A novel bacteriophage CUB19 specific to the bacterial species Stenotrophomonas maltophilia was isolated from hospital sewage and characterized as a new species belonging to a proposed new phage genus ‘Cubvirus’ (Caudoviricetes). Its genome contains a total of 48,301 bp and 79 predicted genes, among which some have been associated with packaging and lysis-associated proteins, structural proteins, or DNA- and metabolism-associated proteins. No lysogeny-associated proteins or known virulence proteins were identified on the phage genome. CUB19 showed stability over a wide range of temperatures (−20 °C–60 °C) and pH values (pH 3–pH 13). Despite its narrow host range, this phage has potent observed antimicrobial and antibiofilm activity. A time-killing curve assay showed significant biofilm reduction after 24 h exposure to CUP19. Isothermal microcalorimetry assays investigating phage-antibiotic combinations revealed the effectiveness of CUB19 during co-administration with increasing antibiotic doses, regardless of the administration approach (simultaneous or staggered). These are encouraging indications for its application as a targeted therapeutic agent against resilient biofilm-associated Stenotrophomonas infections.

This protocol is designed for the extraction of bacteriophage (phage) DNA used in downstream applications such as whole genome sequencing. For this purpose, the phage lysate is treated with DNase I and RNase A to degrade any DNA or RNA that is not enclosed by the viral capsid (e.g., from bacterial origin), and then EDTA and SDS are added to inactivate the DNase I/RNase A along with the addition of proteinase K to digest the viral capsid. Thereafter, protein debris are removed by sequential extraction with phenol-chloroform and precipitation with ethanol, which increases DNA concentration and removes excess salts. Compared to commercial kits, the price per sample is lower for this protocol, without entailing greater complexity or yielding inferior results.