Fecal microbiota transplantation for irritable bowel syndrome: Current evidence and perspectives.

In this editorial we comment on the article published in the recent issue of the World journal of Gastroenterology. We focus specifically on the mechanisms un-derlying the effects of fecal microbiota transplantation (FMT) for irritable bowel syndrome (IBS), the factors which affect the outcomes of FMT in IBS patients, and challenges. FMT has emerged as a efficacious intervention for clostridium difficile infection and holds promise as a therapeutic modality for IBS. The utilization of FMT in the treatment of IBS has undergone scrutiny in numerous randomized controlled trials, yielding divergent outcomes. The current frontier in this field seeks to elucidate these variations, underscore the existing knowledge gaps that necessitate exploration, and provide a guideline for successful FMT imple-mentation in IBS patients. At the same time, the application of FMT as a treatment for IBS confronts several challenges.

Comparison of fidaxomicin, thuricin CD, vancomycin and nisin highlights the narrow spectrum nature of thuricin CD.

Vancomycin and metronidazole are commonly used treatments for Clostridioides difficile infection (CDI). However, these antibiotics have been associated with high levels of relapse in patients. Fidaxomicin is a new treatment for CDI that is described as a narrow spectrum antibiotic that is minimally active on the commensal bacteria of the gut microbiome. The aim of this study was to compare the effect of fidaxomicin on the human gut microbiome with a number of narrow (thuricin CD) and broad spectrum (vancomycin and nisin) antimicrobials. The spectrum of activity of each antimicrobial was tested against 47 bacterial strains by well-diffusion assay. Minimum inhibitory concentrations (MICs) were calculated against a select number of these strains. Further, a pooled fecal slurry of 6 donors was prepared and incubated for 24 h with 100 µM of each antimicrobial in a mini-fermentation system together with a no-treatment control. Fidaxomicin, vancomycin, and nisin were active against most gram positive bacteria tested in vitro, although fidaxomicin and vancomycin produced larger zones of inhibition compared to nisin. In contrast, the antimicrobial activity of thuricin CD was specific to C. difficile and some Bacillus spp. The MICs showed similar results. Thuricin CD exhibited low MICs (<3.1 µg/mL) for C. difficile and Bacillus firmus, whereas fidaxomicin, vancomycin, and nisin demonstrated lower MICs for all other strains tested when compared to thuricin CD. The narrow spectrum of thuricin CD was also observed in the gut model system. We conclude that the spectrum of activity of fidaxomicin is comparable to that of the broad-spectrum antibiotic vancomycin in vitro and the broad spectrum bacteriocin nisin in a complex community.

Defining the phylogenetics and resistome of the major Clostridioides difficile ribotypes circulating in Australia.

Clostridioides difficile infection (CDI) remains a significant public health threat globally. New interventions to treat CDI rely on an understanding of the evolution and epidemiology of circulating strains. Here we provide longitudinal genomic data on strain diversity, transmission dynamics and antimicrobial resistance (AMR) of C. difficile ribotypes (RTs) 014/020 (n=169), 002 (n=77) and 056 (n=36), the three most prominent C. difficile strains causing CDI in Australia. Genome scrutiny showed that AMR was uncommon in these lineages, with resistance-conferring alleles present in only 15/169 RT014/020 strains (8.9 %), 1/36 RT056 strains (2.78 %) and none of 77 RT002 strains. Notably, ~90 % of strains were resistant to MLSB agents in vitro, but only ~5.9 % harboured known resistance alleles, highlighting an incongruence between AMR genotype and phenotype. Core genome analyses revealed all three RTs contained genetically heterogeneous strain populations with limited evidence of clonal transmission between CDI cases. The average number of pairwise core genome SNP (cgSNP) differences within each RT group ranged from 23.3 (RT056, ST34, n=36) to 115.6 (RT002, ST8, n=77) and 315.9 (RT014/020, STs 2, 13, 14, 49, n=169). Just 19 clonal groups (encompassing 40 isolates), defined as isolates differing by ≤2 cgSNPs, were identified across all three RTs (RT014/020, n=14; RT002, n=3; RT056, n=2). Of these clonal groups, 63 % (12/19) comprised isolates from the same Australian State and 37 % (7/19) comprised isolates from different States. The low number of plausible transmission events found for these major RTs (and previously documented populations in animal and environmental sources/reservoirs) points to widespread and persistent community sources of diverse C. difficile strains as opposed to ongoing nationwide healthcare outbreaks dominated by a single clone. Together, these data provide new insights into the evolution of major lineages causing CDI in Australia and highlight the urgent need for enhanced surveillance, and for public health interventions to move beyond the healthcare setting and into a One Health paradigm to effectively combat this complex pathogen.

Genetic diversity and phylogenetic relationships of Clostridium perfringens strains isolated from mastitis and enteritis in Egyptian dairy farms.

BACKGROUND: Clostridium perfringens, a common environmental bacterium, is responsible for a variety of serious illnesses including food poisoning, digestive disorders, and soft tissue infections. Mastitis in lactating cattle and sudden death losses in baby calves are major problems for producers raising calves on dairy farms. The pathogenicity of this bacterium is largely mediated by its production of various toxins. RESULTS: The study revealed that Among the examined lactating animals with a history of mastitis, diarrheal baby calves, and acute sudden death cases in calves, C. perfringens was isolated in 23.5% (93/395) of the total tested samples. Eighteen isolates were obtained from mastitic milk, 59 from rectal swabs, and 16 from the intestinal contents of dead calves. Most of the recovered C. perfringens isolates (95.6%) were identified as type A by molecular toxinotyping, except for four isolates from sudden death cases (type C). Notably, C. perfringens was recovered in 100% of sudden death cases compared with 32.9% of rectal swabs and 9% of milk samples. This study analyzed the phylogeny of C. perfringens using the plc region and identified the plc region in five Egyptian bovine isolates (milk and fecal origins). Importantly, this finding expands the known data on C. perfringens phospholipase C beyond reference strains in GenBank from various animal and environmental sources. CONCLUSION: Phylogenetic analyses of nucleotide sequence data differentiated between strains of different origins. The plc sequences of Egyptian C. perfringens strains acquired in the present study differed from those reported globally and constituted a distinct genetic ancestor.

A commensal protozoan attenuates Clostridioides difficile pathogenesis in mice via arginine-ornithine metabolism and host intestinal immune response.

Antibiotic-induced dysbiosis is a major risk factor for Clostridioides difficile infection (CDI), and fecal microbiota transplantation (FMT) is recommended for treating CDI. However, the underlying mechanisms remain unclear. Here, we show that Tritrichomonas musculis (T.mu), an integral member of the mouse gut commensal microbiota, reduces CDI-induced intestinal damage by inhibiting neutrophil recruitment and IL-1ß secretion, while promoting Th1 cell differentiation and IFN-γ secretion, which in turn enhances goblet cell production and mucin secretion to protect the intestinal mucosa. T.mu can actively metabolize arginine, not only influencing the host's arginine-ornithine metabolic pathway, but also shaping the metabolic environment for the microbial community in the host's intestinal lumen. This leads to a relatively low ornithine state in the intestinal lumen in C. difficile-infected mice. These changes modulate C. difficile's virulence and the host intestinal immune response, and thus collectively alleviating CDI. These findings strongly suggest interactions between an intestinal commensal eukaryote, a pathogenic bacterium, and the host immune system via inter-related arginine-ornithine metabolism in the regulation of pathogenesis and provide further insights for treating CDI.

Clinical outcomes and treatment necessity in patients with toxin-negative Clostridioides difficile stool samples.

PURPOSE: The clinical significance of negative toxin enzyme immunoassays (EIA) for Clostridioides difficile infections (CDIs) is unclear. Our study aimed to investigate the significance of toxin EIA-negative in the diagnosis and prognosis of CDI. METHODS: All stool specimens submitted for C. difficile toxin EIA testing were cultured to isolate C. difficile. In-house PCR for tcdA, tcdB, cdtA, and cdtB genes were performed using C. difficile isolates. Stool specimens were tested with C. difficile toxins A and B using EIA kit (RIDASCREEN Clostridium difficile toxin A/B, R-Biopharm AG, Darmstadt, Germany). Characteristics and subsequent CDI episodes of toxin EIA-negative and -positive patients were compared. RESULTS: Among 190 C. difficile PCR-positive patients, 83 (43.7%) were toxin EIA-negative. Multivariate analysis revealed independent associations toxin EIA-negative results and shorter hospital stays (OR = 0.98, 95% CI 0.96-0.99, p = 0.013) and less high-risk antibiotic exposure in the preceding month (OR = 0.38, 95% CI 0.16-0.94, p = 0.035). Toxin EIA-negative patients displayed a significantly lower white blood cell count rate (11.0 vs. 35.4%, p < 0.001). Among the 54 patients who were toxin EIA-negative and did not receive CDI treatment, three (5.6%) were diagnosed with CDI after 7-21 days without complication. CONCLUSION: Our study demonstrates that toxin EIA-negative patients had milder laboratory findings and no complications, despite not receiving treatment. Prolonged hospitalisation and exposure to high-risk antibiotics could potentially serve as markers for the development of toxin EIA-positive CDI.

Colorectal Disease and the Gut Microbiome: What a Surgeon Needs to Know.

The gut microbiome is defined as the microorganisms that reside within the gastrointestinal tract and produce a variety of metabolites that impact human health. These microbes play an intricate role in human health, and an imbalance in the gut microbiome, termed gut dysbiosis, has been implicated in the development of varying diseases. The purpose of this review is to highlight what is known about the microbiome and its impact on colorectal cancer, inflammatory bowel disease, constipation, Clostridioides difficile infection, the impact of bowel prep, and anastomotic leaks.

Identifying Contact Time Required for Secondary Transmission of Clostridioides difficile Infections by Using Real-Time Locating System.

Considering patient room shortages and prevalence of other communicable diseases, reassessing the isolation of patients with Clostridioides difficile infection (CDI) is imperative. We conducted a retrospective study to investigate the secondary CDI transmission rate in a hospital in South Korea, where patients with CDI were not isolated. Using data from a real-time locating system and electronic medical records, we investigated patients who had both direct and indirect contact with CDI index patients. The primary outcome was secondary CDI transmission, identified by whole-genome sequencing. Among 909 direct and 2,711 indirect contact cases, 2 instances of secondary transmission were observed (2 [0.05%] of 3,620 cases), 1 transmission via direct contact and 1 via environmental sources. A low level of direct contact (113 minutes) was required for secondary CDI transmission. Our findings support the adoption of exhaustive standard preventive measures, including environmental decontamination, rather than contact isolation of CDI patients in nonoutbreak settings.

Unveiling the pathogenic mechanisms of Clostridium perfringens toxins and virulence factors.

Clostridium perfringens causes multiple diseases in humans and animals. Its pathogenic effect is supported by a broad and heterogeneous arsenal of toxins and other virulence factors associated with a specific host tropism. Molecular approaches have indicated that most C. perfringens toxins produce membrane pores, leading to osmotic cell disruption and apoptosis. However, identifying mechanisms involved in cell tropism and selective toxicity effects should be studied more. The differential presence and polymorphisms of toxin-encoding genes and genes encoding other virulence factors suggest that molecular mechanisms might exist associated with host preference, receptor binding, and impact on the host; however, this information has not been reviewed in detail. Therefore, this review aims to clarify the current state of knowledge on the structural features and mechanisms of action of the major toxins and virulence factors of C. perfringens and discuss the impact of genetic diversity of toxinotypes in tropism for several hosts.

Deficiency of IL-22-binding protein enhances the ability of the gut microbiota to protect against enteric pathogens.

Interleukin 22 (IL-22) promotes intestinal barrier integrity, stimulating epithelial cells to enact defense mechanisms against enteric infections, including the production of antimicrobial peptides. IL-22 binding protein (IL-22BP) is a soluble decoy encoded by the Il22ra2 gene that decreases IL-22 bioavailability, attenuating IL-22 signaling. The impact of IL-22BP on gut microbiota composition and functioning is poorly understood. We found that Il22ra2-/- mice are better protected against Clostridioides difficile and Citrobacter rodentium infections. This protection relied on IL-22-induced antimicrobial mechanisms before the infection occurred, rather than during the infection itself. Indeed, the gut microbiota of Il22ra2-/- mice mitigated infection of wild-type (WT) mice when transferred via cohousing or by cecal microbiota transplantation. Indicator species analysis of WT and Il22ra2-/- mice with and without cohousing disclosed that IL22BP deficiency yields a gut bacterial composition distinct from that of WT mice. Manipulation of dietary fiber content, measurements of intestinal short-chain fatty acids and oral treatment with acetate disclosed that resistance to C. difficile infection is related to increased production of acetate by Il22ra2-/--associated microbiota. Together, these findings suggest that IL-22BP represents a potential therapeutic target for those at risk for or with already manifest infection with this and perhaps other enteropathogens.

C. difficile biomarkers, pathogenicity and detection.

BACKGROUND: Clostridioides difficile infection (CDI) is the main etiologic agent of antibiotic-associated diarrhea. CDI contributes to gut inflammation and can lead to disruption of the intestinal epithelial barrier. Recently, the rate of CDI cases has been increased. Thus, early diagnosis of C. difficile is critical for controlling the infection and guiding efficacious therapy. APPROACH: A search strategy was set up using the terms C. difficile biomarkers and diagnosis. The found references were classified into two general categories; conventional and advanced methods. RESULTS: The pathogenicity and biomarkers of C. difficile, and the collection manners for CDI-suspected specimens were briefly explained. Then, the conventional CDI diagnostic methods were subtly compared in terms of duration, level of difficulty, sensitivity, advantages, and disadvantages. Thereafter, an extensive review of the various newly proposed techniques available for CDI detection was conducted including nucleic acid isothermal amplification-based methods, biosensors, and gene/single-molecule microarrays. Also, the detection mechanisms, pros and cons of these methods were highlighted and compared with each other. In addition, approximately complete information on FDA-approved platforms for CDI diagnosis was collected. CONCLUSION: To overcome the deficiencies of conventional methods, the potential of advanced methods for C. difficile diagnosis, their direction, perspective, and challenges ahead were discussed.

Efficacy Assessment of the Co-Administration of Vancomycin and Metronidazole in Clostridioides difficile-Infected Mice Based on Changes in Intestinal Ecology.

Vancomycin (VAN) and metronidazole (MTR) remain the current drugs of choice for the treatment of non-severe Clostridioides difficile infection (CDI); however, while their co-administration has appeared in clinical treatment, the efficacy varies greatly and the mechanism is unknown. In this study, a CDI mouse model was constructed to evaluate the therapeutic effects of VAN and MTR alone or in combination. For a perspective on the intestinal ecology, 16S rRNA amplicon sequencing and non-targeted metabolomics techniques were used to investigate changes in the fecal microbiota and metabolome of mice under the co-administration treatment. As a result, the survival rate of mice under co-administration was not dramatically different compared to that of single antibiotics, and the former caused intestinal tissue hyperplasia and edema. Co-administration also significantly enhanced the activity of amino acid metabolic pathways represented by phenylalanine, arginine, proline, and histidine, decreased the level of deoxycholic acid (DCA), and downregulated the abundance of beneficial microbes, such as Bifidobacterium and Akkermansia. VAN plays a dominant role in microbiota regulation in co-administration. In addition, co-administration reduced or increased the relative abundance of antibiotic-sensitive bacteria, including beneficial and harmful microbes, without a difference. Taken together, there are some risks associated with the co-administration of VAN and MTR, and this combination mode should be used with caution in CDI treatment.

Fecal microbiota transplantation stimulates type 2 and tolerogenic immune responses in a mouse model.

OBJECTIVES: Clostridioides difficile infection (CDI) is the leading hospital-acquired infection in North America. While previous work on fecal microbiota transplantation (FMT), a highly effective treatment for CDI, has focused on colonization resistance mounted against C. difficile by FMT-delivered commensals, the effects of FMT on host gene expression are relatively unexplored. This study aims to identify transcriptional changes associated with FMT, particularly changes associated with protective immune responses. METHODS: Gene expression was assessed on day 2 and day 7 after FMT in mice after antibiotic-induced dysbiosis. Flow cytometry was also performed on colon and mesenteric lymph nodes at day 7 to investigate changes in immune cell populations. RESULTS: FMT administration after antibiotic-induced dysbiosis successfully restored microbial alpha diversity to levels of donor mice by day 7 post-FMT. Bulk RNA sequencing of cecal tissue at day 2 identified immune genes, including both pro-inflammatory and Type 2 immune pathways as upregulated after FMT. RNA sequencing was repeated on day 7 post-FMT, and expression of these immune genes was decreased along with upregulation of genes associated with restoration of intestinal homeostasis. Immunoprofiling on day 7 identified increased colonic CD45+ immune cells that exhibited dampened Type 1 and heightened regulatory and Type 2 responses. These include an increased abundance of eosinophils, alternatively activated macrophages, Th2, and T regulatory cell populations. CONCLUSION: These results highlight the impact of FMT on host gene expression, providing evidence that FMT restores intestinal homeostasis after antibiotic treatment and facilitates tolerogenic and Type 2 immune responses.

Toxin gene detection and antibiotic resistance of Clostridium perfringens from aquatic sources.

Clostridium perfringens is a zoonotic opportunistic pathogen that produces toxins that can cause necrotic enteritis and even "sudden death disease". This bacterium is widely distributed in the intestines of livestock and human, but there are few reports of distribution in aquatic animals (Hafeez et al., 2022). In order to explore the isolation rate of C. perfringens and the toxin genes they carry, 141 aquatic samples, including clams (Ruditapes philippinarum), oysters (Ostreidae), and mud snails (Bullacta exerata Philippi), were collected from the coastal areas of Shandong Province, China. C. perfringens strains were tested for cpa, cpb, etx, iap, cpb2, cpe, netB, and tpeL genes. 45 clam samples were boiled at 100 °C for 5 min before bacteria isolation. 80 strains were isolated from 141 samples with the positive rate being 57 %.And the positive rates of cooked clams was 87 % which was higher than the average. In detection of 8 toxin genes, all strains tested cpa positive, 3 strains netB positive, and 2 cpb and cpe, respectively. 64 strains were selected to analyze the antibiotic resistance phenotype of 10 antibiotics. The average antibiotic resistance rates of the strains to tetracycline, clindamycin, and ampicillin were 45 %, 20 %, and 16 % respectively, and the MIC of 4 strains to clindamycin was ≥128 µg/mL. A high isolation rate of C. perfringens from aquatic animals was shown, and it was isolated from boiled clams for the first time, in which cpe and netB toxin genes were detected for the first time too. The toxin encoded by cpe gene can cause food poisoning of human, thus the discoveries of this study have certain guiding significance for food safety. Antibiotics resistant C. perfringens of aquatic origin may arise from transmission in the terrestrial environment or from antibiotic contamination of the aquaculture environment and is of public health significance.

Increase of healthcare-onset Clostridioides difficile infection in adult population since SARS-CoV-2 pandemic: A retrospective cohort study in a tertiary care hospital from 2019 to 2022.

OBJECTIVES: The aim was to assess the impact of the SARS-CoV-2 pandemic on the prevalence, relative incidence (RI), incidence density (ID), ratio of rate incidence (RRI), rate of incidence density (RID), and relative risks (RR) of healthcare-onset Clostridioides difficile infection (HO-CDI) as well as its correlation with the antibiotic consumption. METHODS: Demographic and analytical data of adult patients exhibiting diarrhoea and testing positive for C. difficile were systematically collected from a tertiary care hospital in Madrid (Spain). The periods analysed included: prepandemic (P0), first pandemic-year (P1), and second pandemic-year (P2). We compared global prevalence, RI of HO-CDI per 1,000-admissions, ID of HO-CDI per 10,000-patients-days, RRI, RID, and RR. Antibiotic consumption was obtained by number of defined daily dose per 100 patient-days. RESULTS: In P0, the prevalence of HO-CDI was 7.4% (IC95%: 6.2-8.7); in P1, it increased to 8.7% (IC95%: 7.4-10.1) (p = 0.2), and in P2, it continued to increase to 9.2% (IC95%: 8-10.6) (p < 0.05). During P1, the RRI was 1.5 and RID was 1.4. However, during P2 there was an increase in RRI to 1.6 and RID to 1.6. The RR also reflected the increase in HO-CDI: at P1, the probability of developing HO-CDI was 1.5 times (IC95%: 1.2-1.9) higher than P0, while at P2, this probability increased to 1.6 times (IC95%: 1.3-2.1). There was an increase in prevalence, RI, ID, RR, RRI, and RID during the two postpandemic periods respect to the prepandemic period. During P2, this increase was greater than the P1. Meropenem showed a statistically significant difference increased consumption (p < 0.05) during the pandemic period. Oral vancomycin HO-CDI treatment showed an increase during the period of study (p > 0.05). CONCLUSIONS: Implementation of infection control measures during the SARS-CoV-2 pandemic did not appear to alleviate the burden of HO-CDI. The escalation in HO-CDI cases did not exhibit a correlation with overall antibiotic consumption, except for meropenem.

Comparison of necrotic enteritis effects on growth performance and intestinal health in two different meat-type chicken strains Athens Canadian Random Bred and Cobb 500.

Chickens have undergone genetic improvements in the past few decades to maximize growth efficiency. However, necrotic enteritis (NE), an enteric disease primarily caused by C. perfringens, remains a significant problem in poultry production. A study investigated the differences in intestinal health between the nonselected meat-type chicken Athens Canadian Random Bred (ACRB) and the modern meat-type Cobb 500 broilers (Cobb) when challenged with experimental NE. The study utilized a 2 × 3 factorial arrangement, consisting of two main effects of chicken strain and NE challenge model (nonchallenged control, NC; NE challenge with 2,500/12,500 Eimeria maxima oocysts + 1 × 109C. perfringens, NE2.5/NE12.5). A total of 432 fourteen-day-old male ACRB and Cobb were used until 22 d (8 d postinoculation with E. maxima on d 14, dpi), and the chickens were euthanized on 6 and 8 dpi for the analysis. All data were statistically analyzed using a two-way ANOVA, and Student's t-test or Tukey's HSD test was applied when P < 0.05. The NE12.5 group showed significant decreases in growth performance and relative growth performance from d 14 to 20, regardless of chicken strain (P < 0.01). The ACRB group exhibited significant decreases in relative body weight and relative body weight gain compared to the Cobb group from d 14 to 22 (P < 0.01). On 6 and 8 dpi, both NE challenge groups showed significant decreases in intestinal villus height to crypt depth ratio, jejunal goblet cell count, and jejunal MUC2 and LEAP2 expression (P < 0.01). Additionally, the NE12.5 group had significantly higher intestinal NE lesion score, intestinal permeability, fecal E. maxima oocyst count, intestinal C. perfringens count, and jejunal IFNγ and CCL4 expression compared to the NC group (P < 0.05). In conclusion, NE negatively impacts growth performance and intestinal health in broilers, parameters regardless of the strain.

Bacillus subtilis HW2 enhances growth performance and alleviates gut injury via attenuation of endoplasmic reticulum stress and regulation of gut microbiota in broilers under necrotic enteritis challenge.

This study investigated the effects of Bacillus subtilis HW2 on the growth performance, immune response, endoplasmic reticulum (ER) stress, and intestinal health in broilers with necrotic enteritis. Three hundred 1-day-old male Cobb 500 broilers (33.88 ± 2.34 g) were randomly allocated to 5 groups including non-infected control (NC group), basal diet + necrotic enteritis challenge (NE group), basal diet + 1 × 106 CFU/g B. subtilis HW2 + necrotic enteritis challenge (L-Pro group), basal diet + 5 × 106 CFU/g B. subtilis HW2 + necrotic enteritis challenge (M-Pro group), and basal diet + 1 × 107 CFU/g B. subtilis HW2 + necrotic enteritis challenge (H-Pro group), with 6 replicates per group. All broilers except NC group were orally given with sporulated coccidian oocysts at day 14 and Clostridium perfringens from days 19 to 21. Results showed that L-Pro and M-Pro groups improved growth performance and intestinal morphology in necrotic enteritis-challenged broilers, and L-Pro, M-Pro, and H-Pro groups improved intestinal barrier function and immune response and decreased ER stress in necrotic enteritis-challenged broilers. Analysis of the gut microbiota revealed that L-Pro group increased the abundances of Alistipes, Coprobacter, Barnesiella, and Limosilactobacillus, decreased Erysipelatoclostridium abundance on day 42 in necrotic enteritis-challenged broilers. M-Pro group increased Turicibacter abundance on day 28 and the abundances of Alistipes, Barnesiella, and Limosilactobacillus on day 42 in necrotic enteritis-challenged broilers. H-Pro group decreased Romboutsia abundance on day 28 and unidentified_Clostridia abundance on day 42 in necrotic enteritis-challenged broilers. Analysis of short-chain fatty acids (SCFAs) revealed higher isobutyric acid and isovaleric acid levels in L-Pro and M-Pro groups than NE group. Correlation analysis revealed the correlations between the biochemical parameters and gut microbiota as well as SCFAs, especially Romboutsia, Barnesiella, Coprobacter, isobutyric acid, and isovaleric acid. Overall, our results indicated that B. subtilis HW2 supplementation could ameliorate necrotic enteritis infection-induced gut injury. The optimal dietary supplementation dosage of Bacillus subtilis HW2 was 5 × 106 CFU/g.

Treatment of chickens with lactobacilli prior to challenge with Clostridium perfringens modifies innate responses and gut morphology.

Necrotic enteritis caused by Clostridium perfringens (CP), is a common enteric disease of poultry that has been previously controlled by in-feed antibiotics. However, due to the rapid emergence of antimicrobial resistance, alternatives to antibiotics such as probiotics have received considerable attention because of their immunomodulatory and intestinal health benefits. The present study investigated the effects of probiotic lactobacilli on gut histomorphology and intestinal innate responses in chickens. Day-old male broiler chickens were treated with 1 × 107 or 1 × 108 colony-forming units (CFU) of a lactobacilli cocktail on days 1, 7, 14, and 20 post-hatch, while control groups were not treated with lactobacilli. On day 21, birds in all groups (except the negative control) were challenged with 3 × 108 CFU of CP for 3 days. Intestinal tissue samples were collected before and after the CP challenge to assess gene expression and for histomorphological analysis. Lactobacilli treatment at a dose of 1 × 108 CFU conferred partial protection against NE by lowering lesion scores, increasing villus height in the ileum and reducing crypt depth in the jejunum. In addition, 1 × 108 CFU of lactobacilli enhanced the expression of Toll-like receptor (TLR) 2, interferon-gamma (IFN-γ), interleukin (IL)-10, IL-12, and IL-13 in both the jejunum and ileum at different timepoints and subsequently decreased the expression of transforming growth factor beta (TGF-ß) and IL-1ß post-CP challenge. In conclusion, the results indicate that treatment with lactobacilli mitigated NE in a dose-dependent manner via improvement of intestinal morphology and modulation of innate immune response in chickens.

Broad host range phages target global Clostridium perfringens bacterial strains and clear infection in five-strain model systems.

Clostridium perfringens is a prevalent bacterial pathogen in poultry, and due to the spread of antimicrobial resistance, alternative treatments are needed to prevent and treat infection. Bacteriophages (phages), viruses that kill bacteria, offer a viable option and can be used therapeutically to treat C. perfringens infections. The aim of this study was to isolate phages against C. perfringens strains currently circulating on farms across the world and establish their virulence and development potential using host range screening, virulence assays, and larva infection studies. We isolated 32 phages of which 19 lysed 80%-92% of our global C. perfringens poultry strain collection (n = 97). The virulence of these individual phages and 32 different phage combinations was quantified in liquid culture at multiple doses. We then developed a multi-strain C. perfringens larva infection model, to mimic an effective poultry model used by the industry. We tested the efficacy of 16/32 phage cocktails in the larva model. From this, we identified that our phage cocktail consisting of phages CPLM2, CPLM15, and CPLS41 was the most effective at reducing C. perfringens colonization in infected larvae when administered before bacterial challenge. These data suggest that phages do have significant potential to prevent and treat C. perfringens infection in poultry. IMPORTANCE: Clostridium perfringens causes foodborne illness worldwide, and 95% of human infections are linked to the consumption of contaminated meat, including chicken products. In poultry, C. perfringens infection causes necrotic enteritis, and associated mortality rates can be up to 50%. However, treating infections is difficult as the bacterium is becoming antibiotic-resistant. Furthermore, the poultry industry is striving toward reduced antibiotic usage. Bacteriophages (phages) offer a promising alternative, and to progress this approach, robust suitable phages and laboratory models that mimic C. perfringens infections in poultry are required. In our study, we isolated phages targeting C. perfringens and found that many lyse C. perfringens strains isolated from chickens worldwide. Consistent with other published studies, in the model systems we assayed here, when some phages were combined as cocktails, the infection was cleared most effectively compared to individual phage use.

Culture-independent detection of low-abundant Clostridioides difficile in environmental DNA via PCR.

Clostridioides difficile represents a major burden to public health. As a well-known nosocomial pathogen whose occurrence is highly associated with antibiotic treatment, most examined C. difficile strains originated from clinical specimen and were isolated under selective conditions employing antibiotics. This suggests a significant bias among analyzed C. difficile strains, which impedes a holistic view on this pathogen. In order to support extensive isolation of C. difficile strains from environmental samples, we designed a detection PCR that targets the hpdBCA-operon and thereby identifies low abundances of C. difficile in environmental samples. This operon encodes the 4-hydroxyphenylacetate decarboxylase, which catalyzes the production of the antimicrobial compound para-cresol. Amplicon-based analyses of diverse environmental samples demonstrated that the designed PCR is highly specific for C. difficile and successfully detected C. difficile despite its absence in general 16S rRNA gene-based detection strategies. Further analyses revealed the potential of the hpdBCA detection PCR sequence for initial phylogenetic classification, which allows assessment of C. difficile diversity in environmental samples via amplicon sequencing. Our findings furthermore showed that C. difficile strains isolated under antibiotic treatment from environmental samples were originally dominated by other strains according to PCR amplicon results. This provided evidence for selective cultivation of under-represented but antibiotic-resistant isolates. Thereby, we revealed a substantial bias in C. difficile isolation and research.IMPORTANCEClostridioides difficile is a main cause of diarrheic infections after antibiotic treatment with serious morbidity and mortality worldwide. Research on this pathogen and its virulence has focused on bacterial isolation from clinical specimens under antibiotic treatment, which implies a substantial bias in isolated strains. Comprehensive studies, however, require an unbiased strain collection, which is accomplished by isolation of C. difficile from diverse environmental samples and avoidance of antibiotic-based enrichment strategies. Thus, isolation can significantly benefit from our C. difficile-specific detection PCR, which rapidly verifies C. difficile presence in environmental samples and further allows estimation of the C. difficile diversity by using next-generation sequencing.