A new class of antimicrobial molecules derived from kefir, effective against Pseudomonas aeruginosa and methicillin resistant Staphylococcus aureus (MRSA) strains.
ABSTRACT: Many studies have linked the antimicrobial properties of kefir with the presence of bacteriocins and organic acids. In the present work, results obtained from bacteriostatic and bactericidal studies, and from RP-HPLC, Mass Spectrometry and proton NMR analysis, show that a sample of milk kefir grains is able to produce an antimicrobial fraction, denoted FK-1000, composed of sugars and amino acids, predominantly polymers of alanine, doublets of tyrosine and phenylalanine. Since this fraction is a lyophilized product whose molecular profile is different from bacteriocins and simple carboxylic acids, its antimicrobial effect cannot be attributed to these molecules, or to alcohols or hydrogen peroxide. The fraction is bactericidal against weak-acid-resistant MRSA and weak-acid resistant P. aeruginosa at pH 5, and is bacteriostatic against both pathogens at pH 7. In combination formulation, the FK-1000 fraction is able to increase fivefold the effect of streptomycin against P. aeruginosa and it is not toxic to human epithelial cells at antimicrobial concentrations. 16 S rRNA microbiota analysis of antimicrobial-producing and non-producing kefir grains demonstrated that they are distinct. In summary, the results indicate that milk kefir grains can produce different classes of molecules with potent antibiotic activity against resistant bacteria.
Project description:The microorganisms that make up kefir grains are well known for lactose fermentation, but the extent to which they hydrolyze and consume milk proteins remains poorly understood. Peptidomics technologies were used to examine the proteolytic activity of kefir grains on bovine milk proteins.Gel electrophoresis revealed substantial digestion of milk proteins by kefir grains, with mass spectrometric analysis showing the release of 609 protein fragments and alteration of the abundance of >1500 peptides that derived from 27 milk proteins. Kefir contained 25 peptides identified from the literature as having biological activity, including those with antihypertensive, antimicrobial, immunomodulatory, opioid and anti-oxidative functions. 16S rRNA and shotgun metagenomic sequencing identified the principle taxa in the culture as Lactobacillus species.The model kefir sample contained thousands of protein fragments released in part by kefir microorganisms and in part by native milk proteases.
Project description:Kefir is a functional beverage that contains lactic and acetic acid bacteria (LAB, AAB) and yeasts. This work's aim was to study the chemical, microbial, and functional characteristics of kefir produced from cow's milk and soy milk. After fermentation, free amino acids were 20.92?mg 100?mL-1 and 36.20?mg 100?mL-1 for cow's milk and soy milk kefir, respectively. Glutamic acid was majority in both, suggesting that microbial proteolysis leads to an increase in free amino acids including glutamic acid. 108-109?CFU?mL-1 LAB, 106-107?CFU?mL-1 AAB, and 106-107?CFU?mL-1 yeasts were counted in cow's milk kefir, whereas soy milk kefir contained greatly lower yeasts and AAB. Lactococcus lactis, Kazachstania unispora, and Saccharomyces cerevisiae were isolated as major microorganisms in both kefirs. Acetobacter orientalis only existed in cow's milk kefir. Cow's milk and soy milk showed ACE inhibitory activity, which significantly increased after fermentation. Both kefirs also exhibited antioxidant activity and bactericidal activity against Escherichia coli, Salmonella Typhimurium, and Staphylococcus aureus.
Project description:Antimicrobial resistance is one of the major clinical concerns, making the discovery of new antimicrobial drugs desirable. Moringin (MOR), the major isothiocyanate produced from Moringa oleifera seeds, could represent an alternative therapeutic strategy to commonly used antibiotics. The aim of our study was to investigate the antimicrobial effect of MOR conjugated with ?-cyclodextrin (MOR/?-CD), a complex with an improved solubility and stability in aqueous solutions. Our data demonstrated that MOR/?-CD was able to exert antimicrobial activity against the S. aureus reference strains (ATCC 25923, ATCC 6538, and ATCC BAA-977). Moreover, MOR/?-CD showed bacteriostatic effects (MIC = minimum inhibitory concentration = 0.5 mg/mL) and bactericidal properties (MBC = minimum bactericidal concentration = 1 mg/mL) against the overall assessed strains. In addition, MOR/?-CD showed bactericidal activity against the S. aureus strain ATCC BAA-977 after treatment with erythromycin (Ery), which induced clindamycin-resistance on the erm (A) gene. This evidence led us to assume that MOR/?-CD could be a promising antimicrobial agent against strains with the clindamycin-resistant phenotype (CC-resistant).
Project description:Staphylococcus aureus is a major pathogen in humans and causes serious problems due to antibiotic resistance. We investigated the antimicrobial effect of glycyrrhetinic acid (GRA) and its derivatives against 50 clinical S. aureus strains, including 18 methicillin-resistant strains. The minimum inhibitory concentrations (MICs) of GRA, dipotassium glycyrrhizate, disodium succinoyl glycyrrhetinate (GR-SU), stearyl glycyrrhetinate and glycyrrhetinyl stearate were evaluated against various S. aureus strains. Additionally, we investigated the bactericidal effects of GRA and GR-SU against two specific S. aureus strains. DNA microarray analysis was also performed to clarify the mechanism underlying the antibacterial activity of GR-SU. We detected the antimicrobial activities of five agents against S. aureus strains. GRA and GR-SU showed strong antibacterial activities compared to the other three agents tested. At a higher concentration (above 2x MIC), GRA and GR-SU showed bactericidal activity, whereas at a concentration of 1x MIC, they showed a bacteriostatic effect. Additionally, GRA and GR-SU exhibited a synergistic effect with gentamicin. The expression of a large number of genes (including transporters) and metabolic factors (carbohydrates and amino acids) was altered by the addition of GR-SU, suggesting that the inhibition of these metabolic processes may influence the degree of the requirement for carbohydrates or amino acids. In fact, the requirement for carbohydrates or amino acids was increased in the presence of either GRA or GR-SU. GRA and GR-SU exhibited strong antibacterial activity against several S. aureus strains, including MRSA. This activity may be partly due to the inhibition of several pathways involved in carbohydrate and amino acid metabolism.
Project description:Although 90% of BRD relapses are reported to receive retreatment with a different class of antimicrobial, studies examining the impact of antimicrobial selection (i.e. bactericidal or bacteriostatic) on retreatment outcomes and the emergence of antimicrobial resistance (AMR) are deficient in the published literature. This survey was conducted to determine the association between antimicrobial class selection for treatment and retreatment of BRD relapses on antimicrobial susceptibility of Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni. Pathogens were isolated from samples submitted to the Iowa State University Veterinary Diagnostic Laboratory from January 2013 to December 2015. A total of 781 isolates with corresponding animal case histories, including treatment protocols, were included in the analysis. Original susceptibility testing of these isolates for ceftiofur, danofloxacin, enrofloxacin, florfenicol, oxytetracycline, spectinomycin, tilmicosin, and tulathromycin was performed using Clinical and Laboratory Standards Institute guidelines. Data were analyzed using a Bayesian approach to evaluate whether retreatment with antimicrobials of different mechanistic classes (bactericidal or bacteriostatic) increased the probability of resistant BRD pathogen isolation in calves. The posterior distribution we calculated suggests that an increased number of treatments is associated with a greater probability of isolates resistant to at least one antimicrobial. Furthermore, the frequency of resistant BRD bacterial isolates was greater with retreatment using antimicrobials of different mechanistic classes than retreatment with the same class. Specifically, treatment protocols using a bacteriostatic drug first followed by retreatment with a bactericidal drug were associated with a higher frequency of resistant BRD pathogen isolation. In particular, first treatment with tulathromycin (bacteriostatic) followed by ceftiofur (bactericidal) was associated with the highest probability of resistant M. haemolytica among all antimicrobial combinations. These observations suggest that consideration should be given to antimicrobial pharmacodynamics when selecting drugs for retreatment of BRD. However, prospective studies are needed to determine the clinical relevance to antimicrobial stewardship programs in livestock production systems.
Project description:The aim of this paper is to describe a new variant of Janthinobacterium lividum - ROICE173, isolated from Antarctic snow, and to investigate the antimicrobial effect of the crude bacterial extract against 200 multi-drug resistant (MDR) bacteria of both clinical and environmental origin, displaying various antibiotic resistance patterns. ROICE173 is extremotolerant, grows at high pH (5.5-9.5), in high salinity (3%) and in the presence of different xenobiotic compounds and various antibiotics. The best violacein yield (4.59?±?0.78?mg·g-1 wet biomass) was obtained at 22?°C, on R2 broth supplemented with 1% glycerol. When the crude extract was tested for antimicrobial activity, a clear bactericidal effect was observed on 79 strains (40%), a bacteriostatic effect on 25 strains (12%) and no effect in the case of 96 strains (48%). A very good inhibitory effect was noticed against numerous MRSA, MSSA, Enterococci, and Enterobacteriaceae isolates. For several environmental E. coli strains, the bactericidal effect was encountered at a violacein concentration below of what was previously reported. A different effect (bacteriostatic vs. bactericidal) was observed in the case of Enterobacteriaceae isolated from raw vs. treated wastewater, suggesting that the wastewater treatment process may influence the susceptibility of MDR bacteria to violacein containing bacterial extracts.
Project description:Milk kefir is a traditional fermented milk product whose consumption is becoming increasingly popular. The natural starter for kefir production is kefir grain, which consists of various bacterial and yeast species. At the industrial scale, however, kefir grains are rarely used due to their slow growth, complex application, bad reproducibility and high costs. Instead, mixtures of defined lactic acid bacteria and sometimes yeasts are applied, which alter sensory and functional properties compared to natural grain-based milk kefir. In order to be able to mimic natural starter cultures for authentic kefir production, it is a prerequisite to gain deep knowledge about the nature of kefir grains, its microbial composition, morphologic structure, composition of strains on grains and the impact of environmental parameters on kefir grain characteristics. In addition, it is very important to deeply investigate the numerous multi-dimensional interactions among different species, which play important roles on the formation and the functionality of grains.
Project description:Backgroud: The growing emergence of antibiotic-resistant pathogens including the most dangerous superbugs requires quick discovery of novel antibiotics/biopreservatives for human health and food safety. Bacteriocins, a subgroup of antimicrobial peptides, have been considered as promising alternatives to antibiotics. Abundant novel bacteriocins are stored in genome sequences of lactic acid bacteria. However, discovery of novel bacteriocins still mainly relies on dubious traditional purification with low efficiency. Moreover, sequence alignment is invalid for novel bacteriocins which have no homology to known bacteriocins in databases. Therefore, an efficient, simple, universal, and time-saving method was needed to discover novel bacteriocins. Methods and Results: Crude bacteriocins from both cell-related and culture supernatant of Lactobacillus crustorum MN047 fermentation were applied to LC-MS/MS for peptidome assay, by which 131 extracellular peptides or proteins were identified in the complete genome sequence of L. crustorum MN047. Further, the genes of suspected bacteriocins were verified by expressed in Escherichia coli BL21 (DE3) pLysS. Thereafter, eight novel bacteriocins and two nonribosomal antimicrobial peptides were identified to be broad-spectrum activity against both Gram-positive and Gram-negative bacteria, including some multidrug-resistant strains. Among them, BM1556 located within predicted bacteriocin gene cluster. The most active bacteriocin BM1122 had low MIC values of 13.7 mg/L against both Staphylococcus aureus ATCC29213 and E. coli ATCC25922. The BM1122 had bactericidal action mode by biofilm-destruction, pore-formation, and membrane permeability change. Conclusions: The combination of complete genome and peptidome is a valid approach for quick discovery of novel bacteriocins without/with-low homology to known ones. This method will contribute to deep exploitation of novel bacteriocins in genome of bacteria submitted to GenBank.
Project description:Artisanal kefir is a traditional fermented dairy product made using kefir grains. Kefir has documented natural antimicrobial activity and health benefits. A typical kefir microbial community includes lactic acid bacteria (LAB), acetic acid bacteria, and yeast among other species in a symbiotic matrix. In the presented work, the 16S rRNA gene sequencing was used to reveal bacterial populations and elucidate the diversity and abundance of LAB species in international artisanal kefirs from Fusion Tea, Britain, the Caucuses region, Ireland, Lithuania, and South Korea. Bacterial species found in high abundance in most artisanal kefirs included Lactobacillus kefiranofaciens, Lentilactobacillus kefiri,Lactobacillus ultunensis, Lactobacillus apis, Lactobacillus gigeriorum, Gluconobacter morbifer, Acetobacter orleanensis, Acetobacter pasteurianus, Acidocella aluminiidurans, and Lactobacillus helveticus. Some of these bacterial species are LAB that have been reported for their bacteriocin production capabilities and/or health promoting properties.
Project description:Kefir is a dairy product that can be prepared from different milk types, such as goat, buffalo, sheep, camel, or cow via microbial fermentation (inoculating milk with kefir grains). As such, kefir contains various bacteria and yeasts which influence its chemical and sensory characteristics. A mixture of two kinds of milk promotes kefir sensory and rheological properties aside from improving its nutritional value. Additives such as inulin can also enrich kefir's health qualities and organoleptic characters. Several metabolic products are generated during kefir production and account for its distinct flavour and aroma: Lactic acid, ethanol, carbon dioxide, and aroma compounds such as acetoin and acetaldehyde. During the storage process, microbiological, physicochemical, and sensory characteristics of kefir can further undergo changes, some of which improve its shelf life. Kefir exhibits many health benefits owing to its antimicrobial, anticancer, gastrointestinal tract effects, gut microbiota modulation and anti-diabetic effects. The current review presents the state of the art relating to the role of probiotics, prebiotics, additives, and different manufacturing practices in the context of kefir's physicochemical, sensory, and chemical properties. A review of kefir's many nutritional and health benefits, underlying chemistry and limitations for usage is presented.