Microbial scout hypothesis, stochastic exit from dormancy, and the nature of slow growers

Abstract

We recently proposed a scout model of the microbial life cycle (S. S. Epstein, Nature 457:1083, 2009), the central element of which is the hypothesis that dormant microbial cells wake up into active (so-called scout) cells stochastically, independently of environmental cues. Here, we check the principal prediction of this hypothesis: under growth-permissive conditions, dormant cells initiate growth at random time intervals and exhibit no species-specific lag phase. We show that a range of microorganisms, including environmental species, Escherichia coli, and Mycobacterium smegmatis, indeed wake up in a seemingly stochastic manner and independently of environmental conditions, even in the longest incubations conducted (months to years long). As is implicit in the model, most of the cultures we obtained after long incubations were not inherently slow growers. Of the environmental isolates that required ≥7 months to form visible growth, only 5% needed an equally long incubation upon subculturing, with the majority exhibiting regrowth within 24 to 48 h. This apparent change was not a result of adaptive mutation; rather, most microbial species that appear to be slow growers were in fact fast growers with a delayed initiation of division. Genuine slow growth thus appears to be less significant than previously believed. Random, low-frequency exit from the nongrowing state may be a key element of a general microbial survival strategy, and the phylogenetic breadth of the organisms exhibiting such exit indicates that it represents a general phenomenon. The stochasticity of awakening can also provide a parsimonious explanation to several microbiological observations, including the apparent randomness of latent infections and the existence of viable-but-nonculturable cells (VBNC).

Fig 1

Temporal pattern of growth resumption of selected microorganisms from soil (A) and marine (B) samples. Values inside the boxes indicate the number of isolates observed at the given time point. Note that the closest cultivated relatives of the marine isolates are all fast growers forming visible colonies within 24 to 94 h (26, 35, and O. Nedashkovskaya, personal communication).

Fig 2

Time (in days) required to form visible growth during initial isolation of marine microorganisms (y axis) and during subculturing (x axis). Note that the overwhelming majority of isolates observed between 45 and 175 days of the initial incubation regrew within 24 h (red squares in the upper left corner of the heat map).

Fig 3

Individual cells of four selected isolates show significant differences in subculture with respect to time of growth initiation. Cells were subcultured in the single-cell format, allowing for the observation of when each cell formed visible growth. Here and in all single-cell format experiments, the percentage of wells showing growth is proportional to the number of cells forming visible biomass but is not the percent recovery, because some wells in microtiter plates were unoccupied due to chance.

Fig 4

Awakening kinetics of dormant E. coli. (A) After antibiotic challenge, individual surviving cells of E. coli do not initiate growth simultaneously but start growing at different time points spread throughout 2 weeks of incubation. Gradual awakening is apparent in single-cell experiments employing microtiter plates and in conventionally plated petri dishes. Recovery at the end of 2 weeks is designated total recovery and is assigned a value of 100%; earlier data points are presented as fractions of this value, with standard deviations indicated. The results represent data from three independent experiments, two of which employed gentamicin and one ofloxacin, all showing similar kinetics of growth initiation. (B) In a single growth experiment involving cells remaining after gentamicin treatment, we extended the incubation period to 2 months and continued to observe new growth. Note a much lower level of cell recovery in petri dishes versus single-cell experiments in microtiter plates. Note also that gradual awakening is not apparent in petri dishes, likely due to overgrowth by colonies developing early in incubation. Incidentally, this may be one reason why the phenomenon was not observed in earlier studies.

Fig 5

Awakening kinetics of dormant M. smegmatis. Four independent long-term experiments show that dormant cells of M. smegmatis initiate growth at apparently random time points, with cumulative growth curves exhibiting no sign of leveling off even after 3 months of incubation. Each well was inoculated with, on average, either 1 (A) or 10 (B) dormant cells of M. smegmatis.