The extra Little Sisters bug is the result of scripted events that trigger a Little Sister and Big Daddy pair for a specific scene – typically a fight with Splicers. The triggers are usually as simple as entering a particular area (i.e. in Neptune's Bounty, it's the Upper Wharf; in Frolic, it's the Atrium after visiting the Plaza the first time; in Arcadia, it's a Gatherer's Garden.) If the player avoids these trigger points until all Little Sisters have been dealt with, then entering the trigger area will spawn an extra Little Sister.