VECTOR-BORNE AND ZOONOTIC DISEASES Volume 15, Number 12, 2015 ª Mary Ann Liebert, Inc. DOI: 10.1089/vbz.2015.1836
ORIGINAL ARTICLE
ERIC-PCR Genotyping of Some Campylobacter jejuni Isolates of Chicken and Human Origin in Egypt Heba A. Ahmed,1 Fatma I. El Hofy,2 Ahmed M. Ammar,3 Ashraf A. Abd El Tawab,2 and Ahmed A. Hefny 4
Abstract
The public health importance of the genus Campylobacter is attributed to several species causing diarrhea in consumers. Poultry and their meat are considered the most important sources of human campylobacteriosis. In this study, 287 samples from chicken (131 cloacal swabs, 39 chicken skin, 78 chicken meat, and 39 cecal parts) obtained from retail outlets as well as 246 stool swabs from gastroenteritis patients were examined. A representative number of the biochemically identified Campylobacter jejuni isolates were identified by real-time PCR, confirming the identification of the isolates as C. jejuni. Genotyping of the examined isolates (n = 31) by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) revealed a high discriminatory index of ERICPCR (D = 0.948), dividing C. jejuni isolates of chicken and human origins into 18 profiles and four clusters. The 18 profiles obtained indicated the heterogeneity of C. jejuni. Dendrogram analysis showed that four clusters were generated; all human isolates fell into clusters I and III. These observations further support the existence of a genetic relationship between human and poultry isolates examined in the present study. In conclusion, the results obtained support the speculation that poultry and poultry meat have an important role as sources of infection in the acquisition of Campylobacter infection in humans. Key Words:
Campylobacter jejuni—ERIC-PCR—Genotyping—Chicken—Human—Egypt.
ship 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.
Introduction
C
ampylobacter 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 (ERICPCR) 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 relation-
Material and Methods Sampling and sample processing
A total of 287 samples were collected from chickens; the samples were comprised of 131 cloacal swabs from living birds, in addition to skin, breast meat, thigh meat, and cecal parts (39 each) from freshly slaughtered chicken at randomly selected 10 retail outlets in Zagazig City, Egypt. Moreover, 246 stool swabs from gastroenteritis patients attending the outpatient clinic of Al-Ahrar General Hospital, Zagazig City, Egypt, were examined. Informed consent for participation in the study was obtained from all the patients. The study was approved by the Research Ethics Committee, Faculty of Veterinary Medicine, Zagazig University, Egypt. The samples were collected between September, 2013, and March,
1
Zoonoses Department, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt. Bacteriology, Immunology, and Mycology Department, Faculty of Veterinary Medicine, Benha University, Banha, Egypt Bacteriology, Immunology, and Mycology Department, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt. 4 Veterinary Hospital, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt. 2 3
1
2
2014. The swabs and 25 grams of the other samples were collected in sterile Preston enrichment broth base containing Campylobacter growth supplement (Oxoid, SR 0232) (Ellerbroek et al. 2010). Bacteriological examination
For the isolation of Campylobacter species, enrichment of the collected samples in Preston enrichment broth was performed at 42C for 24–48 h. After enrichment, Campylobacter spp. were isolated on modified Campylobacter-selective agar base cefoperazone-charcoal-desoxycholate agar (mCCDA; cat. no. CM0739, Oxoid) containing antibiotic supplement (cat. no. SR0155, Oxoid,) under microaerophilic conditions at 42C for 48 h (Vandepitte and Verhaegen 2003). Suspected colonies were subjected to biochemical identification using a catalase test, oxidase test, urea hydrolysis test, hydrogen sulfide (H2S) production, citrate utilization test, and rapid hippurate hydrolysis test (Quinn et al. 1994). After identification, a single colony from each sample was purified and preserved on slope agar for molecular identification. Molecular identification of C. jejuni isolates
DNA extraction from the biochemically identified isolates was performed according to the manufacturer’s guidelines using a Bacterial DNA Extraction Kit (Spin-column; BioTeke Corporation, China). Confirmation of C. jejuni isolates was carried out by a real-time probe-based quantitative PCR reaction. Primer and TaqMan probe sets targeting the hipO gene specific for C. jejuni were used (LaGier et al. 2004). Nontemplate DNA and controls of C. jejuni, C. coli, Escherichia coli, Salmonella serovar Typhimurium, Staphylococcus aureus, and two biochemically identified Campylobacter isolates other than C. jejuni and C. coli were also run to determine the specificity of the reaction.
AHMED ET AL. Genotyping of C. jejuni isolates using ERIC-PCR
Genotypes and the genetic relationship between the molecularly identified C. jejuni isolates from chicken and human sources were determined by ERIC-PCR using a single amplification profile (Versalovic et al. 1991). According to the presence or absence of each band, fingerprinting data were transformed into a binary code. A Jaccard coefficient was used to assess the similarity between profiles ( Jaccard 1912). The dendrogram was generated by the unweighted pair group method with arithmetic average (UPGMA) and sequential hierarchical and nested clustering routine. The cluster analysis and the dendrogram construction were performed with SPSS version 22 (IBM Corp. 2013, Armonk, NY). ERIC-PCR discriminatory power was measured by Simpson’s index of diversity (D), which indicates the average probability that a typing system will assign a different type to two unrelated strains randomly sampled from a population (Hunter 1990). A D value of more than 0.9 indicates good differentiation. 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
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.
C. jejuni GENOTYPING
3 Discussion
Table 1. Discriminatory Power and Profile Numbers Produced by ERIC-PCR Fingerprinting Source of isolates Chicken Human Total
Number of isolates
Number of profiles
Discriminatory index (D)
21 10 31
17 4a 17
0.985 0.78 0.948
a The four profiles of human origin are included within the chicken profiles. ERIC-PCR, enterobacterial repetitive intergenic consensus PCR.
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. 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.
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
Table 2. ERIC-PCR Fingerprinting Profiles, Antibiotic Resistance Profiles and Associated Clusters Profile
Number of isolates
Samples (code number)
1 2 3 4 5 6 7
3 1 1 3 2 1 5
E8
4
E E E E E E E E E E
1 1 1 1 1 1 1 2 1 1
Human (30), human (31), Intestine (18) Swab (19) Skin (14) Swab (11), human (29), swab (10) Swab (8), thigh (15) Swab (7) Human (27), human (28), thigh (13), Human (23), human (25) Human (24), human (26), thigh (16), Human (22) Swab (17) Thigh (21) Swab (20) Intestine (9) Intestine (2) skin (6) Swab (3) Swab (4), swab (5) Swab (12) Swab (1)
E E E E E E E
9 10 11 12 13 14 15 16 17 18
ERIC-PCR, enterobacterial repetitive intergenic consensus PCR.
Cluster Cluster I
Cluster II Cluster III
Individual isolate Individual isolates Individual isolates Individual isolate Cluster IV Individual isolate Individual isolate
4
AHMED ET AL.
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. 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. Acknowledgment
The authors express sincere gratitude to veterinarian Alaa El-Din Saad, assistant lecturer of Zoonoses, Faculty of Veterinary Medicine, Zagazig University, for his valuable support during the bacteriological examination part of the current work.
C. jejuni GENOTYPING Author Disclosure Statement
No competing financial interests exist. References
Anderson J, Horn BJ, Gilpin BJ. The prevalence and genetic diversity of Campylobacter spp. in domestic ’backyard’ poultry in Canterbury, New Zealand. Zoonoses Public Health 2012; 59:52–60. Bednarski M, Wieliczko A, Mazurkiewicz M. Genetic comparison of Campylobacter jejuni isolated from different cattle farms. Pol J Vet Sci 2011; 14:279–281. Behringer M, Miller WG, Oyarzabal OA. Typing of Campylobacter jejuni and Campylobacter coli isolated from live broilers and retail broiler meat by flaA-RFLP, MLST, PFGE and REP-PCR. J Microbiol Methods 2011; 84:194–201. Berrang ME, Buhr RJ, Cason JA, Dickens JA. Broiler carcass contamination with Campylobacter from feces during defeathering. J Food Prot 2001; 64:2063–2066. Cardinale E, Rose V, Perrier Gros-Claude JD, Tall F, et al. Genetic characterization and antibiotic resistance of Campylobacter spp. isolated from poultry and humans in Senegal. J Appl Microbiol 2006; 100:209–217. Denis M, Chidaine B, Laisney MJ, Kempf I, et al. Comparison of genetic profiles of Campylobacter strains isolated from poultry, pig and Campylobacter human infections in Brittany, France. Pathol Biol (Paris) 2009; 57:23–29. Ellerbroek LI, Lienau JA, Klein G. Campylobacter spp. in broiler flocks at farm level and the potential for cross-contamination during slaughter. Zoonoses Public Health 2010; 57:e81–e88. Ghorbanalizadgan M, Bakhshi B, Kazemnejad Lili A, NajarPeerayeh S, et al. A molecular survey of Campylobacter jejuni and Campylobacter coli virulence and diversity. Iran Biomed J 2014; 18:158–164. Giesendorf BA, Goossens H, Niesters HG, Van Belkum A, et al. Polymerase chain reaction-mediated DNA fingerprinting for epidemiological studies on Campylobacter spp. J Med Microbiol 1994; 40:141–147. Hook H, Fattah MA, Ericsson H, Vagsholm I, et al. Genotype dynamics of Campylobacter jejuni in a broiler flock. Vet Microbiol 2005; 106:109–117. Hulton CS, Higgins CF, Sharp PM. ERIC sequences: A novel family of repetitive elements in the genomes of Escherichia coli, Salmonella typhimurium and other enterobacteria. Mol Microbiol 1991; 5:825–834. Hunter PR. Reproducibility and indices of discriminatory power of microbial typing methods. J Clin Microbiol 1990; 28: 1903–1905. Iriarte MP, Owen RJ. Repetitive and arbitrary primer DNA sequences in PCR-mediated fingerprinting of outbreak and sporadic isolates of Campylobacter jejuni. FEMS Immunol Med Microbiol 1996; 15:17–22. Jaccard P. The distribution of the flora in the Alpine zone. New Phytologist 1912; 11:37–50. LaGier MJ, Joseph LA, Passaretti TV, Musser KA, et al. A realtime multiplexed PCR assay for rapid detection and differentiation of Campylobacter jejuni and Campylobacter coli. Mol Cell Probes 2004; 18:275–282. Lindmark H, Harbom B, Thebo L, Andersson L, et al. Genetic characterization and antibiotic resistance of Campylobacter jejuni isolated from meats, water, and humans in Sweden. J Clin Microbiol 2004; 42:700–706. Matsui T, Moore JE, Patterson C, Millar BC, et al. Molecular characterisation of human campylobacteriosis in Northern
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Ireland: Evidence of clonal stability. Ir J Med Sci 2002; 171: 33–36. Millemann Y, Lesage-Descauses MC, Lafont JP, ChaslusDancla E. Comparison of random amplified polymorphic DNA analysis and enterobacterial repetitive intergenic consensus-PCR for epidemiological studies of Salmonella. FEMS Immunol Med Microbiol 1996; 14:129–134. Nadeau E, Messier S, Quessy S. Prevalence and comparison of genetic profiles of Campylobacter strains isolated from poultry and sporadic cases of campylobacteriosis in humans. J Food Prot 2002; 65:73–78. Quinn PJ, Carter ME, Markey B, Carter CR. Campylobacter species. In: Clinical Veterinary Microbiology. London: Wolfe Publishing, 1994:268–272. Rahimi E, Ameri M. Antimicrobial resistance patterns of Campylobacter spp. isolated from raw chicken, turkey, quail, partridge, and ostrich meat in Iran. Food Control 2011; 22:1165–1170. Saito S, Yatsuyanagi J, Harata S, Ito Y, et al. Campylobacter jejuni isolated from retail poultry meat, bovine feces and bile, and human diarrheal samples in Japan: Comparison of serotypes and genotypes. FEMS Immunol Med Microbiol 2005; 45:311–319. Shi ZY, Liu PY, Lau YJ, Lin YH, et al. Comparison of polymerase chain reaction and pulsed-field gel electrophoresis for the epidemiological typing of Campylobacter jejuni. Diagn Microbiol Infect Dis 1996; 26:103–108. Steinhauserova I, Ceskova J, Fojtikova K, Obrovska I. Identification of thermophilic Campylobacter spp. by phenotypic and molecular methods. J Appl Microbiol 2001; 90:470–475. Vandepitte J, Engbaek K, Rohner P, Piot P, Heuck C, et al. Basic Laboratory Procedures in Clinical Bacteriology, 2nd ed. Geneva, Switzerland: World Health Organization, 2003. Versalovic J, Koeuth T, Lupski JR. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res 1991; 19:6823–6831. Wassenaar TM, Geilhausen B, Newell DG. Evidence of genomic instability in Campylobacter jejuni isolated from poultry. Appl Environ Microbiol 1998; 64:1816–1821. Wieczorek K. Relationship between the molecular typing of Campylobacter strains and the prevalence of their virulence genes. Bulletin of the Veterinary Institute in Pulawy 2009; 53:193–198. Wieland B, Wittwer M, Regula G, Wassenaar TM, et al. Phenon cluster analysis as a method to investigate epidemiological relatedness between sources of Campylobacter jejuni. J Appl Microbiol 2006; 100:316–324. Wilson MK, Lane AB, Law BF, Miller WG, et al. Analysis of the pan genome of Campylobacter jejuni isolates recovered from poultry by pulsed-field gel electrophoresis, multilocus sequence typing (MLST), and repetitive sequence polymerase chain reaction (rep-PCR) reveals different discriminatory capabilities. Microb Ecol 2009; 58:843–855. Zorman T, Heyndrickx M, Uzunovic-Kamberovic S, Smole Mozina S. Genotyping of Campylobacter coli and C. jejuni from retail chicken meat and humans with campylobacteriosis in Slovenia and Bosnia and Herzegovina. Int J Food Microbiol 2006; 110:24–33.
Address correspondence to: Heba A. Ahmed Faculty of Veterinary Medicine Alzeraa Street Zagazig University Zagazig, 44511 Egypt E-mail: [email protected]
ORIGINAL ARTICLE
ERIC-PCR Genotyping of Some Campylobacter jejuni Isolates of Chicken and Human Origin in Egypt Heba A. Ahmed,1 Fatma I. El Hofy,2 Ahmed M. Ammar,3 Ashraf A. Abd El Tawab,2 and Ahmed A. Hefny 4
Abstract
The public health importance of the genus Campylobacter is attributed to several species causing diarrhea in consumers. Poultry and their meat are considered the most important sources of human campylobacteriosis. In this study, 287 samples from chicken (131 cloacal swabs, 39 chicken skin, 78 chicken meat, and 39 cecal parts) obtained from retail outlets as well as 246 stool swabs from gastroenteritis patients were examined. A representative number of the biochemically identified Campylobacter jejuni isolates were identified by real-time PCR, confirming the identification of the isolates as C. jejuni. Genotyping of the examined isolates (n = 31) by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) revealed a high discriminatory index of ERICPCR (D = 0.948), dividing C. jejuni isolates of chicken and human origins into 18 profiles and four clusters. The 18 profiles obtained indicated the heterogeneity of C. jejuni. Dendrogram analysis showed that four clusters were generated; all human isolates fell into clusters I and III. These observations further support the existence of a genetic relationship between human and poultry isolates examined in the present study. In conclusion, the results obtained support the speculation that poultry and poultry meat have an important role as sources of infection in the acquisition of Campylobacter infection in humans. Key Words:
Campylobacter jejuni—ERIC-PCR—Genotyping—Chicken—Human—Egypt.
ship 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.
Introduction
C
ampylobacter 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 (ERICPCR) 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 relation-
Material and Methods Sampling and sample processing
A total of 287 samples were collected from chickens; the samples were comprised of 131 cloacal swabs from living birds, in addition to skin, breast meat, thigh meat, and cecal parts (39 each) from freshly slaughtered chicken at randomly selected 10 retail outlets in Zagazig City, Egypt. Moreover, 246 stool swabs from gastroenteritis patients attending the outpatient clinic of Al-Ahrar General Hospital, Zagazig City, Egypt, were examined. Informed consent for participation in the study was obtained from all the patients. The study was approved by the Research Ethics Committee, Faculty of Veterinary Medicine, Zagazig University, Egypt. The samples were collected between September, 2013, and March,
1
Zoonoses Department, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt. Bacteriology, Immunology, and Mycology Department, Faculty of Veterinary Medicine, Benha University, Banha, Egypt Bacteriology, Immunology, and Mycology Department, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt. 4 Veterinary Hospital, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt. 2 3
1
2
2014. The swabs and 25 grams of the other samples were collected in sterile Preston enrichment broth base containing Campylobacter growth supplement (Oxoid, SR 0232) (Ellerbroek et al. 2010). Bacteriological examination
For the isolation of Campylobacter species, enrichment of the collected samples in Preston enrichment broth was performed at 42C for 24–48 h. After enrichment, Campylobacter spp. were isolated on modified Campylobacter-selective agar base cefoperazone-charcoal-desoxycholate agar (mCCDA; cat. no. CM0739, Oxoid) containing antibiotic supplement (cat. no. SR0155, Oxoid,) under microaerophilic conditions at 42C for 48 h (Vandepitte and Verhaegen 2003). Suspected colonies were subjected to biochemical identification using a catalase test, oxidase test, urea hydrolysis test, hydrogen sulfide (H2S) production, citrate utilization test, and rapid hippurate hydrolysis test (Quinn et al. 1994). After identification, a single colony from each sample was purified and preserved on slope agar for molecular identification. Molecular identification of C. jejuni isolates
DNA extraction from the biochemically identified isolates was performed according to the manufacturer’s guidelines using a Bacterial DNA Extraction Kit (Spin-column; BioTeke Corporation, China). Confirmation of C. jejuni isolates was carried out by a real-time probe-based quantitative PCR reaction. Primer and TaqMan probe sets targeting the hipO gene specific for C. jejuni were used (LaGier et al. 2004). Nontemplate DNA and controls of C. jejuni, C. coli, Escherichia coli, Salmonella serovar Typhimurium, Staphylococcus aureus, and two biochemically identified Campylobacter isolates other than C. jejuni and C. coli were also run to determine the specificity of the reaction.
AHMED ET AL. Genotyping of C. jejuni isolates using ERIC-PCR
Genotypes and the genetic relationship between the molecularly identified C. jejuni isolates from chicken and human sources were determined by ERIC-PCR using a single amplification profile (Versalovic et al. 1991). According to the presence or absence of each band, fingerprinting data were transformed into a binary code. A Jaccard coefficient was used to assess the similarity between profiles ( Jaccard 1912). The dendrogram was generated by the unweighted pair group method with arithmetic average (UPGMA) and sequential hierarchical and nested clustering routine. The cluster analysis and the dendrogram construction were performed with SPSS version 22 (IBM Corp. 2013, Armonk, NY). ERIC-PCR discriminatory power was measured by Simpson’s index of diversity (D), which indicates the average probability that a typing system will assign a different type to two unrelated strains randomly sampled from a population (Hunter 1990). A D value of more than 0.9 indicates good differentiation. 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
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.
C. jejuni GENOTYPING
3 Discussion
Table 1. Discriminatory Power and Profile Numbers Produced by ERIC-PCR Fingerprinting Source of isolates Chicken Human Total
Number of isolates
Number of profiles
Discriminatory index (D)
21 10 31
17 4a 17
0.985 0.78 0.948
a The four profiles of human origin are included within the chicken profiles. ERIC-PCR, enterobacterial repetitive intergenic consensus PCR.
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. 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.
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
Table 2. ERIC-PCR Fingerprinting Profiles, Antibiotic Resistance Profiles and Associated Clusters Profile
Number of isolates
Samples (code number)
1 2 3 4 5 6 7
3 1 1 3 2 1 5
E8
4
E E E E E E E E E E
1 1 1 1 1 1 1 2 1 1
Human (30), human (31), Intestine (18) Swab (19) Skin (14) Swab (11), human (29), swab (10) Swab (8), thigh (15) Swab (7) Human (27), human (28), thigh (13), Human (23), human (25) Human (24), human (26), thigh (16), Human (22) Swab (17) Thigh (21) Swab (20) Intestine (9) Intestine (2) skin (6) Swab (3) Swab (4), swab (5) Swab (12) Swab (1)
E E E E E E E
9 10 11 12 13 14 15 16 17 18
ERIC-PCR, enterobacterial repetitive intergenic consensus PCR.
Cluster Cluster I
Cluster II Cluster III
Individual isolate Individual isolates Individual isolates Individual isolate Cluster IV Individual isolate Individual isolate
4
AHMED ET AL.
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. 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. Acknowledgment
The authors express sincere gratitude to veterinarian Alaa El-Din Saad, assistant lecturer of Zoonoses, Faculty of Veterinary Medicine, Zagazig University, for his valuable support during the bacteriological examination part of the current work.
C. jejuni GENOTYPING Author Disclosure Statement
No competing financial interests exist. References
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Address correspondence to: Heba A. Ahmed Faculty of Veterinary Medicine Alzeraa Street Zagazig University Zagazig, 44511 Egypt E-mail: [email protected]
