Identification of uncultured bacteria from abscesses of exotic pet animals using broad-range nested 16S rRNA polymerase chain reaction and Sanger sequencing

• Published in: Veterinary World Vol. 12; no. 10; pp. 1546 - 1553
• Main Authors: Duangurai, T, Siengsanan-Lamont, J, Bumrungpun, C, Kaewmongkol, G, Areevijittrakul, L, Sirinarumitr, T, Fenwick, S G, Kaewmongkol, S
• Format: Journal Article
• Language: English
• Published: India Veterinary World 01-10-2019
• Subjects: Abscess; Abscesses; Aerobes; Anaerobes; Anaerobic bacteria; Analysis; Animals; Bacteria; Bacteroides; DNA sequencing; exotic pet animals; Fusobacterium; Genes; Gliders; Gram-negative bacteria; Next-generation sequencing; Pets; Phylogeny; Polymerase chain reaction; RNA; RpoB protein; rRNA 16S; Sanger sequencing; Technology; Technology application; Thailand; abscesses; Sanger sequencing; anaerobic bacteria; exotic pet animals
• ISSN: 0972-8988; 2231-0916
• DOI: 10.14202/vetworld.2019.1546-1553

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.
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.
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.
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
spp.,
spp.,
spp. Phylogenetic analysis using the
gene revealed that the species for the
was
and for the
was
and
. However, the amplification of the
gene for the
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.
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.