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Evolution of Prophages that carry Antibiotic Resistance Genes (ARGs) and their host-bacteria in response to antibiotics and increased lytic activity

Periodic Reporting for period 1 - ProphARG (Evolution of Prophages that carry Antibiotic Resistance Genes (ARGs) and their host-bacteria in response to antibiotics and increased lytic activity)

Reporting period: 2018-09-01 to 2020-08-31

Antibiotic resistance genes (ARGs) can spread horizontally by mobile genetic elements, such as prophages, i.e. viruses that can incorporate their own genetic material into the bacterial chromosome (then called lysogen).
Prophages can increase their hosts’ fitness through additional genes, such as ARGs and by their ability to kill phage-susceptible competitors. However, spontaneous or environment-dependent prophage induction can also be costly for the lysogen. In addition, ARGs often come with additional costs, especially in the absence of the selective antibiotic.
A better understanding of how prophage-associated fitness effects of antibiotic resistant lysogens vary across environments is key to elucidate how prophages contribute to the ecology and evolution of antimicrobial resistance.
At the start of the project, the I set out to address the following questions: (1) How does the net fitness effect of prophage carriage vary across different antibiotic concentrations? (2) Are these effects specific to prophages that encode ARGs? (3) Are prophages more beneficial in environments where lysis is more frequent? (4) In which environments is the acquisition of ARG-carrying prophages by susceptible strains most likely?
During the fellowship I performed pairwise competitions to investigate fitness benefits derived from prophage encoded ARGs relative to susceptible wild types using four different phage genotypes. Competitions were performed in the presence and absence of three different antibiotics and along an antibiotic gradient while accounting for spontaneous and environment-dependent prophage induction. By using prophages that carry antibiotic resistance genes (ARGs) and ARG-free versions I was able to disentangle the fitness benefit of the prophage encoded ARG from the prophage alone.
We observed that in the presence of antibiotics, the resistance gene was highly beneficial. However, when we added a substance to the competing culture that increased prophage induction and ultimately the amount of free phages, we observed that those phages were more important than the resistance gene in determining the fitness benefit of the lysogen.

These results have been presented by means of oral presentation at the German National Phage Meeting, at the MSCA cluster event on antimicrobial resistance and during the Congress of the European Society for Evolutionary Biology. A manuscript is currently in preparation and will be submitted soon.

I also gave an interview about my results for a local school.
We provide important empirical evidence, that lysogens benefit more from free phages than from phage-encoded resistance genes during direct competition in the presence of antibiotics and in environments where prophage induction is high. By taking phage-mediated drug-resistance into account our study extends previous observations that prophages can enhance lysogen fitness by lysing phage-susceptible competitors (Davies et al 2016, Gama et al 2013). Moreover, we show that sub-inhibitory concentrations of antibiotics favour the horizontal transfer of phage encoded resistance genes to phage- and antibiotic susceptible strains. This suggests that phages, which are a massive reservoir of resistance genes (Calero-Caceres et al 2019), might play a key role in the horizontal transfer of ARGs in bacterial communities. Future studies should investigate how these strains, which recently acquired an ARG-encoding prophage, coevolve with their prophage and in which environments they are most likely to rise in frequency in the community over longer time-scales. This will improve our basic understanding of the mobilization of ARGs by phages and shed new insights into open questions in the current antimicrobial resistance crisis.