(795b) Dormancy Is Not Necessary Or Sufficient for Bacterial Persistence to Antibiotics | AIChE

(795b) Dormancy Is Not Necessary Or Sufficient for Bacterial Persistence to Antibiotics

Authors 

Orman, M. A. - Presenter, Rutgers, The State University of New Jersey
Brynildsen, M. P., Princeton University



Persisters are phenotypic variants that are tolerant to high concentrations of antibiotics, and this phenomena has been attributed to transient dormancy. Evidence in support of this model has been obtained from time-lapse fluorescence microscopy (1-3) and fluorescence-activated cell sorting (FACS) experiments that traced cell division (4) or used markers of growth-rate (5). Although these studies demonstrated that persisters can originate from growth-inhibited cells, it had not been determined to what extent dormancy is a characteristic of persisters, or if persister antibiotic tolerances require growth inhibition. In this context, we sought to determine whether dormancy in replication or metabolism prior to antibiotic treatment was necessary or sufficient for persistence. To quantify the metabolic and cell division distributions of persisters within an exponentially-growing E. coli culture at single cell-level, we used fluorescence-activated cell sorting, a fluorescent indicator of cell division, a fluorescent measure of metabolic activity, and persistence assays. FACS was used to segregate cultures into quantiles based on a fluorescent signal providing information about the cell division or metabolic states, and each quantile was then treated with antibiotics to determine the number of persisters present. First, we verified that fluorescent labeling, flow cytometry, and segregation did not change the level of persisters in the population. Using the cell division reporter, we observed that although the non-growing sub-population was more enriched with persisters (~1%) than the growing sub-population (~0.01%), approximately 20% of all the persisters originated from normally-growing cells. Since the most rapidly-growing sub-population also included persisters, these data demonstrated that growth inhibition prior to antibiotic exposure is not required for persistence. When we employed a fluorescent measure of metabolism, we found that cells with low metabolic activity were ~40 times more likely to be persisters than cells with high metabolic activity. These data, which include the first direct measurements of persister metabolic activity, suggest that persister sub-populations are highly heterogeneous, and in spite of the fact that inhibition of replication or metabolism enhances the likelihood of a cell to be a persister, persistence is far more complex than dormancy and additional characteristics are needed to define the persister phenotype.   

References

1.         Balaban NQ, Merrin J, Chait R, Kowalik L, Leibler S. 2004. Bacterial persistence as a phenotypic switch. Science 305:1622-1625.

2.         Rotem E, Loinger A, Ronin I, Levin-Reisman I, Gabay C, Shoresh N, Biham O, Balaban NQ. 2010. Regulation of phenotypic variability by a threshold-based mechanism underlies bacterial persistence. Proc. Natl. Acad. Sci. U. S. A. 107:12541-12546.

3.         Gefen O, Gabay C, Mumcuoglu M, Engel G, Balaban NQ. 2008. Single-cell protein induction dynamics reveals a period of vulnerability to antibiotics in persister bacteria. Proc. Natl. Acad. Sci. U. S. A. 105:6145-6149.

4.         Roostalu J, Joers A, Luidalepp H, Kaldalu N, Tenson T. 2008. Cell division in Escherichia coli cultures monitored at single cell resolution. BMC Microbiol. 8.

5.         Shah D, Zhang ZG, Khodursky A, Kaldalu N, Kurg K, Lewis K. 2006. Persisters: a distinct physiological state of E-coli. BMC Microbiol. 6.