Ubericin K, a New Pore-Forming Bacteriocin Targeting mannose-PTS.

Bovine mastitis infection in dairy cattle is a significant economic burden for the dairy industry globally. To reduce the use of antibiotics in treatment of clinical mastitis, new alternative treatment options are needed. Antimicrobial peptides from bacteria, also known as bacteriocins, are potential alternatives for combating mastitis pathogens. In search of novel bacteriocins against mastitis pathogens, we screened samples of Norwegian bovine raw milk and found a Streptococcus uberis strain with potent antimicrobial activity toward Enterococcus, Streptococcus, Listeria, and Lactococcus. Whole-genome sequencing of the strain revealed a multibacteriocin gene cluster encoding one class IIb bacteriocin, two class IId bacteriocins, in addition to a three-component regulatory system and a dedicated ABC transporter. Isolation and purification of the antimicrobial activity from culture supernatants resulted in the detection of a 6.3-kDa mass peak by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, a mass corresponding to the predicted size of one of the class IId bacteriocins. The identification of this bacteriocin, called ubericin K, was further confirmed by in vitro protein synthesis, which showed the same inhibitory spectrum as the purified antimicrobial compound. Ubericin K shows highest sequence similarity to the class IId bacteriocins bovicin 255, lactococcin A, and garvieacin Q. We found that ubericin K uses the sugar transporter mannose phosphotransferase (PTS) as a target receptor. Further, by using the pHlourin sensor system to detect intracellular pH changes due to leakage across the membrane, ubericin K was shown to be a pore former, killing target cells by membrane disruption. IMPORTANCE Bacterial infections in dairy cows are a major burden to farmers worldwide because infected cows require expensive treatments and produce less milk. Today, infected cows are treated with antibiotics, a practice that is becoming less effective due to antibiotic resistance. Compounds other than antibiotics also exist that kill bacteria causing infections in cows; these compounds, known as bacteriocins, are natural products produced by other bacteria in the environment. In this work, we discover a new bacteriocin that we call ubericin K, which kills several species of bacteria known to cause infections in dairy cows. We also use in vitro synthesis as a novel method for rapidly characterizing bacteriocins directly from genomic data, which could be useful for other researchers. We believe that ubericin K and the methods described in this work will aid in the transition away from antibiotics in the dairy industry.

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.

Effect of a novel postbiotic containing lactic acid bacteria on the intestinal microbiota and disease resistance of rainbow trout (Oncorhynchus mykiss).

OBJECTIVE: This study was aimed to assess the effect of a novel postbiotic on bacterial community composition and structure within the intestinal ecosystem of rainbow trout (Oncorhynchus mykiss), as well as evaluate its capacity to protect rainbow trout from Lactococcus garvieae infection. RESULTS: After 30 days of dietary postbiotic supplementation, high-throughput 16S rRNA gene sequencing revealed that bacterial community composition, diversity and richness were significantly higher in treated fish than in control fish. The proportion of sequences affiliated to the phylum Tenericutes, and to a lesser extent, the phyla Spirochaetes and Bacteroidetes was increased in fish fed a postbiotic-enriched diet compared to control fish, whereas the abundance of Fusobacteria was higher in control fish. Moreover, the treated fish showed significantly (p < 0.05) improved protection against L. garvieae compared to control fish. CONCLUSIONS: These findings suggest that dietary postbiotic supplementation may represent an environmentally friendly strategy for preventing and controlling diseases in aquaculture.

Short communication: Antimicrobial activity of pequi (Caryocar brasiliense) waste extract on goat Minas Frescal cheese presenting sodium reduction.

Sodium chloride reduction in foods is a significant focus of the dairy industry; however, it can interfere with dairy product quality. Thus, researchers have carried out studies on alternatives to maintain dairy product safety when presenting reduced NaCl content, such as natural antimicrobial addition. Caryocar brasiliense (pequi) is a fruit with high phenolic compound concentrations in the pulp and peel and known antioxidant and antimicrobial properties. This study aimed to define the optimum stage for pequi waste extract addition during cheese manufacturing in order to maintain and prolong the shelf life of reduced-sodium goat Minas Frescal cheese. Four different goat Minas Frescal cheese treatments were carried out: control cheese (without extract; CC), pequi extract addition to milk (CM), pequi extract addition to mass (CS), and cheese immersion in pequi extract (CIE). The treatments were subjected to microbiological (Staphylococcus spp., Escherichia coli, Enterobacteriaceae, coliforms and fecal coliforms, Lactococcus spp., and lactic acid bacteria counts), textural (hardness and consistency), and instrumental color (luminosity, yellow intensity, red intensity, chroma, hue angle, and total color change) analyses. No Enterobacteriaceae, Staphylococcus spp., E. coli, or coliforms and fecal coliforms were detected during storage for any of the assessed samples, including CC. Regarding texture, all samples presented a trend for decreasing rigidity during storage. In addition, lower luminosity values were also observed in cheeses produced with added pequi extract (CM, CS, and CIE) when compared with CC. All cheeses produced with added pequi were stable regarding all evaluated parameters; however, pequi extract addition to milk (CM) was shown to be more efficient, leading to higher textural parameters and better microbiological quality during storage. Thus, the CM treatment is the most recommended for pequi waste extract addition during Minas Frescal cheese manufacture.

Therapeutic effects of beard lichen, Usnea barbata extract against Lactococcus garvieae infection in rainbow trout (Oncorhynchus mykiss).

In this study, therapeutic effects of aqueous methanolic extracts of beard lichen (Usnea barbata) against Lactococcus garvieae (ATCC 43921) in rainbow trout (Onchorhynchus mykiss) were investigated. Six different experimental groups [0 mg/100 µL (Control), 4 mg/100 µL, 8 mg/100 µL, 12 mg/100 µL, 6 mg/100 µL florfenicol (positive control), 6 mg/100 µL erythromycin (positive control)] were set up to determine effects of lichen extract on immune responses and survival rate. In the study, superoxide radical production was increased in fish treated with 12 mg beard lichen extract, florfenicol and erythromycin compared to that of control (P < 0.05). Lysozyme activity was generally decreased (P < 0.05) or no differences were observed in all experimental groups compared to that of control (P > 0.05). Myeloperoxidase was significantly increased in all antibiotic treated groups. No differences were observed in liver histology of experimental groups compared to control. Cytokine gene expressions were elevated in all experimental groups compared to that of control (P < 0.05), except IL-1ß expression at 10th day sampling time. Other immune related genes (IL-8, TGF- ß, IL-12 Beta, TNFα1, IL-10, COX-2, IL-6, TLR5, C3, IGM, MHC-II, iNOS, IgT, IFN1, IFN2, IFN reg) were also elevated in all experimental groups compared to that of control group. The survival rates obtained in 4 mg beard lichen treated group, 8 mg beard lichen treated group and erythromycin treated group were 73.08, 65.38 and 80.77% respectively. Our results suggest that beard lichen methanolic extract could be an effective therapeutic agent to be used against L. garvieae infection in rainbow trout at the dose of 4 mg/17.41 ±â€¯0.3 g body weight/day.

Effects of Eclipta prostrata on gut microbiota of SAMP6 mice with osteoporosis.

PURPOSE: Traditional Chinese medicine (TCM) has been extensively studied for its preventive and treatment properties toward osteoporosis (OP). Pharmacological studies have shown that TCM Eclipta prostrata induce anti-OP effects. Considering the growing evidence demonstrating that gut microbiota (GM) is related to OP, we aimed to study the GM-dependent function and mechanism of E. prostrata for preventing OP in mice. METHODOLOGY: Bone micro-structure was obtained using micro-computed tomography (micro-CT) and bone-relating factors were detected by molecular biological test. High-throughput sequencing of the 16S rRNA V4 region was performed for GM diversity analysis. Growth effects of E. prostrata on potential targeted strains Lactobacillus and Lactococcus were investigated by in vitro bacterial assay. By feeding Lactobacillus and Lactococcus in mice, GM and bone condition were analysed. RESULTS: Bone micro-structure was significantly improved by E. prostrata with a potential mechanism of inhibiting osteoclast, increasing the number of osteoblasts and regulating the dynamic balance of bone absorption and formation. Sequencing results indicated that E. prostrata altered the bacterial community. The abundance of bacteria genera Lactobacillus and Lactococcus was markedly decreased in individuals with OP and positively correlated with high dose of E. prostrata. GM of the low-dose E. prostrata-fed group did not significantly differ from that of the chow-fed OP group, which was consistent with bone structure test results. Moreover, E. prostrata could promote Lactobacillus and Lactococcus growth in vitro. GM was altered and bone condition was improved via bacterial feeding in vivo. CONCLUSION: Our findings suggested that E. prostrata might be a novel therapy for OP prevention by targeting GM.

Phenolic compounds from red wine and coffee are associated with specific intestinal microorganisms in allergic subjects.

The dietary modulation of gut microbiota, suggested to be involved in allergy processes, has recently attracted much interest. While several studies have addressed the use of fibres to modify intestinal microbial populations, information about other components, such as phenolic compounds, is scarce. The aim of this work was to identify the dietary components able to influence the microbiota in 23 subjects suffering from rhinitis and allergic asthma, and 22 age- and sex-matched controls. The food intake was recorded by means of an annual food frequency questionnaire. Dietary fibre tables were obtained from Marlett et al., and the Phenol-Explorer database was used to assess the phenolic compound intake. The quantification of microbial groups was performed using an Ion Torrent 16S rRNA gene-based analysis. The results showed a direct association between the intake of red wine, a source of stilbenes, and the relative abundance of Bacteroides, and between the intake of coffee, rich in phenolic acids, and the abundance of Clostridium, Lactococcus and Lactobacillus genera. Despite epidemiological analyses not establishing causality, these results support the association between polyphenol-rich beverages and faecal microbiota in allergic patients.