Pseudomonas syringae pv. phaseolicola Uses Distinct Modes of Stationary-Phase Persistence To Survive Bacteriocin and Streptomycin Treatments

Abstract

Antimicrobial treatment of bacteria often results in a small population of surviving tolerant cells, or persisters, that may contribute to recurrent infection. Antibiotic persisters are metabolically dormant, but the basis of their persistence in the presence of membrane-disrupting biological compounds is less well understood. We previously found that the model plant pathogen Pseudomonas syringae pv. phaseolicola 1448A (Pph) exhibits persistence to tailocin, a membrane-disrupting biocontrol compound with potential for sustainable disease control. Here, we compared physiological traits associated with persistence to tailocin and to the antibiotic streptomycin and established that both treatments leave similar frequencies of persisters. Microscopic profiling of treated populations revealed that while tailocin rapidly permeabilizes most cells, streptomycin treatment results in a heterogeneous population in the redox and membrane permeability state. Intact cells were sorted into three fractions according to metabolic activity, as indicated by a redox-sensing reporter dye. Streptomycin persisters were cultured from the fraction associated with the lowest metabolic activity, but tailocin persisters were cultured from a fraction associated with an active metabolic signal. Cells from culturable fractions were able to infect host plants, while the nonculturable fractions were not. Tailocin and streptomycin were effective in eliminating all persisters when applied sequentially, in addition to eliminating cells in other viable states. This study identifies distinct metabolic states associated with antibiotic persistence, tailocin persistence, and loss of virulence and demonstrates that tailocin is highly effective in eliminating dormant cells. IMPORTANCE Populations of genetically identical bacteria encompass heterogeneous physiological states. The small fraction of bacteria that are dormant can help the population survive exposure to antibiotics and other stresses, potentially contributing to recurring infection cycles in animal or plant hosts. Membrane-disrupting biological control treatments are effective in killing dormant bacteria, but these treatments also leave persister-like survivors. The current work demonstrates that in Pph, persisters surviving treatment with membrane-disrupting tailocin proteins have an elevated redox state compared to that of dormant streptomycin persisters. Combination treatment was effective in killing both persister types. Culturable persisters corresponded closely with infectious cells in each treated population, whereas the high-redox and unculturable fractions were not infectious. In linking redox states to heterogeneous phenotypes of tailocin persistence, streptomycin persistence, and infection capability, this work will inform the search for mechanisms and markers for each phenotype.

Cooperative colonization of the host and pathogen dissemination involves stochastic and spatially structured expression of virulence traits

“…The apoplastic Flagella ON /T3SS OFF subpopulation is potentially more vulnerable to plant defenses since it elicits but cannot suppress immunity. We used propidium iodide to detect membrane-compromised (not viable) apoplast-extracted bacteria to determine if any of the phenotypic variants was subject to higher killing rates 35,36 . No significant differences were observed between the dead/live ratios of ON versus OFF subpopulations for either fliC , hrpL , hrcU or hopAB1 genes ( Fig.…”

Persistence and viable but non-culturable state induced by streptomycin in Erwinia amylovora

“…However, unculturable cells that were considered to be in the VBNC state after the persistence state were analyzed by staining each individual cell according to its physiological characteristics. We were unable to get the five categories that Patel et al (2021) defined, because in this study, only two fluorescent dyes (the green vitality indicator RSG and red membrane permeability indicator PI) and one mixture fluorescence of them were used. Instead, we identified three classes ( Figure 2C ) and quadrant ( Supplementary Figure 3 ): class 1, redox-active cells with intact membranes being regarded VBNC cells in this experiment (green, Q4 in FITC-A image); class 2, which are redox-active cells with damaged membranes and considered to be true VBNC cells (yellow, Q2 in FITC-A image); class 3, redox-inactive cells with damaged membranes (red, Q1 in FITC-A image); and no defined class, unstaining cells (white, Q3 in FITC-A image).…”

“…Furthermore, in a few cases with copper ( Ordax et al, 2006 ) but not in a majority of cases with antibiotics, results of the fire blight pathogen E. amylovora varied by dormancy, including the induction of the persister and VBNC cells by chemical control agents. Recently, Patel et al (2021) reported that P. syringae pv. phaseolicola has the persistence to survive under streptomycin treatment.…”

“…This was performed to produce a biphasic kill curve within the first few hours of cultivation, which showed a declining growth curve and identified the VBNC state after streptomycin treatment. To determine the proportions of each different physiological state in VBNC, staining with RedoxSensor Green (RSG) and PI was also conducted using the BacLight RSG Green vitality kit (Invitrogen, CA, USA) in accordance with a recommended protocol and previous report ( Patel et al, 2021 ). Each 1 mL of the two case samples contained 1 μL of SYTO9 (3.34 μM) and PI (20 mM) or each of 1 μL RSG (1 mM) and PI and were incubated for 15 min in the dark.…”

Genome Mining Shows Ubiquitous Presence and Extensive Diversity of Toxin-Antitoxin Systems in Pseudomonas syringae

“…Previous studies have reported induction of viable but not culturable (VBNC) state and a concomitant alteration in the gene expression profile upon exposure of P. syringae cells to conditions that mimic oxidative burst of early stage plant infection ( Mock et al, 2015 ; Postnikova et al, 2015 ). We recently reported that a plant pathogenic P. syringae can survive bacteriocin and antibiotics exposure by entering another dormant, tolerant state known as persistence ( Kandel et al, 2020 ; Patel et al, 2021 ). Although several stress response pathways have been identified, it is still not well understood how P. syringae can cope with environmental stresses in epiphytic and free-living conditions or maintain virulence genes in the extended absence of host selection pressure.…”