Microbial scout hypothesis and microbial discovery.

In this study, we examine the temporal pattern of colony appearance during cultivation experiments, and whether this pattern could inform on optimizing the process of microbial discovery. In a series of long-term cultivation experiments, we observed an expected gradual increase over time of the total number of microbial isolates, culminating in a 700-fold colony count increase at 18 months. Conventional thought suggests that long-term incubations result in a culture collection enriched with species that are slow growing or rare, may be unavailable from short-term experiments, and likely are novel. However, after we examined the phylogenetic novelty of the isolates as a function of the time of their isolation, we found no correlation between the two. The probability of discovering either a new or rare species late in the incubation matched that of species isolated earlier. These outcomes are especially notable because of their generality: observations were essentially identical for marine and soil bacteria as well as for spore formers and non-spore formers. These findings are consistent with the idea of the stochastic awakening of dormant cells, thus lending support to the scout model. The process of microbial discovery is central to the study of environmental microorganisms and the human microbiome. While long-term incubation does not appear to increase the probability of discovering novel species, the technology enabling such incubations, i.e., single-cell cultivation, may still be the method of choice. While it does not necessarily allow more species to grow from a given inoculum, it minimizes the overall isolation effort and supplies needed.

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

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).

Biofilms and planktonic cells of Pseudomonas aeruginosa have similar resistance to killing by antimicrobials.

Biofilms are considered to be highly resistant to antimicrobial agents. Strictly speaking, this is not the case-biofilms do not grow in the presence of antimicrobials any better than do planktonic cells. Biofilms are indeed highly resistant to killing by bactericidal antimicrobials, compared to logarithmic-phase planktonic cells, and therefore exhibit tolerance. It is assumed that biofilms are also significantly more tolerant than stationary-phase planktonic cells. A detailed comparative examination of tolerance of biofilms versus stationary- and logarithmic-phase planktonic cells with four different antimicrobial agents was performed in this study. Carbenicillin appeared to be completely ineffective against both stationary-phase cells and biofilms. Killing by this beta-lactam antibiotic depends on rapid growth, and this result confirms the notion of slow-growing biofilms resembling the stationary state. Ofloxacin is a fluoroquinolone antibiotic that kills nongrowing cells, and biofilms and stationary-phase cells were comparably tolerant to this antibiotic. The majority of cells in both populations were eradicated at low levels of ofloxacin, leaving a fraction of essentially invulnerable persisters. The bulk of the population in both biofilm and stationary-phase cultures was tolerant to tobramycin. At very high tobramycin concentrations, a fraction of persister cells became apparent in stationary-phase culture. Stationary-phase cells were more tolerant to the biocide peracetic acid than were biofilms. In general, stationary-phase cells were somewhat more tolerant than biofilms in all of the cases examined. We concluded that, at least for Pseudomonas aeruginosa, one of the model organisms for biofilm studies, the notion that biofilms have greater resistance than do planktonic cells is unwarranted. We further suggest that tolerance to antibiotics in stationary-phase or biofilm cultures is largely dependent on the presence of persister cells.