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(1) The beach areas of Galveston, Texas, USA are heavily used for recreational activities and often experience elevated fecal indicator bacteria levels, representing a potential threat to ecosystem services, human health, and tourism-based economies that rely on suitable water quality. (2) During the span of 15 months (March 2022–May 2023), water samples that exceeded the U.S. Environmental Protection Agency-accepted alternative Beach Action Value (BAV) for enterococci of 104 MPN/100 mL were analyzed via microbial source tracking (MST) through quantitative polymerase chain reaction (qPCR) assays. The Bacteroides HF183 and DogBact as well as the Catellicoccus LeeSeaGull markers were used to detect human, dog, and gull fecal sources, respectively. The qPCR MST data were then utilized in a quantitative microbial risk assessment (QMRA) to assess human health risks. Additionally, samples collected in July and August 2022 were sequenced for 16S rRNA and matched with fecal sources through the Bayesian SourceTracker2 program. (3) Overall, 26% of the 110 samples with enterococci exceedances were positive for at least one of the MST markers. Gull was revealed to be the primary source of identified fecal contamination through qPCR and SourceTracker2. Human contamination was detected at very low levels (<1%), whereas dog contamination was found to co-occur with human contamination through qPCR. QMRA identified Campylobacter from canine sources as being the primary driver for human health risks for contact recreation for both adults and children. (4) These MST results coupled with QMRA provide important insight into water quality in Galveston that can inform future water quality and beach management decisions that prioritize public health risks. Full article

In Brazil, Plasmodium infections in non-human primates (NHPs) have been associated with P. simium and P. brasilianum, which are morphologically and genetically similar to the human-infecting species P. vivax and P. malariae, respectively. Surveillance and monitoring of wild NHPs are crucial for understanding the distribution of these parasites and assessing the risk of zoonotic transmission. This study aimed to detect the presence of Plasmodium spp. genetic material in Platyrrhini primates from 47 municipalities in the states of Bahia and Minas Gerais. The animals were captured using Tomahawk-type live traps baited with fruit or immobilized with tranquilizer darts. Free-ranging individuals were chemically restrained via inhalation anesthesia using VetBag^® or intramuscular anesthesia injection. Blood samples were collected from the femoral vein. A total of 298 blood and tissue samples were collected from 10 primate species across five genera: Alouatta caraya (25), Alouatta guariba clamitans (1), Callicebus melanochir (1), Callithrix geoffroyi (28), Callithrix jacchus (4), Callithrix kuhlii (31), Callithrix penicillata (175), Callithrix spp. hybrids (15), Leontopithecus chrysomelas (16), Sapajus robustus (1), and Sapajus xanthosthernos (1). Molecular diagnosis was performed using a nested PCR targeting the 18S small subunit ribosomal RNA (18S SSU rRNA) gene, followed by sequencing. Of the 298 samples analyzed, only one (0.3%) from Bahia tested positive for Plasmodium brasilianum/P. malariae. This represents the first detection of this parasite in a free-living C. geoffroyi in Brazil. These findings highlight the importance of continued surveillance of Plasmodium infections in NHPs to identify regions at risk for zoonotic transmission. Full article

Vibrio parahaemolyticus is a major foodborne pathogen worldwide, responsible for seafood-associated poisoning. Among its toxin genes, tdh2 is the most critical. To investigate the role of tdh2 in V. parahaemolyticus under gastrointestinal conditions, we constructed tdh2 deletion and complementation strains and compared their survival under acid (pH 3 and 4) and bile stress (2%). The results showed that tdh2 expression was significantly upregulated under cold (4 °C) and bile stress (0.9%). Survival assays and PI staining revealed that the tdh2 mutant strain (VP: △tdh2) was more sensitive to acid and bile stress than the wild-type (WT), and this sensitivity was rescued by tdh2 complementation. These findings suggest that tdh2 plays a protective role in enhancing V. parahaemolyticus tolerance to acid and bile stress. In the VP: △tdh2 strain, seven genes were significantly upregulated and six were downregulated as a result of tdh2 deletion. These genes included VPA1332 (vtrA), VPA1348 (vtrB), VP2467 (ompU), VP0301 and VP1995 (ABC transporters), VP0527 (nhaR), and VP2553 (rpoS), among others. Additionally, LC-MS/MS analysis identified 12 differential metabolites between the WT and VP: △tdh2 strains, including phosphatidylserine (PS) (17:2 (9Z,12Z) /0:0 and 20:1 (11Z) /0:0), phosphatidylglycerol (PG) (17:0/0:0), flavin mononucleotide (FMN), and various nucleotides. The protective mechanism of tdh2 may involve preserving cell membrane permeability through regulation of ompU and ABC transporters and enhancing electron transfer efficiency via regulation of nhaR. The resulting reduction in ATP, DNA, and RNA synthesis—along with changes in membrane permeability and electron transfer due to decreased FMN—likely contributed to the reduced survival of the VP: △tdh2 strain. Meanwhile, the cells actively synthesized phospholipids to repair membrane damage, leading to increased levels of PS and PG. This study provides important insights into strategies for preventing and controlling food poisoning caused by tdh⁺ V. parahaemolyticus. Full article

Neisseria gonorrhoeae, the causative agent of gonorrhea, represents a major public health concern due to its increasing antimicrobial resistance. While often asymptomatic—particularly in extragenital infections—untreated cases can lead to severe complications and further transmission. Despite global efforts to monitor antimicrobial resistance, data on the molecular determinants underlying decreased susceptibility in N. gonorrhoeae remain scarce in Peru. This study aimed to detect mutations in the penA and 23S rRNA genes, which confer decreased susceptibility to cephalosporins and azithromycin resistance. We extracted DNA from 124 N. gonorrhoeae-positive clinical rectal specimens collected in Aptima Combo 2 transport tubes from MSM patients. These DNA samples were then screened using the Mismatch Amplification Mutation Assay-based real-time PCR (MAMA-qPCR) to identify mutations in the 23S rRNA and penA genes. Each sample underwent separate reactions to detect A2059G and C2611T mutations in the 23S rRNA gene, and 86 of these samples were further tested in individual qPCR assays for the penA D345 deletion (D345del) or G545S mutations. Sanger sequencing was performed on all DNA samples positive for 23S rRNA mutations by MAMA-qPCR assay, and on 27 DNA samples that yielded sufficient penA amplicons for additional sequencing. Using the MAMA-qPCR assay for the 23S rRNA gene, 64 of 124 samples amplified in the A2059G reaction: 2 (3.1%) carried the mutation, and 62 were classified as wild type. In the C2611T reaction, 42 of 124 samples amplified, and none of them carried the mutation. Using the MAMA-qPCR assay for the penA gene, we only analyzed 86 samples, as the remaining 38 samples had insufficient DNA yield. A total of 44 of the 86 samples amplified in the D345del reaction: 5 (11.4%) carried the D345del, and 39 were classified as wild type. In the G545S reaction, 4 (6.4%) carried the mutation, and 58 were classified as wild type. Finally, sequencing of the penA gene in the 27 samples revealed mutations related to decreased susceptibility to cephalosporins. This study identified genetic mutations conferring resistance to azithromycin and decreased susceptibility to cephalosporins, providing an overview of the circulating mutations conferring resistance in N. gonorrhoeae strains in Peru. Full article

Background/Objectives: Environmental stressors, including spaceflight and altered gravity, can negatively affect the symbiotic relationship between the gut microbiome and host health. Dietary prebiotics, which alter components of the gut microbiome, show promise as an effective way to mitigate the negative impacts of stressor exposure. It remains unknown, however, if the stress-protective effects of consuming dietary prebiotics will extend to chronic altered-gravity exposure. Methods: Forty female C57BL/6 mice consumed either a control diet or a prebiotic diet containing galactooligosaccharides (GOS) and polydextrose (PDX) for 4 weeks, after which half of the mice were exposed to 3 times the gravitational force of Earth (3g) for an additional 4 weeks. Fecal microbiome samples were collected weekly for 8 weeks, sequenced, and analyzed using 16S rRNA gene sequencing. Terminal physiological endpoints, including immune and red blood cell characteristics, were collected at the end of the study. Results: The results demonstrate that dietary prebiotic consumption altered the gut microbial community structure through changes to β-diversity and multiple genera across time. In addition, consuming dietary prebiotics reduced the neutrophil-to-lymphocyte ratio (NLR) and increased red blood cell distribution width (RDW-CV). Importantly, the prebiotic diet prevented the impacts of altered-gravity on β-diversity and the bloom of problematic genera, such as Clostridium_sensu_stricto_1 and Turicibacter. Furthermore, several prebiotic diet-induced genera-level changes were significantly associated with several host physiological changes induced by 3g exposure. Conclusions: These data demonstrate that the stress-protective potential of consuming dietary prebiotics extends to environmental stressors such as altered gravity, and, potentially, spaceflight. Full article

The abnormal growth of smooth muscle cells (SMCs) contributes to the vascular remodeling associated with coronary artery disease, a leading cause of death in women. Estradiol (E2) mediates cardiovascular protective actions, in part, by inhibiting the abnormal growth (proliferation and migration) of SMCs through various mechanism. Since microRNAs (miRNAs) play a major role in regulating cell growth and vascular remodeling, we hypothesize that miRNAs may mediate the protective actions of E2. Following preliminary leads from E2-regulated miRNAs, we found that platelet-derived growth factor (PDGF)-BB-induced miR-193a in SMCs is downregulated by E2 via estrogen receptor (ER)α, but not the ERβ or G-protein-coupled estrogen receptor (GPER). Importantly, miR-193a is actively involved in regulating SMC functions. The ectopic expression of miR-193a induced vascular SMC proliferation and migration, while its suppression with antimir abrogated PDGF-BB-induced growth, effects that were similar to E2. Importantly, the restoration of miR-193a abrogated the anti-mitogenic actions of E2 on PDGF-BB-induced growth, suggesting a key role of miR-193a in mediating the growth inhibitory actions of E2 in vascular SMCs. E2-abrogated PDGF-BB, but not miR-193a, induced SMC growth, suggesting that E2 blocks the PDGF-BB-induced miR-193a formation to mediate its anti-mitogenic actions. Interestingly, the PDGF-BB-induced miR-193a formation in SMCs was also abrogated by 2-methoxyestradiol (2ME), an endogenous E2 metabolite that inhibits SMC growth via an ER-independent mechanism. Furthermore, we found that miR-193a induces SMC growth by activating the phosphatidylinositol 3-kinases (PI3K)/Akt signaling pathway and promoting the G1 to S phase progression of the cell cycle, by inducing Cyclin D1, Cyclin Dependent Kinase 4 (CDK4), Cyclin E, and proliferating-cell-nuclear-antigen (PCNA) expression and Retinoblastoma-protein (RB) phosphorylation. Importantly, in mice, treatment with miR-193a antimir, but not its control, prevented cuff-induced vascular remodeling and significantly reducing the vessel-wall-to-lumen ratio in animal models. Taken together, our findings provide the first evidence that miR-193a promotes SMC proliferation and migration and may play a key role in PDGF-BB-induced vascular remodeling/occlusion. Importantly, E2 prevents PDGF-BB-induced SMC growth by downregulating miR-193a formation in SMCs. Since, miR-193a antimir prevents SMC growth as well as cuff-induced vascular remodeling, it may represent a promising therapeutic molecule against cardiovascular disease. Full article

The postmortem interval (PMI), defined as the time elapsed between death and the discovery or examination of the body, is a crucial parameter in forensic science for estimating the time of death. There are many ways to measure the PMI, such as Henssge’s nomogram, which uses rectal temperature measurement; livor mortis; rigor mortis; and forensic entomology. However, these methods are usually affected by various conditions in the surrounding environment. The purpose of the present study was to compare molecular genetics and histological changes in the brain and skeletal muscle tissues of SD rats over increasing periods of time after death. For the PMIs, we considered 0 h, 6 h, 12 h, 24 h, 36 h, 48 h, 4 days, 6 days, 8 days, 10 days, 14 days, and 21 days and compared them at 4 °C and 26 °C. Hematoxylin and Eosin (H&E) staining was performed to observe tissue changes. Morphological tissue changes were observed in cells for up to 21 days at 4 °C, and cell destruction was visually confirmed after 14 days at 26 °C. Total RNA (tRNA) was isolated from each tissue sample, and complementary DNA (cDNA) was synthesized. A reverse transcription quantitative PCR (RT-qPCR) SYBR Green assay targeting three types of housekeeping genes, including Gapdh, Sort1, B2m, and 5S rRNA, was performed. The results showed that Gapdh and 5S rRNA were highly stable and could be better RNA targets for estimating the PMI in brain and skeletal muscle tissues. Conversely, Sort1 and B2m showed poor stability and low expression levels. In conclusion, these molecular biomarkers could be used as auxiliary indicators of the PMI in human, depending on the stability of the marker. Full article

The human exposome, including dietary exposures such as pesticides, additives, and environmental contaminants, plays a critical role in shaping the gut microbiota (GM) and long-term health outcomes. While the Mediterranean Diet is known for its health-promoting effects, the role of food quality, specifically organic vs. conventional products, in modulating GM within this dietary pattern remains underexplored. The aim of this study was to evaluate (1) whether an Italian Mediterranean Organic Diet (IMOD) confers additional benefits compared to an Italian Mediterranean non-Organic Diet (IMnOD), and (2) the impact of IMOD and IMnOD versus a free diet (No Diet) on GM and anthropometric parameters. A randomized, controlled trial was conducted on 39 healthy subjects. Eligible subjects were divided into the following groups: (1) 4 weeks No Diet, (2) 4 weeks IMOD, and (3) 4 weeks IMnOD. Microbiota profiling (16S rRNA sequencing), body composition (BIA), and dietary adherence (MEDAS, FFQ) were evaluated. Distinct microbial shifts following both IMOD and IMnOD compared to No Diet were revealed. Several taxa previously associated with short-chain fatty acid (SCFA) biosynthesis (i.e., Anaerobutyricum hallii, Anaerostipes hadrus, and Dorea longicatena) were increased after both Mediterranean Diet interventions, while Parabacteroides distasonis showed a specific increase in the IMOD group. No significant changes in body weight or composition were observed. These findings suggest that adherence to a Mediterranean Diet, regardless of food source, reshapes the gut microbiota, while organic food intake may influence specific microbial trajectories. Our results support the relevance of food quality in dietary interventions. Full article

Carotenoids are widely recognized for their antioxidant and health-beneficial properties, making them attractive for applications in the food, pharmaceutical, medical, and agricultural sectors. Rhodotorula yeasts are considered one of the most suitable alternatives for carotenoid synthesis due to their rapid biomass growth and high pigment yield. During this study, based on the sequences of the ITS region between 18S and 28S rRNA genes, the yeast strains were identified as belonging to Rhodotorula babjevae, R. dairenensis, R. diobovata, R. glutinis, R. graminis, R. ingeniosa, R. kratochvilovae, and R. mucilaginosa. The production of carotenoids by different Rhodotorula yeast strains was analyzed under the combined effects of lighting and temperature. Among all tested strains, the isolate identified as R. ingeniosa exhibited the lowest carotenoid content, ranging from 0.18 to 0.23 mg/g biomass. The highest levels of pigment were accumulated in dark conditions by R. babjevae (0.86 mg/g biomass) and R. graminis (0.76 mg/g biomass) cultivated for 14 days at a constant temperature of 26 °C, and by R. glutinis (0.89 mg/g biomass) after incubation at 4 °C. The majority of yeasts tested produced more carotenoids at a higher temperature. It was observed that in R. babjevae, R. glutinis, and R. graminis, lighting negatively affected the pigment content regardless of incubation temperature. In these strains, the pigment content decreased by 1.2- to 1.4-fold after one week of cultivation under light conditions at 26 °C, compared to cultures grown in the dark. The results suggest that the isolated Rhodotorula strains could be attractive candidates for the efficient synthesis of carotenoids. Full article

The objective of this study is to investigate the degradation characteristics of oat grass in the rumen of Mindong goats and changes in microbial community attached to the grass surface. Four healthy male goats, aged 14 months, with permanent rumen fistula, in eastern Fujian, were selected as experimental animals. The rumen degradation rate of oat grass was measured at 4, 12, 24, 36, 48, and 72 h using the nylon bag method. Surface physical structure changes in oat grass were observed using scanning electron microscopy (SEM), cellulase activity was measured, and bacterial composition was analyzed using high-throughput 16S rRNA gene sequencing technology. The findings of this study indicate that oat grass had effective degradation rates (ED) of 47.94%, 48.69%, 38.41%, and 30.24% for dry matter (DM), crude protein (CP), neutral detergent fiber (NDF), and acidic detergent fiber (ADF), respectively. The SEM was used to investigate the degradation process of oat grass in the rumen. After 24 h, extensive degradation of non-lignified tissue was observed, resulting in the formation of cavities. At 36 h, significant shedding was observed, and by 72 h, only the epidermis and thick-walled tissue, which exhibited resistance to degradation, remained intact. Surface-attached microorganisms produced β-GC, EG, CBH, and NEX enzymes. The activity of these enzymes exhibited a significant increase between 4 and 12 h and showed a positive correlation with the degradation rate of nutrients. However, the extent of correlation varied. Prevotella and Treponema were identified as key genera involved in the degradation of roughage, with their abundance decreasing over time. Principle Coordinate Analysis (PCOA) revealed no significant differences in the rumen microbial structure across different time points. However, Non-Metric Multidimensional Scaling (NMDS) indicated a discernible diversity order among the samples. According to the Spearman correlation coefficient test, Ruminococcus, Fibrobacter, and Saccharoferments exhibited the closest relationship with nutrient degradation rate and surface enzyme activity, displaying a significant positive correlation. In summary, this study delineates a time-resolved correlative framework linking microbial succession to structural and enzymatic dynamics during oat grass degradation. Full article

Gamma-aminobutyric acid (GABA) is a non-protein amino acid playing a significant role in the central nervous system and the gut–brain axis. This study investigated the potential to produce GABA by lactic acid bacteria (LAB) isolated from different varieties of organic tomatoes. The isolated LAB were taxonomically identified by 16S rRNA gene sequencing, the presence of the gadB gene (glutamate decarboxylase) was detected, and GABA production was quantified using HPLC. Levilactobacillus brevis CRAI showed the highest GABA production under optimised fermentation conditions with 4% monosodium glutamate (MSG). The genome sequencing of L. brevis CRAI revealed the presence of gadA and gadB isoforms and assessed the strain’s safety profile. The gene expression analysis revealed that the gadA and gadB genes were upregulated in the presence of 4% MSG. The probiotic potential of L. brevis CRAI was also assessed by functional assays. The strain showed strong antimicrobial activity against representative enteropathogens, i.e., Escherichia coli ETEC, Salmonella choleraesuis, and Yersinia enterocolitica, and anti-inflammatory effect, reducing nitric oxide production in LPS-stimulated RAW264.7 macrophages. In addition, its ability to adhere to intestinal epithelial Caco-2 cells was demonstrated. These results highlight L. brevis CRAI as a promising candidate for the development of GABA-enriched functional foods or probiotic supplements with the perspective to modulate the gut-brain axis. Full article

The major objective was to investigate the protective capabilities of endophytic bacterial strains isolated from a number of medicinal plant species towards Aspergillus spp. secured from the internal tissues of fungi-infected peanuts. Among 32 fungal isolates surveyed for mycotoxin production in various culture media (PDA, RBCA, YES, CA), 10 isolates qualitatively producing AFB₁, besides 10 OTA-producers, were assayed by HPLC for quantitative toxin production. Aspergillus spp. isolate Be 13 produced an extraordinary quantity of 1859.18 μg mL⁻¹ AFB₁, against the lowest toxin level of 280.40 μg mL⁻¹ produced by the fungus isolate IS 4. The estimated amounts of OTA were considerably lower and fell in the range 0.88–6.00 μg mL⁻¹; isolate Sa 1 was superior, while isolate Be 7 seemed inferior. Based on ITS gene sequencing, the highly toxigenic Aspergillus spp. isolates Be 13 and Sa 1 matched the description of A. novoparasiticus and A. ochraceus, respectively, ochraceus, respectively, which are present in GenBank with identity exceeding 99%. According to 16S rRNA gene sequencing, these antagonists labeled Ar6, Ma27 and So34 showed the typical characteristics of Pseudomonas aeruginosa, Bacillus subtilis and Bacillus velezensis, respectively, with similarity percentages of 99–100. The plant growth-promoting activity measurements of the identified endophytes indicated the production of 16.96–80.00 μg/100 mL culture medium of IAA. Phosphate-solubilizing capacity varied among endophytes from 2.50 to 21.38 μg/100 mL. The polysaccharide production pool of bacterial strains ranged between 2.74 and 6.57 mg mL⁻¹. P. aeruginosa Ar6 and B. velezensis successfully produced HCN, but B. subtilis failed. The in vitro mycotoxin biodegradation potential of tested bacterial endophytes indicated the superiority of B. velezensis in degrading both mycotoxins (AFB₁-OTA) with average percentage of 88.7; B. subtilis ranked thereafter (85.6%). The 30-day old peanut (cv. Giza 6) seedlings grown in gnotobiotic system severely injured due to infection with AFB₁/OTA-producing fungi, an effect expressed in significant reductions in shoot and root growth traits. Simultaneous treatment with the endophytic antagonists greatly diminished the harmful impact of the pathogens; B. velezensis was the pioneer, not P. aeruginosa Ar6. In conclusion, these findings proved that several endophytic bacterial species have the potential as alternative tools to chemical fungicides for protecting agricultural commodities against mycotoxin-producing fungi. Full article

Equine gastric disease (EGD) is a common condition in performance horses (Equus caballus), potentially compromising behaviour, performance, and welfare. EGD is often attributed to high-starch, high-sugar feeds and limited forage. Evidence for diet-induced changes on digestive microbiota is lacking. Nine elite showjumping horses were housed at the same performance yard with standardised diet and management throughout the study. Horses were transitioned from a high-sugar and -starch (31%) feed to a low-starch and -sugar (16.5%) concentrate feed. Gastroscopies, blood, and faecal samples were taken pre- and 12 weeks post-diet change. Squamous and glandular ulceration was blindly graded a posteriori using 0–4 scores and faecal microbiota profiled using 16S rRNA gene amplicon sequencing. Total (t_(1,8) = −6.17, p < 0.001; Pre: 4 [0–5], Post: 1 [0–2]), squamous (t_(1,8) = −5.32, p < 0.001; Pre: 1 [0–3], Post: 0 [0–1]), and glandular (t_(1,8) = −2.53, p = 0.04; Pre: 2.5 [0–4], Post: 0 [0–2]) disease improved following the introduction of a low-starch diet. Diet change did not impact microbiota communities (PERMANOVA: F_(1,16) = 1.37, p = 0.15, r² = 0.08), but Firmicute to Bacteroidota (F/B) ratio reduced (t_(1,8) = −3.13, p = 0.01; Pre: 2.07 ± 0.21 vs. Post: 1.29 ± 0.14). Lower F/B ratios were associated with reduced total EGD scores (ChiSq_(1,17) = 3.83, p = 0.05). Low-starch diets did not influence faecal microbiota diversity but aided gastric disease healing and reduced F/B ratios in elite showjumpers during a competition season without medication. Full article

(1) Background: This study aimed to investigate the bacterial microbiomes in the ingredients and final PBM products during a storage period of 28 days at 2–4 °C for food safety and quality. (2) Methods: DNA from raw ingredients (i.e., defatted soy flour, potato starch, wheat gluten, mung bean protein, and duckweed) and three PBM formulations were extracted and sequenced using 16S rRNA gene sequencing. (3) Results: Alpha diversity (Simpson and Shannon) was high in the raw ingredients (p ≤ 0.05). Beta diversity showed dissimilarities between the samples. Firmicutes and Proteobacteria were the core microflora in these ingredients. The heat-stable microbes in PBM (e.g., Nostocaceae in SF and Cyanobacteriale in MB and DW) survived after extrusion. After the ingredients were stored at room temperature, the bacterial communities shifted, with Paucibacter being the majority population in raw ingredients and PBM in the 2nd batch. The predictions of Potential_Pathogens related to the abundance of Aeromonadaceae and Enterobacteriaceae need to be monitored during storage. (4) Conclusions: Our results showed that the bacterial community in PBM containing 30% MB and 3% DW did not drastically change during 28 days of storage at cold temperatures. Uncovering bacterial microbiomes in the ingredients should be emphasized for quality and safety, as ingredients influence the microbiome in the final products. Full article

Rising rates of antimicrobial-resistant infections have prompted increased scrutiny on antimicrobial use (AMU) in livestock agriculture. Dairy farms primarily use antimicrobials to maintain animal health and welfare by treating and preventing infectious diseases. However, the impact of dairy farm AMU practices on the cattle fecal microbiome remains largely unclear, partly due to difficulties in quantifying AMU. This study leveraged quantitative AMU data from 40 large commercial dairy farms to identify farms with low (n = 4) and high (n = 4) AMU. Using 16S rRNA gene amplicon sequencing, we compared the fecal bacterial communities of dairy calves and cows (healthy, cull, sick) by both AMU designation (high/low) and by individual farm AMU, summarized by animal defined daily dose (DDD) and mg/kg. We found significant differences in beta-diversity between cattle from high- and low-AMU groups using either method and found that Corynebacterium and Clostridium abundances increased with farm AMU. Additionally, we found fecal bacterial communities differed across farms within high- and low-AMU groupings, highlighting the need to account for farm-to-farm variation when assessing AMU impacts. These findings suggest that dairy farm AMU influences the fecal microbiome and identifies specific taxa that warrant further investigation as potential reservoirs for antimicrobial resistance genes. Full article