The viable but nonculturable concept, bacteria in urine samples, and Occam's razor.

Discrepancies between the number of bacterial cells that can be detected by a direct counting method such as microscopy and the number that form colonies on standard media are regularly reported and extensively discussed (3). The observations of Anderson and colleagues concerning urine specimens (1) fall into this category and are without doubt of considerable interest and potential importance. The intriguing conclusion that there is an abundant source of nonculturable prokaryotic cells in the urinary tracts of humans and mice seems unavoidable. However, we are concerned that the title of the paper and the assumption that the bacteria detected were in the viable but nonculturable (VBNC) state is not justified by either those authors’ data or the preceding literature. In particular, the implication that the cell populations termed VBNC by Anderson et al. are in a specific physiological state distinct from starvation is highly contentious and potentially misleading. Notwithstanding problems with the term VBNC (2, 4, 9), our principal concern is that undue emphasis has been given to one interpretation of the results of Anderson et al., which refers to a hypothetical bacterial condition at the expense of other interpretations whose physiological basis is extensively validated. Specifically, we dispute the view that excludes the possibility that the populations labeled VBNC were injured, moribund, or even dead. Anderson et al. define viable cells as those fluorescing green with the LIVE/DEAD reagent, and they calculate the size of the VBNC population by determining the difference between the colony count and the green-cell count. They argue that their VBNC populations were “actually viable” on the basis that lethally UV-irradiated cells acquire the “dead” labeling phenotype more quickly than the cells in their samples. However, killing cells by lethal doses of UV does not address the real issue of whether any of the likely processes by which the bacteria in their samples may have been killed could result in a sustained “live” labeling phenotype. Indeed, cells with intact membrane signals have been shown directly to have lost reproductive capacity (6). Most importantly, we and others argue that the term “viable” should be reserved for cells that retain the capacity for reproduction. Andersen et al. make no attempt to address this point and, in doing so, ignore key aspects of the preceding literature. Some attempt should have been made to resuscitate putatively VBNC cells. Moreover, simple tests could have been done to determine the presence of peroxide-sensitive cells (5), injured cells (8), and cells capable of growth only in broth (7). While numerous studies have demonstrated nonculturable bacterial populations that retain cellular indicators of integrity or activity, very few have demonstrated that any of these cells can return to culturability. In the latter instances, the period during which the return to culturability was demonstrated was brief. Furthermore, it has been argued that such results are due to injured cells on a pathway of unrelenting deterioration to complete loss of culturability (4, 5). Thus, we dispute the view that truly viable but temporarily nonculturable cells can “remain in this condition for long periods.” Our central point is that many established and fully validated descriptors might be applied to the nonculturable cells detected in this study. Occam’s razor, which urges us to go with the simplest available explanation that is consistent with the observations at issue, provides a radically different interpretation of Anderson et al.’s data. This simpler explanation is that the observed nonculturable cells are either dead or passing through a brief injured state to death. This interpretation, in our view, renders the VBNC concept redundant.