Elaboration of Nanostructured Levan-Based Colloid System as a Biological Alternative with Antimicrobial Activity for Applications in the Management of Pathogenic Microorganisms.

Considering the documented health benefits of bacterial exopolysaccharides (EPSs), specifically of bacterial levan (BL), including its intrinsic antimicrobial activity against certain pathogenic species, the current study concentrated on the development of active pharmaceutical ingredients (APIs) in the form of colloid systems (CoSs) containing silver nanoparticles (AgNPs) employing in-house biosynthesized BL as a reducing and capping agent. The established protocol of fermentation conditions implicating two species of lactic acid bacteria (LAB), i.e., Streptococcus salivarius K12 and Leuconostoc mesenteroides DSM 20343, ensured a yield of up to 25.7 and 13.7 g L-1 of BL within 72 h, respectively. An analytical approach accomplished by Fourier-transform infrared (FT-IR) spectroscopy allowed for the verification of structural features attributed to biosynthesized BL. Furthermore, scanning electron microscopy (SEM) revealed the crystalline morphology of biosynthesized BL with a smooth and glossy surface and highly porous structure. Molecular weight (Mw) estimated by multi-detector size-exclusion chromatography (SEC) indicated that BL biosynthesized using S. salivarius K12 has an impressively high Mw, corresponding to 15.435 × 104 kilodaltons (kDa). In turn, BL isolated from L. mesenteroides DSM 20343 was found to have an Mw of only 26.6 kDa. Polydispersity index estimation (PD = Mw/Mn) of produced BL displayed a monodispersed molecule isolated from S. salivarius K12, corresponding to 1.08, while this was 2.17 for L. mesenteroides DSM 20343 isolate. The presence of fructose as the main backbone and, to a lesser extent, glucose and galactose as side chain molecules in EPS hydrolysates was supported by HPLC-RID detection. In producing CoS-BL@AgNPs within green biosynthesis, the presence of nanostructured objects with a size distribution from 12.67 ± 5.56 nm to 46.97 ± 20.23 was confirmed by SEM and energy-dispersive X-ray spectroscopy (EDX). The prominent inhibitory potency of elaborated CoS-BL@AgNPs against both reference test cultures, i.e., Pseudomonas aeruginosa, Escherichia coli, Enterobacter aerogenes, and Staphylococcus aureus and those of clinical origin with multi-drug resistance (MDR), was confirmed by disc and well diffusion tests and supported by the values of the minimum inhibitory and bactericidal concentrations. CoS-BL@AgNPs can be treated as APIs suitable for designing new antimicrobial agents and modifying therapies in controlling MDR pathogens.

Genomic Characterization and Initial Insight into Mastitis-Associated SNP Profiles of Local Latvian Bos taurus Breeds.

Latvia has two local Bos taurus breeds-Latvian Brown (LBG) and Latvian Blue (LZG)-characterized by a good adaptation to the local climate, longevity, and high fat and protein contents in milk. Since these are desired traits in the dairy industry, this study investigated the genetic background of the LBG and LZG breeds and identified the genetic factors associated with mastitis. Blood and semen samples were acquired, and whole genome sequencing was then performed to acquire a genomic sequence with at least 35× or 10× coverage. The heterozygosity, nucleotide diversity, and LD analysis indicated that LBG and LZG cows have similar levels of genetic diversity compared to those of other breeds. An analysis of the population structure revealed that each breed clustered together, but the overall differentiation between the breeds was small. The highest genetic variance was observed in the LZG breed compared with the LBG breed. Our results show that SNP rs721295390 is associated with mastitis in the LBG breed, and SNPs rs383806754, chr29:43998719CG>C, and rs462030680 are associated with mastitis in the LZG breed. This study shows that local Latvian LBG and LZG breeds have a pronounced genetic differentiation, with each one suggesting its own mastitis-associated SNP profile.

The Release of Non-Extractable Ferulic Acid from Cereal By-Products by Enzyme-Assisted Hydrolysis for Possible Utilization in Green Synthesis of Silver Nanoparticles.

The present work was undertaken to elucidate the potential contribution of biosynthetically produced ferulic acid (FA) via enzymatic hydrolysis (EH) of rye bran (RB) to the formation of silver nanoparticles (AgNPs) during green synthesis. An analytical approach accomplished by multiple reaction monitoring (MRM) using triple quadrupole mass selective detection (HPLC-ESI-TQ-MS/MS) of the obtained hydrolysate revealed a relative abundance of two isomeric forms of FA, i.e., trans-FA (t-FA) and trans-iso-FA (t-iso-FA). Further analysis utilizing high-performance liquid chromatography with refractive index (HPLC-RID) detection confirmed the effectiveness of RB EH, indicating the presence of cellulose and hemicellulose degradation products in the hydrolysate, i.e., xylose, arabinose, and glucose. The purification process by solid-phase extraction with styrene-divinylbenzene-based reversed-phase sorbent ensured up to 116.02 and 126.21 mg g-1 of t-FA and t-iso-FA in the final eluate fraction, respectively. In the green synthesis of AgNPs using synthetic t-FA, the formation of NPs with an average size of 56.8 nm was confirmed by scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) techniques. The inclusion of polyvinylpyrrolidone (PVP-40) in the composition of NPs during synthesis favorably affected the morphological features, i.e., the size and shape of AgNPs, in which as big as 22.4 nm NPs were engineered. Meanwhile, nearly homogeneous round-shaped AgNPs with an average size of 16.5 nm were engineered using biosynthetically produced a mixture of t-FA and t-iso-FA and PVP-40 as a capping agent. The antimicrobial activity of AgNPs against Gram-positive and Gram-negative bacteria, including Pseudomonas aeruginosa, E. coli, Enterococcus faecalis, Bacillus subtilis, and Staphylococcus aureus was confirmed by the disk diffusion method and additionally supported by values of minimum inhibitory (MIC) and bactericidal (MBC) concentrations. Given the need to reduce problems of environmental pollution with cereal processing by-products, this study demonstrated a technological solution of RB rational use in the sustainable production of AgNPs during green synthesis. The AgNPs can be considered as active pharmaceutical ingredients (APIs) to be used for developing new antimicrobial agents and modifying therapies in treating multi-drug resistant (MDR) pathogens.

Effect of Essential Oils Supplemented with Caprylic Acid and Sodium Chloride against Faecal ESBL-Producing Escherichia coli Isolated from Pigs.

The purpose of the present investigation was to compare the antibacterial activity of six commercial and lab-scale extracted essential oils (EOs) alone or in combination with caprylic acid (CA) and sodium chloride (NaCl) against faecal Escherichia coli with and without extended-spectrum beta-lactamase (ESBL) encoding genes, and of isolates classified as multidrug-resistant (MDR). Gas chromatography−mass spectrometry (GC−MS) was used for the analysis of chemical composition of EOs, while the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays were carried out to elucidate the antibacterial activity of non-supplemented and supplemented EOs against different resistance levels of E. coli strains. The main compounds in commercial EOs were aromatic monoterpenoids (30−56%) and p-cymene (8−35%), while the main compounds in the lab-scale EOs were aromatic monoterpenoids (12−37%) and γ-terpinene (18−22%). Commercial EOs exhibited superior inhibitory activity of E. coli in comparison to lab-scale produced EOs. Antibacterial activity of EOs was significantly enhanced by enrichment of the EOs with NaCl (p < 0.001) or CA (p = 0.012). Most of the non-supplemented EOs exhibited lower activity against MDR and ESBL producing E. coli. In contrast, EOs supplemented with CA and especially NaCl was equally effective against ESBL and non-ESBL as well as MDR and non-MDR E. coli. It was found that supplementation of EOs with NaCl could enhance the antibacterial activity towards ESBL and MDR E. coli isolates. However, additional studies are needed to clarify the potential risks of developing resistance.

The Prevalence and Characterization of Fecal Extended-Spectrum-Beta-Lactamase-Producing Escherichia coli Isolated from Pigs on Farms of Different Sizes in Latvia.

The aim of this study was to determine the prevalence of fecal ESBL-producing Escherichia coli (E. coli) in pigs on large and small farms in Latvia, to characterize beta-lactamase genes and establish an antimicrobial resistance profile. Fecal samples (n = 615) were collected from 4-week, 5-week, 6-week, 8-week, 12-week and 20-week-old piglets, pigs and sows on four large farms (L1, L2, L3, L4) and three small farms (S1, S2, S3) in Latvia. ChromArt ESBL agar and combination disc tests were used for the screening and confirmation of ESBL-producing E. coli. The antimicrobial resistance was determined by the disc diffusion method and ESBL genes were determined by polymerase chain reaction (PCR). Subsequently, ESBL-producing E. coli was confirmed on three large farms, L1 (64.3%), L2 (29.9%), L3 (10.7%) and one small farm, S1 (47.5%); n = 144 (23.4%). The prevalence of ESBL-producing E. coli differed considerably between the large and small farm groups (26.9% vs. 12.7%). Of ESBL E. coli isolates, 96% were multidrug-resistant (MDR), demonstrating there were more extensive MDR phenotypes on large farms. The distribution of ESBL genes was blaTEM (94%), blaCTX-M (86%) and blaSHV (48%). On the small farm, blaSHV dominated, thus demonstrating a positive association with resistance to amoxicillin-clavulanic acid, ceftazidime and cefixime, while on the large farms, blaCTX-M with a positive association to cephalexin and several non-beta lactam antibiotics dominated. The results indicated the prevalence of a broad variety of ESBL-producing E. coli among the small and large farms, putting the larger farms at a higher risk. Individual monitoring of ESBL and their antimicrobial resistance could be an important step in revealing hazardous MDR ESBL-producing E. coli strains and reviewing the management of antibiotic use.

Effect of Probiotics and Herbal Products on Intestinal Histomorphological and Immunological Development in Piglets.

The aim of the study was to evaluate the effect of probiotics and herbal products on the intestinal histomorphological and immunological development in piglets. Accordingly, 2-week-old piglets were allocated in 4 groups: C (basal diet), Pro (basal diet + probiotics), Pro+B (basal diet + probiotics + buckwheat bran), and H (powder of herbs). After 6 weeks of the experiment, 4 piglets from each experimental group were randomly selected and slaughtered at a slaughterhouse. Samples of tissue and digestive content from the jejunum and colon were collected for bacteriological, histological, and immunohistochemical examination. The results showed that probiotics increased the number of Lactobacillus spp. in the small (p < 0.05) and large intestines. The intestinal histomorphology was improved (p < 0.05) in all experimental groups by an increased villus height, VH : CD ration, colon crypt depth, and number of Ki-67+ epithelial cells. A higher number (p < 0.05) of goblet cells and their acidification were observed in group Pro, while the density of goblet cells was decreased by the herbs. Probiotics increased (p < 0.05) the number of intraepithelial lymphocytes (IELs), density of CD3+ cells in Peyer's patches (PPs), and lamina propria (LP). In group H, a dual effect on the CD3+ cell distribution was observed. The herbs reduced (p < 0.05) the number of IELs and CD3+ in LP but increased the distribution of CD3+ cells in PPs. In the colon, herbs increased CD3+ cells in LP as well. It suggests that probiotics and herbs had influence on the intestinal histomorphology and the ability to modulate the mucosal immune system; however, the combination of probiotics and buckwheat bran was not so convincing, probably due to the inhibitory effect of the buckwheat bran on the probiotics used.