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Too Much of a Good Thing: Dangers of Excessive Dietary Zinc


By: Carol A. Rouzer, VICB Communications
Published: October 17, 2016



New studies show that a diet high in zinc increases the susceptibility to and severity of Clostridium difficile infections of the gastrointestinal tract.


Clostridium difficile (Figure 1) is a Gram-positive rod-shaped bacterium that is widely distributed in the environment. It is a common cause of gastrointestinal infections that produce symptoms ranging from mild diarrhea to severe colitis and even death. Although C. difficile infections primarily occur in patients who have been treated with broad spectrum antibiotics, their prevalence among the general population is increasing. This led Vanderbilt Institute of Chemical Biology members Eric Skaar, Walter Chazin, and Richard Caprioli to search for risk factors that contribute to the likelihood of C. difficile gastroenteritis. They now report that excess dietary zinc increases susceptibility to and severity of infection by C. difficile [J. P. Zackular, et al. (2016) Nat. Med., published online September 26, DOI:10.1038/nm.4174].



FIGURE 1. Illustration of a C. difficile bacterium based on photomicrographic data. Figure reproduced from the CDC website, source James Archer. Public domain.



During an infection, bacteria must rely on the host for critical nutrients, including metal ions such as iron, zinc, and manganese. Host cells have evolved mechanisms to sequester such nutrients to suppress bacterial growth, a process called nutritional immunity. However, the presence of high levels of nutrients may override these natural host defenses, enabling bacteria to thrive and grow. This led the Skaar lab investigators to hypothesize that diet might have an effect on C. difficile infection. Prior research had indicated that zinc is important for the production of virulence factors by C. difficile, so the researchers focused on this nutrient to test their hypothesis.


The investigators began by feeding groups of mice three different diets, one containing very low levels, one containing standard levels, and one containing high levels of zinc comparable to those found in the diets of people taking large doses of zinc supplements. After five weeks of diet exposure, the mice exhibited the expected changes in zinc levels in the cecum, colon, and feces, with low and high levels found in samples from mice on low and high zinc diets, respectively (Figure 2). A more interesting finding was that mice fed the high zinc diet exhibited a reduction in the number of different kinds of bacteria in the colon, as well as a change in the species that were dominant. No such changes were present in mice fed the low zinc diet, and none of the diets was associated with any signs of toxicity or histologic changes in the colon.


FIGURE 2. Images of zinc levels in the colons of mice given a control diet (top) or a diet high in zinc (bottom). Images were acquired using laser ablation inductively coupled plasma mass spectrometry. "L" indicates the lumen of the colon. Figure reproduced by permission from Macmillan Publishers Ltd: J. P. Zackular, et al. (2016) Nat. Med., published online September 26, DOI:10.1038/nm.4174. Copyright 2016.



Their initial results suggested that dietary zinc can have an effect on the population dynamics of intestinal bacteria. Consequently, the researchers applied their dietary treatment to a model of C. difficile infection. After exposing mice for five weeks to a low, normal, or high zinc diet, the investigators treated the animals for five days with cefoperazone, a broad spectrum antibiotic. Then, they exposed the mice to C. difficile spores by oral gavage. The results were striking. Mice fed a high zinc diet prior to C. difficile infection exhibited increased inflammation, epithelial damage, and pseudomembrane formation (a collection of protein, inflammatory cells, and necrotic cells characteristic of severe C. difficile colitis) as compared to mice fed a low or normal zinc diet (Figure 3). Although the high zinc diet had no effect on the number of C. difficile bacteria in the gastrointestinal tract of infected mice, it was associated with higher titres of C. difficile toxins in the feces. The high zinc diet was also associated with changes in both pro- and anti-inflammatory cytokines in C. difficile-infected mice. Furthermore, the presence of high numbers and kinds of bacteria in the livers of the infected mice pretreated with the high zinc diet as opposed to the control diet indicated that the high zinc diet led to disruption of the intestinal epithelium's barrier function in the context of C. difficile infection. In contrast, the low zinc diet had no effect on the severity of infection when compared to the control diet. Together, these results suggested that high levels of dietary zinc substantially exacerbate the severity of inflammation and tissue disruption in response to C. difficile.




FIGURE 3. Hematoxylin and eosin-stained tissue sections from the ceca of mice infected with C. difficile strain 630 following pretreatment with a control diet (left) or a high zinc diet (right). A strong inflammatory response is evident in the cecum from the mouse pretreated with the high zinc diet, and the arrow heads indicate the presence of pseudomembranes, a characteristic of C. difficile infection. Figure reproduced by permission from Macmillan Publishers Ltd: J. P. Zackular, et al. (2016) Nat. Med., published online September 26, DOI:10.1038/nm.4174. Copyright 2016.



In their model of C. difficile infection, the investigators treated the mice first with an antibiotic because, as noted above, antibiotic exposure increases susceptibility to the infection in humans. The favored explanation for this phenomenon is that the antibiotics alter the normal balance of intestinal bacteria, enabling C. difficile to become the dominant species. The researchers noted that the high zinc diet alone also altered the dynamics of intestinal bacterial populations, leading them to hypothesize that this effect might reduce the requirement for antibiotic treatment prior to C. difficile infection. They tested this hypothesis by exposing mice to a high or normal zinc diet and then pretreating them with a very low dose of antibiotics prior to C. difficile exposure. Under these conditions, mice fed the high zinc diet exhibited severe infection, while those fed the normal diet did not become infected with C. difficile. These results suggest that a high zinc diet alters the dynamics of intestinal bacteria in such a way as to increase the likelihood of infection with C. difficile, even in the absence of exposure to high doses of antibiotics.


Calprotectin is a zinc- and manganese-binding protein that is produced in large quantities by leukocytes. It suppresses the growth of C. difficile in culture, an effect that is not observed with a mutant protein that does not bind zinc, or with the wild-type protein in the presence of excess zinc. Thus, calprotectin exhibits antibacterial activities associated with its ability to sequester zinc. Calprotectin levels are elevated in the serum and feces of human patients suffering from C. difficile infection, and consistently, the investigators found elevated levels of calprotectin in the ceca of infected mice (Figure 4). This led to the hypothesis that calprotectin plays a significant role in fighting C. difficile infection. Support for that hypothesis came from experiments using mice genetically deficient in calprotectin. Infection of these mice with C. difficile resulted in more severe disease and greater mortality than were observed in infected wild-type mice. Further experiments showed that the calprotectin-deficient mice could be protected, at least in part, from C. difficile by feeding them a zinc-deficient diet.




FIGURE 4. MALDI-MS images of the ceca from an uninfected wild-type mouse, not treated with antibiotics (upper left), an uninfected wild-type mouse treated with antibiotics (upper right), an antibiotic-treated C. difficile-infected wild-type mouse (lower left) and a C. difficile-infected calprotectin-deficient mouse (lower left). The images show the presence of calprotectin (S100A8/S100A9 heterodimer) as detected by MALDI-MS. Figure reproduced by permission from Macmillan Publishers Ltd: J. P. Zackular, et al. (2016) Nat. Med., published online September 26, DOI:10.1038/nm.4174. Copyright 2016.



The findings demonstrate that high levels of dietary zinc increase the susceptibility of mice to C. difficile infection, likely by causing a change in the dynamics of the bacteria colonizing the colon. Excessive zinc also increased the severity of C. difficile infection without changing the actual number of bacterial cells present. This effect could be attributed, at least in part, to augmented bacterial virulence, as demonstrated by increased toxin production. The zinc binding protein calprotectin serves as a natural defense against C. difficile by sequestering zinc from the bacteria; however excessive dietary levels of the metal overwhelm calprotectin's ability to serve its protective function. These experiments were carried out in mice, so it is not clear if their implications also apply to humans. However, they clearly suggest that dietary zinc supplements, which are promoted for a wide range of beneficial effects from fighting colds to preventing cancer and heart disease to improving fertility, should be used with caution.




View Nature Medicine article: Dietary zinc alters the microbiota and decreases resistance to Clostridium difficile infection.










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