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Title: Helicobacter pylori antibiotic eradication coupled with a chemically defined diet in INS-GAS mice triggers dysbiosis and vitamin K deficiency resulting in gastric hemorrhage

Author
item QUINN, LISA - Massachusetts Institute Of Technology
item SHEH, ALEXANDER - Massachusetts Institute Of Technology
item ELLIS, JESSIE - Jean Mayer Human Nutrition Research Center On Aging At Tufts University
item SMITH, DONALD - Jean Mayer Human Nutrition Research Center On Aging At Tufts University
item BOOTH, SARAH - Jean Mayer Human Nutrition Research Center On Aging At Tufts University
item FU, XUEYAN - Jean Mayer Human Nutrition Research Center On Aging At Tufts University
item MUTHUPALANI, SURESHKUMAR - Massachusetts Institute Of Technology
item GE, ZHONGMING - Massachusetts Institute Of Technology
item PUGLISI, DYLAN - Massachusetts Institute Of Technology
item WANG, TIMOTHY - Columbia University - New York
item GONDA, TAMAS - Columbia University - New York
item HOLCOMBE, HILDA - Massachusetts Institute Of Technology
item FOX, JAMES - Massachusetts Institute Of Technology

Submitted to: Gut Microbes
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/2/2020
Publication Date: 1/19/2020
Citation: Quinn, L., Sheh, A., Ellis, J.L., Smith, D., Booth, S.L., Fu, X., Muthupalani, S., Ge, Z., Puglisi, D.A., Wang, T.C., Gonda, T.A., Holcombe, H., Fox, J.G. 2020. Helicobacter pylori antibiotic eradication coupled with a chemically defined diet in INS-GAS mice triggers dysbiosis and vitamin K deficiency resulting in gastric hemorrhage. Gut Microbes. https://doi.org/10.1080/19490976.2019.1710092.
DOI: https://doi.org/10.1080/19490976.2019.1710092

Interpretive Summary: In animal models, antibiotics and increased dietary folate have been demonstrated to be protective against the development of gastric cancer following Helicobacter pylori (H. pylori) infection. The purpose of this study was to determine if protective effects are even greater when antibiotics and folate are given together. Male mice were infected with H. pylori and then treated with increased dietary folate, antibiotics, or both. Unexpectedly, some of the mice became ill with symptoms resembling vitamin K deficiency, particularly the mice given antibiotics. The vitamin K content of the diet was suspected to be inadequate. Tissue vitamin K concentrations of affected mice were low, supporting this diagnosis. Mice were treated with vitamin K injections and diets were reformulated to contain adequate vitamin K, after which clinical condition of the mice improved. Primary results found that antibiotics, but not dietary folate, were protective against H. pylori promotion of gastric cancer. Because more of the mice given antibiotics developed vitamin K deficiency symptoms compared to mice not given antibiotics, despite being on the same initial diet, it was hypothesized that antibiotics altered vitamin K production by bacteria in the mouse gut. Analysis of the fecal microbiota and fecal vitamin K content showed that both were altered with antibiotic treatment. This study highlights the importance of the microbiome and diet in preventing vitamin K deficiency.

Technical Abstract: Background: Helicobacter pylori infection promotes gastric adenocarcinoma development in male INS-GAS mice, but early intervention with antibiotics or increased dietary folate significantly decreases gastric pathology. To determine potential synergistic effects, H. pylori-infected male INS-GAS mice on an amino acid defined (AAD) diet were treated with either increased dietary folate, antibiotics, or both at 18 weeks post-infection (WPI). Unexpectedly, the combination of antibiotics and AAD diet led to increased gastric hemorrhage and mortality. Results: Antibiotic therapy, with or without increased dietary folate, decreased gastric pathology in H. pylori-infected mice, but increasing dietary folate alone had no effect. However, 41% of infected mice given antibiotics (HPab) were moribund or found dead, compared to 8% of infected mice without antibiotics (HPno). Affected mice exhibited acute pallor and a hunched appearance. At necropsy, severe gastric hemorrhage and pale livers were observed. Clinically ill mice developed anemia with decreased hematocrit, red blood cell counts and hemoglobin compared to healthy cagemates, suggesting a vitamin K deficiency associated with antibiotic treatment. Vitamin K levels in the AAD diet were lower than recommended by dietary guidelines. Phylloquinone (PK/vitamin K1) was administered subcutaneously and via a reformulated AAD diet, resulting in an improved clinical condition of all mice with no subsequent mortality. PK treatment improved PK (PRE-PK, 8+/-2 pmol/g vs. POST-PK, 715+/-202 pmol/g) and MK4 (PRE-PK, 5+/-2 pmol/g vs. POST-PK, 13+/-2 pmol/g) levels in the liver, supporting the diagnosis of hypovitamininosis K. Prior to PK treatment, antibiotics mediated the loss of Bacteroidetes and gain of Firmicutes. Shifts in the relative abundances of menaquinone producers (order Bacteroidales) and non-producers (Clostridiales) significantly decreased the levels of menaquinone biosynthetic pathway genes, menA and menD. Compared to HPno mice, HPab mice had reduced MK5 (0.13% HPab vs. 0.33% HPno) and MK6 (2.79% vs. 16.57%), accompanied by increased MK7 (1.84% vs 1.03%) and MK11 (40.9% vs. 28.8%). Conclusions: Antibiotic-mediated loss of menaquinone producers altered enteric production of vitamin K. Marginal dietary vitamin K and antibiotic-reduced MK6 levels led to hypovitaminosis K, gastric hemorrhage, and death in H. pylori-infected mice. This study highlights the importance of the microbiome and diet in preventing hypovitaminosis K.