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Diversity profiling of xenic cultures of Dientamoeba fragilis following systematic antibiotic treatment and prospects for genome sequencing

Published online by Cambridge University Press:  23 September 2019

Rory Gough Open the ORCID record for Rory Gough [Opens in a new window] ,
Joel Barratt Open the ORCID record for Joel Barratt [Opens in a new window] ,
Damien Stark  and
John Ellis
Rory Gough*
Affiliation:
The University of Technology Sydney, Australia St Vincent's Hospital, Sydney, Australia
Joel Barratt
Affiliation:
The University of Technology Sydney, Australia
Damien Stark
Affiliation:
The University of Technology Sydney, Australia St Vincent's Hospital, Sydney, Australia
John Ellis
Affiliation:
The University of Technology Sydney, Australia
*
Author for correspondence: Rory Gough, E-mail: [email protected]
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Abstract

The presence of bacterial DNA in Dientamoeba fragilis DNA extracts from culture poses a substantial challenge to sequencing the D. fragilis genome. However, elimination of bacteria from D. fragilis cultures has proven difficult in the past, presumably due to its dependence on some unknown prokaryote/s. This study explored options for removal of bacteria from D. fragilis cultures and for the generation of genome sequence data from D. fragilis. DNA was extracted from human faecal samples and xenic D. fragilis cultures. Extracts were subjected to 16S ribosomal DNA bacterial diversity profiling. Xenic D. fragilis cultures were then subject to antibiotic treatment regimens that systematically removed bacterial species depending on their membrane structure (Gram-positive or Gram-negative) and aerobic requirements. The impact of these treatments on cultures was assessed by 16S amplicon sequencing. Prior to antibiotic treatment, the cultures were dominated by Gram-negative bacteria. Addition of meropenem to cultures eliminated anaerobic Gram-negative bacteria, but it also led to protozoan death after 5 days incubation. The seeding of meropenem resistant Klebsiella pneumoniae strain KPC-2 into cultures before treatment by meropenem prevented death of D. fragilis cells beyond this 5 day period, suggesting that one or more species of Gram-negative bacteria may be an essential nutritional requirement for D. fragilis. Gram-positive cells were completely eliminated using vancomycin without affecting trophozoite growth. Finally, this study shows that genome sequencing of D. fragilis is feasible following bacterial elimination from cultures as the result of the major advances occurring in bioinformatics. We provide evidence on this fact by successfully sequencing the D. fragilis 28S large ribosomal DNA subunit gene using culture-derived DNA.

Keywords

Dientamoeba fraiglisMicrobial diversityMetagenomics
Type
Research Article
Information
Parasitology , Volume 147 , Issue 1 , January 2020 , pp. 29 - 38
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Copyright
Copyright © Cambridge University Press 2019

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