Figures and data
Infection by bacteriophage ΦStaph1N drives the loss of β-lactam resistance in MRSA.
A. Schematic of the experimental setup. Drug-resistant (AbxR), phage-sensitive (PhageS) bacterial cultures are infected with phage. The population of infected cells is passaged and allowed to recover. The surviving cell population is resistant to phage infection (PhageR) but has evolved sensitivity to antibiotics (AbxS). B. ΦStaph1N infects MRSA strains MRSA252, MW2, and LAC (left panel). Following infection with ΦStaph1N, evolved cultures of the three MRSA strains are resistant to ΦStaph1N (right panel). C. ΦStaph1N-treated, evolved MRSA strains show significant loss of resistance against oxacillin, compared to the parental strains. Loss of resistance is indicated by the area of bacterial clearance surrounding the antibiotic resistance strip. D. ΦStaph1N treatment causes loss resistance against different β-lactams. Plotted are the fold reductions of minimal inhibitory concentration (MIC) between treated and mock-treated cells. OXI = oxacillin; CEF = cefazolin; AMX = amoxicillin; AMX+CA = amoxicillin & clavulanic acid; VANC = vancomycin.
Evo2 is a variant of ΦStaph1N with higher activity against MRSA.
A. Single Evo2 plaques appear inside hazy ΦStaph1N plaques on LAC (Left-most panel). Compared to ΦStaph1N, Evo2 shows comparable infectivity towards MRSA252 but improved infectivity towards MW2 and LAC (Right-most panels). B. Similar to ΦStaph1N, Evo2 infection reduces β-lactam resistance in MRSA.
Phage infection of MRSA strains produces distinct mutational profiles.
A. Coding sequences (CDS) with mutations from the three MRSA strains following phage treatment or mock treatment. For each strain, three isolates were sequenced and their mutations identified. Mutations are color-coded based on the number of occurrences among the three replicates. B. Categories of genes with mutations that arose in each MRSA strain and treatment condition.
Mutated genes in MRSA following infection with phages ΦStaph1N or Evo2
Phage treatment of MRSA results in attenuated virulence phenotypes.
A. MW2 and LAC strains display hemolytic activity on rabbit blood agar plates, while MRSA252 does not. B. Phage-treated MW2 and LAC strains display reduced hemolysis compared to uninfected cells. C. Surviving cultures of MW2 and LAC treated with either ΦStaph1N (blue) or Evo2 (red) show reduced clumping rates compared to mock untreated cells (teal).
Phage sensitivity of MRSA strains.
Efficiencies of plaquing of phages on MRSA252, MW2, and LAC. Phages were 10-fold serially diluted and spotted onto top agar overlays of each strain. Phage Evo2 shows the highest plaquing efficiency on all three strains.
Growth curves of MRSA strains under varying levels of phage infection.
MRSA252, MW2, and LAC cultures were infected with either ΦStaph1N or Evo2 at the indicated multiplicity of infection (MOI). The optical density (OD600) of the cultures was monitored on an automated plate reader. Each condition was tested in 3 independent replicates.
Phage <λNM1ψ6 infection LAC does not drive the loss of β-lactam resistance.
LAC cultures were infected with phage at an MOI 0.1 and allowed to recover. The surviving cells were then assayed for their MICs against different β-lactams and vancomycin using antibiotic strips.
Sequencing analysis of Evo2.
Evo2 is a mutant form of ΦStaph1N with a nonsense mutation in ORF141. The A to C mutation (marked by the arrow) in Evo2 converts Serine 77 of ORF141 into a stop codon.
Types of polymorphisms in MRSA strains following infection by phage or a mock treatment.
Plotted are the polymorphisms that were found in a gene with an assigned COG category.
Effect of phage infection on biofilm formation in MRSA strains.
Cultures were infected or mock-infected with either ΦStaph1N or Evo2. RP62a is a strain of S. epidermidis with known biofilm forming capability, while LM1680 is a derivative of RP62a that has lost biofilm forming ability.51,52 Biofilm biomass was assessed by staining with Crystal Violet. Solubilized crystal violet was quantified by measuring absorbance at 600 nm. Values represent averages and standard deviations of three replicates. Statistical significance was determined with a two-tailed t-test.
Bacterial strains and bacteriophages used in this study
