Induction of a viable‐but‐non‐culturable state in bacteria treated with gas discharge plasma

Sir, The article by Cooper et al. (2010) deals with the use of plasma to inactivate micro-organisms and concludes that plasma induces bacteria to enter a viable-but-non-culturable (VBNC) state, probably due to oxidative stress. The authors correlate viability results with morphological ones, and they propose that longer exposure times may be needed to ensure complete cell death. Their approach is interesting, but the paper does not report novel aspects. In fact, their approach and conclusions are almost identical to previous contributions from our group, although we are not referenced in their paper. The authors state that ‘the assessment is made employing a comparison of the methods of culturability, membrane integrity, bacterial morphology and respiration in present studies on the responses of bacteria under plasma induced stress’. Their assessment techniques are identical to the methods we previously used and reported, being the only difference that instead of Atomic Force Microscopy these authors used another type of microscopy. We are glad that researchers and the scientific community in general are now on our same track, putting the accent on cell viability and physiology instead of focusing only on culturability determinations. Before our group started looking at viability issues, the plasma community used to believe that plasma was effective based solely on culturability results. Our group was a pioneer when in 2006, we hypothesized that plasma induced a VBNC state (Abramzon et al. 2006). Further work published in 2008 and 2009 plus dozens of presentations to meetings supported these ideas (Vandervoort et al. 2008; Joaquin et al. 2009). Our work paved the way to think that viability tests have to be always carried out before concluding that plasma inactivates cells. We were the first discussing these issues, and it took us many international meetings to ‘convince’ the plasma community about it. Therefore, Cooper et al. propose hypotheses that were already proven valid several years ago. Cooper et al. hypothesize that the loss of cell culturability after plasma treatment is induced as one of the responses to oxidative stress. This hypothesis was already proposed in 2006 by Abramzon et al., who demonstrated that although plasma-treated bacteria yielded a very low colony-forming unit counting, other experiments such as colorimetric assays and a spectrophotometric measurement of bacterial extracellular fluid showed that cells were still alive at short exposure times. These authors hypothesized that plasma-assisted cell removal proceeded through a first step in which bacterial cells were not culturable, but entered a (VBNC) stage followed by a second step in which cells were actually killed. Abramzon et al. stated that bacteria enter into this dormant state in response to one or more environmental stresses including oxidative stress and radicals. Cooper et al. stated that correlation of culturability results, with membrane integrity and respiration activity, is indicative of the VBNC state. These authors showed changes in cellular morphology to support their point. The approach from our 2006–2009 papers is supported by the authors in their recent contribution. In 2008, Vandervoort et al. showed that ‘combining the AFM results with data from several other analysis techniques including fluorescence microscopy, cell viability studies, and ATP and DNA assays, a more complete picture emerges and shows that the plasma-treatment process produces interactions that render bacterial cells nonculturable after short plasmaexposure times (e.g. 5 min), but with little change in cell morphology, whereas longer exposure times (e.g. 60 min) result in major cell damage.’ (Vandervoort et al. 2008). Cooper et al. reported the presence of a heterogenous population of plasma-treated Bacillus stratosphericus cells comprising of predominantly elongated larger cells, flattened cells and normal cells. Similar results were reported by Vandervoort et al., showing a heterogenous population of plasma-treated Chromobacterium violaceum cells including mostly intact cells and a few cells exhibiting defects such as flattened cells and rough surfaces. Journal of Applied Microbiology ISSN 1364-5072