Isolation of Persister Cells of Bacillus subtilis and Determination of Their Susceptibility to Antimicrobial Peptides

Abstract

Persister cells are growth-arrested subpopulations that can survive possible fatal environments and revert to wild types after stress removal. Clinically, persistent pathogens play a key role in antibiotic therapy failure, as well as chronic, recurrent, and antibiotic-resilient infections. In general, molecular and physiological research on persister cells formation and compounds against persister cells are much desired. In this study, we firstly demonstrated that the spore forming Gram-positive model organism Bacillus subtilis can be used to generate persister cells during exposure to antimicrobial compounds. Interestingly, instead of exhibiting a unified antibiotic tolerance profile, different number of persister cells and spores were quantified in various stress conditions. qPCR results also indicated that differential stress responses are related to persister formation in various environmental conditions. We propose, for the first time to the best of our knowledge, an effective method to isolate B. subtilis persister cells from a population using fluorescence-activated cell sorting (FACS), which makes analyzing persister populations feasible. Finally, we show that alpha-helical cationic antimicrobial peptides SAAP-148 and TC-19, derived from human cathelicidin LL-37 and human thrombocidin-1, respectively, have high efficiency against both B. subtilis vegetative cells and persisters, causing membrane permeability and fluidity alteration. In addition, we confirm that in contrast to persister cells, dormant B. subtilis spores are not susceptible to the antimicrobial peptides.

Isolation and characterization of persisters of the pathogenic microorganismStaphylococcus aureus

“…This suggests that protein aggregation was likely associated with the formation of these vancomycin persisters. This result aligns with our previous observations that vancomycin persisters showed a high level of protein aggregation [8] and provides a potential explanation for the upregulation of chaperones which are associated with protein disaggregation. However, there was no noticeable increase in cell wall thickness despite the observed upregulation of proteins associated with cell wall synthesis in the proteomics data.…”

“…Previously, we analyzed the formation of S. aureus persisters through 24 hours of vancomycin or enrofloxacin exposure [8]. Although the modes of action of these two drugs are different, they generated similar levels of persisters with a final survival fraction of approximately 0.1 %.…”

“…However, this level of persister generation in antibiotic exposed populations is not universally applicable to any bacterial population or strategies to generate persisters. For example, in our previous work analyzing Bacillus subtilis persisters generated by different antibiotics, we observed that in the final washed population, the percentage of persisters ranged from 25 % to 90 % [10]. Therefore, sorting techniques such as Fluorescence-activated cell sorting (FACS) or Magnetic-activated cell sorting (MACS) combined with selected probes have been conducted to separate persisters [11,12].…”

“…In our previous study, we presented a persister isolation method with a commonly used model strain Bacillus subtilis where metabolism dependent dye 5-(and-6)-Carboxyfluorescein diacetate (CFDA) and PI detecting membrane permeabilization were used to label and isolate persisters [10]. Thereby, persisters can be analyzed at both single cell level and population level as purified cells.…”

Pseudomonas fluorescens Cells’ Recovery after Exposure to BAC and DBNPA Biocides

“…Once the antibiotic/biocide is removed, cells continue to grow and become again susceptible to the same antibiotic/biocide. Unlike resistant bacteria that can grow in the presence of antibiotics/biocides and that rely on mutations, persisters are non-genetically modified [ 3 , 4 , 5 ] cells. VBNC are viable but dormant cells that are not culturable (in conditions known to enable bacterial growth) but that remain metabolically active and retain membrane integrity [ 2 , 6 ].…”