Antibacterial mode of action of garviecin LG34 against Gram-negative bacterium Salmonella typhimurium.

Garviecin LG34 produced by Lactococcus garvieae LG34 exhibits wide-spectrum antibacterial activity against both Gram-positive and Gram-negative bacteria. This work aimed at clarifying the antibacterial mode of action of garviecin LG34 against Gram-negative bacterium Salmonella typhimurium. To determine the concentration for the bacteriocin antimicrobial mode experiments, the minimum inhibitory concentration of garviecin LG34 against S. typhimurium CICC21484 was determined as 0.25 mg/ml. Garviecin LG34 decreased the viable count of S. typhimurium CICC21484 and its antibacterial activity was the dose and time dependant. Garviecin LG34 led to the dissipation of transmembrane potential, the rise in the extracellular conductivity, UV-absorbing material at 260 nm, and LDH level of S. typhimurium CICC21484. Scanning electron micrographs results shown that garviecin LG34 cause dramatic deformation and fragmentation including the flagellum shedding, pores formation in surface, and even completely breakage of S. typhimurium cell. Moreover, garviecin LG34 decreased the intracellular ATP level. The results of this study demonstrated that garviecin LG34 can destroy cell structure, increase membrane permeability of S. typhimurium, thereby might be used as biopreservative for treating food borne and salmonellosis resulting from Gram-negative bacterium S. typhimurium.

Transcriptome responses of Lactococcus paracarnosus to different gas compositions and co-culture with Brochothrix thermosphacta.

Lactococcus (Lc.) paracarnosus and the phylogenetically closely related Lc. carnosus species are common members of the microbiota in meat stored under modified atmosphere and at low temperature. The effect of these strains on meat spoilage is controversially discussed. While some strains are known to cause spoilage, others are being studied for their potential to suppress the growth of spoilage and pathogenic bacteria. In this study, Lc. paracarnosus DSM 111017T was selected based on a previous study for its ability to suppress the growth of meat spoilers, including Brochothrix thermosphacta. The mechanism by which this bioprotective strain inhibits competing bacteria and how it contributes to spoilage are not yet known. To answer these two questions, we investigated the effect of four different headspace gas mixtures (simulated air (21 % O2/79 % N2); HiOx-MAP (70 % O2/30 % CO2); nonOx-MAP (70 % N2/ 30 % CO2); simulated vacuum (100 % N2) and the presence of Brochothrix (B.) thermosphacta TMW 2.2101 on the growth and transcriptional response of Lc. paracarnosus DSM 111017T when cultured on a meat simulation agar surface at 4 °C. Analysis of genes specifically upregulated by the gas mixtures used revealed metabolic pathways that may lead to different levels of spoilage metabolites production. We propose that under elevated oxygen levels, Lc. paracarnosus preferentially converts pyruvate from glucose and glycerol to uncharged acetoin/diacetyl instead of lactate to counteract acid stress. Due to the potential production of a buttery off-flavour, the strain may not be suitable as a protective culture in meat packaged under high­oxygen conditions. 70 % N2/ 30 % CO2, simulated vacuum- and the presence of Lc. paracarnosus inhibited the growth of B. thermosphacta TMW 2.2101. However, B. thermosphacta did not affect gene regulation of metabolic pathways in Lc. paracarnosus, and genes previously predicted to be involved in B. thermosphacta growth suppression were not regulated at the transcriptional level. In conclusion, the study indicates that the gas mixture used in packaging significantly affects the metabolism and spoilage potential of Lc. paracarnosus and its ability to inhibit B. thermosphacta growth.

An innovative approach to improving lactic acid production from food waste using iron tailings.

In this study, the feasibility of promoting the lactic acid (LA) fermentation of food waste (FW) with iron tailings (ITs) addition was explored. The best LA yield was 0.91 g LA/g total sugar when 1 % ITs were added into the system. The mechanisms for promoting LA production were acidification alleviation effects and reduction equivalent supply of ITs. Furthermore, the addition of ITs promoted carbohydrate hydrolysis, and the carbohydrates digestibility reached 88.85 % in the 1 % ITs group. The ITs also affected the microbial communities, Lactococcus gradually replaced Streptococcus as the dominant genus, and results suggested that Lactococcus had a positive correlation with LA production and carbohydrate digestibility. Finally, the complex LAB in FW had significant effects on heavy metal removal from ITs, and the removal efficiency Cr, As, Pb, Cd, and Hg can reach 50.84 %, 26.72 %, 59.65 %, 49.75 % and 78.87 % in the 1 % ITs group, respectively.

Behavioral transcriptomic effects of triploidy and probiotic therapy (Bifidobacterium, Lactobacillus, and Lactococcus mixture) on juvenile Chinook salmon (Oncorhynchus tshawytscha).

Aquaculturists use polyploid fish to maximize production albeit with some unintended consequences including compromised behaviors and physiological function. Given benefits of probiotic therapies (e.g., improved immune response, growth, and metabolism), we explored probiotic supplementation (mixture of Bifidobacterium, Lactobacillus, and Lactococcus), to overcome drawbacks. We first examined fish gut bacterial community composition using 16S metabarcoding (via principal coordinate analyses and PERMANOVA) and determined probiotics significantly impacted gut bacteria composition (p = 0.001). Secondly, we examined how a genomic disruptor (triploidy) and diet supplements (probiotics) impact gene transcription and behavioral profiles of hatchery-reared Chinook salmon (Oncorhynchus tshawytscha). Juveniles from four treatment groups (diploid-regular feed, diploid-probiotic feed, triploid-regular feed, and triploid-probiotic feed; n = 360) underwent behavioral assays to test activity, exploration, neophobia, predator evasion, aggression/sociality, behavioral sensitivity, and flexibility. In these fish, transcriptional profiles for genes associated with neural functions (neurogenesis/synaptic plasticity) and biomarkers for stress response and development (growth/appetite) were (i) examined across treatments and (ii) used to describe behavioral phenotypes via principal component analyses and general linear mixed models. Triploids exhibited a more active behavioral profile (p = 0.002), and those on a regular diet had greater Neuropeptide Y transcription (p = 0.02). A growth gene (early growth response protein 1, p = 0.02) and long-term neural development genes (neurogenic differentiation factor, p = 0.003 and synaptysomal-associated protein 25-a, p = 0.005) impacted activity and reactionary profiles, respectively. Overall, our probiotic treatment did not compensate for triploidy. Our research highlights novel applications of behavioral transcriptomics for identifying candidate genes and dynamic, mechanistic associations with complex behavioral repertoires.

First identification and histopathological analysis of Lactococcus garvieae infection in whiteleg shrimp, Penaeus vannamei cultured in low salinity water.

The isolation and characterization of bacterial species Lactococcus garvieae, previously unreported in whiteleg shrimp, Penaeus vannamei, has now been identified in the species. The pathogen was recovered from an affected shrimp farm in southern Taiwan. Bacterial characterization first identified the isolate as Gram-positive cocci, and biochemical profiles demonstrated that the causative agent of mortality was 97% L. garvieae. The bacterial cell DNA resulted in amplification of 1522 bp with 99.6% confirmation by PCR analysis. The phylogenetic tree revealed 100% evolutionary similarity among previously isolated strains. Experimental infection further confirmed higher susceptibility of whiteleg shrimp to L. garvieae in waters of lower salinity, particularly 5 ppt, than in higher salinity. Histopathological analysis showed severely damaged hepatopancreas with necrotized, elongated, collapsed tubules, dislodged membranes and granuloma formation in infected shrimp. Transmission electron microscopy observation indicated a hyaluronic acid capsular layer surrounding bacterial cell which is a virulence factor of L. garvieae and likely responsible for immunosuppression and higher mortality of shrimp cultured in lower salinity. Collectively, these findings report the first isolation of L. garvieae from whiteleg shrimp and shed new light on the disease that threatens the highly valuable species and accentuates the need for finding a solution.

Lactococcus intestinalis sp. nov., a new lactic acid bacterium isolated from intestinal contents in Alzheimer's disease mice.

A gram-positive, facultatively anaerobic, and coccoid or ovoid-shaped bacterium designated M2458T was isolated from the intestinal contents of APPswe/PSΔE9 mouse model of Alzheimer's disease. With the polyphasic approach, the taxonomic position of the novel isolate was confirmed. Strain M2458T grew well at 37 °C on YCFA agar. Strain M2458T belongs to the family Streptococcaceae and class Bacilli, and it is closed to Lactococcus formosensis NBRC 109475T (97.59% sequence similarity) according to its 16S rRNA gene sequence. In a comparison of two housekeeping genes, rpoA and rpoB, strain M2458T was found to be well separated from Lactococcus formosensis NBRC 109475T. On the basis of whole genome sequences, the DNA G+C content was 38.29 mol%. The phylogentic analysis of the whole genome showed that a different branch was clearly formed in the phylogenetic tree of strain M2458T compared to other strains in the genus Lactococcus. A total of eight genes in strain M2458T are involved in the 'neurodegenerative disease' pathway, which involves an annotated protein (glyceraldehyde 3-phosphate dehydrogenase) involved in Alzheimer's disease. In terms of average nucleotide identity and digital DNA-DNA hybridization, strain M2458T was identified as a novel species of the genus Lacococcus. The major fatty acids (> 10% of the total fatty acids) were C18:1ω9c (39.68%), C16:0 (13.26%) and C18:1ω7c (11.52%). The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and an unidentified phospholipid. As a result of its genotypic and phenotypic characteristics, strain M2458T was considered to be a new species within the genus Lactococcus; the name Lactococcus intestinalis sp. nov. has been proposed, with type strain M2458T (=JCM 35706 = CGMCC 1.60066).

Heat-Killed Lactococcus lactis subsp. cremoris H61 Altered the Iron Status of Young Women: A Randomized, Double-Blinded, Placebo-Controlled, Parallel-Group Comparative Study.

Women are prone to iron deficiency because of increased iron excretion associated with menstruation. This is often treated by oral iron supplementation, although this treatment can cause side effects, such as stomach pain and nausea, with low absorption of ingested iron. Previously, a significant increase in serum iron was observed in association with the consumption of foods containing Lactococcus lactis subsp. cremoris H61 (H61). However, the causal relationship between H61 ingestion and elevated serum iron is still unclear. Therefore, in this study, we aimed to determine the effects of H61 ingestion on the iron status of young women. Healthy young Japanese women (18-25 years of age) ingested either heat-killed H61 or placebo for 4 weeks. Serum iron, transferrin saturation, and ferritin were significantly elevated in the H61 group but remained unchanged in the placebo group. Compared to before the intervention, iron intake remained unchanged during the intervention period, so the change in the iron status of the H61 group was not due to increased iron intake. These results suggest that heat-killed H61 may elevate iron status by enhancing iron absorption.

Lactococcus cremoris subsp. cremoris FC-fermented milk activates protein synthesis and increases skeletal muscle mass in middle-aged mice.

Age-related muscle atrophy is associated with decreased protein anabolic capacity. Dietary intervention is an important strategy for the treatment of age-related muscle atrophy. This study examined the effect of Lactococcus cremoris subsp. cremoris FC-fermented milk on muscle mass and protein anabolic signaling in middle-aged mice. Male C57BL/6J mice (18-month-old) were divided into the control and Lactococcus cremoris subsp. cremoris FC-fermented milk supplementation groups. Mice were administered unfermented or fermented milk (300 µL/day) by gavage every alternate day for 8 weeks; thereafter, muscle weight, protein metabolic signaling factors, and inflammatory factors were investigated. Soleus muscle weight was higher in the fermented milk group than in the control group. Expression of insulin growth factor-1, a typical anabolic factor, and phosphorylation levels of anabolic signaling factors (mTOR and p70S6K) were higher after fermented milk supplementation. Levels of tumor necrosis factor-α, an inhibitor of protein anabolism, were lower in the fermented milk group. These data suggest that the daily intake of Lactococcus cremoris subsp. cremoris FC-fermented milk increased skeletal muscle mass as well as protein synthesis in the middle-aged mice, which may be mediated by reduction in the levels of inflammatory factors. Therefore, accelerated protein synthesis, induced by the consumption of fermented milk, has a potential role in counteracting muscle atrophy.

Gas production by Paucilactobacillus wasatchensis WDCO4 is increased in Cheddar cheese containing sodium gluconate.

Paucilactobacillus wasatchensis can use gluconate (GLCN) as well as galactose as an energy source and because sodium GLCN can be added during salting of Cheddar cheese to reduce calcium lactate crystal formation, our primary objective was to determine if the presence of GLCN in cheese is another risk factor for unwanted gas production leading to slits in cheese. A secondary objective was to calculate the amount of CO2 produced during storage and to relate this to the amount of gas-forming substrate that was utilized. Ribose was added to promote growth of Pa. wasatchensis WDC04 (P.waWDC04) to high numbers during storage. Cheddar cheese was made with lactococcal starter culture with addition of P.waWDC04 on 3 separate occasions. After milling, the curd was divided into six 10-kg portions. To the curd was added (A) salt, or salt plus (B) 0.5% galactose + 0.5% ribose (similar to previous studies), (C) 1% sodium GLCN, (D) 1% sodium GLCN + 0.5% ribose, (E) 2% sodium GLCN, (F) 2% sodium GLCN + 0.5% ribose. A vat of cheese without added P.waWDC04 was made using the same milk and a block of cheese used as an additional control. Cheeses were cut into 900-g pieces, vacuum packaged and stored at 12°C for 16 wk. Each month the bags were examined for gas production and cheese sampled and tested for lactose, galactose and GLCN content, and microbial numbers. In the control cheese, P.waWDC04 remained undetected (i.e., <104 cfu/g), whereas in cheeses A, C, and E it increased to 107 cfu/g, and when ribose was included with salting (cheeses B, D, and F) increased to 108 cfu/g. The amount of gas (measured as headspace height or calculated as mmoles of CO2) during 16 wk storage was increased by adding P.waWDC04 into the milk, and by adding galactose or GLCN to the curd. Galactose levels in cheese B were depleted by 12 wk while no other cheeses had residual galactose. Except for cheese D, the other cheeses with GLCN added (C, E and F) showed little decline in GLCN levels until wk 12, even though gas was being produced starting at wk 4. Based on calculations of CO2 in headspace plus CO2 dissolved in cheese, galactose and GLCN added to cheese curd only accounted for about half of total gas production. It is proposed that CO2 was also produced by decarboxylation of amino acids. Although P.waWDC04 does not have all the genes for complete conversion and decarboxylation of the amino acids in cheese, this can be achieved in conjunction with starter culture lactococcal. Adding GLCN to curd can now be considered another confirmed risk factor for unwanted gas production during storage of Cheddar cheese that can lead to slits and cracks in cheese. Putative risk factors now include having a community of bacteria in cheese leading to decarboxylation of amino acids and release of CO2 as well autolysis of the starter culture that would provide a supply of ribose that can promote growth of Pa. wasatchensis.

Beneficial effects of Lactococcus lactis subsp. cremoris LL95 treatment in an LPS-induced depression-like model in mice.

Clinical evidence suggests that neuroinflammation, activation of the immune system, and the composition of the intestinal microbiota are involved in the pathology of depression. This study evaluated the effectiveness of a probiotic intervention using Lactococcus lactis subsp. cremoris LL95 in ameliorating mood disorders in a lipopolysaccharide (LPS)-induced depression-like mouse model. C57BL/6 mice were randomly divided into four groups and treated with 5 mg/kg LPS via intraperitoneal injection to induce depression-like symptoms, followed by oral administration of LL95 for one week (1â€¯× 109 CFU/mouse). The animals were then subjected to a series of behavioral assessments, including open field, sucrose preference, and forced swimming tests. In addition, we evaluated the levels of reactive oxygen species, tumor necrosis factor-α, and interleukin-1ß in the hippocampal tissues of these animals, and also determined their fecal lactic acid bacteria (LAB) content. LL95 intervention improved LPS-induced depression-like behaviors in mice, including decreased sucrose preference and increased immobility time in the forced swim test. LL95 treatment reversed the LPS-induced increase in hippocampal levels of reactive oxygen species and tumor necrosis factor-α, and of interleukin-1ß to a lesser extent. Furthermore, LL95 intervention increased the fecal LAB content in these animals, suggesting changes in the gut microbiota. These findings suggest that LL95 exerts antidepressant-like effects in LPS-induced depression, which may be attributed to modulation of the oxidative status and pro-inflammatory cytokine expression in the hippocampus and alteration in the LAB content of the gut microbiota.

Essential oil of Mentha suaveolens Ehrh., composition and antibacterial activity against bacterial fish pathogens.

In this study, it was determinated the essential oil of cultivated apple mint, Mentha suaveolens Ehrh. composition and in vitro antibacterial activity of against 11 fish pathogen bacteria including Gram-positive (Staphylococcus warneri, Staphylococcus sp., Lactococcus garvieae, Vagococus salmoninarum) and Gram-negative (Aeromonas hydrophila, Aeromonas sobria Aeromonas cavieae, Vibrio anguillarum, Pseudomonas aeroginosa, Yersinia ruckeri, Edwardsiella tarda) by using agar diffusion assay. The main component of M. suaveolence oil was obtained as piperitenone oxide. The essential oil exhibited strong inhibitory activity such as inhibition zone sizes: 30-50mm at 250-1000 µL mL-1 concentrations against V. anguillarum; 16-20mm at 31.25-125 µL mL-1 concentrations against P. aeroginosa; 15-18mm at 500-1000 µL mL-1 concentrations against A. sobria. However, it was found to be moderately effective against E. tarda (8-15 mm), Y. ruckeri (9-12mm), S. warneri (9-10mm), V. salmoninarum (9mm) and Staphylococcus sp. (8-9mm). The essential oil showed weak inhibitory activity against A. cavieae (5-8), A. hydrophila (6-7mm), L. garvieae (5-7mm). Thus, effect of essential oil of M. suaveolens on immune response and disease resistance against Vibrio anguillarum, A. sobria and P. aeroginosa should be investigated in vivo in cultured fish species in future studies.

Hepatic antioxidant activity, immunomodulation, and pro-anti-inflammatory cytokines manipulation of κ-carrageenan (κ-CGN) in cobia, Rachycentron canadum against Lactococcus garvieae.

The effects of dietary k-Carrageenan (k-CGN) at 10, 20, and 30 g kg-1 on growth rate, hemato-biochemical indices, innate-adaptive parameters and modification of pro- and/or anti-inflammatory cytokines and chemokines pathway in cobia, Rachycentron canadum against Lactococcus garvieae is reported. The weight gain (WG) increased substantially (P < 0.05) in all k-CGN treated groups; the specific growth rate (SGR) was significant in healthy uninfected normal (HuN) and L. garvieae challenged (LaC) groups fed with 20 g kg-1k-CGN diet on 45 and 60 days. The white blood cell (WBC) counts, total protein (TP) level, total anti-oxidant (T-AOC), catalase (CAT), and glutathione (GSH) activities increased significantly when fed with 20 g and 30 g kg-1k-CG diets on 45th and 60th day. The immunological parameters such as phagocytic (PC) index and the activity of phagocytic (PC), respiratory burst (RB), superoxide dismutase (SOD), alternate complement pathway (ACH50), and lysozyme (LZM) were significantly enhanced with all k-CG diets in 45 and 60 days of treatment. No cumulative mortality (CM) in HuN group fed by control or any k-CGN diets. CM was 5% in LaC group fed with 20 g kg-1k-CGN diet whereas in LaC groups fed with 10 g and 30 g kg-1k-CGN diets the CM was 10%. The interleukin 1 beta (IL-1ß) and tumor necrosis factor alpha (TNFα) pro-inflammatory cytokines mRNA transcripts were one-fold high (P < 0.05) in both HuN and LaC group fed all k-CGN enriched diets on 45 and 60 days. Similarly, IL-18 and TLR2 mRNA was one-fold high expression in both groups fed the 20 g and 30 g kg-1k-CGN enriched diets on 45 or 60 days. Interferon gamma (IFNγ) and interferon regulatory factor 3/7 (IRF3/IRF7) mRNA transcripts did not change with any diet. IL-6, IL-10, and IL-11 mRNA were one-fold high expressions in both groups fed the 20 g and 30 g kg-1k-CGN enriched diets on 45 and 60 days. However, the expression of CC1, CC3, and CCR9 pro-inflammatory chemokines mRNA did not vary with any control or k-CGN enriched diets. The results indicate that diet enriched with k-CGN at 20 g kg-1 significantly influences the growth, antioxidant and innate-adaptive immune performance, and pro-anti-inflammatory cytokines and chemokines regulation in cobia against L. garvieae.

Isolation and Identification of Lactic Acid Bacteria from Cow Milk and Milk Products.

Lactic acid bacteria (LAB) have long been consumed by people in several fermented foods such as dairy products. A study was conducted on lactating dairy cows to isolate and characterize LAB from dairy products found in and around Bahir-Dar city, North Western Ethiopia. Milk and milk products were randomly collected from dairy farms, milk vending shops, individual households, and supermarkets for bacteriological investigations. A total of sixteen samples were taken from different sources and cultured on different selective media: de Man, Rogosa, and Sharpe (MRS) agar for Lactobacillus spp.; M17 agar for Lactococcus spp.; Rogasa SL agar for Streptococci spp.; and MRS supplemented with cysteine (0.5%) for Bifidobacteria spp. Different laboratory techniques were implemented for LAB isolation and identification. A total of 41 bacterial isolates were grouped under five different genera of LAB and Bifidobacteria spp. were identified based on the growth morphology on the selective media, growth at a different temperature, gas production from glucose, carbohydrate fermentation, and other biochemical tests. LAB genera such as Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Streptococcus, and Bifidobacterium spp. were isolated and identified from raw milk, cheese, and yogurt. Based on the current study, the majority of the LAB (24.38%) was isolated from cheese and yogurt. Among these, Lactobacillus, Lactococcus (21.94%), Streptococcus (19.51%), Leuconostoc (14.64%), Bifidobacteria (12.19%), and Pediococcus (7.31%) spp. were also identified from these products. Furthermore, based on the bacterial load count and different identification methodologies, our study revealed that Lactobacillus spp. were the dominant LAB isolated from milk and milk products. As a result, since there are few studies on the isolation and identification of lactic acid bacteria from dairy products in Ethiopia, more research studies are needed to complete the identification and characterization to species level and their possible role as probiotics.

Donkey Milk Fermentation by Lactococcus lactis subsp. cremoris and Lactobacillus rhamnosus Affects the Antiviral and Antibacterial Milk Properties.

BACKGROUND: Milk is considered an important source of bioactive peptides, which can be produced by endogenous or starter bacteria, such as lactic acid bacteria, that are considered effective and safe producers of food-grade bioactive peptides. Among the various types of milk, donkey milk has been gaining more and more attention for its nutraceutical properties. METHODS: Lactobacillus rhamnosus 17D10 and Lactococcus lactis subsp. cremoris 40FEL3 were selected for their ability to produce peptides from donkey milk. The endogenous peptides and those obtained after bacterial fermentation were assayed for their antioxidant, antibacterial, and antiviral activities. The peptide mixtures were characterized by means of LC-MS/MS and then analyzed in silico using the Milk Bioactive Peptide DataBase. RESULTS: The peptides produced by the two selected bacteria enhanced the antioxidant activity and reduced E. coli growth. Only the peptides produced by L. rhamnosus 17D10 were able to reduce S. aureus growth. All the peptide mixtures were able to inhibit the replication of HSV-1 by more than 50%. Seventeen peptides were found to have 60% sequence similarity with already known bioactive peptides. CONCLUSIONS: A lactic acid bacterium fermentation process is able to enhance the value of donkey milk through bioactivities that are important for human health.

RiPPMiner-Genome: A Web Resource for Automated Prediction of Crosslinked Chemical Structures of RiPPs by Genome Mining.

RiPPMiner-Genome is a unique bioinformatics resource for identifying Biosynthetic Gene Clusters (BGC) for RiPPs (Ribosomally Synthesized and Post-translationally Modified Peptides) and automated prediction of crosslinked chemical structures of RiPPs starting from genomic sequences. It is a major update of the RiPPMiner webserver, which used only peptide sequence of RiPP precursors as input for predicting RiPP class and crosslinked chemical structures. Other major improvements are, machine learning (ML) based identification of correct RiPP precursor peptide from among multiple small ORFs (Open Reading Frames) in a BGC, prediction of the cleavage site and cross-links in thiopeptides and identification of non-crosslinked modified residues in lanthipeptides. It has been benchmarked on a dataset of 204 experimentally characterized RiPP BGCs. RiPPMiner-Genome also facilitates visualization of the RiPP BGCs and depiction of the chemical structure of crosslinked RiPP. It also has an interface for searching characterized RiPPs, similar to the predicted core peptide sequence or crosslinked chemical structure.

Plant-based, adjuvant-free, potent multivalent vaccines for avian influenza virus via Lactococcus surface display.

Influenza epidemics frequently and unpredictably break out all over the world, and seriously affect the breeding industry and human activity. Inactivated and live attenuated viruses have been used as protective vaccines but exhibit high risks for biosafety. Subunit vaccines enjoy high biosafety and specificity but have a few weak points compared to inactivated virus or live attenuated virus vaccines, especially in low immunogenicity. In this study, we developed a new subunit vaccine platform for a potent, adjuvant-free, and multivalent vaccination. The ectodomains of hemagglutinins (HAs) of influenza viruses were expressed in plants as trimers (tHAs) to mimic their native forms. tHAs in plant extracts were directly used without purification for binding to inactivated Lactococcus (iLact) to produce iLact-tHAs, an antigen-carrying bacteria-like particle (BLP). tHAs BLP showed strong immune responses in mice and chickens without adjuvants. Moreover, simultaneous injection of two different antigens by two different formulas, tHAH5N6 + H9N2 BLP or a combination of tHAH5N6 BLP and tHAH9N2 BLP, led to strong immune responses to both antigens. Based on these results, we propose combinations of plant-based antigen production and BLP-based delivery as a highly potent and cost-effective platform for multivalent vaccination for subunit vaccines.

Bio-Engineered Nisin with Increased Anti-Staphylococcus and Selectively Reduced Anti-Lactococcus Activity for Treatment of Bovine Mastitis.

Bovine mastitis is a significant economic burden for dairy enterprises, responsible for premature culling, prophylactic and therapeutic antibiotic use, reduced milk production and the withholding (and thus wastage) of milk. There is a desire to identify novel antimicrobials that are expressly directed to veterinary applications, do not require a lengthy milk withholding period and that will not have a negative impact on the growth of lactic acid bacteria involved in downstream dairy fermentations. Nisin is the prototypical lantibiotic, a family of highly modified antimicrobial peptides that exhibit potent antimicrobial activity against many Gram-positive microbes, including human and animal pathogens including species of Staphylococcus and Streptococcus. Although not yet utilized in the area of human medicine, nisin is currently applied as the active agent in products designed to prevent bovine mastitis. Over the last decade, we have harnessed bioengineering strategies to boost the specific activity and target spectrum of nisin against several problematic microorganisms. Here, we screen a large bank of engineered nisin derivatives to identify novel derivatives that exhibit improved specific activity against a selection of staphylococci, including mastitis-associated strains, but have unchanged or reduced activity against dairy lactococci. Three such peptides were identified; nisin A M17Q, nisin A T2L and nisin A HTK.

Bacteria in Surgical Smoke: A Self-controlled Laboratory Study Using Porcine Spinal Tissues.

STUDY DESIGN: A laboratory study performed in a self-controlled fashion. OBJECTIVE: The aim of this study was to examine whether "surgical smoke" generated by using the electrocautery on porcine spinal tissues could contain viable bacteria. SUMMARY OF BACKGROUND DATA: The year 2020 has seen a surge in legislation regarding surgical smoke evacuation in the operating room across the United States. Surgical smoke contains numerous toxic and carcinogenic substances, which spine surgeons and operating room staff are regularly exposed to. Although the smoke's chemical toxicity has been extensively studied, little is known about its biohazard level. METHODS: The electrocautery was used to "operate on" 20 segments of porcine spinal tissues on a clean bench. In each operation, the generated smoke was captured with a swab, which was positioned above the surgical site and within the smoke, to obtain bacteria cultures. Two other swabs-a tissue swab (swabbing the porcine tissue itself) and a blank swab (swabbing an empty sterile bowl)-were collected as controls. The culture results were compared among the three groups. An additional experiment was conducted to see whether using a mask can reduce the "infection rate" of the swab. RESULTS: Although all blank swabs tested negative, 95% (19/20) of the smoke swabs tested positive, with a total of nine bacteria species identified. The most prevalent species observed in the smoke swabs included Serratia liquefaciens, Lactococcus garvieae, and Hafnia alvei, and so on. Among the positive smoke swabs, 84% (16/19) had one or more bacteria species that were consistent with the cultures of their corresponding tissue swabs. Using a surgical or N95 mask reduced the "infection rate" of the swab. CONCLUSION: "Surgical smoke" generated by electrocauterization of porcine spinal tissues contains viable bacteria. Further research in actual spine surgery is needed next.Level of Evidence: N/A.

Can sucrose-substitutes increase the antagonistic activity against foodborne pathogens, and improve the technological and functional properties of sheep milk kefir?

The effect of different types of sugar (sucrose, demerara, brown, fructose, coconut sugar, and honey) on sheep milk kefir was evaluated. Microbial counts (Lactobacillus, Lactococcus, Leuconostoc, yeast), antagonistic activity against foodborne pathogens, microstructure (scanning electron microscopy), and antiproliferative activity of cancer cells were evaluated. Furthermore, the antioxidant activity (DPPH), inhibitory activity of angiotensin-converting enzyme (ACE), α-amylase, and α-glucosidase, lactose content, lactic and acetic acids and ethanol, fatty acid profile and volatile organic compounds were determined. The addition of sugars increased the Lactobacillus population (up to 2.24 log CFU/mL), metabolites concentration, antagonistic activity against pathogens, antioxidant activity (11.1 to 24.1%), ACE inhibitory activity (27.5 to 37.6%), α-amylase inhibition (18 to 37.4%), and anti-proliferative activity. Furthermore, it improved the fatty acid profile and volatile compounds. The results suggest that the replacement of sucrose with different types of sugar constitute an interesting option to be used in sheep milk kefir formulations.

Antimicrobial activity and cytotoxicity trait of a bioactive peptide purified from Lactococcus garvieae subsp. bovis BSN307T.

A bioactive peptide of 8595 Da was purified from the cell free supernatant of Lactococcus garvieae subsp. bovis BSN307T . MALDI MS/MS peptide mapping and the data base search displayed no significant similarity to any reported antimicrobial peptide of LAB. This peptide at a dose concentration of 200 µg ml-1 inhibited the growth of both Gram-positive and Gram-negative bacteria by 58-89% and a dose of 500 µg ml-1 scavenged 50% of DPPH-free radicals generated. Interestingly, cytotoxicity assay demonstrated that 17 µg ml-1 of peptide selectively inhibited 50% proliferation of mammalian cancer cell lines HeLa and MCF-7 whereas normal H9c2 cells remained unaffected. Fluorescent microscopic analysis after DAPI nuclear staining of HeLa cells showed characteristics of apoptosis and activation of caspase-3 was ascertained by caspase-3 fluorescence assay.