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.

Bacteriophage and the Innate Immune System: Access and Signaling

“…Could our understanding of phage biology be correct when most experiments investigated an artificial group consisting of only a single type of phage? Consistent with the idea that our experimental approaches may have masked the true nature of phage biology, is the finding by a number of groups that a bacteriophage population is capable of broadening their host range [93][94][95]. Such findings would argue away from the current paradigm that bacteriophage targets only a narrow, fixed range of bacteria.…”

Bioprospecting Staphylococcus Phages with Therapeutic and Bio-Control Potential

“…A potential problem with clinical strains as propagation hosts is that they are likely to contain prophages and may produce enterotoxins, therefore, it is safer to propagate the phages in a food-quality S. xylosus. Even though the Stab phages had distinct host ranges, with Stab20 and Stab21 having a wide and Stab22 and Stab23 a narrow host range, their genomes proved to be closely related to other phages of therapeutic and biocontrol significance such as phage Sb-1 [31,57]. Proteomic analysis depicted uniformity among some of their structural proteins with the Staphylococcus phage K and other members of Kayvirus genus [58].…”

Bacteriophage Therapy: Developments and Directions

“…In vitro evolution (or phage adaptation, phage training) to expand or optimize host range is an approach to modify and improve phages that has been used extensively in former Soviet countries and Eastern Europe, and was recently described in the Western literature [37,[47][48][49]. Alteration of phage host range by this approach can also potentially be used to discover novel molecular mechanisms that can be exploited for genetic modification strategies [50]. Genetic engineering to improve phages as therapeutic candidates has included the alteration or expansion of host range [51], adding factors to enhance therapeutic activity including genes that encode enzymes that degrade biofilms [52] or expressing heterologous genes to increase phage killing potency.…”

“…The design of fixed phage cocktails for different bacterial pathogen species must by nature be centered on the genetic-phenotypic diversity of the pathogen and the characteristics of the lytic phages that infect it. For instance, in the case of Staphylococcus aureus, it is widely accepted that relatively few phage components-or even a single phage, in the case of Eliava's Staphylococcal Phage therapeutic-could be selected to construct therapeutic cocktails that address a significant diversity of the global strains of the pathogen [50,61]. This is because of the relatively broad host range exhibited by Twort and K phages combined with (and perhaps because of) the relatively monomorphic nature of the host species.…”