Persister cell-mediated antimicrobial tolerance in pseudomonas aeruginosa biofilm populations

Abstract

Bacteria preferentially live in biofilms; complex structures of single- or multiple- species of bacteria, surrounded by a self-produced polymeric extracellular matrix and attached to a surface. In this state, they are extremely recalcitrant to antibiotics, in part due to the presence of dormant or very slowly replicating cells called persisters. While lacking specific antibiotic resistance genes, persister cells are able to survive lethal stress due to their slow replication state, and once the antibiotic challenge has been removed, can seed fresh populations of surviving bacteria.

Despite recent progress in understanding molecular determinants of persister cell formation, few studies have examined in vitro the evolutionary and ecological drivers that sustain their presence. A functional role for persister cells is hypothesized in deferring replication within bacterial populations that are too crowded to sustain growth. This model predicts that slow-growing lineages with increased persister allocations will arise among density dependent bacterial populations, and that frequent exposure to lethal and non-lethal stresses will impact persister allocation. In support of the model, it was found that flow-cell biofilm culture rapidly resulted in new P. aeruginosa lineages with increased persistence, and that exposure to antibiotics can enhance this process. These observations have important clinical implications where chronic biofilm infections are treated with long-term antibiotic exposure.

The second results chapter examines the hypothesis that hypermutable phenotypes of P. aeruginosa are a characteristic of persister cell biology. Recent research has shown that key features of the development of bacterial biofilms include increasing mutation rates and increasing numbers of persister cells and the present work addresses whether there may be a relationship between these phenomena. A detailed examination of mutation levels and SOS response-mediated gene expression was carried out in persister cells. Persisters that had been isolated using both DNA damaging (ofloxacin) and non-DNA damaging (ethanol) antimicrobial treatments exhibited significantly increased frequencies of mutation to rifampicin resistance. Gene expression analysis of persister cells isolated following ethanol treatment demonstrated up-regulation of mutS, a DNA mismatch repair gene, suggesting that persister cells may undergo elevated DNA damage and mutation that can occur independently of the SOS response.

The third chapter aimed to study the role of persisters in determining the spatial and temporal pattern of biofilm formation following antibiotic treatment. A key feature of biofilms thought to play a role in antimicrobial tolerance is their ability to develop discrete, differentiated microcolony structures during colonization of a surface - these foci within biofilms are highly recalcitrant towards antimicrobials yet the factors that determine their differentiation and growth are poorly understood. This chapter therefore aimed to study the role of persisters in the initiation of microcolony foci and in mediating regrowth of biofilms. In this work, biofilm initiation was studied under a variety of conditions including with or without exposure to lethal or sub-lethal antibiotic challenge and as expected persister cell populations were able to generate significantly more biomass than in biofilms formed from non-persister populations. Dual labelling experiments were also carried out, where mixed persister and non-persister populations were tagged with either red or green fluorescent proteins. These experiments demonstrated that persister-mediated regrowth predominantly occurs as discrete microcolony foci that do not mix with the surviving non-persister cell population, suggesting that persisters are the progenitors of clonal microcolony foci within biofilms that are structurally distinct from the rest of the population of cells within the biofilm.

In summary, this thesis provides new information on the biology of persister cells in P. aeruginosa biofilms, and contributes to scientific understanding of the emergence of antibiotic tolerance in bacterial biofilm populations.

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Identifiers

ORCID for Jeremy Webb: orcid.org/0000-0003-2068-8589

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