Aggregation assays. S. pyogenes MGAS6180 was initially grown in C medium for 16 h (Lyon et al., 1998) and harvested by centrifugation at 5000 g for 10 min. Bacterial cell pellets were resuspended in either 1 ml C medium or an appropriate concentration of manuka honey (5 and 10 %; w/v) dissolved in C medium and the OD600 of the culture was adjusted to 1.0 (±0.05) if necessary. In both cases, manuka honey at twice the desired concentration was dissolved in double strength TH and diluted to the required concentration using TH; cell pellets were resuspended in either 5 or 10 % (w/v) honey solutions. In an untreated control, manuka honey was replaced with PBS added to maintain the appropriate volume and concentration of TH media. Aggregation assays were carried out in triplicate as described previously (Jakubovics et al., 2005). Briefly, cell suspensions were incubated statically at 37 uC in 1.5 ml semimicrocuvettes and OD600 readings were taken at 30 min intervals over a period of 6 h to monitor the extent of bacterial aggregation. Bacterial aggregation was monitored by observing a reduction on the OD600 over 6 h. Controls containing no manuka honey were analysed simultaneously. Aggregation was expressed as a percentage fall in OD600 relative to the control; triplicate biological samples were assayed and statistical analysis (Students t-test to compare 5 and 10 % honey treatment, individually, to the no honey control) was undertaken using Minitab (version 14).