Clostridium Perfringens as a Rare Cause of Spontaneous Bacterial Peritonitis.

Spontaneous bacterial peritonitis (SBP) is a serious complication in individuals with liver cirrhosis and ascites. In this case report, we present an unusual cause of SBP in loculated ascites caused by an uncommon bacterium, Clostridium perfringens. Although SBP is typically associated with certain common pathogens, it is important to recognize that less frequent organisms can also trigger it. C. perfringens is typically associated with other environmental sources, but in this instance, the infection's origin was suspected to be either nosocomial, from prior paracentesis, or due to a microscopic bowel perforation that was undetectable on imaging. Remarkably, the patient responded well with an improvement of symptoms, and the ascitic fluid bacterial growth resolved on subsequent cultures.

Probiotic Bacillus subtilis QST713 improved growth performance and enhanced the intestinal health of yellow-feather broilers challenged with coccidia and Clostridium perfringens.

In this study, we investigated the effects of dietary supplementation with Bacillus subtilis (QST713) on the performance and intestinal health of yellow feather broilers under Coccidia and Clostridium perfringens (CP) challenge or CP alone. One-day-old yellow-feathered broiler roosters (n = 600) were randomly assigned to 5 groups (6 replicates with 20 roosters per replicate): the Con blank group, the CIC.p group (d24 Coccidia+d28-30 of CP challenge), the CIC.p + BS group (CIC.p +100 mg/kg B. subtilis), the C.p group (d 28-34 of CP challenge), and the C.p +BS group (C.p +100 mg/kg B. subtilis). The experiment lasted 80 d. The birds were evaluated for parameters such as average daily gain (ADG), average daily feed intake (ADFI), feed efficiency (F/G), intestinal lesion score, villus histomorphometry, intestinal tight junctions, inflammatory factors, and cecal microorganisms. The results revealed that 1) C.p. increased the F/G of broilers from 22 to 42 d (P < 0.05), whereas CIC.p. significantly decreased the 42 d and 80 d body weights (BW) and 22-42 d and 1-80 d ADG (P < 0.05) and significantly increased the 22 to 42 d and 1 to 80 d F/G (P < 0.05). The number of intestinal lesions significantly increased at 35 d and 42 d (P < 0.05). CIC.p significantly decreased the jejunum and ileum villus height (VH) and the ileum villus height/crypt depth (P < 0.05) at 35 d. The challenge significantly upregulated the expression of Claudin-1 and IL-4 mRNAs in the jejunum at 35 d and significantly downregulated the expression of IL-10 mRNA in the ileum at 35 d (P < 0.05); the number of unique OTUs in the challenge group decreased significantly after challenge treatment, and the relative abundances of Romboutsia at 35 d and Cladomyces and Lactobacillus at 42 d decreased significantly (P < 0.05). 2) Compared with the challenge groups, the addition of BS decreased the F/G of broilers from 22 to 42 d. Compared with the CIC group, the addition of BS significantly increased the F/G of broilers from 22 to 42 d. Compared with that in the CIC.p group, the addition of BS significantly increased the VH in the jejunum and ileum at 35 d (P < 0.05). Compared with the challenge groups, the BS groups presented significantly lower mRNA expression levels of Claudin-1 (P < 0.05) in the jejunum at 35 d. The Shannon and Chao indices suggested that BS increased the alpha diversity of cecum microorganisms in broilers. Dietary supplementation with B. subtilis can alleviate the damage to intestinal morphology and intestinal barrier function, as well as the altered cecal flora structure in broilers caused by Coccidia and C. perfringens infections.

Molecular characterization of common zoonotic protozoan parasites and bacteria causing diarrhea in dairy calves in Ningxia Hui Autonomous Region, China.

Diarrhea caused by zoonotic pathogens is one of the most common diseases in dairy calves, threatening the health of young animals. Humans are also at risk, in particular children. To explore the pathogens causing diarrhea in dairy calves, the present study applied PCR-based sequencing tools to investigate the occurrence and molecular characteristics of three parasites (Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi) and three bacterial pathogens (Escherichia coli, Clostridium perfringens, and Salmonella spp.) in 343 fecal samples of diarrheic dairy calves from five farms in Lingwu County, Ningxia Hui Autonomous Region, China. The total positive rate of these pathogens in diarrheic dairy calves was 91.0% (312/343; 95% CI, 87.9-94.0), with C. perfringens (61.5%, 211/343; 95% CI, 56.3-66.7) being the dominant one. Co-infection with two to five pathogens was found in 67.3% (231/343; 95% CI, 62.4-72.3) of investigated samples. There were significant differences (p < 0.05) in the positive rates of Cryptosporidium spp. and diarrheagenic E. coli among farms, age groups, and seasons. Two Cryptosporidium species (C. parvum and C. bovis) and five gp60 subtypes of C. parvum (IIdA15G1, IIdA20G1, IIdA19G1, IIdA14G1, and a novel IIdA13G1) were identified. Two assemblages (assemblage E and zoonotic assemblage A) of G. duodenalis and six ITS genotypes of E. bieneusi (J, Henan-IV, EbpC, I, EbpA, and ESH-01) were observed. Four virulence genes (eaeA, stx1, stx2, and st) of diarrheagenic E. coli and one toxin type (type A) of C. perfringens were detected. Our study enriches our knowledge on the characteristics and zoonotic potential of diarrhea-related pathogens in dairy calves.

Title: Caractérisation moléculaire des protozoaires parasites zoonotiques courants et des bactéries responsables de diarrhée chez les veaux laitiers dans la région autonome Hui du Ningxia, en Chine. Abstract: La diarrhée causée par des agents pathogènes zoonotiques est l'une des maladies les plus courantes chez les veaux laitiers, menaçant la santé des jeunes animaux. Ceci est également un risque pour la santé humaine, en particulier les enfants. Pour explorer les agents pathogènes responsables de la diarrhée chez les veaux laitiers, cette étude a utilisé des outils de séquençage basés sur la PCR pour étudier l'occurrence et les caractères moléculaires de trois parasites (Cryptosporidium spp., Giardia duodenalis et Enterocytozoon bieneusi) et de trois agents pathogènes bactériens (Escherichia coli, Clostridium perfringens et Salmonella spp.) dans 343 échantillons fécaux de veaux laitiers diarrhéiques provenant de cinq fermes du comté de Lingwu, région autonome Hui du Ningxia, en Chine. Le taux total positif de ces pathogènes chez les veaux laitiers diarrhéiques était de 91,0 % (312/343; IC à 95 %, 87,9­94,0), et C. perfringens (61,5 %, 211/343; IC à 95 %, 56,3­66,7) était le plus répandu. Une co-infection avec deux à cinq pathogènes a été trouvée dans 67,3 % (231/343; IC à 95 %, 62,4­72,3) des échantillons étudiés. Il y avait des différences significatives (p < 0,05) dans les taux positifs de Cryptosporidium spp. et d'E. coli diarrhéogènes entre les fermes, les groupes d'âge et les saisons. Deux espèces de Cryptosporidium (C. parvum et C. bovis) et cinq sous-types de gp60 de C. parvum (IIdA15G1, IIdA20G1, IIdA19G1, IIdA14G1 et un nouveau, IIdA13G1) ont été identifiés. Deux assemblages (assemblage E et assemblage zoonotique A) de G. duodenalis et six génotypes ITS d'E. bieneusi (J, Henan-IV, EbpC, I, EbpA et ESH-01) ont été observés. Quatre gènes de virulence (eaeA, stx1, stx2 et st) d'E. coli diarrhéogènes et un type de toxine (type A) de C. perfringens ont été détectés. Notre étude enrichit les connaissances sur les caractères et le potentiel zoonotique des agents pathogènes liés à la diarrhée chez les veaux laitiers.

Mechanism of JNK action in oxidative stress-enhanced gut injury by Clostridium perfringens type A infection.

In piglets, oxidative stress can exacerbate gut injury caused by pathogens. C-Jun amino-terminal kinase (JNK) is associated with oxidative stress-induced damage to intestinal epithelial barrier. However, it is unclear whether oxidative stress can increase gut injury by Clostridium perfringens type A (CpA) and whether JNK mediates this process. We aimed to investigate if and how the JNK can regulate the effect of oxidative stress on gut injury induced by CpA infection. In this study, the oxidative stress in IPEC-J2 cells was modeled, and the changes in the susceptibility of IPEC-J2 cells to CpA were examined after treatment of oxidative stressed IPEC-J2 cells with JNK inhibitor (SP600125) and JNK siRNA. Pre-injection with the SP600125 solution was also carried out in oxidative stressed mice, followed by CpA infection. Results indicated that compared to that in the Control group, IPEC-J2 cells under oxidative stress showed reduced transmembrane resistance, degraded tight junction (TJ) proteins, increased membrane permeability, and enhanced CpA infection, all of which were reversed by inhibiting or interfering with JNK expression. Similarly, compared to that in the Control group, mice under oxidative stress showed degradation of jejunal TJ proteins, increased intestinal permeability and barrier damage by CpA, while mice pre-injected with the SP600125 solution showed alleviation of these alterations. These results suggested that oxidative stress enhanced the infection of IPEC-J2 cells and the gut injury caused by CpA, which was mediated by JNK. This study provides important insights regarding the mechanism by which oxidative stress enhanced intestinal damage by CpA.

Biochemical Characterization of Hyaluronate Lyase CpHly8 from an Intestinal Microorganism Clostridium perfringens G1121.

Hyaluronic acid (HA) is an important component of extracellular matrices (ECM) and a linear polysaccharide involved in various physiological and pathological processes within the biological system. Several pathogens exploit HA degradation within the extracellular matrix to facilitate infection. While many intestinal microorganisms play significant roles in HA utilization in the human body, there remains a scarcity of related studies. This paper addressed this gap by screening intestinal microorganisms capable of degrading HA, resulting in the isolation of Clostridium perfringens G1121, which had been demonstrated the ability to degrade HA. Subsequent genome sequencing and analysis of C. perfringens G1121 revealed its utilization of the polysaccharide utilization loci of HA (PULHA), which was obtained by horizontal gene transfer. The PULHA contains a sequence encoding a hyaluronic acid-specific degradation enzyme designated CpHly8, belonging to polysaccharide lyase family 8. The specific activity of CpHly8 towards HA was 142.98 U/mg, with the optimum reaction temperature and pH observed at 50℃ and 6.0, respectively. The final product of HA degradation by CpHly8 was unsaturated hyaluronic acid disaccharide. Moreover, subcutaneous diffusion experiments with trypan blue in mice revealed that CpHly8 effectively promoted subcutaneous diffusion and sustained its effects long-term, suggesting its potential application as an adjunct in drug delivery. Overall, our study enriches our understanding of intestinal microbial degradation of HA, provides new evidence for horizontal gene transfer among intestinal microorganisms, and confirms that CpHly8 is a promising candidate for intestinal microbial hyaluronidase.

Sudden death due to enterotoxemia among Arabian camels (Camelus dromedaries) and associated risk factors.

Background: Sudden death is defined as an unexpected death occurring with no observed antecedent clinical signs. Aim: The current study was performed to notice the tangible causes of sudden death among 51 out of 340 she-camels on a private farm in the eastern region of El Khafgi, Saudi Arabia. Methods: A retrospective cohort study design was conducted to investigate the sudden death of camels through microscopic examination of fecal matter to identify the gastrointestinal parasites, analysis of whole blood thin films to diagnose blood parasites, blood culturing to recognize bacterial infection as Pasteurella multicida, and macroscopic postmortem examination to identify the gastrointestinal adult worm. The quantity and composition of feed were also analyzed. Afterward, a commercial multiscreen Ag-ELISA kit technique determined the toxins of Clostridium perfringens (C. perfringens). Results: The results revealed that the incidence rate of sudden death was 15%. The sudden death occurred due to C. perfringens enterotoxins detected in the rumen, intestinal content, and intestinal wall. The enterotoxins and Alpha toxins were noticed, but the other toxin types, including Beta and Epsilon, could not be detected. All C. perfringens toxins were discovered to be negative in fecal matter. A significant association was reported between sudden death, she-camels age, and feeding habits as risk factors (p = 0.020 and 0.028, respectively). Risk factor assessment by relative risk (RR) revealed that the odds of RR of sudden death occurring among she-camels aged over two years were higher than those less than two years (2.24 CI 95%, 1.093-4.591). Furthermore, the odds RR of sudden death occurring due to exposure of she-camels to a concentrated ration of 18% were higher twice than those not exposed (2.346 CI 95%, 1.039-5.296). Conclusion: Clostridium perfringens enterotoxaemia should be listed as a cause of sudden death in camels and the alteration in diet with 18% concentration feed changes the intestinal environment, which leads to C. perfringens proliferating and yielding potent toxins. More observations and interferences like regular immunization are recommended to reduce the disease and increase the awareness of the farmers of the importance of risk factors.

Effect of anaerobic digestion on pathogens and antimicrobial resistance in the sewage sludge.

Antimicrobial resistance (AMR) is recognized as a global threat. AMR bacteria accumulate in sewage sludge however, knowledge on the persistence of human pathogens and AMR in the sludge line of the wastewater treatment is limited. Sludge can be used, with or without additional treatment, as fertilizer in agricultural fields. The aim of this study is to obtain knowledge about presence of human pathogens and AMR in the sewage sludge, before and after the anaerobic digestion (AD) applying innovative combinations of methods. Fifty sludge samples were collected. Cultivation methods combined with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and Antibiotic Susceptibility Test (AST) were used obtaining knowledge about the microbial community, pathogens, and antibiotic resistant bacteria while the droplet digital Polymerase Chain Reaction (ddPCR) was performed to detect most common AMR genes. In total, 231 different bacterial species were identified in the samples. The most abundant species were spore-forming facultative anaerobic bacteria belonging to Bacillus and Clostridium genera. The AD causes a shift in the microbial composition of the sludge (p = 0.04). Seven pathogenic bacterial species constituting 188 colonies were isolated and tested for susceptibility to Clindamycin, Meropenem, Norfloxacin, Penicillin G, and Tigecycline. Of the Clostridium perfringens and Bacillus cereus isolates 67 and 50 %, respectively, were resistant to Clindamycin. Two B. cereus and two C. perfringens isolates were also resistant to other antibiotics showing multidrug resistance. ARGs (blaOXA, blaTEM, ermB, qnrB, tet(A)-(W), sulI-II) were present at 7-8 Log gene copies/kg of sludge. AD is the main driver of a reduction of some ARGs (1 Log) but resistant bacteria were still present. The results showed the usefulness of the integration of the proposed analytical methods and suggest a decrease in the risk of presence of cultivable pathogens including resistant isolates after AD but a persistent risk of ARGs' horizontal transmission.

Cooling Uncovered Foods at a Depth of ∼5.1 cm (2 in.) or Less Poses Little Risk of Pathogen Growth.

The U.S. Food and Drug Administration has guidelines for cooling cooked foods in retail operations. Data on foodborne illness risk factors in restaurants indicate that cooling is often out of compliance with these guidelines. We sought to identify factors under the control of the operator that had a significant effect on the cooling rates of cooked foods. Minneapolis Minnesota Health Inspectors were trained in standardized operating procedures for cooling data collection. Data loggers set to appropriate time intervals and calibrated to ±0.5 °C (∼1°F) were used in data collection. Analysis was performed using the R statistical computing language version 4.2.2. Preexisting pathogen models were used to predict Log CFU increases of Clostridium perfringens or Bacillus cereus. Data from 224 recipes were recorded by inspectors between October 2018 and October 2019. Food depth had a highly significant effect (p = 8.90E-08) on cooling rate. The use of an ice bath or ice wand was also significant (p < 0.005). There was a significant correlation between container material (metal or plastic) and food depth because foods with a greater depth are often being cooled in plastic containers. Foods at a depth greater than 5.1 cm (2 in.) that cooled faster than 0.23 log(°C)/h were often wholly or partially cooled in blast chillers or freezers, cooled using an ice bath or ice wand (or both), or were composed of protein pieces (e.g., chicken wings) that facilitated more rapid cooling due to air gaps in the food. Foods in shallow containers at a food depth of less than or equal to ∼5.1 cm (2 in.) that cooled more slowly than 0.23 log(°C)/h were being cooled at temperatures greater than 5 °C (41°F) or were partially or wholly covered. These foods also showed little evidence of pathogen growth from predictive models. Our analysis shows that cooling foods in shallow containers at a food depth of less than or equal to ∼5.1 cm (2 in.) poses little risk of significant pathogen growth.

Inhibition of calpain reduces oxidative stress and attenuates pyroptosis and ferroptosis in Clostridium perfringens Beta-1 toxin-induced macrophages.

Clostridium perfringens Beta-1 toxin (CPB1) is a lethal toxin, which can lead to necrotic enteritis, but the pathological mechanism has not been elucidated. We investigated whether reactive oxygen species (ROS) participated in CPB1-induced pyroptosis and ferroptosis, and investigated the effects of calpain on CPB1-induced oxidative stress and inflammation. Scavenging ROS by N-Acetyl-L cysteine (NAC) led to the reduction of ROS, inhibited the death of macrophages, cytoplasmic swelling and membrane rupture, the expression of pyroptosis-related proteins and proinflammatory factor, while increased the expression of anti-inflammatory factors in cells treated with rCPB1. Adenosine triphosphate (ATP) synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1 (ATP5A1) was identified specifically interact with rCPB1. Silencing ATP5A1 inhibited accumulation of ATP and ROS, leaded to less cytoplasmic swelling and membrane rupture, attenuated pyroptosis and inflammation in rCPB1-treated cells. We also found that rCPB1 induces ferroptosis in macrophages, and the level of ferroptosis was similar with H2O2. Of note, H2O2 is a major ROS source, indicated that ROS production may play a major role in the regulation of ferroptosis in macrophages treated with rCPB1. This finding was further corroborated in rCPB1- induced human acute monocytic leukemia cells, which were treated with NAC. In addition, the inhibition of ferroptosis using liproxstatin-1 inhibited the shriveled mitochondrial morphology, increased the expression of glutathione peroxidase 4, nicotinamide adenine dinucleotide (phosphate) hydrogen: quinone oxidoreductase 1 and cysteine/glutamic acid reverse transport solute carrier family 7 members 11, decreased the expression of heme oxygenase 1, nuclear receptor coactivator 4 and transferrin receptor proteins, reduced malondialdehyde and lipid peroxidation levels, and increased intracellular L-glutathione levels in cells treated with rCPB1. Furthermore, calpain inhibitor PD151746 was used to investigate how pyroptosis and ferroptosis were involved simultaneously in rCPB1-treated macrophages. We showed that PD151746 inhibited ATP and ROS production, reversed the representative pyroptosis/ferroptosis indicators and subsequently reduced inflammation. The above findings indicate that rCPB1 might lead to macrophage pyroptosis and ferroptosis through the large and sustained increase in intracellular calpain and oxidative stress, further lead to inflammation.

Investigation of the outbreak of Clostridium perfringens using single nucleotide polymorphism analysis for genotyping in Toyama, Japan, 2023.

Clostridium perfringens, which produces C. perfringens enterotoxin (CPE), is a major causative agent of food poisoning owing to its gastrointestinal symptoms. Genotyping is important for identifying the etiological agent in outbreaks of C. perfringens. We attempted to genotype strains isolated from an outbreak of food poisoning in Toyama in 2023 using single nucleotide polymorphism (SNP) analysis. The strains of C. perfringens were isolated from a piece of curry food consumed by all patients and from the feces of the patients and employees. The cpe gene was detected in isolates from patients and curry food. The cpe-negative isolates were found in patients who consumed curry foods and in employees. The results of the SNP analysis suggest that the patient- and curry-derived isolates were likely from the same source but were unlikely to be related to the employee-derived isolates. The results of the SNP and pulsed-field gel electrophoresis (PFGE) analyses were consistent, indicating that the patient- and curry-derived isolates came from the same source. SNP analysis, a whole-genome-based genotyping method, is a promising alternative to the traditional PFGE method. Further studies are needed to accumulate more experience with genotyping using SNP analysis for the epidemiological investigation of outbreaks of C. perfringens.

Bacillus licheniformis suppresses Clostridium perfringens infection via modulating inflammatory response, antioxidant status, inflammasome activation and microbial homeostasis in broilers.

Pathogenic bacteria infection, especially Clostridium perfringens (C. perfringens), markedly threatened the health of animals, and further caused huge economic loss. In this study, Bacillus licheniformis HJ0135 (BL) was used. Oxford cup bacteriostatic test and inhibitory rate test were conducted to evaluate the antibacterial ability of BL. Results showed the strongest inhibitory role of BL on C. perfringens (P < 0.05). Afterwards, 540 one-day-old yellow-feather broilers (32.7 ± 0.2 g) were randomly allocated into 3 groups, including CON group (basal diet), CP group (basal diet + 1 × 109 CFU C. perfringens in gavage), and BL + CP group (basal diet containing 7.5 × 106 CFU/g BL + 1 × 109 CFU C. perfringens in gavage). At d 70, broilers in the CP and BL + CP groups were treated with C. perfringens by continuously oral administration for 5 d. The experiment lasted for 75 d. The serum, immune organs, jejunal mucosa, and cecal contents were collected for analysis. In vivo experiment showed that BL supplementation markedly improved (P < 0.05) BW, ADG, thymus index, serum immunoglobins and antioxidases, reduced feed conversion ratio (FCR) and serum pro-inflammatory cytokines of C. perfringens-infected broilers. Furthermore, the increased jejunal injury and levels of pro-inflammatory cytokines, decreased gene expressions of tight junction proteins in the jejunal mucosa were significantly alleviated (P < 0.05) by BL. More importantly, the activation of NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome was inhibited (P < 0.05) by BL to further attenuate jejunal damage. Besides, BL supplementation markedly increased (P < 0.05) the cecal isobutyric acid and isovaleric acid. Microbial analysis showed that BL changed the composition and relative abundances of microbiota in the cecal contents (P < 0.05), especially the short chain fatty acids (SCFAs)-producing bacteria including Eubacterium_coprostanoligenes_group, Megamonas, Faecalibacterium, and Lactobacillus, which further protected against C. perfringens-induced jejunal inflammation in broilers. Our study laid a theoretical basis for the application of probiotics in lessening C. perfringens-related diseases in poultry farming.

LAMTOR1/mTORC1 promotes CD276 to induce immunosuppression via PI3K/Akt/MMP signaling pathway in Clostridium perfringens-induced necrotic enteritis of laying hens.

Clostridium perfringens (C. perfringens) causes avian necrotic enteritis, leading to huge economic losses to the poultry industry. This pathogen induces host immunosuppression; however, the molecular mechanism is still unclear. Thus, we established a laying hen infection model to explore this mechanism. We randomly divided 20 one-old-day laying hens into the control and infection groups. The infection group was infected intragastrically with 1 × 109 colony-forming units of C. perfringens in 1 mL of sterile phosphate-buffered saline (PBS) once a day from d 17 to 20; the control group received the same volume of PBS without the bacterium. Twenty-four hours after the last challenge, we sacrificed the laying hens and collected the jejunum for analysis. The infection group presented alterations in blood biochemical parameters and necrotic lesion scores as well as damage to the jejunum. Proteomics revealed 427 upregulated and 291 downregulated proteins in the infection group. In the infection group, CD3, CD4, and CD8 messenger RNA expression (mRNA) expression was decreased; LAMTOR1 and mTORC1 mRNA expression was increased; CD276 protein expression was enhanced; and the PI3K/Akt/MMP pathway was activated in jejunum of laying hens. This is the first study to report CD276 expression in the jejunum related to immunosuppression in a laying hen model of necrotic enteritis. It provides some new key targets to potentially control avian necrotic enteritis.

ParalichenysinDY4, a novel bacteriocin-like substance, is employed to control Clostridium perfringens.

Clostridium perfringens (C. perfringens) is an important pathogen that contributes to human and animal disease. At present, antibiotic therapy is one of the most effective strategies for C. perfringens. However, with the rise of antibacterial resistance, new agents with novel mechanisms of action are urgently needed. Bacteriocins are recognized as a viable alternative to antibiotics. In this study, the bacteriocin-like substance ParalichenysinDY4, derived from the Bacillus paralicheniformis (B. paralicheniformis) DY4 strain, is investigated as a potential alternative for combating Clostridium perfringens. The substance was isolated from B. paralicheniformis DY4 fermentation broth through a series of purification steps including methanol extraction, gel filtration, and high-performance liquid chromatography. Mass spectrometry analysis of ParalichenysinDY4 revealed that the detected peptide sequences did not match any previously known bacteriocins, indicating it is a novel bacteriocin-like substance. The novel bacteriocin-like substance exhibits effective antibacterial activity and broad antimicrobial spectrum against C. perfringens. Subsequent analyses utilizing methodologies including flow cytometry and scanning electron microscopy suggest that its mechanism of action is linked to its effects on the cell membrane. At the same time, due to its exceptional stability, safety, and efficient ability to remove pathogens both in vitro and in vivo, ParalichenysinDY4 holds promise as a valuable natural antimicrobial agent.

Three-mode ratiometric biosensor based on integrated DNA-driven magnetic beads for Clostridium perfringens detection.

A three-mode biosensor incorporated ratiometric (electrochemical/colorimetric, electrochemical/photothermal) into its design was constructed using DNA-driven magnetic beads (DMBs) as a bridge to detect C. perfringens. It further enhances the accuracy of detection results while maintaining compatibility with applications in multiple scenarios. Briefly, the G-quadruplex was combined with aptamer and immobilized onto magnetic beads through amide-bond, resulting in the integration of DMBs. The DMBs and supernatant were separated by magnetic separation when the target was present. Subsequently, the DMBs were utilized to construct the electrochemical biosensor, whereas the supernatant was used to construct colorimetric and photothermal biosensors. The limits of detection the ratiometric biosensor were ultimately reduced to 0.26 and 0.27 lg CFU g-1, respectively, in comparison to the single three-mode biosensor. Moreover, this biosensor had been applied in real-sample assays successfully. The establishment of this platform provides a new method for detecting pathogens in the fields of food safety and environmental monitoring.

Intestinal pathology in goats challenged with Clostridium perfringens type D strain CN1020 wild-type and its genetically modified derivatives.

Clostridium perfringens type D is the causative agent of enterotoxemia in sheep, goats, and cattle. Although in sheep and cattle, the disease is mainly characterized by neurological clinical signs and lesions, goats with type D enterotoxemia frequently have alterations of the alimentary system. Epsilon toxin (ETX) is the main virulence factor of C. perfringens type D, although the role of ETX in intestinal lesions in goats with type D enterotoxemia has not been fully characterized. We evaluated the contribution of ETX to C. perfringens type D enteric pathogenicity using an intraduodenal challenge model in young goats, with the virulent C. perfringens type D wild-type strain CN1020; its isogenic etx null mutant; an etx-complemented strain; and sterile, non-toxic culture medium. The intestinal tract of each animal was evaluated grossly, microscopically, and immunohistochemically for activated caspase-3. Both ETX-producing strains induced extensive enterocolitis characterized by severe mucosal necrosis, apoptosis, and diffuse suppurative infiltrates. No significant gross or microscopic lesions were observed in goats inoculated with the non-ETX-containing inocula. These results confirm that ETX is essential for the production of intestinal lesions in goats with type D disease. Also, our results suggest that the intestinal pathology of type D enterotoxemia in goats is, at least in part, associated with apoptosis.

Heat resistance differences are common between both vegetative cells and spores of Clostridium perfringens type F isolates carrying a chromosomal vs plasmid-borne enterotoxin gene.

Clostridium perfringens type F isolates utilize C. perfringens enterotoxin (CPE) to cause food poisoning (FP) and nonfoodborne gastrointestinal diseases. The enterotoxin gene (cpe) can be located on either the chromosome or plasmids, but most FP isolates carry a chromosomal cpe (c-cpe) gene. Our 2000 article in Applied and Environmental Microbiology (66:3234-3240, 2000, https://doi.org/10.1128/aem.66.8.3234-3240.2000https://doi.org/10.1128/AEM.66.8.3234-3240.2000) determined that vegetative cells and spores of c-cpe isolates are more heat resistant than those of plasmid cpe (p-cpe) isolates, which is favorable for their survival in improperly cooked or held food. However, that 2000 article was recently retracted (90:e00249-24, 2024, https://doi.org/10.1128/aem.00249-24). To our knowledge, the 2000 article remains the only study reporting that heat resistance differences are common between both vegetative cells and spores of type F c-cpe isolates vs type F p-cpe isolates. To confirm and preserve this information in the literature, the heat resistance portion of the 2000 study has been repeated. The 2024 results reproduced the 2000 results by indicating that, relative to the surveyed type F p-cpe isolates, the vegetative cells of surveyed type F c-cpe isolates are ~2-fold more heat resistant and the spores of most surveyed c-cpe isolates are ~30-fold more heat resistant. However, consistent with several reports since our 2000 paper, one surveyed type F c-cpe isolate (which did not appreciably sporulate in 2000 but sporulated in 2024) produced spores with intermediate heat sensitivity, confirming that spores of some type F c-cpe isolates lack exceptional heat resistance.IMPORTANCEClostridium perfringens type F food poisoning (FP), which is the second most common bacterial cause of FP, involves the production of C. perfringens enterotoxin. While the enterotoxin gene (cpe) can be located on either the chromosome or plasmids in type F isolates, most FP cases are caused by chromosomal cpe isolates. The current results support the conclusion that the vegetative cells and spores of type F chromosomal cpe isolates are often more heat resistant than vegetative cells and spores of type F plasmid cpe isolates. Greater heat resistance should favor the survival of the spores and vegetative cells of those chromosomal cpe isolates in temperature-abused food, which may help explain the strong association of type F chromosomal cpe strains with FP.

The presence of differentiated C2C12 muscle cells enhances toxin production and growth by Clostridium perfringens type A strain ATCC3624.

Clostridium perfringens type A causes gas gangrene, which involves muscle infection. Both alpha toxin (PLC), encoded by the plc gene, and perfringolysin O (PFO), encoded by the pfoA gene, are important when type A strains cause gas gangrene in a mouse model. This study used the differentiated C2C12 muscle cell line to test the hypothesis that one or both of those toxins contributes to gas gangrene pathogenesis by releasing growth nutrients from muscle cells. RT-qPCR analyses showed that the presence of differentiated C2C12 cells induces C. perfringens type A strain ATCC3624 to upregulate plc and pfoA expression, as well as increase expression of several regulatory genes, including virS/R, agrB/D, and eutV/W. The VirS/R two component regulatory system (TCRS) and its coupled Agr-like quorum sensing system, along with the EutV/W TCRS (which regulates expression of genes involved in ethanolamine [EA] utilization), were shown to mediate the C2C12 cell-induced increase in plc and pfoA expression. EA was demonstrated to increase toxin gene expression. ATCC3624 growth increased in the presence of differentiated C2C12 muscle cells and this effect was shown to involve both PFO and PLC. Those membrane-active toxins were each cytotoxic for differentiated C2C12 cells, suggesting they support ATCC3624 growth by releasing nutrients from differentiated C2C12 cells. These findings support a model where, during gas gangrene, increased production of PFO and PLC in the presence of muscle cells causes more damage to those host cells, which release nutrients like EA that are then used to support C. perfringens growth in muscle.

Machine learning surveillance of foodborne infectious diseases using wastewater microbiome, crowdsourced, and environmental data.

Clostridium perfringens (CP) is a common cause of foodborne infection, leading to significant human health risks and a high economic burden. Thus, effective CP disease surveillance is essential for preventive and therapeutic interventions; however, conventional practices often entail complex, resource-intensive, and costly procedures. This study introduced a data-driven machine learning (ML) modeling framework for CP-related disease surveillance. It leveraged an integrated dataset of municipal wastewater microbiome (e.g., CP abundance), crowdsourced (CP-related web search keywords), and environmental data. Various optimization strategies, including data integration, data normalization, model selection, and hyperparameter tuning, were implemented to improve the ML modeling performance, leading to enhanced predictions of CP cases over time. Explainable artificial intelligence methods identified CP abundance as the most reliable predictor of CP disease cases. Multi-omics subsequently revealed the presence of CP and its genotypes/toxinotypes in wastewater, validating the utility of microbiome-data-enabled ML surveillance for foodborne diseases. This ML-based framework thus exhibits significant potential for complementing and reinforcing existing disease surveillance systems.

Preparation and Application of Clostridium perfringens Alpha Toxin Nanobodies.

All subtypes of Clostridium perfringens (C. perfringens) produce the alpha toxin (CPA), which can cause enteritis or enterotoxemia in lambs, cattle, pigs, and horses, as well as traumatic clostridial myonecrosis in humans and animals. CPA acts on cell membranes, ultimately leading to endocytosis and cell death. Therefore, the neutralization of CPA is crucial for the prevention and treatment of diseases caused by C. perfringens. In this study, utilizing CPA as an antigen, a nanobody (CPA-VHH) with a half-life of 2.9 h, an affinity constant (KD) of 0.9 nmol/L, and good stability below 60 °C was prepared from a natural nanobody library from alpacas. The biological activity analysis of CPA-VHH revealed its ability to effectively neutralize the phospholipase and hemolytic activity of CPA at a 15-fold ratio. In Vero cells, 9.8 µg/mL CPA-VHH neutralized the cytotoxicity of CPA at two times the half-maximal inhibitory concentration (IC50). In a mouse model, 35.7 ng/g body weight (BW) of CPA-VHH neutralized 90% of the lethality caused by a 2× median lethal dose (LD50) of CPA. It was found that CPA-VHH protected 80% of mice within 30 min at 2 × LD50 CPA, but this dropped below 50% after 2 h and to 0% after 4 h. Rescue trials indicated that using CPA-VHH within 30 min post-infection with 2 × LD50 CPA achieved an 80% rescue rate, which decreased to 10% after 2 h. Furthermore, CPA-VHH effectively mitigated the reduction in the expression levels of zonula occludens-1 (ZO-1), Occludin, and Claudin-1, while also attenuating the upregulation of the pro-inflammatory cytokines interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), tumor necrosis factor α (TNF-α), and interferon-γ (IFN-γ) induced by CPA infection. Overall, this study has identified a specific nanobody, CPA-VHH, that effectively neutralizes CPA toxins in vitro and in animal models, providing a new tool for inhibiting the pathogenicity resulting from these toxins and laying an important foundation for the development of new anti-C. perfringens toxin-related therapeutic products.

Exploring the Interactive Impacts of Citronellol, Thymol, and Trans-Cinnamaldehyde in Broilers: Moving towards an Improved Performance, Immunity, Gastrointestinal Integrity, and Clostridium perfringens Resistance.

AIMS: This study aimed to explore the effectiveness of dietary citronellol, thymol, and trans-cinnamaldehyde (CTC) essential oils blend on broilers` growth performance, immunity, intestinal microbial count, gut integrity, and resistance against Clostridium perfringens utilizing the necrotic enteritis (NE) challenge model. METHODS AND RESULTS: A total of 200 Ross 308 male broiler chicks received either a control diet or diet supplemented with three graded levels of CTC blend including 300, 600, and 900 mg of CTC blend/Kg diet and experimentally infected with C. perfringens strain at 23 days of age. Herein, dietary CTC blend fortifications significantly improved the broilers` growth performance, which was supported by upregulating the expression levels of MUC-2, occludin, and JAM-2 genes. Moreover, dietary CTC blend inclusion significantly enhanced the levels of blood phagocytic percentage and serum IgA, IgG, and MPO, and reduced the values of serum CRP, and NO at 5 days pre-infection, 10-, and 15 days post-infection (dpi) with C. perfringens. At 15 dpi, CTC blend inclusion significantly reduced the intestinal digesta pH, coliforms and C. perfringens loads, and the expression levels of genes related to C. perfringens virulence (cpe, cnaA, and nanI), proinflammatory cytokines (IL-1ß and TNF-α), and chemokines (CCL20), in addition to increasing the count of beneficial total Lactobacillus and total aerobic bacteria, and the expression levels of genes related to anti-inflammatory cytokines (IL-10) and chemokines (AvBD6, and AvBD612). CONCLUSION: Our results point to the growth-provoking, immunostimulant, antibacterial, anti-inflammatory, and antivirulence characteristics of the CTC blend, which improves the broilers' resistance to C. perfringens and ameliorates the negative impacts of NE.