1. Vancomycin-intermediate Staphylococcus aureus isolates are attenuated for virulence when compared with susceptible progenitors 
D.R. Cameron, Y.-H. Lin, S. Trouillet-Assant, V. Tafani, X. Kostoulias, E. Mouhtouris, N. Skinner, K. Visvanathan, S.L. Baines, B. Howden, I.R. Monk, F. Laurent, T.P. Stinear, B.P. Howden, A.Y. Peleg 
Clinical Microbiology and Infection 
Volume 23, Issue 10, Pages (October 2017)
DOI: /j.cmi
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2. Fig. 1
Production of α toxin (Hla) and temporal expression of agr-P3 in clinical pairs of vancomycin-susceptible (VSSA) and vancomycin-intermediate (VISA) Staphylococcus aureus. Hla production was measured by quantitative Western blot. VSSA isolates are represented by black bars, VISA isolates are represented by grey bars. VISA strains that harbour an agr mutation are marked with # [14]. Data shown are mean band intensities (arbitrary units) and bars represent SEM from at least three biological replicates. Statistical significance was determined by two-way analysis of variance with multiple comparisons using the Holm–Sidak method, *p <0.05, **p <0.01, ***p <0.001 (a). agr-P3 activity (bioluminescence, relative light units (RLU)) and growth rate (optical density at 600 nm (OD600)) in heart infusion broth grown at 37°C were measured every 11.6 min for 10 h for A8090/A8094 (b), JKD6009/JKD6008 (c), JKD6052/JKD6051 (d) and A9635/A9639 (e). Data represented in black and grey correspond to VSSA and VISA, respectively. Bacterial growth is represented by crosses (×). Closed circles represent native P3 induction of each strain. Open symbols represent strains supplemented with culture supernatant (10% volume/volume) containing exogenous autoinducing peptides from the specified S. aureus strain (S). Error bars represent SEM for readings taken from three biological replicates.
Clinical Microbiology and Infection  , DOI: ( /j.cmi )
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3. Fig. 2
Intracellular passage and cytotoxicity of vancomycin-susceptible (VSSA) and vancomycin-intermediate (VISA) Staphylococcus aureus isolates within infected osteoblasts. Two independent vancomycin-exposed clinical pairs (A9635/A9639 and A5937/A5940) were used to inoculate MG-63 osteoblast cells at MOI of 100: 1 unless otherwise stated. Osteoblasts inoculated with VSSA are represented by black circles and those infected with VISA are represented by grey squares for each panel. Error bars represent SEM and significance was determined using unpaired t tests with Welch's correction, *p <0.05, **p <0.01, ***p < Adhesion was assessed at 2 h post infection (a). Following lysostaphin treatment, viable intracellular bacteria were quantified 3 h after inoculation (b). Relative cytotoxicity was determined by quantifying lactate dehydrogenase released by damaged MG-63 cells 72 h post infection (c). Interleukin-6 (IL-6) release was determined using ELISA at 72 h post infection (d). To control for initial internalized bacteria, the MOI for A9639 and A5940 were increased to 250: 1 and 500: 1, respectively. This experimental condition is indicated by # and grey triangles (c and d).
Clinical Microbiology and Infection  , DOI: ( /j.cmi )
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4. Fig. 3
Vancomycin-susceptible (VSSA) and vancomycin-intermediate (VISA) Staphylococcus aureus intracellular persistence within infected osteoblasts. MG-63 osteoblast cells were inoculated with vancomycin-exposed pairs A9635/A9639 (a) and A5937/A5940 (b) and viable bacterial cells were quantified following lysostaphin treatment 1, 2, 3 and 7 days post inoculation to compare intracellular persistence capacities. Osteoblasts inoculated with VSSA are represented in black and those infected with VISA are represented in grey. Error bars represent SEM and significance was determined using unpaired t tests with Welch's correction, n = 9, *p <0.05, **p <0.01, ***p <0.001.
Clinical Microbiology and Infection  , DOI: ( /j.cmi )
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5. Fig. 4
Pathogenic consequences of vancomycin-susceptible Staphylococcus aureus (VSSA) versus vancomycin-intermediate S. aureus (VISA) infections in a murine bacteraemia model. (a) Kaplan–Meier curves showing survival of mice infected with vancomycin-exposed clinical pairs (n = 10). In all cases, infection with the VSSA parent strains (black lines) produced significantly greater mortality compared with the paired vancomycin-exposed VISA isolates (grey lines; p <0.01 for each, Log-rank test). (b) Colonization of the liver, 15 h post infection for mice infected with VSSA (black bars) and VISA (grey bars) strains (n = 5). (c) Cytokine stimulation profiles for mice infected with VSSA (black bars) and VISA (grey bars) 8 h post infection. Stimulation of interleukin-6 (IL-6) was reduced in animals infected with VISA (p <0.001, Holm–Sidak multiple comparison test). (d) Colonization of the kidney, 7 days post infection for mice infected with VISA (grey bars) (n = 3).
Clinical Microbiology and Infection  , DOI: ( /j.cmi )
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6. Fig. S1. Representative images of α toxin Western blots
Fig. S1. Representative images of α toxin Western blots. Black strain names are vancomycin-susceptible Staphylococcus aureus, grey strain names are paired vancomycin-intermediate S. aureus.
Clinical Microbiology and Infection  , DOI: ( /j.cmi )
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7. Fig. S2. Bioluminescence indicative of agr-P3 activity was visualized on agar plates after 18 h of growth at 37°C using an IVIS100 imager. Four clinical pairs were assessed; A8090/A8094 (a); JKD6009/JKD6008 (c); JKD6052/JKD6051 (e); and A9635/A9639 (g).
Clinical Microbiology and Infection  , DOI: ( /j.cmi )
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