Panagiotis Sapountzis

Dietary composition is a major driver of the ruminal and intestinal microbiomes in dairy cows and is implicated in digestive disorders such as subacute ruminal acidosis (SARA), which can be triggered by high-concentrate diets. Here, we used a longitudinal shotgun metagenomic approach to investigate the dynamics of ruminal and fecal microbiomes in seven dairy cows subjected to a controlled bidirectional dietary transition from low- to high-concentrate diets and back over two months. Samples were collected at three key stages of dietary modulation, enabling the assessment of temporal changes in microbial composition and function in relation to diet-associated physiological fluctuations. Shotgun metagenomic sequencing enabled a comprehensive characterization of community structure and functional potential across compartments and time, revealing a strong location effect with clear segregation between ruminal and fecal microbiomes, as well as diet-associated differences in diversity and composition. Our results highlight how gradual dietary concentrate transitions induce coordinated taxonomic and functional remodelling of the digestive microbiome in dairy cows and suggest that these responses may not be fully reversible over the course of a short experimental time frame.

The cattle gastrointestinal tract harbors a diverse community of microorganisms, including pathogenic and commensal strains of Escherichia coli . Antimicrobial use in cattle can disrupt the gut microbiome, leading to shifts in bacterial diversity and abundance. Here, we combined shotgun metagenomics and single-cell sequencing to assess how ceftiofur antibiotic treatment impacted microbial diversity and structure. At the start of the experiment, ceftiofur was administered intramuscularly in parallel with the inoculation of a cocktail of extended-beta-lactamase-producing E. coli strains to simulate environmental exposure and acquisition of resistant strains while animals are under antibiotic treatment. Fecal samples were collected from both the antibiotic-treated (ceftiofur and inoculation) and control (inoculation only) calves over the course of 35 days. Read mapping to genome and gene databases showed substantial differences in microbial richness and beta diversity between treatment groups. Treatment group-enriched taxa included Bacteroidaceae and Fibrobacter , which were more abundant in samples that did not receive ceftiofur, and Akkermansia in ceftiofur-treated calves. In ceftiofur-exposed animals, we observed a gradual loss of virulence factors alongside increased abundances of beta-lactam resistance genes, including cfxA5 and cfxA6 , likely encoded by CAG-485 ( Muribaculaceae ). We further profiled individual cells using single-cell sequencing, which revealed a high number of Clostridium carrying macrolide resistance genes lnu (P) and mph (N) in both ceftiofur-treated and control samples. Overall, our complementary approaches reveal distinct remodeling of the calf microbiome following antibiotic and E. coli administration, tied to key functional genes that can be assigned to specific genera or recurrently detected across diverse taxa. IMPORTANCE: Cattle serve as natural reservoirs of zoonotic strains of Escherichia coli , which can cause severe gastrointestinal infections in humans. Antibiotic usage on cattle farms can drive the emergence of antimicrobial-resistant bacterial strains and alter the underlying cattle gastrointestinal microbiome. Consequently, there is a need to understand how antibiotic administration impacts population dynamics of cattle rumen and intestinal microbes. In this study, we combined both shotgun metagenomics and single-cell genomics on feces from ruminating calves to determine microbiome changes following administration of both ceftiofur and E. coli cocktails. We observed considerable variation in the prevalence and abundance of virulence factors, antimicrobial resistance-related genes, and taxa with key roles in animal nutrition and health between the microbiomes of antibiotic-treated and antibiotic-free calves, with potential implications for their subsequent development and overall well-being.

The cattle gastrointestinal tract harbors a diverse community of microorganisms, including pathogenic and commensal strains of Escherichia coli . Antimicrobial use in cattle can disrupt the gut microbiome leading to shifts in bacterial diversity and abundance. Here, we combined shotgun metagenomics and single-cell sequencing to assess how ceftiofur antibiotic treatment impacted microbial diversity and structure. At the start of the experiment, ceftiofur was administered intramuscularly in parallel with the inoculation of a cocktail of extended-beta-lactamase–producing E. coli strains, to simulate environmental exposure and acquisition of resistant strains while animals are under antibiotic treatment. Fecal samples were collected from both the antibiotic-treated (ceftiofur and inoculation) and control (inoculation only) calves over the course of 35 days. Read mapping to genome and gene databases showed substantial differences in microbial richness and beta diversity between treatment groups. Treatment group-enriched taxa included Bacteroidaceae and Fibrobacter , which were more abundant in samples that did not receive ceftiofur, and Akkermansia in ceftiofur-treated calves. In ceftiofur-exposed animals, we observed a gradual loss of virulence factors alongside increased abundances of beta-lactam resistance genes, including cfxA5 and cfxA6 likely encoded by CAG-485 ( Muribaculaceae ). We further profiled individual cells using single-cell sequencing, which revealed a high number of Clostridium carrying macrolide resistance genes lnu (P) and mph (N) in both ceftiofur-treated and control samples. Overall, our complementary approaches reveal distinct remodeling of the calf microbiome following antibiotic and E. coli administration, tied to key functional genes that can be assigned to specific genera or recurrently detected across diverse taxa. IMPORTANCE: Cattle serve as natural reservoirs of zoonotic strains of E. coli , which can cause severe gastrointestinal infections in humans. Antibiotic usage on cattle farms can drive the emergence of antimicrobial resistant bacterial strains and alter the underlying cattle gastrointestinal microbiome. Consequently, there is a need to understand how antibiotic administration impacts population dynamics of cattle rumen and intestinal microbes. In this study, we combined both shotgun metagenomics and single-cell genomics on feces from ruminating calves to determine microbiome changes following administration of both ceftiofur and E. coli cocktails. We observed considerable variation in prevalence and abundance of virulence factors, antimicrobial resistance–related genes, and taxa with key roles in animal nutrition and health between the microbiomes of antibiotic-treated and antibiotic-free calves, with potential implications for their subsequent development and overall well-being.

Dairy farms are major reservoirs of zoonotic bacterial pathogens, which harbor antimicrobial resistance genes (ARGs), and raise critical questions about their dissemination on and off the farm environment. Here, we investigated the role of coprophagous muscid flies (Diptera: Muscidae) as carriers of zoonotic pathogens and antimicrobial resistance. We collected cow manure and flies on a dairy farm and used shotgun metagenomics to identify the presence of clinically relevant bacteria, virulence factors, and ARGs in both environments. Our results reveal that, although the fly microbiome is largely composed of manure-associated taxa, they also harbor specific insect-associated bacteria, which may be involved in nutrient provisioning to the host. Furthermore, we identifed shared ARGs, virulence factors, and zoonotic pathogens enriched within the fly gastrointestinal tract (GIT). Our study illustrates the potential flow of pathogenic microorganisms from manure to coprophagous flies, suggesting that flies may pose an important zoonotic threat on dairy farms.

Stomoxys flies are highly ubiquitous blood-feeding pests on dairy farms and are suspected carriers of pathogenic bacteria due to their close association with both manure reservoirs and cattle hosts. While prior studies using amplicon sequencing and culture dependent methodologies have been used to characterize the composition of the Stomoxys microbiota, little is known about strain level genomic and functional diversity of Stomoxys -associated bacteria. In this study, we address this key gap in knowledge by using whole genome sequencing to provide the first comparative genomic analysis of Stomoxys- derived Escherichia coli , Klebsiella pneumoniae , and Staphylococcaceae isolates. Our results show that fly and manure isolates collected from the same farm system are phylogenetically interspersed, with subsequent pairwise genome alignments revealing near identical strains and plasmids shared between the two sources. We further identified a phylogenetic clade of Mammaliicoccus sciuri , which contains known mastitis agents that were associated with both flies and manure. Functional analysis revealed that this clade was highly enriched in xylose metabolism genes that were rare across other M. sciuri lineages, suggesting potential niche differentiation within the genus. Collectively, our results provide strong evidence for the acquisition of fecal-associated bacterial isolates by adult Stomoxys flies, confirming the link between biting muscid flies and manure habitats. The intermixing of fly and manure isolates in clinically relevant taxonomic groups strongly suggests that flies serve as carriers of opportunistic mastitis-causing or other fecal-borne pathogens, and that flies may serve as important vehicles of pathogen dissemination across the dairy farm environment. Author summary: Blood-feeding Stomoxys flies are a major pest species in dairy operations and have been implicated as carriers of fecal-borne pathogens responsible for bovine mastitis and other important cattle diseases. In a previous study, we generated a large collection of bacterial isolates from manure and Stomoxys flies collected from dairy barns. Most bacterial isolates derived from flies were identified as taxa associated with bovine mastitis, including Escherichia coli , Klebsiella pneumoniae , and non-aureus Staphylococcus ; however, it is unknown if these represent bacterial strains acquired by flies from manure. Here, we examined this possibility directly by performing whole genome sequencing of a subset of potential mastitis pathogens derived primarily from flies and manure and supplemented this analysis with publicly available genomes on NCBI (including mastitis and additional fly and manure strains). Our results reveal significant interrelations between fly, manure, and clinically derived mastitis strains, suggesting that Stomoxys flies participate in the carriage and dissemination of bovine mastitis pathogens acquired from manure. Additionally, this work highlights the functional diversity of bacterial pathogens within the barn environment and greatly expands the available genomic data for understudied mastitis pathogens such as Mammaliicoccus sciuri .

Escherichia coli is a gram-negative bacterium commonly found in mammals and birds’ intestinal microbiota. It can be easily isolated and has been studied for more than nine decades, making it an ideal model organism. It is mainly commensal but occasionally pathogenic, expressing virulence factors, antibiotic-resistance and acid-resistance genes, which contribute to its ability to persist and colonize diverse environments. Its multi-resistance, combined with the virulence of certain pathotypes (e.g. STEC), poses a recurring threat to public health, as treatment for clinical infections is not always straightforward. The remarkable propensity of E. coli for diversification and genomic variability is often referred to as an open pangenome. This project’s objective was to create the ‘Escobase’ database ( Es cherichia co li data base ), which is comprised exclusively of Escherichia coli isolates from cattle farms, allowing us to generate a reference set of genomes that can be used for comparative genomics on samples from cattle and cattle farms. Using the annotated E. coli genomes, we further built a catalog of predicted proteins that help E. coli survive in low pH environments, often encountered when the bacteria traverse through the intestine. We believe these two databases will be of great help to the community, and we plan to keep improving them and publish updated versions in the future.

The mutualistic interaction specificity between attine ants and antibiotic-producing Actinobacteria has been controversial because Pseudonocardia strains cannot always be isolated from worker cuticles across attine ant species, while other actinobacteria can apparently replace Pseudonocardia and also inhibit growth of Escovopsis mycopathogens. Here we report that across field samples of Panamanian species: (i) Cuticular Pseudonocardia were largely restricted to species in the crown of the attine phylogeny and their appearance likely coincided with the first attines colonizing Central/North America. (ii) The phylogenetically basal attines almost always had cuticular associations with other Actinobacteria than Pseudonocardia. (iii) The sub-cuticular glands nourishing cuticular bacteria appear to be homologous throughout the phylogeny, consistent with an ancient general attine-Actinobacteria association. (iv) The basal attine species investigated always had Pseudonocardia as gut symbionts while Pseudonocardia presence appeared mutually exclusive between cuticular and gut microbiomes. (v) Gut-associated Pseudonocardia were phylogenetically ancestral while cuticular symbionts formed a derived crown group within the Pseudonocardia phylogeny. We further show that laboratory colonies often secondarily acquire cuticular Actinobacteria that they do not associate with in the field, suggesting that many previous studies were uninformative for questions of co-adaptation in the wild. An exhaustive literature survey showed that published studies concur with our present results, provided that they analyzed field colonies and that Actinobacteria were specifically isolated from worker cuticles shortly after field collection. Our results offer several testable hypotheses for a better overall understanding of attine-Pseudonocardia interaction dynamics and putative coevolution throughout the Americas.

We describe and discuss the intestinal mycobiota of dairy cows reared in France following variations in dietary regimes and two seasons. Two groups of 21 animals were followed over a summer and winter period, and another group of 28 animals was followed only during the same summer season. The summer diet was based on grazing supplemented with 3–5 kg/d of maize, grass silage and hay, while the winter diet consisted of 30% maize silage, 25% grass silage, 15% hay and 30% concentrate. A total of 69 DNA samples were extracted from the feces of these cows. Amplification and sequencing of the ITS2 region were used to assess mycobiota diversity. Analyses of alpha and beta diversity were performed and compared statistically. The mycobiota changed significantly from summer to winter conditions with a decrease in its diversity, richness and evenness parameters, while beta diversity analysis showed different mycobiota profiles. Of note, the Geotrichum operational taxonomic unit (OTU) was prevalent in the winter group, with a mean relative abundance (RA) of 65% of the total mycobiota. This Geotrichum OTU was also found in the summer group, but to a lesser extent (5%). In conclusion, a summer grazing diet allowed a higher fecal fungal diversity. These data show, for the first time, that a change in diet associated with seasonality plays a central role in shaping hindgut fungal diversity.

Antigen 43 (Ag43) expression induces aggregation and biofilm formation that has consequences for bacterial colonisation and infection. Ag43 is secreted through the Type 5 subtype “a” secretion system (T5aSS) and is a prototypical member of the family of self-associating autotransporters (SAATs). As a T5aSS protein, Ag43 has a modular architecture comprised of (i) a signal peptide, (ii) a passenger domain that can be subdivided into three subdomains (SL, EJ, and BL), (iii) an autochaperone (AC) domain, and (iv) an outer membrane translocator. The cell-surface SL subdomain is directly involved in the “Velcro-handshake” mechanism resulting in bacterial autoaggregation. Ag43 is considered to have a ubiquitous distribution in E. coli genomes and many strains harbour multiple agn43 genes. However, recent phylogenetic analyses indicated the existence of four distinct Ag43 classes exhibiting different propensities for autoaggregation and interactions. Given the knowledge of the diversity and distribution of Ag43 in E. coli genomes is incomplete, we have performed a thorough in silico investigation across bacterial genomes. Our comprehensive analyses indicate that Ag43 passenger domains cluster in six phylogenetic classes associated with different SL subdomains. The diversity of Ag43 passenger domains is a result of the association of the SL subtypes with two different EJ-BL-AC modules. We reveal that agn43 is almost exclusively present among bacterial species of the Enterobacteriaceae family and essentially in the Escherichia genus (99.6%) but that it is not ubiquitous in E. coli. The gene is typically present as a single copy but up to five copies of agn43 with different combinations of classes can be observed. The presence of agn43 as well as its different classes appeared to differ between Escherichia phylogroups. Strikingly, agn43 is present in 90% of E. coli from E phylogroup. Our results shed light on Ag43 diversity and provide a rational framework for investigating its role in E. coli ecophysiology and physiopathology.

Background: The large intestine is a colonization site of beneficial microbes complementing the nutrition of cattle but also of zoonotic and animal pathogens. Here, we present the first global gene catalog of cattle fecal microbiomes, a proxy of the large intestine microbiomes, from 436 metagenomes from six countries. Results: Phylogenomics suggested that the reconstructed genomes and their close relatives form distinct branches and produced clustering patterns that were reminiscent of the metagenomics sample origin. Bacterial taxa had distinct metabolic profiles, and complete metabolic pathways were mainly linked to carbohydrates and amino acids metabolism. Dietary changes affected the community composition, diversity, and potential virulence. However, predicted enzymes, which were part of complete metabolic pathways, remained present, albeit encoded by different microbes. Conclusions: Our findings provide a global insight into the phylogenetic relationships and the metabolic potential of a rich yet understudied bacterial community and suggest that it provides valuable services to the host. However, we tentatively infer that members of that community are not irreplaceable, because similar to previous findings, symbionts of complex bacterial communities of mammals are expendable if there are substitutes that can perform the same task.