Streptomycin and tailocin treatments yield similar frequencies of Pph persisters
We previously found that tailocin exposure results in a stable population of genetically susceptible Pph survivors, which we defined as tailocin persisters (30). In this study, we asked whether Pph exhibits persistence to the antibiotic streptomycin, and whether streptomycin persistence is distinct from tailocin persistence. Pph growth curves were performed to establish the timing of early and late stationary phase (Fig. S1). Survival to 5X MIC streptomycin and tailocin was characterized through kinetic killing curve assays at log phase, early stationary phase (20h), and late stationary phase (96h, Figure 1). In both early and late stationary phase cultures, CFU counts declined to 0.04% of initial values within 3h of streptomycin treatment, remaining stable at subsequent timepoints (Fig. 1A). Similarly, 0.06% of early or late stationary phase Pph cells remained culturable after tailocin exposure, consistent with our previous observations, with the majority of killing occuring within a few minutes (Fig. 1B). Adding either treatment in log phase resulted in a lower proportion of survivors. For each assay, colonies from the surviving population were confirmed to be susceptible upon re-exposure to streptomycin and tailocin. These results demonstrate that stationary phase Pph populations form similar proportions of culturable persisters to both streptomycin and tailocin, although tailocin killing is far more rapid. Subsequent experiments in this study were performed on early stationary phase Pph unless otherwise noted, using the same doses and treatment durations as used for the experiments in Figure 1.
Streptomycin and tailocin treatments have distinct effects on population physiology
Having established that tailocin and streptomycin both eliminate most culturable cells, we next sought to compare their efficiency in eliminating the viable population, inclusive of nonculturable cells. We first measured treatment-induced changes in total cell concentration and culturable frequency. Hemocytometer readings revealed that streptomycin did not cause a reduction in the total concentration of cells in stationary phase or log phase (Fig 2A, Fig. S2A). Tailocin caused a slight reduction in the number of cells, although this was only statistically significant in log phase (Fig 2A, Fig. S2B). Dilution plating confirmed that treatments reduced the proportion of culturable cells from 46% to 0.04% for streptomycin (Fig. 2A) and to 0.05% for tailocin (Fig. 2B), consistent with our earlier measures of survival over the T0 population (Fig. 1). To compare the physiological states of streptomycin- and tailocin-exposed Pph populations, we imaged cells on an agarose pad after staining with a combination of three fluorescent dyes: the vitality indicator Redox Sensor Green (RSG), the red membrane permeability indicator Propidium Iodide (PI), and the blue membrane permeant nucleic acid stain Hoescht 33342. This strategy allowed imaged cells to be classified into five categories (Fig. 2C): 1) redox-active with intact membranes (green/blue), 2) redox-active with compromised membranes (green/red/blue), 3) redox-inactive with compromised membranes (red/blue), 4) redox-inactive with intact nuclear material and membranes (blue), and 5) unstained “ghost” cells with no nucleic acid content, visible in phase-contrast only. The method was first tested on log-phase and ethanol-killed cells to rule out signal interference or overlap between stains (Fig. S3), and we confirmed that the staining combination did not affect culturable rate of streptomycin-treated cells. In preliminary experiments we noted that all Pph cells showed permeabilization and loss of redox activity starting after two hours on the agarose pad, thus all imaging was performed within twenty minutes after placement on the pad.
In untreated stationary phase cultures, over 80% of the population was composed of redox active, or Category 1, cells (Fig. 2D-E, Table S1). After streptomycin treatment, Pph cultures were evenly distributed through Categories 2-4, each comprising an average of 19-25% of the population. Notably, nearly half the treated population were redox-active cells with permeable membranes (Category 2) or redox-inactive cells with intact membranes (Category 4), two states not distinguished by common live-dead staining methods. Cells in Category 2 had a lower green signal intensity than those in Category 1, indicating that membrane-damaged active cells were associated with reduced redox activity (Fig. S4A). A Category 4-like state was previously associated with persister and VBNC cells in E. coli, which were also characterized by increased cell roundness (7); we determined that Category 4 individuals also had significantly increased average roundness compared with the four other categories of treated Pph cells (Fig. S4B).
Unlike streptomycin, tailocin treatment converted most of the stationary phase population to Category 3 (membrane compromised, inactive) within three minutes (Fig. S5). After four hours, only 3% of the remaining cells were in Category 1 (Fig. 2E, Table S1), and Category 4 cells were extremely rare. When the experiments were performed on cultures in log phase, streptomycin and tailocin-induced changes were similar to those in stationary culture, although there was an apparently reduced proportion of redox-active Category 2 cells after tailocin treatment compared to the log phase results (Fig. S2D). Together, the results indicate that streptomycin treatment shifts the majority of the Pph population into diverse physiological states, while tailocin treatment rapidly compromises redox activity and membrane integrity in the vast majority of the population. They also show that after either treatment, the proportion of redox-active cells and other intact cells far exceeds that of culturable persisters.
Streptomycin and tailocin culturable persisters occupy distinct physiological states
While microscopic studies were useful for profiling redox and permeability changes following either treatment, this approach could not determine the culturability of each staining category. Therefore, we applied fluorescence-assisted cell sorting (FACS) to determine whether the culturable and infectious fractions of streptomycin and tailocin-treated populations could be separated according to redox staining characteristics. Because propidium iodide had stained some redox-active Pph cells (i.e., Category 2 cells), we first sought an alternate permeability stain that could provide a distinct live-dead separation in two-color sorting studies. DRAQ7 is a far-red membrane permeant dye that has been validated in eukaryotic cell culture studies (33), but is not widely used for determining viability in bacteria. In microscopic analysis on Pph, DRAQ7 stained redox-inactive cells, but unlike PI, was not observed to co-stain with RSG (Fig. S6). This indicated that DRAQ7 does not permeate cells with redox activity. A triple staining experiment using DRAQ7 instead of PI was performed on treated and untreated Pph to confirm that the stain yielded similar estimates of redox-inactive membrane compromised cells to PI (Table S2).
Flow cytometric analysis of untreated log and stationary phase, ethanol-killed, and unstained cells identified clear patterns associated with death and active growth (Fig. 3A-3D). Consistent with microscopic observations, streptomycin treatment resulted in an apparent increase in both permeabilized cells and low-redox intact cells, but also resulted in a large population of cells with elevated redox signal (Fig. 3E). As expected, tailocin treatment permeabilized all but a small fraction of cells to DRAQ7 (Fig. 3F). To determine how the streptomycin-induced changes compared with the effects of a validated persister induction treatment, we also treated stationary cells with the protonophore CCCP, a highly efficient inducer of multidrug-tolerant persisters in Pseudomonas aeruginosa (34). A killing curve assay demonstrated that 3h CCCP treatment (5x MIC, or 100 µg mL-1) resulted in a stable culturable population representing 13% of the initial count (Fig. S7). CCCP treated cultures showed slightly increased RSG staining in intact cells, with many cells permeabilized to DRAQ7 (Fig. 3G). Histogram analysis supported the finding that streptomycin and CCCP treatments generated populations with increased redox signal, while tailocin treatment largely abolished redox activity (Fig. 3H). For unknown reasons, ethanol-killed cells had a higher level of green fluorescence in cell sorting than tailocin-permeabilized cells (Fig. 3H).
Cells were separated according to physiological state to determine the culturability of each fraction. Optimization assays confirmed that no culturable cells could be recovered from DRAQ7-staining fractions, so we focused on the region of low DRAQ7 intensity. Cells were gated into fractions G1, G2, and G3, corresponding to the highest to lowest green fluorescence intensity (Fig. 3A and 3D-G). Cells intact after streptomycin, tailocin, and CCCP treatments primarily fell into the G1, G3, and G2 gates, respectively (Fig. 3E-G and Table S1). Cells collected from each sorting gate were plated on culture media. In untreated cultures, colonies were recovered from all fractions (Fig. 4A). After streptomycin treatment, over 99.5% of colonies recovered came from the low-redox G3 fraction (Fig. 4B), even though this fraction represented only 13% of gated cells (Fig. 3). In contrast, in the tailocin-treated culture over 99% of colonies were recovered from the G2 fraction (Fig. 4C), despite this fraction containing only 0.2% of the total gated cells (Fig. 3). Colonies were cultured from both G2 and G3 fractions after CCCP treatment (Fig. 4D), but similarly to the other two treatments, nothing was cultured from the G1 fraction. These results demonstrate that while streptomycin persisters occupy a low-redox state consistent with dormancy, tailocin persisters are associated with a state of moderate redox activity. Additionally, diverse treatments resulted in a lack of culturability in cells with high redox activity.
We next asked whether the culturability or redox activity of sorted fractions was associated with infectious capacity of the pathogen. Due to the low volume of sorted inoculum, pathogenicity of the fractions was assessed in a qualitative bean pod inoculation assay, and symptoms of watersoaking or necrosis were observed after five days (Fig 4E-H). No symptoms developed after inoculation from the high-redox G1 fraction of any culture, even without antimicrobial treatment (Fig. 4E). For cultures treated with streptomycin, tailocin, or CCCP, symptoms were observed at sites inoculated with any fraction with a significant culturable population (~104 or greater CFU mL-1, 4F-H). Symptoms were weakest in the tailocin treated cultures, but this may be attributable to the low number of culturable cells obtained through sorting. Notably, for streptomycin and tailocin-treated cultures, the fractions associated with the largest number of membrane-intact cells (G1 and G3, respectively) did not cause symptoms (Fig. 3E-F and 4F-G). These results demonstrate that culturability in media is associated with infection capacity in antimicrobial stressed Pph, and that the highest redox fractions of all cultures were noninfectious.
Streptomycin and tailocin-treated cells colonize the host at the same rates as untreated cells
Because streptomycin persisters were associated with low activity, we hypothesized that streptomycin persisters might colonize the plant at a slower rate than the more active tailocin persisters. Sorting did not yield a sufficient number of cells to perform timepoint analysis, so we instead compared colonization rates of treated and untreated cultures that had been adjusted to contain the same concentration of culturable cells (2.5 ×104 CFU mL-1). Because streptomycin causes a vast decline in culturable cells, the streptomycin-treated inoculum contained roughly 700-fold more RSG-staining cells than the untreated inoculum. The tailocin inoculum contained a similar number of RSG-staining cells to the untreated inoculum, but a far greater number of permeabilized cells. Despite differing viable population sizes and physiologies, streptomycin and tailocin-treated cultures colonized bean leaves at the same rate as untreated cells (Fig. 5A) and were able to cause normal symptoms of leaf spot and chlorosis at 8 days (Fig. 5B). This finding indicates that the distinct physiological states of streptomycin and tailocin persisters do not delay their ability to colonize a susceptible host. It also suggests that in the streptomycin-stressed inoculum, the large populations of high-redox nonculturable cells may not make a significant contribution to early infection, or at least not enough to speed colonization of a susceptible host.
To test the latter hypothesis, we performed a second experiment in which streptomycin and untreated cultures were adjusted to contain the same proportion of redox-active cells, regardless of intensity or culturability. Inocula adjustments were based on microscopic observations of mean RSG staining from Figure 2. In this experiment, both inocula contained 7×104 visibly RSG-staining cells per mL, but the untreated culture contained an estimated 450-fold greater concentration of culturable cells than the streptomycin-treated inoculum. The streptomycin-treated population started growing in the leaf much more slowly than the untreated inoculum, with the population only increasing after a two-day lag (Fig. 5C). This further supports the hypothesis that in a physiologically heterogeneous antibiotic-stressed Pph population, the high-redox unculturable cells do not significantly contribute to early host colonization.
Streptomycin eradicates Pph persisters of tailocin
Having determined that Pph tailocin persisters exist in a distinct physiological state from streptomycin persisters, we next hypothesized that streptomycin could eliminate tailocin persisters and vice-versa. Antibiotic persisters often exhibit multidrug tolerance, so we also asked whether tailocin persisters could be eliminated by two other antibiotics, the bacteriostatic translational inhibitor tetracycline or the DNA replication inhibitor ciprofloxacin. Cross-survival rates of streptomycin persisters were first tested with a sequential treatment of tailocin, tetracycline, or ciprofloxacin. 30 to 70% of the streptomycin persistent population remained culturable after tetracycline or ciprofloxacin treatment, while only 1% remained culturable after tailocin treatment (Fig. 6A). Conversely, when tailocin persisters were washed and treated with tetracycline or ciprofloxacin, means of 7.2 and 11.5% remained culturable after treatment, respectively, while no colonies could be recovered after streptomycin treatment (Fig. 6B). To determine how this compares to whole population survival rates to these antibiotics, we treated stationary phase Pph cultures with tetracycline or ciprofloxacin alone, and measured CFU recovery at 6.5±2.3% and 0.06±0.02% of the initial population, respectively. Thus, compared to untreated Pph cells, tailocin persisters exhibited no survival to streptomycin, a similar survival rate to tetracycline, and a 178-fold increased rate of survival to ciprofloxacin. CCCP-treated cells were also highly multidrug tolerant, but fewer than 0.1% survived tailocin treatment (Fig. 6C).
To further check for elimination of viable unculturable cells, we concentrated and microscopically examined the streptomycin-treated tailocin persisters, and found that all cells stained with PI only or were unstained (Fig. S8). No Category 1, 2, or 4 cells were observed. To rule out the possibility of rare live cells reviving to colonize the host, bean leaves were inoculated with concentrated cultures after the combination treatment. No symptoms developed on leaves (Fig. S8), and no Pph colonies were recovered in leaves collected immediately after inoculation or at days 1-5. In summary, cells surviving antibiotic and CCCP treatments have a high propensity to survive treatment with other antibiotics, but are mostly eliminated by tailocin. Streptomycin treatment is highly effective at eliminating culturable tailocin persisters, while tetracycline and ciprofloxacin are less effective.