Phage stability is important for the successful application of bacteriophages as alternative antibacterial agents. Considering that temperature is a critical factor in phage stability, this study aimed to explore the possibility of improving long-term phage stability through adaptive evolution to elevated temperature. Evolution of three wild-type ancestral phages (Myoviridae phage Wc4 and Podoviridae phages CX5 and P-PSG-11) was induced by subjecting the phages to heat treatment at 60 °C for five cycles. The adapted phages showed better stability than the wild-type ancestral phages when subjected to heat treatment at 60 °C for 1 h and after 60 days of storage at 37 °C. However, the adapted phages could not withstand thermal treatment at 70 °C for 1 h. The infectivity and the lytic properties of the phages were not changed by the evolution process. Whole-genome sequencing revealed that single substitutions in the tail tubular proteins were the only changes observed in the genomes of the adapted phages. This study demonstrates that adaptive evolution could be used as a general method for enhancing the thermal stability of phages without affecting their lytic activity. Sequencing results showed that bacteriophages may exist as a population with minor heterogeneous mutants, which might be important to understand the ecology of phages in different environments.

Bacteriophages exert strong selection on their bacterial hosts to evolve resistance. At the same time, the fitness costs on bacteria following phage resistance may change their virulence, which may affect the therapeutic outcomes of phage therapy. In this study, we set out to assess the costs of phage resistance on the in vitro virulence of priority 1 nosocomial pathogenic bacterium, Acinetobacter baumannii. By subjecting phage-resistant variant Ev5-WHG of A. baumannii WHG40004 to several in vitro virulence profiles, we found that its resistance to phage is associated with reduced fitness in host microenvironments. Also, the mutant exhibited impaired adhesion and invasion to mammalian cells, as well as increased susceptibility to macrophage phagocytosis. Furthermore, the whole-genome sequencing of the mutant revealed that there exist multiple mutations which may play a role in phage resistance and altered virulence. Altogether, this study demonstrates that resistance to phage can significantly alter phenotypes associated with virulence in Acinetobacter baumannii.

African swine fever virus (ASFV) has devastating impacts on swine health and the world economy. Rapid and accurate phylogenetic analysis of ASFV causing outbreaks is important to reveal diversity and evolutionary of ASFV. Because it is time-consuming and needs biosafety laboratories to isolate ASFV, here we present a new way to perform rapid genome-wide phylogenetic analysis of ASFV using an allele calling based on gene by gene approach directly from genome drafts assembled from metagenomic sequences. Using open-accessed chewBBACA software, 41 publicly available ASFV genomes were analyzed to optimize the parameters and find the alleles. Alleles as many as 94 were found for building the phylogenetic trees, which covered more than 56% of the whole genome. Based on the alleles, current ASFV isolates could be divided into two major clades and a few subclades. Then the method is used to analyze two ASFV genome drafts assembled from two metagenomic sequences of a swine whole blood and a swine spleen tissue collected in Wuhan, China. It shows that the two ASFV genomes showed highest similarity to that of Pig/HLJ/2018 strain and DB/LN/2018 strain, which isolated recently in China. This proved that the ASFV in Wuhan originate from the same source causing the earlier outbreaks in Helongjiang and Liaoning province of China. This method makes it possible to analyze phylogenetic analysis of ASFV draft genomes flexibly without the need of ASFV isolation. Furthermore, because it is based on Allele calling, the ASFV-specific genetic markers found could be translated into clinical diagnostics or can be used broadly to identify conserved putative therapeutic candidates.