Isolation and molecular characterization of Campylobacter jejuni from chicken and human stool samples in Egypt

Campylobacteriosis is one of the most well-characterized bacterial foodborne infections worldwide that arises chiefly due to the consumption of poultry and poultry products (Wieczorek et al., 2012). The disease is caused by numerous species within the genus Campylobacter, but Campylobacter jejuni is the most commonly isolated species from established cases of human campylobacteriosis (Eurosurveillance Editorial Team, 2012).


Introduction
Campylobacteriosis is one of the most well-characterized bacterial foodborne infections worldwide that arises chiefly due to the consumption of poultry and poultry products (Wieczorek et al., 2012). The disease is caused by numerous species within the genus Campylobacter, but Campylobacter jejuni is the most commonly isolated species from established cases of human campylobacteriosis (Eurosurveillance Editorial Team, 2012).
Campylobacteriosis causes countless outbreaks and hence a high frequency of hospitalization, occasionally causing death (Taylor et al., 2013).
Campylobacter species are the fundamental cause of the bacterial gastrointestinal malady campylobacteriosis, which causes diarrhoea, dysentery patterns, cramps, pain and fever in developing countries (European Food Safety Authority, 2010).
Foodborne illnesses are largely caused by bacteria, most notably Campylobacter jejuni, which accounts for 77.3% of foodborne illnesses (Doyle & Erickson, 2006).
Foodborne Campylobacter infections occur as a result of the consumption of undercooked or raw poultry, liver or grilled chicken meat (Edwards et al., 2014). This may be due to the thermophilic properties of Campylobacter species, especially C. jejuni, which favours poultry hosts due to their high body temperatures (Verwoerd, 2000).
The intestinal tract of chickens, particularly the caecum and colon, is considered a region of tropism for a vast number of Campylobacter species during processing, especially if the intestinal tract is ruptured and the contents are moved to the skin, prompting further pollution to the carcass (Vinueza-Burgos et al., 2017). Likewise, contact with faecal material on eggs results in egg contamination and the transmission of the bacteria to the inside of the egg, thus initiating the disease after consumption of eggs (Adesiyun et al., 2005).
Campylobacter species are considered the second driving aetiology of paediatric diarrhoea (Rao et al., 2001). In Egypt, it is an endemic disease, and the evaluated percentage of infections in children who are in direct contact with diseased poultry is 12.3% for C. jejuni (El-Tras et al., 2015).
The bacterial isolation and identification of Campylobacter is considered the gold standard for disease identification; however, it is tedious and laborious due to the complex nature of Campylobacter (Li et al., 2014). Thus, molecular techniques, such as polymerase chain reaction (PCR) and sequencing, can permit the simple, fast and exact identification of C. jejuni and reveals its epidemiological characteristics (Miller et al., 2010).
The disease seriousness relies upon the virulence of the strain and on the host's immune state . CadF is one of the reference virulence genes that encodes proteins involved in the attack and attachment of C. jejuni (Elmali & Can, 2019), and this gene is present at a high prevalence in C. jejuni isolates (Andrzejewska et al., 2015).
Thus, this study evaluates the prevalence, virulence gene profile, and molecular and phylogenetic characterization of Campylobacter jejuni isolated from chickens and humans from different governorates in Egypt.

Sampling
A total of 435 samples (Table 1) were used in the present investigation and were gathered between June 2015 and December 2016 from governorates in Egypt (Cairo, Giza, Fayoum, Minya and Qalubia). Additionally, human stool specimens (n = 75) were collected at random from people with diarrhoea who were admitted to different laboratories, people in contact with backyard chickens and slaughterhouses and from diarrheic children admitted to Abul-Riesh hospital for kids in Egypt. Ten grams of each sample (chicken intestine, liver, meat, egg shell swab, cloacal swab, and human stool) was collected in a sterile sample collection vial and transported to the laboratory. All samples were quickly placed at 4 °C and handled to isolate Campylobacter species.
All parts of this study were approved by the Medical Research Ethics Committee, National Research Centre, Giza, Egypt, under registration number 16220.

Isolation and identification of Campylobacter jejuni
C. jejuni was isolated from the inspected samples as recently described (Penner, 1988). Briefly, 10 grams of each sample (meat, liver, intestinal content, inner egg content and stool) were homogenized in sterile thioglycollate broth. Cloacal swabs and swabs from the external egg shell were incubated in tubes containing sterile thioglycollate broth. Broth samples were incubated at 42 °C for 48 hours in a microaerophilic atmosphere (10% CO 2 , 5% O 2 and 85% N 2 ). A loopful of enrichment broth was streaked onto mCCDA plates (Oxoid) and incubated under microaerophilic conditions at 42 °C for 48 hours (Persson & Olsen, 2005). The colonies were then subjected to microscopic examination for the identification of C. jejuni utilizing phase contrast microscopy after a seagull appearance was observed with Gram staining (Vandamme et al., 2008).
Refined colonies were used in the biochemical identification of C. jejuni as previously described (Frost et al., 1998). The recognized colonies were stored at -70 °C in thioglycolate broth containing 15% glycerol for further validation using molecular methods (Sheppard et al., 2009).

Molecular identification
The extraction of DNA from C. jejuni isolates was completed utilizing DNA extraction kits (GF-1, Vivantis, Selangor, Malaysia) as indicated by the producer's guidelines.

Polymerase chain reaction (mapA gene)
The amplification of the mapA gene for C. jejuni was carried out on 10 representative isolates that were biochemically confirmed utilizing the primers listed in Table 2. Amplification conditions were as follows: 6 minutes at 94 °C; 35 cycles of 50 seconds at 94 °C, 40 seconds at 57 °C, and 50 seconds at 72 °C; and a final extension of 3 minutes at 72 °C. The PCR products were analysed using 1.5% agarose gel electrophoresis and inspected with a UV transilluminator ( Figure 1).

Virulence gene characterization of C. jejuni isolates
The biochemically confirmed C. jejuni isolates were characterized for in vitro recognition of the cadF virulence gene by PCR (Konkel et al., 1999) utilizing the primers listed in Table 2.

Phylogenetic tree construction
The positive PCR products were then sequenced by MACROGEN Company (Korea) on 3730XL sequencer (Applied Biosystems, USA). The precision of the data was confirmed by bidirectional sequencing with the forward and reverse primers utilized in PCR.
The nucleotide sequences acquired in this examination were analysed using the BioEdit 7.0.4.1 and Muscle (EMBL's European Bioinformatics Institute, 2020) programs. The subsequent sequences were aligned with the cadF virulence gene of reference sequences of Campylobacter spp. (Table 3) utilizing a neighbour-joining analysis of the aligned sequences executed in the program CLC Genomics Workbench 3.

Results
In this investigation, 435 samples were gathered from Cairo, Giza, Fayoum, Minya and Qalubia in Egypt for the isolation and biochemical identification of C. jejuni from chicken and human stool samples (Table 4). Eighteen (20%), 10 (16.66%) and 6 (12%) C. jejuni isolates were detected in the intestinal content, liver and meat samples of broiler chickens, respectively. Additionally, 7 (17.75%), 6 (15%) and 6 (7.5%) C. jejuni isolates were detected in the intestinal content, cloacal swabs and egg samples of laying chickens, respectively. In the human stool samples, thirteen C. jejuni isolates (17.33%) were distinguished from 75 diarrheic persons, with a total of 66 (15.17%) C. jejuni isolates from the chicken and human stool samples. The prevalence of infection was almost the same in broiler chickens and human stool samples, as presented in Table 4.
In this investigation, ten representative C. jejuni isolates that were biochemically validated were further molecularly characterized through the amplification of the mapA gene specific to C. jejuni. All the isolates demonstrated the specific product (589 bp) for C. jejuni, as shown in Figure 1.
With respect to the virulence properties of the C. jejuni isolates, interestingly, ten isolates (15.15%) carried the virulence-associated CadF gene among the sixty-six C. jejuni isolates (Table 5) and generated the expected product (400 bp), as shown in Figure 2.
From our outcomes, 20.58%, 10.52% and 7.69% of isolates from broiler chickens, layer chickens and human stool samples carried the cadF virulence gene, respectively, with a total percentage of 15.15%. The highest prevalence of the cadF virulence gene was detected in the broiler intestine samples (27.77%), while the lowest was detected in eggs (0%).

Nucleotide sequence accession numbers
Three sequences (Campylobacter jejuni) utilized in this investigation have been deposited in the GenBank database under accession no: MN103378-MN103380. Phylogenetic analysis affirmed that all three isolates were Campylobacter jejuni with homology results of 99-100%. In the phylogenetic tree, all Egyptian isolates formed two separate clusters, as shown in Figure 3. Phylogenetic investigation demonstrated that CJ3 (MN103378) and CJ4 (MN103379), which were isolated from chicken meat and intestine samples, respectively (Table 3), had high homology with the C. jejuni isolates (CP006729) and (CP012216) isolated from human stool samples (Figure 3).
The Egyptian isolate CJ5 (MN103380) isolated from human stool had high homology with the C. jejuni isolate (CP023867), which was also isolated from human stool (Figure 3).
The amino acid sequence alignment of the three Egyptian C. jejuni isolates revealed a mutation in the sequence of two    isolates of zoonotic importance (CJ3 and CJ4) compared to the sequence of the CJ5 isolate (human isolate), where the amino acid arginine (R) number 52 was replaced with lysine (K), as shown in Figure 4.

Discussion
C. jejuni is considered one of the most notable and virulent foodborne pathogens, even in developed countries, and it causes severe bacterial gastroenteritis in humans who have consumed contaminated food, particularly poultry and poultry products (Eurosurveillance Editorial Team, 2012). The outcomes of the present investigation revealed that the intestinal contents of broiler and laying chickens were contaminated with C. jejuni at rates of 20% and 17.75%, respectively. This prevalence could be due to the intestinal tract of chickens, especially the caecum and colon, which is considered an area of tropism for a large number of Campylobacter species (Jokinen et al., 2011). Lower rates of contamination were detected in Egypt in previous studies Omara et al. (2015), Youseef et al. (2017), Elsayed et al. (2019), with C. jejuni isolation rates of 12.8%, 4% and 14.7%, respectively. However, higher rates of isolation (33.3%, 72.1%) were revealed by Abd El-Tawab et al. (2018) and respectively.
In our study, the cloacal swabs from six laying hens yielded C. jejuni at a rate of 17.75%. This finding was in close agreement with the finding (17.8%) of Fonseca et al. (2006). Lower isolation levels of C. jejuni (8%&7%) have been recorded by Chatur (2014) and Akosua et al. (2017) respectively. Higher rates were reported in Italy by Parisi et al. (2007) and others in Egypt (Stojanov et al., 2007;Hedawey &Youssef, 2014) with isolation rates of 21.1%, 26% and 25%, respectively.
Additionally, the egg samples contained internal contents that did not reveal C. jejuni isolates and swabs from eggshells carried C. jejuni at a rate of 15%. This could be due to the poor ability of C. jejuni to penetrate the egg albumin or yolk, while it was confined to the inner egg membranes (Neill et al., 1985). The result obtained from egg content samples was comparable with that of Fonseca et al. (2006), Jones et al. (2012) and Ge et al. (2016), whereas, one isolate (4.28%) of C. jejuni was retrieved from egg samples as reported by Shane et al. (1986). The eggshell sample results were consistent with those (16.66%) reported by Modirrousta et al. (2016). However, Hedawey & Youssef (2014) found a lower isolation rate (1%).
Generally, Campylobacter is the most common bacterium that causes gastroenteritis globally in humans and can be lethal to young children, geriatric patients and immunocompromised patients (Sainato et al., 2018).
Several genes are related to Campylobacter virulence, which could lead to human infection and chicken colonization  (Kalantar et al., 2017). The most virulent is the gene Campylobacter adhesin to fibronectin F (cadF) (Elsayed et al., 2019).
From our results, 20.58%, 10.52% and 7.69% of isolates from broiler chickens, layer chickens and human stool samples carried the cadF virulence gene, respectively, with a total of 15.15%. The highest prevalence of the cadF virulence gene was isolated from the broiler intestine samples (27.77%), while the lowest prevalence was from eggs (0%).Regarding the prevalence of CadF gene, our results were lower than that of Elmali & Can (2019) that reported a prevalence of 41.6% and so lower than that of Al Amri et al.( 2007), Abu-Madi et al.( 2016) and Samad et al.( 2019) who described nearly 100% of CadF gene prevalence.
In the present study, the relatively high prevalence rate of the cadF gene from broiler chicken isolates suggests that many poultry-derived strains have possible pathogenic properties for humans (Frasao et al., 2017;Kalantar et al., 2017).
Our results confirm the extensive prevalence of virulence genes, especially in broiler chickens and human stool samples, which indicates that strict control, public health, and food safety policies are required to prevent consumers from contracting this zoonotic pathogen. The accessibility of pathogen virulence data should increase the awareness of these clinically and economically important pathogen isolates in Egypt.
The detection of Campylobacter species was verified by DNA sequencing of representative samples. The collected sequences were BLAST searched with those in the database, and a phylogenetic analysis was performed (Table 1). Clear sequences of the cadF virulence gene were obtained from three isolates. Homology findings (99-100%) showed that the three isolates were C. jejuni. The neighbour-joining (NJ) phylogenetic analysis based on the cadF gene ( Figure 3) showed that all three Egyptian isolates clustered with specific sequences of human origin and not poultry origin (other clusters), while two of them (CJ3 and CJ4) were isolated from chickens. Human stool was a primary source of infection. This shows the zoonotic importance of these two C. jejuni isolates and the continuous pathogen loop from chicken to human and vice versa. This result is important in understanding the epidemiology of C. jejuni in Egypt.
In addition, the amino acid sequence alignment of the three Egyptian C. jejuni isolates from humans and chickens ( Figure 4) revealed a mutation in the sequence of two isolates of zoonotic significance (CJ3 and CJ4) relative to the sequence of the CJ5 isolate (human isolate), where the amino acid arginine (R) number 52 was substituted with lysine (K). Therefore, when we speak of this virulence gene (Campylobacter adhesin to fibronectin F), the shift in the amino acid sequence of the two C. jejuni isolates (CJ3 and CJ4) compared to the human isolate (CJ5) may be explained by increased pathogen virulence and relatively easier transmission in Egypt between humans and chickens. In the future, this concept will be further researched.

Conclusions
The present results support the possible risk of transmitting C. jejuni as a foodborne pathogen from both broiler and layer chickens due to the high rates of C. jejuni isolation from different chicken samples, as expressed by the percentage of C. jejuni isolated from human stool specimens. The use of PCR and next-generation sequencing is important to ensure that this pathogen is quickly identified, characterized and examined epidemiologically. Active on-farm biosecurity measures in chicken farms and more hygienic efforts in slaughter houses, in local chicken slaughter shops and by those who rear backyard chickens should be made for the effective control of this foodborne disease.