Carriage of Cdt-B Encoding Campylobacter spp., Salmonellaenterica, and Yersinia entercolitica in Patients with Gastroenteritis, Irritable Bowel Syndrome, and Controls

Background: Cytolethal distending toxin (Cdt) is one of the bacterial toxins that present in a variety of Gram-negative human pathogens, such as E. coli, Salmonella spp., and Campylobacter spp. CDT composed of three subunits encoded by three adjacent genes, including cdtA, cdtB and cdtC. It is approved that cdtB had toxic activity and caused DNA damage of the host cell. Despite its presence in different bacterial species, role of Cdt in acute and chronic infections, such as gastroenteritis and irritable bowel syndrome (IBS), is unclear. To analyze this correlation, we studied the prevalence of cdtB among different enteropathogenic bacteria in patients with gastroenteritis and IBS compared with healthy people. Materials and Methods: In this cross-sectional descriptive study, 230 stool samples were collected from patients with gastroenteritis, IBS, and healthy people. The presence of Cdt-B encoding bacteria, including Escherichia coli, Campylobacter spp., Yersinia entercolitica, Providencia alkalifacience, and Salmonella enterica was examined by polymerase chain reaction. Demographic data and type of disease was collected through interview and a questionnaire. Results: Out of 230 stool samples, Cdt-B encoding Campylobacter spp. were found in 34.6% (52/150), 6.25% (5/80), and 4% (2/50) of the patients with gastroenteritis, IBS, and the control group, respectively. Carriage of Cdt-B encoding Salmonella enterica was characterized among 5.3% (8/150) of the patients with gastroenteritis and 17.5% (14/80) of the IBS patients. Although none of the patients carried cdtB of E. coli and Providencia spp., cdtB of Y. enterocolitica was detected in 1 of the patients with gastroenteritis (0.6%). Statistical analysis showed signicant correlation between infection with CdtB-encoding Campylobacter spp. and IBS-D subtype. No signicant correlation was found between infection with CdtB encoding bacteria, and other clinical and demographic data. Conclusions: Our results conrmed relatively higher frequency of Cdt-B encoding bacteria in the intestine of IBS patients and those with gastroenteritis compared with healthy individuals. Regarding the frequency of Cdt-B encoding Salmonella and Campylobacter bacteria, it was proposed that infection with these enteropathogens could be considered as a risk factor for the development or progression of IBS among the Iranian patients. Further studies are needed to establish this involvement.

Many of these Gram-negative bacteria are considered as clinically important human pathogens that are responsible for gastroenteritis [8]. Involvement of these bacteria in development of chronic bowel disorders, such as irritable bowel syndrome (IBS), was reported in different studies [9,10]. Accordingly, 3-30% of people with IBS experience their symptoms after an episode of acute gastroenteritis (Postinfectious IBS, PI-IBS) [11].
Currently, no de nite virulence factors are characterized in these bacteria in association to PI-IBS. Dysbiosis of the gut microbiota and alteration of microbial population in this organ could accelerate growth of more virulent bacteria, which promote functional disorders through their interaction with the host [12][13][14]. This interaction and the functional disorder, including chronic abdominal pain and alerted intestinal habits, could occur by unknown bacterial virulence factors [15,16]. Increased antibody titers against CdtB of Campylobacter as a only proposed virulence factors have been observed in diarrheadominant form of IBS [17,18]. This involvement may cause through cross-reaction of the antibodies with vinculin in the host gut [19][20][21]. Despite wide distribution of this family of toxins in members of the enteric bacteria, little is known about the prevalence of Cdt-B encoding bacteria, their association with distinct types of IBS (IBS with diarrhea, IBS with constipation, and mixed-types), and promotion or exacerbation of the disease. This study was aimed to assess the presence of cdtB gene among different enteric bacteria, including Yersinia entercolitica, Salmonella. enterica, E. coli, Providencia alcalifaciens, Aggregatibacter actinomycetemcomitans, and Campylobacter spp. among patients with IBS and gastroenteritis in compare to healthy people.

Materials And Methods:
Sample collection: This cross-sectional descriptive study was conducted on 230 stool samples that obtained from patients with acute gastroenteritis and IBS. A control group of healthy volunteers (50 samples) was also included in the study. A consent form was lled by all participants. The study was approved by ethical committee of Department of Pathobiology, Tehran university of Medical Sciences, Tehran, Iran (TUMS. Ethics code 92-02-27-22726). Fresh stool samples were collected in clean containers and the samples were immediately transferred to the laboratory under cold chain. Adult patients with functional bowel disorders were interviewed by experienced physicians and ful lled a questionnaire that was designed according to Rome III criteria for IBS [22]. According to symptoms, they was classi ed as either diarrhea-predominant (IBS-D), constipation-predominant (IBS-C), or with alternating stool pattern (mixed IBS). Exclusion criteria were included, intestinal disturbance (Celiac disease and lactose intolerance), recent history of hospitalization (>24 h), antibiotic prescription within the last 3 months, surgery of the gastrointestinal tract, local and systematic in ammatory diseases, de ned diet, food allergy, and pregnancy. Healthy controls were selected from people of the same age, who enrolled in routine medical check-ups in the hospital. These people reported no history of the gastrointestinal disorders and the exclusion criteria described above.

DNA preparation:
Total DNA of the samples was extracted using DNA Stool Kit (Bionner, South Korea) according to the manufacturer's instructions. The concentration of DNA was measured by Nanodrop (Eppendorf-Germany). All DNA extracts were stored at −20 °C until use.

Identi cation of cdtB by PCR:
In this study, speci c primers were designed for characterization of cdtB in Y.entercolitica, S. enterica, E. coli, Providencia alcalifaciens, and Aggregatibacter actinomycetemcomitans (Table 1). Accordingly, homology of cdtB were determined using CLC Sequence Viewer v.6.0, and appropriate regions were selected. Ampli cation of the cdtB were carried out in 25 μl reaction containing 5 μl of DNA template, 0.5 mM of each dATP, dGTP, dCTP and dTTP (gene fanavaran, Iran), PCR (10x) buffer (Gene fanavaran, Iran), 0.3 μl (10 pmol) of each forward and reverse primer, 1x Tag DNA polymerase buffer (Gene fanavaran, Iran), and 0.2 μl (5 U/μl) of Taq DNA polymerase (Fermentase, Germany). Ampli ed products were visualized in 1.5% agarose gels along with a mixed DNA ladder. The results showed that the prevalence of IBS was signi cantly higher in women than in men (56/24) (p < 0.05). The commonest subtype of IBS in female patients was IBS-C (86.2%); while, IBS-M was the most frequent type among males (33.33%). Diarrhea-predominant IBC was the same in men and women.
Anxiety was signi cantly higher among women in compare to men (P < 0.05). Similarly, several other symptoms, including abdominal bloating, cramp, and stress were reported more common in females, which was not statistically signi cant ( Table 2). The most common symptoms associated with constipation in subjects with IBS-C was abdominal bloating (89.65 %), followed by bellyache (75 %), while subjects with IBS-D type felt a higher degree of abdominal cramp (P = 0.01). Additionally, there was a signi cant correlation between IBS-C and IBS-M with anxiety. Anxiety degree was signi cantly associated with bloating (P = 0.02) and abdominal pain in IBS patients (P < 0.01). No statistically signi cant difference was measured between age and IBS disease subtypes.

Diversity ofcdtB gene variants in the fecal DNA extracts
The frequency of cdtB varied between patients with IBS, gastroenteritis, and healthy people. cdtB of Campylobacter showed high-frequency in stool samples of patients with gastroenteritis (34.6%, 52/150); which was higher than those characterized in patients with IBS and healthy people (6.25%, 5/80 and 4%, 2/50, respectively). The difference was statistically signi cant (p < 0.05). Signi cant association also were seen between cdtB of Campylobacter spp. and IBS-D patients. cdtB of S. enterica was detected in 8 (5.3%) and 14 (17.5%) of the patients with gastroenteritis and IBS, respectively. There was a signi cant difference between the presence of cdtB of S. enterica in patients with IBS and gastroenteritis compared with healthy subjects (p < 0.05). However, the presence of cdtB of S. enterica didn't show a correlation with IBS types (24.24%, 13.79 %, and 11.11% in IBS-M, IBS-C, and IBS-D patients, respectively). cdtB of E. coli and P. alkalifacience was not detected in any of the patients with IBS, gastroenteritis, and in the control group. The results also indicated that only 0.6% of the patients with gastroenteritis carried cdtB of Y.entercolitica (Table 3). Statistical analyses showed no signi cant correlation between Cdt-B encoding bacteria and gender or other demographic data among the studied patients with IBS.

Discussion:
The overall objectives of the current study were to determine diversity and frequency of cdtB gene among different enteropathogenic bacteria in subjects with IBS and gastroenteritis and their possible roles in the occurrence of these diseases.
According to our results, higher frequency of cdtB of Campylobacter spp. was detected in patients with IBS (6.25%) and in patients with gastroenteritis (34.6%), compared with healthy people (3.75%). Similar to our results, high prevalence of cdtB was detected among Campylobacter isolates in patients with gastroenteritis [25]. Peter KE et al characterized cdtB gene among 67% of C. jejuni and 19% of C. coli isolates in patients with gastroenteritis. In a study by Burliaeva et al, cdtB encoding Campylobacter strains was detected in 5% of patients with IBS [26], which was relatively similar to our results. In our study, the frequency of Cdt-B encoding Campylobacter spp. was signi cantly higher among IBS-D patients (16.66%). The higher frequency of Cdt-B of Campylobacter among patients with the diarrhea type disease could be explained through function of Campylobacter CdtB with the intestine. Burliaeva et al showed that wild-type Cdt-B encoding C. jejuni strain is able to induce chronic altered bowel patterns and mild chronic rectal in ammation [25,26]. Although, new developing commercial diagnostic kits for detection of anti-CdtB antibodies as speci c biomarker for diarrhea-predominant irritable bowel syndrome is currently in use by some countries [17,18], further studies are needed to establish whether CdtB positive Campylobacter strains are associated with diarrhea in patients with irritable bowel syndrome. It seems that CDT has the ability to attack the cells of intestinal villi, and cause disease [27,28]. In this study, no signi cant association was seen between the Campylobacter encoding cdtB and other clinical symptoms, including abdominal bloating. No signi cant correlation also was found between Cdt-B encoding Campylobacter spp. and gender among IBS patients, while most of IBS patients were female.
cdtB of S. enterica was detected in 14% of patients with IBS and 5.3% of patients with gastroenteritis in current study, while it was not detectable in the control group. Few studies have been done on Salmonella spp.; however, the presence of cdtB in Salmonella is reported both for typhoidal and non-typhoidal S. enterica (NTS) serovars [5,[29][30][31][32]. In a study by Mezal et al., the carriage of cdtB gene was detected among all isolates of Salmonella enterica serovar Javiana isolated from food, environmental, and clinical samples [5]. An increased level of CDT-mediated invasiveness for these isolates was shown in HeLa cell culture, which could support possible involvement of this toxin in different diseases of the gastrointestinal tract. .
Recent evidence demonstrated that Y. entercolitica is linked with chronic gastrointestinal diseases, including IBS, dyspepsia, constipation, and In ammatory bowel disease (IBD) [33]. However, this involvement was not con rmed in our study, since only one Cdt-B encoding Y. entercolitica sample was characterized. Similarly, none of the samples of patients with IBS and gastroenteritis, and healthy people were positive for cdtB of E. coli and P. alcalifacience. In a study by Meza-Segura et al, out of 1306 young children with acute diarrhea, cdt encoding E. coli strains detected in 1% of the patients [34]. In another study, cdtB gene were found in 1.4% of 366 E. coli strains isolated from stool specimens of patients with acute diarrhea in Calcutta, India [35].
In our study, we didn't nd cdtB of P. alcalifacience among our samples. However, Shiam et al. detected cdtB harboring P. alcalifacience among 9.7% of patients with diarrhea [36]. According to our knowledge, no report for this bacterium and CDT exists in patients with IBS, which is consistent to our nding.

Conclusion:
Our data showed high distribution of cdtB gene among Campylobacter spp. and Salmonella spp. in stool samples of patients with IBS and gastroenteritis. These ndings proposed that infection with CdtB encoding bacteria could be a risk factor for development of IBS. In addition, signi cant association was seen between CdtB encoding Campylobacter spp. and diarrhea-predominant irritable bowel syndrome. No signi cant correlation was found between CdtB encoding Campylobacter spp. and S. enterica and gender among the IBS patients. Further studies are needed to establish this correlation.