Identification of uncultured bacteria from abscesses of exotic pet animals using broad-range nested 16S rRNA polymerase chain reaction and Sanger sequencing
Background: The Sanger sequencing technique has been questioned and challenged by advanced high-throughput sequencing approaches. Sanger sequencing seems to be an obsolete technology. However, there are still research problems that could be answered using the Sanger sequencing technology. Fastidious...
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| Format: | Article |
| Language: | English |
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Veterinary World
2019-10-01
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| Series: | Veterinary World |
| Subjects: | |
| Online Access: | http://www.veterinaryworld.org/Vol.12/October-2019/6.pdf |
| _version_ | 1831551978404052992 |
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| author | T. Duangurai J. Siengsanan-Lamont C. Bumrungpun G. Kaewmongkol L. Areevijittrakul T. Sirinarumitr S. G. Fenwick S. Kaewmongkol |
| author_facet | T. Duangurai J. Siengsanan-Lamont C. Bumrungpun G. Kaewmongkol L. Areevijittrakul T. Sirinarumitr S. G. Fenwick S. Kaewmongkol |
| author_sort | T. Duangurai |
| collection | DOAJ |
| description | Background: The Sanger sequencing technique has been questioned and challenged by advanced high-throughput sequencing approaches. Sanger sequencing seems to be an obsolete technology. However, there are still research problems that could be answered using the Sanger sequencing technology. Fastidious obligate anaerobic bacteria are mostly associated with abscesses in animals. These bacteria are difficult to isolate from abscesses and are frequently excluded due to the bias of conventional bacterial culturing.
Aim: This study demonstrated the usefulness of a broad-range polymerase chain reaction (PCR) with Sanger sequencing to identify the majority population of bacteria in abscesses from exotic pet animals.
Materials and Methods: This study performed a pilot investigation of abscesses from 20 clinical cases (17 rabbits, 2 hedgehogs, and 1 sugar glider) using standard culture methods for both aerobes and anaerobes and broad-range nested PCR targeting the 16S rRNA gene followed by the Sanger sequencing technique.
Results: The standard culture and PCR techniques detected bacteria in 9 and 17 of 20 samples, respectively. From the 17 sequencings of the 16S rRNA, 10 PCR products were found to be closely related with obligate anaerobes including Bacteroides spp., Fusobacterium spp., Prevotella spp. Phylogenetic analysis using the rpoB gene revealed that the species for the Bacteroides was thetaiotaomicron and for the Fusobacterium was varium and nucleatum. However, the amplification of the rpoB gene for the Prevotella spp. was unsuccessful. Correlations between the standard culture and PCR techniques were found in 9 (6 positive and 3 negative samples) of 20 samples. Eleven samples were discordant between the standard culture and PCR techniques which were composed of eight samples negative by culture but positive by PCR and three samples had different bacteria by the culture and PCR techniques.
Conclusion: According to this study, broad-range PCR combined with Sanger sequencing might be useful for the detection of dominant anaerobic bacteria in abscesses that were overlooked based on conventional bacterial culture. |
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| id | doaj.art-c6ec502d2b4948dbaf4a2fd2511ea26e |
| institution | Directory Open Access Journal |
| issn | 0972-8988 2231-0916 |
| language | English |
| last_indexed | 2024-12-17T03:19:31Z |
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| spelling | doaj.art-c6ec502d2b4948dbaf4a2fd2511ea26e2022-12-21T22:05:34ZengVeterinary WorldVeterinary World0972-89882231-09162019-10-0112101546155310.14202/vetworld.2019.1546-1553Identification of uncultured bacteria from abscesses of exotic pet animals using broad-range nested 16S rRNA polymerase chain reaction and Sanger sequencingT. Duangurai0J. Siengsanan-Lamont1C. Bumrungpun2G. Kaewmongkol3L. Areevijittrakul4T. Sirinarumitr5S. G. Fenwick6S. Kaewmongkol7Department of Companion Animal Clinical Sciences Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand.Private Consultant, 6 Gundaring Turn, Caning Vale, Western Australia 6155.Department of Companion Animal Clinical Sciences Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand.Department of Companion Animal Clinical Sciences Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand.Exotic Pet Clinic, Faculty of Veterinary Medicine, Veterinary Teaching Hospital, Kasetsart University, Bangkok, Thailand.Department of Pathology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand.Department of Infectious Diseases and Global Health, Cummings School of Veterinary Medicine, Tufts University, Boston, MA, USA.Department of Veterinary Technology, Faculty of Veterinary Technology, Kasetsart University, Bangkok, Thailand.Background: The Sanger sequencing technique has been questioned and challenged by advanced high-throughput sequencing approaches. Sanger sequencing seems to be an obsolete technology. However, there are still research problems that could be answered using the Sanger sequencing technology. Fastidious obligate anaerobic bacteria are mostly associated with abscesses in animals. These bacteria are difficult to isolate from abscesses and are frequently excluded due to the bias of conventional bacterial culturing. Aim: This study demonstrated the usefulness of a broad-range polymerase chain reaction (PCR) with Sanger sequencing to identify the majority population of bacteria in abscesses from exotic pet animals. Materials and Methods: This study performed a pilot investigation of abscesses from 20 clinical cases (17 rabbits, 2 hedgehogs, and 1 sugar glider) using standard culture methods for both aerobes and anaerobes and broad-range nested PCR targeting the 16S rRNA gene followed by the Sanger sequencing technique. Results: The standard culture and PCR techniques detected bacteria in 9 and 17 of 20 samples, respectively. From the 17 sequencings of the 16S rRNA, 10 PCR products were found to be closely related with obligate anaerobes including Bacteroides spp., Fusobacterium spp., Prevotella spp. Phylogenetic analysis using the rpoB gene revealed that the species for the Bacteroides was thetaiotaomicron and for the Fusobacterium was varium and nucleatum. However, the amplification of the rpoB gene for the Prevotella spp. was unsuccessful. Correlations between the standard culture and PCR techniques were found in 9 (6 positive and 3 negative samples) of 20 samples. Eleven samples were discordant between the standard culture and PCR techniques which were composed of eight samples negative by culture but positive by PCR and three samples had different bacteria by the culture and PCR techniques. Conclusion: According to this study, broad-range PCR combined with Sanger sequencing might be useful for the detection of dominant anaerobic bacteria in abscesses that were overlooked based on conventional bacterial culture.http://www.veterinaryworld.org/Vol.12/October-2019/6.pdfanaerobic bacteriaabscessesexotic pet animalsSanger sequencing |
| spellingShingle | T. Duangurai J. Siengsanan-Lamont C. Bumrungpun G. Kaewmongkol L. Areevijittrakul T. Sirinarumitr S. G. Fenwick S. Kaewmongkol Identification of uncultured bacteria from abscesses of exotic pet animals using broad-range nested 16S rRNA polymerase chain reaction and Sanger sequencing Veterinary World anaerobic bacteria abscesses exotic pet animals Sanger sequencing |
| title | Identification of uncultured bacteria from abscesses of exotic pet animals using broad-range nested 16S rRNA polymerase chain reaction and Sanger sequencing |
| title_full | Identification of uncultured bacteria from abscesses of exotic pet animals using broad-range nested 16S rRNA polymerase chain reaction and Sanger sequencing |
| title_fullStr | Identification of uncultured bacteria from abscesses of exotic pet animals using broad-range nested 16S rRNA polymerase chain reaction and Sanger sequencing |
| title_full_unstemmed | Identification of uncultured bacteria from abscesses of exotic pet animals using broad-range nested 16S rRNA polymerase chain reaction and Sanger sequencing |
| title_short | Identification of uncultured bacteria from abscesses of exotic pet animals using broad-range nested 16S rRNA polymerase chain reaction and Sanger sequencing |
| title_sort | identification of uncultured bacteria from abscesses of exotic pet animals using broad range nested 16s rrna polymerase chain reaction and sanger sequencing |
| topic | anaerobic bacteria abscesses exotic pet animals Sanger sequencing |
| url | http://www.veterinaryworld.org/Vol.12/October-2019/6.pdf |
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