Bovine respiratory disease (BRD) remains a significant economic challenge in dairy cattle despite extensive vaccination programs that have been developed and implemented during the last few decades. This study investigated the nasopharyngeal microbiome and resistome of dairy cattle across various life stages to understand the roles of microbial communities associated with BRD. A case-control study was conducted on three commercial dairy farms in Northern California, collecting nasopharyngeal swabs from 69 animals, including preweaned calves, weaned heifers, and lactating cows with and without BRD. Shotgun metagenomic sequencing was used to characterize both microbiome and resistome profiles observed at the time of BRD diagnosis. Results revealed that BRD is associated with distinct microbial community patterns, rather than the increased abundance of a specific pathogen. Age was a critical factor influencing microbial diversity, with adult cows showing the highest diversity and weaned heifers with BRD showing the lowest. A total of 1,164 bacterial species were identified, with BRD cases harboring 14 unique species compared to control animals. BRD cases were characterized by the co-occurrence of multiple respiratory pathogens, including Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mesomycoplasma species, which showed positive correlation with BRD cases but negative correlations in BRD controls, while BRD control animals showed significantly higher abundance of commensal Staphylococcus species. Resistome analysis identified 65 antimicrobial resistance genes, with BRD cases harboring more unique resistance genes than BRD controls. These findings challenge traditional single-pathogen models and demonstrate that BRD is likely the result of complex microbial community interactions and changes in community abundance, providing new potential targets to explore when considering prevention strategies toward promoting microbial communities that prevent or reduce the risk of BRD.IMPORTANCEBovine respiratory disease (BRD) represents one of the most economically challenging conditions in cattle production, with an estimated direct cost that exceeds $165 million annually in the United States alone. Despite decades of vaccination efforts targeting known pathogens, BRD prevalence remains unchanged, indicating an incomplete understanding of disease pathogenesis. This study provides critical insights by shifting focus from individual pathogens to entire microbial communities, revealing that BRD involves complex bacterial interactions, as well as the role of the understudied nasal commensal microbiome in healthy animals. The identification of distinct "pathobiomes" associated with disease and protective commensal communities in healthy animals fundamentally changes approaches to BRD prevention and treatment. The discovery that age significantly influences microbiome stability highlights critical intervention periods. Furthermore, the association between BRD and increased antimicrobial resistance genes raises concerns about current treatment and overall management practices, selecting for drug-resistant communities. This research provides a foundation for developing microbiome-based diagnostic tools and interventions supporting healthy microbial ecosystem development.

Corynebacterium pseudotuberculosis is a facultative intracellular pathogen responsible for chronic infections in livestock, primarily small ruminants and horses, with occasional zoonotic transmission. To investigate the genomic diversity, evolutionary stability, and host adaptation of this species, we analyzed 788 high-quality genomes representing isolates from diverse hosts, geographic regions, and time periods. Comparative population genomics revealed remarkably conserved genome architecture, supporting a closed pangenome with minimal accessory gene variation. Virulence and antimicrobial resistance (AMR) screening across multiple databases confirmed the universal presence of phospholipase D (pld) and the absence of major horizontally acquired AMR determinants, except for APH(3’)-IIa, TEM-116, and APH(3’)-IIIa in a few goat isolates from Brazil. Distinct metabolic features between biovars were conserved, notably nitrate reduction and molybdenum cofactor biosynthesis in biovar equi. However, gene presence/absence alone did not explain host specificity. Instead, machine learning applied to 8,028 core-genome SNPs identified allelic variants associated with host origin, particularly in genes linked to amino-acid biosynthesis and peptide transport (Opp system). These findings demonstrate that host adaptation in this species is driven by fine-scale SNP variation within core metabolic pathways, rather than acquisition of classical virulence or resistance genes, highlighting the species’ exceptional genomic stability and narrow evolutionary flexibility.

Listeria monocytogenes (L. monocytogenes), a foodborne pathogen shed by asymptomatic ruminants, poses a contamination risks in integrated crop-livestock systems (ICLS), where ruminants are introduced to graze cover crops prior to replanting fresh produce in a field. As a follow-up study, we conducted whole genome sequencing of 30 L. monocytogenes isolates obtained from sheep and goat fecal and soil samples collected during our previously published ICLS field trial studies (2019-2022) at organic farms in California and Minnesota. One goat fecal isolate was genetically identical to one soil isolate collected at seven days post-grazing. Most isolates (28/30, 93.3%) belonged to lineage I, specifically to serogroup IVb or IVb-v1, and were classified as CC1 or CC554, both clonal complexes previously associated with human listeriosis outbreaks. The majority of isolates harbored virulence associated genes, including LIPI-1 or LIPI-3 genes, and agrA, associated with biofilm formation and survival in agricultural soils. In antimicrobial susceptibility testing, all isolates were susceptible to antibiotics commonly used to treat ruminant and human listeriosis, including ampicillin and penicillin. These findings suggest that asymptomatic small ruminants in ICLS may introduce pathogenic L. monocytogenes strains into the agricultural environment, posing a potential contamination risk to fresh produce despite the low overall prevalence observed.

Vibrio alginolyticus is a frequently implicated species for vibriosis in humans and diverse wildlife, but it has previously been difficult to identify from the closely related and emerging Vibrio diabolicus. Comparisons of both species, including antimicrobial resistance (AMR) and virulence characterizations, are scarce and impeded by intraspecies diversity, minimal genomes, discordant classification methods, and gene databases with limited utility to understudied species. The species identities of 3,442 public domain genomes (SRA files) within the Harveyi clade were re-evaluated using genomic methods. Public genomes identified as V. diabolicus and V. alginolyticus were combined with previously published genomes isolated from humans, sea otters (Enydra lutris), or coastal environments (V. diabolicus n = 88, V. alginolyticus n = 163, Vibrio parahaemolyticus n = 287) for pangenome-wide association studies to identify species-specific gene clusters (95% identification threshold). Additional genome wide associations with isolation source (humans versus sea otters) were investigated, including AMR and virulence related gene clusters. Genomic reclassification identified 29 of 150 misclassified public domain V. alginolyticus genomes, including 26 reclassified as V. diabolicus. In total, 28 previously misclassified V. diabolicus genomes (n = 37 total) were identified, including 10 human-derived strains. GWAS identified 643 and 477 gene clusters specific to V. alginolyticus and V. diabolicus, respectively, while some multilocus sequencing analysis (MLSA) gene clusters were non-specific. Gene clusters (n = 109) associated with either V. alginolyticus isolated from humans or sea otters were identified including one annotated to a multidrug resistance gene (mdtk_1). No V. diabolicus gene clusters were associated with host species after multiple comparison correction, although pre-correction associations related to antimicrobial resistance were detected (cat_1, ampC). The genomic methods of classification presented provide accurate species identification for V. diabolicus and V. alginolyticus beyond current MLSA/MLST schemes, although target species-specific genes were identified that may be useful for improved future schemes. While limited sample size of V. diabolicus hampered the ability to detect host associated markers, the GWAS approach employed provide a reusable framework for discovering insights into host adaptation and prioritizing target genes for future functional AMR and virulence validation experiments in both species.

Stem cells and organoids have emerged as pivotal biological tools for biologically relevant models. Together, these in vitro models realistically recapitulate structural and functional elements of the in vivo organ, allowing for studies of cellular, molecular, and genetic features that underpin various diseases that are difficult to observe in low-biomass tissues. Stem cells, and more recently organoids, have been applied in vivo as regenerative therapies. The emergence of the microbiome as an occupant throughout different body locales requires new approaches to understand the complex cellular interactions with the host tissue at each site. The success of regenerative medicine strategies and therapeutic development is intricately linked to this understanding and management of host–microbe dynamics. Interactions with the host microbiome and infections can both significantly impair tissue regeneration and compromise the function of stem cell–derived therapies. Therefore, a comprehensive understanding of how pathogens and the microbiome interact with stem cells and organoids is relevant for developing safe and effective regenerative medicine interventions. This review explores the evolving landscape of organoid technology, including a discussion on the importance of stem cell studies and considerations for organoid development that are important for use as models to study microbiome interactions. Additionally, this work describes the pivotal role of cell culture models in advancing host–microbe interaction studies in understudied low-biomass organs such as the stomach and reproductive tract. Through this assessment, we aim to shed light on the potential of these models to transform the approach to studying and managing infectious diseases within the context of regenerative medicine.

Introduction:Dietary prebiotic oligosaccharides, complex carbohydrates that support beneficial bacteria, are ubiquitous on marketplace shelves and in people's diets. Though widely accessible and consumed, little is known about how different prebiotics alter the epithelium and microbes during enteric infections. Methods:Here we show two structurally different prebiotic oligosaccharides, human milk oligosaccharides (HMO) and mannanoligosaccharides (MOS), alter the metabolism of colonic epithelial cells and Salmonella enterica sv. Typhimurium in ways specific to each prebiotic during infection in a focused 'in vitro' model. Results:Initially, HMO and MOS addition decreased S. Typhimurium association with epithelial cells. However, gene expression analysis revealed significantly induced expression of Specific Pathogenicity Island (SPI) 1 (adj. p < 2.0-6) and 2 (adj. p < 3.0-5) with HMO treatment, opposed to increased fimbriae expression (adj.p < 3.0-3) with MOS treatment. Both host and pathogen metabolism were likewise altered with prebiotic addition. MOS treatment induced the expression of genes for amino acid metabolism in both the host cells and in S. Typhimurium, a metabolic shift that was not observed in the HMO treated cells. MOS treatment also altered pathogen-related respiration metabolism in S. Typhimurium toward activity typically seen during gut inflammation. Discussion:The regulation of virulence expression in Salmonella from prebiotic treatment was unexpected and suggests prebiotics act in context-dependent ways to potentiate or attenuate enteric activity.

Dietary prebiotic oligosaccharides are common in people’s diets; however, little is known about how different prebiotics alter the enteric epithelium and microbiome. Here we show two structurally different prebiotic oligosaccharides, human milk oligosaccharides (HMO) and mannanoligosaccharides (MOS), alter the metabolism of colonic epithelial cells and Salmonella enterica sv. Typhimurium in ways specific to each prebiotic. Initially, HMO and MOS addition decreased S. Typhimurium association with epithelial cells. However, gene expression analysis revealed significantly induced expression of Specific Pathogenicity Island (SPI) 1 and 2 with HMO treatment opposed to increased fimbriae expression with MOS treatment. MOS treatment induced the expression of genes for amino acid metabolism in both the host cells and in S. Typhimurium, a metabolic shift that was not observed in the HMO treated cells. MOS treatment also altered respiration metabolism in S. Typhimurium to be more closely aligned to those observed in vivo during gut inflammation, which is opposed to colonization-type expression with HMO. Alteration of virulence observed was found to be prebiotic specific and dose dependent, indicating that some dietary substrates likely alter specific pathogens to change their virulence potential in unanticipated ways that lead to multiple outcomes to potentiate or attenuate enteric infections.

Staphylococcus aureus is a versatile zoonotic pathogen capable of causing a wide range of infections. Due to the organism’s ability to persist, recalcitrant and recurring infections are a major concern for public and animal health. This study investigated the establishment of persistence using two S. aureus strains—ATCC 29740, a bovine mastitis isolate, and USA300, a human clinical isolate—under substrate depletion. This nutritional stress established a persistence phenotype where the strains remained persistent for >120 days at notable concentrations [>2 log10 CFU/mL] and developed persister-like growth, including small colony variant formations. With RT-qPCR, we found the cell density was higher than represented by the plate count while the intracellular ATP remained constant during the persistence phase. These findings indicate that S. aureus has complex survival strategies to support its persistent state, providing a host-specific perspective when addressing recurrent infections in human and animal infectious diseases.

ABSTRACT Antimicrobial resistance in Vibrio species poses risks to both human and marine mammal health. Whole genome sequencing of Vibrio spp. can be utilized to screen for antimicrobial resistance genes and allelic variants to provide mechanistic insights in ways that PCR screening and phenotypic interpretation cannot. Our goals were to (i) characterize antimicrobial resistance patterns of Vibrio spp. pathogens isolated from southern sea otters ( Enhydra lutris nereis ), northern sea otters ( Enhydra lutris kenyoni ), and environmental samples from the central California coast using whole genome sequencing, and (ii) compare the presence of antimicrobial resistance genes with phenotypic interpretation from antibiotic susceptibility testing. Unexpectedly, genomic classification identified an understudied species, Vibrio diabolicus , in sea otter and environmental isolates that were previously identified as Vibrio alginolyticus . A total of 489 Vibrio spp. isolates were sequenced, and frequently detected antimicrobial resistance genes included multidrug efflux pumps and genes associated with resistance to ß-lactams and tetracyclines. Genes associated with resistance to fluoroquinolones, aminoglycosides, chloramphenicol, and sulfonamides were uncommon. Sea otter isolates were phenotypically susceptible to tetracycline despite carrying genes tet34 and tet35 . Both between- and within-species variations in ampicillin resistance were observed despite the ubiquitous presence of bla CARB genes in V. alginolyticus , V. diabolicus , and Vibrio parahaemolyticus . Discordance between phenotypic and genotypic ampicillin resistance was especially noted for V. parahaemolyticus and was partially attributed to the allelic variation of the bla CARB genes. Tetracyclines and fluoroquinolones, but not ß-lactams, are likely to be effective treatments for vibriosis in sea otters. IMPORTANCE Vibriosis (infection with non-cholera Vibrio spp.) is the most common seafood-borne illness globally, with major impacts on public health, food security, and wildlife health. Potential treatments of antimicrobial-resistant Vibrio spp. in humans, aquaculture, and marine wildlife rehabilitation are complicated by current diagnostic challenges regarding bacterial species identification and interpretation of antimicrobial resistance patterns. Unexpected detection of previously misidentified Vibrio diabolicus in sea otters suggests that a broader taxonomic group of Vibrio infect sea otters than previously described. We also determined that the presence of ß-lactamase genes alone in sea otter isolates does not necessarily correlate with an ampicillin-resistant phenotype, likely due to deleterious amino acid substitutions in certain bla CARB alleles. Continued monitoring of Vibrio spp. phenotypes and genotypes in sea otters is warranted to observe biologically relevant changes in antimicrobial resistance.

Aggressive Periodontitis (AP) and Feline Chronic Gingivostomatitis (FCGS) are two oral inflammatory diseases in cats with unknown etiology. Both conditions present with severe inflammation of the oral cavity and in FCGS it is found with additional deterioration of the non-keratinized mucosa. The oral microbiome is increasingly implicated in disease progression, but little is known about shifts in the microbial community during the AP and FCGS progression. To that end, we used deep metagenomic sequencing with total RNA on three longitudinal samples of the oral microbiome in a cat first diagnosed with AP that progressed to FCGS. This deep sequencing approach revealed that increased diversity at both the genus and species levels marked the shift from AP to FCGS, including increases in Porphyromonas and Treponema species, and decreased Streptobacillus species. The metatranscriptomes were then probed for expression of antimicrobial resistance genes and virulence factors. Disease-related genes that include cheY, and ompP5 were expressed in early AP and FCGS, while others like galU were only expressed in one or the other disease state. Both genus and species-level shifts were observed along the longitudinal microbiome samples with a noted increase in species diversity in the FCGS-associated microbiome. Corroborating that functional shifts accompany taxonomic changes, the AMR and virulence factor expression similarly changed between the sampling points. Together, these taxonomic and functional shifts indicate that AP and FCGS are potentially linked and may be marked by changes in the oral microbiome, which supports the development of microbial-based clinical diagnostics and therapeutics.

BACKGROUND:Nocardioform placentitis (NP) is an understudied form of equine placentitis historically attributed to nocardioform bacteria, yet it remains uncertain whether these organisms are the sole pathogens involved. OBJECTIVES:To elucidate the pathophysiology of NP and the host-pathogen interaction. STUDY DESIGN:In vivo clinical multi-omics study. METHODS:Dual RNA sequencing was performed to profile transcriptionally active microbial communities and concurrent placental transcriptome responses in samples from 31 placentas with and without NP. Untargeted metabolomics was performed to study the associated metabolites in the placenta. RESULTS:The most abundant microbial transcripts belonged to Amycolatopsis, Crossiella, Lentzea, Enterococcus, and Mycobacterium. Bacterial gene expression in NP-affected placentas was enriched in pathways related to ribosomal activity and metabolic processes involving amino acid, carbohydrate, and glycosphingolipid metabolism. Concurrently, placental transcripts demonstrated significant upregulation of inflammatory pathways and downregulation of pathways associated with blood vessel formation. Untargeted metabolomics highlighted an elevated abundance of metabolites such as beta-D-fucose, nervonic acid, and zymostenol in the placentitis samples. Significant correlations were found between microbial genes (mraW, rlmB, amy, afuA, and cysC) and host inflammation genes (CXCL14, IL15RA, TASL, and IFIH1). Additionally, elevated beta-D-fucose, a microbe-specific metabolite, showed a strong correlation with microbial genes involved in stress-adaptive metabolism and DNA repair (ydhP, ybgC, serC, puuE, and radA). The bacterial enzymes involved in beta-D-fucose were notably upregulated and predominantly expressed by Amycolatopsis and Lentzea. MAIN LIMITATIONS:Classification based on RNA abundance limited the number of Crossiella cases (n = 3). CONCLUSIONS:Both nocardioform and non-nocardioform bacteria are involved in NP-diagnosed cases, challenging the current generalisation of the term 'nocardioform placentitis' and supporting the need to broaden diagnostic protocols for mucoid placentitis. Multi-omics profiling revealed potential host-microbe interactions mediated by microbial metabolites, offering mechanistic insights and opportunities for improved diagnostic strategies.

Vibriosis is the most important public health threat from seafood consumption and marine recreation. Pathogenic Vibrio spp. employ virulence factors including hemolysins and secretion systems frequently detected in human cases, but virulence data in northern and southern sea otters (Enhydra lutris kenyoni and E. l. nereis, respectively) are limited despite their potential as marine bioindicators. Genomic epidemiology was used to characterize virulence factors of Vibrio spp. genomes (n = 570) including V. alginolyticus ;(n = 55), V. diabolicus ;(n = 52), non-O1/O139 V. cholerae ;(n = 163), and V. parahaemolyticus ;(n = 287) collected in North America (2000-2019). Virulence factors of V. parahaemolyticus were compared between isolation sources: bivalves, environment, humans, and southern and northern sea otters. Hemolysins (tdh, trh) and type III secretion system 2 (T3SS2) gene prevalences were lowest in environmental isolates, while tdh and T3SS2 gene prevalences were higher in human and northern sea otter isolates than those from southern sea otters. A hemolysin allele (trh1) was detected almost exclusively in human and sea otter isolates. Despite V. parahaemolyticus genomic diversity, detected genomic clusters were comprised of highly related and tdh+/trh+ genomes from non-environmental sources including humans and sea otters. Observed pathology in Vibrio spp. positive sea otters frequently included septicemia, enteritis, and moderate to severe melena. Co-occurrence of T3SS2 and T6SS1 in V. parahaemolyticus was associated with pathological findings and ampicillin susceptible genotypes, suggesting a trade-off between virulence and antimicrobial resistance. Based on these findings, V. parahaemolyticus undergoes selection pressures resulting in apparent expansion, i.e. genomic clustering, of tdh+/trh+ virulent strains infecting humans and sea otters.

Antimicrobial resistance (AMR) poses a critical public health threat, with rising multidrug resistance cases compromising treatment effectiveness. Knowledge about the resistome in dairy production systems remains limited, particularly regarding lactating cows. This study investigated the microbiome and resistome across the hospital, fresh, and mid-lactation pens on 18 conventional dairy farms in California and Ohio using shotgun metagenomic sequencing of pooled fecal samples. Pooled fecal pat samples were collected as part of a larger field study using a quasi-experimental design that assigned farms to the training intervention group (six per state) or the control group (three per state). For the training intervention group, farm worker(s), identified as having the task of diagnosing and treating adult cows on the farm, participated in a training program on antimicrobial stewardship practices. Pooled fecal samples (n = 7) were collected at enrollment and 3 months after the intervention was completed on each participating farm (n = 18). A total of 10,221 bacterial species and 345 AMR genes conferring resistance to 22 antimicrobial classes were identified. The hospital pen exhibited a higher AMR gene diversity compared to fresh and mid-lactation pens (p < 0.05). Several AMR genes showed bimodal distribution, suggesting complex transmission mechanisms. Network analysis revealed distinct gene correlation profiles across pens, with the hospital pen showing fewer gene interactions. Our findings suggest that farm-level antimicrobial drug use may not be the sole or primary driver of resistome composition in pooled fecal samples from dairy cattle, highlighting the need to investigate other factors influencing AMR dynamics in livestock systems.

Feline chronic gingivostomatitis (FCGS) is a debilitating inflammatory oral mucosal disease with a multifactorial etiology. The clinical diagnosis of FCGS is made based on inspection of severe inflammatory lesions and histological confirmation rather than a molecular diagnostic outcome. This gap limits the ability to provide an early diagnosis. In this report, we seek to provide additional diagnostic tools using genomics to aid in providing clinically relevant information. The use of in-depth diagnostic tools, like transcriptomics of diseased tissues, to diagnose FCGS and stratify patients into predictive treatment response groups would dramatically improve both clinical decisions and patient outcomes. In this study, we addressed the gap in diagnostic options using transcriptomic analysis of caudal oral mucosal swab specimens coupled to detailed medical record linkage of FCGS-affected cats undergoing tooth extractions and in some cases administration of mesenchymal stromal cells (MSCs). To better identify markers of disease and potential response to treatment, the transcriptomes of FCGS-afflicted cats were compared to those of healthy cats and those with chronic periodontitis to clearly establish diagnostic biomarker signal transduction connections. Phosphatidylinositol 3-kinase/Ak strain transforming (PI3K/AKT) and stress-activated protein kinases/Jun N-terminal kinase (SAP/JNK) signaling pathways were significantly differentially regulated in FCGS-afflicted cats. Activation of these pathways also differed in the treatment response groups. In conjunction, the enzymes Caspase 4 (CASP4), matrix metalloproteinase-8 (MMP8), and prostaglandin-endoperoxide synthase 2 (PTGS2) were identified as potential biomarkers for the prediction of treatment response outcomes. The observations in the case study support the use of transcriptomics of FCGS patients to contribute to improved molecular diagnostics for the diagnosis and treatment of FCGS.

Food-producing animals such as dairy cattle are potential reservoirs of antimicrobial resistance (AMR), with multidrug-resistant (MDR) organisms such as Escherichia coli observed in higher frequency in young calves compared to older cattle. In this study, we characterized the genomes of enteric MDR E. coli from pre-weaned dairy calves with and without diarrhea and evaluated the influence of host-level factors on genomic composition. Whole genome sequence comparative analysis of E. coli (n = 43) revealed substantial genomic diversity that primarily clustered by sequence type and was minimally driven by calf diarrheal disease status (healthy, diarrheic, or recovered), antimicrobial exposure, and dietary zinc supplementation. Diverse AMR genes (ARGs)—including extended-spectrum beta-lactamase genes and quinolone resistance determinants—were identified (n = 40), with unique sets of ARGs co-occurring in gene clusters with large AMR plasmids IncA/C2 and IncFIB(AP001918). Zinc supplementation was not significantly associated with the selection of individual ARGs in E. coli, however analysis of ARG and metal resistance gene pairs identified positive associations between certain aminoglycoside, beta-lactam, sulfonamide, and trimethoprim ARGs with acid, tellurium and mercury resistance genes. Although E. coli in this study lacked the typical virulence factors of diarrheagenic strains, virulence genes overlapping with those in major pathotypes were identified. Among the 103 virulence genes detected, the highest abundance and diversity of genes corresponded to iron acquisition (siderophores and heme uptake). Our findings indicate that the host-level factors evaluated in this study were not key drivers of genomic variability, but that certain accessory genes in enteric MDR E. coli may be enriched. Collectively, this work provides insight into the genomic diversity and host-microbe interface of MDR E. coli from pre-weaned dairy calves.

The objective of this study was to evaluate the impact of dietary zinc supplementation in pre-weaned dairy calves on the phenotypic antimicrobial resistance (AMR) of fecal commensal bacteria. A repository of fecal specimens from a random sample of calves block-randomized into placebo (n = 39) and zinc sulfate (n = 28) groups collected over a zinc supplementation clinical trial at the onset of calf diarrhea, calf diarrheal cure, and the last day of 14 cumulative days of zinc or placebo treatment were analyzed. Antimicrobial susceptibility testing was conducted for Enterococcus spp. (n = 167) and E. coli (n = 44), with one representative isolate of each commensal bacteria tested per sample. Parametric survival interval regression models were constructed to evaluate the association between zinc treatment and phenotypic AMR, with exponentiated accelerated failure time (AFT) coefficients adapted for MIC instead of time representing the degree of change in AMR (MIC Ratio, MR). Findings from our study indicated that zinc supplementation did not significantly alter the MIC in Enterococcus spp. for 13 drugs: gentamicin, vancomycin, ciprofloxacin, erythromycin, penicillin, nitrofurantoin, linezolid, quinupristin/dalfopristin, tylosin tartrate, streptomycin, daptomycin, chloramphenicol, and tigecycline (MR = 0.96–2.94, p > 0.05). In E. coli, zinc supplementation was not associated with resistance to azithromycin (MR = 0.80, p > 0.05) and ceftriaxone (MR = 0.95, p > 0.05). However, a significant reduction in E. coli MIC values was observed for ciprofloxacin (MR = 0.17, 95% CI 0.03–0.97) and nalidixic acid (MR = 0.28, 95% CI 0.15–0.53) for zinc-treated compared to placebo-treated calves. Alongside predictions of MIC values generated from these 17 AFT models, findings from this study corroborate the influence of age and antimicrobial exposure on phenotypic AMR.

Probiotic bacteria have been proposed as an alternative to antibiotics for the control of antimicrobial resistant enteric pathogens. The mechanistic details of this approach remain unclear, in part because pathogen reduction appears to be both strain and ecology dependent. Here we tested the ability of five probiotic strains, including some from common probiotic genera Lactobacillus and Bifidobacterium, to reduce binding of Salmonella enterica sv. Typhimurium to epithelial cells in vitro. Bifidobacterium longum subsp. infantis emerged as a promising strain; however, S. Typhimurium infection outcome in epithelial cells was dependent on inoculation order, with B. infantis unable to rescue host cells from preceding or concurrent infection. We further investigated the complex mechanisms underlying this interaction between B. infantis, S. Typhimurium, and epithelial cells using a multi-omics approach that included gene expression and altered metabolism via metabolomics. Incubation with B. infantis repressed apoptotic pathways and induced anti-inflammatory cascades in epithelial cells. In contrast, co-incubation with B. infantis increased in S. Typhimurium the expression of virulence factors, induced anaerobic metabolism, and repressed components of arginine metabolism as well as altering the metabolic profile. Concurrent application of the probiotic and pathogen notably generated metabolic profiles more similar to that of the probiotic alone than to the pathogen, indicating a central role for metabolism in modulating probiotic-pathogen-host interactions. Together these data imply crosstalk via small molecules between the epithelial cells, pathogen and probiotic that consistently demonstrated unique molecular mechanisms specific probiotic/pathogen the individual associations.

ABSTRACT Escherichia coli has been attributed to playing a major role in a cascade of events that affect the prevalence and severity of uterine disease in cattle. The objectives of this project were to (i) define the association between the prevalence of specific antimicrobial resistance and virulence factor genes in E. coli with the clinical status related to uterine infection, (ii) identify the genetic relationship between E. coli isolates from cows with diarrhea, with mastitis, and with and without metritis, and (iii) determine the association between the phenotypic and genotypic antimicrobial resistance identified on the E. coli isolated from postpartum cattle. Bacterial isolates ( n = 148) were obtained from a larger cross-sectional study. Cows were categorized into one of three clinical groups before enrollment: metritis, cows with purulent discharge, and control cows. For genomic comparison, public genomes ( n = 130) from cows with diarrhea, mastitis, and metritis were included in a genome-wide association study, to evaluate differences between the drug classes or the virulence factor category among clinical groups. A distinct E. coli genotype associated with metritis could not be identified. Instead, a high genetic diversity among the isolates from uterine sources was present. A virulence factor previously associated with metritis ( fim H) using PCR was not associated with metritis. There was moderate accuracy for whole-genome sequencing to predict phenotypic resistance, which varied depending on the antimicrobial tested. Findings from this study contradict the traditional pathotype classification and the unique intrauterine E. coli genotype associated with metritis in dairy cows. IMPORTANCE Metritis is a common infectious disease in dairy cattle and the second most common reason for treating a cow with antimicrobials. The pathophysiology of the disease is complex and is not completely understood. Specific endometrial pathogenic Escherichia coli have been reported to be adapted to the endometrium and sometimes lead to uterine disease. Unfortunately, the specific genomic details of the endometrial-adapted isolates have not been investigated using enough genomes to represent the genomic diversity of this organism to identify specific virulence genes that are consistently associated with disease development and severity. Results from this study provide key microbial ecological advances by elucidating and challenging accepted concepts for the role of Intrauterine E. coli in metritis in dairy cattle, especially contradicting the existence of a unique intrauterine E. coli genotype associated with metritis in dairy cows, which was not found in our study.

Bovine respiratory disease (BRD) is the leading cause of mortality and antimicrobial drug (AMD) use in weaned dairy heifers. Limited information is available regarding antimicrobial resistance (AMR) in respiratory bacteria in this population. This study determined AMR gene presence in 326 respiratory isolates (Pasteurella multocida, Mannheimia haemolytica, and Histophilus somni) from weaned dairy heifers using whole genome sequencing. Concordance between AMR genotype and phenotype was determined. Twenty-six AMR genes for 8 broad classes of AMD were identified. The most prevalent, medically important AMD classes used in calf rearing, to which these genes predict AMR among study isolates were tetracycline (95%), aminoglycoside (94%), sulfonamide (94%), beta-lactam (77%), phenicol (50%), and macrolide (44%). The co-occurrence of AMR genes within an isolate was common; the largest cluster of gene co-occurrence encodes AMR to phenicol, macrolide, elfamycin, β-lactam (cephalosporin, penam cephamycin), aminoglycoside, tetracycline, and sulfonamide class AMD. Concordance between genotype and phenotype varied (Matthew’s Correlation Coefficient ranged from −0.57 to 1) by bacterial species, gene, and AMD tested, and was particularly poor for fluoroquinolones (no AMR genes detected) and ceftiofur (no phenotypic AMR classified while AMR genes present). These findings suggest a high genetic potential for AMR in weaned dairy heifers; preventing BRD and decreasing AMD reliance may be important in this population.

Genetic selection has remarkably helped U.S. dairy farms to decrease their carbon footprint by more than doubling milk production per cow over time. Despite the environmental and economic benefits of improved feed and milk production efficiency, there is a critical need to explore phenotypical variance for feed utilization to advance the long-term sustainability of dairy farms. Feed is a major expense in dairy operations, and their enteric fermentation is a major source of greenhouse gases in agriculture. The challenges to expanding the phenotypic database, especially for feed efficiency predictions, and the lack of understanding of its drivers limit its utilization. Herein, we leveraged an artificial intelligence approach with feature engineering and ensemble methods to explore the predictive power of the rumen microbiome for feed and milk production efficiency traits, as rumen microbes play a central role in physiological responses in dairy cows. The novel ensemble method allowed to further identify key microbes linked to the efficiency measures. We used a population of 454 genotyped Holstein cows in the U.S. and Canada with individually measured feed and milk production efficiency phenotypes. The study underscored that the rumen microbiome is a major driver of residual feed intake ( RFI ), the most robust feed efficiency measure evaluated in the study, accounting for 36% of its variation. Further analyses showed that several alpha-diversity metrics were lower in more feed-efficient cows. For RFI, [Ruminococcus] gauvreauii group was the only genus positively associated with an improved feed efficiency status while seven other taxa were associated with inefficiency. The study also highlights that the rumen microbiome is pivotal for the unexplained variance in milk fat and protein production efficiency. Estimation of the carbon footprint of these cows shows that selection for better RFI could reduce up to 5 kg of diet consumed per cow daily, potentially reducing up to 37.5% of CH 4 . These findings shed light that the integration of artificial intelligence approaches, microbiology, and ruminant nutrition can be a path to further advance our understanding of the rumen microbiome on nutrient requirements and lactation performance of dairy cows to support the long-term sustainability of the dairy community.