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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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Resuscitation of dormant S. aureus SH1000 following addition of spent medium supernatant.Sterile filtered spent medium supernatant was added to fresh minimal medium inoculated with cells from 14 day old dormant cultures in MPN tests, and read at 24%, 25% and 10% concentrations of spent medium supernatant, and 25% boiled or trypsin-digested supernatant are shown. Aliquots of 100 µl of bacterial cultures from the dormant model were used to inoculate 900 µl fresh minimal medium supplemented with spent medium supernatant. The ratio of spent medium supernatant to fresh minimal medium was 100 µl: 800 µl for experiments using 10% spent medium supernatant, 250 µl: 650 µl for experiments using 25% spent medium supernatant and 500 µl: 400 µl for experiments using 50% spent medium supernatant. Trypsin-digested supernatant was prepared by digesting with 50 µg/ml trypsin from bovine pancreas for 30 minutes at 37°C; digestion was stopped by addition of 100 µg/ml type I-S trypsin inhibitor from soybean. Controls used 25% unboiled/undigested supernatant. Averages from three experiments and standard error bars shown in each case.
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pone-0085998-g001: Resuscitation of dormant S. aureus SH1000 following addition of spent medium supernatant.Sterile filtered spent medium supernatant was added to fresh minimal medium inoculated with cells from 14 day old dormant cultures in MPN tests, and read at 24%, 25% and 10% concentrations of spent medium supernatant, and 25% boiled or trypsin-digested supernatant are shown. Aliquots of 100 µl of bacterial cultures from the dormant model were used to inoculate 900 µl fresh minimal medium supplemented with spent medium supernatant. The ratio of spent medium supernatant to fresh minimal medium was 100 µl: 800 µl for experiments using 10% spent medium supernatant, 250 µl: 650 µl for experiments using 25% spent medium supernatant and 500 µl: 400 µl for experiments using 50% spent medium supernatant. Trypsin-digested supernatant was prepared by digesting with 50 µg/ml trypsin from bovine pancreas for 30 minutes at 37°C; digestion was stopped by addition of 100 µg/ml type I-S trypsin inhibitor from soybean. Controls used 25% unboiled/undigested supernatant. Averages from three experiments and standard error bars shown in each case.

Mentions: Resuscitation was observed when supplementing fresh defined media with 10–50% spent medium, with the peak effect using spent medium from cultures grown to an optical density of OD600 between 0.8–1.2. On average using 50 or 25% spent medium an over six-hundred-fold increase in bacterial growth at 24 hours was observed as measured by MPN method (range 200–900-fold), a statistically significant difference (Fig. 1) compared to the negative controls, demonstrating also that dormant cells, which could be cultured only after resuscitation, were present, and therefore that the number of viable bacteria was greater than the number culturable by standard methods. Whilst the difference between the effect with 50% and 25% supernatant was not significant, reducing the proportion of supernatant from 25% to 10% reduced the resuscitation effect, which disappeared completely when less than 10% spent medium supernatant was added. In the control in which spent medium supernatant and fresh minimal medium were incubated without inoculating with dormant cells, no growth was seen (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)

Resuscitation of dormant S. aureus SH1000 following addition of spent medium supernatant.Sterile filtered spent medium supernatant was added to fresh minimal medium inoculated with cells from 14 day old dormant cultures in MPN tests, and read at 24%, 25% and 10% concentrations of spent medium supernatant, and 25% boiled or trypsin-digested supernatant are shown. Aliquots of 100 µl of bacterial cultures from the dormant model were used to inoculate 900 µl fresh minimal medium supplemented with spent medium supernatant. The ratio of spent medium supernatant to fresh minimal medium was 100 µl: 800 µl for experiments using 10% spent medium supernatant, 250 µl: 650 µl for experiments using 25% spent medium supernatant and 500 µl: 400 µl for experiments using 50% spent medium supernatant. Trypsin-digested supernatant was prepared by digesting with 50 µg/ml trypsin from bovine pancreas for 30 minutes at 37°C; digestion was stopped by addition of 100 µg/ml type I-S trypsin inhibitor from soybean. Controls used 25% unboiled/undigested supernatant. Averages from three experiments and standard error bars shown in each case.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0085998-g001: Resuscitation of dormant S. aureus SH1000 following addition of spent medium supernatant.Sterile filtered spent medium supernatant was added to fresh minimal medium inoculated with cells from 14 day old dormant cultures in MPN tests, and read at 24%, 25% and 10% concentrations of spent medium supernatant, and 25% boiled or trypsin-digested supernatant are shown. Aliquots of 100 µl of bacterial cultures from the dormant model were used to inoculate 900 µl fresh minimal medium supplemented with spent medium supernatant. The ratio of spent medium supernatant to fresh minimal medium was 100 µl: 800 µl for experiments using 10% spent medium supernatant, 250 µl: 650 µl for experiments using 25% spent medium supernatant and 500 µl: 400 µl for experiments using 50% spent medium supernatant. Trypsin-digested supernatant was prepared by digesting with 50 µg/ml trypsin from bovine pancreas for 30 minutes at 37°C; digestion was stopped by addition of 100 µg/ml type I-S trypsin inhibitor from soybean. Controls used 25% unboiled/undigested supernatant. Averages from three experiments and standard error bars shown in each case.
Mentions: Resuscitation was observed when supplementing fresh defined media with 10–50% spent medium, with the peak effect using spent medium from cultures grown to an optical density of OD600 between 0.8–1.2. On average using 50 or 25% spent medium an over six-hundred-fold increase in bacterial growth at 24 hours was observed as measured by MPN method (range 200–900-fold), a statistically significant difference (Fig. 1) compared to the negative controls, demonstrating also that dormant cells, which could be cultured only after resuscitation, were present, and therefore that the number of viable bacteria was greater than the number culturable by standard methods. Whilst the difference between the effect with 50% and 25% supernatant was not significant, reducing the proportion of supernatant from 25% to 10% reduced the resuscitation effect, which disappeared completely when less than 10% spent medium supernatant was added. In the control in which spent medium supernatant and fresh minimal medium were incubated without inoculating with dormant cells, no growth was seen (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