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Most antimicrobials sold in the United States are used in food animals. Farm management practices contribute to antibacterial resistance (AR). Controversially, grass-fed diets have been recommended over grain-fed diets to reduce AR in beef cattle. Ionophore feed additives (non-therapeutic antibiotics that enhance feed efficiency) may contribute to AR development. We used shotgun metagenomic sequencing of fecal swabs to prospectively compare the cattle gastrointestinal resistome and microbiota in two different production systems over five periods from pre-weaning to pre-harvest. Cattle were grass-fed and pasture-raised (system A, n = 33) or grain-fed with ionophore additives in feedlots (system B, n = 34). System A cattle averaged 639 lb and 22.8 months of age, and system B cattle averaged 1,173 lb and 12.4 months of age preharvest. In total, 367 antibiotic resistance genes (ARGs) and 329 bacterial species were identified. The resistome of system A cattle had higher alpha diversity than system B cattle over their lifespan (P = 0.008). Beta-diversity estimates indicated overlap in the pre-weaning resistome and microbiota in both systems, which diverged post-weaning, with increases in several medically important ARGs when system B cattle transitioned to a grain diet. Analysis of compositions of microbiomes with bias correction indicated that levels of tetracycline, macrolide, aminoglycoside, beta-lactam, and bacitracin ARGs were significantly higher in system B cattle pre-harvest. Resistome changes were highly correlated with bacterial community changes (Procrustes, M² = 0.958; P = 0.001). Potentially modifiable farm management strategies, including diet and ionophores, may influence abundance and diversity of ARGs in fecal samples from cattle.IMPORTANCEAntibiotic resistance is a One Health threat. More antibiotics are used in agriculture than in human medicine. We compared the relative abundance of antibiotic resistance genes (ARGs) and bacterial species in cattle raised in two different cattle production systems (grass- and grain-fed). Fecal swab samples were collected at five time points spanning pre-weaning and prior to harvest. The antibiotic resistance gene and bacterial communities were relatively similar in the pre-weaning period when cattle in both systems were milking and on pasture. Resistance genes and bacterial communities diverged post-weaning when system B cattle were given a grain diet with feed additives for growth promotion containing non-medically important antibiotics (i.e., ionophores). The levels of medically important ARGs (e.g., macrolides) increased in system B grain-fed cattle post-weaning and were higher than in system A just prior to slaughter. These data provide additional evidence that farm management strategies impact the level of antibiotic resistance.

Lysogenic bacteriophages can integrate their genome into the bacterial chromosome in the form of a prophage and can promote genetic transfer between bacterial strains in vitro. However, the contribution of lysogenic bacteriophages to the incidence of antimicrobial resistance (AMR) in clinical settings is poorly understood. Here, in a set of 186 clinical isolates of Pseudomonas aeruginosa collected from respiratory cultures from 82 patients with cystic fibrosis, we evaluate the links between prophage counts and both genomic and phenotypic resistance to six anti-pseudomonal antibiotics: tobramycin, colistin, ciprofloxacin, meropenem, aztreonam, and piperacillin-tazobactam. We identified 239 different prophages in total. We find that P. aeruginosa isolates contain on average 3.06 ± 1.84 (SD) predicted prophages. We find no significant association between the number of prophages per isolate and the minimum inhibitory concentration for any of these antibiotics. We then investigate the relationship between particular prophages and AMR. We identify a single lysogenic phage associated with phenotypic resistance to the antibiotic tobramycin and, consistent with this association, we observe that AMR genes associated with resistance to tobramycin are more likely to be found when this prophage is present. However, we find that they are not encoded directly on prophage sequences. Additionally, we identify a single prophage statistically associated with ciprofloxacin resistance but do not identify any genes associated with ciprofloxacin phenotypic resistance. These findings suggest that prophages are only infrequently associated with the AMR genes in clinical isolates of P. aeruginosa.IMPORTANCEAntibiotic-resistant infections of Pseudomonas aeruginosa (Pa), a leading pathogen in patients with cystic fibrosis (CF), are a global health threat. While lysogenic bacteriophages are known to facilitate horizontal gene transfer, their role in promoting antibiotic resistance in clinical settings remains poorly understood. In our analysis of 186 clinical isolates of P. aeruginosa from CF patients, we find that prophage abundance does not predict phenotypic resistance to key antibiotics but that specific prophages are infrequently associated with tobramycin resistance genes. In addition, we do not find antimicrobial resistance (AMR) genes encoded directly on prophages. These results highlight that while phages can be associated with AMR, phage-mediated AMR transfer may be rare in clinical isolates and difficult to identify. This work is important for future efforts on mitigating AMR in CFCF and other vulnerable populations affected by Pa infections and advances our understanding of bacterial-phage dynamics in clinical infections.

The bacterial microbiome has a major impact on health and can shape metabolism, host tolerance, immune responses, and the outcome of future infections. The bacterial microbiome is highly variable between individuals. Specific pathogen-free animals have reduced microbiome diversity, making it difficult to evaluate the impact of infection-induced microbiome disruption that would be observed in free-living animals, including people. Mice are commonly used as a preclinical model but unfortunately often fail to predict translation success or failure, particularly for immune and infectious disease-targeting therapies. Here, we utilize pet store mouse cohoused "dirty" mice with diverse microbial experience to explore how host variability and infection may be interacting to drive unique microbiome changes. We found that cohoused animals had significantly increased bacterial diversity in the small intestine and cecum but not in the large intestine. There were differentially abundant taxa between clean and dirty animals in all three tissues. After infection with influenza A virus, samples clustered by both housing condition and infection status in the cecum and large intestine, while small intestine samples clustered predominantly by infection. Altogether, these results highlight the differential impact of housing, infection, and interaction between the two in dictating community composition across the gastrointestinal microbiome.IMPORTANCETraditionally housed pathogen-free mouse models do not fully capture the natural variability observed among human microbiomes, which may underlie their poor translationally predictive value. Understanding the difference between pathogen-induced shifts in the bacterial microbiome and natural microbiome variance is a major hurdle to determining bacterial biomarkers of disease. It is also critical to understand how diverse baseline microbiomes may be differentially impacted by infection and contribute to disease. Pet store cohoused "dirty" mice have diverse microbial experiences and microbiomes, allowing us to evaluate how baseline variation, infection, and interaction between the two impact the microbiome.

The spread of antimicrobial resistance (AMR) is a critical One Health issue. Wildlife could act as reservoirs or vehicles of AMR bacteria (ARBs) and AMR genes (ARGs) but are relatively understudied. We sought to investigate clinically relevant ARGs in golden jackals (Canis aureus) thriving near human settlements in Israel. Fecal samples were collected from 111 jackals across four regions over a 10-month period. Various animal and spatio-temporal metadata were collected. Samples were analyzed by quantitative PCR (qPCR) for beta-lactamases (blaTEM, blaCTX-M15, and blaSHV), qnrS and int1. A subset of samples was subject to shotgun metagenomic sequencing followed by resistome and microbiome analyses. qPCR detected a high prevalence of ARGs, including beta-lactamases (blaTEM-1, 96.4%; blaCTX-M-15, 51.4%, blaSHV, 15.3%), fluoroquinolone resistance (qnrS, 87.4%), and class 1 integrons (Int1, 94.6%). The blaTEM-1 gene was found to be more prevalent in adult jackals compared to younger ones. Metagenomic analysis of a subset of samples revealed a diverse gut microbiome harboring a rich resistome with tetracycline resistance genes being the most prevalent. Metagenome-assembled genome analysis further identified several ARGs associated with clinically relevant bacteria. These findings highlight the potential role of golden jackals as reservoirs for AMR and emphasize the need for ongoing surveillance to better understand AMR transmission dynamics at the wildlife-human interface.

Importance: The research highlights the potential role of the golden jackals as reservoirs for antimicrobial resistance (AMR). The high prevalence of clinically relevant AMR genes in these jackals emphasizes the need for ongoing surveillance and monitoring to better understand AMR transmission dynamics at the wildlife-human interface.

During infection, Toxoplasma gondii tachyzoites must be able to move in order to migrate through tissues, cross biological barriers, and penetrate into and egress from cells they infect. Bradyzoite-stage parasites, which establish infection in naïve hosts, also require motility to escape from cysts after they are ingested and to subsequently migrate to the gut wall, where they either invade cells of the intestinal epithelium or squeeze between these cells to infect the underlying tissue. Little is known about the motility of bradyzoites, which we analyze in detail here and compare to the well-characterized motility and motility-dependent processes of tachyzoites. Unexpectedly, bradyzoites were found to be as motile as tachyzoites in a three-dimensional model extracellular matrix, and they showed increased invasion into and transmigration across monolayers of certain cell types, consistent with their need to establish infection in the gut. The motility of the two stages was inhibited to the same extent by cytochalasin D and KNX-002, compounds known to target the parasite's actomyosin-based motor. Other compounds that impact tachyzoite motility (tachyplegin and enhancer 5) have a reduced effect on bradyzoites. Furthermore, rapid bradyzoite egress from infected cells is not triggered by treatment with calcium ionophores, as it is with tachyzoites. The similarities and differences between these two life cycle stages highlight the need to characterize both tachyzoites and bradyzoites for a more complete understanding of the role of motility in the parasite life cycle and the effect that motility-targeting therapeutics will have on disease establishment and progression.

Importance: Toxoplasma gondii is a parasite that chronically infects around one-third of the world's population. Toxoplasma uses motility for multiple purposes during infection, including extracellular migration, invasion into host cells, and host cell egress. These motility-dependent processes have been extensively studied in the life cycle stage responsible for acute infection, the tachyzoite. In contrast, motility and motility-dependent processes are poorly understood in bradyzoite-stage parasites, which are responsible for both establishing infection after consumption of infected meat and initiating potentially life-threatening reactivated infections in the brains of immunocompromised individuals. We show here that the motility and motility-dependent processes of bradyzoites are similar in many respects to those of tachyzoites but markedly different in others. The results of this study highlight the need to consider both life cycle stages in attempts to develop drugs targeting parasite motility and the signaling processes that regulate motility-dependent processes during infection by these important human pathogens.

Breast cancer is the leading cause of cancer deaths in women worldwide, with about 20,000 cases annually in Argentina. While age, diet, and genetics are known risk factors, most breast cancer cases have unknown causes, necessitating the discovery of new risk factors. The aim of this study was the analysis of the prognostic relevance of the oncobiome in Argentinean breast cancer patients. Sequencing of the V4 region 16S rRNA gene was performed on 34 primary breast tumor samples, using bioinformatic and statistical analyses to identify bacteria and hypothetical pathways. Each sample presented a unique microbial profile, with Proteobacteria being the most abundant phylum. Tumors >2 cm showed greater alpha diversity with increased nucleotide biosynthesis. Moreover, progesterone-receptor tumors showed differences in beta diversity, being progesterone receptor-positive tumors that had the highest expression of Acinetobacter and Moraxella. In disease progression, the phylum Chloroflexi was prevalent in tumors of surviving patients. Acinetobacter and Cloacibacterium genera were significantly higher in patients without events and those without metastasis. We found that nucleotide and cell-structure biosynthesis, and lipid metabolism pathways were enriched in tumors with poor progression, whereas amino-acid degradation was increased in tumors of surviving patients. This finding is an indication that tumor cells are taking advantage of this effect of the microbiome during tumor progression. We conclude that oncobiome is dysbiotic in these patients, with distinct patterns in those with poor progression. Suggesting a link between the oncobiome and cancer progression, paving the way for new therapies to improve patient quality of life and survival.

Importance: This is the first study to investigate the relevance of the oncobiome in the evolution of breast cancer in a cohort of Argentine patients. It also highlights the need for further research in this area to improve our understanding of the role of the microbiome in this disease and potentially identify new therapeutic targets or prognostic indicators. Understanding the complex interaction between the microbiome, the tumor microenvironment, and the pathogenesis of breast cancer holds the promise of more personalized and effective treatment approaches in the future.

Jigar V. Desai works in the field of immunology, studying the mucosal and systemic complement systems and their roles in regulating the immune response. In this mSphere of Influence article, he reflects on how the papers by the Kemper, Kulkarni, and Kasper laboratories made an impact on his ongoing work investigating the cell-intrinsic and extrinsic regulation of complement and studying its impacts on mucosal and systemic immunity.

Colibactin is a genotoxic secondary metabolite produced by certain Enterobacteriaceae strains that populate the intestine and produces a specific mutational signature in human colonocytes. However, the host pathways involved in colibactin response remain unclear. To address this gap, we performed genome-wide CRISPR/Cas9 knockout screens and RNA sequencing utilizing live pks⁺ bacteria and a synthetic colibactin analog. We identified 20 enriched genes with a MAGeCK score of >2.0 in both screens, including proteasomal subunits (e.g., PSMG4 and PSMD4), RNA processing factors (e.g., SF1 and PRPF8), and RNA polymerase III (e.g., CRCP), and validated the role of PSMD4 in colibactin sensitization. PSMD4 knockout in HEK293T and HT-29 cells promoted cell viability and ameliorated G2-M cell cycle arrest but did not affect the amount of phosphorylated H2AX foci after exposure to synthetic colibactin 742. Consistent with these observations, PSMD4^-/- cells had a significantly higher colony formation rate and bigger colony size than control cells after 742 exposure. These findings suggest that PSMD4 regulates cell cycle arrest following colibactin-induced DNA damage and that cells with PSMD4 deficiency may continue to replicate despite DNA damage, potentially increasing the risk of malignant transformation.

Importance: Colibactin has been implicated as a causative agent of colorectal cancer. However, colibactin-producing bacteria are also present in many healthy individuals, leading to the hypothesis that some aspects of colibactin regulation or host response dictate the molecule's carcinogenic potential. Elucidating the host-response pathways involved in dictating cell fate after colibactin intoxication has been difficult, partially due to an inability to isolate the molecule. This study provides the first high-throughput CRISPR/Cas9 screening to identify genes conferring colibactin sensitivity. Here, we utilize both bacterial infection and a synthetic colibactin analog to identify genes directly involved in colibactin response. These findings provide insight into how differences in gene expression may render certain individuals more vulnerable to colibactin-initiated tumor formation after DNA damage.

Diego Huet works in molecular parasitology, focusing on the organellar biology of Toxoplasma gondii. In this mSphere of Influence article, he reflects on how the article "Efficient proximity labeling in living cells and organisms with turboID" (Branon et al., 2018) impacted his research and the strategies used to dissect inter-organellar interactions in T. gondii.