Difficile Colonization Research Articles

The Incidence of Diarrhea and Clostridium Difficile Colonization Due to Antibiotic Intake in Sample of Egyptian Pediatrics

The Incidence of Diarrhea and Clostridium Difficile Colonization Due to Antibiotic Intake in Sample of Egyptian Pediatrics

Mice colonized with the defined microbial community OMM19.1 are susceptible to Clostridioides difficile infection without prior antibiotic treatment.

Diverse gut microorganisms present in humans and mice are essential for the prevention of microbial pathogen colonization. However, antibiotic-induced dysbiosis of the gut microbiome reduces microbial diversity and allows Clostridioides difficile (C. difficile) to colonize the intestine. The Oligo-Mouse-Microbiota 19.1 (OMM19.1) is a synthetic community that consists of bacteria that are taxonomically and functionally designed to mimic the specific pathogen-free mouse gut microbiota. Here, we examined the susceptibility of OMM19.1 colonized mice to C. difficile infection (CDI) at a range of infectious doses (103, 105, and 107 spores) without prior antibiotic treatment. We found that mice colonized with OMM19.1 were susceptible to CDI regardless of the dose. The clinical scores increased with increasing C. difficile dosage. Infection with C. difficile was correlated with a significant increase in Ligilactobacillus murinus and Escherichia coli, while the relative abundance of Bacteroides caecimuris, Akkermansia muciniphila, Extibacter muris, and Turicimonas muris was significantly decreased following CDI. Our results demonstrate that the OMM19.1 community requires additional bacteria to enable C. difficile colonization resistance.IMPORTANCEThe human gut microbiota consists of a wide range of microorganisms whose composition and function vary according to their location and have a significant impact on health and disease. The ability to generate and test the defined microbiota within gnotobiotic animal models is essential for determining the mechanisms responsible for colonization resistance. The exact mechanism(s) by which healthy microbiota prevents Clostridioides difficile infection is unknown, although competition for nutrients, active antagonism, production of inhibitory metabolites (such as secondary bile acids), and microbial manipulation of the immune system are all thought to play a role. Here, we colonized germ-free C57BL/6 mice with a synthetic bacterial community (OMM19.1) that mimics the specific pathogen-free mouse microbiota. Following breeding, to enable immune system development, F1 mice were infected with three different doses of C. difficile. Our research suggests that there are additional essential microbial functions that are absent from the current OMM19.1 model.

Efficacy of grapefruit seed extract and lactic acid bacteria in reducing Clostridioides difficile infection via gut microbiota modulation

Antibiotic-induced dysbiosis is a key factor that causes Clostridioides difficile infection (CDI). The current first-line drugs for initial CDI are antibiotics, and finding an alternate therapy to prevent recurrent CDI is essential. In this study, the anti-C. difficile activities of two isolated probiotic strains (Pediococcus pentosaceus CD5 and Lactobacillus salivarius CD4) and grapefruit seed extract (GSE) were investigated in vitro and in vivo. Specifically, GSE inhibited the growth of C. difficile at a minimal inhibitory concentration (MIC) of 40 ppm. Similarly, live cells of the two probiotics, as well as their cell-free culture supernatants, with or without pH adjustment, substantially suppressed C. difficile growth. In a CDI mouse model, C. difficile colonization in the cecum and colon inflammation were significantly reduced by GSE, followed by the probiotic treatments. A metagenomic analysis of cecal feces revealed that GSE and probiotic treatments modulated gut microbial compositions and enhanced the relative abundances of butyrate-producing bacteria. Therefore, GSE and the novel probiotics can potentially be administered as alternative therapies to alleviate CDI by modifying the gut microbiota.

A Mouse Model of Mild Clostridioides difficile Infection for the Characterization of Natural Immune Responses.

(1) Background: We describe a model of primary mild-Clostridioides difficile infection (CDI) in a naïve host, including gut microbiota analysis, weight loss, mortality, length of colonization. This model was used in order to describe the kinetics of humoral (IgG, IgM) and mucosal (IgA) immune responses against toxins (TcdA/TcdB) and surface proteins (SlpA/FliC). (2) Methods: A total of 105 CFU vegetative forms of C. difficile 630Δerm were used for challenge by oral administration after dysbiosis, induced by a cocktail of antibiotics. Gut microbiota dysbiosis was confirmed and described by 16S rDNA sequencing. We sacrificed C57Bl/6 mice after different stages of infection (day 6, 2, 7, 14, 21, 28, and 56) to evaluate IgM, IgG against TcdA, TcdB, SlpA, FliC in blood samples, and IgA in the cecal contents collected. (3) Results: In our model, we observed a reproducible gut microbiota dysbiosis, allowing for C. difficile digestive colonization. CDI was objectivized by a mean weight loss of 13.1% and associated with a low mortality rate of 15.7% of mice. We observed an increase in IgM anti-toxins as early as D7 after challenge. IgG increased since D21, and IgA anti-toxins were secreted in cecal contents. Unexpectedly, neither anti-SlpA nor anti-FliC IgG or IgA were observed in our model. (4) Conclusions: In our model, we induced a gut microbiota dysbiosis, allowing a mild CDI to spontaneously resolve, with a digestive clearance observed since D14. After this primary CDI, we can study the development of specific immune responses in blood and cecal contents.

Diagnosis and management of Clostridioides difficile in inflammatory bowel disease.

Patients with IBD have an increased risk for Clostridioides difficile infection (CDI), which can lead to worse IBD outcomes. The diagnosis of CDI in IBD patients is complicated by higher C. difficile colonization rates and shared clinical symptoms of intestinal inflammation. Traditional risk factors for CDI, such as antibiotic exposure, may be lacking in IBD patients due to underlying intestinal microbiota dysbiosis. While CDI disproportionately affects people with IBD, patients with IBD are typically excluded from CDI clinical trials creating a knowledge gap in the diagnosis and management of these two diseases. This narrative review aims to provide a comprehensive overview of the diagnosis, treatment, and prevention of CDI in IBD patients. Distinguishing CDI from C. difficile colonization in the setting of an IBD exacerbation is important to avoid treatment delays. When CDI is diagnosed, extended courses of anti-C. difficile antibiotics may lead to better CDI outcomes. Regardless of a diagnosis of CDI, the presence of C. difficile in an IBD patient should prompt a disease assessment of the underlying IBD. Microbiota-based therapies and bezlotoxumab appear to be effective in preventing CDI recurrence in IBD patients. Patients with IBD should be considered high risk for CDI recurrence and evaluated for a preventative strategy when diagnosed with CDI. Ultimately, the co-management of CDI in an IBD patient requires a nuanced, patient-specific approach to distinguish CDI from C. difficile colonization, prevent CDI recurrence, and manage the underlying IBD.

Reduction of Antibiotic-Associated Conditions After Tympanostomy Tube Placement in Children.

Tympanostomy tube placement has been shown to decrease systemic antibiotics usage in patients with recurrent acute otitis media. Systemic antibiotics in children are associated with an increase in antibiotic-associated conditions (asthma, allergic rhinitis, food allergy, atopic dermatitis, celiac disease, overweight/obesity, attention-deficit hyperactivity disorder [ADHD], autism, learning disability, and Clostridium difficile colonization) later in life. The objective of this study is to estimate whether tympanostomy tube placement is associated with a reduction in antibiotic-associated conditions in children with recurrent acute otitis media (RAOM). A retrospective cohort review of electronic medical records from 1991 to 2011 at a large pediatric hospital system was performed identifying 27,584 patients under 18 years old with RAOM, defined by 3 or more episodes of AOM. Antibiotic-associated conditions were defined using ICD-9 and ICD-10 codes. The enrollment population was largely composed of White patients (28.9%), Black patients (30.1%), and Hispanic/Latino patients (16.4%). The number of systemic antibiotics prescribed per encounter was significantly lower in children who pursued tympanostomy tubes (0.14 antibiotics per encounter) versus those who did not (0.23 antibiotics per encounter) (p < 0.001). Patients with RAOM who received tympanostomy tubes were less likely to have diagnoses of overweight/obesity (OR. 0.62 [0.55, 0.68]; p < 0.001), asthma (OR 0.8 [0.74, 0.87]; p < 0.001), allergic rhinitis (OR 0.72 [0.65, 0.81]; p < 0.001), and atopic dermatitis (0.78 [0.71, 0.86]; p < 0.001). Tympanostomy tube placement is associated with less systemic antibiotic administration and a decreased incidence of overweight/obesity, asthma, allergic rhinitis, and atopic dermatitis in children diagnosed with RAOM. 4 Laryngoscope, 2024.

Commensal-pathogen dynamics structure disease outcomes during Clostridioides difficile colonization.

Gastrointestinal colonization by Clostridioides difficile is common in healthcare settings and ranges in clinical presentation from asymptomatic carriage to lethal C. difficile infection (CDI). We used a systems biology approach to investigate why patients colonized with C. difficile have a range of outcomes. Microbiota-humanization of germ-free mice with fecal samples from toxigenic C. difficile carriers revealed a spectrum of virulence among clade 1 lineages and identified commensal Blautia associated with markers of non-pathogenic colonization. Using gnotobiotic mice engrafted with defined human microbiota, we observed strain-specific CDI severity across clade 1 strains. Yet, mice engrafted with a higher diversity community were protected from severe disease across all strains without suppression of C. difficile colonization. These results indicate that when colonization resistance has been breached without overt infection, commensals can attenuate a diversity of virulent strains without inhibiting pathogen colonization, providing insight into determinants of stable C. difficile carriage.

Clostridioides difficile colonization is not mediated by bile salts and requires Stickland fermentation of proline in an in vitro model of infection.

Treatment with antibiotics is a major risk factor for Clostridioides difficile infection, likely due to depletion of the gastrointestinal microbiota. Two microbiota-mediated mechanisms thought to limit C. difficile colonization include conversion of conjugated primary bile salts into secondary bile salts toxic to C. difficile growth, and competition between the microbiota and C. difficile for limiting nutrients. Using a continuous flow model of the distal colon, we investigated how treatment with six clinically-used antibiotics influenced susceptibility to C. difficile infection in 12 different microbial communities cultivated from healthy individuals. Antibiotic treatment reduced microbial richness; disruption varied by antibiotic class and microbiota composition, but did not correlate with C. difficile susceptibility. Antibiotic treatment also disrupted microbial bile salt metabolism, increasing levels of the primary bile salt, cholate, and decreasing levels of the secondary bile salt, deoxycholate. However, decreased levels of deoxycholate did not correlate with increased C. difficile susceptibility. Further, bile salts were not required to inhibit C. difficile colonization. We tested whether amino acid fermentation contributed to persistence of C. difficile in antibiotic-treated communities. C. difficile mutants unable to use proline as an electron acceptor in Stickland fermentation due to disruption of proline reductase (ΔprdB) had significantly lower levels of colonization than wild-type strains in four of six antibiotic-treated communities tested. This data provides further support for the importance of bile salt-independent mechanisms in regulating colonization of C. difficile.

Clostridioides difficile superoxide reductase mitigates oxygen sensitivity.

Clostridioides difficile causes a serious diarrheal disease and is a common healthcare-associated bacterial pathogen. Although it has a major impact on human health, the mechanistic details of C. difficile intestinal colonization remain undefined. C. difficile is highly sensitive to oxygen and requires anaerobic conditions for in vitro growth. However, the mammalian gut is not devoid of oxygen, and C. difficile tolerates moderate oxidative stress in vivo. The C. difficile genome encodes several antioxidant proteins, including a predicted superoxide reductase (SOR) that is upregulated upon exposure to antimicrobial peptides. The goal of this study was to establish SOR enzymatic activity and assess its role in protecting C. difficile against oxygen exposure. Insertional inactivation of sor rendered C. difficile more sensitive to superoxide, indicating that SOR contributes to antioxidant defense. Heterologous C. difficile sor expression in Escherichia coli conferred protection against superoxide-dependent growth inhibition, and the corresponding cell lysates showed superoxide scavenging activity. Finally, a C. difficile SOR mutant exhibited global proteome changes under oxygen stress when compared to the parent strain. Collectively, our data establish the enzymatic activity of C. difficile SOR, confirm its role in protection against oxidative stress, and demonstrate SOR's broader impacts on the C. difficile vegetative cell proteome.IMPORTANCEClostridioides difficile is an important pathogen strongly associated with healthcare settings and capable of causing severe diarrheal disease. While considered a strict anaerobe in vitro, C. difficile has been shown to tolerate low levels of oxygen in the mammalian host. Among other well-characterized antioxidant proteins, the C. difficile genome encodes a predicted superoxide reductase (SOR), an understudied component of antioxidant defense in pathogens. The significance of the research reported herein is the characterization of SOR's enzymatic activity, including confirmation of its role in protecting C. difficile against oxidative stress. This furthers our understanding of C. difficile pathogenesis and presents a potential new avenue for targeted therapies.

Overcoming donor variability and risks associated with fecal microbiota transplants through bacteriophage-mediated treatments

BackgroundFecal microbiota transplantation (FMT) and fecal virome transplantation (FVT, sterile filtrated donor feces) have been effective in treating recurrent Clostridioides difficile infections, possibly through bacteriophage-mediated modulation of the gut microbiome. However, challenges like donor variability, costly screening, coupled with concerns over pathogen transfer (incl. eukaryotic viruses) with FMT or FVT hinder their wider clinical application in treating less acute diseases.MethodsTo overcome these challenges, we developed methods to broaden FVT’s clinical application while maintaining efficacy and increasing safety. Specifically, we employed the following approaches: (1) chemostat-fermentation to reproduce the bacteriophage FVT donor component and remove eukaryotic viruses (FVT-ChP), (2) solvent-detergent treatment to inactivate enveloped viruses (FVT-SDT), and (3) pyronin-Y treatment to inhibit RNA virus replication (FVT-PyT). We assessed the efficacy of these processed FVTs in a C. difficile infection mouse model and compared them with untreated FVT (FVT-UnT), FMT, and saline.ResultsFVT-SDT, FVT-UnT, and FVT-ChP reduced the incidence of mice reaching the humane endpoint (0/8, 2/7, and 3/8, respectively) compared to FMT, FVT-PyT, and saline (5/8, 7/8, and 5/7, respectively) and significantly reduced the load of colonizing C. difficile cells and associated toxin A/B levels. There was a potential elimination of C. difficile colonization, with seven out of eight mice treated with FVT-SDT testing negative with qPCR. In contrast, all other treatments exhibited the continued presence of C. difficile. Moreover, the results were supported by changes in the gut microbiome profiles, cecal cytokine levels, and histopathological findings. Assessment of viral engraftment following FMT/FVT treatment and host-phage correlations analysis suggested that transfer of phages likely were an important contributing factor associated with treatment efficacy.ConclusionsThis proof-of-concept study shows that specific modifications of FVT hold promise in addressing challenges related to donor variability and infection risks. Two strategies lead to treatments significantly limiting C. difficile colonization in mice, with solvent/detergent treatment and chemostat propagation of donor phages emerging as promising approaches.DixucG6bYNjUQ1q5irdn5dVideo

Hospital-Onset Clostridioides difficile infection in chronic kidney disease patients

Introduction: Hospital-onset Clostridioides difficile infection (HO-CDI), reported as laboratory-identified (LabID) event, is common in patients with chronic kidney disease (CKD), especially those with end-stage renal disease (ESRD), and is associated with prolonged length of hospitalization and more severe disease. CKD patients are at increased of developing CDI due to frequent antimicrobial and healthcare exposures. The objective of this study was to assess recent trends of HO-CDI in patients on a nephrology unit at our academic, tertiary care institution. Methods: Retrospective cross-sectional study of patients with HO-CDI who were hospitalized on a nephrology unit between January 2021 to December 2023. Collected variables included: demographic data, characterization of HO-CDI risk factors, infection and diagnosis (including prior history of CDI, toxin versus nucleic acid amplification test [NAAT] positivity, number of loose stools), CDI rate (defined as CDI count/patient days x1000), standardized antimicrobial administration ratio (SAAR) for high-risk for CDI antimicrobials (defined by the National Healthcare Safety Network), and infection prevention and control (IPC) practices, including hand hygiene audit rates. Results: A total of 30 HO-CDI infections were reported on the nephrology unit [Table], with 8 occurring in 2021, 5 in 2022, and 17 in 2023. The median age of patients was 70.8 (range: 37-96) years, and most patients (57%) were female. The majority of patients were admitted from home (73%), and two patients (7%) had a history of CDI in the last 6 months. Among the CDI cases, 60% were NAAT positive and toxin negative, and only 50% had &gt;3 bowel movements (BM) within 24 hours prior to the positive test. Ten percent received promotility agents prior to testing. Most cases (77%) occurred when other CDI patients were on the unit. Hand hygiene compliance rates averaged 81% over the three-year period [Figure 1A]. Eight-four per cent of patients received antibiotics within 30 days of CDI diagnosis; SAAR was &gt;1 for quarters 2 and 4 in 2022, and quarter 1 in 2023 [Figure 1B]. Conclusion: On our nephrology unit, patients often had &lt; 3 BM within 24 hours of CDI diagnosis, and 60% of cases were toxin-negative, NAAT-positive, suggesting possible C. difficile colonization, rather than true infection. In addition, an elevated SAAR correlated with high CDI rates. Multicomponent interventions may be required to reduce the rates of HO-CDI in CKD patients. Opportunities include emphasis on diagnostic and antimicrobial stewardship, environmental cleaning and adherence to IPC practices, including hand hygiene.

Longitudinal Follow Up of Patients Colonized with Clostridioides difficile: a Retrospective Cohort Study

Background: Patients colonized with Clostridioides difficile are at risk of transmitting C. difficile to other patients, and of developing C. difficile infection (CDI). Known risk factors for carriage include previous hospitalization, gastric acid suppression and previous CDI. Data regarding duration of carriage and its predictors are lacking but could be useful to better understand the natural evolution of carriage and better estimate the likelihood of transmission or progression to CDI. Methods: We performed a retrospective cohort study of C. difficile colonized patients with &gt; = 1 admission to a tertiary academic institution between November 2013 and January 2017. Colonization status was determined upon hospital admission by detection of TcdB gene by polymerase chain reaction on a rectal screening swab, as part of a systematic screening program. Overall duration of carriage and predictors of prolonged carriage were explored using Kaplan-Meier methods and Cox regression. Results: There were 134 patients, who after having a positive initial screening test (and therefore identified as colonized with C. difficile), had subsequent testing. The median age was 77 years (IQR, 66 to 85), and 53.6% of the patients were female. After hospital discharge, 26 (19.4%) colonized patients progressed to CDI. Mean duration of follow up was 269 days, with a median of 179 days. Median duration of carriage was 211 days, (95% confidence interval (CI) [157, 264]). Predictors associated with decreased duration of C. difficile colonization included younger age (HR per unit decrease (year), 1.013; 95% CI, 1.025 to 1.001; p=0.03), and receipt of antibiotics in the 3 months prior to first admission (mean days to clearance of patients with and without recent antibiotic use, 252 days vs 372 days, respectively; HR, 1.55; 95% CI, 1.01 to 2.36; p &lt; 0 .04). By contrast, the presence of comorbidities (e.g. heart failure, diabetes, cancer, and chronic kidney disease), the use of proton-pump inhibitors (PPIs), the receipt of antibiotics during the first admission, and the duration of first hospitalization were not associated with significant differences in duration of carriage. Conclusion: This study is the largest cohort of C. difficile carriers with longitudinal follow up of their colonization status. It highlights the extended duration of carrier status especially in older patients and identifies predictors of prolonged carriage. Further studies are needed to understand the underlying relationship with the predictors identified in this study.

Clinical Characteristics and Cycle Thresholds Among Discordant and True Positive Test Results for Clostridiodes difficile

Background: The diagnosis of Clostridioides difficile infection (CDI) is challenging. Despite guideline-directed, multistep testing algorithms and diagnostic stewardship, the treatment of C. difficile colonization persists. The testing algorithm at our system utilizes an initial real-time PCR test (PCR) for Toxin B gene, which if positive, reflexes to an enzyme immunoassay (EIA) for detecting Toxins A and B. Discordant results (PCR +/EIA -) are suggestive of colonization, but the majority of patients with discordant results are treated for CDI. Correlation of C. difficile EIA B polymerase chain reaction (PCR) cycle thresholds (Ct) with the presence of free EIA and disease severity has been observed, but the ability to use Ct in the decision to treat patients with discordant results is unclear. Our study assesses if Ct values and other clinical characteristics favor treatment in select patients with discordant Methods: A retrospective chart review was performed of adult patients (≥ 18-year-old) with positive C. difficile PCR results that were admitted to our health system between June 01 and August 31, 2023. C. difficile PCR and Ct results were obtained by Cepheid GeneXpert and Toxin A and B EIA results were obtained by Meridian Bioscience Immunocard. Patients with discordant (PCR+/EIA) and true positive (PCR+/EIA+) results were compared. We assessed demographics, past medical history, clinical characteristics, severity of illness, PCR Ct values, treatment, and clinical outcomes including: 30-day all-cause mortality and re-admission, and 60-day CDI repeat testing and treatment. Results Of the 122 patients identified, 89 patients had discordant results and 43 had true positive Results: Severity of illness and other clinical and laboratory characteristics were similar between both groups. Mean Ct values were significantly lower for true positive results compared to discordant results, 24.28 vs 29.27, respectively (p26 (p=.08). Of the patients with discordant results, 73 completed treatment for CDI and no difference in clinical outcomes was observed compared to patients with discordant results that were not treated. Conclusion Ct values were lower among patients with true positive results compared to patients with discordant Conclusion: There were no statistically significant different rates of severe or fulminant CDI among patients with discordant results and Ct values &lt; 26, although this finding may be limited by sample size and Ct may be helpful in deciding which discordant patients to treat.

Characterization and dynamics of intestinal microbiota in patients with Clostridioides difficile colonization and infection

Gut microbiota dysbiosis increases the susceptibility to Clostridioides difficile infection (CDI). In this study, we monitored C. difficile colonization (CDC) patients from no CDC status (CDN) to CDC status (CDCp) and CDI patients from asymptomatic status before CDI (PRECDI), CDI status (ONCDI), to asymptomatic status after CDI (POSTCDI). Based on metagenomic sequencing, we aimed to investigate the interaction pattern between gut microbiota and C. difficile. There was no significant difference of microbiota diversity between CDN and CDCp. In CDCp, Bacteroidetes and short-chain fatty acid (SCFA)-producing bacteria increased, with a positive correlation between SCFA-producing bacteria and C. difficile colonization. Compared with PRECDI, ONCDI and POSTCDI showed a significant decrease in microbiota diversity, particularly in Bacteroidetes and SCFA-producing bacteria, with a positive correlation between opportunistic pathogen and C. difficile. Fatty acid metabolism, and amino acid biosynthesis were enriched in CDN, CDCp, and PRECDI, while bile secretion was enriched in ONCDI and POSTCDI. Microbiota and metabolic pathways interaction networks in CDN and CDCp were more complex, particularly pathways in fatty acid and bile acid metabolism. Increasing of Bacteroidetes and SCFA-producing bacteria, affecting amino acid and fatty acid metabolism, is associated with colonization resistance to C. difficile and inhibiting the development of CDI.

72 Effect of In-feed fecal microbiota transplant on growth performance and gut microbiota dynamics of piglet subjected to post-weaning stress

Abstract Weaning is one of the most stressful events for piglets, due to diet change from sow milk to grain-based feed. This diet change causes turnover of the gut microbial which can allow proliferation of pathogens leading to diarrhea. Antibiotics are administered to treat or prevent post-weaning diarrhea (PWD). Increased occurrence of antimicrobial resistance has increased incentives to develop viable antibiotic alternatives. PWD is most often caused by enterotoxigenic Escherichia coli. Similarly, in humans, severe diarrhea can be caused by Clostridium difficile colonization. Use of fecal microbiota transplants (FMT) has been shown to resolve C. difficile-induced diarrhea in 80 to 90% of human cases which may suggest FMT as a potential therapeutic option for post weaning diarrhea. We previously have shown that in feed fecal microbiota transplant (FMT) improved body weight (BW) gain in newly weaned piglets but it is not clear if this benefit will be replicated in piglets subjected to weaning stress. We hypothesize that FMT (in-feed) will increase colonization efficiency, improve growth performance and reduce diarrhea incidence compared with control in piglets subjected to post weaning stress. The objective of this study was to determine the effect of In-feed FMT of diarrhea index, growth performance and gut microbiota dynamics. Piglets (n = 240) subjected to physiological and transport weaning stress were allotted to 5 treatments: In-feed FMT (FMT1), doubles dose of In-feed FMT1 (FMT2), Oral gavage (FMT3), Antibiotic (positive control), and no supplementation (negative control). The donor material was screened for enterotoxigenic E. coli (F18 and F4). FMT was carried out for 14 d. Diarrhea index was observed for the first 14 d and animal BW, and fecal microbiota was collected by rectal swab on d 0, 3, 5, 7, 14, 21, 28 and 32 for analysis of microbiota composition. DNA was extracted from fecal samples and 16S rRNA gene libraries (V4 region) were prepared for bacterial community analysis. Data were analyzed with one-way ANOVA in R. Only positive control had significantly decrease in diarrhea index and significant increase BW gain on d 14, 21, and 28 (P &amp;lt; 0.05) but there was no effect of FMT (P &amp;gt; 0.05). In conclusion, antibiotics but not FMT was efficient in alleviating the negative performance effect in piglets subjected to post-weaning stress.

Impact of the Reverse 2-Step Algorithm for Clostridioides difficile Testing in the Microbiology Laboratory on Hospitalized Patients.

Multistep laboratory testing is recommended for the diagnosis of Clostridioides difficile infection (CDI). The aim of this study was to present the impact of multistep CDI diagnostic testing in an academic hospital system and evaluate the toxin B gene polymerase chain reaction (PCR) cycle threshold (Ct) values of PCR-positive tests. In October 2022, our system began reflex testing all PCR-positive stool samples with the C. DIFF QUIK CHEK COMPLETE (Techlab), an enzyme immunoassay-based test with results for the glutamate dehydrogenase antigen (GDH) and C difficile toxin A/B. Hospital-onset (HO) CDI and CDI antibiotic use before and after testing were tracked. Ct values were obtained from the Infectious Diseases Diagnostic Laboratory. Receiver operating curve analysis was used to examine the sensitivity and specificity for identifying GDH+/toxin+ and GDH-/toxin- at various Ct thresholds. The HO-CDI rate decreased from 0.352 cases per 1000 patient-days to 0.115 cases per 1000 patient-days post-reflex testing (P < .005). Anti-CDI antibiotics use decreased, but the decrease was not commensurate with CDI rates following reflex testing. PCR+/GDH+/toxin+ samples had a lower mean Ct value than PCR+/GDH-/toxin- samples (23.3 vs 33.5, P < .0001). A Ct value of 28.65 could distinguish between those 2 groups. Fifty-four percent of PCR+/GDH+/toxin- samples had a Ct value below that cut-off, suggesting the possibility of CDI with a negative toxin test. Reflex testing for a laboratory diagnosis of CDI results in rapid, systemwide decreases in the rate of HO-CDI. Additional research is needed to distinguish CDI from C difficile colonization in patients with discordant testing.

Engineering probiotic bacteria to express tcdB antigen as an oral vaccine carrier against Clostridium difficile infection

Background Clostridium (now known as Clostridioides) difficile (C. difficile) is a spore-forming, gram-positive organism that can pose serious public health complications. The elderly are especially vulnerable to C. difficile infections, which can be fatal. C. difficile strains cause symptomatic diseases via the release of two toxins; tcdA and tcdB, that induce inflammation and tissue damage. Vaccines targeting any of these toxins may offer an effective strategy against C. difficile infections. Objective This study aimed to use live probiotics as oral vaccines to express the C. difficile toxin B gene. Oral vaccination has many advantages over intramuscular injection, as it has higher compliance, feasibility, and simpler administration. In addition, oral vaccines can stimulate both mucosal and systemic immunity against the target antigen. Probiotic bacteria were chosen as they present ideal candidates for this goal in terms of safety and health promotion. Materials and methods We chose two probiotic strains: Lactobacillus gasseri ATCC 33323 in addition to Enterococcus faecium NM1015, which had previously been identified in our lab and is capable of suppressing C. difficile colonization. The C-terminal of the tcdB gene was amplified by polymerase chain reaction (PCR) from C. difficile, cloned, and transformed into an E. coli EZ strain. The tcdB fragment was digested with BamHI and XhoI (NEB, UK) and subcloned into the bile salt-inducible expression plasmid pLB210 (obtained from INRA, France). The transformation and electroporation procedures were employed to insert cloning and expression plasmids into the target bacteria. Colony PCR was used to confirm the engineering strains. Reverse transcription PCR was used to confirm the expression of the C. difficile tcdB fragment. Results and conclusion The expression vector ‘p210-tcdB’ was constructed, then introduced into the selected probiotic strains by electroporation and confirmed by colony PCR and plasmid extraction. The reverse transcription (RT)-PCR confirmed the expression of the gene by the engineered strains. No significant difference in the survival rate was observed between the engineered strains and their parental types at pH 2.00 and 1% oxygen. Moreover, the strains showed satisfactory plasmid stability for 210 generations. Future work will involve the in vivo evaluation of the engineered probiotic strains as oral vaccines against C. difficile using an animal model.

Rapid discrimination between clinical Clostridioides difficile infection and colonization by quantitative detection of TcdB toxin using a real-time cell analysis system.

It is important to accurately discriminate between clinical Clostridioides difficile infection (CDI) and colonization (CDC) for effective antimicrobial treatment. In this study, 37 stool samples were collected from 17 CDC and 20 CDI cases, and each sample were tested in parallel through the real-time cell analysis (RTCA) system, real-time PCR assay (PCR), and enzyme-linked immunosorbent assay (ELISA). RTCA-measured functional and toxical C. difficile toxin B (TcdB) concentrations in the CDI group (302.58 ± 119.15 ng/mL) were significantly higher than those in the CDC group (18.15 ± 11.81 ng/mL) (p = 0.0008). Conversely, ELISA results revealed no significant disparities in TcdB concentrations between the CDC (26.21 ± 3.57 ng/mL) and the CDI group (17.07 ± 3.10 ng/mL) (p = 0.064). PCR results indicated no significant differences in tcdB gene copies between the CDC (774.54 ± 357.89 copies/μL) and the CDI group (4,667.69 ± 3,069.87 copies/μL) (p = 0.407). Additionally, the functional and toxical TcdB concentrations secreted from C. difficile isolates were measured by the RTCA. The results from the CDC (490.00 ± 133.29 ng/mL) and the CDI group (439.82 ± 114.66 ng/mL) showed no significant difference (p = 0.448). Notably, RTCA-measured functional and toxical TcdB concentration was significantly decreased when mixed with pooled CDC samples supernatant (p = 0.030). This study explored the novel application of the RTCA assay in effectively discerning clinical CDI from CDC cases.

Clostridioides difficile Biofilm.

Clostridioides difficile infection (CDI), previously Clostridium difficile infection, is a symptomatic infection of the large intestine caused by the spore-forming anaerobic, gram-positive bacterium Clostridioides difficile. CDI is an important healthcare-associated disease worldwide, characterized by high levels of recurrence, morbidity, and mortality. CDI is observed at a higher rate in immunocompromised patients after antimicrobial therapy, with antibiotics disrupting the commensal microbiota and promoting C. difficile colonization of the gastrointestinal tract.A rise in clinical isolates resistant to multiple antibiotics and the reduced susceptibility to the most commonly used antibiotic molecules have made the treatment of CDI more complicated, allowing the persistence of C. difficile in the intestinal environment.Gut colonization and biofilm formation have been suggested to contribute to the pathogenesis and persistence of C. difficile. In fact, biofilm growth is considered as a serious threat because of the related antimicrobial tolerance that makes antibiotic therapy often ineffective. This is the reason why the involvement of C. difficile biofilm in the pathogenesis and recurrence of CDI is attracting more and more interest, and the mechanisms underlying biofilm formation of C. difficile as well as the role of biofilm in CDI are increasingly being studied by researchers in the field.Findings on C. difficile biofilm, possible implications in CDI pathogenesis and treatment, efficacy of currently available antibiotics in treating biofilm-forming C. difficile strains, and some antimicrobial alternatives under investigation will be discussed here.

Pulsed dosing and extended daily dosing of oral vancomycin do not facilitate clearance of Clostridioides difficile colonization in mice.

Vancomycin taper and pulse regimens are commonly used to treat recurrent Clostridioides difficile infections, but the mechanism by which these regimens might reduce recurrences is unclear. Here, we used a mouse model to test the hypothesis that pulse dosing of vancomycin after a 10-day treatment course enhances clearance of C. difficile from the intestinal tract. Mice with C. difficile colonization received 10 days of once-daily oral vancomycin followed by 20 days of treatment with saline (controls), daily vancomycin, or pulse dosing of vancomycin every 2 or 3 days. Stool samples were collected to measure the concentration of C. difficile during and after treatment, vancomycin concentrations, and growth of vegetative C. difficile during every 3 days dosing. Pulse dosing of vancomycin was not effective in maintaining suppression of C. difficile (P > 0.05 in comparison to saline controls); growth of vegetative C. difficile occurred between pulse doses when vancomycin decreased to undetectable levels. Daily dosing of vancomycin suppressed C. difficile during treatment, but recurrent colonization occurred after treatment in more than 75% of mice, and by post-treatment day 14, there was no significant difference among the control, pulse dosing, and daily dosing groups (P > 0.05). These findings demonstrate that pulse dosing of vancomycin every 2 or 3 days does not facilitate the clearance of C. difficile spores in mice. Studies are needed to examine the impact of vancomycin taper and pulsed regimens in patients.