Correlation of Host Range Expansion of Therapeutic Bacteriophage Sb-1 with Allele State at a Hypervariable Repeat Locus

Abstract

Staphylococci are frequent agents of health care-associated infections and include methicillin-resistant Staphylococcus aureus (MRSA), which is resistant to first-line antibiotic treatments. Bacteriophage (phage) therapy is a promising alternative antibacterial option to treat MRSA infections. S. aureus-specific phage Sb-1 has been widely used in Georgia to treat a variety of human S. aureus infections. Sb-1 has a broad host range within S. aureus, including MRSA strains, and its host range can be further expanded by adaptation to previously resistant clinical isolates. The susceptibilities of a panel of 25 genetically diverse clinical MRSA isolates to Sb-1 phage were tested, and the phage had lytic activity against 23 strains (92%). The adapted phage stock (designated Sb-1A) was tested in comparison with the parental phage (designated Sb-1P). Sb-1P had lytic activity against 78/90 strains (87%) in an expanded panel of diverse global S. aureus isolates, while eight additional strains in this panel were susceptible to Sb-1A (lytic against 86/90 strains [96%]). The Sb-1A stock was shown to be a mixed population of phage clones, including approximately 4% expanded host range mutants, designated Sb-1M. In an effort to better understand the genetic basis for this host range expansion, we sequenced the complete genomes of the parental Sb-1P and two Sb-1M mutants. Comparative genomic analysis revealed a hypervariable complex repeat structure in the Sb-1 genome that had a distinct allele that correlated with the host range expansion. This hypervariable region was previously uncharacterized in Twort-like phages and represents a novel putative host range determinant.IMPORTANCE Because of limited therapeutic options, infections caused by methicillin-resistant Staphylococcus aureus represent a serious problem in both civilian and military health care settings. Phages have potential as alternative antibacterial agents that can be used in combination with antibiotic drugs. For decades, phage Sb-1 has been used in former Soviet Union countries for antistaphylococcal treatment in humans. The therapeutic spectrum of activity of Sb-1 can be increased by selecting mutants of the phage with expanded host ranges. In this work, the host range of phage Sb-1 was expanded in the laboratory, and a hypervariable region in its genome was identified with a distinct allele state that correlated with this host range expansion. These results provide a genetic basis for better understanding the mechanisms of phage host range expansion.

Keywords: Staphylococcus aureus; adaptation; bacteriophage adaptation; genome analysis; host range expansion; hypervariable DNA region; phage Sb-1; putative host range determinant; therapeutic bacteriophage.

FIG 1

Diversity of S. aureus strains according to PFGE. Strains resistant to the parental Sb-1 therapeutic preparation (Sb-1P) manufactured by Eliava Biopreparations, Ltd., are marked in red. Strains that were also resistant to the adapted Sb-1 (Sb-1A) are marked with yellow highlighting. Sequence type (ST) based on multiple locus sequence typing is also provided in blue type, if available.

FIG 2

Phage adsorption assay. The bars represent the titers of nonadsorbed phage particles in supernatants after precipitation of bacterial cells and phage particles adsorbed to them. Green, Sb-1P; purple, Sb-1M.

FIG 3

Repeat structure analysis of genomic sequence repeat regions. (A) Physical location of the three repeat regions and major indels in the Sb-1 reference genome; (B) annotation of the repeat region 2 locus; (C) complex repeat structure of the region 2 locus illustrating direct and indirect repeats. Boxed regions indicate repeated sequence units. Horizontal lines designate direct repeats.

FIG 4

Region 2 allele diversity in Sb-1P and Sb-1A phage plaques. (A) PCR products of region 2 from Sb-1P clones plated on MRSN8383. (B) PCR products of region 2 from Sb-1A clones plated on MRSN8383. (C) PCR products of region 2 from Sb-1A clones plated on MRSN9832. The control lanes (ctrl) contain the PCR product of region 2 of the Sb-1 production stock (Sb-1P) without any plaque/clone purification.

FIG 5

Dot plot analysis. Dot plots illustrating the region 2 repeat structure of individual clones derived from the dominant genotype of Sb-1A phage plated on the phage-susceptible strain MRSN8383 (A to C) and from the dominant genotype of Sb-1M plated on the previously phage-resistant strain MRSN9832 (D to F).

FIG 6

PCR fragment size variation at the region 2 locus for isolated plaque clones of Sb-1P (A) and of Sb-1M (B). Control lanes (ctrl) show the PCR product of region 2 of the Sb-1P production stock without plaque/clone purification.

FIG 7

Dot plot comparisons of region 2 locus sequences among Sb-1P (A) and Sb-1M (B) plaques.