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Pseudolysogeny and sequential mutations build multiresistance to virulent bacteriophages in Pseudomonas aeruginosa

Abstract : Bacteriophages are obligate parasites of bacteria that can be defined as virulent or temperate according to their lifestyle: virulent phages perform a lytic cycle by injecting their genome in the bacterial cell and immediately multiply. Temperate phages, instead, can either perform a lytic, or a lysogenic cycle by integrating their genome into the bacterial chromosome and persisting in a dormant state until the lytic cycle is resumed. The viral genome can also be maintained in the bacterial cell in an episomal form for an undetermined period of time in a stage known as pseudolysogeny. P. aeruginosa, a bacterium commonly found in the environment and in association with many hosts including plants and animals, is responsible for severe nosocomial infections. A proportion of clinical strains are multidrug-resistant, possessing a high ability to form biofilms which are very difficult to eradicate with conventional treatments. It is therefore essential to find new therapeutic approaches, such as phage therapy. Numerous clinical data obtained in Eastern Europe and Russia attest the effectiveness and safety of phage therapy. However, there remain uncertainties related to their therapeutic use and particularly the high frequency of natural resistance. Our project aimed to better understand the dynamic of phage/bacteria interactions by studying the resistance mechanisms acting in the reference strain P. aeruginosa PAO1, against virulent phages. Infections were performed by combining phages belonging to four different genera: Ab05, a ФKMV-like podovirus, Ab09, a LIT1-like podovirus, Ab27, a PB1-like myovirus and Ab17, a KPP10-like myovirus, all isolated in our laboratory. Single or multiple infections of P. aeruginosa PAO1 were performed, and a collection of phage-resistant variants was isolated and analysed. The frequency of phage-resistant variants selection was 10⁻⁵ for single phage infection, and 10⁻⁶ for infections with cocktails of two or four phages. The phenotype and mobility of the variants was often affected, as compared to the parental strain. The genome of 27 variants was entirely sequenced by Illumina technology in order to identify mutations responsible for the resistance. Other variants were analysed by a candidate gene approach. We identified point mutations or small indels: in total, 27 independent mutations affected 14 genes and 1 regulatory region. The affected genes encode proteins involved in biosynthesis of type IV pili (T4P) and lipopolysaccharide (LPS), frequently used as receptors by the phages. Other mutations were observed in genes necessary for alginate production. Of interest, we found that half of the variants with mutations in genes involved in LPS biosynthesis possessed unstable phase variation mutations, responsible for translation frameshift. In contrast, genes involved in pilus type IV biogenesis were mainly subjected to deletions. Surprisingly, the presence of free phage DNA was found in association with exclusion of superinfection in half of the variants and no chromosomal mutation could be found in three of them. Thus, we showed that pseudolysogeny is a frequent outcome of infection by virulent phages of P. aeruginosa. Moreover, double mutants were selected at high frequency and this could presumably due to evolutionary pressure exerted by re-activation of lytic cycle in some cells of the pseudolysogen population. In conclusion, if phage predation selects for variants with alterations in genes involved in biogenesis or regulation of virulence determinants such as LPS or alginate, the resulting phage-resistant variants could potentially exhibit altered levels of virulence in a beneficial or detrimental way. The use of cocktail does not lower significantly the frequency of phage-resistance and in addition we show that pseudolysogeny is a major actor in the selection of mutations.
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  • HAL Id : tel-01499845, version 1

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Libera Latino. Pseudolysogeny and sequential mutations build multiresistance to virulent bacteriophages in Pseudomonas aeruginosa. Bacteriology. Université Paris-Saclay, 2016. English. ⟨NNT : 2016SACLS274⟩. ⟨tel-01499845⟩

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