Correlating Antibiotic-Induced Dysbiosis to Clostridioides difficile Spore Germination and Host Susceptibility to Infection Using an Ex Vivo 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.

Automated biophysical classification of apoptotic pancreatic cancer cell subpopulations by using machine learning approaches with impedance cytometry.

Unrestricted cell death can lead to an immunosuppressive tumor microenvironment, with dysregulated apoptotic signaling that causes resistance of pancreatic cancer cells to cytotoxic therapies. Hence, modulating cell death by distinguishing the progression of subpopulations under drug treatment from viable towards early apoptotic, late apoptotic, and necrotic states is of interest. While flow cytometry after fluorescent staining can monitor apoptosis with single-cell sensitivity, the background of non-viable cells within non-immortalized pancreatic tumors from xenografts can confound distinction of the intensity of each apoptotic state. Based on single-cell impedance cytometry of drug-treated pancreatic cancer cells that are obtained from tumor xenografts with differing levels of gemcitabine sensitivity, we identify the biophysical metrics that can distinguish and quantify cellular subpopulations at the early apoptotic versus late apoptotic and necrotic states, by using machine learning methods to train for the recognition of each phenotype. While supervised learning has previously been used for classification of datasets with known classes, our advancement is the utilization of optimal positive controls for each class, so that clustering by unsupervised learning and classification by supervised learning can occur on unknown datasets, without human interference or manual gating. In this manner, automated biophysical classification can be used to follow the progression of apoptotic states in each heterogeneous drug-treated sample, for developing drug treatments to modulate cancer cell death and advance longitudinal analysis to discern the emergence of drug resistant phenotypes.

Multiplexed assessment of engineered bacterial constructs for intracellular ß-galactosidase expression by redox amplification on catechol-chitosan modified nanoporous gold.

Synthetic biology approaches for rewiring of bacterial constructs to express particular intracellular factors upon induction with the target analyte are emerging as sensing paradigms for applications in environmental and in vivo monitoring. To aid in the design and optimization of bacterial constructs for sensing analytes, there is a need for lysis-free intracellular detection modalities that monitor the signal level and kinetics of expressed factors within different modified bacteria in a multiplexed manner, without requiring cumbersome surface immobilization. Herein, an electrochemical detection system on nanoporous gold that is electrofabricated with a biomaterial redox capacitor is presented for quantifying ß-galactosidase expressed inside modified Escherichia coli constructs upon induction with dopamine. This nanostructure-mediated redox amplification approach on a microfluidic platform allows for multiplexed assessment of the expressed intracellular factors from different bacterial constructs suspended in distinct microchannels, with no need for cell lysis or immobilization. Since redox mediators present over the entire depth of the microchannel can interact with the electrode and with the E. coli construct in each channel, the platform exhibits high sensitivity and enables multiplexing. We envision its application in assessing synthetic biology-based approaches for comparing specificity, sensitivity, and signal response time upon induction with target analytes of interest.

Apoptotic Bodies in the Pancreatic Tumor Cell Culture Media Enable Label-Free Drug Sensitivity Assessment by Impedance Cytometry.

The ability to rapidly and sensitively predict drug response and toxicity using in vitro models of patient-derived tumors is essential for assessing chemotherapy efficacy. Currently, drug sensitivity assessment for solid tumors relies on imaging adherent cells or by flow cytometry of cells lifted from drug-treated cultures after fluorescent staining for apoptotic markers. Subcellular apoptotic bodies (ABs), including microvesicles that are secreted into the culture media under drug treatment can potentially serve as markers for drug sensitivity, without the need to lift cells under culture. However, their stratification to quantify cell disassembly is challenging due to their compositional diversity, with tailored labeling strategies currently needed for the recognition and cytometry of each AB type. It is shown that the high frequency impedance phase versus size distribution of ABs determined by high-throughput single-particle impedance cytometry of supernatants in the media of gemcitabine-treated pancreatic tumor cultures exhibits phenotypic resemblance to lifted apoptotic cells and enables shape-based stratification within distinct size ranges, which is not possible by flow cytometry. It is envisioned that this tool can be applied in conjunction with the appropriate pancreatic tumor microenvironment model to assess drug sensitivity and toxicity of patient-derived tumors, without the need to lift cells from cultures.

Label-Free Quantification of Cell Cycle Synchronicity of Human Neural Progenitor Cells Based on Electrophysiology Phenotypes.

The ability to coax human-induced pluripotent stem cells (hiPSCs) into human neural progenitor cells (hNPCs) can lead to novel drug discovery and transplant therapy platforms for neurological diseases. Since hNPCs can form organoids that mimic brain development, there is emerging interest in their label-free characterization for controlling cell composition to optimize organoid formation in three-dimensional (3D) cultures. However, this requires the ability to quantify hNPCs in heterogeneous samples with subpopulations of similar phenotype. Using high-throughput (>6000 cells per condition), single-cell impedance cytometry, we present the utilization of electrophysiology for quantification of hNPC subpopulations that are altered in cell cycle synchronicity by camptothecin (CPT) exposure. Electrophysiology phenotypes are determined from impedance magnitude and phase metrics for distinguishing each cell cycle phase, as validated by flow cytometry, for a wide range of subpopulation proportions. Using multishell dielectric models for each cell cycle phase, electrophysiology alterations with CPT dose could be predicted. This label-free detection strategy can prevent loss of cell viability to speed the optimization of cellular compositions for organoid development.

Quantifying bacterial spore germination by single-cell impedance cytometry for assessment of host microbiota susceptibility to Clostridioides difficile infection.

The germination of ingested spores is often a necessary first step required for enabling bacterial outgrowth and host colonization, as in the case of Clostridioides difficile (C. difficile) infection. Spore germination rate in the colon depends on microbiota composition and its level of disruption by antibiotic treatment since secretions by commensal bacteria modulate primary to secondary bile salt levels to control germination. Assessment of C. difficile spore germination typically requires measurement of colony-forming units, which is labor intensive and takes at least 24 h to perform but is regularly required due to the high recurrence rates of nosocomial antibiotic-associated diarrhea. We present a rapid method to assess spore germination by using high throughput single-cell impedance cytometry (>300 events/s) to quantify live bacterial cells, by gating for their characteristic electrophysiology versus spores, so that germination can be assessed after just 4 h of culture at a detection limit of ~100 live cells per 50 µL sample. To detect the phenotype of germinated C. difficile bacteria, we utilize its characteristically higher net conductivity versus that of spore aggregates and non-viable C. difficile forms, which causes a distinctive high-frequency (10 MHz) impedance phase dispersion within moderately conductive media (0.8 S/m). In this manner, we can detect significant differences in spore germination rates within just 4 h, with increasing primary bile salt levels in vitro and using ex vivo microbiota samples from an antibiotic-treated mouse model to assess susceptibility to C. difficile infection. We envision a rapid diagnostic tool for assessing host microbiota susceptibility to bacterial colonization after key antibiotic treatments.

Minimum Bactericidal Concentration of Ciprofloxacin to Pseudomonas aeruginosa Determined Rapidly Based on Pyocyanin Secretion.

Infections due to Pseudomonas aeruginosa (P. aeruginosa) often exhibit broad-spectrum resistance and persistence to common antibiotics. Persistence is especially problematic with immune-compromised subjects who are unable to eliminate the inhibited bacteria. Hence, antibiotics must be used at the appropriate minimum bactericidal concentration (MBC) rather than at minimum inhibitory concentration (MIC) levels. However, MBC determination by conventional methods requires a 24 h culture step in the antibiotic media to confirm inhibition, followed by a 24 h sub-culture step in antibiotic-free media to confirm the lack of bacterial growth. We show that electrochemical detection of pyocyanin (PYO), which is a redox-active bacterial metabolite secreted by P. aeruginosa, can be used to rapidly assess the critical ciprofloxacin level required for bactericidal deactivation of P. aeruginosa within just 2 hours in antibiotic-treated growth media. The detection sensitivity for PYO can be enhanced by using nanoporous gold that is modified with a self-assembled monolayer to lower interference from oxygen reduction, while maintaining a low charge transfer resistance level and preventing electrode fouling within biological sample matrices. In this manner, bactericidal efficacy of ciprofloxacin towards P. aeruginosa at the MBC level and bacterial persistence at the MIC level can be determined rapidly, as validated at later timepoints using bacterial subculture in antibiotic-free media.

Rapid in Vitro Assessment of Clostridioides difficile Inhibition by Probiotics Using Dielectrophoresis to Quantify Cell Structure Alterations.

Clostridioides difficile (C. difficile) infection (CDI) is the primary cause of nosocomial antibiotic-associated diarrhea, with high recurrence rates following initial antibiotic treatment regimens. Restoration of the host gut microbiome through probiotic therapy is under investigation to reduce recurrence. Current in vitro methods to assess C. difficile deactivation by probiotic microorganisms are based on C. difficile growth inhibition, but the cumbersome and time-consuming nature of the assay limits the number of assessed permutations. Phenotypic alterations to the C. difficile cellular structure upon interaction with probiotics can potentially enable rapid assessment of the inhibition without the need for extended culture. Because supernatants from cultures of commensal microbiota reflect the complex metabolite milieu that deactivates C. difficile, we explore coculture of C. difficile with an optimal dose of supernatants from probiotic culture to speed growth inhibition assays and enable correlation with alterations to its prolate ellipsoidal structure. Based on sensitivity of electrical polarizability to C. difficile cell shape and subcellular structure, we show that the inhibitory effect of Lactobacillus spp. supernatants on C. difficile can be determined based on the positive dielectrophoresis level within just 1 h of culture using a highly toxigenic strain and a clinical isolate, whereas optical and growth inhibition measurements require far greater culture time. We envision application of this in vitro coculture model, in conjunction with dielectrophoresis, to rapidly screen for potential probiotic combinations for the treatment of recurrent CDI.

Conductance-Based Biophysical Distinction and Microfluidic Enrichment of Nanovesicles Derived from Pancreatic Tumor Cells of Varying Invasiveness.

Diagnostics based on exosomes and other extracellular vesicles (EVs) are emerging as strategies for informing cancer progression and therapies, since the lipid content and macromolecular cargo of EVs can provide key phenotypic and genotypic information on the parent tumor cell and its microenvironment. We show that EVs derived from more invasive pancreatic tumor cells that express high levels of tumor-specific surface proteins and are composed of highly unsaturated lipids that increase membrane fluidity, exhibit significantly higher conductance versus those derived from less invasive tumor cells, based on dielectrophoresis measurements. Furthermore, through specific binding of the EVs to gold nanoparticle-conjugated antibodies, we show that these conductance differences can be modulated in proportion to the type as well as level of expressed tumor-specific antigens, thereby presenting methods for selective microfluidic enrichment and cytometry-based quantification of EVs based on invasiveness of their parent cell.

Single-cell electro-phenotyping for rapid assessment of Clostridium difficile heterogeneity under vancomycin treatment at sub-MIC (minimum inhibitory concentration) levels.

Current methods for measurement of antibiotic susceptibility of pathogenic bacteria are highly reliant on microbial culture, which is time consuming (requires > 16 hours), especially at near minimum inhibitory concentration (MIC) levels of the antibiotic. We present the use of single-cell electrophysiology-based microbiological analysis for rapid phenotypic identification of antibiotic susceptibility at near-MIC levels, without the need for microbial culture. Clostridium difficile (C. difficile) is the single most common cause of antibiotic-induced enteric infection and disease recurrence is common after antibiotic treatments to suppress the pathogen. Herein, we show that de-activation of C. difficile after MIC-level vancomycin treatment, as validated by microbiological growth assays, can be ascertained rapidly by measuring alterations to the microbial cytoplasmic conductivity that is gauged by the level of positive dielectrophoresis (pDEP) and the frequency spectra for co-field electro-rotation (ROT). Furthermore, this single-cell electrophysiology technique can rapidly identify and quantify the live C. difficile subpopulation after vancomycin treatment at sub-MIC levels, whereas methods based on measurement of the secreted metabolite toxin or the microbiological growth rate can identify this persistent C. difficile subpopulation only after 24 hours of microbial culture, without any ability to quantify the subpopulation. The application of multiplexed versions of this technique is envisioned for antibiotic susceptibility screening.

Innate Immune Response and Outcome of Clostridium difficile Infection Are Dependent on Fecal Bacterial Composition in the Aged Host.

Background: Clostridium difficile infection (CDI) is a serious threat for an aging population. Using an aged mouse model, we evaluated the effect of age and the roles of innate immunity and intestinal microbiota. Methods: Aged (18 months) and young (8 weeks) mice were infected with C difficile, and disease severity, immune response, and intestinal microbiome were compared. The same experiment was repeated with intestinal microbiota exchange between aged and young mice before infection. Results: Higher mortality was observed in aged mice with weaker neutrophilic mobilization in blood and intestinal tissue and depressed proinflammatory cytokines in early infection. Microbiota exchange improved survival and early immune response in aged mice. Microbiome analysis revealed that aged mice have significant deficiencies in Bacteroidetes phylum and, specifically, Bacteroides, Alistipes, and rc4-4 genera, which were replenished by cage switching. Conclusions: Microbiota-dependent alteration in innate immune response early on during infection may explain poor outcome in aged host with CDI.

Label-Free Quantification of Intracellular Mitochondrial Dynamics Using Dielectrophoresis.

Mitochondrial dynamics play an important role within several pathological conditions, including cancer and neurological diseases. For the purpose of identifying therapies that target aberrant regulation of the mitochondrial dynamics machinery and characterizing the regulating signaling pathways, there is a need for label-free means to detect the dynamic alterations in mitochondrial morphology. We present the use of dielectrophoresis for label-free quantification of intracellular mitochondrial modifications that alter cytoplasmic conductivity, and these changes are benchmarked against label-based image analysis of the mitochondrial network. This is validated by quantifying the mitochondrial alterations that are carried out by entirely independent means on two different cell lines: human embryonic kidney cells and mouse embryonic fibroblasts. In both cell lines, the inhibition of mitochondrial fission that leads to a mitochondrial structure of higher connectivity is shown to substantially enhance conductivity of the cell interior, as apparent from the significantly higher positive dielectrophoresis levels in the 0.5-15 MHz range. Using single-cell velocity tracking, we show ∼10-fold higher positive dielectrophoresis levels at 0.5 MHz for cells with a highly connected versus those with a highly fragmented mitochondrial structure, suggesting the feasibility for frequency-selective dielectrophoretic isolation of cells to aid the discovery process for development of therapeutics targeting the mitochondrial machinery.

Rapid detection of Clostridium difficile via magnetic bead aggregation in cost-effective polyester microdevices with cell phone image analysis.

Pathogen detection has traditionally been accomplished by utilizing methods such as cell culture, immunoassays, and nucleic acid amplification tests; however, these methods are not easily implemented in resource-limited settings because special equipment for detection and thermal cycling is often required. In this study, we present a magnetic bead aggregation assay coupled to an inexpensive microfluidic fabrication technique that allows for cell phone detection and analysis of a notable pathogen in less than one hour. Detection is achieved through the use of a custom-built system that allows for fluid flow control via centrifugal force, as well as manipulation of magnetic beads with an adjustable rotating magnetic field. Cell phone image capture and analysis is housed in a 3D-printed case with LED backlighting and a lid-mounted Android phone. A custom-written application (app.) is employed to interrogate images for the extent of aggregation present following loop-mediated isothermal amplification (LAMP) coupled to product-inhibited bead aggregation (PiBA) for detection of target sequences. Clostridium difficile is a pathogen of increasing interest due to its causative role in intestinal infections following antibiotic treatment, and was therefore chosen as the pathogen of interest in the present study to demonstrate the rapid, cost-effective, and sequence-specific detection capabilities of the microfluidic platform described herein.

Cryptosporidium Priming Is More Effective than Vaccine for Protection against Cryptosporidiosis in a Murine Protein Malnutrition Model.

Cryptosporidium is a major cause of severe diarrhea, especially in malnourished children. Using a murine model of C. parvum oocyst challenge that recapitulates clinical features of severe cryptosporidiosis during malnutrition, we interrogated the effect of protein malnutrition (PM) on primary and secondary responses to C. parvum challenge, and tested the differential ability of mucosal priming strategies to overcome the PM-induced susceptibility. We determined that while PM fundamentally alters systemic and mucosal primary immune responses to Cryptosporidium, priming with C. parvum (106 oocysts) provides robust protective immunity against re-challenge despite ongoing PM. C. parvum priming restores mucosal Th1-type effectors (CD3+CD8+CD103+ T-cells) and cytokines (IFNγ, and IL12p40) that otherwise decrease with ongoing PM. Vaccination strategies with Cryptosporidium antigens expressed in the S. Typhi vector 908htr, however, do not enhance Th1-type responses to C. parvum challenge during PM, even though vaccination strongly boosts immunity in challenged fully nourished hosts. Remote non-specific exposures to the attenuated S. Typhi vector alone or the TLR9 agonist CpG ODN-1668 can partially attenuate C. parvum severity during PM, but neither as effectively as viable C. parvum priming. We conclude that although PM interferes with basal and vaccine-boosted immune responses to C. parvum, sustained reductions in disease severity are possible through mucosal activators of host defenses, and specifically C. parvum priming can elicit impressively robust Th1-type protective immunity despite ongoing protein malnutrition. These findings add insight into potential correlates of Cryptosporidium immunity and future vaccine strategies in malnourished children.

Vancomycin Treatment Alters Humoral Immunity and Intestinal Microbiota in an Aged Mouse Model of Clostridium difficile Infection.

BACKGROUND: The elderly host is highly susceptible to severe disease and treatment failure in Clostridium difficile infection (CDI). We investigated how treatment with vancomycin in the aged host influences systemic and intestinal humoral responses and select intestinal microbiota. METHODS: Young (age, 2 months) and aged (age, 18 months) C57BL/6 mice were infected with VPI 10463 after exposure to broad-spectrum antibiotics. Vancomycin was given 24 hours after infection, and treatment was continued for 5 days. At select time points, specimens of serum and intestinal tissue and contents were collected for histopathologic analysis, to measure antibody levels and the pathogen burden, and to determine the presence and levels of select intestinal microbiota and C. difficile toxin. RESULTS: Levels of disease severity, relapse, and mortality were increased, and recovery from infection was slower in aged mice compared to young mice. Serum levels of immunoglobulin M, immunoglobulin A, and immunoglobulin G against C. difficile toxin A were depressed in aged mice, and vancomycin treatment reduced antibody responses in both age groups. While baseline levels of total bacterial load, Bacteroidetes, Firmicutes, and Enterobacteriaceae were mostly similar, aged mice had a significant change in the Firmicutes to Bacteroidetes ratio with vancomycin treatment. CONCLUSIONS: Vancomycin treatment decreases the systemic humoral response to CDI. Increased mortality from and recurrence of CDI in the aged host are associated with an impaired humoral response and a greater susceptibility to vancomycin-induced alteration of intestinal microbiota.

Treatment of Clostridium difficile infection using SQ641, a capuramycin analogue, increases post-treatment survival and improves clinical measures of disease in a murine model.

OBJECTIVES: Clostridium difficile infection (CDI) is a primary cause of antibiotic-associated diarrhoeal illness. Current therapies are insufficient as relapse rates following antibiotic treatment range from 25% for initial treatment to 60% for treatment of recurrence. In this study, we looked at the efficacy of SQ641 in a murine model of CDI. SQ641 is an analogue of capuramycin, a naturally occurring nucleoside-based compound produced by Streptomyces griseus. METHODS: In a series of experiments, C57BL/6 mice were treated with a cocktail of antibiotics and inoculated with C. difficile strain VPI10463. Animals were treated orally with SQ641 for 5 days at a dose range of 0.1-300 mg/kg/day, 20 mg/kg/day vancomycin or drug vehicle. Animals were monitored for disease severity, clostridial shedding and faecal toxin levels for 14 days post-infection. RESULTS: Five day treatment of CDI with SQ641 resulted in higher 14 day survival rates in mice compared with either vancomycin or vehicle alone. CDI survival rates were 100% (13 of 13) and 94% (32 of 34), respectively, in the 1 and 10 mg/kg/day SQ641 treatment groups, 37% (7 of 19) with vancomycin treatment at 20 mg/kg/day and 32% (14 of 44) in the vehicle-only control group. Secondary measures of efficacy, such as prevention of weight loss, decreased disease severity, decreased C. difficile shedding and decreased toxin in faeces, were observed with SQ641 and vancomycin treatment. CONCLUSIONS: SQ641 is effective for CDI treatment with prevention of relapse in the murine model of CDI.

Determining the drift potential of Venturi nozzles compared with standard nozzles across three insecticide spray solutions in a wind tunnel.

BACKGROUND: Previous research has sought to adopt the use of drift-reducing technologies (DRTs) for use in field trials to control diamondback moth (DBM) Plutella xylostella (L.) (Lepidoptera: Plutellidae) in canola (Brassica napus L.). Previous studies observed no difference in canopy penetration from fine to coarse sprays, but the coverage was higher for fine sprays. DBM has a strong propensity to avoid sprayed plant material, putting further pressure on selecting technologies that maximise coverage, but often this is at the expense of a greater drift potential. This study aims to examine the addition of a DRT oil that is labelled for control of DBM as well and its effect on the drift potential of the spray solution. The objectives of the study are to quantify the droplet size spectrum and spray drift potential of each nozzle type to select technologies that reduce spray drift, to examine the effect of the insecticide tank mix at both (50 and 100 L ha(-1) ) application rates on droplet size and spray drift potential across tested nozzle type and to compare the droplet size results of each nozzle by tank mix against the drift potential of each nozzle. RESULTS: The nozzle type affected the drift potential the most, but the spray solution also affected drift potential. The fine spray quality (TCP) resulted in the greatest drift potential (7.2%), whereas the coarse spray quality (AIXR) resulted in the lowest (1.3%), across all spray solutions. The spray solutions mixed at the 100 L ha(-1) application volume rate resulted in a higher drift potential than the same products mixed at the 50 L ha(-1) mix rate. The addition of the paraffinic DRT oil was significant in reducing the drift potential of Bacillus thuringiensis var. kurstkai (Bt)-only treatments across all tested nozzle types. The reduction in drift potential from the fine spray quality to the coarse spray quality was up to 85%. CONCLUSION: The addition of a DRT oil is an effective way to reduce the spray solution drift potential across all nozzle types and tank mixes evaluated in this study. The greatest reduction in drift potential can be achieved by changing nozzle type, which can reduce the losses of the spray to the surrounding environment. Venturi nozzles greatly reduce the drift potential compared with standard nozzles by as much as 85% across all three insecticide spray solutions. Results suggest that a significant reduction in drift potential can be achieved by changing the nozzle type, and can be achieved without a loss in control of DBM. © 2016 Society of Chemical Industry.

Defined Nutrient Diets Alter Susceptibility to Clostridium difficile Associated Disease in a Murine Model.

BACKGROUND: Clostridium difficile is a major identifiable and treatable cause of antibiotic-associated diarrhea. Poor nutritional status contributes to mortality through weakened host defenses against various pathogens. The primary goal of this study was to assess the contribution of a reduced protein diet to the outcomes of C. difficile infection in a murine model. METHODS: C57BL/6 mice were fed a traditional house chow or a defined diet with either 20% protein or 2% protein and infected with C. difficile strain VPI10463. Animals were monitored for disease severity, clostridial shedding and fecal toxin levels. Select intestinal microbiota were measured in stool and C. difficile growth and toxin production were quantified ex vivo in intestinal contents from untreated or antibiotic-treated mice fed with the different diets. RESULTS: C. difficile infected mice fed with defined diets, particularly (and unexpectedly) with protein deficient diet, had increased survival, decreased weight loss, and decreased overall disease severity. C. difficile shedding and toxin in the stool of the traditional diet group was increased compared with either defined diet 1 day post infection. Mice fed with traditional diet had an increased intestinal Firmicutes to Bacteroidetes ratio following antibiotic exposure compared with either a 2% or 20% protein defined nutrient diet. Ex vivo inoculation of cecal contents from antibiotic-treated mice showed decreased toxin production and C. difficile growth in both defined diets compared with a traditional diet. CONCLUSIONS: Low protein diets, and defined nutrient diets in general, were found to be protective against CDI in mice. Associated diet-induced alterations in intestinal microbiota may influence colonization resistance and clostridial toxin production in a defined nutrient diet compared to a traditional diet, leading to increased survival. However, mechanisms which led to survival differences between 2% and 20% protein defined nutrient diets need to be further elucidated.

Zinc deficiency alters host response and pathogen virulence in a mouse model of enteroaggregative Escherichia coli-induced diarrhea.

Enteroaggregative Escherichia coli (EAEC) is increasingly recognized as a major cause of diarrheal disease globally. In the current study, we investigated the impact of zinc deficiency on the host and pathogenesis of EAEC. Several outcomes of EAEC infection were investigated including weight loss, EAEC shedding and tissue burden, leukocyte recruitment, intestinal cytokine expression, and virulence expression of the pathogen in vivo. Mice fed a protein source defined zinc deficient diet (dZD) had an 80% reduction of serum zinc and a 50% reduction of zinc in luminal contents of the bowel compared to mice fed a protein source defined control diet (dC). When challenged with EAEC, dZD mice had significantly greater weight loss, stool shedding, mucus production, and, most notably, diarrhea compared to dC mice. Zinc deficient mice had reduced infiltration of leukocytes into the ileum in response to infection suggesting an impaired immune response. Interestingly, expression of several EAEC virulence factors were increased in luminal contents of dZD mice. These data show a dual effect of dietary zinc in benefitting the host while impairing virulence of the pathogen. The study demonstrates the critical importance of zinc and may help elucidate the benefits of zinc supplementation in cases of childhood diarrhea and malnutrition.