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

Abstract

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.

Molecular viability testing of viable but non‐culturable bacteria induced by antibiotic exposure

“…Also, the step of having a 7 day starvation before the stimulation could induce VBNC cells because they have intact cell membranes, low metabolic activity and continued mRNA synthesis (Lleo et al ., 2000). It has been known that many species of bacteria enter VBNC state when they are subjected to various environmental stressors, such as UV disinfection, osmotic pressures, high/low temperature and starvation (Colwell et al ., 1985; Byrd et al ., 1991; Oliver et al ., 2005; Pawlowski et al ., 2011). Interestingly, it has been demonstrated that VBNC and persister cells coexist after antibiotic treatment and can be resuscitated back to culturable cells when the existing stress is removed (Ayrapetyan et al ., 2015b).…”

The importance of the viable but non-culturable state in human bacterial pathogens

“…35 S-labelled methionine, 3 H-labelled leucine and 35 S-labelled cysteine/methionine have been used to detect viable cells in Shigella dysenteriae , E. faecalis and Y. pestis , respectively (Rahman et al, 1994; Lleò et al, 1998; Pawlowski et al, 2011). …”

Current Perspectives on Viable but Non-Culturable (VBNC) Pathogenic Bacteria

“…These include confirmation of cellular membrane integrity, uptake of labeled amino acids, and protection of genomic DNA (5). For V .…”

“…Several factors such as antibiotic pressure, high/low temperature, starvation, chlorination, change in the pH, and oxygen stress can induce VBNC state of any bacteria (5, 35, 40). The culture supernatant of ameba Hartmannella vermiformis was shown to induce the VBNC state of Legionella pneumophila (41).…”