Biology subjects

Petersen, M. E.

Publications and source records attributed to Petersen, M. E..

3 recordsLinked to original sources

bioRxiv2024

Mitomycin C Retains Efficacy after Adaptive Laboratory Evolution of Staphylococcus aureus

Antibiotic resistance is one of the greatest threats against human health and the misuse and overuse of antibiotics is a key factor driving resistance development. During prolonged antibiotic treatment of chronic infections, the antimicrobial pressure facilitates selection of antibiotic resistance mutations. It has been suggested that using antibiotics in combinations may reduce the emergence of resistance. Furthermore, antibiotic tolerant persister cells may be a reservoir for resistance development, so targeting persister cells with anti-persister drugs could also reduce the emergence of resistance. In this study, we conducted a 42-day adaptive laboratory evolution experiment using Staphylococcus aureus exposed to common antibiotics and the anti-persister drug mitomycin C, either alone or in combination. We monitored susceptibility daily and assessed phenotypic changes in growth and biofilm formation in evolved strains. Whole-genome sequencing revealed mutations linked to antibiotic resistance and phenotypic shifts. Resistance developed rapidly against rifampicin, while ciprofloxacin and daptomycin showed slower resistance emergence. Treatments with vancomycin or mitomycin C resulted in minimal changes in susceptibility. Combination therapies generally delayed resistance, though resistance was not fully prevented. Notably, mitomycin C combined with rifampicin effectively suppressed rifampicin resistance. Sub-inhibitory antibiotic concentrations were associated with both known and novel mutations, including in the nucleotide excision repair system and azoreductase, following mitomycin C treatment--mutations not previously reported. While combination therapy delayed resistance, mitomycin Cs efficacy and ability to prevent rifampicin resistance highlights its potential in combating antibiotic resistance. Further investigation is needed to evaluate the broader application of anti-persister drugs in resistance prevention.

microbiology↗

bioRxiv2023

A novel high-throughput assay identifies small molecules with activity against persister cells

Persister cells are a subpopulation of transiently antibiotic tolerant bacteria, which are believed to be the main cause of relapsing bacterial infections. Due to the importance of persister cells in human infections, there is a need for new antibiotics that kill bacteria independently of their activity. However, high-throughput assays to screen for drugs with such activity are missing. This is partly due to the transient nature of the phenotype, which makes it is difficult to prepare a concentrated population of persister cells that remain inactive during incubation with antibiotics in standard growth media. The purpose of this study was to develop a simple and high-throughput assay to identify compounds with antimicrobial activity against persister cells during a 24 h incubation period. Subsequently, this assay was used to screen a selection of small molecules with hypothesized antimicrobial activity. The fraction of S. aureus that tolerate bactericidal concentrations of ciprofloxacin were defined as persister cells. We first quantified how the cell concentration, growth phase, antibiotic concentration, duration of antibiotic exposure, and presence/absence of nutrients during antibiotic exposure affected the fraction of persister cells in a population. After optimizing these parameters, we compared our approach to generate persister cells, to a process that generated persister cells by a short exposure to rifampicin. Finally, we used the optimised protocol to identify molecular structures that have anti-persister activity by performing screening on initially compound fragments and then selecting compounds that incorporated the fragments that displayed activity. We show that exponential- and stationary-phase cultures transferred to nutrient-rich media only contain a small fraction (0.001 to 0.07 %) of persister cells that tolerated 10, 50 and 100 x MIC ciprofloxacin. Exponential-phase cultures displayed a bi-phasic time-kill curve, which plateaued after 5 h exposure, while stationary phase cultures displayed a low, but constant death rate at 50 and 100 x MIC ciprofloxacin. Inducing the persister phenotype with a short rifampicin treatment resulted in 100% persister cells when evaluated after [≤]5 h exposure to ciprofloxacin. However, after longer incubation times, cells resumed activity and lost their tolerance to ciprofloxacin. Tolerance was only maintained in the majority of the population for the full 24 h incubation period if cells were transferred to a carbon-free minimal medium before exposure to ciprofloxacin. We conclude that keeping cells starved in a carbon-free medium enables generation of high concentrations of S. aureus cells that tolerate 50 x MIC ciprofloxacin, and we find this protocol easily applicable for rapid screening of anti-persister drugs that act on dormant or non-dividing cells.

microbiology↗

bioRxiv2023

Toxin/Antitoxin Systems Induce Persistence and Work in Concert with Restriction/Modification Systems to Inhibit Phage

Myriad bacterial anti-phage systems have been described and often the mechanism of programmed cell death is invoked for phage inhibition. However, there is little evidence of suicide under physiological conditions for these systems. Instead of death to stop phage propagation, we show here that persister cells, i.e., transiently-tolerant, dormant, antibiotic-insensitive cells, are formed and survive using the Escherichia coli C496_10 tripartite toxin/antitoxin system MqsR/MqsA/MqsC to inhibit T2 phage. Specifically, MqsR/MqsA/MqsC inhibited T2 phage by one million-fold and reduced T2 titers by 500-fold. During T2 phage attack, in the presence of MqsR/MqsA/MqsC, evidence of persistence include the single-cell physiological change of reduced metabolism (via flow cytometry), increased spherical morphology (via transmission electron microscopy), and heterogeneous resuscitation. Critically, we found restriction-modification systems (primarily EcoK McrBC) work in concert with the toxin/antitoxin system to inactivate phage, likely while the cells are in the persister state. Phage attack also induces persistence in Klebsiella and Pseudomonas spp. Hence, phage attack invokes a stress response similar to antibiotics, starvation, and oxidation, which leads to persistence, and this dormant state likely allows restriction/modification systems to clear phage DNA.

molecular biology↗