Bacterial Strains and Growth Conditions used in this Study

We used two different strains of S. aureus in this study depending on the growth context. NewHG (SJF 3663) (Newman with saeSL allele from strain RN1 [24]) is well characterised and was used for the zebrafish infection studies. The laboratory MSSA strain Newman has a mutation resulting in a constitutively active saeSP which leads to increased virulence due to activation of certain virulence-related genes [24]. Replacement of the mutated allele to give strain NewHG restored parental virulence determinant levels.

UAMS-1 is a clinical osteomyelitis isolate that is well characterised for its biofilm growth [25].

Both strains NewHG and UAMS-1 are sensitive to oxacillin and tetracycline. Unless otherwise stated, S. aureus was grown at 37°C in tryptone soy broth (TSB, Oxoid), made using 30 g/l in deionised water and sterilised by autoclaving. Bacteriological agar (VWR) was added at 1.5 % w/v to TSB to make tryptone soy agar (TSA).

Plasmids used in this Study

pCM29 [26] was transformed into UAMS-1 for constitutive cytosolic GFP expression in order to quantify area coverage in the Bioflux experiments.

In vitro Biofilm Growth and Imaging

The model for dynamic S. aureus biofilm, as previously described [27], was used for the assessment of biofilm treatment by tetracycline and oxacillin. The Bioflux 1000 (Fluxion biosciences Inc.) system and Bioflux 1000 48-well plates (Fluxion Biosciences Inc.) and sterile 50% TSB were used for all experiments. 250-ml conical flasks containing 25 ml sterile TSB were inoculated from overnight cultures of UAMS-1 pCM29 to OD600 0.05 (approximately 1x107 CFU/ml) and incubated at 37°C, with shaking at 250 rpm until an OD600 0.8 was reached. Biofilm growth channels were primed by adding 200 µl media to output wells and using reverse flow for 2 minutes at 10.0 dynes cm2. A 300 µl volume of fresh media was added to input wells and media in output wells replaced with S. aureus inoculum. The growth channels were then seeded by applying reverse flow of 2.0 dynes cm2 for ~2 seconds using guidance via microscope. The seeded plate was incubated for 1 hour at 37° C on the heated stage of the Bioflux 1000 system to allow attachment of S. aureus cells to the growth channels. 1 ml of fresh media was added to input wells and the remaining inoculum was aspirated from the output wells. Forward flow of 0.6 dynes cm2 was applied to all channels in the plate for 12 hours. After 12-h flow of media was paused, the manifold was removed from plate in situ and 1 ml of media added to input wells containing 1.2 x concentration of antibiotic to give a final volume of 1.2 ml of media containing a final concentration of antibiotic as detailed. Manifold was replaced and forward flow was resumed at 0.6 dynes cm2 for 12 hours. Bright-field and epifluorescence images were taken at 5-minute intervals for a total of 289 timepoints. All epifluorescence images monitoring GFP fluorescence were taken with a fluorescein isothiocyanate (FITC) filter. All biofilm treatments were repeated for a minimum of three independent experiments containing at least two technical replicates as has been previously established [28].

Quantification of Acquired Biofilm Images

Images were reviewed using Bioflux Montage software (Fluxion Biosciences Inc.). Bright-field and epifluorescence images were calibrated to 0.32 µm/pixel. The open-source image processing software Fiji [29] was used for the quantification of area coverage. Area coverage was calculated from cytosolic GFP expression, using a macro to apply the same conditions to all timepoints for every field of view acquired across all experiments. Background signal was removed by thresholding the image to a minimum of 200 AU and all pixels between 200 and 63270 were included in the area measurement.

Growth Conditions of S. aureus for Zebrafish Infection

Frozen stocks of S. aureus were streaked out onto TSA plates and incubated overnight at 37°C. A single colony was picked from the plate and inoculated into 10 ml TSB and incubated overnight at 37°C, with shaking at 250 rpm. The culture was diluted the following day to OD600 0.05 in 50 ml TSB and grown at 37°C, with shaking at 250 rpm until it reached OD600 1 after approximately 2 hours. The culture was prepared for injection into zebrafish as detailed in [20].

In vivo Zebrafish Infection Experiments

Animal work was carried out according to guidelines and legislation set out in UK law in the Animals (Scientific Procedures) Act 1986, under Project License PPL 40/3574. Larvae were infected with 1500 CFU NewHG S. aureus at 33 hours post fertilisation (hpf) as previously described in [20] and survival monitored until 92 hours post infection (hpi), at which point larvae were culled. Larvae were maintained in E3 buffer prepared from a 10x stock solution diluted in deionised water, resulting in a final concentration of 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4 with methylene blue added as an antifungal agent to a final concentration of 0.00005% w/v. The solution was autoclaved to sterilise and cooled to 28.5°C before use. Bacterial burden was determined by homogenising a sample of living larvae and all dead larvae in 100 µl E3 in sterile homogenisation tubes (AlphaLaboratories) containing sterilised 1.4 mm ceramic beads (Peqlab). Larvae were frozen or kept on ice to cool before being homogenised for 20 seconds via a FastPrep-24 homogeniser (MP Biomedicals). 20 µl homogenate was added to 180 µl PBS and serially diluted before being plated out as 10 µl spots onto tryptone soy agar (TSA) plates. Plates were incubated at 37°C overnight. The mean number of CFUs per 10 µl spot was determined to calculate CFU per larva.

Treatment with Antibiotics

Larvae treated at 1 day post infection (dpi) were put into 96-well plates with 250 µl E3 per well, and then 50 µl E3 containing the antibiotic at 6x concentration was pipetted into the well. Larvae treated at 0 hpi in the morpholino experiment were immersed in E3 containing the antibiotics at the required concentrations and then dispersed into 96-well plates.

Zebrafish Morpholino Experiments

Tg(BACmpx:gfp)i114 zebrafish embryos at the 1-cell stage were injected with either 0.5 pmole (in 0.5 nl) pu.1 morpholino (ZDB-MRPHLNO-050224-1) or standard control morpholino (GeneTools) (whereby phagocytes develop normally). Morphants were infected with a target dose of 1500 CFU S. aureus NewHG at 33 hpf and left untreated as a control, or treated with 50 µg ml-1 tetracycline and 32 µg ml-1 oxacillin at 1 hpi. The following day at 18 hpi, survival was calculated for each group and morphants were homogenised to determine the bacterial load. Treatment was given early, as without phagocytes (as in the untreated group), the infection quickly overwhelms the embryos. It has been previously shown that embryos injected with pu.1 morpholino but then uninfected have no survival defect [22].

Statistical Analysis

Statistics were carried out using GraphPad Prism (versions 9 and 10). Biofilm area coverage comparisons were made by Mann–Whitney test for the final timepoints (10.5 h). Zebrafish survival is represented using Kaplan–Meier survival curves, with comparisons between curves performed using the Log-Rank (Mantel-Cox) test. For a reduction in mortality from 40 % to 80 % survival, required sample size is 20 per group (confidence 80%, significance 5%, Chi squared). For the morpholino data to detect a reduction in proportions expected from 90% to 60%, 29 are required in each group (confidence 80%, significance 0.05, Chi squared).