The Care Commission has advised Careshare, which operates 21 nurseries across Scotland, that it will face a series of spot checks to ensure staff are doing everything they can to prevent the spread of infection.
The move comes as four children who attended Careshare's nursery at Lauder College remain at the Royal Hospital for Sick Children in Glasgow suffering from kidney failure after contracting the E.coli infection.
Altogether there have been nine confirmed cases and 26 possible cases. Two of the confirmed cases also attended other nurseries - one at Lynburn primary school nursery and one at Maclean nursery, both in Dunfermline.
Ronnie Hill, the Care Commission's director of children's services regulations, said this week that a review of 19 Careshare nurseries inspected in the past year showed that nine had been criticised on hygiene or infection control issues.
Mr Hill said: "While the source of the outbreak has not yet been confirmed, this should serve as a wake-up call to all care services that they have a very serious responsibility to adhere to the highest possible standards in infection control.
"Regulation on its own cannot prevent outbreaks, but provides a snapshot at any given time. It is the very clear responsibility of all care providers to adopt best practices and ensure they are followed."
Environmental health officers this week raised concerns that the Lauder nursery's sinks and taps might cause dirty water to splash on to other surfaces.
In a separate outbreak in Aberdeenshire, two children who attend the Dreams Daycare centre in Insch (not part of the Careshare group) have been released from hospital.
The two outbreaks have highlighted growing concerns over the much greater prevalence of E.coli 0157 in Scotland - twice the rate in England and Wales and almost 40 times that in Germany.