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Article

Comparative Virulence Gene Profiling of Campylobacter jejuni and Campylobacter coli Isolates from Avian and Human Sources in Egypt

by
Amr Mekky
1,2,*,
Mohamed R. Issa
3,
Amro Hashish
1,4,
Wafaa Hassan
1,
Ali Wahdan
5,
Islam Hisham
1,
Shymaa Enany
6 and
Mohamed Enany
5
1
Reference Laboratory (RLQP), Animal Health Research Institute, Agriculture Research Center (ARC), Giza 12618, Egypt
2
Department of Gastroenterology and Hepatology, Translational Research Institute (TRI), Henan Provincial People’s Hospital, Zhengzhou University, Zhengzhou 450003, China
3
Oncology Department, Comprehensive Cancer Center Mayo Clinic, Rochester, MN 55901, USA
4
Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA
5
Bacteriology, Immunology and Mycology Department, Faculty of Veterinary Medicine, Suez Canal University, Ismailia 41522, Egypt
6
Microbiology and Immunology Department, Faculty of Pharmacy, Suez Canal University, Ismailia 41522, Egypt
*
Author to whom correspondence should be addressed.
Microbiol. Res. 2025, 16(9), 209; https://doi.org/10.3390/microbiolres16090209
Submission received: 11 May 2025 / Revised: 20 July 2025 / Accepted: 29 July 2025 / Published: 18 September 2025
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Abstract

Campylobacter species are considered to be the leading bacterial cause of human gastroenteritis globally. Consumption of undercooked or contaminated food, such as chicken, is the main cause of human campylobacteriosis. Despite this significant zoonotic link, comparative data on virulence determinants in Campylobacter isolates across avian and human sources remain limited. This study aimed to characterize the prevalence and expression of virulence determinants in Campylobacter jejuni and Campylobacter coli isolates from chicken and human sources in Ismailia governorate, Egypt. A total of twenty C. jejuni and C. coli isolates (ten of each species) were screened for 14 virulence genes using PCR. All isolates harbored virB11, iam, racR, and tetO. Chicken isolates exhibited a significantly higher prevalence: C. jejuni (chicken): pldA, dnaJ, flaA (100%), cdtB (80%), ciaB (60%), and wlaN (0%); C. coli (chicken): pldA, dnaJ (100%), flaA (60%), cdtB (60%), ciaB (40%), and wlaN (20%). In contrast, human isolates showed a markedly lower prevalence: C. jejuni (human): dnaJ, flaA, and cdtB (20%); C. coli (human): dnaJ, flaA, and cdtB (40%). Crucially, pldA, ciaB, and wlaN were absent in all human isolates. plda and dnaJ genes showed statistically significant prevalence differences. qPCR revealed a significant upregulation (p < 0.05) of dnaJ, virB11, flaA, and iam in chicken isolates compared to human isolates, with log2 fold changes of 3.52, 2.84, 2.43, and 1.90 for C. jejuni and 3.06, 2.38, 1.51, and 1.32 for C. coli. Differential expressions of racR, cdtB, and tetO were not significant, with log2 fold changes ranging from −0.51 to 0.14. Ganglioside mimicry genes (Cst11, wlaN, Waac, ggt, and cgtB) and the carbon storage regulator gene (csrA) were absent in all human isolates. These findings underscore the significant variability in virulence gene profiles in chicken and human C. jejuni and C. coli isolates and highlight the importance of molecular characterization in the risk assessment and epidemiological surveillance of Campylobacter infections.

1. Introduction

Campylobacter is one of the four most important causes of global diarrheal diseases, with an estimated 96 million cases and 37,600 deaths annually, predominantly in developing countries [1]. In developing countries, campylobacteriosis in children is frequent and sometimes results in death [2]. Mainly, C. jejuni and C. coli are well-recognized causes of human campylobacteriosis with symptoms ranging from mild watery diarrhea to serious neuropathies, such as Guillain–Barré syndrome [3].
Campylobacter species are Gram-negative, microaerophilic bacteria characterized by their spiral shape and motility, which is facilitated by a polar flagellum. Thermophilic Campylobacter species can thrive at higher temperatures (42 °C), making avian hosts, particularly poultry, an ideal reservoir for C. jejuni and C. coli. Notably, these bacteria colonize poultry asymptomatically, unlike in humans, where they cause disease [4]. The high body temperature of poultry species provides an optimal environment for the growth of thermophilic Campylobacter spp., particularly C. jejuni and C. coli, making poultry the main source of human campylobacteriosis [5].
Despite the significant burden of disease, the molecular basis of Campylobacter pathogenicity remains incompletely understood. However, several virulence factors have been identified based on in vitro and in vivo studies. For example, flaA encodes the major flagellin protein—the main component of extracellular flagellar protein—and seems to be highly conserved among Campylobacter isolates. Flagella are not only responsible for motility and chemotaxis but also play a crucial role in attachment to intestinal epithelial cells and secretion of virulence proteins, autoagglutination, microcolony formation, and avoidance of innate immune response [6]. The RNA-binding protein csrA plays an important virulence role in regulating several phenotypes like motility, biofilm formation, and oxidative stress resistance. csrA also directly regulates flaA gene expression in a growth-phase-dependent manner and affects autoagglutination without causing gross flagellar structure defect [7]. virB11 is a Pvir plasmid located on the gene responsible for the type IV secretion system [8]. The invasion-associated marker (iam) gene is responsible for invasion of the host cell [9]. ciaB and pldA genes of the pVir plasmid are involved in host cell invasion [10]. Cytolethal distending toxin (CDT) genes—cdtA, cdtB, and cdtC—are responsible for the expression of Campylobacter cytolethal distending toxin and eventually cell death, as the CcdtB subunit is the active toxic unit. CcdtA and CcdtC are required for CDT binding to target cells and the delivery of CcdtB into the cell interior [11]. Lipo-oligosaccharide-associated genes—cgtB, waaC, and wlaN—are responsible for β-1,3 galactosyltransferase production. The phospholipase A enzyme (pldA) gene is essential for the pathogenicity of Campylobacter species via hydrolysis of the phospholipids of the host cell membrane and facilitating the invasion of host intestinal cells [12]. The ciaB gene is essential for bacterial invasion of host cells. It functions as a key virulence factor by facilitating the secretion of effector proteins required for host cell internalization during infection. Mutant strains lacking ciaB exhibit significantly reduced invasion efficiency compared to the wild-type strain, highlighting its critical role in Campylobacter pathogenesis [13]. The dnaJ virulence gene enables Campylobacter species to cope with diverse physiological stresses and is also considered a chaperone protein [14]. The reduced ability to colonize (racR) is essential in avian host intestinal colonization and growth at 42 °C (the body temperature of chickens) [15]. The cst-II gene has been linked to the invasiveness of C. jejuni for intestinal epithelial cells [16]. The ggt gene encoding the periplasmic gamma-glutamyltranspeptidase (GGT) seems to play a pivotal role in enteric colonization. In a chicken model, GGT has been shown to be important in long-lasting gut colonization [17]. Invasion-associated genes (ciaB, pldA, and virB11) and antibiotic resistance genes (tetO, tetA, and gyrB) were investigated by Rizal et al. [18] and Awad et al. [19]. In Egypt, the majority of Campylobacter isolates obtained from poultry were multidrug-resistant (MDR), particularly against antibiotics commonly employed as first-line therapies. Notably, the tet(O) resistance genes identified in Campylobacter jejuni strains isolated from chickens, humans, and drinking water were closely related and traced back to the same farm environment [20]. There is strong evidence of recent local acquisition and sharing of antimicrobial resistance (AMR) genes among Egyptian Campylobacter isolates [21].
Systematic surveillance and molecular analysis are essential for understanding epidemiological patterns and developing effective strategies in Campylobacter control in Egypt [20,22]. This study addresses a gap by providing comparative data based on molecular surveillance of virulence gene prevalence and expression in C. jejuni and C. coli isolates from chicken and human samples and supports understanding of zoonotic transmission, using a combined PCR and quantitative PCR (qPCR) approach suitable for low-resource settings.

2. Materials and Methods

2.1. Sample Preparation and Bacterial Isolation

A total of 200 chicken broilers and human fecal samples (100 each) were collected as follows: 100 chicken ceca from ten different poultry commercial slaughterhouses and 100 human stool samples from medical laboratories in Ismailia governorate during 2018. All samples were collected in sterile polyethylene cups and transported in an ice box with refrigerants to be examined within two to four hours for isolation of Campylobacter species at the Reference Laboratory for Veterinary Quality Control on Poultry Production (RLQP), Ismailia. All samples were prepared as follows. From chicken, fecal samples were obtained by removal of the cecal wall using sterile scissors and taking one gram of cecal contents. One gram from each sample was added to 9 mL Thioglycolate broth (Hi media®-M 908, Pennsylvania, PA 19348, USA) to form a fecal suspension (1/10 dilution). For human samples, one gram from each fecal sample was added to 9 mL Thioglycolate broth (1/10 dilution) to form a fecal suspension. All samples were examined by passive filtration method [23].
Approximately 0.1 mL from the freshly prepared fecal suspension was carefully and aseptically layered onto a 0.45 µm cellulose nitrate filter (Sartorius®, Göttingen, Germany), which had been previously placed on top of a freshly prepared non-selective blood agar plate (7% sheep blood) (OxoidTM, Hampshire, UK). Care must be taken not to allow the inoculum to spill over the edge of the filter. The bacteria were allowed to migrate through the filter for 30–45 min at 37 °C or at room temperature. The filter was aseptically removed, and then the fluid that passed through the filter was streaked on the blood agar plate using a sterile bacteriological loop and then incubated micro-aerobically in darkness (5% O2, 10% CO2, and 85% N2) using CampyGen sachets 2.5 L (OxoidTM, Hampshire, UK), anaerobic jar 2.5 L and incubated at 42 °C for 48 h.
Phenotypic characterization of Campylobacter species has been performed using colony morphology, Gram staining, and Biochemical identification (Catalase, Oxidase, and Sodium hippurate hydrolysis). One suspected colony was suspended in a drop of sterile distilled water to make a film, stained with Gram stain, and examined under a light Microscope. Campylobacter cells can be identified as Gram-negative short spirally curved rods: S-shape or sea gull wing shape. Old culture becomes cocoid in shape. A catalase test has been performed by taking one colony by loop and adding one drop of 3% H2O2 and examining immediately for evolution of gas. For the oxidase test, one colony was taken by loop and spread on oxidase detection strip and observed for up to 5 s. The positive result was observed as a deep blue or violet color. A loopful of colonies was suspended in 0.4 mL of 1% Na hippurate solution and incubated at 37 °C for 2 h in a water bath. Then 200 μL of 2.5% ninhydrin solution was slowly added on the inner side of the tube to form an over layer, then the tube was re-incubated for 10 min as mentioned. The presence of dark purple/blue color is reported as a positive result, while the negative result appears as clear or gray color.

2.2. Molecular Typing and Virulence Gene Detection of Campylobacter Species

Molecular typing of Campylobacter species using conventional duplex PCR for detection of mapA gene for C. jejuni (589 bp band) and ceuE gene for C. coli (462 bp band) (Supplementary Table S1) has been performed to confirm Campyloabacetr spp. identification [24,25,26,27].
A total of twenty Campylobacter isolates were randomly selected using stratification by host and species for molecular detection of 15 different virulence genes, comprising 10 isolates from chicken samples (5 C. jejuni and 5 C. coli) and 10 isolates from human samples (5 C. jejuni and 5 C. coli). DNA was extracted using the QIAamp DNA Mini Kit (QIAGEN, Germany) following the manufacturer’s instructions.
Oligonucleotide primers were supplied from Metabion (Planegg, Germany). Primer sequences and cycling conditions for confirmation and typing of Campylobacter spp. were performed as reported previously in [27,28]. PCR assays were used for amplification of various genes (Supplementary Tables S2–S7), including flaA and csrA genes, responsible for adhesion [29], virB11, ciaB, pldA, iam genes, responsible for invasion [10], cdtB gene, responsible for cytotoxicity [10], dnaJ and racR genes, responsible for Campylobacter resistance to stress conditions [10], waaC, wlaN, cstII, cgtB, and Ggt genes, responsible for Campylobacter ganglioside mimicry [30], and tetO gene, responsible for tetracycline resistance [31]. For all assays, Emerald Amp GT PCR master mix (Takara Bio Inc., Shiga, Japan) was used. PCR products were separated by electrophoresis using a Gelpilot 100 bp DNA Ladder (Qiagen GmbH, Hilden, Germany) and visualized with a gel documentation system. Images of the gels were captured, and data were analyzed using dedicated software to confirm the presence of the targeted genes.

2.3. Gene Expression of Seven Campylobacter Virulence Genes in Chicken Samples Versus Human Samples

The expression levels of seven virulence genes (dnaJ, virB11, flaA, iam, racR, cdtB, and tetO) in C. jejuni and C. coli were analyzed in isolates from chicken and human samples, with 23S rRNA serving as the reference housekeeping gene [32]. The 23srRNA gene is highly conserved and stably expressed across different conditions and strains of C. jejuni and C. coli. Its consistent expression makes it a reliable internal control for relative quantification. Total RNA was extracted using the RNeasy Mini Kit (QIAGEN, Germany) following the manufacturer’s protocol. Extracted RNA purity was evaluated using a NanoDrop Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Complementary DNA (cDNA) synthesis was performed using RevertAid Reverse Transcriptase (Thermo Fisher, 200 U/µL). Quantitative polymerase chain reaction (qPCR) was conducted using the Quantitect SYBR Green PCR Kit (QIAGEN). Specific oligonucleotide primers for each target gene were synthesized by Metabion (Germany). Cycling conditions for qPCR are detailed in Supplementary File Table S8. Amplifications were performed on the Stratagene MX3005P Real-Time PCR System (Agilent Technologies Germany GmbH & Co. KG, Waldbronn, Germany), a fully integrated platform for quantitative PCR detection and data analysis.

2.4. Analysis of the SYBR Green RT-PCR for Gene Expression

Amplification curves and cycle threshold (CT) values were generated using the Stratagene MX3005P software version 3.2. To estimate the variation of gene expression on the RNA of the different samples, the CT of each sample was compared with that of the control group according to the “ΔΔCT” method stated previously [33] using the following ratio: (2−ΔΔct), whereas ΔΔCT = ΔCT reference (23srRNA) − ΔCT target (for each gene).
ΔCT target (for each gene) = CT control (Human) − CT treatment (chicken)
ΔCT reference (23srRNA) = CT control (Human) − CT treatment (chicken).

2.5. Statistical Analysis

The prevalence and correlation of the virulence genes of Campylobacter species isolated from human and chicken fecal samples were analyzed using Fisher’s exact test using GraphPad Prism 9.0 for Windows (GraphPad Software, San Diego, CA, USA, www.graphpad.com (accessed on 3 May 2025) with two-sided α = 0.05.

2.6. Ethical Statement

All procedures involving the collection of human fecal samples were conducted with prior informed consent from participants, ensuring anonymity and confidentiality, in compliance with ethical guidelines and institutional approval.

3. Results

3.1. Isolation and Molecular Typing of Campylobacter Species

Out of 100 chicken tested samples, 69 were positive for Campylobacter spp. (C. jejuni & C. coli), while out of 100 human tested samples, 23 were positive using the passive filtration method. All positive samples were catalase, oxidase, and microscopically positive for Campylobacter identification. C. jejuni are hippurate-positive, while C. coli are negative. Molecular typing of Campylobacter spp. using conventional duplex PCR confirmed the identification of C. jejuni from ten and eight of the chicken and human samples, and C. coli from 59 and 15 of human fecal samples, respectively.

3.2. Prevalence of Campylobacter Virulence Genes and tetO Gene

The prevalence of virulence genes in both chicken and human samples could be categorized into the following groups: Group no. 1, genes that were detected in 100% of tested chicken and human samples (virB11, iam, racR and tetO), Group no. 2, genes that were detected in chicken and human samples with different ratios (dnaJ, flaA and cdtB), Group no. 3, genes that were detected in chicken samples and not in human samples (pldA, ciaB and wlaN), and Group no. 4, genes that were not detected in any of tested samples, neither chicken nor human (Cst11, csrA, Waac, ggt and cgtB), as shown in Table 1. Human C. jejuni and C. coli isolates harbored seven virulence and resistance genes (virB11, iam, racR, dnaJ, flaA, cdtB, and tetO), whereas chicken-derived C. jejuni isolates carried nine genes, including two additional virulence factors (pldA and ciaB). C. coli isolates from chickens exhibited the highest gene content, possessing ten genes with an additional gene (wlaN) not detected in the human isolates (Table 1). These findings clarify the criteria for multi-virulence by highlighting the number and type of virulence-associated genes present in each isolate group. For chicken samples, pldA and dnaJ genes were detected in all C. jejuni and C. coli samples, and the flaA gene was detected in all C. jejuni and 60% of C. coli samples. The cdtB gene was detected in 80% and 60% of C. jejuni and C. coli samples, respectively, and the ciaB gene was detected in 60% and 40% of C. jejuni and C. coli samples, respectively. The Wlan gene was detected in 20% of C. coli and not detected in any of the C. jejuni samples. For human samples, dnaJ, flaA, and cdtB were detected in 20% of C. jejuni and 40% of C. coli samples, while pldA, ciaB, and wlan were not detected in any sample from a human source, as shown in Table 2, Figure 1 and Supplementary Photos S1–S17.
Fisher’s exact tests were performed for each gene of interest to assess the distribution of virulence genes between C. jejuni and C. coli in human and chicken hosts. The analysis revealed no significant differences in the frequency of all the virulence genes between the two species in the same host (Supplementary Figure S1). Meanwhile, comparing host-specific distributions, pldA and dnaJ genes were significantly more frequent in chicken-derived C. jejuni isolates than in human-derived isolates (p = 0.0079 and 0.0476, respectively), suggesting potential host adaptation or selective pressure. Similarly, the pldA gene was significantly more frequent in chicken-derived C. coli isolates than in human-derived isolates (p = 0.0079) (Figure 1).
The correlation study of virulence gene expression in C. jejuni and C. coli strains demonstrated unique co-expression patterns, indicating functional and regulatory interactions among certain genes. Significant positive associations were identified among flaA, dnaJ, cdtB, and pldA (r > 0.7), suggesting a possible virulence module. Moderate correlations (0.4 < r ≤ 0.7), exemplified by the relationships between ciaB and cdtB, as well as flaA and ciaB, indicate partial co-regulation. Conversely, weak or indeterminate relationships among genes such as wlaN, waaC, and cgtB indicate independent expression or restricted variability within the dataset (Figure 2).
Genes are clustered based on their pairwise correlation values, with blue indicating strong positive correlation and red indicating negative correlation.

3.3. Expression of Seven Campylobacter Virulence Genes in Chicken Samples Versus Human Samples

The expression of seven virulence genes from C. jejuni and C. coli isolates obtained from chicken and human samples is presented in Table 2, Figure 2 and amplification plots in Supplementary Photos S18–S25.
Genes dnaJ, virB11, flaA, and iam show strong upregulation in chickens for both C. jejuni and C. coli (Supplementary Figure S2). dnaJ is the most upregulated gene in both C. jejuni and C. coli, suggesting a potential role in host adaptation or stress response. cdtB and racR are downregulated in chickens, indicating diminished expression or potential host-specific regulation. Gene tetO exhibited negligible variation, indicating stable expression among hosts (Figure 3).
This figure depicts the log2-transformed fold change in the expression of seven virulence genes in C. jejuni and C. coli between avian and human hosts. Positive numbers signify overexpression in chickens, whilst negative values denote downregulation. The comparison underscores host-specific expression patterns that may indicate adaptive or harmful mechanisms.

3.4. Hierarchical Clustering Heatmap of Prevalence of 15 Virulence Genes in Different Isolates

The hierarchical clustering heatmap reveals distinct virulence gene profiles in Campylobacter isolates. Chicken isolates (C. jejuni and C. coli) form a tight cluster characterized by high prevalence of invasion-associated genes (pldA, dnaJ, and cdtB; 60–100%), reflecting enhanced adaptation to chicken. In contrast, human C. jejuni and C. coli isolates cluster separately with minimal expression of these genes (0–40%), relying instead on universal core genes (virB11, iam, racR, tetO; 100% in all groups) for basic pathogenicity and tetracycline resistance. Genes like flaA show moderate activity in both hosts, while ggt, cgtB, cstII, csrAc, and waaC are universally absent. This apparent host-based clustering demonstrates Campylobacter’s adaptive specialization of virulence mechanisms, with chicken isolates evolving enhanced colonization capabilities (Figure 4).

4. Discussion

Poultry, especially chicken, comprises the main source of human campylobacteriosis. Campylobacter jejuni can colonize the caecum of chickens in extremely high numbers of up to 109 CFU/g of fecal matter; even though this pathogen is present in such high quantities, the chicken rarely exhibits disease symptoms [34]. Variable virulence gene detection in Campylobacter spp. isolated from chicken and human fecal samples has been reported [35]. Wieczorek, K. and Osek, J. [36] emphasized that the molecular profiling of Campylobacter spp. can enhance microbial risk assessment by providing insights into the genetic relatedness of strains implicated in foodborne outbreaks.
In the present study, flaA, a major structural protein of the flagellum, plays a critical role in Campylobacter motility, chemotaxis, and initial adhesion to epithelial cells. This motility is essential for reaching and colonizing the mucus layer of the intestinal tract [37]. In the current study, flaA was detected in 20% and 40% in human C. jejuni and C. coli, and in 60% and 100% in chicken C. jejuni and C. coli, respectively. These findings align with a previous study that reported the flaA gene in 20% of human C. jejuni isolates in Indonesia [38]. In contrast, the flaA gene was detected in 100% of C. jejuni and C. coli isolates from dogs and humans in Gyeongnam and Busan in Korea [39]. The variable flaA gene detection in different studies may be due to the flaA gene containing many short variable regions (SVRs) that lead to diverse allelic forms resulting in varying falA gene sequence among strains from different countries [40]. Our study revealed higher expression of the flaA gene in chicken C. jejuni (5.39) and C. coli (2.84) compared to human isolates. This supports the findings of Lima et al. [41], who also reported a higher frequency of the flaA gene in C. jejuni strains from poultry than from human sources. Additionally, Stintzi revealed a 1.5- to 2-fold upregulation of the flaA gene at 5 and 10 min after a temperature increase from 37 to 42 °C [42]. These data suggested that C. jejuni and C. coli from chicken exhibit higher flagellar biosynthesis and subsequently enhanced colonization capabilities in chicken compared to the human gastrointestinal tract. The global regulator csrA (Carbon storage regulator) gene acts as a global post-transcriptional regulator. It has been shown to influence several virulence-related traits, including motility, oxidative stress resistance, and the ability to invade epithelial cells by modulating mRNA stability of multiple target genes [43]. However, it remains relatively uncharacterized in the genus Campylobacter. Previously reported data illustrate the requirement for csrA in several virulence-related phenotypes of C. jejuni strain 81–176, indicating that the csr pathway is important for Campylobacter pathogenesis [44]. The csrA mutant exhibits changes in several virulence-related properties, including oxidative stress resistance, motility, adherence, and invasion. In the present study, we could not detect csrA gene in any of the examined human or chicken fecal samples using conventional PCR. In contrast, a separate study detected the csrA gene in 100% C. jejuni isolates from human sources and 87.3% of chicken meat, linking the csrA gene with oxidative stress responses, biofilm formation, and cell adhesion [30]. The absence of csrA could have been compensated by other virulence genes such as htrB and clpP genes [45].
Many of the virulence factors connected with Campylobacter invasiveness are placed on the pVir plasmid, for example, the virB11 gene that encodes the IV secretory system protein [8]. In the present study, results revealed that all C. jejuni and C. coli isolates tested positive for the virB11 gene. These findings align with Elgazzar et al., who observed a higher prevalence of virB11 in C. jejuni and C. coli from poultry cecal contents than from human origin in Egypt [46]. In contrast, Wieczorek and Osek reported a lower prevalence of virB11, with 32.7% of C. jejuni and 92.9% of C. coli isolates from poultry carcasses and 65% of C. jejuni and 40% of C. coli isolates from poultry fecal samples [47]. Another study investigated the virB11 gene in C. jejuni and C. coli from different sources: 13.6% and 9.1% in chicken, 41.7% and 0% in pigs, 14.3% and 100% in dogs, and 16.7% and 0% in children’s samples [48]. In contrast, the virB11 gene was not detected in any of the thirty-seven C. jejuni or eight C. coli isolates from poultry and poultry by-products in a study conducted by Jribi et al. in Tunisia [49]. The role of the protein encoded by the virB11 gene in the colonization and invasion of eukaryotic cells by Campylobacter spp. has not been elucidated [48]. However, 100% detection of the virB11 gene highlights the important role of the pVir plasmid and its virulence factors in Campylobacter colonization and invasiveness in chickens and humans. Reported data show higher expression of the virB11 gene in C. jejuni (7.16) and C. coli (5.21) from chicken compared to human samples.
The ciaB gene plays a significant role in Campylobacter pathogenicity by encoding the ciaB 73 kDa protein, essential for epithelial cell invasion and intestinal colonization in avian species [50]. In the present study, the ciaB gene was detected in 60% and 40% of C. jejuni and C. coli isolates from chicken samples and it was absent in all human samples. In South Africa, the ciaB gene was detected in 47% C. jejuni and in 10% C. coli isolates from chicken fecal samples and in 45% C. jejuni and in 43% of C. coli from human clinical samples [35]. Conversely, in Brazil, the ciaB gene was detected in all C. jejuni isolates from human and chicken fecal samples [51].
The invasion-associated marker (iam) gene is one of the most important factors responsible for Campylobacter invasion of host cells, and it was detected in 85% of invasive strains and 20% of non-invasive strains [52]. In this study, we detect the iam gene in 100% of C. jejuni and C. coli from chicken and human fecal samples. This is similar to what was observed before in Canada, where the iam gene was detected in 92.31% of human clinical samples [50]. A 100% detection of invasion-associated marker iam gene indicates its important role in Campylobacter invasion of host cells, and its higher expression in chicken than human indicates higher colonization of Campylobacter spp. in chicken than human gastrointestinal tract.
The pldA gene is also related to cell invasion and is responsible for the synthesis of an outer membrane phospholipase that is important for cecal colonization [35]. Interestingly, the distribution of the pldA gene in this study is dissimilar among the two hosts investigated; in chickens, C. jejuni and C. coli showed 100% positivity, while in humans, no positive sample was found. In South Africa, they detected the pldA gene in 57% and 100% of chicken fecal C. jejuni and C. coli isolates, respectively, while human fecal samples showed lower detection, with rates of 49% and 57% for C. jejuni and C. coli, respectively [35]. Another group reported an increase from 88% to 100% in the presence of the pldA gene in C. jejuni isolates from chickens, with the age of broilers as a major contributing factor [10]. In this study, the detection of the cdtB gene is dissimilar among the two hosts; Campylobacter species, with a detection rate of 20% and 40% in human C. jejuni and C. coli, respectively, was observed, while detection rates of 80% and 60% in chicken C. jejuni and C. coli, respectively, were obtained. These data align partially with a group who detected cdtB in 20% and 14% in human C. jejuni and C. coli, respectively, and 53% and 60% in chicken C. jejuni and C. coli, respectively [35]. In contrast, other studies detected cdtB in 100% of human clinical and chicken fecal C. jejuni and C. coli samples [10,30].
The study elucidates that the dnaJ gene was detected in 100% of chicken C. jejuni and C. coli, while in human samples, detection was 20% and 40% of C. jejuni and C. coli samples, respectively. Results of chicken samples were in accordance with Datta et al., who detected dnaJ in 100% of all chicken fecal samples examined, while they found a difference in human samples with a detection rate of 98% [10]. On the other hand, our results in human samples were closely similar to those who detected the dnaJ gene in 46% and 50% of human C. jejuni and C. coli samples, respectively, although there are some differences in the results of dnaJ from chicken samples, which came at rates of 69% and 70% for C. jejuni and C. coli, respectively [35]. Others detected the dnaJ gene in 100% of human C. jejuni and C. coli [39]. Higher expression of the dnaJ gene in chicken C. jejuni group (11.5) and C. coli (8.34) than human isolates (control groups) was observed in our study. These results represent a significant role of dnaJ in Campylobacter colonization in chickens and its high environmental persistence, although it has been assumed to be a delicate bacteria. Coherently, Stintzi confirmed the upregulation of dnaJ upon temperature stress and demonstrated the temperature-responsive regulation of many other heat shock proteins [42].
Herein, results reported that the racR gene was detected in 100% of human and chicken C. jejuni and C. coli samples, and these data confirmed the importance of racR gene in C. jejuni and C. coli colonization. Likewise, the racR gene was detected in 98.2% and 100% of C. jejuni isolates from human and chicken, respectively [10]. This also partially agrees with findings by [50], who reported racR in 94.2% of C. jejuni from human clinical samples. The consistent detection of racR underscores its critical role in Campylobacter colonization and environmental adaptation. In our study, we could not detect any of the Lipo-oligosaccharide (LOS)-associated genes in any of the examined human or chicken fecal samples using conventional PCR, except wlaN gene, found in 20% of chicken C. coli. wlaN gene was detected in 25%, 23.8%, and 4.7% of C. jejuni isolates from human, poultry meat, and chicken feces, respectively [10], while cst-II and ggt genes were detected in 83.6% and 32.7% of 55 examined C. jejuni human-origin isolates and in 40% and 5.5% of 55 C. jejuni broiler chicken meat-origin isolates in Chile [30].
Tetracycline resistance in Campylobacter spp. is primarily mediated by a ribosomal protection protein (tetO), which is transferred as a plasmid-encoded gene [49]. The tetO gene encodes a ribosomal protection protein that dislodges tetracycline from its binding site on the bacterial ribosome, conferring resistance and enabling Campylobacter to survive tetracycline treatment [53]. Our data showed that all human and chicken C. jejuni and C. coli tested samples were positive for the tetO gene. These results agreed with Hassanain, who reported that the rate of tetracycline resistance in C. jejuni isolates from humans was 75% [54]. These data indicated that tetO is widespread among Campylobacter spp., probably due to conjugative plasmids. Lower results of tetracycline resistance, with a rate of 52.5% and 27.1% of human and poultry isolated C. jejuni, respectively, and 34.8% and 56.7% of human and poultry isolated C. coli, respectively, were reported [55]. A 100% detection of tetO indicates high tetracycline resistance of Campylobacter in both chicken and human. This emphasizes the critical need to interrupt the transmission pathways of Campylobacter across interconnected ecosystems in order to mitigate potential public health threats. Furthermore, it highlights the importance of implementing antimicrobial stewardship practices, particularly regarding the use of tetracyclines in poultry production, to curb the emergence and spread of tetracycline-resistant Campylobacter spp.

5. Conclusions

This study demonstrates a higher gastrointestinal colonization potential in C. jejuni and C. coli isolates from chicken- compared to human-derived isolates. Significant upregulation of the dnaJ, virB11, flaA, and iam genes in chicken-derived C. jejuni and C. coli isolates compared to human isolates indicates host-specific expression patterns and implicates chicken as a primary reservoir for human campylobacteriosis. Conversely, no significant expression differences were noted for the tetO, racR, and cdtB genes across the two host groups. The detection of the wlaN gene in 20% of C. coli chicken isolates, coupled with the absence of other ganglioside mimicry genes (Cst11, csrA, waaC, ggt, and cgtB) in both chicken and human samples, highlights the limited distribution of Guillain–Barré syndrome-related virulence genes in the cohort. Collectively, these findings underscore the extensive genetic diversity of C. jejuni and C. coli and emphasize the necessity for comprehensive molecular characterization of virulence genes for epidemiological evaluations and public health risk assessments of Campylobacter infections in Egypt. Furthermore, the presence of the antibiotic resistance gene, tetO, in all chicken and human isolates highlights the urgency of expanding antimicrobial resistance profiling to guide effective intervention strategies.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microbiolres16090209/s1, Photo S1: Agarose gel electrophoresis for detection of mapA gene for C. jejuni (589 bp fragment) and ceuE gene for C. coli (462 bp fragment) in 20 Broiler fecal samples examined directly by conventional duplex PCR showing amplification of 650 bp. fragment. Photo S2: Agarose gel electrophoresis for detection of mapA gene for C. jejuni (589 bp fragment) and ceuE gene for C. coli (462 bp fragment) in 20 Human fecal samples examined directly by conventional duplex PCR showing amplification of 650 bp. fragment. Photo S3: Agarose gel electrophoresis for detection of flaA virulence gene for Campylobacter spp. in 20 human and chicken fecal samples examined directly by conventional PCR showing amplification of 217 bp. fragment. Photo S4: Agarose gel electrophoresis for detection of csrA virulence gene for Campylobacter spp. in 20 human and chicken fecal samples examined directly by conventional PCR showing amplification of 878 bp. fragment. Photo S5: Agarose gel electrophoresis for detection of virB11 virulence gene for Campylobacter spp. in 20 human and chicken fecal samples examined directly by conventional PCR showing amplification of 494 bp. fragment. Photo S6: Agarose gel electrophoresis for detection of ciaB virulence gene for Campylobacter spp. in 20 human and chicken fecal samples examined directly by conventional PCR showing amplification of 527 bp. fragment. Photo S7: Agarose gel electrophoresis for detection of pldA virulence gene for Ca mpylobacter spp. in 20 human and chicken fecal samples examined directly by conventional PCR showing amplification of 385 bp. fragment. Photo S8: Agarose gel electrophoresis for detection of iam virulence gene for Campylobacter spp. in 20 human and chicken fecal samples examined directly by conventional PCR showing amplification of 518 bp. fragment. Photo S9: Agarose gel electrophoresis for detection of cdtB virulence gene for Campylobacter spp. in 20 human and chicken fecal samples examined directly by conventional PCR showing amplification of 620 bp. fragment. Photo S10: Agarose gel electrophoresis for detection of dnaJ virulence gene for Campylobacter spp. in 20 human and chicken fecal samples examined directly by conventional PCR showing amplification of 177 bp. fragment. Photo S11: Agarose gel electrophoresis of conventional PCR for detection of racR virulence gene for Campylobacter spp. in 20 human and chicken fecal samples examined directly by cPCR showing amplification of 584 bp. fragment. Photo S12: Agarose gel electrophoresis for detection of WlaN virulence gene for Campylobacter spp. in 20 human and chicken fecal samples examined directly by conventional PCR showing amplification of 672 bp. fragment. Photo S13: Agarose gel electrophoresis for detection of WaaC virulence gene for Campylobacter spp. in 20 human and chicken fecal samples examined directly by conventional PCR showing amplification of 971 bp. fragment. Photo S14: Agarose gel electrophoresis for detection of cstII virulence gene for Campylobacter spp. in 20 human and chicken fecal samples examined directly by conventional PCR showing amplification of 570 bp. fragment. Photo S15: Agarose gel electrophoresis for detection of cgtB virulence gene for Campylobacter spp. in 20 human and chicken fecal samples examined directly by conventional PCR showing amplification of 562 bp. fragment. Photo S16: Agarose gel electrophoresis for detection of ggt virulence gene for Campylobacter spp. in 20 human and chicken fecal samples examined directly by conventional PCR showing amplification of 419 bp. fragment. Photo S17: Agarose gel electrophoresis for detection of tetO virulence gene for Campylobacter spp. in 20 human and chicken fecal samples examined directly by conventional PCR showing amplification of 559 bp. fragment. Photo S18: 23srRNA (Housekeeping gene) amplification plots for 4 chicken samples; 42 and 49 (C. jejuni) & 23 and 45 (C. coli). And 3 Human Samples; 65 (C. jejuni) & 63 and 68 (C. coli). Photo S19: flaA amplification plots for 4 chicken samples; 42 and 49 (C. jejuni) & 23 and 45 (C. coli). And 3 Human Samples; 65 (C. jejuni) & 63 and 68 (C. coli). Photo S20: virB11 amplification plots for 4 chicken samples; 42 and 49 (C. jejuni) & 23 and 45 (C. coli). And 3 Human Samples; 65 (C. jejuni) & 63 and 68 (C. coli). Photo S21: iam amplification plots for 4 chicken samples; 42 and 49 (C. jejuni) & 23 and 45 (C. coli). And 3 Human Samples; 65 (C. jejuni) & 63 and 68 (C. coli). Photo S22: cdtB amplification plots for 4 chicken samples; 42 and 49 (C. jejuni) & 23 and 45 (C. coli). And 3 Human Samples; 65 (C. jejuni) & 63 and 68 (C. coli). Photo S23: dnaJ amplification plots for 4 chicken samples; 42 and 49 (C. jejuni) & 23 and 45 (C. coli). And 3 Human Samples; 65 (C. jejuni) & 63 and 68 (C. coli). Photo S24: racR amplification plots for 4 chicken samples; 42 and 49 (C. jejuni) & 23 and 45 (C. coli). And 3 Human Samples; 65 (C. jejuni) & 63 and 68 (C. coli). Photo S25: tetO amplification plots for 4 chicken samples; 42 and 49 (C. jejuni) & 23 and 45 (C. coli). And 3 Human Samples; 65 (C. jejuni) & 63 and 68 (C. coli). Figure S1: Frequency of virulence genes in C. jejuni and C. coli isolated in human vs chicken fecal samples. Figure S2: Mean fold changes of seven virulence genes in C. jejuni and C. coli in chicken versus human group (control). Table S1. Oligonucleotide primers sequences for Campylobacter identification genes. Table S2. Oligonucleotide primers sequences for two of genes responsible for Campylobacter adhesion. Table S3. Oligonucleotide primers sequences for four of genes responsible for Campylobacter invasion. Table S4. Oligonucleotide primers sequences for the main gene responsible for Campylobacter cytotoxicity. Table S5. Oligonucleotide primers sequences for the main two genes responsible for Campylobacter resistance to stress conditions. Table S6. Oligonucleotide primers sequences for the five genes responsible for Campylobacter ganglioside Mimicry. Table S7. Oligonucleotide primers sequences for one of genes responsible for Campylobacter tetracycline resistance. Table S8. Cycling conditions of qPCR assays. References [56,57,58,59,60,61] are cited in the Supplementary Materials.

Author Contributions

Conceptualization, M.E.; Methodology, A.M.; analysis and interpretation of results, A.M.; writing—original draft, A.M.; writing—reviewing and editing, A.M., M.R.I., A.H., W.H., A.W., S.E. and M.E.; statistical analysis, A.M. and I.H.; practical work supervision and investigation, W.H.; Principal Investigator, M.E. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

All procedures involving the collection of human fecal samples were conducted with prior informed consent from participants, ensuring anonymity and confidentiality, in compliance with ethical guidelines and institutional approval.

Informed Consent Statement

In our study, the collection of human fecal samples did not involve any invasive procedures, and institutional guidelines required obtaining informed consent from participants rather than formal ethics committee approval. As such, we ensured that all participants were fully informed about the study and provided their voluntary verbal consent before sample collection, respecting cultural norms and practices.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Kirk, M.D.; Pires, S.M.; Black, R.E.; Caipo, M.; Crump, J.A.; Devleesschauwer, B.; Döpfer, D.; Fazil, A.; Fischer-Walker, C.L.; Hald, T.; et al. World Health Organization estimates of the global and regional disease burden of 22 foodborne bacterial, protozoal, and viral diseases, 2010: A data synthesis. PLoS Med. 2015, 12, e1001921. [Google Scholar] [CrossRef]
  2. Kotloff, K.L.; Nataro, J.P.; Blackwelder, W.C.; Nasrin, D.; Farag, T.H.; Panchalingam, S.; Wu, Y.; Sow, S.O.; Sur, D.; Breiman, R.F.; et al. Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): A prospective, case-control study. Lancet 2013, 382, 209–222. [Google Scholar] [CrossRef]
  3. Khoshbakht, R.; Tabatabaei, M.; Shirzad Aski, H.; Hosseinzadeh, S. Occurrence of virulence genes and strain diversity of thermophilic campylobacters isolated from cattle and sheep faecal samples. Iran. J. Vet. Res. 2014, 15, 138–144. [Google Scholar]
  4. OIE. Chapter 2.9.3. Infection with Campylobacter jejuni and C. coli. In OIE Terrestrial Manual; WOAH: Paris, France, 2024. [Google Scholar]
  5. Noormohamed, A.; Fakhr, M.K. Prevalence and antimicrobial susceptibility of Campylobacter spp. in Oklahoma conventional and organic retail poultry. Open Microbiol. J. 2014, 8, 130–137. [Google Scholar] [CrossRef][Green Version]
  6. Sierra-Arguello, Y.M.; Perdoncini, G.; Rodrigues, L.B.; dos Santos, L.R.; Borges, K.A.; Furian, T.Q.; Salle, C.T.P.; Moraes, H.L.d.S.; Gomes, M.J.P.; Nascimento, V.P.D. Identification of pathogenic genes in Campylobacter jejuni isolated from broiler carcasses and broiler slaughterhouses. Sci. Rep. 2021, 11, 4588. [Google Scholar] [CrossRef]
  7. Fields, J.A.; Li, J.; Gulbronson, C.J.; Hendrixson, D.R.; Thompson, S.A. Campylobacter jejuni CsrA Regulates Metabolic and Virulence Associated Proteins and Is Necessary for Mouse Colonization. PLoS ONE 2016, 11, e0156932. [Google Scholar] [CrossRef] [PubMed]
  8. Bacon, D.J.; Alm, R.A.; Burr, D.H.; Hu, L.; Kopecko, D.J.; Ewing, C.P.; Guerry, P. Involvement of a plasmid in virulence of Campylobacter jejuni 81-176. Infect. Immun. 2000, 68, 4384–4390. [Google Scholar] [CrossRef] [PubMed]
  9. Andrzejewska, M.; Grudlewska-Buda, K.; Śpica, D.; Skowron, K.; Ćwiklińska-Jurkowska, M.; Szady-Grad, M.; Indykiewicz, P.; Wiktorczyk-Kapischke, N.; Klawe, J.J. Genetic relatedness, virulence, and drug susceptibility of Campylobacter isolated from water and wild birds. Front. Cell. Infect. Microbiol. 2022, 12, 1005085. [Google Scholar] [CrossRef]
  10. Datta, S.; Niwa, H.; Itoh, K. Prevalence of 11 pathogenic genes of Campylobacter jejuni by PCR in strains isolated from humans, poultry meat and broiler and bovine faeces. J. Med. Microbiol. 2003, 52, 345–348. [Google Scholar] [CrossRef]
  11. Guerra, L.; Cortes-Bratti, X.; Guidi, R.; Frisan, T. The biology of the cytolethal distending toxins. Toxins 2011, 3, 172–190. [Google Scholar] [CrossRef]
  12. Grant, K.A.; Belandia, I.U.; Dekker, N.; Richardson, P.T.; Park, S.F. Molecular characterization of pldA, the structural gene for a phospholipase A from Campylobacter coli, and its contribution to cell-associated hemolysis. Infect. Immun. 1997, 65, 1172–1180. [Google Scholar] [CrossRef]
  13. Konkel, M.E.; Klena, J.D.; Rivera-Amill, V.; Monteville, M.R.; Biswas, D.; Raphael, B.; Mickelson, J. Secretion of virulence proteins from Campylobacter jejuni is dependent on a functional flagellar export apparatus. J. Bacteriol. 2004, 186, 3296–3303. [Google Scholar] [CrossRef]
  14. Chansiripornchai, N.; Sasipreeyajan, J. PCR detection of four virulence-associated genes of Campylobacter jejuni isolates from Thai broilers and their abilities of adhesion to and invasion of INT-407 cells. J. Vet. Med. Sci. 2009, 71, 839–844. [Google Scholar] [CrossRef] [PubMed]
  15. Brás, A.M.; Chatterjee, S.; Wren, B.W.; Newell, D.G.; Ketley, J.M. A novel Campylobacter jejuni two-component regulatory system important for temperature-dependent growth and colonization. J. Bacteriol. 1999, 181, 3298–3302. [Google Scholar] [CrossRef] [PubMed]
  16. Louwen, R.; Heikema, A.; van Belkum, A.; Ott, A.; Gilbert, M.; Ang, W.; Endtz, H.P.; Bergman, M.P.; Nieuwenhuis, E.E. The sialylated lipooligosaccharide outer core in Campylobacter jejuni is an important determinant for epithelial cell invasion. Infect. Immun. 2008, 76, 4431–4438. [Google Scholar] [CrossRef]
  17. Barnes, I.H.; Bagnall, M.C.; Browning, D.D.; Thompson, S.A.; Manning, G.; Newell, D.G. Gamma-glutamyl transpeptidase has a role in the persistent colonization of the avian gut by Campylobacter jejuni. Microb. Pathog. 2007, 43, 198–207. [Google Scholar] [CrossRef] [PubMed][Green Version]
  18. Rizal, A.; Kumar, A.; Vidyarthi, A.S. Prevalence of pathogenic genes in Campylobacter jejuni isolated from poultry and human. Internet J. Food Saf. 2010, 12, 29–34. [Google Scholar][Green Version]
  19. Awad, A.; Yeh, H.Y.; Ramadan, H.; Rothrock, M.J. Genotypic characterization, antimicrobial susceptibility and virulence determinants of Campylobacter jejuni and Campylobacter coli isolated from pastured poultry farms. Front. Microbiol. 2023, 14, 1271551. [Google Scholar] [CrossRef]
  20. Abd El-Hamid, M.I.; Abd El-Aziz, N.K.; Samir, M.; El-Naenaeey, E.-s.Y.; Abo Remela, E.M.; Mosbah, R.A.; Bendary, M.M. Genetic diversity of Campylobacter jejuni isolated from avian and human sources in Egypt. Front. Microbiol. 2019, 10, 2353. [Google Scholar] [CrossRef]
  21. Mouftah, S.F.; Pascoe, B.; Calland, J.K.; Mourkas, E.; Tonkin, N.; Lefevre, C.; Deuker, D.; Smith, S.; Wickenden, H.; Hitchings, M.D. Local accessory gene sharing among Egyptian Campylobacter potentially promotes the spread of antimicrobial resistance. Microb. Genom. 2022, 8, 834. [Google Scholar] [CrossRef]
  22. Sainato, R.; ElGendy, A.; Poly, F.; Kuroiwa, J.; Guerry, P.; Riddle, M.S.; Porter, C.K. Epidemiology of Campylobacter Infections among Children in Egypt. Am. J. Trop. Med. Hyg. 2018, 98, 581–585. [Google Scholar] [CrossRef]
  23. Steele, T.W.; McDermott, S.N. The use of membrane filters applied directly to the surface of agar plates for the isolation of Campylobacter jejuni from feces. Pathology 1984, 16, 263–265. [Google Scholar] [CrossRef]
  24. Barakat, A.M.A.; El-Razik, K.A.A.; Elfadaly, H.A.; Rabie, N.S.; Sadek, S.A.S.; Almuzaini, A.M. Prevalence, molecular detection, and virulence gene profiles of Campylobacter species in humans and foods of animal origin. Vet. World 2020, 13, 1430–1438. [Google Scholar] [CrossRef] [PubMed]
  25. Elbrissi, A.; Sabeil, Y.A.; Khalifa, K.A.; Enan, K.; Khair, O.M.; El Hussein, A.M. Isolation, identification and differentiation of Campylobacter spp. using multiplex PCR assay from goats in Khartoum State, Sudan. Trop. Anim. Health Prod. 2017, 49, 575–581. [Google Scholar] [CrossRef]
  26. Gonzalez, I.; Grant, K.A.; Richardson, P.T.; Park, S.F.; Collins, M.D. Specific identification of the enteropathogens Campylobacter jejuni and Campylobacter coli by using a PCR test based on the ceuE gene encoding a putative virulence determinant. J. Clin. Microbiol. 1997, 35, 759–763. [Google Scholar] [CrossRef]
  27. Shin, E.; Lee, Y. Comparison of three different methods for Campylobacter isolation from porcine intestines. J. Microbiol. Biotechnol. 2009, 19, 647–650. [Google Scholar]
  28. Wang, G.; Clark, C.G.; Taylor, T.M.; Pucknell, C.; Barton, C.; Price, L.; Woodward, D.L.; Rodgers, F.G. Colony multiplex PCR assay for identification and differentiation of Campylobacter jejuni, C. coli, C. lari, C. upsaliensis, and C. fetus subsp. fetus. J. Clin. Microbiol. 2002, 40, 4744–4747. [Google Scholar] [CrossRef]
  29. Zheng, J.; Meng, J.; Zhao, S.; Singh, R.; Song, W. Adherence to and invasion of human intestinal epithelial cells by Campylobacter jejuni and Campylobacter coli isolates from retail meat products. J. Food Prot. 2006, 69, 768–774. [Google Scholar] [CrossRef]
  30. González-Hein, G.; Huaracán, B.; García, P.; Figueroa, G. Prevalence of virulence genes in strains of Campylobacter jejuni isolated from human, bovine and broiler. Braz. J. Microbiol. 2013, 44, 1223–1229. [Google Scholar] [CrossRef] [PubMed]
  31. Pratt, A.; Korolik, V. Tetracycline resistance of Australian Campylobacter jejuni and Campylobacter coli isolates. J. Antimicrob. Chemother. 2005, 55, 452–460. [Google Scholar] [CrossRef] [PubMed]
  32. Rivera-Mendoza, D.; Martínez-Flores, I.; Santamaría, R.I.; Lozano, L.; Bustamante, V.H.; Pérez-Morales, D. Genomic analysis reveals the genetic determinants associated with antibiotic resistance in the zoonotic pathogen Campylobacter spp. Distributed Globally. Front. Microbiol. 2020, 11, 513070. [Google Scholar] [CrossRef]
  33. Yang, C.; Jiang, Y.; Huang, K.; Zhu, C.; Yin, Y. Application of real-time PCR for quantitative detection of Campylobacter jejuni in poultry, milk and environmental water. FEMS Immunol. Med. Microbiol. 2003, 38, 265–271. [Google Scholar] [CrossRef]
  34. Thibodeau, A.; Fravalo, P.; Yergeau, É.; Arsenault, J.; Lahaye, L.; Letellier, A. Chicken caecal microbiome modifications induced by Campylobacter jejuni colonization and by a non-antibiotic feed additive. PLoS ONE 2015, 10, e0131978. [Google Scholar] [CrossRef] [PubMed]
  35. Reddy, S.; Zishiri, O.T. Genetic characterisation of virulence genes associated with adherence, invasion and cytotoxicity in Campylobacter spp. isolated from commercial chickens and human clinical cases. Onderstepoort J. Vet. Res. 2018, 85, e1–e9. [Google Scholar] [CrossRef]
  36. Kinga Wieczorek, J.O. Molecular characterization of Campylobacter spp. isolated from poultry faeces and carcasses in Poland. Acta Vet. Brno 2011, 80, 19–27. [Google Scholar] [CrossRef]
  37. Guerry, P. Campylobacter flagella: Not just for motility. Trends Microbiol. 2007, 15, 456–461. [Google Scholar] [CrossRef] [PubMed]
  38. Murphy, H.M.; Prioleau, M.D.; Borchardt, M.A.; Hynds, P.D. Epidemiological evidence of groundwater contribution to global enteric disease, 1948–2015. Hydrogeol. J. 2017, 25, 981–1001. [Google Scholar] [CrossRef]
  39. Cho, H.-H.; Kim, S.-H.; Min, W.; Ku, B.-K.; Kim, Y.-H. Prevalence of virulence and cytolethal distending toxin (CDT) genes in thermophilic Campylobacter spp. from dogs and humans in Gyeongnam and Busan, Korea. Korean J. Vet. Res. 2014, 54, 39–48. [Google Scholar] [CrossRef]
  40. El-Adawy, H.; Hotzel, H.; Tomaso, H.; Neubauer, H.; Taboada, E.N.; Ehricht, R.; Hafez, H.M. Detection of genetic diversity in Campylobacter jejuni isolated from a commercial turkey flock using flaA typing, MLST analysis and microarray assay. PLoS ONE 2013, 8, e51582. [Google Scholar] [CrossRef]
  41. Lima, L.M.; Perdoncini, G.; Borges, K.A.; Furian, T.Q.; Salle, C.T.P.; Moraes, H.L.D.S.; Nascimento, V.P.D. Prevalence and distribution of pathogenic genes in Campylobacter jejuni isolated from poultry and human sources. J. Infect. Dev. Ctries. 2022, 16, 1466–1472. [Google Scholar] [CrossRef]
  42. Stintzi, A. Gene expression profile of Campylobacter jejuni in response to growth temperature variation. J. Bacteriol. 2003, 185, 2009–2016. [Google Scholar] [CrossRef] [PubMed]
  43. Fields, J.A.; Thompson, S.A. Campylobacter jejuni CsrA Mediates Oxidative Stress Responses, Biofilm Formation, and Host Cell Invasion. J. Bacteriol. 2008, 190, 3411–3416. [Google Scholar] [CrossRef]
  44. Fields, J.A.; Thompson, S.A. Campylobacter jejuni CsrA complements an Escherichia coli csrA mutation for the regulation of biofilm formation, motility and cellular morphology but not glycogen accumulation. BMC Microbiol. 2012, 12, 233. [Google Scholar] [CrossRef]
  45. Otigbu, A.C.; Clarke, A.M.; Fri, J.; Akanbi, E.O.; Njom, H.A. Antibiotic sensitivity profiling and virulence potential of Campylobacter jejuni isolates from estuarine water in the Eastern Cape Province, South Africa. Int. J. Environ. Res. Public Health 2018, 15, 925. [Google Scholar] [CrossRef] [PubMed]
  46. Aljazzar, A.; Abd El-Hamid, M.I.; El-Malt, R.M.S.; El-Gharreb, W.R.; Abdel-Raheem, S.M.; Ibrahim, A.M.; Abdelaziz, A.M.; Ibrahim, D. Prevalence and Antimicrobial Susceptibility of Campylobacter Species with Particular Focus on the Growth Promoting, Immunostimulant and Anti-Campylobacter jejuni Activities of Eugenol and Trans-Cinnamaldehyde Mixture in Broiler Chickens. Animals 2022, 12, 905. [Google Scholar] [CrossRef]
  47. Wieczorek, K.; Osek, J. Identification of virulence genes in Campylobacter jejuni and C. coli isolates by PCR. Bull. Vet. Inst. Puławy 2008, 52, 211–216. [Google Scholar]
  48. Krutkiewicz, A.; Klimuszko, D. Genotyping and PCR detection of potential virulence genes in Campylobacter jejuni and Campylobacter coli isolates from different sources in Poland. Folia Microbiol. 2010, 55, 167–175. [Google Scholar] [CrossRef] [PubMed]
  49. Jribi, H.; Sellami, H.; Hassena, A.B.; Gdoura, R. Prevalence of putative virulence genes in Campylobacter and Arcobacter species isolated from poultry and poultry by-products in Tunisia. J. Food Prot. 2017, 80, 1705–1710. [Google Scholar] [CrossRef]
  50. Biswas, D.; Hannon, S.J.; Townsend, H.; Potter, A.; Allan, B.J. Genes coding for virulence determinants of Campylobacter jejuni in human clinical and cattle isolates from Alberta, Canada, and their potential role in colonization of poultry. Int. Microbiol. 2011, 14, 25–32. [Google Scholar]
  51. Frazão, M.R.; Medeiros, M.I.C.; da Silva Duque, S.; Falcão, J.P. Pathogenic potential and genotypic diversity of Campylobacter jejuni: A neglected food-borne pathogen in Brazil. J. Med. Microbiol. 2017, 66, 350–359. [Google Scholar] [CrossRef]
  52. Carvalho, A.C.; Ruiz-Palacios, G.M.; Ramos-Cervantes, P.; Cervantes, L.-E.; Jiang, X.; Pickering, L.K. Molecular characterization of invasive and noninvasive Campylobacter jejuni and Campylobacter coli isolates. J. Clin. Microbiol. 2001, 39, 1353–1359. [Google Scholar] [CrossRef] [PubMed]
  53. Li, W.; Atkinson, G.C.; Thakor, N.S.; Allas, U.; Lu, C.C.; Chan, K.Y.; Tenson, T.; Schulten, K.; Wilson, K.S.; Hauryliuk, V.; et al. Mechanism of tetracycline resistance by ribosomal protection protein Tet(O). Nat. Commun. 2013, 4, 1477. [Google Scholar] [CrossRef]
  54. Hassanain, N.A. Antimicrobial resistant Campylobacter jejuni isolated from humans and animals in Egypt. Glob. Vet. 2011, 6, 195–200. [Google Scholar]
  55. Bardoň, J.; Pudová, V.; Koláčková, I.; Karpíšková, R.; Röderová, M.; Kolář, M. Virulence and antibiotic resistance genes in Campylobacter spp. in the Czech Republic. Epidemiol. Mikrobiol. Imunol. 2017, 66, 59–66. [Google Scholar]
  56. Godschalk, P.C.; van Belkum, A.; van den Braak, N.; van Netten, D.; Ang, C.W.; Jacobs, B.C.; Gilbert, M.; Endtz, H.P. PCR-restriction fragment length polymorphism analysis of Campylobacter jejuni genes involved in lipooligosaccharide biosynthesis identifies putative molecular markers for Guillain-Barré syndrome. J. Clin. Microbiol. 2007, 45, 2316–2320. [Google Scholar] [CrossRef]
  57. Kordinas, V.; Nicolaou, C.; Ioannidis, A.; Papavasileiou, E.; John Legakis, N.; Chatzipanagiotou, S. Prevalence of four virulence genes in Campylobacter jejuni determined by PCR and sequence analysis. Mol. Diagn. 2005, 9, 211–215. [Google Scholar] [CrossRef]
  58. Linton, D.; Gilbert, M.; Hitchen, P.G.; Dell, A.; Morris, H.R.; Wakarchuk, W.W.; Gregson, N.A.; Wren, B.W. Phase variation of a β-1, 3 galactosyltransferase involved in generation of the ganglioside GM1-like lipo-oligosaccharide of Campylobacter jejuni. Mol. Microbiol. 2000, 37, 501–514. [Google Scholar] [CrossRef] [PubMed]
  59. Nahar, N.; Rashid, R. Genotypic Analysis of the Virulence and Antibiotic Resistance Genes in Campylobacter species in silico. J. Bioanal. Biomed. 2018, 10, 13–23. [Google Scholar] [CrossRef]
  60. Talukder, K.A.; Aslam, M.; Islam, Z.; Azmi, I.J.; Dutta, D.K.; Hossain, S.; Nur-E-Kamal, A.; Nair, G.B.; Cravioto, A.; Sack, D.A. Prevalence of virulence genes and cytolethal distending toxin production in Campylobacter jejuni isolates from diarrheal patients in Bangladesh. J. Clin. Microbiol. 2008, 46, 1485–1488. [Google Scholar] [CrossRef] [PubMed]
  61. Gibreel, A.; Tracz, D.M.; Nonaka, L.; Ngo, T.M.; Connell, S.R.; Taylor, D.E. Incidence of antibiotic resistance in Campylobacter jejuni isolated in Alberta, Canada, from 1999 to 2002, with special reference to tet (O)-mediated tetracycline resistance. Antimicrob. Agents Chemother. 2004, 48, 3442–3450. [Google Scholar] [CrossRef]
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Figure 1. Frequency of virulence genes in C. jejuni and C. coli isolated in human vs. chicken fecal samples. (A) Frequency of virulence genes in C. jejuni in human vs. chicken samples; (B) frequency of virulence genes in C. coli in human vs. chicken samples; (C) frequency of virulence genes in C. jejuni and C. coli in human samples; (D) frequency of virulence genes in C. jejuni and C. coli in chicken samples. * mild statistical significance; ** moderate statistical significance.
Figure 1. Frequency of virulence genes in C. jejuni and C. coli isolated in human vs. chicken fecal samples. (A) Frequency of virulence genes in C. jejuni in human vs. chicken samples; (B) frequency of virulence genes in C. coli in human vs. chicken samples; (C) frequency of virulence genes in C. jejuni and C. coli in human samples; (D) frequency of virulence genes in C. jejuni and C. coli in chicken samples. * mild statistical significance; ** moderate statistical significance.
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Figure 2. Correlation matrix of virulence gene expression in C. jejuni and C. coli isolates.
Figure 2. Correlation matrix of virulence gene expression in C. jejuni and C. coli isolates.
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Figure 3. Log2 fold change in the expression of seven virulence genes between chicken and human hosts.
Figure 3. Log2 fold change in the expression of seven virulence genes between chicken and human hosts.
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Figure 4. Hierarchical clustering heatmap of prevalence of 15 virulence genes in different C. jejuni and C. coli isolates. Heatmap analysis of virulence gene prevalence among C. jejuni and C. coli isolates across both human and chicken isolates. tetO, racR, virB11, iam, flaA, and dnaJ demonstrate conserved prevalence. In contrast, ciaB and wlaN exhibit low detection frequencies. pldA and cdtB show significantly increased prevalence in chicken-derived isolates. CJ: C. jejuni; CC: C. coli.
Figure 4. Hierarchical clustering heatmap of prevalence of 15 virulence genes in different C. jejuni and C. coli isolates. Heatmap analysis of virulence gene prevalence among C. jejuni and C. coli isolates across both human and chicken isolates. tetO, racR, virB11, iam, flaA, and dnaJ demonstrate conserved prevalence. In contrast, ciaB and wlaN exhibit low detection frequencies. pldA and cdtB show significantly increased prevalence in chicken-derived isolates. CJ: C. jejuni; CC: C. coli.
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Table 1. Frequency of virulence genes among Campylobacter isolates obtained from human and chicken fecal samples.
Table 1. Frequency of virulence genes among Campylobacter isolates obtained from human and chicken fecal samples.
GeneC. coli (%)p-ValueC. jejuni (%)p-Value
HumanChickenHumanChicken
flaA2 (40)5 (100)0.16671 (20)3 (60)0.5238
csrA00>0.999900>0.9999
virB115 (100)5 (100)>0.99995 (100)5 (100)>0.9999
iam5 (100)5 (100)>0.99995 (100)5 (100)>0.9999
pldA05 (100)0.007905 (100)0.0079
ciaB02 (40)0.444403 (60)0.1667
cdtB2 (40)3 (60)>0.99991 (20)4 (80)0.2063
dnaJ2 (40)5 (100)0.16671 (20)5 (100)0.0476
racR5 (100)5 (100)>0.99995 (100)5 (100)>0.9999
wlaN01 (20)>0.999900>0.9999
waaC00>0.999900>0.9999
cstII00>0.999900>0.9999
cgtB00>0.999900>0.9999
ggt00>0.999900>0.9999
tetO5 (100)5 (100)>0.99995 (100)5 (100)>0.9999
Table 2. Log2 fold change in the expression of seven virulence genes between chicken and human hosts.
Table 2. Log2 fold change in the expression of seven virulence genes between chicken and human hosts.
GeneC. jejuniC. coli
flaA2.431.51
virB112.842.38
iam1.91.32
cdtB−0.51−0.32
dnaJ3.523.06
racR−0.3−0.22
tetO0.080.14
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Mekky, A.; Issa, M.R.; Hashish, A.; Hassan, W.; Wahdan, A.; Hisham, I.; Enany, S.; Enany, M. Comparative Virulence Gene Profiling of Campylobacter jejuni and Campylobacter coli Isolates from Avian and Human Sources in Egypt. Microbiol. Res. 2025, 16, 209. https://doi.org/10.3390/microbiolres16090209

AMA Style

Mekky A, Issa MR, Hashish A, Hassan W, Wahdan A, Hisham I, Enany S, Enany M. Comparative Virulence Gene Profiling of Campylobacter jejuni and Campylobacter coli Isolates from Avian and Human Sources in Egypt. Microbiology Research. 2025; 16(9):209. https://doi.org/10.3390/microbiolres16090209

Chicago/Turabian Style

Mekky, Amr, Mohamed R. Issa, Amro Hashish, Wafaa Hassan, Ali Wahdan, Islam Hisham, Shymaa Enany, and Mohamed Enany. 2025. "Comparative Virulence Gene Profiling of Campylobacter jejuni and Campylobacter coli Isolates from Avian and Human Sources in Egypt" Microbiology Research 16, no. 9: 209. https://doi.org/10.3390/microbiolres16090209

APA Style

Mekky, A., Issa, M. R., Hashish, A., Hassan, W., Wahdan, A., Hisham, I., Enany, S., & Enany, M. (2025). Comparative Virulence Gene Profiling of Campylobacter jejuni and Campylobacter coli Isolates from Avian and Human Sources in Egypt. Microbiology Research, 16(9), 209. https://doi.org/10.3390/microbiolres16090209

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