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Dormant cells of Staphylococcus aureus are resuscitated by spent culture supernatant.

Pascoe B, Dams L, Wilkinson TS, Harris LG, Bodger O, Mack D, Davies AP - PLoS ONE (2014)

Bottom Line: Supplementing cultures with 25-50% spent medium resulted in a >600-fold increase in bacterial growth.Supernatant also effected a reduction in lag phase of dormant cultures.SEM demonstrated the presence of small coccoid cells in dormant cultures.

View Article: PubMed Central - PubMed

Affiliation: Institute of Life Science, Swansea University College of Medicine, Swansea, United Kingdom.

ABSTRACT
We describe the first in vitro model of dormancy in Staphylococcus aureus, showing that cells are generated which can be resuscitated by addition of spent medium supernatant taken from cultures of the same organism. Over 30 days, culturable counts in dormant cultures of S. aureus SH1000 fell from 10(6)-10(7) cfu/ml to <10 cfu/ml as measured by the Most Probable Number method in liquid culture, while total counts as determined by microscopy, and supported by data from RT-qPCR, remained around 10(6)-10(7) cells/ml. Supplementing cultures with 25-50% spent medium resulted in a >600-fold increase in bacterial growth. Resuscitation was a specific effect, greatly reduced by boiling or addition of trypsin to the spent supernatant. Supernatant also effected a reduction in lag phase of dormant cultures. SEM demonstrated the presence of small coccoid cells in dormant cultures. The results are similar to those seen with resuscitation promoting factors (Rpfs) in actinobacteria. This is the first time resuscitation has been demonstrated in Staphylococcus aureus, which is an important human pathogen. A better understanding of control and reactivation of dormant cells could lead to major improvements in managing staphylococcal infections; resuscitation could be an important step in restoring susceptibility to antibiotic treatment.

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Bacterial counts in resuscitated cultures.Dotted lines: total count by microscopy (▴); genomic DNA content as determined by qPCR using 16S rRNA (□), NucA (*) and Sa442 (○) primer sets. Solid lines: total culturable counts, in liquid culture by MPN tests (▪) and on solid agar (•). Bars represent the estimated viable count in MPN tests after resuscitation –a figure obtained by multiplying the counts in MPN tests without resuscitation by the fold change in growth following resuscitation with 25% spent medium (averages from three experiments with standard error bars).
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pone-0085998-g003: Bacterial counts in resuscitated cultures.Dotted lines: total count by microscopy (▴); genomic DNA content as determined by qPCR using 16S rRNA (□), NucA (*) and Sa442 (○) primer sets. Solid lines: total culturable counts, in liquid culture by MPN tests (▪) and on solid agar (•). Bars represent the estimated viable count in MPN tests after resuscitation –a figure obtained by multiplying the counts in MPN tests without resuscitation by the fold change in growth following resuscitation with 25% spent medium (averages from three experiments with standard error bars).

Mentions: It was observed that only cultures of a particular age could be resuscitated. By taking aliquots and testing cultures throughout their incubation period it was demonstrated that cultures aged around 14 days were most responsive to resuscitation (Fig. 2). At 14 days, there were c.105 viable cells; c.107 genome equivalents were detected, meaning that c. 1% of the genome equivalents were viable. 99–99.9% of the cells could be resuscitated but were not culturable (Fig. 3). Continuing culture up to 70 days showed that cultures displayed no further resuscitation up to that time-point (data not shown).


Dormant cells of Staphylococcus aureus are resuscitated by spent culture supernatant.

Pascoe B, Dams L, Wilkinson TS, Harris LG, Bodger O, Mack D, Davies AP - PLoS ONE (2014)

Bacterial counts in resuscitated cultures.Dotted lines: total count by microscopy (▴); genomic DNA content as determined by qPCR using 16S rRNA (□), NucA (*) and Sa442 (○) primer sets. Solid lines: total culturable counts, in liquid culture by MPN tests (▪) and on solid agar (•). Bars represent the estimated viable count in MPN tests after resuscitation –a figure obtained by multiplying the counts in MPN tests without resuscitation by the fold change in growth following resuscitation with 25% spent medium (averages from three experiments with standard error bars).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3921112&req=5

pone-0085998-g003: Bacterial counts in resuscitated cultures.Dotted lines: total count by microscopy (▴); genomic DNA content as determined by qPCR using 16S rRNA (□), NucA (*) and Sa442 (○) primer sets. Solid lines: total culturable counts, in liquid culture by MPN tests (▪) and on solid agar (•). Bars represent the estimated viable count in MPN tests after resuscitation –a figure obtained by multiplying the counts in MPN tests without resuscitation by the fold change in growth following resuscitation with 25% spent medium (averages from three experiments with standard error bars).
Mentions: It was observed that only cultures of a particular age could be resuscitated. By taking aliquots and testing cultures throughout their incubation period it was demonstrated that cultures aged around 14 days were most responsive to resuscitation (Fig. 2). At 14 days, there were c.105 viable cells; c.107 genome equivalents were detected, meaning that c. 1% of the genome equivalents were viable. 99–99.9% of the cells could be resuscitated but were not culturable (Fig. 3). Continuing culture up to 70 days showed that cultures displayed no further resuscitation up to that time-point (data not shown).

Bottom Line: Supplementing cultures with 25-50% spent medium resulted in a >600-fold increase in bacterial growth.Supernatant also effected a reduction in lag phase of dormant cultures.SEM demonstrated the presence of small coccoid cells in dormant cultures.

View Article: PubMed Central - PubMed

Affiliation: Institute of Life Science, Swansea University College of Medicine, Swansea, United Kingdom.

ABSTRACT
We describe the first in vitro model of dormancy in Staphylococcus aureus, showing that cells are generated which can be resuscitated by addition of spent medium supernatant taken from cultures of the same organism. Over 30 days, culturable counts in dormant cultures of S. aureus SH1000 fell from 10(6)-10(7) cfu/ml to <10 cfu/ml as measured by the Most Probable Number method in liquid culture, while total counts as determined by microscopy, and supported by data from RT-qPCR, remained around 10(6)-10(7) cells/ml. Supplementing cultures with 25-50% spent medium resulted in a >600-fold increase in bacterial growth. Resuscitation was a specific effect, greatly reduced by boiling or addition of trypsin to the spent supernatant. Supernatant also effected a reduction in lag phase of dormant cultures. SEM demonstrated the presence of small coccoid cells in dormant cultures. The results are similar to those seen with resuscitation promoting factors (Rpfs) in actinobacteria. This is the first time resuscitation has been demonstrated in Staphylococcus aureus, which is an important human pathogen. A better understanding of control and reactivation of dormant cells could lead to major improvements in managing staphylococcal infections; resuscitation could be an important step in restoring susceptibility to antibiotic treatment.

Show MeSH
Related in: MedlinePlus