The usefulness of the MALDI-TOF MS technique in the determination of dairy samples' microbial composition: comparison of the new EXS 2600 system with MALDI Biotyper platform.

This study compared the EXS 2600 system with the MALDI Biotyper for identifying microorganisms in dairy samples. Of the 196 bacterial isolates from milk, whey, buttermilk, cream, and dairy wastewater, the species and genus consistent identification between two systems showed 74% and 99%, respectively. However, the level of species identification rate exhibited a difference, which was higher in Zybio than in Bruker-76.0% and 66.8%, respectively. Notably, the EXS 2600 system performed better with certain yeast species and H. alvei, while the Biotyper excelled with Pseudomonas bacteria. Unique microbial compositions were found in 85% of dairy samples, with whey and buttermilk having the highest diversity. This research highlights the EXS 2600's potential as a reliable dairy microbial identification tool and underscores the need for a more diverse and comprehensive spectral database, despite the database's focus on clinical applications (as announced).

Antifungal Properties of Bio-AgNPs against D. pinodes and F. avenaceum Infection of Pea (Pisum sativum L.) Seedlings.

Ascochyta blight and Fusarium root rot are the most serious fungal diseases of pea, caused by D. pinodes and F. avenaceum, respectively. Due to the lack of fully resistant cultivars, we proposed the use of biologically synthesized silver nanoparticles (bio-AgNPs) as a novel protecting agent. In this study, we evaluated the antifungal properties and effectiveness of bio-AgNPs, in in vitro (poisoned food technique; resazurin assay) and in vivo (seedlings infection) experiments, against D. pinodes and F. avenaceum. Moreover, the effects of diseases on changes in the seedlings' metabolic profiles were analyzed. The MIC for spores of both fungi was 125 mg/L, and bio-AgNPs at 200 mg/L most effectively inhibited the mycelium growth of D. pinodes and F. avenaceum (by 45 and 26%, respectively, measured on the 14th day of incubation). The treatment of seedlings with bio-AgNPs or fungicides before inoculation prevented the development of infection. Bio-AgNPs at concentrations of 200 mg/L for D. pinodes and 100 mg/L for F. avenaceum effectively inhibited infections' spread. The comparison of changes in polar metabolites' profiles revealed disturbances in carbon and nitrogen metabolism in pea seedlings by both pathogenic fungi. The involvement of bio-AgNPs in the mobilization of plant metabolism in response to fungal infection is also discussed.

Characterization of the salivary microbiome before and after antibiotic therapy via separation technique.

In the present research, the MALDI-TOF MS technique was applied as a tool to rapidly identify the salivary microbiome. In this fact, it has been monitored the changes occurred in molecular profiles under different antibiotic therapy. Significant changes in the composition of the salivary microbiota were noticed not only in relation to the non antibiotic (non-AT) and antibiotic treatment (AT) groups, but also to the used media, the antibiotic therapy and co-existed microbiota. Each antibiotic generates specific changes in molecular profiles. The highest number of bacterial species was isolated in the universal culture medium (72%) followed by the selective medium (48% and 38%). In the case of non-AT patients, the prevalence of Streptococcus salivarius (25%), Streptococcus vestibularis (19%), Streptococcus oralis (13%), and Staphylococcus aureus (6%) was identified while in the case of AT, Streptococcus salivarius (11%), Streptococcus parasanguinis (11%), Staphylococcus epidermidis (12%), Enterococcus faecalis (9%), Staphylococcus hominis (8%), and Candida albicans (6%) were identified. Notable to specified that the Candida albicans was noticed only in AT samples, indicating a negative impact on the antibiotic therapy. The accuracy of the MALDI-TOF MS technique was performed by the 16S rRNA gene sequencing analysis-as a reference method. Conclusively, such an approach highlighted in the present study can help in developing the methods enabling a faster diagnosis of disease changes at the cellular level before clinical changes occur. Once the MALDI tool allows for the distinguishing of the microbiota of non-AT and AT, it may enable to monitor the diseases treatment and develop a treatment regimen for individual patients in relation to each antibiotic. KEY POINTS: The salivary microbiota of antibiotic-treated patients was more bacteria variety MALDI-TOF MS is a promising tool for recording of reproducible molecular profiles Our data can allow to monitor the treatment of bacterial diseases for patients.

Multi-Instrumental Analysis Toward Exploring the Diabetic Foot Infection Microbiota.

The polymicrobial nature of diabetic foot infection (DFI) makes accurate identification of the DFI microbiota, including rapid detection of drug resistance, challenging. Therefore, the main objective of this study was to apply matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF MS) technique accompanied by multiply culture conditions to determine the microbial patterns of DFIs, as well as to assess the occurrence of drug resistance among Gram-negative bacterial isolates considered a significant cause of the multidrug resistance spread. Furthermore, the results were compared with those obtained using molecular techniques (16S rDNA sequencing, multiplex PCR targeting drug resistance genes) and conventional antibiotic resistance detection methods (Etest strips). The applied MALDI-based method revealed that, by far, most of the infections were polymicrobial (97%) and involved many Gram-positive and -negative bacterial species-19 genera and 16 families in total, mostly Enterobacteriaceae (24.3%), Staphylococcaceae (20.7%), and Enterococcaceae (19.8%). MALDI drug-resistance assay was characterized by higher rate of extended-spectrum beta-lactamases (ESBLs) and carbapenemases producers compared to the reference methods (respectively 31% and 10% compared to 21% and 2%) and revealed that both the incidence of drug resistance and the species composition of DFI were dependent on the antibiotic therapy used. MALDI approach included antibiotic resistance assay and multiply culture conditions provides microbial identification at the level of DNA sequencing, allow isolation of both common (eg. Enterococcus faecalis) and rare (such as Myroides odoratimimus) bacterial species, and is effective in detecting antibiotic-resistance, especially those of particular interest-ESBLs and carbapenemases.

Metal tolerance and Cd phytoremoval ability in Pisum sativum grown in spiked nutrient solution.

In the presented study, the effects of cadmium (Cd) stress and silicon (Si) supplementation on the pea plant (Pisum sativum L.) were investigated. The tendency to accumulate cadmium in the relevant morphological parts of the plant (roots and shoots respectively)-bioaccumulation, the transfer of this element in the plant (translocation) and the physiological parameters of the plant through indicators of oxidative stress were determined. Model studies were carried out at pH values 6.0 and 5.0 plant growth conditions in the hydroponic cultivation. It was shown that Cd accumulates mostly in plant roots at both pH levels. However, the Cd content is higher in the plants grown at lower pH. The Cd translocation factor was below 1.0, which indicates that the pea is an excluder plant. The contamination of the plant growth environment with Cd causes the increased antioxidant stress by the growing parameters of the total phenolic content (TPC), polyphenol oxidase activity (PPO), the malondialdehyde (MDA) and lipid peroxidation (LP). The results obtained showed that the supplementation with Si reduces these parameters, thus lowering the oxidative stress of the plant. Moreover, supplementation with Si leads to a lower content of Cd in the roots and reduces bioaccumulation of Cd in shoots and roots of pea plants.

Removal of the Basic and Diazo Dyes from Aqueous Solution by the Frustules of Halamphora cf. salinicola (Bacillariophyta).

Industrial wastes with hazardous dyes serve as a major source of water pollution, which is considered to have an enormous impact on public health. In this study, an eco-friendly adsorbent, the porous siliceous frustules extracted from the diatom species Halamphora cf. salinicola, grown under laboratory conditions, has been identified. The porous architecture and negative surface charge under a pH of 7, provided by the various functional groups via Si-O, N-H, and O-H on these surfaces, revealed by SEM, the N2 adsorption/desorption isotherm, Zeta-potential measurement, and ATR-FTIR, respectively, made the frustules an efficient mean of removal of the diazo and basic dyes from the aqueous solutions, 74.9%, 94.02%, and 99.81% against Congo Red (CR), Crystal Violet (CV), and Malachite Green (MG), respectively. The maximum adsorption capacities were calculated from isotherms, as follows: 13.04 mg g-1, 41.97 mg g-1, and 33.19 mg g-1 against CR, CV, and MG, respectively. Kinetic and isotherm models showed a higher correlation to Pore diffusion and Sips models for CR, and Pseudo-Second Order and Freundlich models for CV and MG. Therefore, the cleaned frustules of the thermal spring-originated diatom strain Halamphora cf. salinicola could be used as a novel adsorbent of a biological origin against anionic and basic dyes.

Comprehensive Study of Si-Based Compounds in Selected Plants (Pisum sativum L., Medicago sativa L., Triticum aestivum L.).

This review describes the role of silicon (Si) in plants. Methods of silicon determination and speciation are also reported. The mechanisms of Si uptake by plants, silicon fractions in the soil, and the participation of flora and fauna in the Si cycle in terrestrial ecosystems have been overviewed. Plants of Fabaceae (especially Pisum sativum L. and Medicago sativa L.) and Poaceae (particularly Triticum aestivum L.) families with different Si accumulation capabilities were taken into consideration to describe the role of Si in the alleviation of the negative effects of biotic and abiotic stresses. The article focuses on sample preparation, which includes extraction methods and analytical techniques. The methods of isolation and the characterization of the Si-based biologically active compounds from plants have been overviewed. The antimicrobial properties and cytotoxic effects of known bioactive compounds obtained from pea, alfalfa, and wheat were also described.

Viability study of clinical bacterial strains by capillary electrophoresis and flow cytometry approaches.

One of the challenges medicine faces is the constantly growing resistance of pathogens to various classes of antibiotics. In this study, we investigated the use of capillary electrophoresis (CE) to characterize and assess the physiological states of three clinical bacterial strains-methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA), and Escherichia coli extended-spectrum ß-lactamases (ESßL)-exposed to different antibiotics. All chosen bacteria are the leading causes of healthcare-associated and hospital-acquired invasive infections in adults. In the first part of the research, it was determined the optimal incubation time of the tested strains with antibiotics, represented as an optimal time of 24 h. In the second part, we have compared two approaches: flow cytometry (FC) as a standard method and CE as a proposed alternative approach. The viability of clinical strains treated with different class antibiotics calculated in CE measurements was strongly correlated (>0.83 for MSSA, >0.92 for ESßL and MRSA) with the viability obtained on the basis of FC measurements. As a result, CE has a chance to become a modern diagnostic method used in clinical practice. The CE cutoff was found to be 50%; above this value, the strain shows resistance to the action of the antibiotic.

CE-DAD-MS/MS in the simultaneous determination and identification of selected antibiotic drugs and their metabolites in human urine samples.

In this study, a new analytical method was developed and validated for the simultaneous analysis of antibiotic drugs (amoxicillin, cefotaxime, ciprofloxacin, clindamycin, linezolid, metronidazole) and their metabolites (amoxycilloic acid, amoxicillin diketopiperazine, 3-desacetyl cefotaxime lactone, clindamycin sulfoxide, ciprofloxacin piperazinyl-N4-sulfate, linezolid N-oxide, metronidazole-OH) in human urine. Capillary electrophoresis (CE) along with the tandem mass spectrometry (MS/MS) was used to determine and identify all analytes. Appropriate conditions for MS/MS measurements along with the use of the central composite design were optimized. The effects of different analytical conditions (the composition, the concentration, and the pH value of the background electrolyte, the time and pressure of the injection, the capillary temperature and influence of the organic modifier) on the migration and separation of antibiotic drugs and metabolites were examined using the CE-DAD. The analytical procedure was linear for concentrations ranging from 20 to 1000 ng/mL, with determination coefficients higher than 0.99 for all the analytes. The validated analytical procedure was then applied to the measurement of antibiotic drugs and their metabolites in human urine samples.

New sources of lactic acid bacteria with potential antibacterial properties.

In the face of the growing demand for functional food, the search for new sources of lactic acid bacteria (LAB) becomes a priority. In our research, we used multiplied culture conditions followed by identification via the matrix-assisted laser desorption ionization-time of flight mass spectrometry for seeking LAB strains in plant- and animal-derived sources. Furthermore, the selected LAB isolates were examined for their proteolytic activity as well as antimicrobial action against different bacterial pathogens. The applied method appeared to be useful tool for searching LAB strains within different types of the biological matrices. The best source of the LABs was from calf. Comparing properties of the two selected LABs, those isolated from calf demonstrated the greatest proteolytic and antibacterial properties suggesting that gastrointestinal microbiota are the most valuable LAB source. Nevertheless, second selected strain derived from pickled cucumber juice may be also treated as a promising source of potential probiotic strains.

Diatom biosilica: Source, physical-chemical characterization, modification, and application.

Growing research interest in the use of diatomaceous biosilica results from its unique properties such as chemical inertness, biocompatibility, high mechanical and thermal stability, low thermal conductivity, and homogeneous porous structure with a large specific surface. Unlike the production of synthetic silica materials with a micro- or nanoscale structure in an expensive conventional manufacturing process, diatomaceous biosilica can be produced in huge quantities without significant expenditure of energy and materials. This fact makes it an unlimited, easily accessible, natural, inexpensive, and renewable material. Moreover, the production of biosilica is extremely environmental friendly, as there is essentially no toxic waste and the process does not require more energy compared to the production of synthetic silica-based materials. For all these reasons, diatoms are an intriguing alternative to synthetic materials in developing cheap biomaterials used in a different branches of industry. In this review, the state-of-art of biosilica materials, their characteristics approaches, and possible ways of application have been reported.

Synthesis and physicochemical characterization of zinc-lactoferrin complexes.

One trend of the modern world is the search for new biologically active substances based on renewable resources. Milk proteins can be a solution for such purposes as they have been known for a long time as compounds that can be used for the manufacturing of multiple food and non-food products. Thus, the goal of the work was to investigate the parameters of Zn-bovine lactoferrin (bLTF) interactions, which enables the synthesis of Zn-rich protein complexes. Zinc-bLTF complexes can be used as food additives or wound-healing agents. Methodology of the study included bLTF characterization by sodium dodecyl sulfate-PAGE, MALDI-TOF, and MALDI-TOF/TOF mass spectrometry as well Zn-bLTF interactions by attenuated total reflection-Fourier-transform infrared, Raman spectroscopy, scanning and transmission microscopy, and zeta potential measurements. The obtained results revealed that the factors that affect Zn-bLTF interactions most significantly were found to be pH and ionic strength of the solution and, in particular, the concentration of Zn2+. These findings imply that these factors should be considered when aiming at the synthesis of Zn-bLTF metallocomplexes.

In Vivo Efficacy of Wound Healing under External (Bio)AgNCs Treatment: Localization Case Study in Liver and Blood Tissue.

The present study reports on the in vivo application of (Bio)silver nanocomposite formulations (LBPC-AgNCs) on wound healing. Additionally, the present study emphasizes the limited uptake of silver by liver and blood tissues as well as the high viability of PBMCs following external LBPC-AgNCs treatment. The wound closure was monitored via stereoscopic microscope, a localization case study in liver and blood tissue was carried out by (Inductively Coupled Plasma-Mass Spectrometers (ICP/MS), and peripheral blood mononuclear cells (PMBC) viability was determined via flow cytometry technique. The silver formulation was applied externally on the site of the wound infection for a period of ten days. At the beginning of the experiment, a moderate decrease in body weight and atypical behavior was observed. However, during the last period of the experiment, no abnormal mouse behaviors were noticed. The wound-healing process took place in a gradual manner, presenting the regeneration effect at around 30% from the fourth day. From the seventh day, the wounds treated with the silver formulation showed 80% of the wound healing potential. The viability of PBMCs was found to be 97%, whereas the concentrations of silver in the liver and blood samples were determined to be 0.022 µg/g and 9.3 µg/g, respectively. Furthermore, the present report becomes a pilot study in transferring from in vitro to in vivo scale (e.g., medical field application) once LBPC-AgNCs have demonstrated a unique wound healing potential as well as a non-toxic effect on the liver and blood.

"Omic" Approaches to Bacteria and Antibiotic Resistance Identification.

The quick and accurate identification of microorganisms and the study of resistance to antibiotics is crucial in the economic and industrial fields along with medicine. One of the fastest-growing identification methods is the spectrometric approach consisting in the matrix-assisted laser ionization/desorption using a time-of-flight analyzer (MALDI-TOF MS), which has many advantages over conventional methods for the determination of microorganisms presented. Thanks to the use of a multiomic approach in the MALDI-TOF MS analysis, it is possible to obtain a broad spectrum of data allowing the identification of microorganisms, understanding their interactions and the analysis of antibiotic resistance mechanisms. In addition, the literature data indicate the possibility of a significant reduction in the time of the sample preparation and analysis time, which will enable a faster initiation of the treatment of patients. However, it is still necessary to improve the process of identifying and supplementing the existing databases along with creating new ones. This review summarizes the use of "-omics" approaches in the MALDI TOF MS analysis, including in bacterial identification and antibiotic resistance mechanisms analysis.

The Study of Protein-Cyclitol Interactions.

Investigation of interactions between the target protein molecule and ligand allows for an understanding of the nature of the molecular recognition, functions, and biological activity of protein-ligand complexation. In the present work, non-specific interactions between a model protein (Bovine Serum Albumin) and four cyclitols were investigated. D-sorbitol and adonitol represent the group of linear-structure cyclitols, while shikimic acid and D-(-)-quinic acid have cyclic-structure molecules. Various analytical methods, including chromatographic analysis (HPLC-MS/MS), electrophoretic analysis (SDS-PAGE), spectroscopic analysis (spectrofluorimetry, Fourier transform infrared spectroscopy, and Raman spectroscopy), and isothermal titration calorimetry (ITC), were applied for the description of protein-cyclitol interactions. Additionally, computational calculations were performed to predict the possible binding places. Kinetic studies allowed us to clarify interaction mechanisms that may take place during BSA and cyclitol interaction. The results allow us, among other things, to evaluate the impact of the cyclitol's structure on the character of its interactions with the protein.

Study on Biogenic Spindle-Shaped Iron-Oxide Nanoparticles by Pseudostaurosira trainorii in Field of Laser Desorption/Ionization Applications.

Nanostructures-assisted laser desorption/ionization mass spectrometry (NALDI-MS) is gaining attention for the analysis of a wide range of molecules. In this present investigation, Pseudostaurosira trainorii mediated biosynthesized iron-oxide nanoparticles (IONPs) have been utilized as nanostructures assisting ionization and desorption for laser desorption/ionization mass spectrometry (LDI-MS). The chain forming diatom, P. trainorii showed efficiency in the production of IONPs against 0.01 M Fe+3 (pH 2) aqueous solution at the intracellular and extracellular level. The whole biomass and external media turned dark orange in color after 3 days of reaction with Fe3+ solution. Scanning electron microscopic (SEM) images illustrated that the surface of Fe3+ exposed frustules of P. trainorii were entirely covered by synthesized nanostructures contrasting with the natural surface ornamentation of control cells. The IONPs loaded frustules also exhibited catalytic properties by decolorizing yellow colored nitrophenol after 3 h of reaction. Transmission electron microscopic (TEM) images confirmed that the produced particles are spindle-shaped with ~50-70 nm length and ~10-30 nm width. The biogenic IONPs were utilized as an inorganic matrix in LDI-MS and showed high sensitivity towards small molecules as glucose, alanine and triacylglycerols at nano- and picomolar level per spot, respectively. The presented biocompatible technique offers new perspectives in nanobiotechnology for the production of spindle-shaped IONPs that can be applied in future for the preparation of NALDI plates.

Synthesis, Physicochemical Characterization, and Antibacterial Performance of Silver-Lactoferrin Complexes.

Antibiotic-resistant bacteria pose one of the major threats to human health worldwide. The issue is fundamental in the case of chronic wound treatment. One of the latest trends to overcome the problem is the search for new antibacterial agents based on silver. Thus, the aim of this research was to synthesize the silver-lactoferrin complex as a new generation of substances for the treatment of infected wounds. Moreover, one of the tasks was to investigate the formation mechanisms of the respective complexes and the influence of different synthesis conditions on the features of final product. The batch-sorption study was performed by applying the Langmuir and Freundlich isotherm models for the process description. Characterization of the complexes was carried out by spectroscopy, spectrometry, and separation techniques, as well as with electron microscopy. Additionally, the biological properties of the complex were evaluated, i.e., the antibacterial activity against selected bacteria and the impact on L929 cell-line viability. The results indicate the formation of a heterogeneous silver-lactoferrin complex that comprises silver nanoparticles. The complex has higher antibacterial strength than both native bovine lactoferrin and Ag+, while being comparable to silver toxicity.

The Effect of Bio-Synthesized Silver Nanoparticles on Germination, Early Seedling Development, and Metabolome of Wheat (Triticum aestivum L.).

Changes in the metabolome of germinating seeds and seedlings caused by metal nanoparticles are poorly understood. In the present study, the effects of bio-synthesized silver nanoparticles ((Bio)Ag NPs) on grains germination, early seedlings development, and metabolic profiles of roots, coleoptile, and endosperm of wheat were analyzed. Grains germinated well in (Bio)Ag NPs suspensions at the concentration in the range 10-40 mg/L. However, the growth of coleoptile was inhibited by 25%, regardless of (Bio)Ag NPs concentration tested, whereas the growth of roots gradually slowed down along with the increasing concentration of (Bio)Ag NPs. The deleterious effect of Ag NPs on roots was manifested by their shortening, thickening, browning of roots tips, epidermal cell death, progression from apical meristem up to root hairs zone, and the inhibition of root hair development. (Bio)Ag NPs stimulated ROS production in roots and affected the metabolic profiles of all tissues. Roots accumulated sucrose, maltose, 1-kestose, phosphoric acid, and some amino acids (i.e., proline, aspartate/asparagine, hydroxyproline, and branched-chain amino acids). In coleoptile and endosperm, contrary to roots, the concentration of most metabolites decreased. Moreover, coleoptile accumulated galactose. Changes in the concentration of polar metabolites in seedlings revealed the affection of primary metabolism, disturbances in the mobilization of storage materials, and a translocation of sugars and amino acids from the endosperm to growing seedlings.

A new approach to identifying pathogens, with particular regard to viruses, based on capillary electrophoresis and other analytical techniques.

Fast determination, identification and characterization of pathogens is a significant challenge in many fields, from industry to medicine. Standard approaches (e.g., culture media and biochemical tests) are known to be very time-consuming and labor-intensive. Conversely, screening techniques demand a quick and low-cost grouping of microbial isolates, and current analysis call for broad reports of pathogens, involving the application of molecular, microscopy, and electromigration techniques, DNA fingerprinting and also MALDI-TOF methods. The present COVID-19 pandemic is a crisis that affects rich and poor countries alike. Detection of SARS-CoV-2 in patient samples is a critical tool for monitoring disease spread, guiding therapeutic decisions and devising social distancing protocols. The goal of this review is to present an innovative methodology based on preparative separation of pathogens by electromigration techniques in combination with simultaneous analysis of the proteome, lipidome, and genome using laser desorption/ionization analysis.

Combination of electrochemical unit and ESI-MS in fragmentation of flavonoids.

INTRODUCTION: Predictive approaches on the activity of natural compounds based on the fragmentation by instrumental techniques are important for consideration of such molecules as drug candidates and defining new structures with promising properties. Since flavonoids are well-known antioxidants, their redox properties can be related to their pharmacological activity. OBJECTIVES: In this work, the potential of electrochemical unit coupled to electrospray ionisation mass spectrometry (ESI-MS) was assessed for fragmentation activity relationships studies of selected flavonoids. METHODOLOGY: Methodology of this research included electrochemical conversion of standards of flavonoids at different pH values and their further analysis with the use of ESI-MS. In addition, signals obtained from the blank samples were also identified and used for interpretation due to electrochemical nature of the ESI source. Half maximal inhibitory concentration (IC50 ) values of flavonoids for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) antioxidant activity assays were analysed for possible correlation with the structures of flavonoids and products of electrochemical conversion. RESULTS: Fragmentation activity relationships were suggested using the proposed approach and for some of the flavonoids it was not specific enough to determine the input of a particular structural feature to the activity, but for others they were in agreement with those found in the literature. Obtained results showed potential of the proposed approach for application in plant sciences as a fast pre-screening tool for newly isolated bioactive compounds.