Sheep blood agar plates

The following S. aureus strains were used in this study: methicillin-resistant strains LUH14616 (sequence type 247), a kind gift of dr. S. Croes [30 (link)]; LUH15051 (ST 239) obtained from dr. M.E.O.C. Heck, (Laboratory of Infectious Diseases and Screening, RIVM, Bilthoven, The Netherlands); USA300 strain Sac042w (ST 8) described earlier [31 (link)]; a strain derived from an impetigo patient LUH15091 (ST121) within the Erasmus Medical Center and NCTC 8325–4 (ST 8). All strains were typed using multi locus sequence typing (MLST) [27 (link),32 (link)]. Before usage the strains were grown on sheep blood agar plates (Biomerieux).

Methicillin-resistant Staphylococcus aureus (MRSA) LUH14616 sequence type 247, was collected by a nasal swab from a patient without an infection. It was preserved in nutrient broth supplemented with 20% glycerol at −80°C. Prior to experiments, inocula from the frozen stocks were grown overnight at 37°C on sheep blood agar plates (BioMerieux). Thereafter, bacteria were cultured to mid-log phase in tryptic soy broth for 2.5 h at 37°C. Finally, the bacteria were harvested by centrifugation (1,000 × g for 10 min) and then resuspended in PBS to the desired inoculum concentration (10⁷ CFU/mL), based on the optical density at 600 nm.

In vitro biofilm formation capacity was determined for each isolate using a crystal violet assay. Briefly, all bacterial strains were incubated overnight on 5% sheep blood agar plates (bioMérieux, France) at 37°C and suspended in tryptic soy broth (TSB; Oxoid, ThermoFisher Scientific) to a concentration equivalent to a 0.5 McFarland standard. Ninety-six well polystyrene flat-bottomed microtiter plates (Cellstar®, Greiner bio-one®, Frickenhausen, Germany) were filled with 200 μL each and incubated at 37°C for 24 h. Planktonic cells were removed using phosphate-buffered saline (PBS), and biofilm was heat-fixed for 10 min at 60°C and subsequently fixed with 150 μl methanol (Merck, Darmstadt, Germany). Plates were dried at room temperature, stained for 20 min using 150 μl 1% crystal violet (Merck KGaA®, Darmstadt, Germany), and washed with tap water. For improved quantification, 150 μl 33% acetic acid (AnalaR Normapur, Prolabo®, VWR International®, USA) was placed in each well, and plates were incubated at 37°C and 50% humidity for 1 h. Plates were measured using a microplate reader (Sunrise, Tecan, Switzerland) at 595 nm with a reference measurement at 405 nm. The mean OD for each isolate was determined by measuring 14 replicates in two separate plates (7 replicates each).

A modified coagulase typing was performed based on the methods described by Hookey et al. (1998 (link)). Briefly, acquired isolates were incubated overnight on 5% sheep blood agar plates (bioMérieux, France) at 37°C and bacterial colonies were suspended in 200 μL water. Samples were heated up to 95°C for 10 min and sonicated for 15 min. A PCR for the coa-gene was conducted using Taq-polymerase (Applied Biological Materials Inc., Canada). Samples were analyzed immediately using gel electrophoresis and restriction fragment length polymorphism (RFLP) analysis using AluI (Roche Diagnostics, Germany). Clotting factors were determined for each isolate using a multiplex PCR, and the vWbp gene was determined using a singleplex PCR as described by Ghasemian et al. (2015 (link)) and Sukhumungoon et al. (2014 (link)), respectively (Sukhumungoon et al., 2014 (link); Ghasemian et al., 2015 (link)). Primers, PCR and RFLP conditions used in the present study are described in the Supplementary Material (see Table S1 and Supplementary Data). For fragment detection, a 2% agarose gel with PepGreen (Peqlab, Germany) was used. Gels were assessed by three independent investigators and experiments were repeated if one of the three investigators observed another result. The standardized S. aureus ATCC33592 served as a positive control.

The haemolytic activity of the PDD isolates was classified on the basis of the diameter of the haemolytic halo observed on 5% sheep blood agar plates (bioMérieux, Craponne, France), following overnight incubation of a single colony, as previously described (Rivas et al., 2013b (link)). Isolates were classified into four distinct groups based on the haemolytic phenotypes: large haemolysis (LH, halo diameter ≥ 7 mm), medium haemolysis (MH, halo diameter from 5 to 7 mm), small haemolysis (SH, halo diameter from 2 to 4 mm) and no-haemolysis (NH).

Planarians were collected and homogenized in 100 µL of sterile phosphate buffered saline (PBS) (Life Technologies Corporation, Grand Island, USA). The lysate was passed five times through a sterile syringe with a 29G needle to disrupt planarian tissue clumps. 10 µL of ten-fold serial dilutions in one mL of PBS were plated onto 5% sheep-blood agar plates (bioMérieux) and incubated at 37 °C under a 5% CO₂ atmosphere for 24 h. CFUs were then counted using an automatic Scan 1200 scanner (Interscience international, Saint Nom la Bretèche, France).