A "terminal" case of glycan catabolism: Structural and enzymatic characterization of the sialidases of Clostridium perfringens.

Sialic acids are commonly found on the terminal ends of biologically important carbohydrates, including intestinal mucin O-linked glycans. Pathogens such as Clostridium perfringens, the causative agent of necrotic enteritis in poultry and humans, have the ability to degrade host mucins and colonize the mucus layer, which involves removal of the terminal sialic acid by carbohydrate-active enzymes (CAZymes). Here, we present the structural and biochemical characterization of the GH33 catalytic domains of the three sialidases of C. perfringens and probe their substrate specificity. The catalytically active domains, which we refer to as NanHGH33, NanJGH33, and NanIGH33, displayed differential activity on various naturally occurring forms of sialic acid. We report the X-ray crystal structures of these domains in complex with relevant sialic acid variants revealing the molecular basis of how each catalytic domain accommodates different sialic acids. NanHGH33 displays a distinct preference for α-2,3-linked sialic acid, but can process α-2,6-linked sialic acid. NanJGH33 and NanIGH33 both exhibit the ability to process α-2,3- and α-2,6-linked sialic acid without any significant apparent preference. All three enzymes were sensitive to generic and commercially available sialidase inhibitors, which impeded sialidase activity in cultures as well as the growth of C. perfringens on sialylated glycans. The knowledge gained in these studies can be applied to in vivo models for C. perfringens growth and metabolism of mucin O-glycans, with a view toward future mitigation of bacterial colonization and infection of intestinal tissues.

Isolation of a Virulent Clostridium perfringens Strain from Elaphurus davidianus and Characterization by Whole-Genome Sequence Analysis.

Clostridium perfringens (C. perfringens) is an important veterinary pathogen and a noteworthy threat to human and animal health. Recently, there has been a significant rise in the number of moose fatalities caused by this rare, endemic species in China. Currently, there is an increasing trend in conducting whole-genome analysis of C. perfringens strains originating from pigs and chickens, whereas fewer studies have been undertaken on Elaphurus davidianus-originating strains at the whole-genome level. Our laboratory has identified and isolated five C. perfringens type A from affected Elaphurus davidianus. The current study identified the most potent strain of C. perfringens, which originated from Elaphurus davidianus, and sequenced its genome to reveal virulence genes and pathogenicity. Our findings show that strain CX1-4 exhibits the highest levels of phospholipase activity, hemolytic activity, and mouse toxicity compared to the other four isolated C. perfringens type A strains. The chromosome sequence length of the CX1-4 strain was found to be 3,355,389 bp by complete genome sequencing. The current study unveils the genomic characteristics of C. perfringens type A originating from Elaphurus davidianus. It provides a core foundation for further investigation regarding the prevention and treatment of such infectious diseases in Elaphurus davidianus.

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.

Using reverse vaccinology techniques combined with B-cell epitope prediction to screen potential antigenic proteins of the bovine pathogen Clostridium perfringens type A.

Clostridium perfringens type A frequently causes necrohaemorrhagic enteritis in cattle, a rapidly progressing disease with a high mortality rate, thus inflicting substantial economic losses in the cattle industry. Effective prevention and control of this disease rely on rapid detection and vaccination strategies, making the screening of antigenic proteins with diagnostic and vaccine potential particularly crucial. In this study, we conducted a pangenomic analysis of 15 bacterial strains, grounded in traditional reverse vaccinology and supplemented with B-cell linear and conformational epitope analysis tools. This approach led to the identification of 2304 core genes and 3606 accessory genes, among which 58 surface-exposed proteins, encoded by core genes, were identified Proteins lacking tertiary structure information were predicted via AlphaFold2, ultimately identifying four target proteins and 14 candidate proteins enriched with linear and conformational epitopes, including virulence proteins such as alpha-toxin, theta-toxin, and alpha-clostripain, and extracellular solute-binding proteins, rhodanese-like proteins, and the accessory gene-encoded lysozyme inhibitor LprI family protein. Our findings demonstrate that the combined use of multiple B-cell epitope analysis tools can help overcome the limitations of any single tool. The proteins selected in this study offer valuable references for rapid diagnostics and the development of genetically engineered vaccines.

Cryo-EM structure of the octameric pore of Clostridium perfringens ß-toxin.

Clostridium perfringens is one of the most widely distributed and successful pathogens producing an impressive arsenal of toxins. One of the most potent toxins produced is the C. perfringens ß-toxin (CPB). This toxin is the main virulence factor of type C strains. We describe the cryo-electron microscopy (EM) structure of CPB oligomer. We show that CPB forms homo-octameric pores like the hetero-oligomeric pores of the bi-component leukocidins, with important differences in the receptor binding region and the N-terminal latch domain. Intriguingly, the octameric CPB pore complex contains a second 16-stranded ß-barrel protrusion atop of the cap domain that is formed by the N-termini of the eight protomers. We propose that CPB, together with the newly identified Epx toxins, is a member a new subclass of the hemolysin-like family. In addition, we show that the ß-barrel protrusion domain can be modified without affecting the pore-forming ability, thus making the pore particularly attractive for macromolecule sensing and nanotechnology. The cryo-EM structure of the octameric pore of CPB will facilitate future developments in both nanotechnology and basic research.

STING-dependent trained immunity contributes to host defense against Clostridium perfringens infection via mTOR signaling.

Clostridium perfringens (C. perfringens) infection is recognized as one of the most challenging issues threatening food safety and perplexing agricultural development. To date, the molecular mechanisms of the interactions between C. perfringens and the host remain poorly understood. Here, we show that stimulator of interferon genes (STING)-dependent trained immunity protected against C. perfringens infection through mTOR signaling. Heat-killed Candida albicans (HKCA) training elicited elevated TNF-α and IL-6 production after LPS restimulation in mouse peritoneal macrophages (PM). Although HKCA-trained PM produced decreased levels of TNF-α and IL-6, the importance of trained immunity was demonstrated by the fact that HKCA training resulted in enhanced bacterial phagocytic ability and clearance in vivo and in vitro during C. perfringens infection. Interestingly, HKCA training resulted in the activation of STING signaling. We further demonstrate that STING agonist DMXAA is a strong inducer of trained immunity and conferred host resistance to C. perfringens infection in PM. Importantly, corresponding to higher bacterial burden, reduction in cytokine secretion, phagocytosis, and bacterial killing were shown in the absence of STING after HKCA training. Meanwhile, the high expression levels of AKT/mTOR/HIF1α were indeed accompanied by an activated STING signaling under HKCA or DMXAA training. Moreover, inhibiting mTOR signaling with rapamycin dampened the trained response to LPS and C. perfringens challenge in wild-type (WT) PM after HKCA training. Furthermore, STING­deficient PM presented decreased levels of mTOR signaling-related proteins. Altogether, these results support STING involvement in trained immunity which protects against C. perfringens infection via mTOR signaling.

Necrotic enteritis in chickens: a comprehensive review of vaccine advancements over the last two decades.

Necrotic enteritis (NE) is a severe gastrointestinal disease that poses a significant threat to poultry, leading to progressive deterioration of the small intestine, reduced performance, and increased mortality rates, causing economic losses in the poultry industry. The elimination of antimicrobial agents from chicken feed has imposed a need to explore alternative approaches for NE control, with vaccination emerging as a promising strategy to counteract the detrimental consequences associated with NE. This comprehensive study presents an overview of the extensive efforts made in NE vaccination from 2004 to2023. The study focuses on the development and evaluation of vaccine candidates designed to combat NE. Rigorous evaluations were conducted in both laboratory animals and broiler chickens, the target population, to assess the vaccines' capacity to elicit an immune response and provide substantial protection against toxin challenges and experimental NE infections. The review encompasses the design of vaccine candidates, the antigens employed, in vivo immune responses, and the efficacy of these vaccines in protecting birds from experimental NE infection. This review contributes to the existing knowledge of NE vaccination strategies, offering valuable insights for future research and development in this field.

Efficacy of Bacillus subtilis probiotic in preventing necrotic enteritis in broilers: a systematic review and meta-analysis.

Probiotics can enhance broiler chicken health by improving intestinal microbiota, potentially replacing antibiotics. They protect against bacterial diseases like necrotic enteritis (NE) in poultry. Understanding their role is crucial for managing bacterial diseases, including NE. This study conducted a meta-analysis to assess the effects of Bacillus subtilis probiotic supplementation on feed conversion ratio (FCR), NE lesion score, and mortality. Additionally, a systematic review analysed gut microbiota changes in broilers challenged with Clostridium perfringens with or without the probiotic supplementation. Effect sizes from the studies were estimated in terms of standardized mean difference (SMD). Random effect models were fitted to estimate the pooled effect size and 95% confidence interval (CI) of the pooled effect size between the control [probiotic-free + C. perfringens] and the treatment [Bacillus subtilis supplemented + C. perfringens] groups. Overall variance was computed by heterogeneity (Q). The meta-analysis showed that Bacillus subtilis probiotic supplementation significantly improved FCR and reduced NE lesion score but had no effect on mortality rates. The estimated overall effects of probiotic supplementation on FCR, NE lesion score and mortality percentage in terms of SMD were -0.91 (CI = -1.34, -0.49; P < 0.001*); -0.67 (CI = -1.11, -0.22; P = 0.006*), and -0.32 (CI = -0.70, 0.06; P = 0.08), respectively. Heterogeneity analysis indicated significant variations across studies for FCR (Q = 69.66; P < 0.001*) and NE lesion score (Q = 42.35; P < 0.001*) while heterogeneity was not significant for mortality (Q = 2.72; P = 0.74). Bacillus subtilis probiotic supplementation enriched specific gut microbiota including Streptococcus, Butyricicoccus, Faecalibacterium, and Ruminococcus. These microbiotas were found to upregulate expression of various genes such as TJ proteins occluding, ZO-1, junctional adhesion 2 (JAM2), interferon gamma, IL12-ß and transforming growth factor-ß4. Moreover, downregulated mucin-2 expression was involved in restoring the intestinal physical barrier, reducing intestinal inflammation, and recovering the physiological functions of damaged intestines. These findings highlight the potential benefits of probiotic supplementation in poultry management, particularly in combating bacterial diseases and promoting intestinal health.

Exploring the interactive impacts of citronellol, thymol, and trans-cinnamaldehyde in broilers: moving toward 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.

Emergence of genetic diversity and multi-drug resistant Clostridium perfringens from wild birds.

BACKGROUND: Clostridium perfringens (C. perfringens) is an important zoonotic microorganism that can cause animal and human infections, however information about the prevalence status in wild birds of this pathogenic bacterium is currently limited. RESULT: In this study, 57 strains of C. perfringens were isolated from 328 fecal samples of wild birds. All the isolates were identified as type A and 70.18% of the isolates carried the cpb2 gene. Antimicrobial susceptibility testing showed that and 22.80% of the isolates were classified as multidrug-resistant strains. The MLST analysis of the 57 isolates from wild birds was categorized into 55 different sequence types (STs) and clustered into eight clonal complexes (CCs) with an average of 20.1 alleles and the Simpson Diversity index (Ds) of 0.9812, and revealed a high level of genetic diversity within the C. perfringens populations. Interestingly, the isolates from swan goose were clustered in the same CC while isolates from other bird species were more scattered suggesting that a potential difference in genetic diversity among the C. perfringens populations associated with different bird species. CONCLUSION: C. perfringens exhibits a wide range of host adaptations, varying degrees of antimicrobial resistance, and a high degree of genetic diversity in wild birds. Understanding the prevalence, toxin type, antimicrobial resistance, and genetic diversity of C. perfringens in wildlife populations is essential for developing effective strategies for disease control and management.

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.

Clostridium and Cryptosporidium outbreak linked to a splash pad.

BACKGROUND: . Splash pads for recreational purposes are widespread. Using these pads can pose a health risk if they lack installation regulation and water quality supervision. Our aim was to describe a waterborne disease outbreak caused by Clostridium perfringens and Cryptosporidium spp. in a Barcelona district and the measures taken for its control. METHODS: . On August 2018, 71 cases of acute gastroenteritis were detected, affecting people who used a splash pad or were in contact with a user. Microbiological and environmental investigations were carried out. A descriptive analysis of the sample and Poisson regression models adjusted for age and sex were performed, obtaining frequencies, median values, and adjusted prevalence ratios with their 95% confidence intervals. RESULTS: The median age of the cases was 6.7 years, 27 (38%) required medical care, and three (4.2%) were hospitalized. The greater the number of times a person entered the area, the greater the number of symptoms and their severity. Nineteen (76%) of the 25 stool samples collected from cases showed the presence of one or both pathogens. Environmental investigations showed deficiencies in the facilities and identified the presence of both species in the splash pad. Health education and hygiene measures were carried out, and 14 days after the closure of the facilities, no more cases related to the pad were recorded. CONCLUSIONS: . Specific regulations are needed on the use of splash pads for recreational purposes. Until these regulations are in place, these types of facility should comply with the regulations that apply to swimming pools and spas, including those related to the design of the tanks, water recirculation systems, and adequate disinfection systems.

Prevalence, toxin-genotype distribution, and transmission of Clostridium perfringens from the breeding and milking process of dairy farms.

This study aimed to elucidate the distribution, transmission, and cross-contamination of Clostridium perfringens during the breeding and milking process from dairy farms. The prevalence of 22.3% (301/1351) yielded 494 C. perfringens isolates; all isolates were type A, except for one type D, and 69.8% (345/494) of the isolates carried atyp. cpb2 and only 0.6% (3/494) of the isolates carried cons. cpb2. C. perfringens detected throughout the whole process but without type F. 150 isolates were classified into 94 pulsed-field gel electrophoresis (PFGE) genotypes; among them, six clusters contained 34 PFGE genotypes with 58.0% isolates which revealed epidemic correlation and genetic diversity; four PFGE genotypes (PT57, PT9, PT61, and PT8) were the predominant genotypes. The isolates from different farms demonstrated high homology. Our study confirmed that C. perfringens demonstrated broad cross-contamination from nipples and hides of dairy cattle, followed by personnel and tools and air-introduced raw milk during the milking process. In conclusion, raw milk could serve as a medium for the transmission of C. perfringens, which could result in human food poisoning. Monitoring and controlling several points of cross-contamination during the milking process are essential as is implementing stringent hygiene measures to prevent further spread and reduce the risk of C. perfringens infection.

The small acid-soluble proteins of spore-forming organisms: similarities and differences in function.

The small acid-soluble proteins are found in all endospore-forming organisms and are a major component of spores. Through their DNA binding capabilities, the SASPs shield the DNA from outside insults (e.g., UV and genotoxic chemicals). The absence of the major SASPs results in spores with reduced viability when exposed to UV light and, in at least one case, the inability to complete sporulation. While the SASPs have been characterized for decades, some evidence suggests that using newer technologies to revisit the roles of the SASPs could reveal novel functions in spore regulation.

Approaching toxigenic Clostridia from a One Health perspective.

Spore-forming pathogens have a unique capacity to thrive in diverse environments, and with temporal persistence afforded through their ability to sporulate. Their prevalence in diverse ecosystems requires a One Health approach to identify critical reservoirs and outbreak-associated transmission chains, given their capacity to freely move across soils, waterways, foodstuffs and as commensals or infecting pathogens in human and animal populations. Among anaerobic spore-formers, genomic resources for pathogens including C. botulinum, C. difficile, and C. perfringens enable our capacity to identify common and unique factors that support their persistence in diverse reservoirs and capacity to cause disease. Publicly available genomic resources for spore-forming pathogens at NCBI's Pathogen Detection program aid outbreak investigations and longitudinal monitoring in national and international programs in public health and food safety, as well as for local healthcare systems. These tools also enable research to derive new knowledge regarding disease pathogenesis, and to inform strategies in disease prevention and treatment. As global community resources, the continued sharing of strain genomic data and phenotypes further enhances international resources and means to develop impactful applications. We present examples showing use of these resources in surveillance, including capacity to assess linkages among clinical, environmental, and foodborne reservoirs and to further research investigations into factors promoting their persistence and virulence in different settings.

A subanalysis of Clostridium perfringens bloodstream infections from a 5-year retrospective nationwide survey (ITANAEROBY).

Clostridium perfingens bloodstream infections (BSIs) can be associated with high mortality rates. We performed a subanalysis of all C. perfringens BSIs enrolled during a multicentric retrospective observational study (ITANAEROBY). Data were collected from January 2016 to December 2020. C. perfringens BSIs were 134 (134/1960, 6.8 %). The highest resistance rate was observed for clindamycin (26/120, 21.6 %), penicillin (11/71, 15.4 %) and metronidazole (14/131, 10.7 %). In conclusion, C. perfringens reduced susceptibility phenotype to first-line therapy.

Mechanisms of intestinal epithelial cell damage by Clostridiumperfringens.

Clostridium perfringens, a Gram-positive bacterium, causes intestinal diseases in humans and livestock through its toxins, related to alpha toxin (CPA), beta toxin (CPB), C. perfringens enterotoxin (CPE), epsilon toxin (ETX), Iota toxin (ITX), and necrotic enteritis B-like toxin (NetB). These toxins disrupt intestinal barrier, leading to various cell death mechanisms such as necrosis, apoptosis, and necroptosis. Additionally, non-toxin factors like adhesins and degradative enzymes contribute to virulence by enhancing colonization and survival of C. perfringens. A vicious cycle of intestinal barrier breach, misregulated cell death, and subsequent inflammation is at the heart of chronic inflammatory and infectious gastrointestinal diseases. Understanding these mechanisms is essential for developing targeted therapies against C. perfringens-associated intestinal diseases.

Injury of Macrophages Induced by Clostridium perfringens Type C Exotoxins.

Clostridium perfringens is a kind of anaerobic Gram-positive bacterium that widely exists in the intestinal tissue of humans and animals. And the main virulence factor in Clostridium perfringens is its exotoxins. Clostridium perfringens type C is the main strain of livestock disease, its exotoxins can induce necrotizing enteritis and enterotoxemia, which lead to the reduction in feed conversion, and a serious impact on breeding production performance. Our study found that treatment with exotoxins reduced cell viability and triggered intracellular reactive oxygen species (ROS) in human mononuclear leukemia cells (THP-1) cells. Through transcriptome sequencing analysis, we found that the levels of related proteins such as heme oxygenase 1 (HO-1) and ferroptosis signaling pathway increased significantly after treatment with exotoxins. To investigate whether ferroptosis occurred after exotoxin treatment in macrophages, we confirmed that the protein expression levels of antioxidant factors glutathione peroxidase 4/ferroptosis-suppressor-protein 1/the cystine/glutamate antiporter solute carrier family 7 member 11 (GPX4/FSP1/xCT), ferroptosis-related protein nuclear receptor coactivator 4/transferrin/transferrin receptor (NCOA4/TF/TFR)/ferritin and the level of lipid peroxidation were significantly changed. Based on the above results, our study suggested that Clostridium perfringens type C exotoxins can induce macrophage injury through oxidative stress and ferroptosis.

Antimicrobial Activity of Synthetic Enterocins A, B, P, SEK4, and L50, Alone and in Combinations, against Clostridium perfringens.

Multidrug-resistant Clostridium perfringens infections are a major threat to the poultry industry. Effective alternatives to antibiotics are urgently needed to prevent these infections and limit the spread of multidrug-resistant bacteria. The aim of the study was to produce by chemical synthesis a set of enterocins of different subgroups of class II bacteriocins and to compare their spectrum of inhibitory activity, either alone or in combination, against a panel of twenty C. perfringens isolates. Enterocins A, P, SEK4 (class IIa bacteriocins), B (unsubgrouped class II bacteriocin), and L50 (class IId leaderless bacteriocin) were produced by microwave-assisted solid-phase peptide synthesis. Their antimicrobial activity was determined by agar well diffusion and microtitration methods against twenty C. perfringens isolates and against other pathogens. The FICINDEX of different combinations of the selected enterocins was calculated in order to identify combinations with synergistic effects. The results showed that synthetic analogs of L50A and L50B were the most active against C. perfringens. These peptides also showed the broadest spectrum of activity when tested against other non-clostridial indicator strains, including Listeria monocytogenes, methicillin-resistant Staphylococcus aureus, Streptococcus suis, Streptococcus pyogenes, Enterococcus cecorum, Enterococcus faecalis, as well as Gram-negative bacteria (Campylobacter coli and Pseudomonas aeruginosa), among others. The selected synthetic enterocins were combined on the basis of their different mechanisms of action, and all combinations tested showed synergy or partial synergy against C. perfringens. In conclusion, because of their high activity against C. perfringens and other pathogens, the use of synthetic enterocins alone or as a consortium can be a good alternative to the use of antibiotics in the poultry sector.

The Effects of Encapsulation on the In Vitro Anti-Clostridial Activity of Olive Mill Wastewater Polyphenolic Extracts: A Promising Strategy to Limit Microbial Growth in Food Systems.

Despite the technologies applied to food production, microbial contamination and chemical deterioration are still matters of great concern. In order to limit these phenomena, new natural approaches should be applied. In this context, the present study aimed to assess the antioxidant and anti-Clostridial effects of two different polyphenolic extracts derived from olive mill vegetation water, one liquid (LE) and one encapsulated (EE). The extracts have been preliminary characterized using Liquid Chromatography Quadrupole Time-Of Flight spectrometry. The Oxygen Radical Absorbance Capacity method was used to determine the antioxidant capacity, registering a higher value for EE compared to that for LE (3256 ± 85 and 2446 ± 13 µgTE/g, respectively). The antibacterial activity against C. perfringens, C. botulinum and C. difficile was studied by the agar well diffusion method, MIC and MBC determination and a time-kill test. The results confirm that EE and LE are able to limit microbial growth, albeit with minor effects when the phenolic compounds are encapsulated. Further studies are needed to evaluate the possible application of these extracts in food systems.