Increased risk of intestinal dysfunction has been reported in patients after
infection (CDI). Enteric glial cells (EGCs), a component of the enteric nervous system (ENS), contribute to gut ...homeostasis. Previous studies showed that adenosine receptors, A2A and A2B, modulate inflammation during CDI. However, it is unknown how these receptors can modulate the EGC response to the
toxins (TcdA and TcdB). We investigated the effects of these toxins on the expression of adenosine receptors in EGCs and the role of these receptors on toxin-induced EGC death. Rat EGCs line were incubated with TcdA or TcdB alone or in combination with adenosine analogues 1h prior to toxins challenge. After incubation, EGCs were collected to evaluate gene expression (adenosine receptors and proinflammatory markers) and cell death.
, WT, A2A, and A2B KO mice were infected with
, euthanized on day 3 post-infection, and cecum tissue was processed. TcdA and TcdB increased A2A and A3 transcripts, as well as decreased A2B. A2A agonist, but not A2A antagonist, decreased apoptosis induced by TcdA and TcdB in EGCs. A2B blocker, but not A2B agonist, diminished apoptosis in EGCs challenged with both toxins. A3 agonist, but not A3 blocker, reduced apoptosis in EGCs challenged with TcdA and TcdB. Inhibition of protein kinase A (PKA) and CREB, both involved in the main signaling pathway driven by activation of adenosine receptors, decreased EGC apoptosis induced by both toxins. A2A agonist and A2B antagonist decreased S100B upregulation induced by
toxins in EGCs.
, infected A2B KO mice, but not A2A, exhibited a decrease in cell death, including EGCs and enteric neuron loss, compared to infected WT mice, reduced intestinal damage and decreased IL-6 and S100B levels in cecum. Our findings indicate that upregulation of A2A and A3 and downregulation of A2B in EGCs and downregulation of A2B in intestinal tissues elicit a protective response against C.
toxins. Adenosine receptors appear to play a regulatory role in EGCs death and proinflammatory response induced by TcdA and TcdB, and thus may be potential targets of intervention to prevent post-CDI intestinal dysmotility.
Clostridioides difficile infection (CDI) is the fifth leading cause of death from nonmalignant gastrointestinal disease in the United States. The contribution of resistance to C. difficile-active ...antibiotics to the outcomes of CDI is unclear. We evaluated the antimicrobial susceptibility of C. difficile isolates in a U.S. hospital and determined associations of clinical variables and binary toxin positivity with antibiotic resistance. C. difficile spores were cultured from fecal specimens of adult patients with CDI for genotyping and antimicrobial susceptibility assay (for clindamycin CLI, fidaxomicin FDX, metronidazole MTZ, moxifloxacin MXF, tigecycline TGC, and vancomycin VAN). Electronic medical records were reviewed for clinical data extraction. Ninety-seven of 130 (75%) fecal samples grew toxigenic C. difficile in culture. Most of the isolates were
(80.4%), and 18.6% and 1% were
and
, respectively. Susceptibility to VAN, MTZ, FDX, TGC, MXF, and CLI was 96%, 94%, 100%, 100%, 8%, and 79%, respectively. Six isolates, all
positive and belonging to the 027 ribotype, were resistant to VAN and/or MTZ. Higher MICs were found in isolates with a mutation in the VAN-related resistance gene
, but not
. In addition,
isolates exhibited higher MICs of VAN, MTZ, TGC, CLI, and MXF compared to
strains. Patients with greater intestinal inflammation or severe disease were more likely to be infected with
strains. Decreased susceptibility to antibiotics is not directly associated with either severe or recurrent CDI. However, antimicrobial susceptibility of C. difficile is decreased in strains positive for the binary toxin gene.
Abstract
Background
Clostridioides difficile infection (CDI) affects nearly half a million patients in the US annually. Previous studies have associated delirium and dementia with poor outcomes of ...CDI in older adults. We examined how CDI may affect the brain and induce neuroinflammation in the mouse model of CDI.
Methods
6 and 18-month-old mice were infected with 105 CFU of C.difficile, monitored daily (body weight and diarrhea score), and euthanized on day 3, day 7, and day 10 post-infection (p.i.). Cecum and colon tissues were processed for histopathology. Hippocampus, prefrontal cortex, and cecum samples were analyzed for pro-inflammatory cytokine levels (S100B, IL-6, IL-1β, and MPO) by ELISA and for phosphorylated NFκB by western blot.
Results
Infected mice developed diarrhea and weight loss, resulting in maximum weight loss around day 3 p.i., which resolved around day 7 and day 10 p.i. for 6 and 18-month-old mice respectively. Inflammatory marker analysis showed elevated levels of IL-1β (p=0.04) and myeloperoxidase (MPO, p=0.004) levels in the cecum, as well as higher histopathology scores in the cecal (p=0.04) and colon (p=0.009) tissue on day 3 p.i. By day 7 and day 10 post p.i., the measures of intestinal inflammation were no longer elevated, including IL-1β and MPO levels. The inflammatory markers in the hippocampus were not significantly elevated on day 3 p.i., but on day 7 p.i., levels of hippocampal S100B (p=0.003) and IL-6 (p= 0.03) were elevated in the 6-month-old mice, indicating that CDI is associated with an inflammatory response in the brain after the initial intestinal inflammation. Moreover, hippocampal phosphorylated NFκB levels were detectable on day 7 p.i. 6-month-old infected mice by western blot, pointing to an NFκB pathway as a mechanism by which the neuroinflammation is activated post-CDI. In addition to the hippocampus, the prefrontal cortex also showed a trend towards elevation of MPO levels on day 7 and day 10 p.i.
Conclusion
Our findings demonstrate that CDI promotes neuroinflammation which occurs after the peak of intestinal inflammation. NFκB signaling may be involved in the upregulation of the pro-inflammatory cytokines contributing to this neuroinflammatory effect; however, further investigation is needed.
Disclosures
Cirle A. Warren, MD, Ser-109 Coactuate: Advisor/Consultant|Ser-109 Coactuate: Honoraria
