title: Culture independent analysis using gnd as a target gene to assess Escherichia coli diversity and community structure.
abstract: Current culture methods to investigate changes in Escherichia coli community structure are often slow and laborious. Genes such as gnd (6-phosphogluconate dehydrogenase) have a highly variable nucleotide sequence and may provide a target for E. coli microbiome analysis using culture-independent methods. Metabarcoded PCR primers were used to generate separate libraries from calf faecal samples for high throughput sequencing. Although a total of 348 separate gnd sequence types (gSTs) were identified, 188 were likely to be due to sequencing errors. Of the remaining 160 gSTs, 92 did not match those in a database of 319 separate gnd sequences. 'Animal' was the main determinant of E. coli diversity with limited impact of sample type or DNA extraction method on intra-host E. coli community variation from faeces and recto-anal mucosal swab samples. This culture-independent study has addressed the difficulties of quantifying bacterial intra-species diversity and revealed that, whilst individual animals may harbour >50 separate E. coli strains, communities are dominated by <10 strains alongside a large pool of subdominant strains present at low abundances. This method will be useful for characterising the diversity and population structure of E. coli in experimental studies designed to assess the impact of interventions on the gut microbiome.
authors.affiliation: AgResearch Limited, Hopkirk Research Institute, Palmerston North, New Zealand. adrian.cookson@agresearch.co.nz.|mEpiLab, Hopkirk Research Institute, Massey University, Palmerston North, New Zealand. adrian.cookson@agresearch.co.nz.
authors.forename: Adrian L
authors.initials: AL
authors.lastname: Cookson
authors.affiliation: mEpiLab, Hopkirk Research Institute, Massey University, Palmerston North, New Zealand.|Massey Genome Service, New Zealand Genomics Limited, Massey University, Palmerston North, New Zealand.
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Biggs
authors.affiliation: mEpiLab, Hopkirk Research Institute, Massey University, Palmerston North, New Zealand.|Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand.
authors.forename: Jonathan C
authors.initials: JC
authors.lastname: Marshall
authors.affiliation: AgResearch Limited, Hopkirk Research Institute, Palmerston North, New Zealand.
authors.forename: Angela
authors.initials: A
authors.lastname: Reynolds
authors.affiliation: AgResearch Limited, Hopkirk Research Institute, Palmerston North, New Zealand.
authors.forename: Rose M
authors.initials: RM
authors.lastname: Collis
authors.affiliation: mEpiLab, Hopkirk Research Institute, Massey University, Palmerston North, New Zealand.
authors.forename: Nigel P
authors.initials: NP
authors.lastname: French
authors.affiliation: AgResearch Limited, Hopkirk Research Institute, Palmerston North, New Zealand.
authors.forename: Gale
authors.initials: G
authors.lastname: Brightwell
mesh_terms: D000818:Animals; D002417:Cattle / Q000382:microbiology; D004926:Escherichia coli / Q000235:genetics* / Q000472:pathogenicity; D029968:Escherichia coli Proteins / Q000235:genetics*; D000069196:Gastrointestinal Microbiome; D010734:Phosphogluconate Dehydrogenase / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide
chemical_list: D029968:Escherichia coli Proteins; D010734:Phosphogluconate Dehydrogenase