in intestinal swabs is not a reliable predictor of successful in vitro replication (Figure 3). Intestinal swabs containing DNA polymerase inhibitors were excluded from this analysis because bacterial load could not be determined.

Interestingly, 24 isolates of C. suis were obtained. However, four Chlamydia LPS-positive cell culture harvests, originating from two different farms, were negative by C. suis-specific PCR, indicating the presence of another Chlamydia species.

3.3. MIC and MBC Determination

Due to the emergence of tetracycline-resistant C. suis strains, tetracycline susceptibility was determined for all C. suis isolates (n = 24). The bacterial load was standardized across isolates (10^7^ IFU/mL) to ensure equal input for MIC/MBC determination. Details of MIC/MBC values are presented in Table A3.

Tetracycline-resistant C. suis strains were identified on two of 30 farms (6.67%), corresponding to three of 24 examined pigs (12.5%) carrying tetracycline-resistant C. suis in either the ileum (n = 1) or colon (n = 2). C. suis strains with an intermediate tetracycline susceptibility phenotype were detected on two additional farms, corresponding to three of 24 examined pigs (12.5%) harboring tetracycline-intermediate strains in either the jejunum (n = 1) or ileum (n = 2). All remaining isolates (n = 18) were tetracycline-sensitive, corresponding to 18 of 24 examined pigs (75%) carrying tetracycline-sensitive C. suis in the jejunum (n = 5), ileum (n = 6), or colon (n = 7). Notably, both sensitive and intermediate phenotypes were detected on the same farm (Farm ID 8), as were sensitive and resistant phenotypes on another (Farm ID 14) (Table 3). At farm level, there were no significant differences in the number of susceptible, intermediate, and resistant isolates. Likewise, no significant differences were observed in the numbers of tetracycline-susceptible, intermediate, and resistant isolates among the jejunum, ileum and colon.

MIC and MBC values for jejunal isolates ranged from 0.06 to 2.0 µg/mL and 0.125 to 2.0 µg/mL, respectively. For ileal isolates, MIC and MBC values ranged from 0.125 to 4.0 µg/mL. Colonic isolates exhibited MIC values ranging from 0.06 to 4.0 µg/mL and MBC values from 0.125 to 4.0 µg/mL. Overall, MBC values were generally 0 to 0.75 µg/mL higher than the corresponding MIC values (Table A3). The MIC and MBC values of the isolates did not differ significantly between the jejunum, ileum, and colon.

To evaluate whether the bacterial load of the intestinal swabs influenced tetracycline susceptibility, the calculated bacterial load (IFU/mL) of the swabs was compared to susceptible, intermediate, and resistant isolates for each intestinal segment (Figure 4). The distribution of bacterial loads indicated no correlation between bacterial load and tetracycline susceptibility. However, interpretation of this finding is limited by the low number of phenotypes classified as tetracycline-resistant or intermediate. Intestinal swabs with DNA polymerase inhibitors were excluded from this analysis, as no bacterial load could be calculated.

3.4. Background Information Farms

Information on clinical disease and antibiotic use during the fattening period was obtained from 20 of 30 farms (66.7%). Only four of these 20 farms (20%) reported administering tetracycline. None of the farms reported observing specific clinical symptoms. For one farm from which a tetracycline-resistant phenotype was recovered, no information on antimicrobial use was available because the producer declined to participate in this part of the study. The remaining two resistant phenotypes were both isolated from pigs originating from the same farm. Notably, this farm reported no use of tetracycline and no administration of any other antibiotics during the fattening period.

4. Discussion

Chlamydia suis is an obligate intracellular bacterium commonly found in pigs worldwide. In swine production, it is associated with a range of clinical manifestations, including mainly conjunctivitis, reproductive disorders, and reduced growth performance, although infections can also be subclinical [33,34,35]. In fact, C. suis is often isolated from clinically healthy animals, and it is therefore generally not considered a primary pathogen in swine [4]. However, the bacterium’s widespread presence in commercial herds can contribute to economic losses due to impaired weight gain, increased veterinary interventions, and reduced overall herd health. Beyond its impact on animal health, C. suis has been identified as having zoonotic potential [2], particularly for those in close contact with pigs, raising occupational health concerns for farm workers and veterinarians [2,3].

Experimental infections in gnotobiotic pigs have clarified the pathogenesis of C. suis [14,15,16,36]. Following oral or intra-gastric infection, C. suis infects the gut, but not uniformly. It primarily affects certain parts of the small intestine, particularly the jejunum and ileum, where it invades enterocytes and undergoes its life cycle, while replication in the duodenum, caecum, and colon is minimal or occurs to a far lesser extent. However, little is known about its segment-specific distribution under field conditions, and no study has systematically combined PCR, culture, and antimicrobial susceptibility testing across different gut regions. In this study, we sampled 30 commercial pig farms, analyzing 20 jejunum samples from 10 farms, 20 ileum samples from another 10 farms, and 20 colon samples from an additional 10 farms. Thus, each intestinal segment represents an independent dataset derived from separate animals and farms. This approach allowed us to investigate segment-specific prevalence without relying solely on fecal shedding, providing a more accurate picture of bacterial colonization in naturally infected pigs. Differences in gut physiology, microbiota composition, and immune activity may influence colonization and shedding patterns, making segment-specific data of naturally infected pigs particularly interesting for understanding infection dynamics at the population level.

The emergence of tetracycline-resistant C. suis strains is a growing concern in swine production [18,19,20,37]. These strains carry the tetracycline resistance gene tet(C), which encodes an antibiotic efflux pump [38]. Tetracyclines are widely used to treat bacterial infections, as they are mostly effective and relatively inexpensive. Resistance can compromise therapy and contribute to broader antimicrobial resistance issues. By determining the minimum inhibitory and bactericidal concentrations (MIC/MBC) of isolates from different gut segments, this study provides information on resistance patterns in field isolates, complementing prevalence data and informing rational antimicrobial use.

In the first part of the study, C. suis real-time PCR was performed on intestinal swabs. PCR revealed C. suis DNA in 16.3% of all intestinal swabs and in 70% of the examined farms. This prevalence is lower than that reported in our previous studies using PCR on rectal swabs. For example, De Puysseleyr et al. [3] examined 10 pigs from each of 10 Belgian farrow-to-slaughter farms and found 52% of PCR-positive pigs upon arrival at the slaughterhouse, with all farms testing positive. In another study, the same group analyzed 10 finisher pigs (mean age 5 months) and five sows (mean age 3 years) across nine Belgian farms and reported a higher overall positivity rate of 69%, with C. suis DNA also detected on all farms [39]. The higher detection of C. suis in rectal swabs compared to the intestinal swabs used in the present study may reflect either a genuinely lower infection rate in Belgian pigs or, more likely, the fact that rectal swabs primarily detect bacteria being shed, rather than bacteria actively colonizing the tissue at the sampling site. C. suis replicates in the enterocytes of the small and large intestine, and shed organisms can accumulate in the feces. Consequently, rectal swabs may contain bacteria from multiple gut regions. Similar studies in other countries using C. suis PCR on fecal or rectal swabs also reported higher prevalences in pigs and farms than the ones found in the present study [20,40,41,42,43].

Thus, PCR on rectal swabs is more suitable for assessing C. suis prevalence, while intestinal segment swabs provide more accurate information on bacterial colonization sites under field conditions, which was one of the objectives of the present study. At the farm level, all colonic swabs were PCR-positive, whereas detection rates were significantly lower in jejunal and ileal swabs. At the individual swab level, colon samples exhibited the highest PCR positivity (40%), with significantly higher bacterial loads compared to jejunal and ileal swabs. These findings indicate that C. suis DNA is particularly abundant in colonic samples at the population level. The colon acts as a storage and fermentation compartment, where material from the small intestine remains present for long periods. Consequently, organisms and/or their DNA originating from proximal intestinal regions accumulate in higher concentrations in the colon [4,20,41].

However, PCR quantification in the colon may overestimate actual local colonization. This is why we integrated molecular detection with culture to assess the spatial distribution of C. suis colonization and identify predominant sites of active intracellular replication under field conditions. As mentioned earlier, C. suis colonization of the GI tract is receiving increasing attention, as the GI tract may serve as an important site of colonization and a potential reservoir for infection [6,7,8,9]. In addition, a study on C. trachomatis, phylogenetically highly related to C. suis [44], found C. trachomatis DNA in appendix and colon biopsies taken during colonoscopy [45]. Additionally, C. trachomatis is frequently detected in anorectal samples from men and women [46,47]. According to these studies and others, its presence in the GI tract might impact mucosal immune regulation, augmenting the risk for inflammatory bowel disease (IBD) [48]. C. suis can cause intestinal inflammation and epithelial damage under experimental conditions [12]. However, there is no evidence that C. suis causes a chronic, self-sustaining inflammatory bowel disease equivalent in pigs.

Thus, in the second part of the study, culture was performed on all C. suis PCR-positive swabs. Culture-based isolation confirmed the presence of viable C. suis in all intestinal segments, but we only obtained 24 isolates. At the farm level, viable organisms were detected in 40% of jejunal, 50% of ileal, and 60% of colonic samples, with no statistically significant differences among segments. At the individual swab level, isolation success differed. Viable C. suis was recovered significantly more often from ileal swabs than from jejunal or colonic swabs, and significantly more times from jejunal than from colonic swabs. Ileal isolates were characterized by significantly lower Ct values and higher calculated bacterial loads than jejunal and colonic isolates, strongly suggesting that C. suis preferentially replicates in the ileum following a natural infection. However, paired sampling within naturally infected animals needs to be done to confirm this.

Indeed, the present study only provides insights into the distribution of C. suis across the different gut segments in fattening pigs at the population level. Unfortunately, we could not examine the segment-specific prevalence of C. suis within the same individual animal. Such an approach would allow direct correlations between gut segments and offer a clearer understanding of intra-host variation. However, the collection of jejunum, ileum, and colon samples from 20 pigs per slaughter batch is technically challenging due to the limited time window during evisceration and the need for accurate anatomical identification of intestinal segments. Such sampling would require multiple trained personnel, precise carcass tracking per farm, and, in particular, a dedicated workspace adjacent to the slaughter line to prevent interference with routine operations. In the present study, this approach proved feasible, as insufficient space was available alongside the slaughter line to accommodate multiple samplers or to temporarily store intestinal packages without disrupting line speed.

The culture data for the different intestinal segments differ from the PCR data. While C. suis DNA was detected significantly more frequently in the colon than in the ileum and jejunum, viable C. suis was recovered significantly more frequently from the ileum or jejunum than from the colon. This finding suggests a similar pathogenesis under field conditions to that observed in experimentally infected pigs, as immunohistochemistry analysis of experimentally infected, euthanized gnotobiotic pigs revealed a higher and more sustained presence of C. suis in the small intestine than in the large intestine [15]. This finding also raises the question of what these many C. suis PCR-positive samples in the colon represent. They can be non-viable, or viable but non-culturable C. suis. C. suis strains are difficult to grow, and they do not replicate equally well in cell culture, as De Puysseleyr et al. observed a strain-dependent preference for certain cell culture types [11]. Thus, some C. suis organisms perhaps did not grow in McCoy cells. On the other hand, previous studies have also shown that C. suis and C. trachomatis can adopt, under non-optimal conditions, aberrant forms in the GI tract of naturally and experimentally infected pigs (small intestine examined) [36,49]. Aberrant bodies are metabolically active but non-replicating, resulting in a prolonged relationship with the host cell. Aberrant bodies are thus viable but non-culturable. They can regain their replicative state when conditions improve, potentially serving as a reservoir of infection. Therefore, part of the colonic PCR-positive samples and culture negative samples could have been aberrant bodies.

Interestingly, bacterial load in the original intestinal swabs did not predict isolation success. Neither higher nor lower C. suis DNA loads were associated with successful recovery of viable organisms, consistent with the obligate intracellular nature of chlamydia and other host- or sample-dependent factors affecting replication. This contradicts a previous study, which reported that the isolation success in fecal swabs is influenced by the C. suis DNA loads [4]. This discrepancy cannot be explained by differences in bacterial load, as the previous study reported copy numbers that cannot be directly compared with the bacterial loads determined in the present study. Differences in experimental setup between studies, including the use of LLC-MK2 cells for isolation in the previous study, may have contributed to the contrasting results.

A total of 24 C. suis isolates were recovered. Additionally, the detection of four chlamydial LPS-positive but C. suis PCR-negative isolates highlights the presence of other Chlamydia species in the porcine intestine, which is not new, as others have already found C. abortus and C. pecorum in the porcine gut [33,34,36,37,38,39,40,41]. However, these species are only sporadically detected in the intestinal tract, and their biological relevance in the porcine gut therefore appears limited compared to C. suis, which is considered the predominant chlamydial species colonizing the porcine intestine.

In the final part of our study, C. suis isolates were phenotypically characterized for tetracycline susceptibility as the presence of the tet(C) gene does not always translate into a tetracycline resistant phenotype [4,31]. In addition, a tet(C) PCR directly on swabs may give false positives, as the gene can also be present in other pathogens or even in bacteria belonging to the porcine gut microbiota [4,18,20,50].

MIC and MBC values varied among isolates, but no statistically significant differences were observed between jejunal, ileal, and colonic isolates. Most isolates (75%) were tetracycline-sensitive, while intermediate and resistant phenotypes were each detected in 12.5% of isolates, occurring in the jejunum, ileum and colon across different farms. MBC values were generally slightly higher than MICs, consistent with the primarily bacteriostatic activity of tetracycline against C. suis. These findings align with a previous report [4]. Statistical analysis revealed no association between bacterial load and tetracycline susceptibility, with resistant, intermediate, and susceptible phenotypes occurring across a wide range of bacterial loads.

Phenotypic susceptibility testing determines whether bacterial growth is inhibited by an antibiotic but does not provide information on the underlying resistance mechanisms, nor does it indicate the presence or absence of the tet(C) gene. Moreover, susceptibility to tetracycline does not necessarily imply the absence of tet(C), as the gene may be present but not expressed or phenotypically masked. In addition, isolates were not plaque-purified to assess whether multiple strains were present within a single isolate. Consequently, the prevalence of the tet(C) gene in the analyzed samples may have been underestimated.

The detection of tetracycline-resistant strains is related to the continued use of tetracyclines and their derivatives in veterinary medicine. However, since 2011, a total reduction of 58.2% in antibiotic sales for veterinary use has been recorded in Belgium. In 2022, penicillin constituted the most sold class of veterinary antibiotics (49.4 tons), followed by tetracyclines (24.2 tons). Sales of fluoroquinolones, antibiotics that may serve as alternatives for tetracycline-resistant C. suis strains, increased slightly by 0.4% between 2021 and 2022 [51]. Despite these reductions, tetracycline-resistant C. suis strains continue to be reported worldwide. Tetracycline-resistant C. suis isolates have been reported on pig farms in the United States since the 1990s [37], and more recently in Belgium, China, Cyprus, Estonia, Germany, Israel, Italy, and Switzerland [18,19,20]. Importantly, previous in vitro studies have demonstrated that the tet(C) resistance gene can be horizontally transferred to C. trachomatis, underscoring the potential public health impact of tetracycline-resistant C. suis strains [24,25,26,52].

For one farm, from which a tetracycline-resistant phenotype was recovered, information on antimicrobial use was unavailable as the producer declined to participate in this part of the study. The remaining resistant phenotypes were both isolated from the same farm, which reported no use of tetracyclines and no administration of any other antibiotics during the fattening period. This might be true, as a study by Dewulf et al. [53] showed that a substantial proportion of the European pig production is already able to raise pigs without any group of treatments, indicating that it is possible to rear pigs without systematic use of antibiotics. Our finding contrasts with previous studies, which have found a higher proportion of tetracycline-resistant isolates in farms where tetracycline is commonly administered [4,19]. However, we received limited information from only a few farmers, so we cannot draw a firm conclusion on the impact of antibiotic treatment and the occurrence of tetracycline-resistant isolates.

These findings support the use of porcine gut organoids to study host-pathogen interactions, bridging the gap between simple cell cultures and complex in vivo pig models [54]. Gut organoids derived from the ileum, and perhaps to a lesser extent the jejunum, are particularly suitable, as these intestinal regions represent the primary sites of active C. suis replication.

5. Conclusions

This study provides a comprehensive field-based assessment of the intestinal distribution, viability, and tetracycline susceptibility of C. suis in commercial pig herds. By combining real-time PCR, culture, and phenotypic antimicrobial testing across distinct gut segments, we extend previous knowledge derived mainly from fecal or rectal sampling and experimental infections in gnotobiotic pigs. The PCR data confirmed that C. suis is widespread at the farm level, with colonic samples showing the highest detection rates and bacterial loads, likely reflecting accumulation of shed organisms rather than true local replication. In contrast, the culture-based results demonstrated that viable C. suis was more frequently recovered from the small intestine, particularly the ileum, indicating that this segment is the most important site of active replication under natural field conditions, while the colon contains non-viable and/or viable but non-culturable aberrant forms of C. suis. The latter suggests that the colon might act as a reservoir of reactivatable C. suis. However, this hypothesis requires confirmation by transmission electron microscopy.

Phenotypic antimicrobial susceptibility testing revealed that most isolates were tetracycline-sensitive, but the detection of intermediate and resistant strains across all gut segments and on farms without reported tetracycline use confirms that tetracycline resistance remains present in Belgian pig production. Given the zoonotic potential of C. suis and the documented ability of the tet(C) gene to transfer in vitro to C. trachomatis, this finding reinforces concerns about antimicrobial resistance at the animal–human interface.

These results highlight the need for continued monitoring, judicious antimicrobial use, and further studies incorporating paired sampling within individual animals to better understand intra-host dynamics and transmission risks.