Growth Medium Research Articles

The comparison of eight different common in vitro and ex vivo environments with in vivo conditions applying model collagen samples: Correlation possibilities and their limits

New biomaterials are routinely evaluated for their degradation behaviour in the real body environment. Following the 3R strategy, in vitro simulated body conditions are often preferred. No studies that simultaneously compare such conditions with the real body environment have been conducted to date. Model porous collagen scaffolds were exposed for 21 days to eight different environments: simple salt-based and enzymatic media, human blood plasma, cell culture media with and without human fibroblasts and ex vivo model cortical bone, and subsequently compared with an in vivo environment represented by a pig peritoneum. The mechanical properties of the scaffolds were then determined via uniaxial compression testing, and the structural properties via the micro-CT, weight loss, infrared spectroscopy, X-ray diffraction and histological methods. Interestingly, the various analysed simulated body conditions caused differing alterations in the collagen scaffold characteristics when compared with the real body environment. The mechanical properties were similar during the first 7 days of incubation but diverged after 14 and 21 days. The structural properties varied significantly after just 7 days of incubation. The histological evaluation of the scaffolds exposed to the cellular, ex vivo and in vivo conditions revealed the poor ability of cells to completely populate the scaffolds, accompanied by the massive ingrowth of connective tissue into the in vivo exposed scaffolds, which resulted in their variable global behaviour. In conclusion, the value of in vitro simulated body environments lies in their screening capacity and feasibility; however, direct extrapolation to real body conditions needs to be verified going forward.

Inactivation of Escherichia coli O157:H7 in tomato juice by combined treatment of ethanolic clove extract and mild heat

The combination of plant extracts with mild heat treatment is a promising strategy for obtaining microbiologically and chemically safe food products. In this study, we evaluated the antimicrobial activity of ethanolic clove extract against Escherichia coli O157: H7 in nutrient broth, buffer, and tomato juice. Clove extract at 0.4% concentration showed significant E. coli O157: H7 population reduction in all media tested. E. coli O157: H7 was most sensitive to the ethanolic clove extract in nutrient-deficient media but demonstrated relative resistance in nutrient-rich media and tomato juice. Moreover, we evaluated the potential of clove extract as a preservative in tomato juice stored at cold storage temperatures of 4 °C and 15 °C. The clove extract exhibited stronger inhibitory effects against the pathogen in tomato juice stored at 15 °C than at 4 °C. Additionally, we assessed the antimicrobial efficacy of clove extract in combination with mild heat treatment against E. coli O157: H7 in tomato juice. The combined treatment of 0.05% clove extract with mild heat at 60 °C for 30 min achieved a 5-log reduction in the E. coli population in tomato juice. Our findings present a potential hurdle technology against E. coli O157: H7 in tomato juice processing by employing a combination of naturally sourced antimicrobial agents from clove and mild heat treatment. This approach can be effectively adopted by large-scale industries and small-scale local juice vendors to ensure the safety of tomato juice products.

Cisplatin-resistance and aggressiveness are enhanced by a highly stable endothelin-converting enzyme-1c in lung cancer cells

BackgroundLung cancer constitutes the leading cause of cancer mortality. High levels of endothelin-1 (ET-1), its cognate receptor ETAR and its activating enzyme, the endothelin-converting enzyme-1 (ECE-1), have been reported in several cancer types, including lung cancer. ECE-1 comprises four isoforms, which only differ in their cytoplasmic N-terminus. Protein kinase CK2 phosphorylates the N-terminus of isoform ECE-1c, increasing its stability and leading to enhanced invasiveness in glioblastoma and colorectal cancer cells, which is believed to be mediated by the amino acid residue Lys-6, a conserved putative ubiquitination site neighboring the CK2-phosphorylated residues Ser-18 and Ser-20. Whether Lys-6 is linked to the acquisition of a cancer stem cell (CSC)-like phenotype and aggressiveness in human non–small cell lung cancer (NSCLC) cells has not been studied.MethodsIn order to establish the role of Lys-6 in the stability of ECE-1c and its involvement in lung cancer aggressiveness, we mutated this residue to a non-ubiquitinable arginine and constitutively expressed the wild-type (ECE-1cWT) and mutant (ECE-1cK6R) proteins in A549 and H1299 human NSCLC cells by lentiviral transduction. We determined the protein stability of these clones alone or in the presence of the CK2 inhibitor silmitasertib, compared to ECE-1cWT and mock-transduced cells. In addition, the concentration of secreted ET-1 in the growth media was determined by ELISA. Expression of stemness genes were determined by Western blot and RT-qPCR. Chemoresistance to cisplatin was studied by MTS viability assay. Migration and invasion were measured through transwell and Matrigel assays, respectively, and the side-population was determined using flow cytometry.ResultsECE-1cK6R displayed higher stability in NSCLC cells compared to ECE-1cWT-expressing cells, but ET-1 secreted levels showed no difference up to 48 h. Most importantly, ECE-1cK6R promoted expression of the stemness genes c-Myc, Sox-2, Oct-4, CD44 and CD133, which enhance cellular self-renewal capability. Also, the ECE-1cK6R-expressing cells showed higher cisplatin chemoresistance, correlating with an augmented side-population abundance due to the increased expression of the ABCG2 efflux pump. Finally, the ECE-1cK6R-expressing cells showed enhanced invasiveness, which correlated with the regulated expression of known EMT markers.ConclusionsOur findings suggest an important role of ECE-1c in lung cancer. ECE-1c is key in a non-canonical ET-1-independent mechanism which triggers a CSC-like phenotype, leading to enhanced lung cancer aggressiveness. Underlying this mechanism, ECE-1c is stabilized upon phosphorylation by CK2, which is upregulated in many cancers. Thus, phospho-ECE-1c may be considered as a novel prognostic biomarker of recurrence, as well as the CK2 inhibitor silmitasertib as a potential therapy for lung cancer patients.

M6A-modified exosome-derived circHIF1α binding to KH domain of IGF2BP3 mediates DNA damage and arrests G1/S transition phase to resists bacterial infection in bacteremia

BackgroundAnimal and human health are seriously threatened by bacterial infections, which can lead to bacteremia and extremely high rates of morbidity and mortality. Recently, there have been reports indicating the involvement of exosomal circular RNAs (circRNAs) in a range of human disorders and tumor types. However, the role of exosomal circRNAs in bacterial infection remains elusive.MethodsWe extracted and identified exosomes from the culture medium of PIEC cells infected with or without Glaesserella parasuis. RNA sequencing analysis was performed on the exosomes to screen and identify circRNAs (circHIF1α) associated with Glaesserella parasuis infection. PIEC cells were infected with Staphylococcus aureus or Streptococcus suis 2 to further determine whether exosome-derived circHIF1α was the crucial circHIF1α associated with bacterial infections. The transmission process of exosomes and their circHIF1α between cells was clarified via exosome tracing and co-culture assay. Moreover, the mechanism of circHIF1α being packaged into exosomes was explored, and the effects of exosomes and their circHIF1α on cell proliferation, DNA damage and cell cycle were analyzed. In addition, the binding mode and site of interacting proteins with circHIF1α were further determined. In vivo and in vitro, the role of exosomes and their circHIF1α in host resistance to bacterial infection was confirmed.ResultsWe first discovered a new circHIF1α that was very stable and detectable, encapsulated into exosomes by hnRNPA2B1, and whose expression in exosomes of bacterially infected PIEC cells significantly decreased. Additionally, exosomal circHIF1α reduced bacterial infection both in vitro and in vivo and suppressed the growth of reception cells. Mechanistically, the circHIF1α interacted with the KH domain of IGF2BP3 in an m6A-modified manner, which mediated DNA damage to arrest the cells at the G1/S phase through the interaction between the regulator of Chromosome Condensation 2 (RCC2) and γ-H2AX protein. Exosomal circHIF1α is a unique therapeutic target for bacterial infection since this work highlights its critical function in fighting bacterial infection.

Stereorandomized Oncocins with Preserved Ribosome Binding and Antibacterial Activity.

We recently showed that solid-phase peptide synthesis using racemic amino acids yields stereorandomized peptides comprising all possible diastereomers as homogeneous, single-mass products that can be purified by HPLC and that stereorandomization modulates activity, toxicity, and stability of membrane-disruptive cyclic and linear antimicrobial peptides (AMPs) and dendrimers. Here, we tested if stereorandomization might be compatible with target binding peptides with the example of the proline-rich AMP oncocin, which inhibits the bacterial ribosome. Stereorandomization of up to nine C-terminal residues preserved ribosome binding and antibacterial effects including activities against drug-resistant bacteria and protected against serum degradation. Surprisingly, fully stereorandomized oncocin was as active as L-oncocin in dilute growth media stimulating peptide uptake, although it did not bind the ribosome, indicative of an alternative mechanism of action. These experiments show that stereorandomization can be compatible with target binding peptides and can help understand their mechanism of action.

Reappraisal of different agro-industrial waste for the optimization of cellulase production from Aspergillus niger ITV02 in a liquid medium using a Box-Benkhen design.

High-value metabolites, such as enzymes and biofuels, can be produced from various agro-industrial waste containing high percentages of cellulose and hemicellulose. Aspergillus niger ITV02 demonstrates high potential in cellulases production, the key enzyme for converting lignocellulosic materials into fermentable sugars to produce second-generation bioethanol (bioethanol 2G). This study evaluated five lignocellulosic residues of agricultural importance: sugarcane bagasse (SCB), sorghum bagasse (SB), corn stubble (CS), barley straw (BS) and rice husk (RH) as substrates for cellulase production. The temperature, pH and stirring conditions were optimized using a Box-Behnken design to identify the most suitable conditions for cellulase production while minimizing nitrogen concentrations. The results indicate that the best way of propagation A. niger ITV02 is through the use of spores as an inoculant, in conjunction with the use of materials with a high cellulose/lignin ratio, such as CS and SB for the generation of cellulases. These conditions promote the expression of cellulases towards β-glucosidase production, unlike materials with lower cellulose/lignin ratios like BS and RH, which exhibited lower cellulase activity. The optimal conditions for cellulase production by A. niger ITV02 were determined to be 33 °C, pH 5.3, and 200rpm, resulting in a 1.7-fold increase in Exoglucanase (FPase activity) (from 0.127 to 0.215 U/mL). These findings demonstrate the potential to enhance FPase activity by utilizing substrates with high cellulose/lignin content and implementing optimal operational conditions without the need to raise the nitrogen content of the basal medium, thus mitigating the economic impact of cellulase production.

Effect of Fermentation by Probiotic Bacteria on the Bioaccessibility of Bioactive Compounds from the Fruit of the Juçara Palm (Euterpe edulis Martius)

The underexplored fruit from the juçara palm tree (Euterpe edulis Martius) has bioactive compounds with antioxidant activities, such as phenolic acids and anthocyanins. This fruit’s pulp presents itself as an appropriate fermentation medium for probiotic bacteria growth. Therefore, this study was conducted to evaluate the effects of fermentation by Limosilactobacillus reuteri LR92 (JLR) and Bifidobacterium animalis ssp. lactis BB-12 (JBB) on the bioactive compound contents of the juçara pulp, before and after a gastrointestinal simulation. The pulp of the juçara fruit showed probiotic counts of 8.70 ± 0.07 log UFC/mL for JLR and 8.44 ± 0.09 log UFC/mL for JBB, after 24 h of fermentation. Fermentation with the strains used modified the proportions of fatty acids (fatty acids esters were quantified using a gas chromatography equipment) and fibers when compared to the non-fermented pulp. The antioxidant capacity determined by DPPH, FRAP and ABTS showed significant reduction after the gastrointestinal simulation for samples. Phenolic compound analysis by UPLC-MS/MS showed, after fermentation, a greater amount of ferulic, protocatechuic and catechin acids in the samples. These results show changes in the bioactive compounds due to the fermentation of the juçara pulp by probiotics. However, these compounds showed bioactive potential and were bioaccessible after the gastrointestinal simulation, with the pulp being a potential means for bacteria growth, which may bring health benefits.

Characterization of Extracellular Vesicles by Sulfophosphovanillin Colorimetric Assay and Raman Spectroscopy.

Detailed characterization of extracellular vesicles (EVs) is crucial for their application in medical diagnostics. However, the complexity of their chemical composition and the heterogeneity of EV populations make their characterization challenging. Here we describe two analytical procedures that can help overcome this challenge. Small EVs were isolated from conditioned cell culture media using ultracentrifugation and characterized using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Raman spectroscopy was used to assess the overall composition of the isolated samples and lipids extracted from them. Sulfophosphovanillin (SPV) colorimetric assay was used to quantify the contents of lipid. Six samples of EVs were characterized. The lipid contents measured using SPV assay was in reasonable agreement with the quantitative estimates based on the particle size and concentration measured using NTA. The most peaks observed in the Raman spectra could be attributed to either proteins or lipids, and their origins was confirmed by lipid extraction. The protein-to-lipid ratio was estimated based on the Raman spectra. The experiential procedures described in this study will help to overcome the challenge of quick and highly informative characterization of the EVs.

Fpa (YlaN) is an iron(II) binding protein that functions to relieve Fur-mediated repression of gene expression in Staphylococcus aureus.

Iron (Fe) is a trace nutrient required by nearly all organisms. As a result of the demand for Fe and the toxicity of non-chelated cytosolic ionic Fe, regulatory systems have evolved to tightly balance Fe acquisition and usage while limiting overload. In most bacteria, including the mammalian pathogen Staphylococcus aureus, the ferric uptake regulator (Fur) is the primary transcriptional regulator controlling the transcription of genes that code for Fe uptake and utilization proteins. Fpa (formerly YlaN) was demonstrated to be essential in Bacillus subtilis unless excess Fe is added to the growth medium, suggesting a role in Fe homeostasis. Here, we demonstrate that Fpa is essential in S. aureus upon Fe deprivation. Null fur alleles bypassed the essentiality of Fpa. The absence of Fpa abolished the derepression of Fur-regulated genes during Fe limitation. Bioinformatic analyses suggest that fpa was recruited to Gram-positive bacteria and, once acquired, was maintained in the genome as it co-evolved with Fur. Consistent with a role for Fpa in alleviating Fur-dependent repression, Fpa and Fur interacted in vivo, and Fpa decreased the DNA-binding ability of Fur in vitro. Fpa bound Fe(II) in vitro using oxygen or nitrogen ligands with an association constant that is consistent with a physiological role in Fe homeostasis. These findings have led to a model wherein Fpa is an Fe(II) binding protein that influences Fur-dependent regulation through direct interaction.IMPORTANCEIron (Fe) is an essential nutrient for nearly all organisms. If Fe homeostasis is not maintained, Fe may accumulate in the cytosol, which can be toxic. Questions remain about how cells efficiently balance Fe uptake and usage to prevent overload. Iron uptake and proper metalation of proteins are essential processes in the mammalian bacterial pathogen Staphylococcus aureus. Understanding the gene products involved in the genetic regulation of Fe uptake and usage and the physiological adaptations that S. aureus uses to survive in Fe-depleted conditions provides insight into pathogenesis. Herein, we demonstrate that the DNA-binding activity of the ferric uptake regulator transcriptional repressor is alleviated under Fe limitation, but uniquely, in S. aureus, alleviation requires the presence of Fpa.

Enhancing the volatile organic compound and biomass production by three biocontrol potential bacteria in corn steep liquor growth medium and development of cell freeze-drying process.

This study investigates the traits of three plant growth-promoting (PGP) and antagonistic bacteria, Pseudomonas protegens MP12, Bacillus sp. 3R4, and Bacillus sp. T22, to assess their potential application as biocontrol agents by using the ecofriendly and low-cost substrate Corn Steep Liquor (CSL) medium. Analyses of antagonism through volatile organic compounds (VOCs) production, biofilm formation, and growth performance were carried out. Dual antagonism assay showed that all strains displayed significant antagonistic activity against Botrytis cinerea through VOCs. Gas chromatography demonstrated that strains in the CSL exhibited higher VOCs production than Nutrient medium. Moreover, enhanced biofilm formation analysed by Calgary Biofilm Device, growth and biomass were noted in CSL cultures. Pseudomonas protegens MP12, which showed higher cell concentration and biomass yield, was selected for freeze-drying treatments. Storage cell viability assays evidenced that it can be effectively preserved for nearly 7 months at 4°C. The results here obtained showed that CLS medium enhance VOCs production, biofilm formation, growth and biomass of the antagonistic bacteria of the three strains. Eventually, the more effective strain P. protegens MP12 can be stored for nearly 7 months at 4°C.

Individual and combined effects of ionophore antibiotics monensin and lasalocid on growth, oxidative stress and antioxidant responses of Microcystis aeruginosa

ABSTRACT To assess the impact of monensin, lasalocid, and a 1:1 (w/w) mixture of monensin and lasalocid on the formation of Microcystis aeruginosa-based harmful algal blooms (HABs), laboratory experiments were conducted [temperature 20 ± 1°C, illumination PAR 30 ± 4 μmol/m2 ·s (12-h light/dark cycle), growth medium BG-11] at concentrations of 100, 200, 500, 1,000, and 2,000 μg/L. Measurements included optical density, cell protein content, chlorophyll ‘a’ (Chl. a) content, oxidative stress, catalase activity (CAT), and guaiacol peroxidase activity (GPX). Monensin treatment showed a dose-dependent positive effect on M. aeruginosa growth at concentrations ≤500 μg/L and a gradual dose-dependent growth inhibition at concentrations of 1,000 and 2,000 μg/L. The results indicated that monensin has a significant positive effect on the formation of HABs by M. aeruginosa. Lasalocid treatment showed a growth reduction of M. aeruginosa at concentrations ≤500 μg/L and a growth increase at higher concentrations (1,000 and 2,000 μg/L). The monensin and lasalocid 1:1 (w/w) mixture test showed an intermediate response, as indicated by the individual treatments signifying the potential interactive effects of these antibiotics on the growth of M. aeruginosa. Furthermore, alterations in Chl. ‘a’, oxidative stress, CAT, and GPX measurements provided evidence of the impact of these antibiotics on the existence of M. aeruginosa.

Investigation of FF-MAS oxysterole’s role in follicular development and its relation to hedgehog signal pathway

The Hedgehog signaling pathway plays a crucial role in folliculogenesis; however, the association between FF-MAS oxysterol activity in folliculogenesis and the Hedgehog signaling pathway has not been revealed. The evaluation of FF-MAS activity in polycystic ovary syndrome (PCOS) with folliculogenesis disorder might provide a new approach to tackle follicular and oocyte maturation failure. The question is: does FF-MAS oxysterol affect granulosa cell (GC) proliferation? If so, is this effect facilitated through the Hedgehog pathway? To answer these questions, GCs were isolated from follicle fluids obtained from patients undergoing oocyte retrieval during in vitro fertilization (IVF) treatment. After the isolated GCs were incubated in different cell culture media, the levels of Hedgehog pathway components (SMO, Gli1) were measured by using immunohistochemical methods, cytoELISA, and qRT-PCR. Meanwhile, cell proliferation rates were determined. Significant increases (p < 0.001) in SMO and Gli1 expressions and cell proliferation were observed in the FF-MAS-treated subgroups of both PCOS and male factor participants compared to the FF-MAS deficient subgroup. Remarkably, FF-MAS positively affected the pathway components despite the pathway inhibitor cyclopamine. Although the increase in Hedgehog pathway components was slightly higher in the male factor group (MF), it was not statistically significant. In our study, we demonstrated for the first time the molecular effect of FF-MAS on human GCs in folliculogenesis. Since FF-MAS is already used in assisted reproductive techniques in animals and is known to be synthesized in the human body, it could be considered a new approach in human IVF treatments.

Choline chloride and N-allylglycine promote plant growth by increasing the efficiency of photosynthesis.

We previously reported that choline chloride and N-allylglycine stimulate photosynthesis in wheat protoplasts. Treatment of Arabidopsis thaliana and Brassica rapa plants with both compounds promoted growth and photosynthesis. To clarify the relationship between the enhancement of photosynthesis and increased growth, A. thaliana T87 cells, which show photosynthesis-dependent growth, and YG1 cells, which use sugar in the medium for growth, were treated with choline chloride or N-allylglycine. Only the T87 cells showed increased growth, suggesting that choline chloride and N-allylglycine promote growth by increasing photosynthetic activity. Transcriptome analysis using choline chloride and N-allylglycine-treated plants showed that the most abundant transcripts corresponded to photosynthetic electron transfer-related genes among the genes upregulated by both compounds. Furthermore, the compounds also upregulated genes encoding transcription factors that may control the expression of these photosynthetic genes. These results suggest that choline chloride and N-allylglycine promote photosynthesis through increased expression of photosynthetic electron transfer-related genes.

Assessment of the Quality, Bioactive Compounds, and Antimicrobial Activity of Egyptian, Ethiopian, and Syrian Black Cumin Oils.

The oils obtained from the seeds of Nigella sativa, also named black cumin, are rich in bioactive compounds that strengthen immunity and support human health. This study aimed to compare Nigella sativa oils pressed from Egyptian (Eg-NSSO), Ethiopian (Et-NSSO), and Syrian (Sy-NSSO) seeds. The analyzed oils were obtained from a local company. The content of phenolic compounds, tocochromanols, phytosterols, volatile compounds, triglycerides, and fatty acids composition was determined using chromatographic methods. The oxidative stability was determined by Rancimat technique as well as the determination of DPPH and ABTS scavenging activity. As an assessment of bioactivity, the antimicrobial and anti-aflatoxigenic properties of oils were evaluated. Ethiopian oil had highest content of phenolic compounds, flavonoids, phytosterols, and tocochromanols and was characterized by the longest induction period (IP = 7.89 h). The share of thymoquinone was the highest in Ethiopian oil (34.84%), followed by Egyptian (27.36%), then Syrian (22.59%). Ethiopian oil recorded a high antibacterial activity, while Egyptian oil showed a unique antifungal activity against toxigenic fungi. Aflatoxins' secretion into liquid medium containing NSSO was reduced, especially with Egyptian oil.

High-Throughput Discovery of Synthetic Siderophores for Trojan Horse Antibiotics.

To cause infection, bacterial pathogens must overcome host immune factors and barriers to nutrient acquisition. Reproducing these aspects of host physiology in vitro has shown great promise for antibacterial drug discovery. When used as a bacterial growth medium, human serum replicates several aspects of the host environment, including innate immunity and iron limitation. We previously reported that a high-throughput chemical screen using serum as the growth medium enabled the discovery of novel growth inhibitors overlooked by conventional screens. Here, we report that a subset of compounds from this high-throughput serum screen display an unexpected growth enhancing phenotype and are enriched for synthetic siderophores. We selected 35 compounds of diverse chemical structure and quantified their ability to enhance bacterial growth in human serum. We show that many of these compounds chelate iron, suggesting they were acting as siderophores and providing iron to the bacteria. For two different pharmacophores represented among these synthetic siderophores, conjugation to the β-lactam antibiotic ampicillin imparted iron-dependent enhancement in antibacterial activity. Conjugation of the most potent growth-enhancing synthetic siderophore with the monobactam aztreonam produced MLEB-22043, a broad-spectrum antibiotic with significantly improved activity against Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa. This synthetic siderophore-monobactam conjugate uses multiple TonB-dependent transporters for uptake into P. aeruginosa. Like aztreonam, MLEB-22043 demonstrated activity against metallo-β-lactamase expressing bacteria, and, when combined with the β-lactamase inhibitor avibactam, was active against clinical strains coexpressing the NDM-1 metallo-β-lactamase and serine β-lactamases. Our work shows that human serum is an effective bacterial growth medium for the high-throughput discovery of synthetic siderophores, enabling the development of novel Trojan Horse antibiotics.

Valorization of abattoir water discharge through phycoremediation for enhanced biomass and biodiesel production

Phycoremediation has been discussed as a multipurpose strategy to refine various waste streams for sustainable algal biomass, value-added compounds, and biodiesel production. While phycoremediation has been explored for various wastewaters, there is still a research gap in systematical understanding the impact of abattoir water discharge (AWD) on both algal growth and biodiesel quality. The present study evaluated the potential of Chlorella sorokiniana to grow at different ratios of AWD, 30–90 %, v/v with conventional growth medium. Results showed that nutrient removal efficiency after 12 days of cultivation reached up to 85 % for total nitrogen, 78 % for total phosphorus, 70 % for potassium, and 65 % for sodium. Thus, all algae-treated AWD samples showed Kelly ratio (KR) values less than 1, confirming their suitability for direct application in soil irrigation. In addition, there were insignificant variations in algal growth and biomass yield across all treatments. Interestingly, carotenoids yield showed significant increase in all AWD ratios, with the highest recorded value of 0.750 mg L−1 at day 12 using 90 % AWD, compared to 0.523 mg L−1 in the control. Among various treatments, C. sorokiniana grown in 90 % AWD exhibited the highest lipid content of 201.6 mg g−1 algal dw (comparing to 180.6 mg g−1 dw in the control). The biodiesel derived from C. sorokiniana at different AWD ratios demonstrated promising characteristics due to enhancement of saturated fatty acids proportion, which resulted in lower iodine value, longer oxidation stability, and higher cetane number than the control.

Whole genome sequence analysis of multi-drug resistant and biofilm-forming Staphylococcus haemolyticus isolated from bovine milk

BackgroundMilk is an excellent growth medium for microorganisms due to its nutritive composition. Microorganisms have been implicated in bovine mastitis (BM) in dairy cows as well as causing infections in animals and humans. Despite extensive endeavours to manage BM, this condition continues to persist as the most prevalent and economically burdensome problem affecting dairy cattle on a global scale. Non-aureus staphylococci (NAS) species such as Staphylococcus haemolyticus, S. epidermidis, and S. xylosus are currently the predominant microbiological agents identified as the main cause of subclinical udder infections and are also considered opportunistic pathogens in cases of clinical mastitis in dairy cows. Therefore, it is crucial to elucidate the genetic profile of these species. The primary objective of this study was to characterise three phenotypically determined multidrug-resistant NAS environmental strains (NWU MKU1, NWU MKU2, and NWU MKS3) obtained from dairy cows milk via whole-genome sequencing.ResultsThe results confirmed that the three isolates were S. haemolyticus with genome sizes of 2.44, 2.56, and 2.56 Mb and a G + C content of 32.8%. The genomes contained an array of antibiotic resistance genes that may potentially confer resistance to a range of antibiotic classes, such as macrolides, fluoroquinolones, aminoglycosides, cephalosporins, tetracyclines, peptides, and phenicol. Furthermore, all the genomes carried virulence genes, which are responsible for several functions, such as adhesion, enzyme and toxin production. The genomes of these organisms contained signatures encoding mobile genetic elements such as prophages and insertion sequences.ConclusionThese findings indicate there is a need for diligent monitoring with improved management practices and quality control strategies on farms to safeguard milk production systems and human health.

Formation of treatment regimens in newly diagnosed patients before the diagnosis of pulmonary tuberculosis verification

Abstract. One of the basic principles of treatment of tuberculosis patients is the appointment of treatment taking into account the drug resistance of Mycobacterium tuberculosis (MBT). However, during treatment, it may be necessary to change the intake regimen when new data on drug resistance become available. The search for ways to choose the optimal treatment method seems relevant.Aim: To analyze the prescribed regimens and the reason for their correction in newly identified patients before verifying the diagnosis of tuberculosis.Material and Methods. The following methods were used to detect MBT: luminescent microscopy, molecular genetic methods and seeding on liquid and solid media. Tests for drug sensitivity were conducted in parallel. The study group consisted of patients with established bacterial excretion (n = 79), whose diagnosis was confirmed by cultural methods. A retrospective method was used in the analysis.Results. At the beginning of treatment with bacterioscopy and polymerase chain reaction methods MBT was detected in 65/79 (82.2%) cases, the remaining 14 cases were detected only by culture methods. Forced change of the therapy regimen during treatment after receiving the results was in 25/79 (31.6%) cases, of which 1/25 (4%) with isoniazid resistance, 7/25 (28%) with multidrug-resistant tuberculosis, 17/25 (68%) with the pre-extensive drug resistance regimen. The latter was prescribed only after receiving results of tests for drug sensitivity on liquid and solid media after 1–3 months. The multidrug-resistant tuberculosis treatment regimen decreased after receiving data on resistance to fluoroquinolones in accordance with the seeding data, but the largest number of patients still remained on this regimen – 41/79 (51.9%). Among these patients a change in chemotherapy regimen from multidrug-resistant tuberculosis to pre-extensive drug resistance during the intensive phase of treatment occurred in 17/25 (68%).Conclusion. When prescribing a regimen, it is recommended to assess the risk of changing the regimen and, if there are predictors, apply a chemotherapy regimen before treatment.

Antibacterial activity of Savory (Satureja hortensis) extract against pure culture strains of

The paper presents the results of a study of the antibacterial activity of supercritical carbon dioxide extract of the aboveground part of Savory (Satureja hortensis) against Escherichia coli, Salmonella spp. and Staphylococcus aureus bacteria.To study the antibacterial properties of Savory extract (Satureja hortensis) against infections caused by Staphylococcus aureus, Escherichia coli and Salmonella spp.In the course of the experiment, Savory extract obtained by supercritical carbon dioxide extraction was studied for antimicrobial activity against these bacteria. The component composition of the extract was determined by chromatography–mass spectrometry. To determine the antibacterial activity, we used the method of dilution in a liquid medium with subsequent measurement of the growth of the suspension culture biomass. The activity of the extract was compared with certified drugs included in the protocols for the treatment of bacterial infections. DMSO and potassium phosphate buffer were used as a negative control.The studies showed that the extract exhibits high antibacterial activity against all the studied bacterial strains and has a direct positive correlation with its concentration. Chromatographic mass‐spectrometry analysis showed that the extract of garden Savory contains 28 substances, the main ones being thymol (30.51 %), gamma‐terpinene (15.27 %), para‐ cymene (14.25 %) and Carvacrol (9.18 %), which both individually and in combination could exhibit antibacterial activity.Supercritical carbon dioxide extract exhibits pronounced antimicrobial activity against reference strains of S. aureus, Escherichia coli and Salmonella spp.

Combining culture optimization and synthetic biology to improve production and detection of secondary metabolites in Myxococcus xanthus: application to myxoprincomide.

Microbial secondary metabolites play crucial ecological roles in governing species interactions and contributing to their defense strategies. Their unique structures and potent bioactivities have been key in discovering antibiotics and anticancer drugs. Genome sequencing has undoubtedly revealed that myxobacteria constitute a huge reservoir of secondary metabolites as the well-known producers, actinomycetes. However, because most secondary metabolites are not produced in the laboratory context, the natural products from myxobacteria characterized to date represent only the tip of the iceberg. By combining the engineering of a dedicated Myxococcus xanthus DZ2 chassis strain with a two-step growth medium protocol, we provide a new approach called two-step Protocol for Resource Integration and Maximization-Biomolecules Overproduction and Optimal Screening Therapeutics (2PRIM-BOOST) for the production of non-ribosomal peptides synthetases (NRPS)/polyketides synthases (PKS) secondary metabolites from myxobacteria. We further show that the 2PRIM-BOOST strategy will facilitate the screening of secondary metabolites for biological activities of medical interest. As proof of concept, using a constitutive strong promoter, the myxoprincomide from M. xanthus DZ2 has been efficiently produced and its biosynthesis has been enhanced using the 2PRIM-BOOST approach, allowing the identification of new features of myxoprincomide. This strategy should allow the chances to produce and discover new NRPS, PKS, and mixed NRPS/PKS hybrid natural metabolites that are currently considered as cryptic and are the most represented in myxobacteria.IMPORTANCEMicrobial secondary metabolites are important in species interactions and are also a prolific source of drugs. Myxobacteria are ubiquitous soil-dwelling bacteria constituting a huge reservoir of secondary metabolites. However, because most of these molecules are not produced in the laboratory context, one can estimate that only one-tenth have been characterized to date. Here, we developed a new strategy called two-step Protocol for Resource Integration and Maximization-Biomolecules Overproduction and Optimal Screening Therapeutics (2PRIM-BOOST) that combines the engineering of a dedicated Myxococcus xanthus chassis strain together with growth medium optimization. By combining these strategies with the insertion of a constitutive promoter upstream the biosynthetic gene cluster (BGC), the production of myxoprincomide, a characterized low-produced secondary metabolite, was successfully and significantly increased. The 2PRIM-BOOST enriches the toolbox used to produce previously cryptic metabolites, unveil their ecological role, and provide new molecules of medical interest.