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Advanced sample preparation for the molecular quantification of Staphylococcus aureus in artificially and naturally contaminated milk.

Aprodu, Iuliana; Walcher, Georg; Schelin, Jenny LU ; Hein, Ingeborg; Norling, Börje; Rådström, Peter LU ; Nicolau, Anca and Wagner, Martin (2011) In International Journal of Food Microbiology 145. p.61-65
Abstract
Sample treatment is an essential element when using real-time PCR for quantification of pathogens directly on food samples. This study comparatively evaluated three different principles of sample treatment, i.e. immunomagnetic separation based on phage-derived cell wall binding molecules, matrix solubilization and flotation, in order to establish their suitability for quantifying low numbers of Staphylococcus aureus in milk. All three procedures succeeded to remove S. aureus from the milk matrix, either raw or pasteurized, and, as a result of the concentration of the target cells, minimized the effect of milk associated PCR inhibitors. Sample preparation based on immunomagnetic separation albeit of being user friendly, specific and rapid,... (More)
Sample treatment is an essential element when using real-time PCR for quantification of pathogens directly on food samples. This study comparatively evaluated three different principles of sample treatment, i.e. immunomagnetic separation based on phage-derived cell wall binding molecules, matrix solubilization and flotation, in order to establish their suitability for quantifying low numbers of Staphylococcus aureus in milk. All three procedures succeeded to remove S. aureus from the milk matrix, either raw or pasteurized, and, as a result of the concentration of the target cells, minimized the effect of milk associated PCR inhibitors. Sample preparation based on immunomagnetic separation albeit of being user friendly, specific and rapid, failed to allow quantification of low and medium numbers (<10(4)CFU) of S. aureus. In a mastitic milk model cell wall binding domain (CBD)-based target cell extraction revealed results most closely matching those derived from culture-based quantification. Both matrix lysis and flotation allowed quantification of S. aureus at a level of 1-10 cells per ml. Both methods resulted in higher numbers of bacterial cell equivalents (bce) than plating could reveal. Since both methods harvest cells that have been subjected to either mechanical and chemical stresses before quantification, we concluded that the higher bce numbers resulted from a disaggregation of S. aureus clusters initially present in the inoculum. Conclusively, since likely each S. aureus cell of a toxigenic strain contributes to enterotoxin production, molecular quantification could provide an even more realistic impact assessment in outbreak investigations than plating does. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
International Journal of Food Microbiology
volume
145
pages
61 - 65
publisher
Elsevier
external identifiers
  • wos:000289819900011
  • pmid:21035221
  • scopus:79953034873
ISSN
0168-1605
DOI
10.1016/j.ijfoodmicro.2010.09.018
language
English
LU publication?
yes
id
99250c4f-177c-4d02-8ce9-2b66cb29aef2 (old id 1732556)
date added to LUP
2010-12-16 14:20:41
date last changed
2017-01-01 03:45:38
@article{99250c4f-177c-4d02-8ce9-2b66cb29aef2,
  abstract     = {Sample treatment is an essential element when using real-time PCR for quantification of pathogens directly on food samples. This study comparatively evaluated three different principles of sample treatment, i.e. immunomagnetic separation based on phage-derived cell wall binding molecules, matrix solubilization and flotation, in order to establish their suitability for quantifying low numbers of Staphylococcus aureus in milk. All three procedures succeeded to remove S. aureus from the milk matrix, either raw or pasteurized, and, as a result of the concentration of the target cells, minimized the effect of milk associated PCR inhibitors. Sample preparation based on immunomagnetic separation albeit of being user friendly, specific and rapid, failed to allow quantification of low and medium numbers (&lt;10(4)CFU) of S. aureus. In a mastitic milk model cell wall binding domain (CBD)-based target cell extraction revealed results most closely matching those derived from culture-based quantification. Both matrix lysis and flotation allowed quantification of S. aureus at a level of 1-10 cells per ml. Both methods resulted in higher numbers of bacterial cell equivalents (bce) than plating could reveal. Since both methods harvest cells that have been subjected to either mechanical and chemical stresses before quantification, we concluded that the higher bce numbers resulted from a disaggregation of S. aureus clusters initially present in the inoculum. Conclusively, since likely each S. aureus cell of a toxigenic strain contributes to enterotoxin production, molecular quantification could provide an even more realistic impact assessment in outbreak investigations than plating does.},
  author       = {Aprodu, Iuliana and Walcher, Georg and Schelin, Jenny and Hein, Ingeborg and Norling, Börje and Rådström, Peter and Nicolau, Anca and Wagner, Martin},
  issn         = {0168-1605},
  language     = {eng},
  pages        = {61--65},
  publisher    = {Elsevier},
  series       = {International Journal of Food Microbiology},
  title        = {Advanced sample preparation for the molecular quantification of Staphylococcus aureus in artificially and naturally contaminated milk.},
  url          = {http://dx.doi.org/10.1016/j.ijfoodmicro.2010.09.018},
  volume       = {145},
  year         = {2011},
}