Duality of the lytic phages Sb-1 and NP-3 : How well does co-predation work against Staphylococcus aureus and Pseudomonas aeruginosa under varying environmental conditions?

Simon, Kevin; Horz, Hans-Peter (Thesis advisor); Panstruga, Ralph (Thesis advisor)

Aachen : RWTH Aachen University (2022, 2023)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2022

Abstract

In the area of the antibiotic-crisis we fish in troubled water how to counter the uprising number of multi-drug resistant bacteria (MDR). Phage therapy could be an alternative for topical treatment of chronic wound infections where typically Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) together drive the infection. In contrast to the high number of mono-culture studies, this study investigated the simultaneous challenge of a bacterial co-culture of SA and PA with the SA-phage Sb-1, the PA-phage NP-3 and the antibiotic meropenem (MP). Accurate species-specific enumeration of both bacteria and phages was neither achieved with fluorescence proteins encoding plasmids, nor the fluorescence compound pyoverdine naturally produced by PA, nor fluorescence activated cell sorting (FACS). Instead, quantitative PCR (qPCR) along with bacterial plating and phage spotting was highly precise with no interferences between the bacterial species, the two phages and MP. Liquid culture assays with Sb-1 or NP 3 in combination with MP showed that phages and β-lactam antibiotics work mostly beneficial in clearing bacterial monocultures. In contrast, in co-cultures, both phages interfered with each other while preying on their different hosts. The downregulation of bacterial and phage gene expression caused a prolonged phage propagation and a delayed antibacterial killing. Conversely, the addition of MP not only restored, but also enhanced phage based bacterial clearance by the strong upregulation of phage gene expression. All these phage-host antibiotic interactions were largely comparable between culture medium, equine serum and in a pig skin model, but occurred over different time scales. In addition, it was found, that the phages "hibernated" in metabolic inactive bacterial cells. They latently replicated in their host but started to produce new phage progenies only when the infected hosts switched back to active metabolism. As a conclusion, the assessments of phage compatibilities in cocktails are vital for generating synthetic phage communities with meaningful effects against bacterial pathogens in mixed infections. Notably, antibiotics which have become useless against MDR, are still of therapeutic value, as they not only assist in overcoming negative phage interactions, but boost phage based bacterial killing. Understanding the underlying molecular mechanisms of predator-prey relationships beyond bacterial monocultures takes us one important step further towards phage-based therapeutic concepts against MDR.

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