BACKGROUND: Non-obese diabetic (NOD) mice develop type 1 diabetes (T1D) spontaneously and serve as a good model for investigating the underlying pathological mechanisms and devising novel treatment procedures. Although acid water consumption has been reported to exaggerate or reduce diabetes incidence in female NOD mice by two groups, the causative bacteria responsible for these contrasting changes remain unclear. On the contrary, we and others failed to observe the effect of acid water consumption on diabetes incidence. This study aimed to determine whether the consumption of low-pH drinking water could alter the frequencies of prominent bacterial groups independent of diabetes manifestation. METHODS: Six-week-old female NOD mice maintained on acidified drinking water at the Jackson Laboratories were transferred to neutral pH water or continuously provided with low pH drinking water at our facility. Diabetes was monitored weekly using a glucometer. Using the 454-pyrosequencing methodology, we profiled the gut microbiome of mice transferred to neutral water and developed diabetes. Further, we performed quantitative real-time polymerase chain reactions (qRT-PCR) using primers specific for prominent 16S rRNA genes on the fecal DNA of mice provided with low pH or neutral water and displayed diabetes similarly. RESULTS: Consistent with our earlier report, the incidence of T1D was robust (80-100%) regardless of whether female NOD mice consumed acid (~pH 2.9) or neutral water. The 454-pyrosequencing of fecal DNA indicated no substantial influence of transferring mice to neutral pH drinking water on the gut microbiome. To validate these findings, we conducted qRT-PCR on the fecal DNA of mice longitudinally from six weeks of age to adulthood that consumed acidic or neutral pH water and developed diabetes similarly. Among the 15 selected bacterial groups examined, the frequency of sp. remained consistently lower ( < 0.05) throughout the life of NOD mice compared to that found in young (6-week-old) mice, regardless of the pH of the drinking water. The relative frequencies of the and the members sp. and sp. increased significantly ( < 0.05) during the transition to the overtly diabetic stage irrespective of the ionic strength of the drinking water. Interestingly, the members , , and and the members sp. and sp. remained unchanged throughout the analysis irrespective of the pH of the drinking water. Paradoxically, the representations of and the segmented filamentous bacteria implicated in diabetes protection did not differ regardless of the age or the ionic strength of the drinking water. CONCLUSIONS: The data presented herein validate the lack of influence of acidic drinking water on T1D development in female NOD mice. Diabetes was associated with the lower representation of sp. throughout life, which was not influenced by the differing pH of the drinking water. Significantly, segmented filamentous bacteria and , previously implicated in protection against T1D, were not perturbed by the varying pH of the water consumed. These data indicate that although acidified water consumption was reported previously to diminish specific gastrointestinal pathogens, it failed to perturb gut commensals that influence diabetes development.