Figure 1. Illustration of the mutant selection procedure. Growth curves of a randomly selected persistence mutant (triangles) and of PA14 wild type (circles) incubated in an automated optical density (OD) plate reader, both after control treatment (open symbols) and after treatment with ofloxacin (closed symbols). ΔT (calculated as ΔT = Δt wild type (dashed line) – Δt mutant (straight line)) was used as an estimation for the difference in lag phase between ofloxacin-treated cultures of mutant and wild type and was used as a parameter for statistical selection of mutants.
Figure 2. Mean relative persister fraction of the nine selected P. aeruginosa mutants. The persister fraction is defined as the number of surviving cells after treatment with ofloxacin, divided by the number of cells of the control condition. The relative persister fraction for each mutant is the persister fraction of the mutant divided by that of the wild type (a value of 1 in the y-axis).
Genetic determinants of persistence in Pseudomonas aeruginosa
Even though P. aeruginosa persistence is of great clinical relevance, the organism has been the subject of few molecular studies on this matter. When we started our studies on P. aeruginosa persistence, the only persistence genes that had been discovered in P. aeruginosawere the global regulators spoT, relA, dksA and rpoS.
In 2009, we carried out and published the first large-scale screening of a P. aeruginosa mutant library to identify genes that contribute to the persistence phenomenon in this pathogen (De Groote et al., 2009). A screening procedure was designed to select, in a high-throughput manner, mutants exhibiting a significant difference in persister fraction after prolonged treatment with the fluoroquinolone antibiotic ofloxacin.
Several mutants with either an increased or a decreased number of surviving persister cells were identified and subjected to sequence analysis.
Following up on the screening, we characterized in more detail the interplay between fosfomycin resistance mechanisms and persistence (De Groote et al., 2011).
A library of 5000 P. aeruginosa PA14 mutants was constructed by random insertion of the pTnMod-OGm plasposon and screened for persistence mutants in a high-throughput manner. To select mutants affected in genes that play a role in persistence, the selection procedure was based on differential survival after prolonged exposure to ofloxacin compared with the wild type.
After treatment, the cells were diluted in growth medium and growth curves originating from the surviving cells were generated by automated microtiter plate measurements. The selection for persistence mutants was based on a significant difference in lag phase compared with the wildtype cells. Mutants with a lower or a higher number of persister cells will display a longer or a shorter lag phase, respectively, when they are recultured after ofloxacin treatment compared with the wild type (see Fig. 1).
After an initial round of screening, independent confirmation by plate counts and determination of MIC50 values to exclude resistant mutants, nine mutants consistently displaying an altered persister fraction were retained.
Four mutants showed a decreased persister fraction, ranging from a 2.8- to a 16-fold reduction compared with the wild-type strain.
Five mutants exhibited a higher number of persister cells after treatment, between 2.6- and 18.4-fold the number of persister cells compared with the wild type (see Fig. 2).
Sequence analysis revealed the plasposon insertion site in the selected mutants to be located in genes of diverse functional classes, such as enzymes and regulators involved in various cellular processes: algR, dinG, pheA, pilH, spuC, ycgM, PA_13680 and PA_66140 (Table 1). Further analysis of these newly discovered persistence genes may, in the future, lead to new candidate targets for improved drug development.
Liebens V., Defraine V., Van der Leyden A., De Groote V.N., Fierro C., Beullens S., Verstraeten N., Kint C., Jans A., Frangipani E., Visca P., Marchal K., Versées W., Fauvart M., Michiels J. (2014) A putative de-N-acetylase of the PIG-L superfamily affects fluoroquinolone tolerance in Pseudomonas aeruginosa. Pathog. Dis. 71:39-54 -- PubMed -- PDF
Fauvart M., De Groote V.N., Michiels J. (2011). Role of persister cells in chronic infections: clinical relevance and perspectives on anti-persister therapies. J. Med. Microbiol. 60:699-709 -- PubMed -- PDF -- LabNews feature
De Groote V.N., Fauvart M., Kint C.I., Verstraeten N., Jans A., Cornelis P., Michiels J. (2011). Pseudomonas aeruginosa fosfomycin resistance mechanisms affect non-inherited fluoroquinolone tolerance. J. Med. Microbiol. -- PubMed -- PDF -- JMM Editorial -- SGM Press Release -- De Standaard (piece in Belgian newspaper) -- NRC Handelsblad (piece in Dutch newspaper)
De Groote, V.N., Verstraeten, N., Fauvart, M., Kint, C.I., Verbeeck, A.M., Beullens, S., Cornelis, P., Michiels, J. (2009). Novel persistence genes in Pseudomonas aeruginosaidentified by high-throughput screening. FEMS Microbiol. Lett. -- PubMed -- PDF -- Faculty of 1000 evaluation
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