Lactobacillus crispatus Limits Bladder Uropathogenic E. coli Infection by Triggering a Host Type I Interferon Response.

Many urinary tract infections (UTIs) are recurrent because uropathogens persist within the bladder epithelial cells (BECs) for extended periods between bouts of infection. Because persistent uropathogens are intracellular, they are often refractive to antibiotic treatment. The recent discovery of endogenous Lactobacillus spp. in the bladders of healthy humans raised the question of whether these endogenous bacteria directly or indirectly impact intracellular bacterial burden in the bladder. Here, we report that in contrast to healthy women, female patients experiencing recurrent UTIs have a bladder population of Lactobacilli that is markedly reduced. Exposing infected human BECs to L. crispatus in vitro markedly reduced the intracellular uropathogenic Escherichia coli (UPEC) load. The adherence of Lactobacilli to BECs was found to result in increased type I interferon (IFN) production, which in turn enhanced the expression of cathepsin D within lysosomes harboring UPECs. This lysosomal cathepsin D-mediated UPEC killing was diminished in germ-free mice and type I IFN receptor-deficient mice. Secreted metabolites of L. crispatus seemed to be responsible for the increased expression of type I IFN in human BECs. Intravesicular administration of Lactobacilli into UPEC-infected murine bladders markedly reduced their intracellular bacterial load suggesting that components of the endogenous microflora can have therapeutic effects against UTIs.

Vibrio cholerae FeoB contains a dual nucleotide-specific NTPase domain essential for ferrous iron uptake.

The Feo ferrous iron transporter is widely distributed among bacteria and archaea, but its mechanism of transport has not been fully elucidated. In Vibrio cholerae, the transport system requires three proteins: the small cytosolic proteins FeoA and FeoC and a large cytoplasmic-membrane-associated protein FeoB, which has an N-terminal G-protein domain. We show that, in contrast to Escherichia coli FeoB, which is solely a GTPase, the V. cholerae and Helicobacter pylori FeoB proteins have both GTPase and ATPase activity. In V. cholerae, mutation of the G4 motif, responsible for hydrogen bonding with the guanine base, abolished the GTPase activity but not ATPase activity. The ATPase activity of the G4 motif mutants was sufficient for Feo function in the absence of GTPase. We show that the serine and asparagine residues in the G5 motif likely play a role in the ATPase activity, and substitution of these residues with those found in the corresponding positions in E. coli FeoB resulted in similar nucleotide hydrolysis activity in the E. coli protein. These results add significantly to our understanding of the NTPase domain of FeoB and its role in Feo function.

Biochemical characterization of bacterial FeoBs: A perspective on nucleotide specificity.

Iron is an essential requirement for the survival and virulence of most bacteria. The bacterial ferrous iron transporter protein FeoB functions as a major reduced iron transporter in prokaryotes, but its biochemical mechanism has not been fully elucidated. In the present study, we compared enzymatic properties of the cytosolic portions of pathogenic bacterial FeoBs to elucidate each bacterial strain-specific characteristic of the Feo system. We show that bacterial FeoBs are classified into two distinct groups that possess either a sole GTPase or an NTPase with a substrate promiscuity. This difference in nucleotide preference alters cellular requirements for monovalent and divalent cations. While the hydrolytic activity of the GTP-dependent FeoBs was stimulated by potassium, the action of the NTP-dependent FeoBs was not significantly affected by the presence of monovalent cations. Mutation of Asn11, having a role in potassium-dependent GTP hydrolysis, changed nucleotide specificity of the NTP-dependent FeoB, resulting in loss of ATPase activity. Sequence analysis suggested a possible association of alanine in the G5 motif for the NTP-dependent activity in FeoBs. This demonstration of the distinct enzymatic properties of bacterial FeoBs provides important insights into mechanistic details of Feo iron transport processes, as well as offers a promising species-specific anti-virulence target.

Xylose utilization in Saccharomyces cerevisiae during conversion of hydrothermally pretreated lignocellulosic biomass to ethanol.

With growing interest in alternative fuels to minimize carbon and particle emissions, research continues on the production of lignocellulosic ethanol and on the development of suitable yeast strains. However, great diversities and continued technical advances in pretreatment methods for lignocellulosic biomass complicate the evaluation of developed yeast strains, and strain development often lags industrial applicability. In this review, recent studies demonstrating developed yeast strains with lignocellulosic biomass hydrolysates are compared. For the pretreatment methods, we highlight hydrothermal pretreatments (dilute acid treatment and autohydrolysis), which are the most commonly used and effective methods for lignocellulosic biomass pretreatment. Rather than pretreatment conditions, the type of biomass most strongly influences the composition of the hydrolysates. Metabolic engineering strategies for yeast strain development, the choice of xylose-metabolic pathway, adaptive evolution, and strain background are highlighted as important factors affecting ethanol yield and productivity from lignocellulosic biomass hydrolysates. A comparison of the parameters from recent studies demonstrating lignocellulosic ethanol production provides useful information for future strain development.

Deletion of the neural tube defect-associated gene Mthfd1l disrupts one-carbon and central energy metabolism in mouse embryos.

One-carbon (1C) metabolism is a universal folate-dependent pathway essential for de novo purine and thymidylate synthesis, amino acid interconversion, universal methyl-donor production, and regeneration of redox cofactors. Homozygous deletion of the 1C pathway gene Mthfd1l encoding methylenetetrahydrofolate dehydrogenase (NADP+-dependent) 1-like, which catalyzes mitochondrial formate production from 10-formyltetrahydrofolate, results in 100% penetrant embryonic neural tube defects (NTDs), underscoring the central role of mitochondrially derived formate in embryonic development and providing a mechanistic link between folate and NTDs. However, the specific metabolic processes that are perturbed by Mthfd1l deletion are not known. Here, we performed untargeted metabolomics on whole Mthfd1l-null and wildtype mouse embryos in combination with isotope tracer analysis in mouse embryonic fibroblast (MEF) cell lines to identify Mthfd1l deletion-induced disruptions in 1C metabolism, glycolysis, and the TCA cycle. We found that maternal formate supplementation largely corrects these disruptions in Mthfd1l-null embryos. Serine tracer experiments revealed that Mthfd1l-null MEFs have altered methionine synthesis, indicating that Mthfd1l deletion impairs the methyl cycle. Supplementation of Mthfd1l-null MEFs with formate, hypoxanthine, or combined hypoxanthine and thymidine restored their growth to wildtype levels. Thymidine addition alone was ineffective, suggesting a purine synthesis defect in Mthfd1l-null MEFs. Tracer experiments also revealed lower proportions of labeled hypoxanthine and inosine monophosphate in Mthfd1l-null than in wildtype MEFs, suggesting that Mthfd1l deletion results in increased reliance on the purine salvage pathway. These results indicate that disruptions of mitochondrial 1C metabolism have wide-ranging consequences for many metabolic processes, including those that may not directly interact with 1C metabolism.

Comparative global metabolite profiling of xylose-fermenting Saccharomyces cerevisiae SR8 and Scheffersomyces stipitis.

Bioconversion of lignocellulosic biomass into ethanol requires efficient xylose fermentation. Previously, we developed an engineered Saccharomyces cerevisiae strain, named SR8, through rational and inverse metabolic engineering strategies, thereby improving its xylose fermentation and ethanol production. However, its fermentation characteristics have not yet been fully evaluated. In this study, we investigated the xylose fermentation and metabolic profiles for ethanol production in the SR8 strain compared with native Scheffersomyces stipitis. The SR8 strain showed a higher maximum ethanol titer and xylose consumption rate when cultured with a high concentration of xylose, mixed sugars, and under anaerobic conditions than Sch. stipitis. However, its ethanol productivity was less on 40 g/L xylose as the sole carbon source, mainly due to the formation of xylitol and glycerol. Global metabolite profiling indicated different intracellular production rates of xylulose and glycerol-3-phosphate in the two strains. In addition, compared with Sch. stipitis, SR8 had increased abundances of metabolites from sugar metabolism and decreased abundances of metabolites from energy metabolism and free fatty acids. These results provide insights into how to control and balance redox cofactors for the production of fuels and chemicals from xylose by the engineered S. cerevisiae.

Deletion of PHO13 improves aerobic L-arabinose fermentation in engineered Saccharomyces cerevisiae.

Pentose sugars are increasingly being used in industrial applications of Saccharomyces cerevisiae. Although L-arabinose is a highlighted pentose that has been identified as next-generation biomass, arabinose fermentation has not yet undergone extensive development for industrial utilization. In this study, we integrated a heterologous fungal arabinose pathway with a deletion of PHO13 phosphatase gene. PHO13 deletion increased arabinose consumption rate and specific ethanol productivity under aerobic conditions and consequently depleted sedoheptulose by activation of the TAL1 gene. Global metabolite profiling indicated upregulation of the pentose phosphate pathway and downstream effects such as trehalose accumulation and downregulation of the TCA cycle. Our results suggest that engineering of PHO13 has ample potential for arabinose conversion to ethanol as an industrial source for biofuels.

Mitochondrial MTHFD2L is a dual redox cofactor-specific methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase expressed in both adult and embryonic tissues.

Mammalian mitochondria are able to produce formate from one-carbon donors such as serine, glycine, and sarcosine. This pathway relies on the mitochondrial pool of tetrahydrofolate (THF) and several folate-interconverting enzymes in the mitochondrial matrix. We recently identified MTHFD2L as the enzyme that catalyzes the oxidation of 5,10-methylenetetrahydrofolate (CH2-THF) in adult mammalian mitochondria. We show here that the MTHFD2L enzyme is bifunctional, possessing both CH2-THF dehydrogenase and 5,10-methenyl-THF cyclohydrolase activities. The dehydrogenase activity can use either NAD(+) or NADP(+) but requires both phosphate and Mg(2+) when using NAD(+). The NADP(+)-dependent dehydrogenase activity is inhibited by inorganic phosphate. MTHFD2L uses the mono- and polyglutamylated forms of CH2-THF with similar catalytic efficiencies. Expression of the MTHFD2L transcript is low in early mouse embryos but begins to increase at embryonic day 10.5 and remains elevated through birth. In adults, MTHFD2L is expressed in all tissues examined, with the highest levels observed in brain and lung.

Investigation of phenyllactic acid as a potent tyrosinase inhibitor produced by probiotics.

Melanogenesis is responsible for skin pigmentation and the enzymatic browning of foods. Tyrosinases play a major role in melanin synthesis, and many attempts have been made to identify new natural tyrosinase inhibitors, but few have sought to do in microbes. Postbiotics are bioactive compounds produced by the metabolism of probiotics and have been reported to be safe and effective. In this study, we evaluated the tyrosinase inhibitory effects of culture supernatants of probiotics and discovered novel bacterial metabolites that can be used as a potent tyrosinase inhibitor based on metabolomics. Cultures of Bifidobacterium bifidum IDCC 4201 and Lactiplantibacillus plantarum IDCC 3501 showed effective anti-tyrosinase, reduced melanin synthesis, and altered protein expression associated with the melanogenesis pathway. Comparative metabolomics analyses conducted by GC-MS identified metabolites commonly produced by B. bifidum and L. plantarum. Of eight selected metabolites, phenyllactic acid exhibited significant tyrosinase-inhibitory activity. Our findings suggest that applications of probiotic culture supernatants containing high amounts of phenyllactic acid have potential use as anti-melanogenesis agents in food and medicines.

Bifidobacterium lactis IDCC 4301 Exerts Anti-Obesity Effects in High-Fat Diet-Fed Mice Model by Regulating Lipid Metabolism.

SCOPE: Chronic hypernutrition promotes lipid accumulation in the body and excessive lipid accumulation leads to obesity. An increase in the number and size of adipocytes, a characteristic of obesity is closely associated with adipose dysfunction. Recent in vitro and in vivo studies have shown that probiotics may prevent this dysfunction by regulating lipid metabolism. However, the mechanisms of action of probiotics in obesity are not fully understood and their usage for treating obesity remains limited. METHODS AND RESULTS: Bifidobacterium lactis IDCC 4301 is selected for its anti-obesity potential after evaluating inhibitory activity of pancreatic lipase and cholesterol reducing activity. Next, this study investigates the roles of B. lactis IDCC 4301 on lipid metabolism in 3T3-L1 preadipocytes and high-fat diet (HFD)-fed mice. B. lactis IDCC 4301 inhibits cell differentiation and lipid accumulation by suppressing the expression of adipogenic enzymes in 3T3-L1 cells. Moreover, the administration of B. lactis IDCC 4301 decreases body and adipose tissue weight, improves serum lipid levels, and downregulates adipogenic mRNA expression in HFD-fed mice. Additionally, metabolomic analysis suggests that 2-ketobutyrate should be a possible target compound against obesity. CONCLUSIONS: B. lactis IDCC 4301 may be used as an alternative treatment for obesity.

Portable Colorimetric Hydrogel Beads for Point-of-Care Antimicrobial Susceptibility Testing.

Sepsis is a life-threatening condition with systemic inflammatory responses caused by bacterial infections. Considering the emergence of antibiotic-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), sepsis is a great threat to public health. The gold standard methods for antimicrobial susceptibility testing (AST), however, take at least approximately 3 days to implement the entire blood culture, pure culture, and AST processes. To overcome the time-consuming nature of conventional AST, a method employing a chromatic biosensor composed of poly(diacetylene), alginate, and LB broth (PAL) is introduced in this study. Compared to the gold standards, AST with PAL biosensors can be completed within a time frame as short as 16 h. Such a significant reduction in time is possible because the consecutive cultures and AST are carried out simultaneously by encapsulating the bacterial nutrients and detection molecules into a single component. The bead-like hydrogel sensors were used in their freeze-dried form, which endows them with portability and stability, thus making them adequate for point-of-care testing. The PAL biosensor yields minimum inhibitory concentrations comparable to those from the Clinical and Laboratory Standards Institute, and the applicability of the biosensor is further shown in MRSA-infected mice.

Alleviation of DSS-induced colitis via bovine colostrum-derived extracellular vesicles with microRNA let-7a-5p is mediated by regulating Akkermansia and ß-hydroxybutyrate in gut environments.

IMPORTANCE: Even though studying on the possible involvement of extracellular vesicles (EVs) in host-microbe interactions, how these relationships mediate host physiology has not clarified yet. Our current findings provide insights into the encouraging benefits of dietary source-derived EVs and microRNAs (miRNAs) on organic acid production and ultimately stimulating gut microbiome for human health, suggesting that supplementation of dietary colostrum EVs and miRNAs is a novel preventive strategy for the treatment of inflammatory bowel disease.

Red ginseng dietary fiber promotes probiotic properties of Lactiplantibacillus plantarum and alters bacterial metabolism.

Korean red ginseng has been widely used as an herbal medicine. Red ginseng dietary fiber (RGDF) is a residue of the processed ginseng product but still contains bioactive constituents that can be applied as prebiotics. In this study, we evaluated changes on fermentation profiles and probiotic properties of strains that belong to family Lactobacillaceae with RGDF supplementation. Metabolomic analyses were performed to understand specific mechanisms on the metabolic alteration by RGDF and to discover novel bioactive compounds secreted by the RGDF-supplemented probiotic strain. RGDF supplementation promoted short-chain fatty acid (SCFA) production, carbon source utilization, and gut epithelial adhesion of Lactiplantibacillus plantarum and inhibited attachment of enteropathogens. Intracellular and extracellular metabolome analyses revealed that RGDF induced metabolic alteration, especially associated with central carbon metabolism, and produced RGDF-specific metabolites secreted by L. plantarum, respectively. Specifically, L. plantarum showed decreases in intracellular metabolites of oleic acid, nicotinic acid, uracil, and glyceric acid, while extracellular secretion of several metabolites including oleic acid, 2-hydroxybutanoic acid, hexanol, and butyl acetate increased. RGDF supplementation had distinct effects on L. plantarum metabolism compared with fructooligosaccharide supplementation. These findings present potential applications of RGDF as prebiotics and bioactive compounds produced by RGDF-supplemented L. plantarum as novel postbiotic metabolites for human disease prevention and treatment.

Modified Korean MIND Diet: A Nutritional Intervention for Improved Cognitive Function in Elderly Women through Mitochondrial Respiration, Inflammation Suppression, and Amino Acid Metabolism Regulation.

SCOPE: Mild cognitive impairment is associated with a high prevalence of dementia. The study examines the benefits of a modified Korean MIND (K-MIND) diet and explores biomarkers using multi-omics analysis. METHODS AND RESULTS: The K-MIND diet, tailored to the elderly Korean population, includes perilla oil, milk, or fermented milk, and avoids alcohol consumption. As a result, the K-MIND diet significantly improves subjects "orientation to place" in the Korean version of the Mini-Mental State Examination, 2nd edition test. According to multi-omics analysis, the K-MIND diet upregulates genes associated with mitochondrial respiration, including ubiquinone oxidoreductase, cytochrome C oxidase, and ATP synthase, and immune system processes, and downregulates genes related to nuclear factor kappa B activity and inflammatory responses. In addition, K-MIND affects the metabolic pathways of glycine, serine, threonine, tryptophan, and sphingolipids, which are closely linked to cognitive function through synthesis of neurotransmitters and structures of brain cell membranes. CONCLUSION: The findings imply that the K-MIND diet improves cognitive function by upregulating key genes involved in oxidative phosphorylation and downregulating pro-inflammatory cytokines.

Psychosocial Characters and Their Behavioural Indexes for Evaluation in Secondary School Physical Education Classes and Sports Club Activities.

The purpose of this study was to explore students' psychosocial characteristics presumably nurtured in school physical education (PE) and school sports club activities in Korea. In addition, this study attempted to investigate what actual behaviours for each characteristic are observed and could be evaluated. Previous studies related to secondary students' character development in school sports clubs and school PE classes were investigated at the initial stage, and then a panel of 3 experts and 4 host researchers reviewed and selected 9 characteristics and 30 behaviours. Two replicates of a modified Delphi analysis and the Analytic Hierarchy Process (AHP) with 25 and 50 PE teachers respectively were performed and reached 7 characteristics and 21 behavioural indexes and their hierarchy. The content validity ratio (CVR) for seven characteristics in two replicates of a modified Delphi analysis was 0.93. The highest CVR was 1.00 while the lowest was 0.68. The highest CVR among 21 behaviour indexes was 1.00 while the lowest was 0.52, which implied that all the characteristics and the behaviour indexes are valid. In the stage of AHP for each characteristic's hierarchy, "Earnestness" was ranked highest with a weight of 0.215, while "Leadership" was ranked lowest at 0.044 (consistency index and consistency ratio < 0.1). 'Disengaged observation/late in class,' 'helping peer,' and 'opinion coordination' shared the highest score at 0.091 while 'taking initiatives' was placed lowest with 0.010 in the list of 21 behaviour indexes. The results helped infer that PE teachers consider development of interpersonal characteristics and the level of articulation for behaviour indexes important.

Molecular characterization of gut microbiome in weaning pigs supplemented with multi-strain probiotics using metagenomic, culturomic, and metabolomic approaches.

BACKGROUND: Probiotics have been reported to exhibit positive effects on host health, including improved intestinal barrier function, preventing pathogenic infection, and promoting nutrient digestion efficiency. These internal changes are reflected to the fecal microbiota composition and, bacterial metabolites production. In accordance, the application of probiotics has been broadened to industrial animals, including swine, which makes people to pursue better knowledge of the correlation between changes in the fecal microbiota and metabolites. Therefore, this study evaluated the effect of multi-strain probiotics (MSP) supplementation to piglets utilizing multiomics analytical approaches including metagenomics, culturomics, and metabolomics. RESULTS: Six-week-old piglets were supplemented with MSP composed of Lactobacillus isolated from the feces of healthy piglets. To examine the effect of MSP supplement, piglets of the same age were selected and divided into two groups; one with MSP supplement (MSP group) and the other one without MSP supplement (Control group). MSP feeding altered the composition of the fecal microbiota, as demonstrated by metagenomics analysis. The abundance of commensal Lactobacillus was increased by 2.39%, while Clostridium was decreased, which revealed the similar pattern to the culturomic approach. Next, we investigated the microbial metabolite profiles, specifically SCFAs using HPLC-MS/MS and others using GC-MS, respectively. MSP supplement elevated the abundance of amino acids, including valine, isoleucine and proline as well as the concentration of acetic acid. According to the correlation analyses, these alterations were found out to be crucial in energy synthesizing metabolism, such as branched-chain amino acid (BCAA) metabolism and coenzyme A biosynthesis. Furthermore, we isolated commensal Lactobacillus strains enriched by MSP supplement, and analyzed the metabolites and evaluated the functional improvement, related to tight junction from intestinal porcine enterocyte cell line (IPEC-J2). CONCLUSIONS: In conclusion, MSP administration to piglets altered their fecal microbiota, by enriching commensal Lactobacillus strains. This change contributed amino acid, acetic acid, and BCAA concentrations to be increased, and energy metabolism pathway was also increased at in vivo and in vitro. These changes produced by MSP supplement suggests the correlation between the various physiological energy metabolism functions induced by health-promoting Lactobacillus and the growth performance of piglets.

Effects of Dietary Carbohydrases on Fecal Microbiome Composition of Lactating Sows and Their Piglets.

Corn-soybean meal diets are commonly used in the pork industry as a primary source of energy and protein. However, such a diet generally contains non-starch polysaccharides (NSPs) which present a challenge in finding ways to improve their availability and digestibility. Dietary multi-carbohydrases (MCs) have been proposed as an efficient approach to utilize NSPs, and can result in improved growth performance and host intestinal fitness. In this study, we evaluated the effects of MC in lactation diets on gut microbiota composition of lactating sows and their litters. The experimental design contained two dietary treatments, a diet based on corn-soybean meal (CON), and CON supplemented with 0.01% multigrain carbohydrases (MCs). Sow and piglet fecal samples were collected on days 7 and 28 after farrowing. Based on the results from 16S rRNA gene amplicon sequencing, MC led to changes in species diversity and altered the microbial compositions in lactating sows and their piglets. Specifically, the MC treatment induced an increase in the proportions of Lactobacillus in piglets. Clostridium and Spirochaetaceae showed a significantly reduced proportion in MC-treated sows at day 28. Our results support the beneficial effects of dietary carbohydrases and their link with improved production due to better host fitness outcomes and gut microbiota composition.

Vanadate inhibits Feo-mediated iron transport in Vibrio cholerae.

Iron is an essential element for Vibrio cholerae to survive, and Feo, the major bacterial system for ferrous iron transport, is important for growth of this pathogen in low-oxygen environments. To gain insight into its biochemical mechanism, we evaluated the effects of widely used ATPase inhibitors on the ATP hydrolysis activity of the N-terminal domain of V. cholerae FeoB. Our results showed that sodium orthovanadate and sodium azide effectively inhibit the catalytic activity of the N-terminal domain of V. cholerae FeoB. Further, sodium orthovanadate was the more effective inhibitor against V. cholerae ferrous iron transport in vivo. These results contribute to a more comprehensive biochemical understanding of Feo function, and shed light on designing effective inhibitors against bacterial FeoB proteins.

Evaluation of Probiotic Characteristics of Newly Isolated Lactic Acid Bacteria from Dry-Aged Hanwoo Beef.

Dry aging is a traditional method that improves meat quality, and diverse microbial communities are changed during the process. Lactic acid bacteria (LAB) are widely present in fermented foods and has many beneficial effects, such as immune enhancement and maintenance of intestinal homeostasis. In this study, we conducted metagenomic analysis to evaluate the changes in the microbial composition of dry-aged beef. We found that lactic acid bacterial strains were abundant in dry-aged beef including Lactobacillus sakei and Enterococcus faecalis. We investigated their abilities in acid and bile tolerance, adhesion to the host, antibiotic resistance, and antimicrobial activity as potential probiotics, confirming that L. sakei and E. faecalis strains had remarkable capability as probiotics. The isolates from dry-aged beef showed at least 70% survival under acidic conditions in addition to an increase in the survival level under bile conditions. Antibiotic susceptibility and antibacterial activity assays further verified their effectiveness in inhibiting all pathogenic bacteria tested, and most of them had low resistance to antibiotics. Finally, we used the Caenorhabditis elegans model to confirm their life extension and influence on host resistance. In the model system, 12D26 and 20D48 strains had great abilities to extend the nematode lifespan and to improve host resistance, respectively. These results suggest the potential use of newly isolated LAB strains from dry-aged beef as probiotic candidates for production of fermented meat.

Identification of a New Antimicrobial Agent against Bovine Mastitis-Causing Staphylococcus aureus.

Staphylococcus aureus RF122 is a major pathogen that causes bovine mastitis, which is the most prevalent and costly disease in the milk and dairy industry. S. aureus expresses various virulence factors that are especially highly associated with iron metabolism, and the bacterial ferrous iron transport system Feo is important for bacterial growth or virulence in mammalian hosts. In this study, we evaluated a new antimicrobial agent, PHT-427, targeting the S. aureus RF122 Feo system for the prevention of bovine mastitis. Various analyses on in vitro enzymatic assays, growth inhibition, virulence expressions, and toxicity of animal model systems were conducted to characterize the inhibition properties of PHT-427. This small molecule efficiently inhibited enzyme activity of FeoB and bacterial growth. PHT-427 attenuated various virulence factors related to milk quality, including staphyloxanthin production, biofilm formation, and coagulation. Considering the high frequency of antibiotic-resistant S. aureus in bovine mastitis isolates, PHT-427 synergistically enhanced bacterial antibiotic susceptibility and further inhibited global Gram-positive bacterial growth. Unlike its effects on bacteria, the inhibitor did not show any toxicity on animal model systems. These results indicate that the S. aureus Feo system represents a good target for antimicrobial strategies, and this new antimicrobial agent may represent a promising biotechnological application for preventing S. aureus-induced bovine mastitis in the milk and dairy industry.