Antimicrobial resistance and virulence associated genes in Campylobacter jejuni isolated from chicken in Côte d'Ivoire.

INTRODUCTION
Campylobacter jejuni is one of the major causes of gastroenteritis worldwide of the last century. The aim of this study was to investigate the antibiotics profiles and the virulence gene in C. jejuni strains isolated from chicken in Côte d'Ivoire.


METHODOLOGY
A total of 336 chicken ceaca samples recovered from market of two municipality of Abidjan were examined by conventional microbiological methods and molecular test using PCR. The antibiotic susceptibility tests of the isolates were determined by disk diffusion method. The presence of virulence genes was examined using simple PCR method.


RESULTS
Among of 336 samples, 168 (50%) were positives for C. jejuni. Among the C. jejuni isolates, 159 strains (94.64%) were resistant to one or more antimicrobial agents. The highest percentage of antimicrobial resistance was found for Nalidixic acid (85.33%), Tétracyclin (71.76%) and Ciprofloxacin (55.65%). Moreover, MDR including 3, 4, 5 and 6 antibiotics families was detected in 16.66% of isolates. On the other hand, detection of virulence putative gene shows presence of cadF in 100% of tested strains. In addition, cdtA, cdtB and cdtC genes were detected in 100%; 89.51% and 90.32% respectively of C. jejuniisolates.


CONCLUSION
Because of the key role of broiler chicken in human campylobacteriosis infection, it will important in first time to monitoring using of antibiotics in chicken farms and in second time to verify presence of campylobactériosis in country.


Introduction
Campylobacter is a food-borne zoonotic pathogen, and is considered to be one of the most common causes of bacterial gastroenteritis both in developed and developing countries [1,2]. Indeed, World Health Organization (WHO) [3] estimated that Campylobacter caused more than 37000 deaths per year worldwide. In developing countries, Campylobacter has been associated to 11.3 to 21% of diarrhea episodes in children under the age of two years [4]. Moreover, absence of epidemiological data could lead to the underestimation of the burden of Campylobacter infections in these regions [4]. Among all species of Campylobacter, Campylobacter jejuni is the mostly associated to humans campylobactériosis with more 85% cases worldwide. In generally, C. jejuni pathogenicity is due to various factors such as cytotoxin production [5], intestinal cell invasion [6], extraintestinal adherence and translocation [7]. Several genes have been linked to this pathogenicity but the most important are Campylobacter adhesin to fibronectin F (cadF), Campylobacter invasion protein B (ciaB), which is vital for invasion and cytolethal distending toxin (cdtA, B and C), which disrupts mucosal barriers by causing host cell death [1,8]. Human campylobacteriosis due to Campylobacter in its acute phase is characterized by diarrhea, fever, abdominal cramps, and vomiting [9]. Moreover, C. jejuni infection may lead to extragastrointestinal manifestations, including bacteremia, lung infections, brain abscesses, meningitis, and reactive arthritis, in individual cases and small cohorts of patients [10]. In general, Campylobacter infections do not require antibiotic treatment, however the use of erythromycin, tetracycline, and quinolones is recommended in severe cases [3,11]. However, emergence of Campylobacter strains resistant to antibiotics since several years could make it difficult to treat human campylobacteriosis. Some studies reported that the misuse of antibiotics and the lack of control over their usage in poultry production systems promote the development of resistant and even multidrug-resistant bacteria [12].
Indeed, antibiotics are widely used to prevent, control, and treat bacterial infections as well as growth promoters in a large number of countries during poultry production [13]. These facts are of special relevance in developing countries where misuse of antibiotics and the lack of control over their usage is a problem to be addressed [12]. In Côte d'Ivoire, increased rates of antimicrobial resistant Campylobacter isolated from poultry have been reported [14,15].
Despite of the importance of C. jejuni as a foodborne pathogen [16] and the fact that chicken meat is frequently consumed and its demand increased over the years, little is known about C. jejuni strains contaminated poultry farms and slaughterhouses in the main market in Côte d'Ivoire. Yet, this information may help in first time to establish surveillance programs and intervention measures regarding to the presence and antimicrobial resistance of Campylobacter in Ivoirian poultry and in a second time to monitor human infections with Campylobacter in Côte d'Ivoire. The aim of this study was to investigate the prevalence, antimicrobial resistance and virulence genes profiles of C. jejuni in broilers slaughtered in two municipalities in Abidjan.

Isolation and identification of Campylobacter jejuni
This study was conducted in two municipality of Abidjan including Adjamé and Abobo. Adjame is the principal market of Abidjan with more than 3 million visitors per day and Abobo is a commune with a large part of population compared to other in Côte d'Ivoire. During 2009 to 2010, samples were collected at the largest poultry slaughter sites in each municipality. A total of 366 samples of chicken caeca were collected over a period of one year and analyzed for Campylobacter isolation.
Isolation of Campylobacter was performed with passive filtration method preceded by enrichment as proposed by [14]. Briefly, 1 g of caeca contents was transferred to 9 mL of Preston enrichment broth base (OXOID LTD., Basingstoke, Hampshire, UK) and 7% (v/v) defibrinated sheep blood. Incubation was performed in an anaerobic jar containing a packet generator microaerophilic atmosphere (5% oxygen, 10% carbon dioxide, 85% nitrogen) type CAMPYGen (Oxoid, Basingstoke, Hampshire, UK) during 24 hours at 37 •C. After enrichment, 300 µL of broth was filtered through acetate cellulose filter (0.45 µm) on Columbia agar (Sharlau; Barcelona, Spain) containing 5% (v/v) fresh sheep blood at aerobic conditions during 45 minutes. After this time, filter was removed and plates were incubated at 42°C during 2-5 days under microaerobic conditions. After incubation, five (5) presumptive Campylobacter colonies from each agar plate were identified as previously described [14]. After biochemical identification, each Campylobacter colony was cultured on Columbia agar supplemented with 5% of sheep blood and incubating at 42°C during 48 hours. The subculture from each positive sample was stored in 25% glycerol at -70°C until using for molecular identification and detection of virulence genes.

DNAextraction and molecular identification
DNA was extracted from freshly grown colonies on Columbia agar supplemented with sheep blood using Biomerieux DNA Bacteria Kits (Biomerieux, Marcy l'Etoile, France). In brief, colonies from on Columbia agar plates were suspended in 1mL of bidistilled sterile water and centrifuged at 14000 trs/minute for 10 minutes at 4 °C. After this, the pellet was suspended in 200 µL of bidistilled sterile water and incubated at -20°C for 1 hour. Then, the tube was immediately put at +95 °C during 10 minutesby usingdry block incubator (Thermo Scientific, Dardilly, France). Samples were allowed to cool, centrifuged at 13000 × g for 5 minutes and then 100 µL of each sample was transferred in 1.5 ml microcentrifuge tubes and stored at -20°C for future use.
Molecular identification was performed as previously described [14] using PCR amplification of the hipo gene encoding C. jejuni hippurase. Sequences Antimicrobial Susceptibility C. jejuni susceptibility was tested by using the disk diffusion method and the following antimicrobial disks (BioRad, Marnes-la-Coquette France) were included: Tetracycline (30 μg), Erythromycin (15 μg), Ciprofloxacin (5 µg), Azithromicin (15 µg), Gentamicin (10 µg) and Nalidixic acid (30 μg); Amoxicillin (20 µg). Susceptibility testing to all antimicrobial agents was carried out on Mueller-Hinton agar supplemented with sheep blood that were spread with a 0.5 McFarland standard suspension of C. jejuni in trypton saline buffered (Biorad, Marnes-la-Coquette, France) and incubated for 48 h at 37°C under microaerobic conditions. Zones of inhibition were measured and three isolates were classified as sensitive, intermediate, and resistant according to the clinical and laboratory standards institute (CLSI) [18] guidelines. Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were used as reference strains.

Potential virulence factors genes
A simple PCR method was used to detect the presence of four genes (cadF, cdtA, cdtB and cdtC), which are involved mainly in adhesion and cytotoxin production capacities. The primers used in this study were provided in Table 1.For cadF gene, amplification program consist of an initial denaturation 5 minutes followed by 30 cycles of denaturation at 94 °C for 1 minute, annealing at 49 °C for 1 minute and polymerization at 72 °C for 1 minute . A final extension was performed at 72 °C for 7 minutes. in case of cdt genes, PCR reactions were performed in same conditions except the annealing step which carried out at 42°C during 2 minutes.

Antimicrobial susceptibility testing
Antimicrobial susceptibility was tested for all the isolates and antimicrobial resistance profiles were defined (Table 2 and Figure 1). Among 168 tested isolates, a total of 159 strains (94.64%) were resistant to one or more antimicrobial agents. The highest percentage of antimicrobial resistance was found for nalidixic acid (85.33%), Tetracyclin (71.76%) and  Ciprofloxacin (55.65%). Comparatively, resistance levels observed to the other antibiotics were low with 20.12%; 18.5%; 16.51% and 6.71% for amoxicillin, Erythromicin, Azithromicin and Gentamicin respectively. On the other hand, 55.55% C. jejuni isolates were cross-resistant to ciprofloxacin and Nalidixic acid. Moreover, 32.25% of tested strains were also resistant to both Erythromicin and Azithromicin which belonging to macrolide antibiotics family. The multiple drugs resistance (MDR)was detected in 16.66% of the tested strains. Moreover, the MDR including three antibiotics families was detected 9.43% of these strains while 4.40% of them were resistant to four drugs families and 1.25% were resistant to five drugs families.

Prevalence of virulence gene
In this study, presence of genes coding for putative virulence factors in the 168 C. jejuni strains have been tested by PCR. As expected, cadF was detected in 100% of tested strains. Moreover, toxin virulence factors cdtA, cdtB and cdtC were present in 100%; 89.51% and 90.32% respectively of the C. jejuni isolates in this study.

Discussion
In this study, Campylobacter were isolated in 80.87% chickens ceaca analyzed and 56.75% of these were positive for C. jejuni. Studies from other countries showed also a high prevalence rates ranged from 77 to 100% [21,22]. Chicken's ceaca contamination by Campylobacter can occur during poultry production including breeding and transportation to slaughterhouse [23][24][25][26]. Furthermore, C. jejuni which contaminate chicken's Ceaca could also contaminate poultry carcass at the end of the slaughter process because of the noticeable lack of hygiene practice in these slaughterhouses. Indeed, some study reported that if some step of poultry slaughter process (scalding) would contribute to lowering C. jejuni contamination rate, others steps (plucking, evisceration and carcass washing) are important factors of the poultry meat campylobacter contamination at the end of the processing. In traditional slaughterhouses in general in Abidjan, poultry slaughterers are illiterate and have no knowledge of hygienic practices. For example, the plucking is carried out on the ground, the slaughtering equipment is not disinfected and many carcasses washing is carried out in the same water basin [21,[27][28][29]. In these conditions, all poultry carcasses from these slaughterhouses will probably be contaminated with C. jejuni.
Moreover, theseC. jejuni strains belonging high rate resistance to several antibiotics mainly with nalidixic acid, Tetracyclin and Ciprofloxacin. These high resistances obtained in our study are comparable to those observed in many countries [11,[30][31][32][33]. On the other hand, low resistance has been also observed with amoxicillin, Erythromicin, Azithromicin and Gentamicin by Rivera et al. [34], in strains isolated in Chile and by Vinueza-Burgos et al. [11] in Ecuador.
The multiple drugs resistance (MDR) corresponding to resistance to three or more families of antimicrobial agents [35] was detected in 16.66%, 9.43, 4.40% and in 1.25% of the tested strains. Those MDR strains were recovered from broilers worldwide [36]. The appearance of these combinations of resistance is a direct consequence of the uncontrolled use of antibiotics in the poultry sector in Côte d'Ivoire [37]. The antibiotics tested being the most used molecules in cases of Campylobacter infection in humans, so existence of these resistances could constitute a major problem of public health given the risks of therapeutic failure [11]. Therefore, the search for new approaches to limit the spread of these strains in poultry farming in first time and also on products derived from poultry likely to be contaminated is required. Indeed, data collected through the world indicate that poultry products are responsible for a significant proportion (20-40%) of Campylobacter human infections and many cases of others enteropathogens diseases such as salmonellosis [1].
In addition, virulence genes cadF and cdtA were detected in all tested strains while, cdtB, and cdtC were isolated in 89.51% and 90.32% respectively in these isolates. This high prevalence of these four (4) genes isin agreement with several studies that found it in nearly all of theCampylobacterisolates [1,38]. The cadF gene is coding for a putative OmpA-like protein that mediates bacterial adhesion by binding to host fibronectin. This ability of Campylobacter to bind to host epithelial cells is a key step in expression of pathogenicity especially since this ability contributes to colonization and invasion of host organism [39,40]. The high prevalence of this gene in our study could indicate this decisive role of the cadF gene in the virulence capacity of C. jejuni. However, Iglesias-Torrens et al. [38] studies suggest that cadF may not be essential for the ability of Campylobacter to cause infection in humans. Therefore, we hypothesize that the high prevalence of the virulence factors in our finding is more indispensable for these strain in the colonization of the digestive tract of broilers chicken as reported by Ziprin et al. [41] and Konkel et al. [42].
Regarding the genes cdt coding for synthesis and deliver of the cytolethal distending toxin, our findings are similar with those reported by Bang et al. [20], Datta et al. [43] and Al-Mahmeed et al. [44]. In our knowledge, activity of CDT toxin (consisting CDTA, CDTB and CDTC) lead to increase of intestinal epithelial cell volume by stopping cell division cycle at G2 step causing cell destruction [38,[45][46]. Indeed, after adhesion to fibronectin, C. jejuni colonizes intestinal cells and then invasion occurs simultaneously with production of CDT toxin causing inflammatorytype diarrhea in the host organism [47]. Indeed, Purdy et al. [48] showed that in a mutant cdt; the invasion capacity of the mouse epithelial cells is considerably reduced compared to the non-mutant strain. These data show the key role of CDT toxin in the occurrence of Campylobacter infection. Thus, presence of these putative virulence factors in these C. jejuni strains may constitute a major health problem since customers of artisanal slaughterhouses generally consist of restaurant owners and the monitoring sanitary systems are nonefficient in Côte d'Ivoire.

Conclusion
This study revealed presence of C. jejuni with virulence gene including cad F, cdtA, cdtB and cdtC. Moreover, our results indicated a high level of resistance of tested strains to antibiotics generally used in treatment of diarrheal cases in Côte d'Ivoire. Because C. jejuni is the most common cause of bacterial gastroenteritis, it is crucial to investigate a thorough and reliable monitoring program to reduce the availability of contaminated chicken's products in our country.