Generation of Persister Cells of Pseudomonas aeruginosa and Staphylococcus aureus by Chemical Treatment and Evaluation of Their Susceptibility to Membrane-Targeting Agents

Abstract

Persister cells (PCs) are a subset of dormant, phenotypic variants of regular bacteria, highly tolerant to antibiotics. Generation of PCs in vivo may account for the recalcitrance of most chronic infections to antimicrobial treatment and demands for the identification of new antimicrobial agents able to target such cells. The present study explored the possibility to obtain in vitro PCs of Pseudomonas aeruginosa and Staphylococcus aureus at high efficiency through chemical treatment, and to test their susceptibility to structurally different antimicrobial peptides (AMPs) and two clinically used peptide-based antibiotics, colistin and daptomycin. The main mechanism of action of these molecules (i.e., membrane-perturbing activity) renders them potential candidates to act against dormant cells. Exposure of stationary-phase cultures to optimized concentrations of the uncoupling agent cyanide m-chlorophenylhydrazone (CCCP) was able to generate at high efficiency PCs exhibiting an antibiotic-tolerant phenotype toward different classes of antibiotics. The metabolic profile of CCCP-treated bacteria was investigated by monitoring bacterial heat production through isothermal microcalorimetry and by evaluating oxidoreductase activity by flow cytometry. CCCP-pretreated bacteria of both bacterial species underwent a substantial decrease in heat production and oxidoreductase activity, as compared to the untreated controls. After CCCP removal, induced persisters showed a delay in heat production that correlated with a lag phase before resumption of normal growth. The metabolic reactivation of bacteria coincided with their reversion to an antibiotic-sensitive phenotype. Interestingly, PCs generated by CCCP treatment resulted highly sensitive to three different membrane-targeting AMPs at levels comparable to those of CCCP-untreated bacteria. Colistin was also highly active against PCs of P. aeruginosa, while daptomycin killed PCs of S. aureus only at concentrations 32 to 64-fold higher than those of the tested AMPs. In conclusion, CCCP treatment was demonstrated to be a suitable method to generate in vitro PCs of medically important bacterial species at high efficiency. Importantly, unlike conventional antibiotics, structurally different AMPs were able to eradicate PCs suggesting that such molecules might represent valid templates for the development of new antimicrobials active against persisters.

Viable but non‐culturable and persistence describe the same bacterial stress state

“…We have verified seven ways that these rifampicin pre-treated cells are persister cells (multidrug tolerance, easy conversion from persister state to non-persister state, metabolic dormancy, no change in the minimum inhibitory antibiotic concentration compared with exponential cells, no resistance phenotype, similar morphology to natural persisters and similar resuscitation as natural persisters) (Kim and Wood, 2018). Critically, this method of creating a high concentration of persister cells, by pretreating with one of the compounds we utilized for E. coli, carbonyl cyanide mchlorophenylhydrazone (Kwan et al, 2013), has been validated for Pseudomonas aeruginosa and S. aureus (Grassi et al, 2017); pretreating with rifampicin and tetracyline to generate E. coli persisters has also been verified by an independent group (Cui et al, 2018). We also compared the rates at which individual cells resuscitate by using agarose pads and light microscopy as well as compared cell morphology via transmission electron microscopy (TEM).…”

Single cell observations show persister cells wake based on ribosome content

“…Based on these eight independent lines of evidence, these rifampicin-induced persisters are bona fide persister cells. Furthermore, this method of generating a high concentration of persister cells has been recently validated for Pseudomonas aeruginosa and Staphylococcus aureus (Grassi et al, 2017).…”

Persister cells resuscitate via ribosome modification by 23S rRNA pseudouridine synthase RluD

“…Furthermore, our method has been used to (i) determine that persister cells wake via ribosome activation (Kim et al, 2018b) and (ii) by activating chemotaxis (Yamasaki et al, 2019), (iii) show that the cells capable of resuscitation in a viable but not culturable population are equivalent to persister cells (Kim et al, 2018a), (iv) identify compounds that kill persister cells and (v) reveal that the alarmone ppGpp directly creates persister cells by stimulating ribosome dimerization . In addition, our method to generate a high population of persister cells has been utilized by at least six independent groups (Grassi et al, 2017;Cui et al, 2018;Narayanaswamy et al, 2018;Sulaiman et al, 2018;Tkhilaishvili et al, 2018;Pu et al, 2019).…”

“…Yamasaki et al, 2019). RluD is involved in the synthesis and assembly of 70S ribosomes as well as their function based on its post-transcriptional modification of 23S rRNA to form three pseudouridine (5-ribosyl-uracil) nucelosides at positions 1911, 1915, and 1917(Gutgsell et al, 2005. In pseudouridine, uracil is attached via a carbon-carbon bond to the sugar base rather than through a carbon-nitrogen bond.…”