Entry of Yersinia pestis into the Viable but Nonculturable State in a Low-Temperature Tap Water Microcosm

Yersinia pestis, the causative agent of plague, has caused several pandemics throughout history and remains endemic in the rodent populations of the western United States. More recently, Y. pestis is one of several bacterial pathogens considered to be a potential agent of bioterrorism. Thus, elucidating potential mechanisms of survival and persistence in the environment would be important in the event of an intentional release of the organism. One such mechanism is entry into the viable but non-culturable (VBNC) state, as has been demonstrated for several other bacterial pathogens. In this study, we showed that Y. pestis became nonculturable by normal laboratory methods after 21 days in a low-temperature tap water microcosm. We further show evidence that, after the loss of culturability, the cells remained viable by using a variety of criteria, including cellular membrane integrity, uptake and incorporation of radiolabeled amino acids, and protection of genomic DNA from DNase I digestion. Additionally, we identified morphological and ultrastructural characteristics of Y. pestis VBNC cells, such as cell rounding and large periplasmic spaces, by electron microscopy, which are consistent with entry into the VBNC state in other bacteria. Finally, we demonstrated resuscitation of a small number of the non-culturable cells. This study provides compelling evidence that Y. pestis persists in a low-temperature tap water microcosm in a viable state yet is unable to be cultured under normal laboratory conditions, which may prove useful in risk assessment and remediation efforts, particularly in the event of an intentional release of this organism

Prokaryotic Hydrocarbon Degraders

Hydrocarbons have been part of the biosphere for millions of years, and a diverse group of prokaryotes has evolved to use them as a source of carbon and energy. To date, the vast majority of formally defined genera are eubacterial, in 7 of the 24 major phyla currently formally recognized by taxonomists (Tree of Life, http://tolweb.org/Eubacteria. Accessed 1 Sept 2017, 2017); principally in the Actinobacteria, the Bacteroidetes, the Firmicutes, and the Proteobacteria. Some Cyanobacteria have been shown to degrade hydrocarbons on a limited scale, but whether this is of any ecological significance remains to be seen – it is likely that all aerobic organisms show some basal metabolism of hydrocarbons by nonspecific oxygenases, and similar “universal” metabolism may occur in anaerobes. This chapter focuses on the now quite large number of named microbial genera where there is reasonably convincing evidence for hydrocarbon metabolism. We have found more than 320 genera of Eubacteria, and 12 genera of Archaea. Molecular methods are revealing a vastly greater diversity of currently uncultured organisms – Hug et al. (Nat Microbiol 1:16048, 2016) claim 92 named bacterial phyla, many with almost totally unknown physiology – and it seems reasonable to believe that the catalog of genera reported here will be substantially expanded in the future