The Impact of pH on Clostridioides difficile Sporulation and Physiology

Abstract

Clostridioides difficile is a pathogenic bacterium that infects the human colon to cause diarrheal disease. Growth of the bacterium is known to be dependent on certain bile acids, oxygen levels, and nutrient availability in the intestine, but how the environmental pH can influence C. difficile is mostly unknown. Previous studies indicated that C. difficile modulates the intestinal pH, and prospective cohort studies have found a strong association between a more alkaline fecal pH and C. difficile infection. Based on these data, we hypothesized that C. difficile physiology can be affected by various pH conditions. In this study, we investigated the impact of a range of pH conditions on C. difficile to assess potential effects on growth, sporulation, motility, and toxin production in the strains 630Δerm and R20291. We observed pH-dependent differences in sporulation rate, spore morphology, and viability. Sporulation frequency was lowest under acidic conditions, and differences in cell morphology were apparent at low pH. In alkaline environments, C. difficile sporulation was greater for strain 630Δerm, whereas R20291 produced relatively high levels of spores in a broad range of pH conditions. Rapid changes in pH during exponential growth impacted sporulation similarly among the strains. Furthermore, we observed an increase in C. difficile motility with increases in pH, and strain-dependent differences in toxin production under acidic conditions. The data demonstrate that pH is an important parameter that affects C. difficile physiology and may reveal relevant insights into the growth and dissemination of this pathogen. IMPORTANCE Clostridioides difficile is an anaerobic bacterium that causes gastrointestinal disease. C. difficile forms dormant spores which can survive harsh environmental conditions, allowing their spread to new hosts. In this study, we determine how intestinally relevant pH conditions impact C. difficile physiology in the two divergent strains, 630Δerm and R20291. Our data demonstrate that low pH conditions reduce C. difficile growth, sporulation, and motility. However, toxin production and spore morphology were differentially impacted in the two strains at low pH. In addition, we observed that alkaline environments reduce C. difficile growth, but increase cell motility. When pH was adjusted rapidly during growth, we observed similar impacts on both strains. This study provides new insights into the phenotypic diversity of C. difficile grown under diverse pH conditions present in the intestinal tract, and demonstrates similarities and differences in the pH responses of different C. difficile isolates.

Species richness determinesC. difficileinvasion outcome in synthetic human gut communities

“…Our results demonstrated that communities can inhibit growth of C. difficile by acidifying the environment. We showed that communities that reduce the external pH below 6.2 inhibit C. difficile in a pH-dependent manner, consistent with studies showing that C. difficile has lower viability and rates of sporulation in acidic environments 33,34 . While our in vitro system lacks the pH-buffering secretion of bicarbonate by host intestinal epithelial cells, the amount of bicarbonate buffer in our media (4.8 mM) is within the estimated range in the gastrointestinal tract (2-20mM) 45 , suggesting the observed pH changes in our media could be physiologically relevant.…”

“…2a). Since environmental pH has been shown to influence C. difficile's growth in previous studies 33,34 , we turned next to investigate how biotic modification of the environment alters the growth of C. difficile. To this end, we grew the set of 15, 3-4 member communities for six hours and then invaded with low or high initial densities of C. difficile.…”

Systematic Evaluation of Parameters Important for Production of Native Toxin A and Toxin B from Clostridioides difficile

“…These results are supported by a previous study showing that TcdB production was highest at pH 7.5 when cultivating the C. difficile strain VPI 10463 in liquid Gifu Anaerobic Medium ranging from pH 4.5 to 8.5 [50]. In contrast, there is a recent paper by Wetzel and McBride et al [49], which found that the C. difficile strain R20291 (same strain as the one in this study) produced the highest amount of TcdA at pH 5.5 (measured by western blot) when cultivated on solid media, more than 2-fold higher than at pH 7.0 and 7.5. However, the findings of the study showing that pH 5.5 was superior at supporting toxin production likely is due to the cultivation on solid media known to induce high levels of sporulation, since sporulation and toxin expression are known to be coregulated in C. difficile [51][52][53].…”

“…We further found that buffering the TY for medium with 100 mM PBS was slightly better at inducing toxin production compared to TY for mixed in deionized water ( Table 4 ). The increased toxin production by using PBS was not significant, however we decided to continue buffering the medium with PBS and possibly prevent the previously reported acidification of the medium during cultivation [ 49 ]. Interestingly, we found that adding 5 g/L bicarbonate to the PBS-buffered TY for medium had a dramatic negative effect on the toxin yield with a 3-fold decrease, without affecting the growth.…”

The Positive Association between Proton Pump Inhibitors and Clostridium Difficile Infection

“…25,26 The study results indicated that sporulation frequency was the lowest under acidic pH, while alkaline colonic pH increased C. difficile growth for both strains. 27 Together, these may explain the high prevalence of CDIs in patients with alkaline stools, as C. difficile spores are unable to sporulate with low gastric pH whereas high gastric pH and alkaline pH of the intestine facilitates C. difficile survival allows for the sporulation and germination of vegetative form of the bacterium. This evidence suggests that acid suppressive therapies, such as PPIs, facilitate vegetative C. difficile survival and growth, increasing the likelihood of acquiring CDI.…”