Summary
This study investigated the inhibition of Clostridium species, isolated from cheeses with late‐blowing defects, by lactic acid bacteria (LAB). Four batches of Turkish white cheeses were ...produced; control cheese, cheese sample containing Lactococcus lactis subsp. lactis (PY91K) and Lactiplantibacillus plantarum (Y48) as protective cultures (LAB), a cheese sample containing the protective culture and inoculated with Clostridium sporogenes spores (CL‐LAB) and finally a cheese sample containing only Cl. sporogenes spores (CL). White cheese's microbiological and physicochemical properties were also determined throughout the 90‐day storage period at 4 °C. Approximately 1 log reduction in Cl. sporogenes (ZMP‐1‐4) spores was observed in the LAB co‐inoculated Turkish white cheese samples.
Lactococcus lactis subsp. lactis (PY91K) and Lactiplantibacillus plantarum (Y48) was effective as protective cultures (LAB) to control Cl. sporogenes in Turkish white cheese samples.
Background: Clostridium sporogenes is part of a highly diverse group of Gram positive, spore forming, anaerobic bacteria. C. sporogenes can be used as a delivery vehicle for chemotherapeutics in ...cancer treatment due to the inactive spore form of C. sporogenes only germinating in the microenvironment of the hypoxic tumour. The directed enzyme pro-drug therapy (DEPT) strategy has been tested with several different delivery vectors, and Clostridial DEPT (CDEPT) has previously been developed with one pro-drug converting enzyme (nitroreductase). In this treatment the non-toxic pro-drugs are administered to the patient, then a pro-drug converting enzyme is delivered through genetically altered C. sporogenes. This enzyme breaks the pro-drug down into a toxic component. The aim of this project is to develop the system further with an alternative pro-drug converting enzyme which digests its potential substrate into a 100 fold more toxic subcomponent.Materials and Methods: To efficiently implement the use of this enzyme in the CDEPT strategy, the delivery system needed to be optimised to allow for its effective export and binding to the cell wall of the bacteria to maintain site specificity. To do this, the use of a panel of different signal peptides was investigated. Concurrently to that different sortase signal motifs were identified in Clostridium species through psiBLAST, and their use to anchor the protein to the cell wall was investigated. Successful attachment and protein export was judged by assaying the presence of enzyme activity in the different fractions of bacterial cultures.Conclusion: Results showed an enhanced export of the enzyme into the culture supernatant, and significant cell wall anchoring. This data gives a good basis from which development of an optimized CDEPT system should be achievable, which would result in a highly site specific, hypoxic tumour therapy system.
The antimicrobial activity of blend of essential oils and their nanoemulsions against Clostridium sporogenes inoculated in mortadella with sodium nitrite reduction (75 ppm) was evaluated. In ...addition, physicochemical analysis such as pH, residual nitrite, lipid oxidation, aw and color were realized. Sensory characteristics of mortadella containing essential oils and their nanoemulsions were evaluated through a focus group and control difference test. Nanoemulsions were more effective than individual essential oils in reducing vegetative cells of C. sporogenes. The combinations of essential oils and their nanoemulsions acted as an antioxidant in the meat product and did not modify the other technological characteristics of the product. Although mortadella added with a natural preservative is an attraction, flavor and color variability must be considered. The present study showed good results for the application of essential oil nanoemulsions as a preservative in meat products and encourages further studies in this regard.
•The blends of essential oils were effective against vegetative cells of C. sporogenes.•It was possible to make nanoemulsion from combinations of essential oils.•Nanoemulsions reduced the microbial growth in mortadella.•The blends of essential oils and nanoemulsions were antioxidants in the meat product.
The gut microbiota synthesize hundreds of molecules, many of which influence host physiology. Among the most abundant metabolites are the secondary bile acids deoxycholic acid (DCA) and lithocholic ...acid (LCA), which accumulate at concentrations of around 500 μM and are known to block the growth of Clostridium difficile
, promote hepatocellular carcinoma
and modulate host metabolism via the G-protein-coupled receptor TGR5 (ref.
). More broadly, DCA, LCA and their derivatives are major components of the recirculating pool of bile acids
; the size and composition of this pool are a target of therapies for primary biliary cholangitis and nonalcoholic steatohepatitis. Nonetheless, despite the clear impact of DCA and LCA on host physiology, an incomplete knowledge of their biosynthetic genes and a lack of genetic tools to enable modification of their native microbial producers limit our ability to modulate secondary bile acid levels in the host. Here we complete the pathway to DCA and LCA by assigning and characterizing enzymes for each of the steps in its reductive arm, revealing a strategy in which the A-B rings of the steroid core are transiently converted into an electron acceptor for two reductive steps carried out by Fe-S flavoenzymes. Using anaerobic in vitro reconstitution, we establish that a set of six enzymes is necessary and sufficient for the eight-step conversion of cholic acid to DCA. We then engineer the pathway into Clostridium sporogenes, conferring production of DCA and LCA on a nonproducing commensal and demonstrating that a microbiome-derived pathway can be expressed and controlled heterologously. These data establish a complete pathway to two central components of the bile acid pool.
Amino acid-fermenting Clostridia have undesirable effects in agricultural systems, which can be mitigated by antibiotics, but resistance necessitates alternatives. Here, we demonstrate the efficacy ...of cannabidiol on growth and ammonia inhibition of five agriculturally relevant Clostridia: Clostridium sporogenes, Peptostreptococcus spp., Clostridioides difficile, Acetoanaerobium sticklandii, and Clostridium aminophilum.
•Cannabidiol inhibits the growth of some animal-associated members of Class Clostridia.•Cannabidiol inhibits the growth of Clostridium sporogenes.•Cannabidiol reduces ammonia production of agriculturally important Clostridia.
Despite considerable clinical success, the potential of cancer immunotherapy is restricted by a lack of tumour-targeting strategies. Treatment requires systemic delivery of cytokines or antibodies at ...high levels to achieve clinically effective doses at malignant sites. This is exacerbated by poor penetration of tumour tissue by therapeutic antibodies. High-grade immune-related adverse events (irAEs) occur in a significant number of patients (5-15%, cancer- and therapeutic-dependent) that can lead to lifelong issues and can exclude from treatment patients with pre-existing autoimmune diseases. Tumour-homing bacteria, genetically engineered to produce therapeutics, is one of the approaches that seeks to mitigate these drawbacks. The ability of
Clostridium sporogenes
to form spores that are unable to germinate in the presence of oxygen (typical of healthy tissue) offers a unique advantage over other vectors. However, the limited utility of existing gene editing tools hinders the development of therapeutic strains. To overcome the limitations of previous systems, expression of the Cas9 protein and the gRNA was controlled using tetracycline inducible promoters. Furthermore, the components of the system were divided across two plasmids, improving the efficiency of cloning and conjugation. Genome integrated therapeutic genes were assayed biochemically and in cell-based functional assays. The potency of these strains was further improved through rationally-conceived gene knock-outs. The new system was validated by demonstrating the efficient addition and deletion of large sequences from the genome. This included the creation of recombinant strains expressing two pro-inflammatory cytokines, interleukin-2 (IL-2) and granulocyte macrophage-colony stimulating factor (GM-CSF), and a pro-drug converting enzyme (PCE). A comparative, temporal
in vitro
analysis of the integrant strains and their plasmid-based equivalents revealed a substantial reduction of cytokine activity in chromosome-based constructs. To compensate for this loss, a 7.6 kb operon of proteolytic genes was deleted from the genome. The resultant knock-out strains showed an 8- to 10-fold increase in cytokine activity compared to parental strains.
Although it has long been recognized that the enteric community of bacteria that inhabit the human distal intestinal track broadly impacts human health, the biochemical details that underlie these ...effects remain largely undefined. Here, we report a broad MS-based metabolomics study that demonstrates a surprisingly large effect of the gut "microbiome" on mammalian blood metabolites. Plasma extracts from germ-free mice were compared with samples from conventional (conv) animals by using various MS-based methods. Hundreds of features were detected in only 1 sample set, with the majority of these being unique to the conv animals, whereas almost equal to10% of all features observed in both sample sets showed significant changes in their relative signal intensity. Amino acid metabolites were particularly affected. For example, the bacterial-mediated production of bioactive indole-containing metabolites derived from tryptophan such as indoxyl sulfate and the antioxidant indole-3-propionic acid (IPA) was impacted. Production of IPA was shown to be completely dependent on the presence of gut microflora and could be established by colonization with the bacterium Clostridium sporogenes. Multiple organic acids containing phenyl groups were also greatly increased in the presence of gut microbes. A broad, drug-like phase II metabolic response of the host to metabolites generated by the microbiome was observed, suggesting that the gut microflora has a direct impact on the drug metabolism capacity of the host. Together, these results suggest a significant interplay between bacterial and mammalian metabolism.
Microwave processing has been a promising technology in improving quality and ensuring microbial safety of food products, while non-thermal effects of microwave is in controversy. The objective of ...this study was to investigate the non-thermal effects of microwave electromagnetic (EM) fields on Clostridium Sporogenes (surrogate of Clostridium botulinum) inoculated in salmon fillets using same time-temperature profiles method. The inoculated salmon fillets were processed by a single-mode pilot microwave processing system with different radiation time (2, 3 and 4 min) and holding time (0, 2 and 4 min). Corresponding water bath treatments were designed for each microwave processing to obtain matching time-temperature profiles. Results showed that the same time-temperature profiles method was successfully achieved to evaluate the microwave non-thermal effects in salmon fillets. With equal or lower thermal lethality value, microwave processing brought significantly higher (P < 0.05) reduction values than water bath treatment. This determined the non-thermal effects of microwave EM fields. And the non-thermal effects increased with longer microwave radiation time and was intensified by higher temperature. However, the following holding section will weaken the additional inactivation efficiency of microwave non-thermal effects. Results suggested that microwave processing could provide better bacteria control with lower thermal processing level.
•Non-thermal effects of microwave on bacterial in salmon fillets were investigated.•Same time-temperature profiles method was used to estimate non-thermal effects.•Microwave non-thermal effects brought additional inactivation efficiency on bacteria.•Microwave processing provided better bacteria control due to non-thermal effects.
The study explored the effects of electric field strength, temperature, and frequency on the inactivation of Clostridium sporogenes PA3679 spores (a surrogate of Clostridium botulinum) within buffer. ...The experiments were conducted at three different field strengths (30, 40, and 50 V/cm), temperatures (110, 120, and 130 °C), and frequencies (60, 1000, and 5000 Hz). Ohmic heating resulted in significantly greater reduction of C. sporogenes compared to conventional heating. Electric field strength significantly influenced spore inactivation. It was found that lower frequencies led to quicker rates of inactivation. Given that high field strengths have dual benefits of increasing both heating and spore kill rate, the possibility exists to process foods to sterility without a hold. This is expected to result in improved product quality, reduced process times and increased efficiency.
•Ohmic heating results in higher bacterial spore inactivation than conventional heating.•This study also confirms the existence of non-thermal effects of ohmic heating.•Higher field strength offered significantly higher inactivation of spores.•60Hz and 5000 Hz offered highest and lowest inactivation of Clostridium sporogenes.
Late blowing is a type of microbiological spoilage that causes unwanted changes in the taste, odour and texture of cheese due to the metabolic activities of Clostridium contaminating raw milk. This ...study aimed to develop a multiplex quantitative real-time polymerase chain reaction (PCR) analysis method for the rapid and simultaneous detection of Clostridium butyricum, Clostridium sporogenes and Clostridium tyrobutyricum using specific primers and probes. The optimised method was determined to work with high sensitivity and specificity to quantify late-blowing Clostridium species in cheese samples. The lowest detectable gene copy number was 4.9 × 101, 7.8 × 101 and 8.5 × 101 for C. tyrobutyricum, C. butyricum and C. sporogenes, respectively. These results demonstrated that this method is a powerful tool for detecting and quantifying late-blowing agents in cheese. This is also the first multiplex qPCR study involving C. butyricum for detecting Clostridia, a late-blowing agent in cheese.
•Late-blowing is one of the most important problems of cheese producers.•The culture method does not provide quantitative results at species level.•This qPCR method was able to detect Clostridium species with high sensitivity.•This assay has low detection limit ̴ log10 1.0 CFU/mL.•This assay has the potential as a molecular tool for analyses cheese by rapidly.
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