Role of Daptomycin in the Induction and Persistence of the Viable but Non-Culturable State of Staphylococcus Aureus Biofilms
Abstract
:1. Introduction
2. Results and Discussion
2.1. Biofilm Production, Stress Exposure and Non-Culturability
2.2. Persistence of the VBNC State
Stress Condition | Days Since Achievement of Non-Culturability | Cells/mL | |
---|---|---|---|
Total | Viable | ||
Nutrient depletion | 0 | 1.6×107 | 3.8 × 104 |
7 | 1.9 × 107 | 1.7 × 104 | |
15 | 1.7 × 107/1.5 × 108 * | 1.6 × 104/8.2 × 104 * | |
60 | 2.0 × 107 | 4.4 × 104 | |
90 | 2.3 × 107 | 2.6 × 103 | |
150 | 2.0 × 107 | <60 | |
Nutrient depletion+ daptomycin | 0 | 1.4 × 107 | 2.7 × 104 |
7 | 1.1 × 107 | 3.9 × 104 | |
15 | 1.6 × 107/1.4 × 108 * | 1.2 × 104/7.6 × 104 * | |
60 | 1.4 × 107 | 2.7 × 104 | |
90 | 2.1 × 107 | 5.3 × 103 | |
150 | 1.8 × 107 | 9.8 × 103 |
2.3. Gene Expression of VBNC Cells
Stress Condition | Time from non Culturability | Gene Analysis | |||
---|---|---|---|---|---|
16SrDNA | glt | nuc | mecA | ||
Nutrient depletion | T0 | + | ND | ND | ND |
T7 | + | - | - | + | |
T15 | + | - | - | + | |
T60 | + | - | - | + | |
Nutrient depletion + daptomycin | T0 | + | ND | ND | ND |
T7 | + | + | - | + | |
T15 | + | + | - | + | |
T60 | + | - | - | + |
3. Experimental Section
3.1. Bacterial Strains, Media, Antibiotics and Enzymes
3.2. MIC Determination
3.3. Biofilm Production, Stress Exposure and Culturability Assays
3.4. Epifluorescence Microscopy and Flow Cytometry
3.5. Real-Time RT–PCR Assays
Target Gene | Gene Function | Primer Pair (5ʹ-3ʹ) | Annealing Temperature (°C) | Product Size (bp) | Reference |
---|---|---|---|---|---|
16S rDNA | Housekeeping | F-TGGAGCATGTGGTTTAATTCGA R-TGCGGGACTTAACCCAACA | 60 | 159 | [32] |
glt | Species specific, coding for glutamate synthase | F-AATCTTTGTCGGTACACGATATTCTTCACG R-CGTAATGAGATTTCAGTAGATAATACAACA | 58 | 108 | [33] |
nuc | Virulence factor, coding for thermonuclease | F-GACTATTATTGGTTGATCCACCTG R- GCCTTGACGAACTAAAGCTTCG | 60 | 218 | [34] |
mecA | Methicillin resistance | F-TCCAGATTACAACTTCACCAGG R-CCACTTCATATCTTGTAACG | 57 | 162 | [35] |
4. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Pasquaroli, S.; Citterio, B.; Cesare, A.D.; Amiri, M.; Manti, A.; Vuotto, C.; Biavasco, F. Role of Daptomycin in the Induction and Persistence of the Viable but Non-Culturable State of Staphylococcus Aureus Biofilms. Pathogens 2014, 3, 759-768. https://doi.org/10.3390/pathogens3030759
Pasquaroli S, Citterio B, Cesare AD, Amiri M, Manti A, Vuotto C, Biavasco F. Role of Daptomycin in the Induction and Persistence of the Viable but Non-Culturable State of Staphylococcus Aureus Biofilms. Pathogens. 2014; 3(3):759-768. https://doi.org/10.3390/pathogens3030759
Chicago/Turabian StylePasquaroli, Sonia, Barbara Citterio, Andrea Di Cesare, Mehdi Amiri, Anita Manti, Claudia Vuotto, and Francesca Biavasco. 2014. "Role of Daptomycin in the Induction and Persistence of the Viable but Non-Culturable State of Staphylococcus Aureus Biofilms" Pathogens 3, no. 3: 759-768. https://doi.org/10.3390/pathogens3030759