In vitro screening and characterization of lactic acid bacteria from Lithuanian fermented food with potential probiotic properties.

The present work aimed to identify probiotic candidates from Lithuanian homemade fermented food samples. A total of 23 lactic acid bacteria were isolated from different fermented food samples. Among these, only 12 showed resistance to low pH, tolerance to pepsin, bile salts, and pancreatin. The 12 strains also exhibited antimicrobial activity against Staphylococcus aureus ATCC 29213, Salmonella Typhimurium ATCC 14028, Streptococcus pyogenes ATCC 12384, Streptococcus pyogenes ATCC 19615, and Klebsiella pneumoniae ATCC 13883. Cell-free supernatants of isolate 3A and 55w showed the strongest antioxidant activity of 26.37 µg/mL and 26.06 µg/mL, respectively. Isolate 11w exhibited the strongest auto-aggregation ability of 79.96% as well as the strongest adhesion to HCT116 colon cells (25.671 ± 0.43%). The selected strains were tested for their synbiotic relation in the presence of a prebiotic. The selected candidates showed high proliferation in the presence of 4% as compared to 2% galactooligosaccharides. Among the strains tested for tryptophan production ability, isolate 11w produced the highest L-tryptophan levels of 16.63 ± 2.25 µm, exhibiting psychobiotic ability in the presence of a prebiotic. The safety of these strains was studied by ascertaining their antibiotic susceptibility, mucin degradation, gelatin hydrolysis, and hemolytic activity. In all, isolates 40C and 11w demonstrated the most desirable probiotic potentials and were identified by 16S RNA and later confirmed by whole genome sequencing as Lacticaseibacillus paracasei 11w, and Lactiplantibacillus plantarum 40C: following with the harboring plasmid investigation. Out of all the 23 selected strains, only Lacticaseibacillus paracasei 11w showed the potential and desirable probiotic properties.

Corrigendum to: Draft genome sequence dataset of Latilactobacillus curvatus PN39MY isolated from fermented vegetables Data in Brief, 49 (2023) 109436.

[This corrects the article DOI: 10.1016/j.dib.2023.109436.].

Draft genome sequence dataset of Latilactobacillus curvatus PN39MY isolated from fermented vegetables.

Here we report the draft genome sequence of the Latilactobacillus curvatus PN39MY strain. The strain was isolated from Lithuanian traditionally (homemade) fermented cucumber. The genome consisted of 83 contigs with a total size of 1,899,018 bp, an N50 of 40562 and a GC% of 42.1. After sequence trimming, 83 contigs were annotated and 1910 genes were coding sequences. The average nucleotide identity (ANI) between PN39MY and Latilactobacillus curvatus_ZJUNIT8 was 99.45% identifying the strain as Latilactobacillus curvatus. No genes related to antimicrobial resistance or virulence factors were found. The data presented here can be used in comparative genomics to identify antimicrobial resistant genes, plasmids and/or virulence factors that may be present in related Latilactobacillus species. The draft genome sequence data was deposited at NCBI under Bioproject with the accession number PRJNA941180.

Insights in MICP dynamics in urease-positive Staphylococcus sp. H6 and Sporosarcina pasteurii bacterium.

Microbially induced calcite precipitation (MICP) is an efficient and eco-friendly technique that has attracted significant interest for resolving various problems in the soil (erosion, improving structural integrity and water retention, etc.), remediation of heavy metals, production of self-healing concrete or restoration of different concrete structures. The success of most common MICP methods depends on microorganisms degrading urea which leads to the formation of CaCO3 crystals. While Sporosarcina pasteurii is a well-known microorganism for MICP, other soil abundant microorganisms, such as Staphylococcus bacteria have not been thoroughly studied for its efficiency in bioconsolidation though MICP is a very important proccess which can ensure soil quality and health. This study aimed to analyze MICP process at the surface level in Sporosarcina pasteurii and a newly screened Staphylococcus sp. H6 bacterium as well as show the possibility of this new microorganism to perform MICP. It was observed that Staphylococcus sp. H6 culture precipitated 157.35 ± 3.3 mM of Ca2+ ions from 200 mM, compared to 176 ± 4.8 mM precipitated by S. pasteurii. The bioconsolidation of sand particles was confirmed by Raman spectroscopy and XRD analysis, which indicated the formation of CaCO3 crystals for both Staphylococcus sp. H6 and S. pasteurii cells. The water-flow test suggested a significant reduction in water permeability in bioconsolidated sand samples for both Staphylococcus sp. H6 and S. pasteurii. Notably, this study provides the first evidence that CaCO3 precipitation occurs on the surface of Staphylococcus and S. pasteurii cells within the initial 15-30 min after exposure to the biocementation solution. Furthermore, Atomic force microscopy (AFM) indicated rapid changes in cell roughness, with bacterial cells becoming completely coated with CaCO3 crystals after 90 min incubation with a biocementation solution. To our knowledge, this is the first time where atomic force microscopy was used to visualize the dynamic of MICP on cell surface.

Effect of Biosynthesized Silver Nanoparticles on the Growth of the Green Microalga Haematococcus pluvialis and Astaxanthin Synthesis.

Silver nanoparticles (AgNPs) are widely known for their antimicrobial activity in various systems from microorganisms to cell cultures. However, the data on their effects on microalgae are very limited. Unicellular green algae Haematococcus pluvialis is known for its ability to accumulate large amounts of astaxanthin under stress conditions. Therefore, it can be used as a suitable model system to test the influence of AgNPs on stress induction in unicellular algae, with visible phenotypic effects, such as astaxanthin synthesis and cell morphology. This study tested different AgNP concentrations (0-8 mg/L) effects on different growth stages (red and green) of H. pluvialis culture. Effects on cell morphology, culture productivity, and astaxanthin synthesis were evaluated. Data showed that the addition of high concentrations of AgNPs to the growing culture had a significant negative impact on culture productivity. Green-stage (HpG) cultures productivity was reduced by up to 85% by increasing AgNPs concentration to 8 mg/L while the impact on red-stage (HpR) culture was lower. Astaxanthin concentration measurements showed that AgNPs do not have any effect on astaxanthin concentration in HpG culture and caused decreased astaxanthin production rate in HpR culture. HpG culture astaxanthin concentration stayed constant at ~0.43% dry weight, while HpR culture astaxanthin concentration was significantly reduced from 1.89% to 0.60% dry weight by increasing AgNP concentration. AgNPs in the media lead to significant changes in cell morphology in both HpG and HpR cultures. Cell deformations and disrupted cytokinesis, as well as AgNPs and induced sexual reproduction, were observed.

Biosynthesis of Silver Nanoparticles Produced Using Geobacillus spp. Bacteria.

Silver nanoparticles (AgNPs) are well known for their unique physical and chemical properties, which can be incorporated into a wide range of applications. The growing resistance of microorganisms to antimicrobial compounds promoted the use of AgNPs in antimicrobial therapy. AgNPs can be obtained using physical and chemical methods, but these technologies are highly unfriendly to nature and produce large amounts of side compounds (for example, sodium borohydride and N,N-dimethylformamide). Therefore, alternative technologies are required for obtaining AgNPs. This report focuses on the biosynthesis of silver nanoparticles through the reduction of Ag+ with the cell-free secretomes of four Geobacillus bacterial strains, namely, 18, 25, 95, and 612. Only a few studies that involved Geobacillus bacteria in the synthesis of metal nanoparticles, including AgNPs, have been reported to date. The silver nanoparticles synthesized through bio-based methods were characterized using UV-Vis spectroscopy, scanning electron microscopy (SEM), dynamic light scattering (DLS), and zeta potential measurements. UV-Vis spectroscopy showed a characteristic absorbance peak at 410-425 nm, indicative of AgNPs. SEM analysis confirmed that most nanoparticles were spherical. DLS analysis showed that the sizes of the obtained AgNPs were widely distributed, with the majority less than 100 nm in diameter, while the zeta potential values ranged from -25.7 to -31.3 mV and depended on the Geobacillus spp. strain.

Mono- and combinational drug therapies for global viral pandemic preparedness.

Broadly effective antiviral therapies must be developed to be ready for clinical trials, which should begin soon after the emergence of new life-threatening viruses. Here, we pave the way towards this goal by reviewing conserved druggable virus-host interactions, mechanisms of action, immunomodulatory properties of available broad-spectrum antivirals (BSAs), routes of BSA delivery, and interactions of BSAs with other antivirals. Based on the review, we concluded that the range of indications of BSAs can be expanded, and new pan- and cross-viral mono- and combinational therapies can be developed. We have also developed a new scoring algorithm that can help identify the most promising few of the thousands of potential BSAs and BSA-containing drug cocktails (BCCs) to prioritize their development during the critical period between the identification of a new virus and the development of virus-specific vaccines, drugs, and therapeutic antibodies.

Phosphate Solubilizing Microorganism Bacillus sp. MVY-004 and Its Significance for Biomineral Fertilizers' Development in Agrobiotechnology.

In this study, a phosphate solubilizing microorganism was isolated from the soil of an agricultural field in Lithuania. Based on 16S rRNA gene sequence analysis, the strain was identified as Bacillus sp. and submitted to the NCBI database, Sector of Applied Bio-catalysis, University Institute of Biotechnology, Vilnius, Lithuania and allocated the accession number KY882273. The Bacillus sp. was assigned with the number MVY-004. The culture nutrient medium and growth conditions were optimized: molasses was used as a carbon source; yeast extract powder was used as an organic source; NH4H2PO4 was used as a nitrogen source; the culture growth temperature was 30 ± 0.5 °C; the initial value of pH was 7.0 ± 0.5; the partial pressure of oxygen (pO2) was 60 ± 2.0; the mixer revolutions per minute (RPM) were 25-850, and the incubation and the fermentation time was 48-50 h. Analysis using Liquid Chromatography Time-of-Flight Mass Spectrometry (LC-TOF/MS) results showed that Bacillus sp. MVY-004 produced organic acids such as citric, succinic, 2-ketogluconic, gluconic, malic, lactic, and oxalic acids. Furthermore, the experiment showed that Bacillus sp. MVY-004 can also produce the following phytohormones: indole-3-acetic (IAA), jasmonic (JA), and gibberellic (GA3) acids. In the climate chamber, the experiment was performed using mineral fertilizer (NPS-12:40:10 80 Kg ha-1) and mineral fertilizers in combination with Bacillus sp. MVY-004 cells (NPS-12:40:10 80 Kg ha-1 + Bacillus sp. MVY-004) in loamy soil. Analysis was performed in three climate conditions: normal (T = 20 °C; relative humidity 60%); hot and dry (T = 30 °C; relative humidity 30%); hot and humid (T = 30 °C; relative humidity 80%).

Synergistic Interferon-Alpha-Based Combinations for Treatment of SARS-CoV-2 and Other Viral Infections.

BACKGROUND: There is an urgent need for new antivirals with powerful therapeutic potential and tolerable side effects. METHODS: Here, we tested the antiviral properties of interferons (IFNs), alone and with other drugs in vitro. RESULTS: While IFNs alone were insufficient to completely abolish replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), IFNα, in combination with remdesivir, EIDD-2801, camostat, cycloheximide, or convalescent serum, proved to be more effective. Transcriptome and metabolomic analyses revealed that the IFNα-remdesivir combination suppressed SARS-CoV-2-mediated changes in Calu-3 cells and lung organoids, although it altered the homeostasis of uninfected cells and organoids. We also demonstrated that IFNα combinations with sofosbuvir, telaprevir, NITD008, ribavirin, pimodivir, or lamivudine were effective against HCV, HEV, FLuAV, or HIV at lower concentrations, compared to monotherapies. CONCLUSIONS: Altogether, our results indicated that IFNα can be combined with drugs that affect viral RNA transcription, protein synthesis, and processing to make synergistic combinations that can be attractive targets for further pre-clinical and clinical development against emerging and re-emerging viral infections.

The Impact of Intensive Fish Farming on Pond Sediment Microbiome and Antibiotic Resistance Gene Composition.

Aquaculture is a fast-growing animal food sector, and freshwater fish farming is particularly common in Central and Eastern Europe. As the biodiversity of fishery ponds is changed toward fulfilling the industrial needs, precautions should be taken to keep the system sustainable and protect the adjacent environment from possible damage. Due to risk of infectious diseases, antibiotics are used in aquaculture production systems. The constant exposure to antimicrobials can contribute to the rise of antibiotic resistance in aquaculture products and the adjacent ecosystems, with possibility of dissemination to the wider environment as well as between animals and humans. Even though previous studies have found antibiotic resistance genes in the sediments and water of farming ponds, the tendency and direction of spreading is not clear yet. The objective of this project was to evaluate the influence of intensive fish farming on the condition of water bodies used for the aquaculture and the environment, concentrating on the impact of the aquaculture on the surrounding water ecosystems as well as the possibility of transferring the pollutants and antibiotic resistance genes to both environment and the human hosts. Combined measurement of antibiotic and heavy metal contamination, toxicity assessment, microorganism diversity, and the detection of common antibiotic resistance genes was performed in the sediments of one fishery farm ponds as well as sampling points upstream and downstream. All the tested sediment samples did not show significantly elevated heavy metal concentrations and no substantial veterinary antibiotic pollution. From the antibiotic resistance genes tested, the presence of aminoglycoside and ß-lactam resistance determinants as well as the presence of integrons could be of concern for the possibility of transfer to humans. However, despite the lack of heavy metal and antibiotic pollution, the sediments showed toxicity, the cause of which should be explored more.

Microbial and Antimicrobial Resistance Profiles of Microbiota in Common Carps (Cyprinus carpio) from Aquacultured and Wild Fish Populations.

In this study we analyzed differences in microbial composition and antimicrobial resistance profiles in common carp living in two different environments: fish ponds, where carp have been kept under the same growing conditions over the last 50 years, and from the wild. The results demonstrated that wild fish carry a great variety of bacterial species (448 species with a prevalence of at least 0.01% from the total number of reads). Aquacultured individuals harbored 2.56 times fewer species in their gut. Significant microbial differences were observed in all taxonomic ranks, including bacterial classes and phyla. Besides bacterial variety, it was determined that aquacultured fish harbored more bacteria that are considered pathogens or opportunistic pathogens, such as Moraxellaceae, Flavobacteriaceae, and Staphylococcaceae. The frequency of antimicrobial resistance in bacterial indicators was more common in aquacultured fish than in wild fish, therefore fish farming may be a potential source of environmental contamination with antimicrobial resistant bacteria.

Study of individual domains' functionality in fused lipolytic biocatalysts based on Geobacillus lipases and esterases.

The prospects of industrial uses of microbial enzymes have increased greatly during the 21st century. Fused lipolytic enzymes (where one or both fused domains possess lipolytic activity) is a rapidly growing group of industrial biocatalysts. However, the most effective fusion strategy, catalytic behavior of each domain and influence of added linkers on physicochemical and kinetic characteristics of such biocatalysts has not been yet explored. In this study the functionality of individual domains in fused lipolytic enzymes, while using GDEst-lip, GDLip-lip and GDEst-est enzymes as a model system, is analyzed for the first time. Analysis of mutant GDEst-lip, GDLip-lip and GDEst-est variants, where one domain is inactive, showed that both domains retained their activity, although the reduction in specific activity of individual domains has been detected. Moreover, experimental data proposed that the N-terminal domain mostly influenced the thermostability, while the C-terminal domain was responsible for thermal activity. GDEst-lip variants fused by using rigid (EAAELAAE) and flexible (GGSELSGG) linkers indicated that a unique restriction site or a rigid linker is the most preferable fusion strategy to develop new chimeric biocatalysts with domains of Geobacillus lipolytic enzymes.

New engineered Geobacillus lipase GD-95RM for industry focusing on the cleaner production of fatty esters and household washing product formulations.

This study presents a new microbial lipolytic enzyme GD-95RM designed via random mutagenesis using previously characterized GD-95 lipase as a template. The improvement in activity of GD-95 lipase was caused by E100K, F154V and V174I mutations. Compared with GD-95 lipase, the GD-95RM lipase had 1.3-fold increased specific activity (2000 U/mg), demonstrated resistance to higher temperatures (75-85 °C), had fourfold increased Vmax towards p-NP dodecanoate and showed 2.5-fold lower KM for p-NP butyrate. It retained > 50% of its lipolytic activity when hydrolyzing short, medium and long acyl chain substrates at 30 °C and 55 °C reaction temperatures after 20 days' incubation with 25% of ethanol. GD-95RM also displayed long-term tolerance (40 d) to 5% NaCl, trisodium citrate, sodium perborate, urea, 0.1% boric acid, citric acid and Triton X-100. Moreover, oil hydrolysis and transesterification results revealed the capability of GD-95RM lipase to produce fatty acids or fatty acid esters through eco-friendly hydrolysis and transesterification reactions using a broad range of vegetable and fish oils, animal fat and different alcohols as substrates. GD-95RM lipase was successfully applied in synthesis reactions for ethyl oleate, octyl oleate and isoamyl oleate without giving to use additional reaction compounds or special reaction conditions.

Development of a new Geobacillus lipase variant GDlip43 via directed evolution leading to identification of new activity-regulating amino acids.

In this study three lipases GD-28, GD-95 and GD-66 (all 43 kDa in size), isolated from Geobacillus spp. were subjected to directed evolution experiments to yield a new synthetic lipolytic enzyme. This new lipase, obtained by DNA shuffling and epPCR, was named GDlip43 (also 43 kDa in size). It demonstrated increased thermoactivity, thermostability, an ability to hydrolyze short and long acyl chain p-NP esters and was activated by different organic solvents. Different activity of GDlip43 raised the hypothesis of new candidate amino acids which could be important for the activity of Geobacillus lipases. Based on the sequence alignment of parental and GDlip43 lipase, three candidate amino acids were selected. The importance of these amino acids, localized at positions 153, 154 and 247 (all of which are distant from the catalytic center of Geobacillus lipases) was investigated using site-directed mutagenesis. Directed evolution experiments also yielded another new lipase - GDlip30 (30 kDa in size). This low molecular mass derivative of GDlip43 had clearly detectable lipolytic activity (40 U/mg) and is the smallest currently known active Geobacillus lipase variant.

Geobacillin 26 - high molecular weight bacteriocin from a thermophilic bacterium.

Bacteriocins are ribosomally synthesized peptides/proteins produced by bacteria. These compounds have antibacterial activity against other bacteria that are usually closely related to the producer strain. Here we describe bacteriocin geobacillin 26 from a thermophilic Gram-positive bacterium Geobacillus stearothermophilus 15. We have purified native bacteriocin, determined its amino acid sequence and heterologously expressed in Gram-negative Escherichia coli. Geobacillin 26 is a heat-labile, high molecular weight antibacterial protein belonging to class III bacteriocins. It has a narrow antibacterial spectrum against other thermophilic bacteria. Our study suggests that this bacteriocin is not a cell wall hydrolyzing enzyme as most of high molecular weight bacteriocins. In addition, geobacillin 26 has no amino acid sequence similarities to other known function proteins. No other class III bacteriocin from a thermophilic bacterium has been reported and well characterized before. Geobacillin 26 as a natural antibacterial agent has a great potential in industry where contamination with other thermophilic bacteria is unwanted. Moreover, this study may prompt to disclose more novel geobacillin 26-like antibacterial proteins, which could find their applications in food industry or medicine.

Low concentrations of acetic and formic acids enhance the inactivation of Staphylococcus aureus and Pseudomonas aeruginosa with pulsed electric fields.

BACKGROUND: Skin infections, particularly caused by drug-resistant pathogens, represent a clinical challenge due to being a frequent cause of morbidity and mortality. The objectives of this study were to examine if low concentrations of acetic and formic acids can increase sensitivity of Staphylococcus aureus and Pseudomonas aeruginosa to pulsed electric field (PEF) and thus, promote a fast and efficient treatment methodology for wound treatment. RESULTS: We have shown that the combination of PEF (10-30 kV/cm) with organic acids (0.1% formic and acetic acids) increased the bactericidal properties of treatment. The effect was apparent for both acids. The proposed methodology allowed to reduce the energy of electrical pulses and the inhibitory concentrations of acids, while still maintain high efficiency of bacteria eradication. CONCLUSIONS: Application of weak organic acids as bactericidal agents has many advantages over antibiotics because they do not trigger development of drug-resistance in bacteria. The combination with PEF can make the treatment effective even against biofilms. The results of this study are particularly useful for the development of new methodologies for the treatment of extreme cases of wound infections when the chemical treatment is no longer effective or hinders wound healing.

Nanosecond duration pulsed electric field together with formic acid triggers caspase-dependent apoptosis in pathogenic yeasts.

Antifungal substances that are used for the treatment of candidiasis have considerable side effects and Candida yeasts are known to obtain drug resistance. The multidrug resistance cases are promoting the search for the new alternative methods and pulsed electric field (PEF) treatment could be the alternative or could be used in combination with conventional therapy for the enhancement of the effect. We have shown that nanosecond range PEF is capable to induce apoptosis in the S. cerevisiae as well as in the drug resistant C. lusitaniae and C. guilliermondii. Supplementing the PEF procedure with formic acid (final concentration 0.05%) resulted in improvement of the inactivation efficacy and the induction of apoptosis in the majority of the yeast population. After the treatment yeast were displaying the DNA strand brakes, activation of yeast metacaspase and externalization of phosphatidylserine. Apoptotic phenotypes were registered already after 30 kV/cm × 250 ns × 50 pulses treatment. The highest number of apoptotic yeast cells (>60%) was obtained during the 30 kV/cm × 750 ns × 50 pulses protocol when combined with 0.05% formic acid. The results of our study are useful for development of new non-toxic and effective protocols to induce programed cell death in different yeast species and thus minimize inflammation of the tissue.

Non-invasive nanosecond electroporation for biocontrol of surface infections: an in vivo study.

Invasive infections caused by drug-resistant bacteria are frequently responsible for fatal sepsis, morbidity and mortality rates. In this work, we propose a new methodology based on nanosecond high frequency electric field bursts, which enables successful eradication of bacteria in vivo. High frequency (15 kHz) 15-25 kV/cm 300-900 ns pulsing bursts were used separately and in combination with acetic acid (0.1-1%) to treat Pseudomonas aeruginosa in a murine model. Acetic acid 1% alone was effective resulting in almost 10-fold reduction of bacteria viability, however combination of nanosecond electric field and acetic acid 1% treatment was the most successful showing almost full eradication (0.01% survival compared to control) of the bacteria in the contaminated area. The short duration of the pulses (sub-microsecond) and high frequency (kHz range) of the burst enabled reduction of the muscle contractions to barely detectable level while the proposed applicators ensured predominantly topical treatment, without electroporation of deeper tissues. The results of our study have direct application for treatment of wounds and ulcers when chemical treatment is no longer effective.

Usage of GD-95 and GD-66 lipases as fusion partners leading to improved chimeric enzyme LipGD95-GD66.

Lipases are used as biocatalysts in industrial processes mainly because of their stability at broad temperature and pH range, resistance to organic solvents and wide spectrum of substrates. The usage of several lipolytic domains, each with different activity and resistance profiles, enables both the flexibility and efficiency of industrial processes. In this study, GD-95 and GD-66 lipases produced by Geobacillus sp. 95 and Geobacillus sp. 66, respectively, were used as fusion partners to create a new fused lipolytic enzyme LipGD95-GD66. Chimeric LipGD95-GD66 lipase displayed tenfold increase in activity (200 U/mg) compared to parental GD-66 lipase, improved Vmax (10 µmol/min mg-1) and catalytic efficiency (2 ∗ 105 min-1 mM-1) for p-NP palmitate as a substrate and increased activity at 70-75 °C compared to both parental lipases. All three lipases also retained >50% of their lipolytic activity after incubation with methanol, n-hexane, ethanol and DMF for longer than three weeks, highlighting a great prospect for application in industrial processes. Moreover, transesterification results revealed the capability of parental GD-95 lipase to be the most promising biocatalyst for production of methyl and ethyl esters through eco-friendly transesterification using argan oil and ethanol/methanol as acceptors of acyl group.

Induction of Different Sensitization Patterns of MRSA to Antibiotics Using Electroporation.

Treatment of bacteria-associated infections is complicated and antibiotic treatment alone is often inadequate to overcome biofilm infections. Physical methods allow overcoming this problem and propose solutions that are non-dependent on drug resistance. In this work, we investigated the feasibility of pulsed electric fields for sensitization of MRSA to common antibiotics. We analyzed the efficacy of inactivation of methicillin-resistant Staphylococcus aureus in 5⁻20 kV/cm electric field separately and in combination with gentamicin, doxycycline, ciprofloxacin, sulfamethoxazole, and vancomycin. Combined treatment allowed using up to 1000-fold smaller concentrations of antibiotics to induce the same inactivation of S. aureus.