Fate of pathogenic bacteria in five full-scale biogas plants monitored using cultivation, dPCR, and shotgun metagenomics: Insights from each approach.

The effects of drinking water with essential oils-organic acids blend replacing preventive antibiotics during withdrawal period on growth performance and gut health of broilers in a commercial farm.

Enterococcus cecorum has become a significant pathogen of broilers and broiler breeders worldwide, and there are few effective prevention, treatment, or control measures. Improved, preferably nonantibiotic tools are needed to control the disease. Two pen trials were conducted to assess the effectiveness of a day-of-age probiotic and a continuous direct fed microbial (DFM) in preventing colonization of the spleen and free thoracic vertebra after challenge with pathogenic E. cecorum in commercial broiler chickens. In Experiment 1, three treatment groups were administered either no treatment (challenge controls), a day-of-age probiotic gel plus DFM 1 continuously in the feed, or the probiotic plus DFM 2, and challenged orally on Day 0 with a known pathogenic strain of E. cecorum (SA3). Both treatments resulted in a significant decrease in prevalence of colonization of spleens and free thoracic vertebrae at Day 42 compared to the challenge controls, with no effects on performance parameters or total mortality. In Experiment 2, four treatment groups were administered either no treatment (challenge controls), the day-of-age probiotic gel alone, the probiotic gel plus DFM 1, or DFM 1 alone. At 42 days of age, both the probiotic alone and the probiotic plus DFM 1 resulted in a significant decrease in colonization of the free thoracic vertebrae compared to the challenge controls, with the DFM alone being statistically intermediate. There were no impacts on performance parameters, livability, or lameness compared to the challenge controls. These results suggest that the day-of-age probiotic and the DFM may be a useful tool in controlling enterococcal spondylitis, and further larger-scale trials are warranted to assess impacts on clinical signs and performance.

Modulation of gut immunity and microbiota by Bacteroides thetaiotaomicron confers dose-dependent protection against necrotic enteritis in broiler chickens.

BackgroundZearalenone (ZEN), a common mycotoxin in ruminant diets, could disturb the rumen ecosystem and impair rumen fermentation. Noticeably, ZEN has been shown to reduce the relative abundances of specific bacterial taxa that potentially possess quorum sensing (QS) functions, which are deemed essential for the microbial interactions and adaptations during rumen fermentation. Nonetheless, whether QS communications participate in the responses of rumen microbial fermentation to ZEN remains unknown. Therefore, the present trial was performed to explore the potential roles of QS during the alterations of rumen microbial fermentation by ZEN through a rumen simulation technique (RUSITEC) system, in a replicated 4 × 4 Latin square design.ResultsZEN significantly (P < 0.05) reduced QS signal autoinducer-2 (AI-2), and tended to (P = 0.051) downregulate QS signal C4-homoserine lactone (HSL). ZEN also significantly (P < 0.05) decreased total volatile fatty acid (TVFA), acetate, propionate, isobutyrate, isovalerate, organic matter disappearance (OMD), neutral detergent fiber disappearance (NDFD), and acid detergent fiber disappearance (ADFD) in different manners. The linear discriminant analysis effect size (LEfSe) analysis indicated significantly (P < 0.05) differential enrichments of a series of bacterial taxa such as Butyrivibrio_sp_X503, Rhizobium daejeonense, Hoylesella buccalis, Ezakiella coagulans, Enterococcus cecorum, Ruminococcus_sp_zg-924, Polystyrenella longa, and Methylacidimicrobium fagopyrum across different treatments. The phylogenetic investigation of communities by reconstruction of unobserved states 2 (PICRUSt2) analysis suggested that QS were predicted to be significantly (P < 0.05) affected by ZEN. The metabolomics analysis detected considerable significantly (P < 0.05) differing metabolites and implied that ZEN challenge significantly (P < 0.05) influenced the indole alkaloid biosynthesis, biosynthesis of alkaloids derived from shikimate pathway, and sesquiterpenoid and triterpenoid biosynthesis. Significant (P < 0.05) interconnections of QS molecules with the differential rumen fermentation traits, differential bacterial taxa, and differential metabolites were exhibited by Spearman analysis.ConclusionsZEN negatively affected the QS signals of AI-2 and C4-HSL, which was found to correlate with the fluctuations in specific rumen fermentation characteristics, ruminal bacterial populations, and ruminal metabolisms. These interrelationships implied the potential involvement of QS in the reactions of rumen microbiota to ZEN contamination, and probably contributed to the inhibition of rumen fermentation.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40104-025-01337-z.

Development of gene manipulation methods for Enterococcus cecorum.

Avian coccidiosis is an intestinal disease caused by Eimeria spp. infection. A deeper understanding of the interaction between host gut microbiota and the Eimeria parasite is crucial for developing alternative therapies to control avian coccidiosis. Here, we used full-length sequencing of 16S ribosomal RNA amplicons to compare changes in the gut microbiota of chickens infected with Eimeria tenella, Eimeria maxima, and Eimeria necatrix, aiming to identify both species-specific and common alterations in gut microbiota at 4 and 10 days post-infection. The result revealed that infection with all three Eimeria species led to a decrease in the abundance of the microbial genera Limosilactobacillus, Streptococcus, Alistipes, Lactobacillus and Phocaeicola, while the abundance of Bacteroides, Escherichia and Ligilactobacillus increased. Escherichia and Enterococcus were most abundant in the jejunum of the E. necatrix-infected group and in the cecum of the E. tenella-infected group, whereas Megamonas abundance was highest in the E. maxima-infected group. LEfSe analysis showed that infection with all three Eimeria species significantly reduced the abundance of 13 bacterial species, including Acetilactobacillus jinshanensis, Bacteroides ndongoniae, Barnesiella viscericola, Christensenella minuta, Enterocloster clostridioformis, Gemella haemolysans_A, Granulicatella adiacens, Lawsonibacter sp000177015, Limosilactobacillus reuteri, Limosilactobacillus reuteri_D, Limosilactobacillus vaginalis_A, Limosilactobacillus caviae, Limosilactobacillus vaginalis. Infection with E. tenella significantly increased the abundance of five bacterial species, including Bacteroides fragilis, Enterococcus cecorum, Helicobacter pylori, Methylovirgula ligni, and Phocaeicola sp900066445. Infection with E. maxima significantly increased the abundance of seven bacterial species, including Clostridioides difficile, Faecalibacterium prausnitzii, Mediterraneibacter torques, Muribaculum intestinale, Mediterraneibacter massiliensis, Phascolarctobacterium faecium, and Phocaeicola plebeius. Infection with E. necatrix significantly increased the abundance of seven bacterial species, including Alistipes sp900290115, Anaerotignum faecicola, Bacteroides fragilis_A, Escherichia coli, Harryflintia acetispora, Pseudoclostridium thermosuccinogenes, and Tidjanibacter inops_A. The results showed that Eimeria infection causes significant species- and time-dependent changes in the gut microbiota of chickens. These findings enhance our understanding of coccidiosis pathogenesis and offer potential targets for developing probiotics.

Pathogenic bacteria can inhabit the chicken intestinal tract and potentially cause disease and mortality in chickens. Proteinogenic amino acids are major nutrients available in the intestine that have diverse growth-modulating effects on bacteria. However, limited knowledge exists about the direct association between amino acids and the growth of bacteria in the microbiome of the chicken's intestinal tract. The present study evaluated the growth-modulating effects of the 20 proteinogenic amino acids on chicken pathogens. Seven bacterial species were tested, including avian pathogenic Escherichia coli (APEC), Salmonella enterica., Staphylococcus aureus, Clostridium perfringens, Enterococcus cecorum, Enterococcus faecalis, and Enterococcus faecium. All bacteria were tested in minimal nutrient medium (MNM) in an in vitro model, with or without the addition of each amino acid. Bacterial growth was measured by absorbance (OD600) and visualised as growth curves. l-cysteine significantly (p ≤ 0.05) inhibited the growth of APEC, extended the lag phase and increased the maximum density of C. perfringens, Salmonella enterica. and S. aureus. In contrast, l-glutamine and l-glutamic acid significantly shortened the lag phase and increased both the growth rate and maximum density of C. perfringens, Glycine and l-serine significantly (p ≤ 0.05) increased the maximum density of S. aureus. The effect of amino acids was insignificant (p ≥ 0.05) on the total growth of Enterococcus spp. These findings suggested that l-cysteine has inhibiting effects on APEC, C. perfringens, S. enterica and S. aureus. l-glutamine and l-glutamic acid promoted the growth of C. perfringens, while glycine and l-serine promoted the growth of S. aureus.

Assessing the impact of broiler genotype on cecal and tracheal microbiome composition using full-length 16S rRNA sequencing

Pampus argenteus (Zobaidy) is an important fish in Kuwait and the Gulf region due to its economic value in the fish industry. Analyzing the gut microbiome of Zobaidy can help determine the health status of the fish and its responses to environmental changes. In this study, we investigated the microbiome composition of the intestinal tract among seven wild-caught silver pomfret specimens sampled in the Arabian gulf. The 16S rRNA was sequenced using the Illumina platform; then, sequences were analyzed using several bioinformatics tools to identify the microbial diversity, taxonomical status, and functional aspects. The results were 5933 operational taxonomic units (OTUs) categorized into 35 phyla. Proteobacteria, Firmicutes, Bacteroidota, and Actinobacterota were most abundant in the Zobaidy and water samples. At the genus level, we found high relative abundances of Acinetobacter. The results indicated that Lactococcus piscium, Enterococcus cecorum, Psychrobacter arenosus, Vagococcus salmoninarum, and Carnobacterium maltaromaticum are the most commonly present species in the analyzed Zobaidy samples. A heatmap analysis indicated notable differences in the functional categories of intestinal microflora within the Zobaidy2 sample compared to other Zobaidy samples. It should be noted that microbiome studies can provide novel ways to enhance the overall welfare of fish, strengthen disease prevention, and increase sustainability in aquaculture production.

This study aimed to characterize the fecal microbiome of highly productive laying hens to identify microbial signatures linked with enhanced egg production performance. Six commercial layer farms were enrolled in the study. Farms with an average monthly egg production rate (amEPR) above 80% over six consecutive months were classified as the high-production group (D group; n=3), while those with amEPR below 60% were designated as the low-production group (P group; n=3). All hens were raised in open-type housing systems without a history of forced molting. From each farm, 36 fecal samples were randomly collected and subjected to full-length 16S ribosomal RNA gene sequencing to characterize the microbiome. The D group showed significantly higher Firmicutes-to-Bacteroidetes ratios (p<0.0001) and lower species richness (Menhinick index, p<0.05) compared with the P group. Multivariate analyses (Adonis, ANOSIM, and MRPP; all p<0.001) revealed distinct microbial community structures between the groups. Taxonomic profiling indicated that the D group harbored higher relative abundances of Enterococcus cecorum and Lactobacillus kitasatonis (both p<0.05). In contrast, the P group had elevated levels of Bacteroides eggerthii and Bacteroides coprosuis (p<0.001 and p<0.05, respectively). Principal component analysis and Linear discriminant analysis effect size further identified Enterococcus cecorum and Lactobacillus kitasatonis as biomarkers associated with superior egg production. In Hy-Line hens, Lactobacillus gallinarum and Lactobacillus salivarius were also identified as biomarkers linked to high productivity. The fecal microbiome of highly productive laying hens is characterized by an enrichment of Enterococcus cecorum and lactic acid bacteria, particularly Lactobacillus kitasatonis, Lactobacillus gallinarum, and Lactobacillus salivarius. Their targeted supplementation may represent a promising probiotic strategy to improve egg production efficiency in commercial laying hens.

BackgroundHypertension and hyperlipidemia are interconnected conditions that heighten cardiovascular risk, yet their intricate multi-scale molecular signatures remain inadequately mapped. This study aimed to conduct an integrated multi-omics investigation to unravel the key pathways and biomarkers underlying hypertension, hyperlipidemia, and both conditions.MethodsMetabolomic analysis was performed on serum samples and metagenomic analysis on fecal samples collected from individuals with hypertension (n = 16), hyperlipidemia (n = 19), or both conditions concurrently (n = 20). In addition, 20 healthy individuals were recruited as controls.ResultsMetabolomics uncovered altered levels of sphingolipids, phosphatidylcholines, glycylprolines, and nucleic acid metabolites, which may be associated with changes in vascular tone, lipid and protein homeostasis, and thyroid signaling. Metagenomics showed depletion in the abundance of the Fibrobacteres phylum. Altered abundances of Escherichia coli and Bacteroides vulgatus were also observed, which were correlated with deviations in lipid and carbohydrate metabolism. Sphingomyelin d18:1/16:0 and sphingomyelin d18:1/24:1(15Z) were the key metabolites that were identified as potential diagnostic biomarkers across conditions. Microbial taxa such as Enterococcus cecorum, Lachnospiraceae bacterium, Prevotella histicola, and Flavobacterium discriminated these diseases. Pathway analysis revealed glycoxylate, amino acid, purine, and sphingolipid metabolism alterations intersecting hypertension and hyperlipidemia.ConclusionsThis multi-omics landscape of comorbid disease pathways and biomarkers lays the foundation for precision diagnosis and treatment of prevalent cardiovascular conditions.

The rate of Enterococcus cecorum infections within poultry (e.g. chickens, racing pigeons and Pekin ducks) has rapidly increased since they were initially reported in the UK and the Netherlands in 2002. E. cecorum can infect the free thoracic vertebra of chickens, often leading to carcass rejection at processing plants, incurring large economic costs to farmers. The environmental spread of this pathogen has been facilitated by high levels of antimicrobial resistance, as well as the emergence of divergent clonal lines with a higher pathogenic potential within poultry. Previous genomic studies have explored these characteristics within E. cecorum populations in both the USA and France. However, whether E. cecorum isolates in other countries show the same genomic traits remains unknown. In the present study, we investigate the properties of 190 E. cecorum isolates collected from UK broiler farms between 2021 and 2022. We report the MICs and epidemiological cutoff values for 32 antibiotics and analyse the genome sequence of 39 representative organisms from diseased or healthy broiler farms. We show that UK isolates present phenotypic signs of resistance for 26 antibiotics tested and show that clinical isolates are enriched in a single clade. These strains share properties with pathogenic strains from France and the USA, including conserved cell wall biosynthesis loci, as well as mobile genetic elements involved in the propagation of antibiotic resistance genes. Our results support the idea of a cross-continental spread of virulent E. cecorum.

Background:Raw milk and unpasteurized dairy products are still widely consumed in Iraq, particularly in rural areas. These products may carry harmful bacteria, including those resistant to antibiotics. Although many studies have focused on common pathogens, few have used molecular methods to detect and compare local bacterial strains with international strains. This lack of data creates a gap in understanding the public health risk and genetic diversity of these bacteria in Iraqi dairy products.Aim:This study aimed to detect bacteria in local dairy products from Al-Diwaniyah City, Iraq.Methods:This study was observational. Milk, yogurt, and cheese samples were taken. A total of 150 samples were collected: 60 milk, 45 yogurts, and 45 cheese samples. They were collected from farms, markets, and shops. Each sample was tested in the laboratory. The objectives of this study were to detect and identify the presence of bacteria in local dairy products, including milk, yogurt, and cheese, collected from Al-Diwaniyah City, Iraq. This study aimed to use both biochemical methods, such as the VITEK®2 system, and molecular tools, such as polymerase chain reaction and 16S rRNA sequencing, to accurately classify the bacterial species. Another goal was to compare the genetic sequences of local isolates with those of international strains using Basic Local Alignment Search Tool analysis and phylogenetic tree construction. In addition, we evaluated the protein patterns of the bacterial isolates using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to understand their enzymatic activity. These objectives were designed to highlight the risks of contamination in unpasteurized dairy products and support better food safety practices using molecular evidence.Results:Klebsiella pneumoniae, Klebsiella aerogenes, Enterobacter cloacae, Escherichia coli, and Enterococcus cecorum were found. These bacteria were very similar to those from other countries, such as India and Egypt. Gene sequences were matched with those in the GenBank database. Some gene regions were the same in all bacteria. Other regions exhibited changes. SDS-PAGE showed clear protein bands between 20 and 70 kDa, confirming diverse peptide profiles among the bacterial isolates. These changes help to separate the bacterial species.Conclusion:Dairy products can carry serious bacteria. These bacteria can be studied by examining their genes. Regular checking of food is needed. Molecular tools can help identify and track these bacteria.

Necrotic enteritis (NE), caused by Clostridium perfringens, poses significant economic challenges to the global poultry industry. The widening ban on in-feed antibiotics in livestock production underscores the need for alternative strategies to combat NE. Deoxycholic acid (DCA), a secondary bile acid, has shown promise in NE mitigation. However, its protective mechanism remains largely unexplored. A total of 120 newly hatched, male Cobb broilers were randomly divided into four treatments to investigate the impact of DCA on host response and intestinal microbiome in both healthy and NE-infected chickens. The results demonstrated that the dietary supplementation of 1.5 g/kg DCA significantly improved animal survival, reversed growth inhibition, and alleviated intestinal lesions (p < 0.01). Furthermore, DCA selectively inhibited the NE-induced proliferation of C. perfringens and other pathobionts such as Escherichia and Enterococcus cecorum. Concurrently, DCA markedly enriched dominant lactic acid bacteria like Lactobacillus johnsonii in both the ileum and cecum of NE-infected chickens. However, DCA had a marginal effect on the jejunal transcriptomic response in both mock- and NE-infected chickens. Therefore, we conclude that DCA protects chicken from NE mainly through the targeted inhibition of pathobionts including C. perfringens, with minimum impact on the host. These findings elucidate the protective mechanisms of DCA, supporting its development as a promising antibiotic alternative for NE mitigation.

BackgroundEgg production and performance in commercial layer flocks can be impacted by gastrointestinal health. Probiotics are considered potential alternatives to antibiotics in poultry, and have been proven to have positive impacts on gut health. The objective of this study was to isolate and characterise Lactobacillus strains isolated from high production layer flocks for probiotic properties.ResultsEleven different Lactobacillus (L.) species were isolated from faecal samples collected from layer hens in late lay. Species included L. agilis, L. crispatus, L. gallinarum, L. ingluviei, L. johnsonii, L. kitasatonis, L. mucosae, L. oris, L. reuteri, L. saerimneri and L. salivarius. Enterococcus cecorum and Enterococcus faecium as well as Streptococcus alactolyticus were also isolated. Phenotypic properties of the eleven Lactobacillus species were then evaluated to assess their probiotic potential. Bacterial culturability at low pH, under high concentrations of bile salt and sodium chloride, as well as in simulated gastric juice were characterised for each species. Auto- and co-aggregation capacity, adhesion to the intestinal epithelium as well as inhibition of Salmonella Typhimurium were also evaluated. All the Lactobacillus spp. exhibited high culturability in sodium chloride at different temperatures, and exhibited strong adhesion to chicken epithelial cells. L. reuteri, L. salivarius, L. saerimneri, and L. mucosae displayed higher culturability in low pH conditions, and L. johnsonii displayed higher culturability in high concentration of bile salt. Only L. crispatus was culturable in simulated gastric juice after 24 h incubation. L. kitasatonis exhibited strong auto-aggregation and co-aggregation with wild-type Salmonella Typhimurium. All Lactobacillus species exhibited strong inhibition activity against Salmonella Typhimurium.ConclusionsThe Lactobacillus species isolated in this study exhibited probiotic properties. Further in vivo experiments are required to evaluate their capacity to colonise and persist in the host as well as evaluate their potential effects on the gut microbiota and overall health parameters of layer hens.

Full-length 16S rRNA sequencing revealed an altered microbiome diversity and composition of the jejunum and cecum in chicken infected with Eimeria necatrix.

ABSTRACT Field observations suggest genotype-associated differences in susceptibility to infection with Enterococcus cecorum (EC). We hypothesized that slow-growing (SG) chickens show less clinical signs and reduced lesions compared to fast-growing (FG) chickens after experimental challenge with EC. At 1-day post-hatch (dph), 97 FG and 97 SG chickens were randomly assigned to four groups: control, SG (CSG), control, FG (CFG), EC-inoculated, SG (ESG), and EC-inoculated, FG (EFG). After oral inoculation with 107 colony forming units EC or mock inoculation with physiological saline, chickens were monitored for clinical signs and samples were collected weekly for further analysis via ELISA, real-time PCR, and flow cytometric analysis. The final necropsy of 40 chickens per group was carried out at 43/44 dph. Clinical signs and pathological findings were observed in a reduced number of chickens in group ESG compared to EFG. Extra-intestinal colonization with EC at 43/44 dph was significantly reduced in group ESG compared to EFG (P < 0.05). Circulating heterophils and monocytes were significantly increased in groups ESG and EFG compared to the control groups (P < 0.05). Additionally, circulating monocytes were increased and heterophil/lymphocyte (H/L) ratios were decreased in SG chickens compared to FG chickens. We may speculate that SG chickens were less affected by the EC-associated disease due to a more efficient innate immune response and may more vigorously control extra-intestinal colonization after translocation from the intestine. Overall, the use of slow-growing chickens may reduce the incidence of the EC-associated disease in meat-type chickens and therefore increase overall health performance.

Since the early 2000s, skeletal effects, specifically enterococcal spondylitis, related to pathogenic Enterococcus cecorum (EC), have been observed in older broiler chicken flocks. This skeletal involvement has typically been associated with persistent EC infections in the free thoracic vertebrae leading to paralysis. However, the emergence of virulent EC in young broiler chicken flocks causing clinical septicemia requires further investigation. The purpose of this review is to provide an update on EC-related research and pending industry needs.

Osteomyelitis caused by Enterococcus cecorum is an emerging disease in broiler chickens in Canada. Other Enterococcus species have been reported as causative agents in certain outbreaks. The epidemiology of this disease is unknown, but contaminated barns are affected by recurring episodes. A broiler chicken flock located in Quebec, Canada, exhibited osteomyelitis lesions positive for E. cecorum and Enterococcus faecalis. Surprisingly, the following lot, in the same barn, revealed the presence of E. faecalis- and Enterococcus raffinosus-positive lesions but no E. cecorum. To better understand the epidemiology of these two outbreaks, verify the persistence of pathogenic isolates in the barn, and identify the possible transfer of genetic material between the Enterococcus species isolated from both events, 16 isolates (1 E. cecorum, 13 E. faecalis, and 2 E. raffinosus isolates) were sequenced, and their genomes were compared. Interestingly, more than one Enterococcus species could be isolated from the same lesion, while other lesions also revealed several nonclonal isolates from the same species. This might suggest the opportunistic nature of Enterococcus spp. as there was no predominant isolate in the lesions. The number of virulence genes varied from 1 to 34 across three Enterococcus species with no common virulence gene. The number and nature of antimicrobial resistance genes among those isolates were worrisome because they indicate the presence of multidrug resistance on the farm. Both plasmids and phages were shared by different Enterococcus species, which suggests potential horizontal gene transfer of mobile genetic elements within this enterococci population.