Dielectrophoretic Monitoring and Interstrain Separation of Intact <i>Clostridium difficile</i> Based on Their S(Surface)-Layers

<i>Clostridium difficile</i> (<i>C. difficile</i>) infection (CDI) rates have exhibited a steady rise worldwide over the last two decades and the infection poses a global threat due to the emergence of antibiotic resistant strains. Interstrain antagonistic interactions across the host microbiome form an important strategy for controlling the emergence of CDI. The current diagnosis method for CDI, based on immunoassays for toxins produced by pathogenic <i>C. difficile</i> strains, is limited by false negatives due to rapid toxin degradation. Furthermore, simultaneous monitoring of nontoxigenic <i>C. difficile</i> strains is not possible, due to absence of these toxins, thereby limiting its application toward the control of CDI through optimizing antagonistic interstrain interactions. Herein, we demonstrate that morphological differences within the cell wall of particular <i>C. difficile</i> strains with differing S-layer proteins can induce systematic variations in their electrophysiology, due alterations in cell wall capacitance. As a result, dielectrophoretic frequency analysis can enable the independent fingerprinting and label-free separation of intact microbials of each strain type from mixed <i>C. difficile</i> samples. The sensitivity of this contact-less electrophysiological method is benchmarked against the immunoassay and microbial growth rate methods for detecting alterations within both, toxigenic and nontoxigenic <i>C. difficile</i> strains after vancomycin treatment. This microfluidic diagnostic platform can assist in the development of therapies for arresting clostridial infections by enabling the isolation of individual strains, optimization of antibiotic treatments and the monitoring of microbiomes

Correlating Antibiotic-Induced Dysbiosis to <i>Clostridioides difficile</i> Spore Germination and Host Susceptibility to Infection Using an <i>Ex Vivo</i> Assay

Antibiotic-induced microbiota disruption and its persistence create conditions for dysbiosis and colonization by opportunistic pathogens, such as those causing Clostridioides difficile (C. difficile) infection (CDI), which is the most severe hospital-acquired intestinal infection. Given the wide differences in microbiota across hosts and in their recovery after antibiotic treatments, there is a need for assays to assess the influence of dysbiosis and its recovery dynamics on the susceptibility of the host to CDI. Germination of C. difficile spores is a key virulence trait for the onset of CDI, which is influenced by the level of primary vs secondary bile acids in the intestinal milieu that is regulated by the microbiota composition. Herein, the germination of C. difficile spores in fecal supernatant from mice that are subject to varying degrees of antibiotic treatment is utilized as an ex vivo assay to predict intestinal dysbiosis in the host based on their susceptibility to CDI, as determined by in vivo CDI metrics in the same mouse model. Quantification of spore germination down to lower detection limits than the colony-forming assay is achieved by using impedance cytometry to count single vegetative bacteria that are identified based on their characteristic electrical physiology for distinction vs aggregated spores and cell debris in the media. As a result, germination can be quantified at earlier time points and with fewer spores for correlation to CDI outcomes. This sets the groundwork for a point-of-care tool to gauge the susceptibility of human microbiota to CDI after antibiotic treatments

Figure 3

<p>A. Representative ileal histology from orally <i>Cryptosporidium parvum</i> infected mice. Wild-type, APOE knock-out, and APOE targeted replacement mice (APOE 3/3 TR and APOE 4/4 TR) were fed with a low protein diet during 7 days then infected with 10⁷-unexcysted <i>Cryptosporidium parvum</i> oocysts and euthanized seven days after infection. H&E ×400. Scale bar 10 µM. <b>B</b>. Ileal villus height; <b>C</b>. crypt depth, and <b>D</b>. villus-crypt ratio from wild-type, APOE knock-out, and APOE targeted replacement mice (APOE 3/3 TR and APOE 4/4 TR). Morphometrics was done from hematoxylin and eosin stained-sections in at least four animals per group at low magnification. Data are presented as mean±SEM. Comparisons were performed by Students unpaired <i>T</i> test. Villi and crypts were measured only when their full longitudinal axis was found.</p

The loss of PPARγ in T cells regulates colonic cytokine expression of mice infected with <i>Clostridium difficile</i>.

<p>Colonic expression of interleukin 10 (IL-10) (A), interleukin 17 (IL-17) (B), monocyte chemoattractant protein 1 (MCP-1) (C) and tumor necrosis factor (TNF-α) (D) were assessed by real-time quantitative RT-PCR in wild type and T cell PPARγ null mice infected with <i>C. difficile</i> (n = 8). Data are represented as mean ± standard error. Points with an asterisk are significantly different when compared to the wild type control group (<i>P</i><0.05).</p

Lipid profile of experimental undernourished mice following <i>Cryposporidium parvum</i> infection (mice orally infected with 10⁷ unexcysted oocysts).

<p>*p<0.001 ApoE Ko vs all;</p><p>** p = 0.05 ApoE TR 4/4 vs ApoE TR 3/3;</p><p>*** p<0.001 ApoE Ko vs all;</p>#<p>p<0.05 ApoE Ko vs ApoE TR 4/4 by Student <i>t</i> Test.</p

Quantitative real-time PCR assays from experimental mice for the following ileal mRNA transcripts: (A) cationic amino acid transporter (CAT-1); (B) arginase 1; (C) Toll-like receptor 9 (TLR9); and (D) Inducible nitric oxide synthase (iNOS).

<p>Experimental mice were challenged by a compounded malnutrition and <i>Cryptosporidium parvum</i> insult and samples were harvested on day 7 post-<i>C. parvum inoculum</i>. Wild-type, APOE knockout, and APOE targeted replacement mice (APOE 3/3 TR and APOE 4/4 TR) were orally inoculated with 10⁷- unexcysted oocysts diluted in 100 µl of PBS. Groups have at least 4 per groups and the results are shown as mean ±SEM and expressed after β-actin normalization.</p

Modeling the Role of Peroxisome Proliferator-Activated Receptor γ and MicroRNA-146 in Mucosal Immune Responses to <em>Clostridium difficile</em>

<div><p><em>Clostridium difficile</em> is an anaerobic bacterium that has re-emerged as a facultative pathogen and can cause nosocomial diarrhea, colitis or even death. Peroxisome proliferator-activated receptor (PPAR) γ has been implicated in the prevention of inflammation in autoimmune and infectious diseases; however, its role in the immunoregulatory mechanisms modulating host responses to <em>C. difficile</em> and its toxins remains largely unknown. To characterize the role of PPARγ in <em>C. difficile</em>-associated disease (CDAD), immunity and gut pathology, we used a mouse model of <em>C. difficile</em> infection in wild-type and T cell-specific PPARγ null mice. The loss of PPARγ in T cells increased disease activity and colonic inflammatory lesions following <em>C. difficile</em> infection. Colonic expression of IL-17 was upregulated and IL-10 downregulated in colons of T cell-specific PPARγ null mice. Also, both the loss of PPARγ in T cells and <em>C. difficile</em> infection favored Th17 responses in spleen and colonic lamina propria of mice with CDAD. MicroRNA (miRNA)-sequencing analysis and RT-PCR validation indicated that miR-146b was significantly overexpressed and nuclear receptor co-activator 4 (NCOA4) suppressed in colons of <em>C. difficile</em>-infected mice. We next developed a computational model that predicts the upregulation of miR-146b, downregulation of the PPARγ co-activator NCOA4, and PPARγ, leading to upregulation of IL-17. Oral treatment of <em>C. difficile</em>-infected mice with the PPARγ agonist pioglitazone ameliorated colitis and suppressed pro-inflammatory gene expression. In conclusion, our data indicates that miRNA-146b and PPARγ activation may be implicated in the regulation of Th17 responses and colitis in <em>C. difficile</em>-infected mice.</p> </div

Fecal shedding of parasites in weaned undernourished C57BL/6 mice orally inoculated with 10⁷-unexcysted <i>Cryptosporidium parvum</i> oocysts per mouse (given in100 µL of PBS) on day 7 after the onset of the low protein diet.

<p>Results are shown in a log scale a mean±SEM. <i>Cryptosporidium parvum</i> stool oocyst shedding was determined by qRT PCR. Data were expressed as number of parasites per miligram of stool and percentage and number of infected mice with measurable oocyst shedding per day after <i>Cryptosporidium parvum</i> challenge. N above the bars means the number of mice still showing oocyst shedding.</p

Figure 1

<p>A. Body weight gain (% initial weight) from experimental uninfected and undernourished groups under a low protein diet. APOE 4/4 targeted replacement (APOE 4/4 TR) mice (n = 17) showed 643 a better growth response in comparison with APOE 3/3 targeted replacement (APOE 3/3 TR) mice (n = 8). <b>B.</b> Body weight gain (% initial infection weight) from experimental mice challenged by a compounded malnutrition and <i>Cryptosporidium parvum</i> insult. Undernourished mice were orally inoculated with 10⁷- unexcysted oocysts diluted in 100 µl of PBS. APOE deficient mice show impaired growth following <i>Cryptosporidium parvum</i> infection as compared to the other groups. Results are shown as mean ±SEM.</p

The loss of PPARγ in T cells and <i>Clostridium difficile</i> infection enhances Th17 responses in spleen and lamina propria of mice.

<p>Splenocytes and lamina propria lymphocytes from wild type and T cell PPARγnull mice infected with <i>C. difficile</i> (n = 6) were immunophenotyped to identify immune cell subsets by flow cytometry. Data are represented as mean ± standard error. Points with an asterisk are significantly different when compared to the control group (<i>P</i><0.05).</p