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    Culture and genome-based analysis of four soil Clostridium isolates reveal their potential for antimicrobial production
    (BioMed Central Ltd, 2021-12) Pahalagedara ASNW; Jauregui R; Maclean P; Altermann E; Flint S; Palmer J; Brightwell G; Gupta TB
    BACKGROUND: Soil bacteria are a major source of specialized metabolites including antimicrobial compounds. Yet, one of the most diverse genera of bacteria ubiquitously present in soil, Clostridium, has been largely overlooked in bioactive compound discovery. As Clostridium spp. thrive in extreme environments with their metabolic mechanisms adapted to the harsh conditions, they are likely to synthesize molecules with unknown structures, properties, and functions. Therefore, their potential to synthesize small molecules with biological activities should be of great interest in the search for novel antimicrobial compounds. The current study focused on investigating the antimicrobial potential of four soil Clostridium isolates, FS01, FS2.2 FS03, and FS04, using a genome-led approach, validated by culture-based methods. RESULTS: Conditioned/spent media from all four Clostridium isolates showed varying levels of antimicrobial activity against indicator microorganism; all four isolates significantly inhibited the growth of Pseudomonas aeruginosa. FS01, FS2.2, and FS04 were active against Bacillus mycoides and FS03 reduced the growth of Bacillus cereus. Phylogenetic analysis together with DNA-DNA hybridization (dDDH), average nucleotide identity (ANI), and functional genome distribution (FGD) analyses confirmed that FS01, FS2.2, and FS04 belong to the species Paraclostridium bifermentans, Clostridium cadaveris, and Clostridium senegalense respectively, while FS03 may represent a novel species of the genus Clostridium. Bioinformatics analysis using antiSMASH 5.0 predicted the presence of eight biosynthetic gene clusters (BGCs) encoding for the synthesis of ribosomally synthesized post-translationally modified peptides (RiPPs) and non-ribosomal peptides (NRPs) in four genomes. All predicted BGCs showed no similarity with any known BGCs suggesting novelty of the molecules from those predicted gene clusters. In addition, the analysis of genomes for putative virulence factors revealed the presence of four putative Clostridium toxin related genes in FS01 and FS2.2 genomes. No genes associated with the main Clostridium toxins were identified in the FS03 and FS04 genomes. CONCLUSIONS: The presence of BGCs encoding for uncharacterized RiPPs and NRPSs in the genomes of antagonistic Clostridium spp. isolated from farm soil indicated their potential to produce novel secondary metabolites. This study serves as a basis for the identification and characterization of potent antimicrobials from these soil Clostridium spp. and expands the current knowledge base, encouraging future research into bioactive compound production in members of the genus Clostridium.
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    Antimicrobial potential of Clostridium and closely related species derived from farm environmental samples : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Manawatū, New Zealand
    (Massey University, 2021) Weligala Pahalagedara, Amila Srilal Nawarathna
    The exploration of antimicrobial compounds from natural sources such as bacteria, has been fast tracked by the development of antimicrobial resistance to existing antimicrobials and the increasing consumer demand for natural food preservatives. So far, antimicrobial discovery has been biased towards aerobic and facultative anaerobic bacteria and fungi. Strict anaerobes such as Clostridium species have not been thoroughly investigated for their antimicrobial potential. The objective of the current study was to evaluate the antimicrobial potential of Clostridium and closely related species against bacteria associated with food spoilage, food safety, and human health. Tests on culture media inoculated with Clostridium and closely related species from farm samples (conditioned media/CMs) showed various degrees of antimicrobial activity. Farm 4 soil conditioned medium (F4SCM) showed potential for further investigation in the search for potent antimicrobials with its promising antimicrobial activity. Bacterial isolates (FS01, FS2.2, FS03, and FS04) belonging to Clostridium and closely related spp. associated with F4SCM showed antimicrobial potential as evident by culture-based and genome-based methods. F4SCM and FS03CM (CM prepared from FS03) metabolomes showed the presence of several putative antimicrobial metabolites. Among them, 2-hydroxyisocaproic acid (HICA) showed antimicrobial activity against a wide range of bacteria associated with food spoilage and safety indicating its potential as a bio-preservative agent in food products. The cell cytoplasmic membrane is a likely target of the HICA’s antimicrobial activity. Overall, this study demonstrates that anaerobic bacterial species, Clostridium, and closely related species can produce antimicrobial metabolites, that have potential applications in food preservation and human health. The knowledge obtained in this study will help future investigations to identify and characterize antimicrobials from these Clostridium and closely related bacteria and expands the understanding of the potential to produce antimicrobial compounds from the genus Clostridium and closely related species.
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    Germination of psychrotolerant clostridia responsible for red meat spoilage : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand
    (Massey University, 2012) Adam, Katharine Helen
    Psychrotolerant clostridia are responsible for spoilage of fresh chilled vacuum-packed red meat (beef, lamb and venison). Red meat is one of New Zealand’s primary exports, and spoilage results in financial loss. Spoilage by psychrotolerant clostridia is difficult to control due to the ability of these bacteria to grow at cold temperatures, down to -1.5 °C. They can also form spores that have increased resistance to heat, chemicals, oxygen and desiccation compared to vegetative bacterial cells. As clostridia are strict anaerobes, it is considered highly likely that initial contamination of meat is primarily with spores. The main objective of this work was to determine the triggers of germination of spores, of those psychrophilic and psychrotrophic clostridia, associated with spoilage of New Zealand red meat. Germination of psychrotolerant clostridia was studied using a range of techniques including molecular, in vitro, and on meat methods. In this study in vitro germinant systems were identified for Clostridium frigidicarnis, and a New Zealand species designated LA1, consisting of lactate in combination with an amino acid. Some of the amino acids identified, including valine and cysteine, are naturally present on the surface of red meat. Failure to chill to, or maintain meat at, the recommended temperature, of -1.5 °C and a pH of above 5.5 were identified as being important factors leading to spoilage by Cl. frigidicarnis. Germination in Clostridium estertheticum was extremely poor in media, compared with meat slurry or fresh meat, preventing the identification of a specific germinant system(s), and indicating a non-nutrient factor may be involved. Two distinct nonchemical interventions, hot water wash (HWW) and cold water wash (CWW), were found to reduce spoilage of vacuum-packed chilled lamb inoculated with spores of Cl. estertheticum. Vegetative cells of psychrotolerant clostridia survived exposure to air longer than expected, upwards of seven days in the case of Cl. estertheticum subsp. estertheticum, suggesting that they play a greater role in initial contamination of meat than originally thought. From an industry point of view the results highlight the importance of preventing initial contamination and proper chilling, as well as the need for further investigation of HWW and CWW interventions.
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    Occurrence of genes encoding spore germination in Clostridium species that cause meat spoilage.
    (2022-02) Burgess SA; Palevich FP; Gardner A; Mills J; Brightwell G; Palevich N
    Members of the genus Clostridium are frequently associated with meat spoilage. The ability for low numbers of spores of certain Clostridium species to germinate in cold-stored vacuum-packed meat can result in blown pack spoilage. However, little is known about the germination process of these clostridia, despite this characteristic being important for their ability to cause spoilage. This study sought to determine the genomic conditions for germination of 37 representative Clostridium strains from seven species (C. estertheticum, C. tagluense, C. frigoris, C. gasigenes, C. putrefaciens, C. aligidicarnis and C. frigdicarnis) by comparison with previously characterized germination genes from C. perfringens, C. sporogenes and C. botulinum. All the genomes analysed contained at least one gerX operon. Seven different gerX operon configuration types were identified across genomes from C. estertheticum, C. tagluense and C. gasigenes. Differences arose between the C. gasigenes genomes and those belonging to C. tagluense/C. estertheticum in the number and type of genes coding for cortex lytic enzymes, suggesting the germination pathway of C. gasigenes is different. However, the core components of the germination pathway were conserved in all the Clostridium genomes analysed, suggesting that these species undergo the same major steps as Bacillus subtilis for germination to occur.