Pathogen Clostridium Perfringens Research Articles

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.

Application of Hazard Analysis Critical Control Point (HACCP) principles to beef mortadella production

A mortadella is a cooked sausage made from raw pork, beef, chicken or lamb. It is popular in Jordan and neighboring countries for its taste, texture, nutritional value and ease of inclusion in sandwiches. The product is sold in plastic packaging, in circular rolls or vacuum packed. Although it poses no serious threat to human health, the heat can be dangerous if used improperly. A HACCP system is used to manage the production process, with a focus on quality control (CCP) throughout the manufacturing phase. Safety risks are identified at all stages of production, from raw materials to storage and distribution. Foodborne illnesses are primarily caused by pathogenic microorganisms, and these raw materials include pathogenic plant cells, Bacillus cereus, Clostridium botulinum and Clostridium perfringens that survive after cooking and contamination detected after monitoring. Post-manufacturing controls are performed through Good Manufacturing Practices (GMPs). The objectives of the study were to identify potential hazards associated with the production of beef mortadella, determine the CCPs to be implemented, develop a monitoring plan and establish corrective action on a case-by-case basis. The HACCP plan was developed in collaboration with local stakeholders and follows a process flow diagram to identify potential hazards and preventive measures. The system includes the steps for obtaining and storing the ingredients, preparing the meat, adding non-meat ingredients, and transferring the raw mixture to an incubator. The meat is weighed and filled with the mixture to improve cleanliness and avoid excess nitrite. The storage time between incubation and cooking is controlled to allow bacteria to multiply. To kill plant pathogens and reduce the total number of microorganisms, the product is cooked in a steam oven at 75°C for 5 minutes. The aim of the cooking process is to reduce microbial growth before lethal temperatures have been reached and uniform heating of the product has been ensured. Cooking suitability is checked by thermographic records and visual inspection of the cooked product. The plan also includes personnel in charge of every stage of production, ensuring a clean and hygienic environment. Key words: HACCP, Mortadella, meat processing, hygiene control, food safety

Occurrence of 40 sanitary indicators in French digestates derived from different anaerobic digestion processes and raw organic wastes from agricultural and urban origin.

This study investigated the sanitary quality of digestates resulting from the mesophilic anaerobic digestion (AD) of urban and agricultural organic wastes (OWs). 40 sanitary indicators, including pathogenic bacteria, antimicrobial resistance genes, virulence factor genes, and mobile genetic elements were evaluated using real-time PCR and/or droplet digital PCR. 13 polycyclic aromatic hydrocarbons (PAHs) and 13 pharmaceutical products (PHPs) were also measured. We assessed agricultural OWs from three treatment plants to study the effect of different AD processes (feeding mode, number of stages, pH), and used three laboratory-scale reactors to study the effect of different feed-supplies (inputs). The lab-scale reactors included: Lab1 fed with 97% activated sludge (urban waste) and 3% cow manure; Lab2 fed with 85% sludge-manure mixture supplemented with 15% wheat straw (WS); and Lab3 fed with 81% sludge-manure mixture, 15% WS, and 4% zeolite powder. Activated sludge favored the survival of the food-borne pathogens Clostridium perfringens and Bacillus cereus, carrying the toxin-encoding genes cpe and ces, respectively. Globally, the reactors fed with fecal matter supplemented with straw (Lab2) or with straw and zeolite (Lab3) had a higher hygienization efficiency than the reactor fed uniquely with fecal matter (Lab1). Three pathogenic bacteria (Enterococcus faecalis, Enterococcus faecium, and Mycobacterium tuberculosis complex), a beta-lactam resistance gene (bla TEM), and three mobile genetic elements (intI1, intI2, and IS26) were significantly decreased in Lab2 and Lab3. Moreover, the concentrations of 11 PAHs and 11 PHPs were significantly lower in Lab2 and Lab3 samples than in Lab1 samples. The high concentrations of micropollutants, such as triclosan, found in Lab1, could explain the lower hygienization efficiency of this reactor. Furthermore, the batch-fed reactor had a more efficient hygienization effect than the semi-continuous reactors, with complete removal of the ybtA gene, which is involved in the production of the siderophore yersiniabactin, and significant reduction of intI2 and tetO. These data suggest that it is essential to control the level of chemical pollutants in raw OWs to optimize the sanitary quality of digestates, and that adding co-substrate, such as WS, may overcome the harmful effect of pollutants.

Absent in Melanoma 2 Mediates Inflammasome Signaling Activation against Clostridium perfringens Infection.

Absent in melanoma 2 (AIM2), a key component of the IFI20X/IFI16 (PYHIN) protein family, is characterized as a DNA sensor to detect cytosolic bacteria and DNA viruses. However, little is known about its immunological role during pathogenic Clostridium perfringens (C. perfringens) infection, an extracellular bacterial pathogen. In a pathogenic C. perfringens gas gangrene model, Aim2-/- mice are more susceptible to pathogenic C. perfringens soft tissue infection, revealing the importance of AIM2 in host protection. Notably, Aim2 deficiency leads to a defect in bacterial killing and clearance. Our in vivo and in vitro findings further establish that inflammasome signaling is impaired in the absence of Aim2 in response to pathogenic C. perfringens. Mechanistically, inflammasome signaling downstream of active AIM2 promotes pathogen control. Importantly, pathogenic C. perfringens-derived genomic DNA triggers inflammasome signaling activation in an AIM2-dependent manner. Thus, these observations uncover a central role for AIM2 in host defense and triggering innate immunity to combat pathogenic C. perfringens infections.

Comparative prebiotic potential of galacto- and fructo-oligosaccharides, native inulin, and acacia gum in Kenyan infant gut microbiota during iron supplementation.

Iron fortification to prevent anemia in African infants increases colonic iron levels, favoring the growth of enteropathogens. The use of prebiotics may be an effective strategy to reduce these detrimental effects. Using the African infant PolyFermS gut model, we compared the effect of the prebiotics short-chain galacto- with long-chain fructo-oligosaccharides (scGOS/lcFOS) and native inulin, and the emerging prebiotic acacia gum, a branched-polysaccharide-protein complex consisting of arabinose and galactose, during iron supplementation on four Kenyan infant gut microbiota. Iron supplementation did not alter the microbiota but promoted Clostridioides difficile in one microbiota. The prebiotic effect of scGOS/lcFOS and inulin was confirmed during iron supplementation in all investigated Kenyan infant gut microbiota, leading to higher abundance of bifidobacteria, increased production of acetate, propionate, and butyrate, and a significant shift in microbiota composition compared to non-supplemented microbiota. The abundance of the pathogens Clostridium difficile and Clostridium perfringens was also inhibited upon addition of the prebiotic fibers. Acacia gum had no effect on any of the microbiota. In conclusion, scGOS/lcFOS and inulin, but not acacia gum, showed a donor-independent strong prebiotic potential in Kenyan infant gut microbiota. This study demonstrates the relevance of comparing fibers in vitro prior to clinical studies.

Moringa oleifera as a potential antimicrobial against pathogenic Clostridium perfringens isolates in farm animals.

Clostridium perfringens (CP) is an emerging anaerobic pathogen that can aggravate severe fatal infections in different hosts and livestock. This paper was designed to monitor the antibacterial efficacy of Moringa oleifera (M. oleifera) plant against different CP isolates of variant toxin genotypes comparing that with commercial antibiotics in the veterinary field. A total of 200 examined fecal, intestinal, and liver samples from cattle, sheep, and goats were investigated bacteriologically and biochemically for CP. Then, the isolates were examined by polymerase chain reaction (PCR) for toxin gene typing. Thereafter, the antimicrobial susceptibility testing as well as the antibacterial efficacy of M. oleifera were evaluated and statistically analyzed against recovered isolates. The prevalence rate of CP was 51% (102/200); of which 54.5% was from cattle, 50% from sheep, and 40% from goat. Moreover, all CP isolates were highly resistant to tetracycline and lincomycin drugs; meanwhile, they were of the least resistance against ciprofloxacin (8.3%-16.7%), cefotaxime (16.7%-25%), and gentamycin (26.7%-33.3%). For M. oleifera, high antibacterial efficacy with greater inhibition zones of the plant was recorded with its oil (20-24 mm) and ethanolic extracts (16-20 mm) against CP than the aqueous extract (≤ 10 mm). A good correlation was stated between M. oleifera oil and toxin type of CP isolates particularly type A followed by D and B types. Interestingly, the oil and ethanolic extracts of M. oleifera gave higher antibacterial efficacy than most commercial antibiotics against the recovered isolates. This study highlighted the potent antibacterial properties of M. oleifera for suppressing CP isolated from farm animals; hence, more investigations on M. oleifera are suggested to support its use as a medical herbal plant substituting antibiotics hazards and resistance problems worldwide.

Identification of <em>Clostridium perfringens</em> and <em>Fusobacterium necrophorum</em> isolates

The article is devoted to the differential diagnosis of strains of Clostridium perfringens and Fusobacterium necrophorum in the studied material of cattle. The research topic is relevant, since rational, timely and accurate diagnosis of the etiology of infectious diseases in cattle in the conditions of industrial cattle breeding prevents the occurrence and transmission of infectious agents, thereby ensuring cost-effective uninterrupted production of highly nutritious products and raw materials. Timely and accurate diagnosis allows veterinarians to prescribe an effective treatment and prevention plan to prevent the occurrence and spread of infectious diseases in cattle, including intestinal diseases [2,6]. One of the causes of hemorrhagic enteritis, reduced productivity and mortality from 25% of young animals may be enterotoxemia associated with toxin-producing strains of Clostridium perfringens. Differential diagnosis is an obligatory step in a comprehensive diagnosis, taking into account epizootological data, clinical signs of sick and conditionally sick animals, pathoanatomical data of forcedly killed and dead animals, laboratory diagnostic methods, including bacteriological and molecular genetic methods. As a result of the study, optimal methods of laboratory diagnostics were applied, including bacterioscopy, the method of pure cultures and polymerase chain reaction (PCR), which made it possible to identify strains of Fusobacterium necrophorum and Clostridium perfringens. Accurate and rapid identification of pathogens Fusobacterium necrophorum and Clostridium perfringens will allow veterinarians to develop a plan of treatment and preventive measures in a short time.

Utilization of Porcine Livers through the Formation of Zn-Protoporphyrin Pigment Optimized by a Response Surface Methodology.

There is a growing demand for clean-label products. This study aimed to obtain a food-grade coloring ingredient for meat products based on the formation of Zn-protoporphyrin from porcine livers, thus contributing to the development of nitrite-free products. First, the effects of sodium disulfite and acetic, ascorbic, and lactic acids on the formation of Zn-protoporphyrin and the total microbial count were studied. The combination of ascorbic and acetic acids resulted in a higher Zn-protoporphyrin content than acetic acid alone, and microbial levels were maintained (ca. 3 log CFU/mL). Second, a response surface methodology was used to maximize Zn-protoporphyrin while maintaining microbiological food standards. To that end, the effects of pH (4.2-5.4), incubation time (3-30 h), and temperature (25-50 °C) were studied. The selected conditions for Zn-protoporphyrin formation involved anaerobic incubation at pH 4.8 and 45 °C for 24 h. The safety was validated through challenge testing for relevant pathogens (Listeria monocytogenes, Salmonella spp., and Clostridium perfringens). A significant reduction (>6 log units) was observed in the selected conditions for L. monocytogenes and Salmonella, whereas C. perfringens spores remained at the inoculated levels. The optimized procedure is proven to be microbiologically safe, and may improve the color of nitrite-free meat products.

Clostridium perfringens virulence factors are nonredundant activators of the NLRP3 inflammasome

Inflammasome signaling is a central pillar of innate immunity triggering inflammation and cell death in response to microbes and danger signals. Here, we show that two virulence factors from the human bacterial pathogen Clostridium perfringens are nonredundant activators of the NLRP3 inflammasome in mice and humans. C. perfringens lecithinase (also known as phospolipase C) and C. perfringens perfringolysin O induce distinct mechanisms of activation. Lecithinase enters LAMP1+ vesicular structures and induces lysosomal membrane destabilization. Furthermore, lecithinase induces the release of the inflammasome‐dependent cytokines IL‐1β and IL‐18, and the induction of cell death independently of the pore‐forming proteins gasdermin D, MLKL and the cell death effector protein ninjurin‐1 or NINJ1. We also show that lecithinase triggers inflammation via the NLRP3 inflammasome in vivo and that pharmacological blockade of NLRP3 using MCC950 partially prevents lecithinase‐induced lethality. Together, these findings reveal that lecithinase activates an alternative pathway to induce inflammation during C. perfringens infection and that this mode of action can be similarly exploited for sensing by a single inflammasome.

TcpA from the Clostridium perfringens plasmid pCW3 is more closely related to the DNA translocase FtsK than to coupling proteins

Conjugative DNA transfer is a major factor in the dissemination of antibiotic resistance and virulence genes. In the Gram-positive pathogen Clostridium perfringens, the majority of conjugative plasmids share the conserved tcp locus that governs the assembly of the transfer system. Here, we describe multiple structures of the coupling protein TcpA, an essential ATPase that is suggested to provide the mechanical force to propel the DNA through the transfer apparatus. The structures of TcpA in the presence and absence of nucleotides revealed conformational rearrangements and highlight a crucial role for the unstructured C terminus. Our findings reveal that TcpA shares most structural similarity with the FtsK DNA translocase, a central component of the bacterial cell division machinery. Our structural data suggest that conjugation in C.perfringens may have evolved from the bacterial chromosome segregation system and, accordingly, suggest the possibility that double-stranded DNA is transferred through the Tcp conjugation apparatus.

Antimicrobial Properties of Bacillus Probiotics as Animal Growth Promoters.

Antibiotic growth promoters (AGPs) suppress the growth of infectious pathogens. These pathogens negatively impact agricultural production worldwide and often cause health problems if left untreated. Here, we evaluate six Bacillus strains (BPR-11, BPR-12, BPR-13, BPR-14, BPR-16 and BPR-17), which are known for their ability to survive harsh environmental conditions, as AGP replacements in animal feed. Four of these Bacillus strains (BPR-11, BPR-14, BPR-16 and BPR-17) showed antimicrobial activity against the pathogenic strains Clostridium perfringens, Escherichia coli and Staphylococcus aureus at 25 μg/mL, with BPR-16 and BPR-17 also able to inhibit Pseudomonas aeruginosa and Salmonella enterica at 100 μg/mL. Further chemical investigation of BPR-17 led to the identification of eight metabolites, namely C16, C15, C14 and C13 surfactin C (1-4), maculosin (5), maculosine 2 (6), genistein (7) and daidzein (8). Purified compounds (1-4) were able to inhibit all the tested pathogens with MIC values ranging from 6.25 to 50 μg/mL. Maculosin (5) and maculosine 2 (6) inhibited C. perfringens, E. coli and S. aureus with an MIC of 25 μg/mL while genistein (7) and daidzein (8) showed no activity. An animal trial involving feeding BPR-11, BPR-16 and BPR-17 to a laboratory poultry model led to an increase in animal growth, and a decrease in feed conversion ratio and mortality. The presence of surfactin C analogues (3-4) in the gut following feeding with probiotics was confirmed using an LC-MS analysis. The investigation of these Bacillus probiotics, their metabolites, their impacts on animal performance indicators and their presence in the gastrointestinal system illustrates that these probiotics are effective alternatives to AGPs.

Battling Enteropathogenic Clostridia: Phage Therapy for Clostridioides difficile and Clostridium perfringens.

The pathogenic Clostridioides difficile and Clostridium perfringens are responsible for many health care-associated infections as well as systemic and enteric diseases. Therefore, they represent a major health threat to both humans and animals. Concerns regarding increasing antibiotic resistance (related to C. difficile and C. perfringens) have caused a surge in the pursual of novel strategies that effectively combat pathogenic infections, including those caused by both pathogenic species. The ban on antibiotic growth promoters in the poultry industry has added to the urgency of finding novel antimicrobial therapeutics for C. perfringens. These efforts have resulted in various therapeutics, of which bacteriophages (in short, phages) show much promise, as evidenced by the Eliava Phage Therapy Center in Tbilisi, Georgia (https://eptc.ge/). Bacteriophages are a type of virus that infect bacteria. In this review, the (clinical) impact of clostridium infections in intestinal diseases is recapitulated, followed by an analysis of the current knowledge and applicability of bacteriophages and phage-derived endolysins in this disease indication. Limitations of phage and phage endolysin therapy were identified and require considerations. These include phage stability in the gastrointestinal tract, influence on gut microbiota structure/function, phage resistance development, limited host range for specific pathogenic strains, phage involvement in horizontal gene transfer, and—for phage endolysins—endolysin resistance, -safety, and -immunogenicity. Methods to optimize features of these therapeutic modalities, such as mutagenesis and fusion proteins, are also addressed. The future success of phage and endolysin therapies require reliable clinical trial data for phage(-derived) products. Meanwhile, additional research efforts are essential to expand the potential of exploiting phages and their endolysins for mitigating the severe diseases caused by C. difficile and C. perfringens.

Antibacterial and cytotoxic evaluation of sequential extract of Moringa oleifera leaves

Moringa Oleifera is an interesting plant used in Asian traditional medicine. In this study, in vitro antibacterial and cytotoxic evaluation of sequential extracts (aqueous, ethanol, and chloroform) of Moringa Oleifera was carried out. The antibacterial analysis was estimated with minimum inhibitory concentration (MIC) by micro dilution of Moringa Oleifera against common poultry pathogens Clostridium perfringens type A and Escherichia coli, while cytotoxic evaluation was estimated by the reduction in cell viability due to apoptosis or necrosis by metabolic events in the presence and absence of crude extracts or tested component. The aqueous extract shows the highest percentage yield (45/50gm) succeeded by ethanol extract (5.5gm/50gm) and chloroform extract (0.2gm/50gm). In our study, the zone of inhibition of sequential extracts of Moringa Oleifera against Haemophilus species are highest for chloroform (17mm), intermediate for ethanol (13mm), and lowest for aqueous extract (12.3mm). For chloroform extract the CSP was calculated at 10 different concentrations, 2000 µg/ml, 1000µg/ml, 500µg/ml, 250µg/ml, 125µg/ml, 62.5µg/ml, 31.25µg/ml, 15.63µg/ml, 7.81µg/ml and 3.91µg/ml .The results for cell survival percentage (CSP) in the present research are 26%, 46%, 58%, 55%, 60%, 63%, 62%, 59%, 68% and 82% respectively. The CSP results of chloroform extract indicated that it is toxic for cells at ≥1000µg/ml. At 1000µg/ml concentration CSP was 46% which is > 50% and therefore it is cytotoxic. At higher concentrations, chloroform is more cytotoxic than hexane because at > 1000µg/ml the cell survival percentage was recorded to be < 50%. For ethanol extract CSP was calculated at 10 concentrations, 6000µg/ml, 3000µg/ml, 1500µg/ml, 750µg/ml, 375µg/ml, 187.5µg/ml, 93.75µg/ml, 46.85µg/ml, 23.43µg/ml and 11.71µg/ml. The CSP values are 18%, 48%, 60%, 58%, 69%, 56%, 59%, 74%, 57% and 78% respectively which indicate that at concentrations ≥3000µg/ml the chloroform extract is toxic for cells. At a concentration less than 3000µg/ml, the CSP is more than 50%. So, as compared to hexane and chloroform, ethanol extract is less toxic at higher concentrations. Cytotoxicity of aqueous extract was calculated at 10 concentrations, 5000µg/ml, 2500µg/ml, 1250µg/ml, 625µg/ml, 312.5µg/ml, 156.25µg/ml, 78.125µg/ml, 39.06µg/ml, 19.53µg/ml and 9.76µg/ml. The CSP values are 8%, 18%, 42%, 56%, 54%, 59%, 55%, 62% 59% and 66% respectively. At a concentration ≥625µg/ml, the aqueous extract is toxic for cells. The CSP at 625µg/ml is 42%, hence toxic for cells. The cell survival percentage is more than 50% at a concentration > 625µg/ml, indicating that aqueous extract is more toxic to the cell than the rest of the three (Hexane, Chloroform, Ethanol extracts) at higher concentrations.

Mechanical limitations of bacterial motility mediated by growing cell chains

Many bacterial species are able to expedite colony expansion through active motility of the cells. A unique mode of colony expansion on a substrate surface recently discovered in a pathogenic bacterium Clostridium perfringens, surprisingly, does not depend on motility in single cells. Specifically, daughter cells maintain end-to-end connections after cell division, amounting to long chains of cells that continuously elongate as individual cells grow and divide. In principle, such cell growth-driven motility would accelerate as cell divisions increase the number of cells in a chain, and the tip of a long chain could potentially reach a very high speed.

Effects of feeding diets containing increasing content of purified lignocellulose supplied by sugarcane bagasse to early-weaned pigs on growth performance and intestinal health

A total of 96 weaned piglets, with mean age of 21 days and initial body weight (BW) of 6.67 ± 0.63 kg, were used in a 42-day experiment to evaluate the effects of supplementation of dietary purified lignocellulose (LNC) on growth performance and intestinal health. Piglets were blocked by initial BW and allotted to one of 32 pens, each pen containing three pigs. Dietary treatments consisted of a corn-soybean meal-based control diet with no added LNC and three dietary inclusion rates (10, 20, and 30 g/kg) of LNC. Average daily feed intake (ADFI), average daily gain (ADG), and gain-to-feed ratio (G:F) were determined over 42 days postweaning. On day 14 postweaning, one pig from each pen was euthanized to determine gastrointestinal tract organ weight, small intestinal morphology, and digesta pH, viscosity, fermentation end-products, and bacterial enumeration. Total tract retention time was measured by adding 10 g/kg ferric oxide in the morning feed on day 42. From day 1–14 postweaning, G:F tended to linearly increase (P = 0.088) as the dietary LNC contents increased. Although increasing the dietary LNC supplementation quadratically increased (P = 0.008) ADFI and tended to quadratically decrease (P = 0.072) G:F from day 30–42 postweaning, overall growth performance was unaffected by the treatments. The weight of the small intestine adjusted for kg of BW on day 14 postweaning increased quadratically (P = 0.041) as the dietary LNC contents increased. Increasing the dietary LNC supplementation linearly increased (P = 0.027) crypt depth and tended to linearly reduce (P = 0.075) villus:crypt ratio in the jejunum. Dietary treatments did not affect total tract retention time as well as pH and viscosity of both cecum and colonic digesta. Increasing the dietary LNC supplementation quadratically increased (P = 0.001) cecal butyrate formation, whereas cecal acetate and propionate concentrations did not differ among treatments. Although ileal counts of Lactobacillus spp. and Escherichia coli were similar among treatments, the pathogenic Clostridium perfringens population exhibited a quadratic decrease (P = 0.041) as the dietary LNC contents increased. These results indicate that feeding pig diets containing up to 30 g/kg purified LNC enhanced gut health through the inhibition of harmful bacteria proliferation and the expansion of butyrate producers, without compromising overall growth performance.

Occurrence of Clostridium perfringens vegetative cells and spores throughout an industrial production process of black soldier fly larvae (Hermetia illucens)

The main use of black soldier fly larvae (Hermetia illucens) is currently as an animal feed ingredient. While the bacterial community of the larvae has been characterised repeatedly via sequencing, microbiological safety assessment based on culture-dependent techniques is still scarce. This study focused on the occurrence of the spore-forming foodborne pathogen Clostridium perfringens during rearing and consecutive processing of the larvae, based on observations in a single rearing facility. C. perfringens vegetative cells and spores were determined, in addition to total viable counts, total aerobic spore counts and intrinsic parameters including pH, water activity and moisture content. All samples were obtained from an industrial production plant. In a preliminary experiment, substrate ingredients and dried larvae were analysed, but the larvae were produced with a previous batch of the substrate mixture. A second, more detailed, experiment was performed where all samples were collected sequentially from the same production run (substrate ingredients, substrate mixture, starting larvae, harvested larvae, residue, dried larvae and stored dried larvae). In the two experiments, (presumptive) C. perfringens, as determined on tryptose sulphite cycloserine agar, was found at low numbers in the ingredients and in the second experiment it was also found in the substrate mixture. Over the two experiments, total C. perfringens counts (i.e. vegetative cells plus spores) ranged between 3.0±0.1 and <1.2±0.5 log cfu/g and C. perfringens spores ranged between 2.5±0.1 and <1.0±0.0 log cfu/g. Interestingly, vegetative cells and spores of C. perfringens were below the detection limit in all larvae samples. Therefore, it appears that at this production site and based on the samples investigated, the pathogen did not colonise the larvae. However, these results indicate that insect producers should monitor this pathogen among others, and install good hygiene practices to avoid contamination.

Identification of an Intestinal Microbiota Signature Associated With the Severity of Necrotic Enteritis.

Necrotic enteritis (NE), an economically devastating disease of poultry caused by pathogenic Clostridium perfringens, is known to induce small intestinal lesions and dysbiosis. However, the intestinal microbes that are associated with NE severity are yet to be characterized. Here, we investigated the link between the ileal microbiota and disease severity in a chicken model of clinical NE using 16S rRNA gene sequencing. Our results indicated that richness and Shannon Index of the ileal microbiota were drastically reduced (p<0.01) as NE was exacerbated. While the relative abundance of C. perfringens increased from 0.02% in healthy chickens to 58–70% in chickens with severe infection, a majority of the ileal microbes were markedly diminished, albeit varying in their sensitivity to NE. Compositionally, a large group of ileal microbes showed a significant correlation with NE severity. Firmicutes, such as group A and B Lactobacillus, Lactobacillus reuteri, Subdoligranulum variabile, Mediterraneibacter, and Staphylococcus as well as two genera of Actinobacteria (Corynebacterium and Kocuria) and two highly related Cyanobacteria were progressively declined as NE was aggravated. Other Firmicutes, such as Weissella, Romboutsia, Kurthia, Cuneatibacter, Blautia, and Aerococcus, appeared much more sensitive and were rapidly abolished in chickens even with mild NE. On the other hand, Enterococcus cecorum and two Escherichia/Shigella species were only enriched in the ileal microbiota of chickens with extremely severe NE, while several other species such as Streptococcus gallolyticus and Bacteroides fragilis remained unaltered by NE. Functionally, secondary bile acid biosynthesis was predicted to be suppressed by NE, while biosynthesis of aromatic and branched-amino acids and metabolism of a majority of amino acids were predicted to be enhanced in the ileum of NE-afflicted chickens. These intestinal microbes showing a strong correlation with NE severity may provide important leads for the development of novel diagnostic or therapeutic approaches to NE and possibly other enteric diseases.

Prebiotic Effects of a Cranberry Beverage in a Randomized, Placebo-Controlled, Crossover Clinical Trial

ObjectivesThe objective was to evaluate the prebiotic effects of a milled whole cranberry beverage on modulating the gut microbiota in young adults. MethodsAdults (n = 17; ages 18–42 y; BMI 30.5 ± 3.1 kg/m2) were enrolled in a 60-d, two-period, randomized, placebo-controlled, crossover clinical study. Throughout the study, participants were fed a standardized 10-d cycle menu on site. During each 20-d treatment period, participants consumed twice daily a whole cranberry or placebo beverage (240 mL per serving). Treatment periods were separated by an 11-wk washout period and preceded by 10-d run-in periods on the controlled study diet. Fecal samples were collected before and after the dietary intervention for bacterial compositional analysis and short-chain fatty acid analysis by LC-MS/MS. The V5-V6 region of the 16S rRNA gene in fecal DNA was amplified and sequenced. Taxonomy was assigned using the q2-feature-classifier in QIIME2 and matched against the Greengenes 13_8 database. Differential abundance was analyzed using ANCOM2 in R. Alpha-diversity was assessed using Faith’s PD, Shannon diversity, and observed OTU richness generated by QIIME 2 and compared between treatments using Mann-Whitney U test. Beta-diversity was compared between treatments using PERMANOVA of the weighted and unweighted UniFrac distances between samples generated by QIIME 2. ResultsCoriobacteriaceae was significantly more abundant after participants consumed the cranberry as compared with the placebo beverage (ANCOM W > 0.7). The clinically-important pathogen Clostridium perfringens was present after consumption of the placebo beverage, but was a structural zero (not present) after consumption of the cranberry beverage. Alpha-diversity, beta-diversity, and fecal short-chain fatty acid concentrations did not differ between treatments. ConclusionsDaily consumption of a whole cranberry beverage resulted in favorable change in the composition of the gut microbiota and thus showed prebiotic potential. Funding SourcesOcean Spray Cranberries, Inc.

Essential oil combinations against Clostridium perfringens and Clostridium septicum - the causative agents of gas gangrene.

The inhibitory and bactericidal effect of a wide range of essential oils, and their selected combinations against two pathogens (Clostridium perfringens and Clostridium septicum), the causative pathogens of gas gangrenous infections were investigated. Fractional inhibitory indices were also calculated to determine the interactions. The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) assays were used to determine the efficacy of the essential oils. Santalum austrocaledonicum demonstrated the highest activity inhibiting both Clostridial pathogens at the lowest concentration of 0·02mgml-1 . Santalum austrocaledonicum combined with Cymbopogon martinii had the strongest inhibition against C. perfringens (MIC 0·02mgml-1 ) and C. septicum (MIC 0·01mgml-1 ). Selected combinations demonstrated synergy (ΣFIC≤0·50) in combination against both pathogens tested. Antagonism was also observed in many combinations. Selected essential oils, when studied either individually or in combination, have high inhibitory and bactericidal effects against both Clostridial strains. Nine combinations have proven to be synergistic with 23 combinations additive; 96 indifferent and 77 having an antagonistic effect against the pathogenic strains. Some combinations demonstrated extreme antagonism and as such, careful consideration needs to be given to essential oil selection against these pathogens. Very few essential oils have been antimicrobially screened (MIC and MBC) against Clostridial strains and furthermore, the efficacies in combination are not known.

Identification of the Clostridial cellulose synthase and characterization of the cognate glycosyl hydrolase, CcsZ.

Biofilms are community structures of bacteria enmeshed in a self-produced matrix of exopolysaccharides. The biofilm matrix serves numerous roles, including resilience and persistence, making biofilms a subject of research interest among persistent clinical pathogens of global health importance. Our current understanding of the underlying biochemical pathways responsible for biosynthesis of these exopolysaccharides is largely limited to Gram-negative bacteria. Clostridia are a class of Gram-positive, anaerobic and spore-forming bacteria and include the important human pathogens Clostridium perfringens, Clostridium botulinum and Clostridioides difficile, among numerous others. Several species of Clostridia have been reported to produce a biofilm matrix that contains an acetylated glucan linked to a series of hypothetical genes. Here, we propose a model for the function of these hypothetical genes, which, using homology modelling, we show plausibly encode a synthase complex responsible for polymerization, modification and export of an O-acetylated cellulose exopolysaccharide. Specifically, the cellulose synthase is homologous to that of the known exopolysaccharide synthases in Gram-negative bacteria. The remaining proteins represent a mosaic of evolutionary lineages that differ from the described Gram-negative cellulose exopolysaccharide synthases, but their predicted functions satisfy all criteria required for a functional cellulose synthase operon. Accordingly, we named these hypothetical genes ccsZABHI, for the Clostridial cellulose synthase (Ccs), in keeping with naming conventions for exopolysaccharide synthase subunits and to distinguish it from the Gram-negative Bcs locus with which it shares only a single one-to-one ortholog. To test our model and assess the identity of the exopolysaccharide, we subcloned the putative glycoside hydrolase encoded by ccsZ and solved the X-ray crystal structure of both apo- and product-bound CcsZ, which belongs to glycoside hydrolase family 5 (GH-5). Although not homologous to the Gram-negative cellulose synthase, which instead encodes the structurally distinct BcsZ belonging to GH-8, we show CcsZ displays specificity for cellulosic materials. This specificity of the synthase-associated glycosyl hydrolase validates our proposal that these hypothetical genes are responsible for biosynthesis of a cellulose exopolysaccharide. The data we present here allowed us to propose a model for Clostridial cellulose synthesis and serves as an entry point to an understanding of cellulose biofilm formation among class Clostridia.