Characterization of novelStaphylococcus aureuslytic phage and defining their combinatorial virulence using the OmniLog® system

Abstract

Skin and soft tissue infections (SSTI) caused by methicillin resistant Staphylococcus aureus (MRSA) are difficult to treat. Bacteriophage (phage) represent a potential alternate treatment for antibiotic resistant bacterial infections. In this study, 7 novel phage with broad lytic activity for S. aureus were isolated and identified. Screening of a diverse collection of 170 clinical isolates by efficiency of plating (EOP) assays shows that the novel phage are virulent and effectively prevent growth of 70–91% of MRSA and methicillin sensitive S. aureus (MSSA) isolates. Phage K, which was previously identified as having lytic activity on S. aureus was tested on the S. aureus collection and shown to prevent growth of 82% of the isolates. These novel phage group were examined by electron microscopy, the results of which indicate that the phage belong to the Myoviridae family of viruses. The novel phage group requires β-N-acetyl glucosamine (GlcNac) moieties on cell wall teichoic acids for infection. The phage were distinct from, but closely related to, phage K as characterized by restriction endonuclease analysis. Furthermore, growth rate analysis via OmniLog® microplate assay indicates that a combination of phage K, with phage SA0420ᶲ1, SA0456ᶲ1 or SA0482ᶲ1 have a synergistic phage-mediated lytic effect on MRSA and suppress formation of phage resistance. These results indicate that a broad spectrum lytic phage mixture can suppress the emergence of resistant bacterial populations and hence have great potential for combating S. aureus wound infections.

Development and Use of Personalized Bacteriophage-Based Therapeutic Cocktails To Treat a Patient with a Disseminated Resistant Acinetobacter baumannii Infection

“…This subset of 98 phages was initially selected for screening because previous work had demonstrated that, collectively, these bacteriophages are highly active against a broad range of MDR A. baumannii isolates (data not shown). Within 18 h of receipt of the first clinical isolate, selection of an appropriate therapeutic cocktail for the original isolate, named TP1, was achieved via an OmniLog-based time-kill assay system (19,20). The bacterial isolates from this study are summarized in Table 1.…”

“…Finally, small-scale phage amplifications on their corresponding host bacterium were performed to prepare the A. baumannii-specific phage library, which was subsequently stored at 4°C until required. The growth of A. baumannii clinical isolates TP1, TP2, and TP3 in the presence of phage was evaluated via spot testing and also in a Biolog imaging system (Biolog, Hayward, CA) (19,20). In this case, one covered 96-well plate was used per phage strain and incubated at 37°C for 24 h, with wells positive only for bacteria and test wells with a single phage added at a multiplicity of infection (MOI) of approximately 100.…”

Salvage Bacteriophage Therapy for a Chronic MRSA Prosthetic Joint Infection

“…The Navy's medical research center (NMRC) plaque purified the selected bacteriophage three times in preparation for the two-step amplification process [9]. The plaque purified bacteriophage was used to first infect a 100 mL culture of a producer S. aureus strain SaGR51 grown to~0.1 OD600 and infected at a MOI of~0.1.…”

“…Bacteriophage particles extracted from the CsCl bands were dialyzed four times and 0.22 µm filtered. Approximately, seven (7) mL of this lysate was transferred to APT for further processing [9].…”

Characterizing a Lytic Bacteriophage Infecting Methicillin-Resistant Staphylococcus aureus (MRSA) Isolated From Burn Patients

“…Also, community-acquired MRSA infections have increasingly been identified in the past two decades (10). This microorganism has been declared as an international concern by the World Health Organization (1,15,16). It is estimated that 70% of mortality in burn units is related to infections.…”