Antonie van Leeuwenhoek

, Volume 108, Issue 4, pp 907–917 | Cite as

Effect of negative pressure on growth, secretion and biofilm formation of Staphylococcus aureus

  • Tongtong Li
  • Guoqi Wang
  • Peng Yin
  • Zhirui Li
  • Licheng Zhang
  • Jianheng Liu
  • Ming Li
  • Lihai ZhangEmail author
  • Li HanEmail author
  • Peifu TangEmail author
Original Paper


Negative pressure wound therapy (NPWT) has gained popularity in the management of contaminated wounds as an effective physical therapy, although its influence on the bacteria in the wounds remains unclear. In this study, we attempted to explore the effect of negative pressure conditions on Staphylococcus aureus, the most frequently isolated pathogen during wound infection. S. aureus was cultured in Luria–Bertani medium at subatmospheric pressure of −125 mmHg for 24 h, with the bacteria grown at ambient pressure as the control. The application of negative pressure was found to slow down the growth rate and inhibit biofilm development of S. aureus, which was confirmed by static biofilm assays. Furthermore, decreases in the total amount of virulence factors and biofilm components were observed, including α-hemolysin, extracellular adherence protein, polysaccharide intercellular adhesin and extracellular DNA. With quantitative RT-PCR analysis, we also revealed a significant inhibition in the transcription of virulence and regulatory genes related to wound infections and bacterial biofilms. Together, these findings indicated that negative pressure could inhibit the growth, virulence and biofilm formation of S. aureus. A topical subatmospheric pressure condition, such as NPWT, may be a potential antivirulence and antibiofilm strategy in the field of wound care.


Negative pressure Staphylococcus aureus Virulence Biofilm formation 



This research was supported by the National Natural Science Foundation of China (No. 81472112). The authors declare that they have no conflicts of interest.

Supplementary material

10482_2015_545_MOESM1_ESM.tif (2.6 mb)
Fig. S1 Colonial morphology and growth curves of S. aureus in response to negative pressure treatment. Negative pressure conditions resulted in smaller colony sizes and decreased pigmentation compared to the controls. (a) ambient pressure group; (b) negative pressure group; (c) measurement of colony diameter. *p < 0.001. (d) Growth curves of S. aureus by measuring OD600 values. When exposed to negative pressure, the time needed to reach post-exponential phase was 2 h longer than the control group. scale bar in (a) and (b) = 5 mm (TIFF 2698 kb)


  1. Assadian O, Assadian A, Stadler M, Diab-Elschahawi M, Kramer A (2010) Bacterial growth kinetic without the influence of the immune system using vacuum-assisted closure dressing with and without negative pressure in an in vitro wound model. Int Wound J 7:283–289. doi: 10.1111/j.1742-481X.2010.00686.x CrossRefPubMedGoogle Scholar
  2. Athanasopoulos AN et al (2006) The extracellular adherence protein (Eap) of Staphylococcus aureus inhibits wound heating by interfering with host defense and repair mechanisms. Blood 107:2720–2727. doi: 10.1182/blood-2005-08-3140 PubMedCentralCrossRefPubMedGoogle Scholar
  3. Bernardo K et al (2004) Subinhibitory concentrations of linezolid reduce Staphylococcus aureus virulence factor expression. Antimicrob Agents Chemother 48:546–555PubMedCentralCrossRefPubMedGoogle Scholar
  4. Boles BR, Horswill AR (2011) Staphylococcal biofilm disassembly. Trends Microbiol 19:449–455. doi: 10.1016/j.tim.2011.06.004 PubMedCentralCrossRefPubMedGoogle Scholar
  5. Bradley BH, Cunningham M (2013) Biofilms in chronic wounds and the potential role of negative pressure wound therapy: an integrative review. J Wound, Ostomy, Cont Nurs 40:143–149. doi: 10.1097/WON.0b013e31827e8481 CrossRefGoogle Scholar
  6. Castro SL, Nelman-Gonzalez M, Nickerson CA, Ott CM (2011) Induction of attachment-independent biofilm formation and repression of Hfq expression by low-fluid-shear culture of Staphylococcus aureus. Appl Environ Microbiol 77:6368–6378. doi: 10.1128/aem.00175-11 PubMedCentralCrossRefPubMedGoogle Scholar
  7. Cazander G, van Veen KE, Bouwman LH, Bernards AT, Jukema GN (2009) The influence of maggot excretions on PAO1 biofilm formation on different biomaterials. Clin Orthop Relat Res 467:536–545. doi: 10.1007/s11999-008-0555-2 PubMedCentralCrossRefPubMedGoogle Scholar
  8. Cazander G, van de Veerdonk MC, Vandenbroucke-Grauls CM, Schreurs MW, Jukema GN (2010) Maggot excretions inhibit biofilm formation on biomaterials. Clin Orthop Relat Res 468:2789–2796. doi: 10.1007/s11999-010-1309-5 PubMedCentralCrossRefPubMedGoogle Scholar
  9. Cheng HT, Hsu YC, Wu CI (2013) Risk of infection with delayed wound coverage by using negative-pressure wound therapy in Gustilo Grade IIIB/IIIC open tibial fracture: an evidence-based review. J Plast, Reconstr Aesthet Surg 66:876–878. doi: 10.1016/j.bjps.2012.11.030 CrossRefGoogle Scholar
  10. Costello JP et al (2014) Negative pressure wound therapy for sternal wound infections following congenital heart surgery. J Wound Care 23:31–36. doi: 10.12968/jowc.2014.23.1.31 CrossRefPubMedGoogle Scholar
  11. Diaz L et al (2015) Changes in lipopolysaccharide profile of Porphyromonas gingivalis clinical isolates correlate with changes in colony morphology and polymyxin B resistance. Anaerobe 33:25–32. doi: 10.1016/j.anaerobe.2015.01.009 CrossRefPubMedGoogle Scholar
  12. Gui Z, Wang H, Ding T, Zhu W, Zhuang X, Chu W (2014) Azithromycin reduces the production of alpha-hemolysin and biofilm formation in Staphylococcus aureus. Indian J Microbiol 54:114–117. doi: 10.1007/s12088-013-0438-4 PubMedCentralCrossRefPubMedGoogle Scholar
  13. Gurjala AN et al (2011) Development of a novel, highly quantitative in vivo model for the study of biofilm-impaired cutaneous wound healing. Wound Repair Regen 19:400–410. doi: 10.1111/j.1524-475X.2011.00690.x CrossRefPubMedGoogle Scholar
  14. Harrison-Balestra C, Cazzaniga AL, Davis SC, Mertz PM (2003) A wound-isolated Pseudomonas aeruginosa grows a biofilm in vitro within 10 hours and is visualized by light microscopy. Dermatol Surg 29:631–635PubMedGoogle Scholar
  15. Holmes CJ, Plichta JK, Gamelli RL, Radek KA (2015) Dynamic role of host stress responses in modulating the cutaneous microbiome: implications for wound healing and infection. Adv Wound Care 4:24–37. doi: 10.1089/wound.2014.0546 CrossRefGoogle Scholar
  16. Hsu CC, Tsai WC, Chen CP, Lu YM, Wang JS (2010) Effects of negative pressures on epithelial tight junctions and migration in wound healing. Am J Physiol Cell Physiol 299:C528–C534. doi: 10.1152/ajpcell.00504.2009 CrossRefPubMedGoogle Scholar
  17. Hsu CC, Chow SE, Chen CP, Tsai WC, Wang JS, Yu SY, Lee SC (2013) Negative pressure accelerated monolayer keratinocyte healing involves Cdc42 mediated cell podia formation. J Dermatol Sci 70:196–203. doi: 10.1016/j.jdermsci.2013.03.007 CrossRefPubMedGoogle Scholar
  18. Huang C, Leavitt T, Bayer LR, Orgill DP (2014) Effect of negative pressure wound therapy on wound healing. Curr Probl Surg 51:301–331. doi: 10.1067/j.cpsurg.2014.04.001 CrossRefPubMedGoogle Scholar
  19. Islam N, Kim Y, Ross JM, Marten MR (2014) Proteomic analysis of Staphylococcus aureus biofilm cells grown under physiologically relevant fluid shear stress conditions. Proteome Sci 12:21. doi: 10.1186/1477-5956-12-21 PubMedCentralCrossRefPubMedGoogle Scholar
  20. Kairinos N, Solomons M, Hudson DA (2009) Negative-pressure wound therapy I: the paradox of negative-pressure wound therapy. Plast Reconstr Surg 123:589–598; discussion 599–600. doi: 10.1097/PRS.0b013e3181956551
  21. Kobayashi SD et al (2011) Comparative analysis of USA300 virulence determinants in a rabbit model of skin and soft tissue infection. J Infect Dis 204:937–941. doi: 10.1093/infdis/jir441 PubMedCentralCrossRefPubMedGoogle Scholar
  22. Krishna S, Miller LS (2012) Host-pathogen interactions between the skin and Staphylococcus aureus. Curr Opin Microbiol 15:28–35. doi: 10.1016/j.mib.2011.11.003 PubMedCentralCrossRefPubMedGoogle Scholar
  23. Lee K, Lee JH, Ryu SY, Cho MH, Lee J (2014) Stilbenes reduce Staphylococcus aureus hemolysis, biofilm formation, and virulence. Foodborne Pathog Dis 11:710–717. doi: 10.1089/fpd.2014.1758 CrossRefPubMedGoogle Scholar
  24. Li G, Qiao M, Guo Y, Wang X, Xu Y, Xia X (2014) Effect of subinhibitory concentrations of chlorogenic acid on reducing the virulence factor production by Staphylococcus aureus. Foodborne Pathog Dis 11:677–683. doi: 10.1089/fpd.2013.1731 CrossRefPubMedGoogle Scholar
  25. Lindsay D, von Holy A (2006) Bacterial biofilms within the clinical setting: what healthcare professionals should know. J Hosp Infect 64:313–325. doi: 10.1016/j.jhin.2006.06.028 CrossRefPubMedGoogle Scholar
  26. Liu Y et al (2011) RNAIII activates map expression by forming an RNA-RNA complex in Staphylococcus aureus. FEBS Lett 585:899–905. doi: 10.1016/j.febslet.2011.02.021 CrossRefPubMedGoogle Scholar
  27. Mann EE et al (2009) Modulation of eDNA release and degradation affects Staphylococcus aureus biofilm maturation. PLoS One 4:e5822. doi: 10.1371/journal.pone.0005822 PubMedCentralCrossRefPubMedGoogle Scholar
  28. Moet GJ, Jones RN, Biedenbach DJ, Stilwell MG, Fritsche TR (2007) Contemporary causes of skin and soft tissue infections in North America, Latin America, and Europe: report from the SENTRY antimicrobial surveillance program (1998–2004). Diagn Microbiol Infect Dis 57:7–13. doi: 10.1016/j.diagmicrobio.2006.05.009 CrossRefPubMedGoogle Scholar
  29. Nakimbugwe D, Masschalck B, Atanassova M, Zewdie-Bosuner A, Michiels CW (2006) Comparison of bactericidal activity of six lysozymes at atmospheric pressure and under high hydrostatic pressure. Int J Food Microbiol 108:355–363. doi: 10.1016/j.ijfoodmicro.2005.11.021 PubMedGoogle Scholar
  30. Ngo QD, Vickery K, Deva AK (2012) The effect of topical negative pressure on wound biofilms using an in vitro wound model. Wound Repair Regen 20:83–90. doi: 10.1111/j.1524-475X.2011.00747.x CrossRefPubMedGoogle Scholar
  31. Otto M (2008) Staphylococcal biofilms. Curr Top Microbiol Immunol 322:207–228PubMedCentralPubMedGoogle Scholar
  32. Qiu J et al (2010) Subinhibitory concentrations of thymol reduce enterotoxins A and B and alpha-hemolysin production in Staphylococcus aureus isolates. PLoS One 5:e9736. doi: 10.1371/journal.pone.0009736 PubMedCentralCrossRefPubMedGoogle Scholar
  33. Reissig JL, Storminger JL, Leloir LF (1955) A modified colorimetric method for the estimation of N-acetylamino sugars. J Biol Chem 217:959–966PubMedGoogle Scholar
  34. Rosado H, Doyle M, Hinds J, Taylor PW (2010) Low-shear modelled microgravity alters expression of virulence determinants of Staphylococcus aureus. Acta Astronaut 66:408–413. doi: 10.1016/j.actaastro.2009.06.007 CrossRefGoogle Scholar
  35. Rosado H, O’Neill AJ, Blake KL, Walther M, Long PF, Hinds J, Taylor PW (2012) Rotating wall vessel exposure alters protein secretion and global gene expression in Staphylococcus aureus. Int J Astrobiol 11:71–81. doi: 10.1017/s1473550411000346 CrossRefGoogle Scholar
  36. Sadovskaya I, Vinogradov E, Flahaut S, Kogan G, Jabbouri S (2005) Extracellular carbohydrate-containing polymers of a model biofilm-producing strain, Staphylococcus epidermidis RP62A. Infect Immun 73:3007–3017. doi: 10.1128/iai.73.5.3007-3017.2005 PubMedCentralCrossRefPubMedGoogle Scholar
  37. Sadovskaya I, Chaignon P, Kogan G, Chokr A, Vinogradov E, Jabbouri S (2006) Carbohydrate-containing components of biofilms produced in vitro by some staphylococcal strains related to orthopaedic prosthesis infections. FEMS Immunol Med Microbiol 47:75–82. doi: 10.1111/j.1574-695X.2006.00068.x CrossRefPubMedGoogle Scholar
  38. Saxena V, Hwang CW, Huang S, Eichbaum Q, Ingber D, Orgill DP (2004) Vacuum-assisted closure: microdeformations of wounds and cell proliferation. Plast Reconstr Surg 114:1086–1096; discussion 1097–1088Google Scholar
  39. Soromou LW et al (2013) Subinhibitory concentrations of pinocembrin exert anti-Staphylococcus aureus activity by reducing alpha-toxin expression. J Appl Microbiol 115:41–49. doi: 10.1111/jam.12221 CrossRefPubMedGoogle Scholar
  40. Steingrimsson S, Gottfredsson M, Gudmundsdottir I, Sjogren J, Gudbjartsson T (2012) Negative-pressure wound therapy for deep sternal wound infections reduces the rate of surgical interventions for early re-infections. Interact CardioVasc Thorac Surg 15:406–410. doi: 10.1093/icvts/ivs254 PubMedCentralCrossRefPubMedGoogle Scholar
  41. Thoendel M, Kavanaugh JS, Flack CE, Horswill AR (2011) Peptide signaling in the staphylococci. Chem Rev 111:117–151. doi: 10.1021/cr100370n PubMedCentralCrossRefPubMedGoogle Scholar
  42. Ulrich M et al (2007) The staphylococcal respiratory response regulator SrrAB induces ica gene transcription and polysaccharide intercellular adhesin expression, protecting Staphylococcus aureus from neutrophil killing under anaerobic growth conditions. Mol Microbiol 65:1276–1287. doi: 10.1111/j.1365-2958.2007.05863.x CrossRefPubMedGoogle Scholar
  43. Valente PM, Deva A, Ngo Q, Vickery K (2014) The increased killing of biofilms in vitro by combining topical silver dressings with topical negative pressure in chronic wounds. Int Wound J. doi: 10.1111/iwj.12248 PubMedGoogle Scholar
  44. Watters C, Everett JA, Haley C, Clinton A, Rumbaugh KP (2014) Insulin treatment modulates the host immune system to enhance Pseudomonas aeruginosa wound biofilms. Infect Immun 82:92–100. doi: 10.1128/iai.00651-13 PubMedCentralCrossRefPubMedGoogle Scholar
  45. White NT, Delitto A, Manal TJ, Miller S (2015) The American Physical Therapy Association’s top five choosing wisely recommendations. Phys Ther 95:9–24. doi: 10.2522/ptj.20140287 CrossRefPubMedGoogle Scholar
  46. Wu X, Wang Y, Tao L (2011) Sulfhydryl compounds reduce Staphylococcus aureus biofilm formation by inhibiting PIA biosynthesis. FEMS Microbiol Lett 316:44–50. doi: 10.1111/j.1574-6968.2010.02190.x CrossRefPubMedGoogle Scholar
  47. Wu Y et al (2015) Staphylococcus epidermidis SrrAB regulates bacterial growth and biofilm formation differently under oxic and microaerobic conditions. J Bacteriol 197:459–476. doi: 10.1128/jb.02231-14 PubMedCentralCrossRefPubMedGoogle Scholar
  48. Yang Z et al (2014) Functions and mechanisms of intermittent negative pressure for osteogenesis in human bone marrow mesenchymal stem cells. Mol Med Rep 9:1331–1336. doi: 10.3892/mmr.2014.1968 PubMedGoogle Scholar
  49. Zhang YG, Yang Z, Zhang H, Wang C, Liu M, Guo X, Xu P (2010) Effect of negative pressure on human bone marrow mesenchymal stem cells in vitro. Connect Tissue Res 51:14–21. doi: 10.3109/03008200902855891 CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2015

Authors and Affiliations

  1. 1.Medical CollegeNankai UniversityTianjinChina
  2. 2.Department of OrthopedicsChinese PLA General HospitalBeijingChina
  3. 3.Center for Hospital Infection ControlChinese PLA Institute for Disease Control and PreventionBeijingChina

Personalised recommendations