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Diversity of Campylobacter jejuni and Campylobacter coli Genotypes from Human and Animal Sources from Rio de Janeiro, Brazil
Research in Veterinary Science 88 (2010) 214–217
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Diversity of Campylobacter jejuni and Campylobacter coli Genotypes from Human and Animal Sources from Rio de Janeiro, Brazil M.H.C. Aquino a,*, A.L.L. Filgueiras b, R. Matos c, K.R.N. Santos d, T. Ferreira a, M.C.S. Ferreira d, L.M. Teixeira d, A. Tibana d a
Faculdade de Veterinária, Universidade Federal Fluminense, Niterói, RJ, Brazil Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil Instituto Nacional de Saúde Dr. Ricardo Jorge, Lisboa, Portugal d Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil b c
a r t i c l e
i n f o
Article history: Accepted 18 August 2009
Keywords: Campylobacter jejuni Campylobacter coli Genotypes
a b s t r a c t To compare the genotypes of Campylobacter jejuni and Campylobacter coli isolates of human and animal origin collected in Rio de Janeiro City, 30 C. jejuni and 35 C. coli isolates from animal sources (n = 45) and human patients with gastroenteritis (n = 20) were genotyped by PCR-based techniques, namely random amplified polymorphic DNA (RAPD-PCR) and enterobacterial repetitive intergenic consensus sequence (ERIC-PCR). RAPD-PCR identified 50 types and ERIC-PCR identified 22 genotypes, among the 65 Campylobacter isolates. Both PCR methods discriminated the C. jejuni and C. coli groups of isolates. Combining the results of both methods, no single genotype was shared between isolates from human and animal sources. Two groups of two C. coli isolates each with identical genotypes were found among poultry and pig isolates. A high level of genetic diversity observed among the Campylobacter isolates suggests lack of overlap between isolates from different sources. Ó 2009 Elsevier Ltd. All rights reserved.
Campylobacter jejuni and Campylobacter coli are recognized as important causative agents of acute human diarrheal diseases world-wide. However, the sources and transmission routes of human campylobacteriosis are not fully understood. Consuming and handling poultry meat is the most consistent risk factor, and other meat-producing animals and pet animals are suggested as potential sources for human infection (Steinbruecker et al., 2001; Zorman et al., 2006). This confusing epidemiological evidence is partly because of the sporadic nature of the disease, along with the organism’s wide distribution, and high levels of genetic and antigenic diversity (Zorman et al., 2006). Several molecular typing methods have been used to support studies on the epidemiology of Campylobacter infections. Although pulsed-field gel electrophoresis and multilocus sequence typing are highly standardized (Zorman et al., 2006), polymerase chain reaction based methods, like arbitrarily primed PCR are also successfully applied for the discrimination of C. jejuni and C. coli strains (Zorman et al., 2006; Madden et al., 2007). In this study we describe a molecular epidemiological investigation using PCR-based DNA fingerprinting of C. jejuni and C. coli isolates from human enteritis and animal reservoirs. The 65 Campylobacter isolates included in this study were collected over a 12-month period, in Rio de Janeiro City. * Corresponding author. Tel./fax: +55 21 26299534. E-mail address: [email protected] (M.H.C. Aquino). 0034-5288/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2009.08.005
They comprised 45 animal isolates, including eight C. jejuni and 16 C. coli strains from poultry carcasses from different flocks; 10 C. coli strains from slaughter pigs from different herds; six C. jejuni from a single sheep herd collected in one visit; three C. jejuni from dogs; one C. jejuni and one C. coli from Rhesus monkeys housed in caging. Twelve C. jejuni and eight C. coli strains from human patients with clinically confirmed gastroenteritis collected during the same period were also incorporated. All the Campylobacter isolates were recovered from stool samples, except those from rinsed poultry carcasses. C. jejuni ATCC 33291 and C. coli ATCC 43485 were included as control strains. The Campylobacter strains were cultured on 5% Columbia blood agar plates (Oxoid Ltd.) in a microaerophilic atmosphere at 37 °C for 48 h. The strains were identified to the species level by biochemical tests for Campylobacter (Stern et al., 1992) and by the use of a multiplex PCR test (Harmon et al., 1997). For the molecular analysis, freshly cultured cells were inoculated in 10 mL of Brucella broth (Oxoid Ltd.) and incubated at 37 °C in a microaerophilic atmosphere for 24–48 h. From these cultures, 3 mL aliquots were centrifuged (13,000g for 1 min) and the pellets were used for DNA extraction according to Pitcher et al. (1989). Random amplified polymorphic DNA (RAPD) typing was conducted using the method of Akopyanz et al. (1992) and ERIC-PCR was performed as described previously by Giesendorf et al. (1994). The profiles were analysed in duplicate to highlight the
M.H.C. Aquino et al. / Research in Veterinary Science 88 (2010) 214–217
reproducibility of the described RAPD protocol as suggested by Hilton et al. (1997). Amplified products were analysed on agarose 2% gels, stained with ethidium bromide, and photographed under UV translumination. The /X-174 RF DNA-Hae III Digest (Amersham Pharmacia Biotech) and 1-kb DNA ladder (Gibco BRL) were used as molecular weight markers for RAPD-PCR and ERIC-PCR, respectively. All genotypes were analysed by using the Molecular Analyst Fingerprinting Plus software package version 1.12 (Bio-Rad). Gels were analysed and compared by using band matching with UPGMA (Unweighted Pair Group Method using Arithmetic Averages) clustering using the Dice coefficient, and 1.2% tolerance. Two dendrograms were constructed to assess genetic relationship among the isolates investigated (Figs. 1 and 2). RAPD-PCR identified a total of 50 types among the 65 isolates, using a cut off of 90%, and 26 and 24 patterns were identified for C. jejuni (cluster I) and for C. coli (cluster II), respectively. Amplification reactions by ERIC-PCR identified 8 and 14 patterns among C. coli (cluster I- with two exceptions, H5 and PO7/11 identified as C. jejuni) and C. jejuni (cluster II- with one exception, H1103 identified as C. coli) isolates, respectively.
215
The results of this study showed extensive genomic diversity among the C. jejuni and C. coli isolates, most notably among C. jejuni as observed in both PCR typing methods. Combining the results of both methods, no identical pattern was identified among human isolates and no pattern observed for any animal isolate matched those of human origin. This significant genomic diversity is probably due to the lack of overlap of the different sources sampled. Earlier investigations also reported extreme heterogeneity of Campylobacter isolates (Madden et al., 2007; Ridley et al., 2008). Although Campylobacter spp. have a natural ability for transformation, genomic rearrangements most likely explain this increase in genetic diversity (Ridley et al., 2008). Reliable tracing of the source of Campylobacter strains by molecular typing is therefore very difficult. Besides the extreme heterogeneity of the isolates, mainly for C. jejuni, other reasons include contamination of meat samples with multiple strains and cross-contamination (Zorman et al., 2006). In this study, C. jejuni sheep isolates were cultured from rectal swabs collected from animals belonging to the same herd and no identical pattern was identified among them. However, two identical C. coli patterns from poultry carcasses purchased from
Fig. 1. Dendrogram of the cluster analysis of C. jejuni (cluster I) and C. coli(cluster II) isolates based on PCR/RAPD patterns from human (H), poultry (PO), pig (P), sheep (S), dog (D), and monkey (M) origin with their degree of relatedness.
216
M.H.C. Aquino et al. / Research in Veterinary Science 88 (2010) 214–217
except
except
Fig. 2. Dendrogram of the cluster analysis of C. coli (cluster I) and C. jejuni (cluster II) isolates based on PCR/ERIC patterns from human (H), poultry (PO), pig (P), sheep (S), dog (D), and monkey (M) origin with their degree of relatedness.
different abattoirs at different times were identified. Besides, two identical C. coli isolates cultured from rectal swabs obtained from pigs in different herds at different times were observed. C. coli strains not epidemiologically connected, showing identical PFGE and fla-RFLP types, were also previously reported by Zorman et al. (2006) showing a lower genetic diversity when compared to C. jejuni. Our findings support a wide DNA diversity existing among C. jejuni and C. coli isolates recovered from different sources in Rio de Janeiro City. The PCR-based methods used in this study provided discriminating and reproducible means for differentiating C. jejuni and C. coli strains and RAPD-PCR is preferred as it was the most discriminatory. Since sufficient technical consideration is given to the genomic diversity existing in these microorganisms, these molecular typing techniques might be more useful in defining localized outbreaks or elucidating the transmission pathways in investigations of less diversified sources of isolation.
Acknowledgements This study was supported by a Grant from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). The authors thank Antônio Carlos dos Santos for technical assistance.
References Akopyanz, N., Bukanov, N.O., Westblom, T.U., Kresovich, S., Berg, D.E., 1992. DNA diversity among clinical isolates of Helicobacter pylori detected by PCR-based RAPD fingerprinting. Nucleic Acids Research 20, 5137–5142. Giesendorf, B.A.J., Goossens, H., Niestters, H.G.M., van Belkun, A., Koeken, A., Endtz, H.P., Stegeman, H., Quint, W.G.V., 1994. Polymerase chain reaction-mediated DNA fingerprinting for epidemiological studies on Campylobacter spp. Journal of Medical Microbiology 40, 141–147. Harmon, K.M., Ranson, G.M., Wesley, I.V., 1997. Differentiation of Campylobacter. Cellular Probes 11, 195–200. Hilton, A.C., Mortiboy, D., Banks, J.G., Penn, C.W., 1997. RAPD analysis of environmental food and clinical isolates of Campylobacter spp. FEMS Immunology and Medical Microbiology 18, 119–124.
M.H.C. Aquino et al. / Research in Veterinary Science 88 (2010) 214–217 Madden, R.H., Moran, L., Scates, P., 2007. Diversity of Campylobacter coli genotypes in the lower porcine gastrointestinal tract at time of slaughter. Letters in Applied Microbiology 45, 575–580. Pitcher, D.G., Saunders, N.A., Owen, R.J., 1989. Rapid extraction of bacterial DNA with guanidium thiocyanate. Letters in Applied Microbiology 8, 1511–1516. Ridley, A.M., Toszegery, M.J., Cawthraw, S.A., Wassenaar, T.M., Newell, D.G., 2008. Genetic instability is associated with changes in the colonization potential of Campylobacter jejuni in the avian intestine. Journal of Applied Microbiology 105, 95–104. Steinbruecker, B., Ruberg, F., Kist, M., 2001. Bacterial genetic fingerprint: a reliable factor in the study of the epidemiology of human Campylobacter enteritis? Journal of Clinical Microbiology 39, 4155–4159.
217
Stern, N.J., Patton, C.M., Doyle, M.P., 1992. Campylobacter. In: Microbiological Examination of Foods, second ed. American Public Health Association, pp. 475– 489 (Copyright, 1219 p.cap. 29). Zorman, T., Heyndrickx, M., Uzunovic-Kamberovic, S., Smole Mozina, S., 2006. Genotyping of Campylobacter coli and C. Jejuni from retail chicken meat and human with campylobacteriosis in Slovenia and Bosnia and Herzegovina. International Journal of Food Microbiology 110, 24–33.
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Diversity of Campylobacter jejuni and Campylobacter coli Genotypes from Human and Animal Sources from Rio de Janeiro, Brazil M.H.C. Aquino a,*, A.L.L. Filgueiras b, R. Matos c, K.R.N. Santos d, T. Ferreira a, M.C.S. Ferreira d, L.M. Teixeira d, A. Tibana d a
Faculdade de Veterinária, Universidade Federal Fluminense, Niterói, RJ, Brazil Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil Instituto Nacional de Saúde Dr. Ricardo Jorge, Lisboa, Portugal d Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil b c
a r t i c l e
i n f o
Article history: Accepted 18 August 2009
Keywords: Campylobacter jejuni Campylobacter coli Genotypes
a b s t r a c t To compare the genotypes of Campylobacter jejuni and Campylobacter coli isolates of human and animal origin collected in Rio de Janeiro City, 30 C. jejuni and 35 C. coli isolates from animal sources (n = 45) and human patients with gastroenteritis (n = 20) were genotyped by PCR-based techniques, namely random amplified polymorphic DNA (RAPD-PCR) and enterobacterial repetitive intergenic consensus sequence (ERIC-PCR). RAPD-PCR identified 50 types and ERIC-PCR identified 22 genotypes, among the 65 Campylobacter isolates. Both PCR methods discriminated the C. jejuni and C. coli groups of isolates. Combining the results of both methods, no single genotype was shared between isolates from human and animal sources. Two groups of two C. coli isolates each with identical genotypes were found among poultry and pig isolates. A high level of genetic diversity observed among the Campylobacter isolates suggests lack of overlap between isolates from different sources. Ó 2009 Elsevier Ltd. All rights reserved.
Campylobacter jejuni and Campylobacter coli are recognized as important causative agents of acute human diarrheal diseases world-wide. However, the sources and transmission routes of human campylobacteriosis are not fully understood. Consuming and handling poultry meat is the most consistent risk factor, and other meat-producing animals and pet animals are suggested as potential sources for human infection (Steinbruecker et al., 2001; Zorman et al., 2006). This confusing epidemiological evidence is partly because of the sporadic nature of the disease, along with the organism’s wide distribution, and high levels of genetic and antigenic diversity (Zorman et al., 2006). Several molecular typing methods have been used to support studies on the epidemiology of Campylobacter infections. Although pulsed-field gel electrophoresis and multilocus sequence typing are highly standardized (Zorman et al., 2006), polymerase chain reaction based methods, like arbitrarily primed PCR are also successfully applied for the discrimination of C. jejuni and C. coli strains (Zorman et al., 2006; Madden et al., 2007). In this study we describe a molecular epidemiological investigation using PCR-based DNA fingerprinting of C. jejuni and C. coli isolates from human enteritis and animal reservoirs. The 65 Campylobacter isolates included in this study were collected over a 12-month period, in Rio de Janeiro City. * Corresponding author. Tel./fax: +55 21 26299534. E-mail address: [email protected] (M.H.C. Aquino). 0034-5288/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2009.08.005
They comprised 45 animal isolates, including eight C. jejuni and 16 C. coli strains from poultry carcasses from different flocks; 10 C. coli strains from slaughter pigs from different herds; six C. jejuni from a single sheep herd collected in one visit; three C. jejuni from dogs; one C. jejuni and one C. coli from Rhesus monkeys housed in caging. Twelve C. jejuni and eight C. coli strains from human patients with clinically confirmed gastroenteritis collected during the same period were also incorporated. All the Campylobacter isolates were recovered from stool samples, except those from rinsed poultry carcasses. C. jejuni ATCC 33291 and C. coli ATCC 43485 were included as control strains. The Campylobacter strains were cultured on 5% Columbia blood agar plates (Oxoid Ltd.) in a microaerophilic atmosphere at 37 °C for 48 h. The strains were identified to the species level by biochemical tests for Campylobacter (Stern et al., 1992) and by the use of a multiplex PCR test (Harmon et al., 1997). For the molecular analysis, freshly cultured cells were inoculated in 10 mL of Brucella broth (Oxoid Ltd.) and incubated at 37 °C in a microaerophilic atmosphere for 24–48 h. From these cultures, 3 mL aliquots were centrifuged (13,000g for 1 min) and the pellets were used for DNA extraction according to Pitcher et al. (1989). Random amplified polymorphic DNA (RAPD) typing was conducted using the method of Akopyanz et al. (1992) and ERIC-PCR was performed as described previously by Giesendorf et al. (1994). The profiles were analysed in duplicate to highlight the
M.H.C. Aquino et al. / Research in Veterinary Science 88 (2010) 214–217
reproducibility of the described RAPD protocol as suggested by Hilton et al. (1997). Amplified products were analysed on agarose 2% gels, stained with ethidium bromide, and photographed under UV translumination. The /X-174 RF DNA-Hae III Digest (Amersham Pharmacia Biotech) and 1-kb DNA ladder (Gibco BRL) were used as molecular weight markers for RAPD-PCR and ERIC-PCR, respectively. All genotypes were analysed by using the Molecular Analyst Fingerprinting Plus software package version 1.12 (Bio-Rad). Gels were analysed and compared by using band matching with UPGMA (Unweighted Pair Group Method using Arithmetic Averages) clustering using the Dice coefficient, and 1.2% tolerance. Two dendrograms were constructed to assess genetic relationship among the isolates investigated (Figs. 1 and 2). RAPD-PCR identified a total of 50 types among the 65 isolates, using a cut off of 90%, and 26 and 24 patterns were identified for C. jejuni (cluster I) and for C. coli (cluster II), respectively. Amplification reactions by ERIC-PCR identified 8 and 14 patterns among C. coli (cluster I- with two exceptions, H5 and PO7/11 identified as C. jejuni) and C. jejuni (cluster II- with one exception, H1103 identified as C. coli) isolates, respectively.
215
The results of this study showed extensive genomic diversity among the C. jejuni and C. coli isolates, most notably among C. jejuni as observed in both PCR typing methods. Combining the results of both methods, no identical pattern was identified among human isolates and no pattern observed for any animal isolate matched those of human origin. This significant genomic diversity is probably due to the lack of overlap of the different sources sampled. Earlier investigations also reported extreme heterogeneity of Campylobacter isolates (Madden et al., 2007; Ridley et al., 2008). Although Campylobacter spp. have a natural ability for transformation, genomic rearrangements most likely explain this increase in genetic diversity (Ridley et al., 2008). Reliable tracing of the source of Campylobacter strains by molecular typing is therefore very difficult. Besides the extreme heterogeneity of the isolates, mainly for C. jejuni, other reasons include contamination of meat samples with multiple strains and cross-contamination (Zorman et al., 2006). In this study, C. jejuni sheep isolates were cultured from rectal swabs collected from animals belonging to the same herd and no identical pattern was identified among them. However, two identical C. coli patterns from poultry carcasses purchased from
Fig. 1. Dendrogram of the cluster analysis of C. jejuni (cluster I) and C. coli(cluster II) isolates based on PCR/RAPD patterns from human (H), poultry (PO), pig (P), sheep (S), dog (D), and monkey (M) origin with their degree of relatedness.
216
M.H.C. Aquino et al. / Research in Veterinary Science 88 (2010) 214–217
except
except
Fig. 2. Dendrogram of the cluster analysis of C. coli (cluster I) and C. jejuni (cluster II) isolates based on PCR/ERIC patterns from human (H), poultry (PO), pig (P), sheep (S), dog (D), and monkey (M) origin with their degree of relatedness.
different abattoirs at different times were identified. Besides, two identical C. coli isolates cultured from rectal swabs obtained from pigs in different herds at different times were observed. C. coli strains not epidemiologically connected, showing identical PFGE and fla-RFLP types, were also previously reported by Zorman et al. (2006) showing a lower genetic diversity when compared to C. jejuni. Our findings support a wide DNA diversity existing among C. jejuni and C. coli isolates recovered from different sources in Rio de Janeiro City. The PCR-based methods used in this study provided discriminating and reproducible means for differentiating C. jejuni and C. coli strains and RAPD-PCR is preferred as it was the most discriminatory. Since sufficient technical consideration is given to the genomic diversity existing in these microorganisms, these molecular typing techniques might be more useful in defining localized outbreaks or elucidating the transmission pathways in investigations of less diversified sources of isolation.
Acknowledgements This study was supported by a Grant from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). The authors thank Antônio Carlos dos Santos for technical assistance.
References Akopyanz, N., Bukanov, N.O., Westblom, T.U., Kresovich, S., Berg, D.E., 1992. DNA diversity among clinical isolates of Helicobacter pylori detected by PCR-based RAPD fingerprinting. Nucleic Acids Research 20, 5137–5142. Giesendorf, B.A.J., Goossens, H., Niestters, H.G.M., van Belkun, A., Koeken, A., Endtz, H.P., Stegeman, H., Quint, W.G.V., 1994. Polymerase chain reaction-mediated DNA fingerprinting for epidemiological studies on Campylobacter spp. Journal of Medical Microbiology 40, 141–147. Harmon, K.M., Ranson, G.M., Wesley, I.V., 1997. Differentiation of Campylobacter. Cellular Probes 11, 195–200. Hilton, A.C., Mortiboy, D., Banks, J.G., Penn, C.W., 1997. RAPD analysis of environmental food and clinical isolates of Campylobacter spp. FEMS Immunology and Medical Microbiology 18, 119–124.
M.H.C. Aquino et al. / Research in Veterinary Science 88 (2010) 214–217 Madden, R.H., Moran, L., Scates, P., 2007. Diversity of Campylobacter coli genotypes in the lower porcine gastrointestinal tract at time of slaughter. Letters in Applied Microbiology 45, 575–580. Pitcher, D.G., Saunders, N.A., Owen, R.J., 1989. Rapid extraction of bacterial DNA with guanidium thiocyanate. Letters in Applied Microbiology 8, 1511–1516. Ridley, A.M., Toszegery, M.J., Cawthraw, S.A., Wassenaar, T.M., Newell, D.G., 2008. Genetic instability is associated with changes in the colonization potential of Campylobacter jejuni in the avian intestine. Journal of Applied Microbiology 105, 95–104. Steinbruecker, B., Ruberg, F., Kist, M., 2001. Bacterial genetic fingerprint: a reliable factor in the study of the epidemiology of human Campylobacter enteritis? Journal of Clinical Microbiology 39, 4155–4159.
217
Stern, N.J., Patton, C.M., Doyle, M.P., 1992. Campylobacter. In: Microbiological Examination of Foods, second ed. American Public Health Association, pp. 475– 489 (Copyright, 1219 p.cap. 29). Zorman, T., Heyndrickx, M., Uzunovic-Kamberovic, S., Smole Mozina, S., 2006. Genotyping of Campylobacter coli and C. Jejuni from retail chicken meat and human with campylobacteriosis in Slovenia and Bosnia and Herzegovina. International Journal of Food Microbiology 110, 24–33.