Liquid chromatography-tandem mass spectrometry profile and antioxidant, antimicrobial, antiproliferative, and enzyme activities of Thymus pectinatus and Thymus convolutus: in vitro and in silico approach.

BACKGROUND: The objective of this study is to investigate the antiproliferative, antioxidant, antimicrobial, and enzyme activity capacities and phytochemical compositions of Thymus pectinatus (TP), Thymus convolutus (TC), which are endemic to Türkiye. Quantitative analysis of phenolic compounds in the extracts was conducted using liquid chromatography-tandem mass spectrometry, targeting 53 phenolic compounds. RESULTS: Rosmarinic acid, quinic acid, and cynaroside were identified as the major compounds, exhibiting quantitative variation in both extracts. The extracts had a high total phenolic content, with 113.57 ± 0.58 mg gallic acid equivalents (GAE)/g extract for TP and 130.52 ± 1.05 mg GAE/g extract for TC. Furthermore, although both extracts exhibited high total flavonoid content; the TP extract (75.12 ± 1.65 mg quercitin equivalents (QE)/g extract) displayed a higher flavonoid content than the TC extract (30.24 ± 0.74 mg QE/g extract) did. The extracts had a promising antiproliferative effect on C6, HeLa, and HT29 cancer cell lines with a less cytotoxic effect (10.5-14.2%) against normal cells. Both extracts exhibited very potent inhibitory activity against the xanthine oxidase enzyme, with half-maximal inhibitory concentration values of respectively 2.07 ± 0.03 µg mL-1 and 2.76 ± 0.06 µg mL-1 and moderate activity against tyrosinase and α-glucosidase. Docking simulations proved that rosmarinic acid and cynaroside, the major components of the extracts, were the most potent inhibitors of xanthine oxidase. According to antimicrobial activity results, the TC extract exhibited moderate activity against Staphylococcus aureus, and the TP extract had strong activity against both Enterococcus faecium and S. aureus. CONCLUSION: These findings emphasize the beneficial effects of the two endemic Thymus species on human health and suggest their potential use as plant-derived bioactive agents. © 2024 Society of Chemical Industry.

Investigating the Ecotoxicity of Select Emerging Organic Contaminants Toward the Marine Copepod Gladioferens pectinatus.

Estuarine ecosystems are recipients of anthropogenic stressors released from land-based activities. The aim of the present study was to investigate the ecotoxicological hazards of organic contaminants toward the estuarine copepod Gladioferens pectinatus using acute and chronic testing. Most chemicals demonstrated acute toxicity and influenced development of the copepods. Further research should be conducted to investigate these chemicals and their mixtures using long-term, multigenerational testing to characterize mechanisms of toxicity. Environ Toxicol Chem 2022;41:792-799. © 2022 SETAC.

First transcriptome of the copepod Gladioferens pectinatus subjected to chronic contaminant exposures.

Contaminants are often at low concentrations in ecosystems and their effects on exposed organisms can occur over long periods of time and across multiple generations. Alterations to subcellular mechanistic pathways in response to exposure to contaminants can provide insights into mechanisms of toxicity that methods measuring higher levels of biological may miss. Analysis of the whole transcriptome can identify novel mechanisms of action leading to impacts in exposed biota. The aim of this study was to characterise how exposures to copper, benzophenone and diclofenac across multiple generations altered molecular expression pathways in the marine copepod Gladioferens pectinatus. Results of the study demonstrated differential gene expression was observed in cultures exposure to diclofenac (569), copper (449) and benzophenone (59). Pathways linked to stress, growth, cellular and metabolic processes were altered by exposure to all three contaminants with genes associated with oxidative stress and xenobiotic regulation also impacted. Protein kinase functioning, cytochrome P450, transcription, skeletal muscle contraction/relaxation, mitochondrial phosphate translocator, protein synthesis and mitochondrial methylation were all differentially expressed with all three chemicals. The results of the study also suggested that using dimethyl sulfoxide as a dispersant influenced the transcriptome and future research may want to investigate it's use in molecular studies. Data generated in this study provides a first look at transcriptomic response of G. pectinatus exposed to contaminants across multiple generations, future research is needed to validate the identified biomarkers and link these results to apical responses such as population growth to demonstrate the predictive capacity of molecular tools.

Pectinatus spp. - Unpleasant and recurrent brewing spoilage bacteria.

Traditionally, beer has been recognised as a beverage with high microbiological stability because of the hostile growth environment posed by beer and increasing attention being paid to brewery hygiene. However, the microbiological risk has increased in recent years because of technological advances toward reducing oxygen in beers, besides the increase in novel beer styles production, such as non-pasteurised, flash pasteurised, cold sterilised, mid-strength, and alcoholic-free beer, that are more prone to spoilage bacteria. Moreover, using innovative beer ingredients like fruits and vegetables is an added cause of microbial spoilage. To maintain quality and good brand image, beer spoilage microorganisms are a critical concern for breweries worldwide. Pectinatus and Megasphaera are Gram-negative bacteria mostly found in improper brewing environments, leading to consumer complaints and financial losses. Because of the lack of compiled scientific knowledge on Pectinatus spoilage ability, this review provides a comprehensive overview of the occurrence, survival mechanisms, and the factors affecting beer spoilage Pectinatus species in the brewing process.

Comparative genetic and physiological characterisation of Pectinatus species reveals shared tolerance to beer-associated stressors but halotolerance specific to pickle-associated strains.

Obligate anaerobic bacteria from the genus Pectinatus have been known to cause beer spoilage for over 40 years. Whole genome sequencing was performed on eleven beer spoilage strains (nine Pectinatus frisingensis, one Pectinatus cerevisiiphilus and one Pectinatus haikarae isolate), as well as two pickle spoilage species (Pectinatus brassicae MB591 and Pectinatus sottacetonis MB620) and the tolerance of all species to a range of environmental conditions was tested. Exploration of metabolic pathways for carbohydrates, amino acids and vitamins showed little difference between beer spoilage- and pickle spoilage-associated strains. However, genes for certain carbohydrate- and sulphur-containing amino acid-associated enzymes were only present in the beer spoilage group and genes for specific transporters and regulatory genes were uniquely found in the pickle spoilage group. Transporters for compatible solutes, only present in pickle-associated strains, likely explain their experimentally observed higher halotolerance compared to the beer spoilers. Genes involved in biofilm formation and ATP Binding Cassette (ABC) transporters potentially capable of exporting hop-derived antimicrobial compounds were found in all strains. All species grew in the presence of alcohol up to 5% alcohol by volume (ABV) and hops extract up to 80 ppm of iso-α-acids. Therefore, the species isolated from pickle processes may pose novel hazards in brewing.

Regression model, artificial neural network, and cost estimation for biosorption of Ni(II)-ions from aqueous solutions by Potamogeton pectinatus.

This study investigated the application of Potamogeton pectinatus for Ni(II)-ions biosorption from aqueous solutions. FTIR spectra showed that the functional groups of -OH, C-H, -C = O, and -COO- could form an organometallic complex with Ni(II)-ions on the biomaterial surface. SEM/EDX analysis indicated that the voids on the biosorbent surface were blocked due to Ni(II)-ions uptake via an ion exchange mechanism. For Ni(II)-ions of 50 mg/L, the adsorption efficiency recorded 63.4% at pH: 5, biosorbent dosage: 10 g/L, and particle-diameter: 0.125-0.25 mm within 180 minutes. A quadratic model depicted that the plot of removal efficiency against pH or contact time caused quadratic-linear concave up curves, whereas the curve of initial Ni(II)-ions was quadratic-linear convex down. Artificial neural network with a structure of 5 - 6 - 1 was able to predict the adsorption efficiency (R2: 0.967). The relative importance of inputs was: initial Ni(II)-ions > pH > contact time > biosorbent dosage > particle-size. Freundlich isotherm described well the adsorption mechanism (R2: 0.974), which indicated a multilayer adsorption onto energetically heterogeneous surfaces. The net cost of using P. pectinatus for the removal of Ni(II)-ions (4.25 ± 1.26 mg/L) from real industrial effluents within 30 minutes was 3.4 $USD/m3.

A laboratory exercise for detecting microbial contaminants.

The development and evaluation of a 6-hours laboratory class, based on capillary electrophoresis (CE) and the detection of microbial contaminants, is described. It can be easily scaled up or down, to suit class sizes up to 188 and completed in a shorter time scale. CE uses narrow-bore fused-silica capillaries to separate a complex array of large and small molecules. A laboratory exercise has been devised to illustrate how CE-based genetic analysis system processes DNA fragment analysis to detect three microbial contaminants. The protocol is relatively inexpensive and uses standard molecular biology reagents and equipment. © 2018 by The International Union of Biochemistry and Molecular Biology, 46(3):279-284, 2018.

Development of a polymerase chain reaction assay for the rapid detection of the oral pathogenic bacterium, Selenomonas noxia.

BACKGROUND: In recent studies, periodontal health has been linked to being overweight and/or obese. Among common oral bacteria, Selenomonas noxia has been implicated in converting periodontal health to disease, and Selenomonas species have also been found in gastric ulcers. The objective of this study was to develop and validate a quantitative polymerase chain reaction (qPCR) assay for the specific and rapid detection of S. noxia. METHODS: Two oligonucleotide primer pairs and one probe were designed and tested to determine optimal amplification signal with three strains of S. noxia. The PCR assay was tested against fourteen non-target organisms, including closely related oral Selenomonads, one phylogenetically closely related bacterium, and two commonly isolated oral bacteria. RESULTS: One of the primer sets was more sensitive at detecting the target organism and was selected for optimization and validation experiments. The designed primers and probe amplified the target organism with 100% specificity. PCR inhibition was observed with an internal positive control, and inhibition was resolved by diluting the DNA extract. CONCLUSIONS: The qPCR assay designed in this study can be used to specifically detect S. noxia in the clinical setting and in future research involving the enhanced detection of S. noxia. The assay can also be used in epidemiological studies for understanding the role of S. noxia in disease processes including, but not limited to, oral health and obesity of infectious origin.

Lipidomics as an important key for the identification of beer-spoilage bacteria.

Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) was used for characterizing intact plasmalogen phospholipid molecules in beer-spoilage bacteria. Identification of intact plasmalogens was carried out using collision-induced dissociation and the presence of suitable marker molecular species, both qualitative and quantitative, was determined in samples containing the anaerobic bacteria Megasphaera and Pectinatus. Using selected ion monitoring (SIM), this method had a limit of detection at 1 pg for the standard, i.e. 1-(1Z-octadecenyl)-2-oleoyl-sn-glycero-3-phosphoethanolamine and be linear in the range of four orders of magnitude from 2 pg to 20 ng. This technique was applied to intact plasmalogen extracts from the samples of contaminated and uncontaminated beer without derivatization and resulted in the identification of contamination of beer by Megasphaera and Pectinatus bacteria. The limit of detection was about 830 cells of anaerobic bacteria, i.e. bacteria containing natural cyclopropane plasmalogenes (c-p-19:0/15:0), which is the majority plasmalogen located in both Megasphaera and Pectinatus. The SIM ESI-MS method has been shown to be useful for the analysis of low concentration of plasmalogens in all biological samples, which were contaminated with anaerobic bacteria, e.g. juice, not only in beer. Significance and impact of the study: Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) using collision-induced dissociation was used to characterize intact plasmalogen phospholipid molecules in beer-spoilage anaerobic bacteria Megasphaera and Pectinatus. Using selected ion monitoring (SIM), this method has a detection limit of 1 pg for the standard 1-(1Z-octadecenyl)-2-oleoyl-sn-glycero-3-phosphoethanolamine and is linear within four orders of magnitude (2 pg to 20 ng). The limit of detection was about 830 cells of bacteria containing natural cyclopropane plasmalogen (c-p-19:0/15:0). SIM ESI-MS method is useful for analyzing low concentrations of plasmalogens in biological samples contaminated with anaerobic bacteria, e.g. beer or juice.

In situ production of human ß defensin-3 in lager yeasts provides bactericidal activity against beer-spoiling bacteria under fermentation conditions.

AIMS: To examine the use of a natural antimicrobial peptide, human ß-defensin-3 (HBD3), as a means of preventing spoilage from bacterial contamination in brewery fermentations and in bottled beer. METHODS AND RESULTS: A chemically synthesised HBD3 peptide was tested for bactericidal activity against common Gram-positive and Gram-negative beer-spoiling bacteria, including species of Lactobacillus, Pediococcus and Pectinatus. The peptide was effective at the µmol l(-1) range in vitro, reducing bacterial counts by 95%. A gene construct encoding a secretable form of HBD3 was integrated into the genome of the lager yeast Saccharomyces pastorianus strain CMBS-33. The integrated gene was expressed under fermentation conditions and was secreted from the cell into the medium, but a significant amount remains associated with yeast cell surface. We demonstrate that under pilot-scale fermentation conditions, secreted HBD3 possesses bactericidal activity against beer-spoiling bacteria. Furthermore, when added to bottled beer, a synthetic form of HBD3 reduces the growth of beer-spoiling bacteria. CONCLUSIONS: Defensins provide prophylactic protection against beer-spoiling bacteria under brewing conditions and also in bottled beer. SIGNIFICANCE AND IMPACT OF THE STUDY: The results have direct application to the brewing industry where beer spoilage due to bacterial contamination continues to be a major problem in breweries around the world.

Detection of beer spoilage bacteria Pectinatus and Megasphaera with acridinium ester labelled DNA probes using a hybridisation protection assay.

DNA probes specific for rRNA of selected target species were utilised for the detection of beer spoilage bacteria of the genera Pectinatus and Megasphaera using a hybridisation protection assay (HPA). All the probes were modified during synthesis by addition of an amino linker arm at the 5' end or were internally modified by inserting an amine modified thymidine base. Synthesised probes then were labelled with acridinium ester (AE) and purified using reverse phase HPLC. The internally AE labelled probes were able to detect target RNA within the range of 0.016-0.0032pmol. All the designed probes showed high specificity towards target RNA and could detect bacterial contamination within the range of ca. 5×10(2)1×10(3) CFU using the HPA. The developed assay was also compatible with MRS, NBB and SMMP beer enrichment media, routinely used in brewing laboratories.

Identification of plasmalogen cardiolipins from Pectinatus by liquid chromatography-high resolution electrospray ionization tandem mass spectrometry.

High resolution electrospray ionization tandem mass spectrometry (HR-ESI-MS/MS) was used to analyze cardiolipins (Ptd2Gro) including their plasmalogen forms from three species of the anaerobic beer-spoilage bacterial genus Pectinatus. Cardiolipins including their plasmalogens were analyzed by HR-ESI-MS/MS on Orbitrap and almost 100 cardiolipins (i.e. tetra-acyl--Ptd2Gro, plasmenyl-tri-acyl--PlsPtd2Gro, and di-plasmenyl-di-acyl--Pls2Ptd2Gro) of three classes were identified. The structures of the molecular species that consist of various regioisomers and structurally similar compounds were revealed in detail. The high resolution mass spectrometry allowed the unambiguous structural assignment of Ptd2Gro, PlsPtd2Gro, and Pls2Ptd2Gro in the three species of Pectinatus, which contain predominantly odd numbered fatty acids.

Dynamics of cathode-associated microbial communities and metabolite profiles in a glycerol-fed bioelectrochemical system.

Electrical current can be used to supply reducing power to microbial metabolism. This phenomenon is typically studied in pure cultures with added redox mediators to transfer charge. Here, we investigate the development of a current-fed mixed microbial community fermenting glycerol at the cathode of a bioelectrochemical system in the absence of added mediators and identify correlations between microbial diversity and the respective product outcomes. Within 1 week of inoculation, a Citrobacter population represented 95 to 99% of the community and the metabolite profiles were dominated by 1,3-propanediol and ethanol. Over time, the Citrobacter population decreased in abundance while that of a Pectinatus population and the formation of propionate increased. After 6 weeks, several Clostridium populations and the production of valerate increased, which suggests that chain elongation was being performed. Current supply was stopped after 9 weeks and was associated with a decrease in glycerol degradation and alcohol formation. This decrease was reversed by resuming current supply; however, when hydrogen gas was bubbled through the reactor during open-circuit operation (open-circuit potential) as an alternative source of reducing power, glycerol degradation and metabolite production were unaffected. Cyclic voltammetry revealed that the community appeared to catalyze the hydrogen evolution reaction, leading to a +400-mV shift in its onset potential. Our results clearly demonstrate that current supply can alter fermentation profiles; however, further work is needed to determine the mechanisms behind this effect. In addition, operational conditions must be refined to gain greater control over community composition and metabolic outcomes.

Pectinatus sottacetonis sp. nov., isolated from a commercial pickle spoilage tank.

A strictly anaerobic, Gram-stain-negative, non-spore-forming, motile bacterium, designated strain FSRU B0405(T), was isolated from a commercial pickle spoilage tank and characterized by biochemical, physiological and molecular biological methods. Analyses of the 16S rRNA gene sequence of strain FSRU B0405(T) showed affiliation to the class Negativicutes in the phylum Firmicutes, with the closest relatives being the type strains of Pectinatus haikarae (96 %) and Pectinatus brassicae (95 %). In maximum-likelihood and neighbour-joining phylogenetic trees, strain FSRU B0405(T) clustered definitively (in 100 % of bootstrapped trees) within the genus Pectinatus, but not specifically with any characterized species within this genus. Strain FSRU B0405(T) was a slightly curved rod, varying from 3 to 30 µm in length, motile with a distinctive X-wise movement, having flagella only on the concave side of the cell. The isolate produced acetate and propionate from fructose and glucose as major metabolites similar to type strains of species of the genus Pectinatus. The major fatty acids were C11 : 0, C13 : 0, C15 : 0, C13 : 0 3-OH, C17 : 1 and C18 : 1ω11t. Strain FSRU B0405(T) differed from the pickle wastewater strain, Pectinatus brassicae TY(T), due to its lack of susceptibility to vancomycin, acetoin production, growth temperature range, acid production from adonitol, erythritol, glycerol, inositol, lactose, maltose, mannose, ribose, salicin, sorbitol, trehalose and xylitol and lack of hydrolysis of milk. Strain FSRU B0405(T) could be differentiated from other species of the genus Pectinatus both phenotypically and genetically. The results indicate that strain FSRU B0405(T) represents a novel species of the genus Pectinatus, for which the name Pectinatus sottacetonis sp. nov. is proposed. The type strain is FSRU B0405(T) ( = ATCC BAA-2501(T) = VTT E-113163(T)). An emended description of the genus Pectinatus is also provided.

Characterization of cucumber fermentation spoilage bacteria by enrichment culture and 16S rDNA cloning.

UNLABELLED: Commercial cucumber fermentations are typically carried out in 40000 L fermentation tanks. A secondary fermentation can occur after sugars are consumed that results in the formation of acetic, propionic, and butyric acids, concomitantly with the loss of lactic acid and an increase in pH. Spoilage fermentations can result in significant economic loss for industrial producers. The microbiota that result in spoilage remain incompletely defined. Previous studies have implicated yeasts, lactic acid bacteria, enterobacteriaceae, and Clostridia as having a role in spoilage fermentations. We report that Propionibacterium and Pectinatus isolates from cucumber fermentation spoilage converted lactic acid to propionic acid, increasing pH. The analysis of 16S rDNA cloning libraries confirmed and expanded the knowledge gained from previous studies using classical microbiological methods. Our data show that Gram-negative anaerobic bacteria supersede Gram-positive Fermincutes species after the pH rises from around 3.2 to pH 5, and propionic and butyric acids are produced. Characterization of the spoilage microbiota is an important first step in efforts to prevent cucumber fermentation spoilage. PRACTICAL APPLICATION: An understanding of the microorganisms that cause commercial cucumber fermentation spoilage may aid in developing methods to prevent the spoilage from occurring.

Pectinatus brassicae sp. nov., a Gram-negative, anaerobic bacterium isolated from salty wastewater.

A novel Gram-negative, non-spore-forming, strictly anaerobic, heterotrophic bacterium, strain TY(T), was isolated from salty pickle wastewater. Cells were rod-shaped with comb-like flagella, slightly curved and very variable in length. Optimal growth occurred at 28 °C and pH 6.5. Cells were resistant to up to 50 g NaCl l(-1). Strain TY(T) produced acid from glycerol, sucrose, glucose, fructose and mannitol. The main fermentation products from glucose were acetic and propionic acids. Tests for acid phosphatase and naphthol-AS-BI-phosphohydrolase activities were positive. The major fatty acids were C(14 : 0) DMA (18.7 %), C(15 : 0) (15.4 %), anteiso-C(18 : 1) (15.2 %), C(11 : 0) (13.3 %) and summed feature 5 (C(17 : 1)ω7c and/or C(17 : 2)) (11.0 %). The DNA G+C content was 35.9 mol%. 16S rRNA gene sequence-based phylogenetic analysis indicated that strain TY(T) represented a novel species of the genus Pectinatus (sequence similarity to other members of the genus ranged from 93.2 to 94.8 %). Based on its phenotypic, genotypic and phylogenetic characteristics, strain TY(T) is proposed to represent a novel species, named Pectinatus brassicae sp. nov. (type strain TY(T) = JCM 17499(T) = DSM 24661(T)).

Hydrophilic interaction liquid chromatography: ESI-MS/MS of plasmalogen phospholipids from Pectinatus bacterium.

Liquid chromatography-electrospray tandem mass spectrometry (LC/ESI-MS/MS) was used to analyze phospholipids from three species of the anaerobic beer-spoilage bacterial genus Pectinatus. Analysis of total lipids by HILIC (Hydrophilic Interaction Liquid Chromatography) column succeeded in separating diacyl- and plasmalogen phospholipids. Plasmalogens were then analyzed by means of the ESI-MS/MS and more than 220 molecular species of four classes of plasmalogens (PlsCho (choline plasmalogen), PlsEtn (ethanolamine plasmalogen), PlsGro (glycerol plasmalogen), and PlsSer (serine plasmalogen)) were identified. Major molecular species were c-p19:0/15:0 PlsEtn and PlsSer, which accounted for more than 4% of the total lipids.

Modified multiplex PCR methods for comprehensive detection of Pectinatus and beer-spoilage cocci.

Specific PCR primers were designed based on the 16S rRNA genes of recently proposed beer-spoilage species, Pectinatus haikarae, Megasphaera sueciensis, and M. paucivorans, and two sets of our previously reported multiplex PCR methods for Pectinatus spp. and beer-spoilage cocci were reconstructed. Each modified multiplex PCR method was found specifically to detect beer-spoilage species of Pectinatus and cocci, including new species.

Oligonucleotide microarrays for the detection and identification of viable beer spoilage bacteria.

AIMS: The design and evaluation of an oligonucleotide microarray in order to detect and identify viable bacterial species that play a significant role in beer spoilage. These belong to the species of the genera Lactobacillus, Megasphaera, Pediococcus and Pectinatus. METHODS AND RESULTS: Oligonucleotide probes specific to beer spoilage bacteria were designed. In order to detect viable bacteria, the probes were designed to target the intergenic spacer regions (ISR) between 16S and 23S rRNA. Prior to hybridization the ISR were amplified by combining reverse transcriptase and polymerase chain reactions using a designed consenus primer. The developed oligonucleotide microarrays allows the detection of viable beer spoilage bacteria. CONCLUSIONS: This method allows the detection and discrimination of single bacterial species in a sample containing complex microbial community. Furthermore, microarrays using oligonucleotide probes targeting the ISR allow the distinction between viable bacteria with the potential to grow and non growing bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The results demonstrate the feasibility of oligonucleotide microarrays as a contamination control in food industry for the detection and identification of spoilage micro-organisms within a mixed population.

The status of the species Pectinatus portalensis Gonzalez et al. 2005. Request for an Opinion.

On the basis of 16S rRNA gene sequence analysis and several key phenotypic features, it was ascertained that the cultures cited as the type strain of the species Pectinatus portalensis, CECT 5841(T) and LMG 22865(T), do not conform to the description, [Gonzalez, J. M., Jurado, V., Laiz, L., Zimmerman, J., Hermosin, B. & Saiz-Jimenez, C. (2004). Antonie van Leeuwenhoek 86, 241-248]. The type strain does not exist in any other established culture collection or with the authors who described this species. Therefore, it cannot be included in any scientific study. It is proposed that the Judicial Commission place the name Pectinatus portalensis on the list of rejected names if a suitable replacement type strain is not found or a neotype is not proposed within two years following the publication of this Request for an Opinion.