Browsing by Author "Palmer J"

Now showing 1 - 5 of 5
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    An improved MTT colorimetric method for rapid viable bacteria counting
    (Elsevier BV, 2023-11) Xu W; Shi D; Chen K; Palmer J; Popovich DG
    The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay has been employed in the analysis of bacterial growth. In comparison to experiments conducted on mammalian cells, the MTT bacterial assay encounters a greater number of interfering factors and obstacles that impact the accuracy of results. In this study, we have elucidated an improved MTT assay protocol and put forth an equation that establishes a correlation between colony-forming units (CFU) and the amount of formazan converted by the bacteria, drawing upon the fundamental principle of the MTT assay. This equation is represented as CFU=kF. Furthermore, we have explicated a methodology to determine the scale factor "k" by employing S. aureus and E. coli as illustrative examples. The findings indicate that S. aureus and E. coli reduce MTT by a cyclic process, from which the optimal reduction time at room temperature was determined to be approximately 30 mins. Furthermore, individual E. coli exhibits an MTT reduction capacity approximately four times greater than that of S. aureus. HPLC analysis proves to be the most accurate method for mitigating interferences during the dissolution and quantification of formazan. Additionally, this study has identified a new constraint related to the narrow linear range (0-125 μg/mL) of formazan concentration-absorbance and has presented strategies to circumvent this limitation.
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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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    Effect of Fluidized Bed Drying, Matrix Constituents and Structure on the Viability of Probiotic Lactobacillus paracasei ATCC 55544 during Storage at 4 °C, 25 °C and 37 °C
    (MDPI (Basel, Switzerland), 2022-01) Poddar D; Palmer J; Das S; Gaare M; Nag A; Singh H; Succi M; Sorrentino E
    The stabilization of probiotics for application in non-refrigerated food products is a challenging task. In the present study, probiotic Lactobacillus paracasei (Lacticaseibacillus paracasei) ATCC 55544 cells were immobilized in a dairy matrix comprising of whole milk powder, skim milk powder, or milk protein isolate using fluidized bed drying technology. The samples were taken out at different drying stages, with an apparent water activity (aw) of aw 0.5, aw 0.4, and aw 0.3, respectively, and vacuum-packed to maintain the aw and stored at three different temperatures of 4 °C, 25 °C, and 37 °C. The study evaluated the impact of matrix constituents, milk fat, protein, and carbohydrate on the viability of encapsulated probiotic L . paracasei ATCC 55544 during storage for 1 month. The whole milk powder matrix provided superior protection to the bacteria. Confocal Laser Scanning Microscopy (CLSM) was used to investigate the structure of the immobilizing matrix and the location of the probiotic L. paracasei cells embedded within the matrix. The CLSM study revealed that the probiotic bacterial cells are mostly embedded as clusters beneath the top layer. We hypothesize that the biofilm-like structure, together with the protective whole milk powder matrix, helps to retain the superior viability of probiotic cells during storage at non-refrigerated storage conditions of 25 °C and 37 °C.
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    Non-Targeted Metabolomic Profiling Identifies Metabolites with Potential Antimicrobial Activity from an Anaerobic Bacterium Closely Related to Terrisporobacter Species.
    (MDPI (Basel, Switzerland), 2023-02-09) Pahalagedara ASNW; Flint S; Palmer J; Brightwell G; Luo X; Li L; Gupta TB; Eisenreich W
    This work focused on the metabolomic profiling of the conditioned medium (FS03CM) produced by an anaerobic bacterium closely related to Terrisporobacter spp. to identify potential antimicrobial metabolites. The metabolome of the conditioned medium was profiled by two-channel Chemical Isotope Labelling (CIL) LC-MS. The detected metabolites were identified or matched by conducting a library search using different confidence levels. Forty-eight significantly changed metabolites were identified with high confidence after the growth of isolate FS03 in cooked meat glucose starch (CMGS) medium. Some of the secondary metabolites identified with known antimicrobial activities were 4-hydroxyphenyllactate, 3-hydroxyphenylacetic acid, acetic acid, isobutyric acid, valeric acid, and tryptamine. Our findings revealed the presence of different secondary metabolites with previously reported antimicrobial activities and suggested the capability of producing antimicrobial metabolites by the anaerobic bacterium FS03.
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    The effect of variations in cold plasma conditions on the detoxification of Aflatoxin M1 and degradation products
    (Elsevier B.V., 2024-10-16) Nguyen T; Palmer J; Pedley J; Petcu M; Newson HL; Keener K; Flint S
    The aim of this study was to explore the chemical reactive species of different operating gases, and their effect on the degradation of aflatoxin M1 (AFM1) by cold plasma by measuring the reactive species concentration. Helium, at 80, 90 or 95%, was used mixed with oxygen, nitrogen and air. The efficacy of cold plasma on aflatoxin M1 (AFM1) reduction was improved when decreasing the ratio of helium in the gas mixture. The ratio of the gas mixtures changed the cold plasma chemistry believed to be due to the differences in the concentrations of the reactive species. The degradation products of AFM1 after cold plasma treatment using a helium/air gas mixture and the degradation pathway were identified by LCMS. AFM1 was oxidised by reactive species in the cold plasma to produce degradant products with, theoretically, lower toxicity than AFM1.