Assessment of PCR-DGGE for the identification of diverse Helicobacter species, and application to faecal samples from zoo animals to determine Helicobacter prevalence.
Helicobacter species are fastidious bacterial pathogens that are difficult to culture by standard methods. A PCR-denaturing gradient gel electrophoresis (PCR-DGGE) technique for detection and identification of different Helicobacter species was developed and evaluated. The method involves PCR detection of Helicobacter DNA by genus-specific primers that target 16S rDNA and subsequent differentiation of Helicobacter PCR products by use of DGGE. Strains are identified by comparing mobilities of unknown samples to those determined for reference strains; sequence analysis can also be performed on purified amplicons. Sixteen DGGE profiles were derived from 44 type and reference strains of 20 Helicobacter species, indicating the potential of this approach for resolving infection of a single host by multiple Helicobacter species. Some more highly related species were not differentiated whereas in highly heterogeneous species, sequence divergence was observed and more than one PCR-DGGE profile was obtained. Application of the PCR-DGGE method to DNA extracted from faeces of zoo animals revealed the presence of Helicobacter DNA in 13 of 16 samples; a correlation was seen between the mobility of PCR products in DGGE analysis and DNA sequencing. In combination, this indicated that zoo animals are colonized by a wide range of different Helicobacter species; seven animals appeared to be colonized by multiple Helicobacter species. By this approach, presumptive identifications were made of Helicobacter bilis and Helicobacter hepaticus in a Nile crocodile, Helicobacter cinaedi in a baboon and a red panda, and Helicobacter felis in a wolf and a Taiwan beauty snake. All of these PCR products ( approximately 400 bp) showed 100 % sequence similarity to 16S rDNA sequences of the mentioned species. These results demonstrate the potential of PCR-DGGE-based analysis for identification of Helicobacter species in complex ecosystems, such as the gastrointestinal tract, and could contribute to a better understanding of the ecology of helicobacters and other pathogens with a complex aetiology.
Al-Soud
Waleed Abu
WA
Department of Medical Microbiology, Dermatology and Infection, Lund University, Sölvegatan 23, SE-223 62 Lund, Sweden 2Department of Clinical Microbiology 7806, National University Hospital, Righospitalet, Tagensvej 20, DK-2200, Copenhagen, Denmark 3Danish Veterinary Institute, Bülowsvej 27, DK-1790, Copenhagen, Denmark 4Laboratorium voor Microbiologie, University of Ghent, Belgium.
Bennedsen
Mads
M
Department of Medical Microbiology, Dermatology and Infection, Lund University, Sölvegatan 23, SE-223 62 Lund, Sweden 2Department of Clinical Microbiology 7806, National University Hospital, Righospitalet, Tagensvej 20, DK-2200, Copenhagen, Denmark 3Danish Veterinary Institute, Bülowsvej 27, DK-1790, Copenhagen, Denmark 4Laboratorium voor Microbiologie, University of Ghent, Belgium.
On
Stephen L W
SLW
Department of Medical Microbiology, Dermatology and Infection, Lund University, Sölvegatan 23, SE-223 62 Lund, Sweden 2Department of Clinical Microbiology 7806, National University Hospital, Righospitalet, Tagensvej 20, DK-2200, Copenhagen, Denmark 3Danish Veterinary Institute, Bülowsvej 27, DK-1790, Copenhagen, Denmark 4Laboratorium voor Microbiologie, University of Ghent, Belgium.
Ouis
Ibn-Sina
IS
Department of Medical Microbiology, Dermatology and Infection, Lund University, Sölvegatan 23, SE-223 62 Lund, Sweden 2Department of Clinical Microbiology 7806, National University Hospital, Righospitalet, Tagensvej 20, DK-2200, Copenhagen, Denmark 3Danish Veterinary Institute, Bülowsvej 27, DK-1790, Copenhagen, Denmark 4Laboratorium voor Microbiologie, University of Ghent, Belgium.
Vandamme
Peter
P
Department of Medical Microbiology, Dermatology and Infection, Lund University, Sölvegatan 23, SE-223 62 Lund, Sweden 2Department of Clinical Microbiology 7806, National University Hospital, Righospitalet, Tagensvej 20, DK-2200, Copenhagen, Denmark 3Danish Veterinary Institute, Bülowsvej 27, DK-1790, Copenhagen, Denmark 4Laboratorium voor Microbiologie, University of Ghent, Belgium.
Nilsson
Hans-Olof
HO
Department of Medical Microbiology, Dermatology and Infection, Lund University, Sölvegatan 23, SE-223 62 Lund, Sweden 2Department of Clinical Microbiology 7806, National University Hospital, Righospitalet, Tagensvej 20, DK-2200, Copenhagen, Denmark 3Danish Veterinary Institute, Bülowsvej 27, DK-1790, Copenhagen, Denmark 4Laboratorium voor Microbiologie, University of Ghent, Belgium.
Ljungh
Åsa
Å
Department of Medical Microbiology, Dermatology and Infection, Lund University, Sölvegatan 23, SE-223 62 Lund, Sweden 2Department of Clinical Microbiology 7806, National University Hospital, Righospitalet, Tagensvej 20, DK-2200, Copenhagen, Denmark 3Danish Veterinary Institute, Bülowsvej 27, DK-1790, Copenhagen, Denmark 4Laboratorium voor Microbiologie, University of Ghent, Belgium.
Wadström
Torkel
T
Department of Medical Microbiology, Dermatology and Infection, Lund University, Sölvegatan 23, SE-223 62 Lund, Sweden 2Department of Clinical Microbiology 7806, National University Hospital, Righospitalet, Tagensvej 20, DK-2200, Copenhagen, Denmark 3Danish Veterinary Institute, Bülowsvej 27, DK-1790, Copenhagen, Denmark 4Laboratorium voor Microbiologie, University of Ghent, Belgium.
D000818:Animals; D000836:Animals, Zoo / Q000382:microbiology*; D004269:DNA, Bacterial / Q000032:analysis; D004275:DNA, Ribosomal / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005243:Feces / Q000382:microbiology; D016998:Helicobacter / Q000145:classification* / Q000235:genetics; D016481:Helicobacter Infections / Q000453:epidemiology / Q000382:microbiology / Q000662:veterinary*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D015995:Prevalence; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S