AimsStarter lactic acid bacteria in Cheddar cheese face physico-chemical stresses during manufacture and ageing that alter their abilities to survive and to interact with other bacterial populations. Nonstarter bacteria are derived from milk handling, cheese equipment and human contact during manufacture. Probiotic bacteria are added to foods for human health benefits that also encounter physiological stresses and microbial competition that may mitigate their survival during ageing. We added probiotic Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei and Bifidobacterium animalis subsp. lactis to full-fat, reduced-fat and low-fat Cheddar cheeses, aiming to study their survival over 270days of ageing and to determine the role of the cheese matrix in their survival. Methods and ResultsProbiotic and other lactic acid bacterial populations were enumerated by quantitative PCR using primers specifically targeting the different bacterial genera or species of interest. Bifidobacteria were initially added at 10(6)CFU g(-1) cheese and survived variably in the different cheeses over the 270-day ageing process. Probiotic lactobacilli that were added at 10(7)CFU g(-1) cheese and incident nonstarter lactobacilli (initially at 10(8)CFU g(-1) cheese) increased by 10- to 100-fold over 270days. Viable bacterial populations were differentiated using propidium monoazide followed by species-specific qPCR assays, which demonstrated that the starter and probiotic microbes survived over ageing, independent of cheese type. Addition of probiotic bacteria, at levels 100-fold below that of starter bacteria, modified starter and nonstarter bacterial levels. ConclusionsWe demonstrated that starter lactococci, nonstarter lactobacilli and probiotic bacteria are capable of surviving throughout the cheesemaking and ageing process, indicating that delivery via hard cheeses is possible. Probiotic addition at lower levels may also alter starter and nonstarter bacterial survival. Significance and Impact of the StudyWe applied qPCR to study multispecies survival and viability and distinctly enumerated bacterial species in commercial-scale Cheddar cheese manufacture.