INVESTIGADORES
RAJAL Veronica Beatriz
artículos
Título:
Evaluation of detachment methods for the enumeration of Bacteroides fragilis in sediments via propidium monoazide-quantitative PCR in comparison to Enterococcus faecalis and Escherichia coli
Autor/es:
MINJI KIM; DOLORES GUTIÉRREZ CACCIABUE; ALEXANDER SCHRIEWER; VERÓNICA B. RAJAL; STEFAN WUERTZ
Revista:
JOURNAL OF APPLIED MICROBIOLOGY
Editorial:
WILEY-BLACKWELL PUBLISHING, INC
Referencias:
Lugar: Londres; Año: 2014 vol. 117 p. 1513 - 1522
ISSN:
1364-5072
Resumen:
Aims: The aim was to develop an optimized detachment method for
separating Bacteroidales from sediments to allow enumeration via PMA-qPCR.
The effectiveness of four different detachment treatments in removing
Bacteroides fragilis was compared as a function of time as well as in relation to
Enterococcus faecalis and Escherichia coli as detected by cultivation and qPCR.
Methods and Results: Cells were inoculated into four sediments from sea water
(SW) and freshwater (FW) beaches. Sediment samples were taken on days 1 and 7
and subjected to four different treatments for separation of micro-organisms. On
day 1, the detachment treatments performed equally well in removing intact
Bact. fragilis cells. In contrast, 7 days later the detachment treatment with Tween 80
and handshaking (TH) resulted in up to eightfold higher 16S rRNA gene
concentrations of intact and total Bact. fragilis cells compared to other detachment
treatments. Total Ent. faecalis cells based on the 23S rRNA gene were also
preferentially recovered by treatment TH. Cultivable Ent. faecalis or E. coli numbers
detached from sediments were similar for all methods in most sediments tested.
Conclusions: Handshaking and 1% Tween 80/NaOH (pH 70) eluant was the
most efficient technique to recover intact as well as total Bact. fragilis cells in
sediment samples with different salinities and after prolonged sediment cell
contact time.
Significance and Impact of the Study: The optimized detachment method
enables the application of PMA-qPCR to sediment samples to detect the presence
of Bacteroidales cells and their DNA in future microbial source tracking studies.