Isolation and characterization of novel bacteriophages targeting Stenotrophomonas maltophilia

Abstract

Stenotrophomonas maltophilia is a bacterium often resistant to antibiotics and is a significant cause of nosocomial infections, particularly in immunocompromised patients. Phage therapy has shown promise as a potential treatment for such difficult-to-treat bacterial infections, but research on phages targeting this bacterium is very limited. In this study, we isolated 34 phages using four clinical strains of S. maltophilia and evaluated their infectivity and bactericidal activity. While some phages infected all four strains, many exhibited strain-specific infectivity. We investigated the bacterial growth curves in response to three phages, named Yut1, Yut2, and Yut4, and found that all phages exhibited potent lytic activity against the clinical strains even at low doses. Genome analysis found that the phages did not carry any lysogeny genes, virulence factors, or antibiotic resistance genes, suggesting their high potential as therapeutic phages. Furthermore, phylogenetic analysis suggested that Yut1 and Yut4 belong to a novel phage lineage. These results highlight the therapeutic potential of our novel phages to combat the growing antibiotic resistance problem.

Fig. 1

Heatmap of spot assay for 34 phages on S. maltophilia. A heatmap was generated based on the spot assay results of 34 isolated phages on the four S. maltophilia strains. The vertical axis represents the bacterial strains, and the horizontal axis represents the phage names. “S” indicates that the plaque size is smaller compared to the original host strain used for phage isolation.

Fig. 2

Bactericidal activity of isolated phages Yut1, Yut2 and Yut4. Line graphs depicting the bactericidal activity of phages Yut1, 2, 4 in liquid culture. Host bacteria (used for phage isolation) were inoculated with each phage at MOIs of 0.01, 0.1, 1, and 10, and bacterial growth curves were measured. Data represents the mean of three independent experiments (n = 3), with error bars indicating standard deviation.

Fig. 3

Genome maps of Yut1, Yut2, and Yut4. Circular genome maps of Yut1, Yut2, and Yut4 generated using Pharokka. The outermost circle represents predicted CDSs, with gene functional categories color-coded as indicated in the legend. The innermost circle shows the GC skew, while the middle circle represents the GC content.

Fig. 4

Phylogenetic analysis of Yut1, Yut2, and Yut4. Phylogenetic tree constructed using the VICTOR web tool^, based on whole-genome sequences of phages registered in the ICTV database and the three phages (Yut1, Yut2, and Yut4) isolated in this study. The phages isolated in this study are highlighted in red. Taxonomic classifications at the family, genus, and species levels, GC content, and genome length indicated by the bar graph are shown on the right for each phage. Taxonomic classifications were inferred based on calculated intergenomic distances.

Fig. 5

Transmission electron microscopy (TEM) images of phages Yut1, Yut2, and Yut4. Transmission electron microscope image of Yut1, Yut2, and Yut4 observed at ×50,000 magnification. Representative phages are shown.