ERIC-PCR Genotyping of Some Campylobacter jejuni Isolates of Chicken and Human Origin in Egypt

Introduction

Campylobacter species are Gram-negative bacteria within the family Campylobacteriaceae, requiring microaerobic conditions for growth (Rahimi and Ameri 2011). Among thermophilic campylobacters, C. jejuni and C. coli are most frequently isolated from patients with diarrhea in both industrialized and developing countries. Consumption and handling of contaminated chicken meat products during slaughter and processing are considered the most common transmission routes to humans (Berrang et al. 2001).

Enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) has been developed for typing different Gram-negative bacteria, including Campylobacter spp. (Versalovic et al. 1991, Millemann et al. 1996). ERIC-PCR genotyping produces strain-specific fingerprinting to evaluate the genetic diversity of C. jejuni (Hulton et al.1991). The reported high reproducibility and discriminatory power of the ERIC-PCR technique in typing thermophilic Campylobacter species recommends its application in studying the genetic relationship between isolates of diverse origins (Steinhauserova et al. 2001). The aim of the current study was to investigate the genotypes of C. jejuni isolates of chicken and human origin using ERIC-PCR fingerprinting.

Material and Methods

Results

According to phenotypic and biochemical identification, 52 C. jejuni strains were isolated from cecal parts, neck skin, thigh meat, cloacal swabs, and breast meat with the isolation rates of 81.3%, 50%, 46.7%, 45.7%, and 41.7%, respectively. Moreover, of the 246 human stool samples, 10 (4.1%) were identified as containing C. jejuni. A representative number of the 62 biochemically identified C. jejuni isolates were selected (21 isolates of chicken origin and 10 isolates from humans) according to their antimicrobial resistance profiles (data not shown) and subjected to qPCR targeting the hipO gene, which is specific for C. jejuni for confirmation. The PCR results confirmed the classification of the biochemically identified isolates as C. jejuni. The specificity of the reaction was characterized because the primer and probe sets specific for C. jejuni did not amplify DNA from C. coli and other positive controls.

The ERIC-PCR fingerprinting patterns of 31 C. jejuni isolates (21 of chicken origin and 10 of human origin) were investigated using a single amplification profile. ERIC-PCR profiles were discriminated by the number and position of the amplified fragments. Visual comparison of the banding patterns revealed multiple DNA fragments ranging in size between 108 and 3327 bp (Fig. 1). ERIC-PCR primer sets produced 17 profiles (referred to as E1 to E17). The discriminatory power of the ERIC-PCR and the profiles produced of the chicken and human isolates are summarized in Table 1.

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FIG. 1.

Sample of ERIC-PCR fingerprinting of C. jejuni isolates in a 1.2% agarose gel. Lanes L, 100-bp ladder; lane 1, negative control; lanes 2–17, C. jejuni isolates; lane 18, positive control.

The dendrogram analysis of the isolates examined (n = 31) showed four clusters and five separate isolates (Table 2 and Fig. 2). One isolate from chicken skin, three isolates from cloacal swabs, one isolate from a cecal sample, and three human isolates were located in the same cluster (cluster I). Seven human isolates, three chicken meat isolates, and two cloacal swabs' isolates were located in cluster III. The similarity between human and chicken isolates of the same profile was calculated by Jaccard coefficient and showed 100% similarity.

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FIG. 2.

Dendrogram showing the relatedness of C. jejuni samples isolated from humans and poultry as determined by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) fingerprinting using the SPSS computer software program. Int, sample from cecal part.

Discussion

Campylobacter spp. are ubiquitous foodborne pathogens that colonize the intestinal tract of most mammals and birds, especially poultry (Rahimi and Ameri 2011). A wide range of molecular genotyping methods, such as pulsed-field gel electrophoresis (PFGE), restriction fragment length polymorphism (RFLP), and multilocus sequence typing (MLST), have been developed for C. jejuni typing (Zorman et al. 2006). However, PCR fingerprinting-based techniques, such as repetitive sequence-based PCR (rep-PCR), are more suitable, because other typing methods are complex and unsuitable for routine application (Giesendorf et al. 1994). In addition, rep-PCR techniques are also used to characterize interspecies variability and to clarify the genetic relatedness of specific bacteria in humans and animals on the basis of the diversity or similarity of the genotypes (Wilson et al. 2009, Behringer et al. 2011). In the current study, ERIC-PCR was used for the genotyping of 31 C. jejuni isolates of chicken (n = 21) and human (n = 10) origin. The choice of ERIC-PCR over other molecular typing methods was based on its previously reported reliability, reproducibility, rapidity, and high discrimination index (Wilson et al. 2009).

The results obtained showed a high discriminatory index of ERIC-PCR (D = 0.948), dividing C. jejuni isolates of chicken and human origins into 18 profiles and four clusters (Table 2 and Fig. 2). Similarly, a discrimination index of 0.95 was obtained when ERIC-PCR was used for genotyping of Campylobacter spp. isolated from chicken carcasses and meat (Wieczorek 2009). These results support the assumption that the value of any typing method depends mainly on the discriminatory power of the technique (Giesendorf et al. 1994).

The 18 profiles obtained for the isolates examined (n = 31) indicated the heterogeneity of C. jejuni. In accordance, similar findings were also reported for C. jejuni isolates of cattle origin in Poland (Bednarski et al. 2011) and isolates of human origin in Iran (Ghorbanalizadgan et al. 2014). The high genomic diversity of C. jejuni could be a consequence of the rapid adaptive changes within the genome during infection or colonization cycles, leading to the production of new genotypes (Wieczorek 2009). Such changes include, for example, natural transformation, genomic rearrangements, and chromosomal point mutations (Wassenaar et al. 1998, Wieczorek 2009). The aforementioned hypothesis highlights the relative genotypic instability of the genus resulting from highly plastic genome of Campylobacter spp. (Hook et al. 2005, Matsui et al. 2002).

Human isolates showed a D value of only 0.78, and they were classified into four profiles, compared to 18 profiles obtained from the genotyping of the chicken isolates (Tables 1 and 2). Likewise, only 13 genotypes from 272 human isolates using ERIC-PCR have been reported (Matsui et al. 2002). These authors attributed their results to the existence of a narrow source of human campylobacteriosis in Northern Ireland. However, the presence of 18 (Iriarte and Owen 1996) and 15 (Shi et al. 1996) ERIC-PCR profiles from 25 and 17 C. jejuni isolates of human origin, respectively, have been documented. The results of the present study could be attributed to the low number of C. jejuni isolates of human origin, resulting in limited profiles.

The human isolates under investigation during the present study produced four profiles by ERIC-PCR, namely, E1, E4, E7, and E8 (Table 2). Within each profile, the human isolates showed 100% similarity with the relevant chicken isolates. These results highlighted the existence of a genetic relationship between human and chicken isolates. Likewise, the presence of indistinguishable Campylobacter isolates from poultry and humans have been reported in France (Denis et al. 2009).

The dendrogram analysis showed the existence of C. jejuni isolates of chicken and human origin within the same clusters, namely clusters I and III (Table 2 and Fig. 2). In Sweden (Lindmark et al. 2004), Switzerland (Wieland et al. 2006), and France (Denis et al. 2009), human and poultry isolates were also shown to be present within the same clusters. Moreover, in Canada, 20% of Campylobacter spp. genotypes of human origin were genetically related to poultry genotypes (Nadeau et al. 2002). Common genotypes of C. jejuni isolated from humans and poultry in Senegal (Cardinale et al. 2006) and Japan (Saito et al. 2005) have also been documented.

The results obtained in the current study support the hypothesis that human participants were exposed to the same genotypes of poultry origin, indicating the risk of contracting infection by contact and consumption of poultry (Anderson et al. 2012). In conclusion, our results and the aforementioned reported ones support the speculation that poultry and poultry meat have an important role as sources of infection for acquiring Campylobacter infection in humans.