Abstract: Background and Objectives: Infection with Campylobacter jejuni is one of the most common causes of bacterial gastroenteritis. Infections are mostly acquired due to consumption of raw or undercooked poultry. The aim of this pilot study is to determine the prevalence and the sequence types (STs) distribution of C. jejuni isolated from broiler meat in Egypt. Materials and Methods: A total of 190 broiler meat samples were collected from retail chicken shops located at Mansoura, Egypt and examined bacteriologically for the presence of Campylobacter spp. The biochemically identified Campylobacter isolates were confirmed by Multiplex PCR (m-PCR). In addition, multilocus sequencing typing (MLST) was used for genotyping of C. jejuni isolates. Results: Thirty two Campylobacter isolates divided into C. coli (25 isolates) and C. jejuni (7 isolates) were recovered. Multiplex PCR results found to be 100% in line with biochemical identification. Out of 7 C. jejuni isolates genotyped by MLST, 4 isolates were assigned to ST21, 2 isolates were assigned to ST48 and one isolate was assigned to ST464. Conclusion: This study provides valuable information concerning the prevalence of thermophilic Campylobacter spp. and sequence types distribution of C. jejuni recovered from broiler meat for the first time in Egypt. The identified sequence types from this study were frequently reported in human illnesses. Thus, the present results highlight the importance of the retail broiler meat as a significant source for human Campylobacter infection.
INTRODUCTION
Campylobacter has been documented as one of the significant bacterial agents causing food-borne disease worldwide1. Among the 25 species and 8 sub-species of Campylobacter identified2, C . jejuni subsp. jejuni or C. coli are associated with more than one host and have zoonotic potential in avian species. Recently, the incidence of human gastrointestinal due to C. jejuni infection has been increasing3. The previous studies confirmed that Campylobacter infection is still endemic in Africa, Asia and Middle East, although epidemiological data from these areas are still incomplete4.
Poultry poses as the main reservoir for Campylobacter spp. harbor them without clinical manifestations and considered an important source of human illness 5. Hence, the principle risk factors linked with Campylobacteriosis infection in human is the transmission of Campylobacter to humans either by handling or consumption of contaminated chicken meat and its products6-8. Campylobacter usually colonization the intestinal tract of chicken one week after hatching9, however, the contamination of chicken meat contributed to cross contamination by intestinal contents at the slaughterhouse10. The ability of Campylobacter to colonize the chicken varies significantly not only among Campylobacter strains but also depending on the original source of the infecting isolate11.
A wide range of genotypic methods with a high discriminatory power have been developed for Campylobacter typing12. Pulsed-field gel electrophoresis (PFGE) and MLST are the most widely used genotyping methods by laboratories worldwide for better understanding the epidemiology of Campylobacter. The MLST is considered one of the most important techniques elucidating the genetic diversity of Campylobacter isolated from animals and providing important information on transmission routes from different sources13.
The MLST technique depends on sequencing of seven housekeeping genes to study the changes in ST distribution worldwide14. Subsequently, sequence data submitted to MLST database for assignment of the sequence type (ST) and clonal complex (CC) and to assign patient isolates to possible sources. In various studies conducting MLST, chicken was found to be the most frequent source of campylobacteriosis worldwide, representing from 38-77%, followed by cattle with an attribution rates varying from 16-54% 5.
Currently, detailed epidemiological information that determines the prospective sources of human campylobacteriosis in Egypt is not available and it is unclear whether same strains of higher risk are responsible for disease in Egypt. Thus, this study was designed to assess the frequency of thermophilic Campylobacter in broiler meat and to discover sequence types (ST) distribution of C. jejuni in broiler meat.
MATERIAL AND METHODS
Samples collection: A total of 190 broiler meat samples (chicken meat with the skin from breast, neck and thigh) were collected in the period between January and March, 2017 from six retail shops located at Mansoura city, Egypt. Each individual sample was separately packaged and transferred to the laboratory in an ice box within 1 h for bacteriological examination.
Bacteriological examination: Isolation of Campylobacter was conducted according15 to ISO 10272-1. In brief, about 25 g from each chicken meat sample was pre-enrichmented in 225 mL Bolton Broth (Oxoid) supplied with SR0183 (Oxoid) for selective growth of Campylobacter. The inoculated broth was incubated firstly at 37°C for 4-6 h under microaerophilic condition by using CampyGen (Oxoid) and then at 42°C for 48 h. A loopful of the previously inoculated broth was plated on the surface of Modified Charcoal Cefoperazone Deoxycholate Agar (CM0739; Oxoid) supplied with SR0155 (Oxoid) and incubated for 48 h at 42°C under microaerophilic condition. Purification of Campylobacter colonies were performed onto Columbia Blood Agar (ASC; Biolife, Milan, Italy) containing 5% defibrinated horse blood and incubated at traces of oxygen (CampyGen, Oxoid) for 48 h at 41.5±1°C. Presumptive colonies were picked and stained with gram stain and tested with catalase, oxidase and motility tests.
DNA isolation: Campylobacter colonies were suspended in PrepMan Ultra (Applied Biosystems, Foster City, USA), the suspension heated at 95°C for 10 min for cell lysis followed by centrifugation. The supernatant containing bacterial DNA was transferred to a new tube and kept at - 20°C to be used as a DNA template for m-PCR16.
Molecular characterization of C. jejuni isolates: Multiplex PCR assay (m-PCR) were developed16 for detection of both C. jejuni and C. coli simultaneously. Three sets of primers were designed for detection of the following loci: 16SrRNA gene for co-identification of C. coli and C. jejuni, MapA gene specific for C. jejuni and CeuE gene encoding lipoprotein of enterochelin transport system characteristic for C. coli (Table 1). The PCR reaction and cyclic condition were performed following the protocol illustrated by Denis et al.17.
| Table 1: | Oligonucleotide primers used in Multiplex PCR |
| Table 2: | Oligonucleotide primers sequences used in MLST for C. jejuni |
Multilocus sequence typing: The PCR was performed for amplification of seven housekeeping genes according to Dingle et al.21. The PCR reaction was performed in 100 μL using the Applied Biosystems 96 well thermal cycler using cyclic conditions reported by Dingle et al.21 (Table 2). The PCR products were purified using QIAquick purification kit (Qiagen, Germany) and sent for sequencing. Sequence data were analyzed by submitting the sequences to Campylobacter MLST website (http://pubmlst.org/campylobacter) for assigning sequence types and clonal complexes.
RESULTS
In this study, the prevalence rate of C. coli from the examined broiler meat samples was 13.15% (25/190) while, C. jejuni was 3.68% (7/190) with overall prevalence of 16.84%. Among the identified Campylobacter spp., C. coli frequency found to be higher than C. jejuni.
By MLST genotyping, among the seven C. jejuni isolates, 3 STs were identified (ST21, ST48 and ST464). These STs were assigned to 3 CCs (CC21, CC48 and CC464) already described before (Table 3). Amongst the identified sequence types, ST21 was identified in 4 isolates and predominating among C. jejuni isolates identified in this study, while, ST48 was identified in two isolates and one isolate was assigned to ST464.
DISCUSSION
The incidence of Campylobacter infection has increased worldwide in the past decade. Understanding the epidemiology of Campylobacter species aids in reducing the disease burden4.
| Table 3: | Allelic profiles, sequence types (STs) and clonal complexes (CCs) for C. jejuni isolates |
Therefore, in this study we aimed to characterize Campylobacter for better understanding the epidemiology of Campylobacter in our area with focusing on broiler meat as a most important source for human cases. Thermophilic Campylobacter species including C. coli and C. jejuni are frequently isolated from poultry. Poultry meat represents a potential source for human campylobacteriosis via consumption of contaminated poultry meat22. In the current study, the prevalence of C. jejuni was slightly lower if compared with other Egyptians surveys23,24,25. Compering with other surveys worldwide, the prevalence rate of Campylobacter spp. from retail chicken meat was 29% in Pakistan26. However, Campylobacter spp. has been stated as the second most frequent bacteria from chicken meat in Europe with a prevalence rate of 33.3%27. Presence of Campylobacter spp. in chicken meat may be caused by the contamination of carcasses with feces and rinsing due to unhygienic slaughtering and processing operations. Differences in the prevalence rate of Campylobacter from different countries may arise from differences in the area, sampling, transportation and sensitivity of the detection methodologies.
Among the identified Campylobacter spp. from this study, C. coli frequency found to be higher than C. jejuni. In agreement with this observation, higher percentages of C. coli have been reported in many previous studies by Nobile et al.28 and Mezher et al.29. Conversely, a higher C. jejuni isolates was reported in other studies by Hafez et al.25 and Wassenaar and Newell30. In general, many surveys worldwide reported this variability in the percentage between C. coli and C. jejuni in broiler meat6.
Campylobacter is characterized by heterogeneity and there are many typing techniques were developed for its typing31. In the current study MLST was used to determine the diversity of C. jejuni isolates. The MLST is widely used for genotyping of Campylobacter worldwide but it is still not commonly used in Egypt with lacking of information about the Campylobacter species Sts.
The MLST is an important technique having a high discriminatory power32,33 used in population studies of Campylobacter spp. Furthermore, MLST sequence data can be easy to interpret via submitting of sequence data to the Campylobacter MLST website and it readily compared between laboratories32. On the other hand, it is expensive and complex technique, labor-intensive to perform in comparison with other typing techniques34.
In this study, ST21, ST48 and ST464 were identified. Amongst the identified sequence types, ST21 was identified in four isolates and predominating among C. jejuni isolates identified in this study, while, ST48 was identified in two isolates and one isolate was assigned to ST464. The prevalence of C. jejuni in broiler meat differs widely between countries, in our study, CC-21 was the highest CC assigned followed by CC-48. These findings go in line with these reported in Belgium35,36. On ST-level, ST21 was the dominant sequence type among the tested C. jejuni isolates. Similarly, ST-21 was reports as the most frequent sequence type form broiler meat worldwide37-39. In contrast to these findings, CC21, ST21 was more common in dairy cattle than broiler sources in Lithuania16. In addition, ST464 has been identified in our study and it has been also identified from broilers in Spain and Belgium40,41.
In many previous studies, ST-21, ST-48 and ST-464 were reported amongst the most common sequence types identified in both human and broiler carcasses isolates39,41 which may poses a potential risk for human. Further studies should be performed to evaluate the risk factors of Campylobacter contamination in the Egyptian poultry production chain.
CONCLUSION
This study has determined the percentage of contamination of broilers meat with Campylobacter spp. in Egypt with predominance of C. coli. In addition, by performing MLST on C. jejuni isolates, the obtained results showed that the identified sequence types were frequently reported in human illnesses which may pose a potential risk for the consumers. To the best of our knowledge, this study considered the first study in Egypt providing information on the distribution C. jejuni STs from poultry sources.
SIGNIFICANCE STATEMENT
This study highlights the importance of broiler meat as a significant source of human campylobacteriosis. In addition, genotyping of Campylobacter with MLST helps in better understanding the epidemiology and the transmission pathways of Campylobacter to decrease the disease burden.
ACKNOWLEDGMENT
This study is one of Competitive Funding Projects provided and funded by Scientific Research Unit, Mansoura University, Egypt