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Original Research ARTICLE Provisionally accepted The full-text will be published soon. Notify me

Front. Microbiol. | doi: 10.3389/fmicb.2019.01992

Oxygen Restriction Generates Difficult-To-Culture P. aeruginosa

  • 1Costerton Biofilm Center, University of Copenhagen, Denmark
  • 2Department of Clinical Microbiology, Rigshospitalet, Denmark
  • 3Statens Serum Institut (SSI), Denmark
  • 4Department of Infectious Diseases, Rigshospitalet, Denmark

Induction of a non-culturable state has been demonstrated for many bacteria, e.g. E. coli and various Vibrio spp. In a clinical perspective, the lack of growth due to these non-culturable bacteria can have major consequences for the treatment of patients. Here we show how anoxic conditioning (restriction of molecular oxygen, O2) generates difficult-to-culture (DTC) bacteria during biofilm growth. A significant subpopulation of Pseudomonas aeruginosa entered a DTC state after anoxic conditioning, ranging from 5 to 90 % of the total culturable population, in both planktonic and biofilm models. Anoxic conditioning also generated DTC subpopulations of Staphylococcus aureus and Staphylococcus epidermidis (89 and 42 % of the total culturable population, respectively). Growth of the DTC populations were achieved by substituting O2 with 10 mM NO3- as an alternative electron acceptor for anaerobic respiration or, in the case of P. aeruginosa, by adding sodium pyruvate or catalase as scavengers against reactive oxygen species (ROS) during aerobic respiration. An increase in normoxic plating due to addition of catalase suggests the molecule hydrogen peroxide as a possible mechanism for induction of DTC P. aeruginosa. Anoxic conditioning also generated a true viable but non-culturable (VBNC) population of P. aeruginosa that was not resurrected by substituting O2 with NO3- during anaerobic respiration.
These results demonstrate that habituation to an anoxic micro-environment could complicate diagnostic culturing of bacteria, especially in the case of chronic infections where oxygen is restricted due to the host immune response.

Keywords: P. aeruginosa, S. aureus, S. epiderimidis, Anoxia, Biofilm, reactive oxygen species, difficult-to-culture, Viable but non culturable (VBNC)

Received: 14 May 2019; Accepted: 13 Aug 2019.

Copyright: © 2019 Kvich, Fritz, Crone, Kragh, Kolpen, Sønderholm, Andersson, Koch, Jensen and Bjarnsholt. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence:
PhD. Peter Ø. Jensen, Costerton Biofilm Center, University of Copenhagen, Copenhagen, Denmark,
Prof. Thomas Bjarnsholt, Costerton Biofilm Center, University of Copenhagen, Copenhagen, Denmark,