Dose-dependent impact of enrofloxacin on broiler chicken gut resistome is mitigated by synbiotic application.

Fluoroquinolone agents are considered critical for human medicine by the World Health Organization (WHO). However, they are often used for the treatment of avian colibacillosis in poultry production, creating considerable concern regarding the potential spread of fluoroquinolone resistance genes from commensals to pathogens. Therefore, there is a need to understand the impact of fluoroquinolone application on the reservoir of ARGs in poultry gut and devise means to circumvent potential resistome expansion. Building upon a recent dose optimization effort, we used shotgun metagenomics to investigate the time-course change in the cecal microbiome and resistome of broiler chickens receiving an optimized dosage [12.5 mg/kg body weight (bw)/day], with or without synbiotic supplementation (PoultryStar®, BIOMIN GmbH), and a high dosage of enrofloxacin (50 mg/kg bw/day). Compared to the high dose treatment, the low (optimized) dose of enrofloxacin caused the most significant perturbations in the cecal microbiota and resistome of the broiler chickens, demonstrated by a lower cecal microbiota diversity while substantially increasing the antibiotic resistance genes (ARGs) resistome diversity. Withdrawal of antibiotics resulted in a pronounced reduction in ARG diversity. Chickens receiving the synbiotic treatment had the lowest diversity and number of enriched ARGs, suggesting an alleviating impact on the burden of the gut resistome. Some Proteobacteria were significantly increased in the cecal metagenome of chickens receiving enrofloxacin and showed a positive association with increased ARG burden. Differential abundance (DA) analysis revealed a significant increase in the abundance of ARGs encoding resistance to macrolides-lincosamides-streptogramins (MLS), aminoglycosides, and tetracyclines over the period of enrofloxacin application, with the optimized dosage application resulting in a twofold higher number of affected ARG compared to high dosage application. Our results provide novel insights into the dose-dependent effects of clinically important enrofloxacin application in shaping the broiler gut resistome, which was mitigated by a synbiotic application. The contribution to ameliorating the adverse effects of antimicrobial agents, that is, lowering the spread of antimicrobial resistance genes, on the poultry and potentially other livestock gastrointestinal microbiomes and resistomes merits further study.

Storage media and RNA extraction approaches substantially influence the recovery and integrity of livestock fecal microbial RNA.

Background: There is growing interest in understanding gut microbiome dynamics, to increase the sustainability of livestock production systems and to better understand the dynamics that regulate antibiotic resistance genes (i.e., the resistome). High-throughput sequencing of RNA transcripts (RNA-seq) from microbial communities (metatranscriptome) allows an unprecedented opportunity to analyze the functional and taxonomical dynamics of the expressed microbiome and emerges as a highly informative approach. However, the isolation and preservation of high-quality RNA from livestock fecal samples remains highly challenging. This study aimed to determine the impact of the various sample storage and RNA extraction strategies on the recovery and integrity of microbial RNA extracted from selected livestock (chicken and pig) fecal samples. Methods: Fecal samples from pigs and chicken were collected from conventional slaughterhouses. Two different storage buffers were used at two different storage temperatures. The extraction of total RNA was done using four different commercially available kits and RNA integrity/quality and concentration were measured using a Bioanalyzer 2100 system with RNA 6000 Nano kit (Agilent, Santa Clara, CA, USA). In addition, RT-qPCR was used to assess bacterial RNA quality and the level of host RNA contamination. Results: The quantity and quality of RNA differed by sample type (i.e., either pig or chicken) and most significantly by the extraction kit, with differences in the extraction method resulting in the least variability in pig feces samples and the most variability in chicken feces. Considering a tradeoff between the RNA yield and the RNA integrity and at the same time minimizing the amount of host RNA in the sample, a combination of storing the fecal samples in RNALater at either 4 °C (for 24 h) or -80 °C (up to 2 weeks) with extraction with PM kit (RNEasy Power Microbiome Kit) had the best performance for both chicken and pig samples. Conclusion: Our findings provided a further emphasis on using a consistent methodology for sample storage, duration as well as a compatible RNA extraction approach. This is crucial as the impact of these technical steps can be potentially large compared with the real biological variability to be explained in microbiome and resistome studies.

Metatranscriptomic Analysis of the Chicken Gut Resistome Response to In-Feed Antibiotics and Natural Feed Additives.

The emergence of resistance against common antibiotics in the gut microbiota is a major issue for both human and livestock health. This highlights the need for understanding the impact of such application on the reservoir of antibiotic resistance genes in poultry gut and devising means to circumvent the potential resistome expansion. Phytogenic feed additives (PFAs) are potential natural alternative to antibiotic to improve animal health and performance, supposedly via positively affecting the gut microbial ecosystem, but there is little systematic information available. In this time-course study, we applied a shotgun meta-transcriptomics approach to investigate the impact of a PFA product as well as the commonly used antibiotic, zinc bacitracin either at AGP concentration or therapeutic concentration on the gut microbiome and resistome of broiler chickens raised for 35 days. Over the course of the trial, PFA treatments increased the abundance of Firmicutes such as Lactobacillus and resulted in a lower abundance of Escherichia, while the latter group increased significantly in the feces of chickens that received either AGP or AB doses of bacitracin. Tetracycline resistance and aminoglycoside resistance were the predominant antibiotic resistance gene (ARG) classes found, regardless of the treatment. PFA application resulted in a decrease in abundance of ARGs compared to those in the control group and other antibiotic treatment groups. In summary, the findings from this study demonstrate the potential of phytogenic feed additives could be an alternative to antibiotics in poultry farming, with the added benefit of counteracting antimicrobial resistance development.

Effects of Fusarium metabolites beauvericin and enniatins alone or in mixture with deoxynivalenol on weaning piglets.

The impact of the Fusarium-derived metabolites beauvericin, enniatin B and B1 (EB) alone or in combination with deoxynivalenol (DON) was investigated in 28-29 days old weaning piglets over a time period of 14 days. The co-application of EB and DON (EB + DON) led to a significant decrease in the weight gain of the animals. Liver enzyme activities in plasma were significantly decreased at day 14 in piglets receiving the EB + DON-containing diet compared to piglets receiving the control diet. All mycotoxin-contaminated diets led to moderate to severe histological lesions in the jejunum, the liver and lymph nodes. Shotgun metagenomics revealed a significant effect of EB-application on the gut microbiota. Our results provide novel insights into the harmful impact of emerging mycotoxins alone or with DON on the performance, gut health and immunological parameters in pigs.

Genome-Resolved Metagenomics of the Chicken Gut Microbiome.

Increasing evidence shows that the chicken gastrointestinal microbiota has a major effect on the modulation of metabolic functions and is correlated with economic parameters, such as feed efficiency and health. Some of these effects derive from the capacity of the chicken to digest carbohydrates and produce energy-rich metabolites such as short-chain fatty acids (SCFA) and from host-microbe interactions. In this study, we utilized information from metagenomic assembled genomes (MAGs) from chicken gastrointestinal tract (GIT) samples, with detailed annotation of carbohydrate-active enzymes (CAZymes) and genes involved in SCFA production, to better understand metabolic potential at different ages. Metagenomic sequencing of 751 chicken GIT samples was performed to reconstruct 155 MAGs, representing species which belong to six phyla, primarily Firmicutes followed by Proteobacteria. MAG diversity significantly (p < 0.001) increased with age, with early domination of Lachnospiraceae, followed by other families including Oscillospiraceae. Age-dependent shifts were observed in the abundance of genes involved in CAZyme and SCFA production, exemplified by a significant increase in glycosyltransferases (GTs) and propionic acid production pathways (p < 0.05), and a lower abundance of glycoside hydrolases (GHs) (p < 0.01). Co-occurrence analysis revealed a large cluster highly interconnected by enzymes from GT2_2 and GH3 families, underscoring their importance in the community. Furthermore, several species were identified as interaction hubs, elucidating associations of key microbes and enzymes that more likely drive temporal changes in the chicken gut microbiota, and providing further insights into the structure of the complex microbial community. This study extends prior efforts on the characterization of the chicken GIT microbiome at the taxonomic and functional levels and lays an important foundation toward better understanding the broiler chicken gut microbiome helping in the identification of modulation opportunities to increase animal health and performance.

Toward Best Practice in Livestock Microbiota Research: A Comprehensive Comparison of Sample Storage and DNA Extraction Strategies.

Understanding the roles of microorganisms in the animal gastrointestinal microenvironment is highly important for the development of effective strategies to manage and manipulate these microbial communities. In order to guide future animal gut microbiota research projects and standardization efforts, we have conducted a systematic comparison of 10 currently used sample preservation and DNA extraction approaches for pig and chicken microbiota samples and quantified their effects on bacterial DNA yield, quality, integrity, and on the resulting sequence-based bacterial composition estimates. The results showed how key stages of conducting a microbiota study, including the sample storage and DNA extraction, can substantially affect DNA recovery from the microbial community, and therefore, biological interpretation in a matrix-dependent manner. Our results highlight the fact that the influence of storage and extraction methods on the resulting microbial community structure differed by sample type, even within the same species. As the effects of these technical steps are potentially large compared with the real biological variability to be explained, standardization is crucial for accelerating progress in the area of livestock microbiota research. This study provided a framework to assist future animal gut microbiota research projects and standardization efforts.

The dynamics of the antibiotic resistome in the feces of freshly weaned pigs following therapeutic administration of oxytetracycline.

In this study, shotgun metagenomics was employed to monitor the effect of oxytetracycline, administered at a therapeutic dose, on the dynamics of the microbiota and resistome in the feces of weaned pigs. Sixteen weaning pigs were assigned to one of two treatments including standard starter diet for 21 days or antibiotic-supplemented diet (10 g oxytetracycline/100 kg body weight/day) for 7 days, followed by 14 days of standard starter diet. Feces were collected from the pigs on days 0, 8, and 21 for microbiota and resistome profiling. Pigs receiving oxytetracycline exhibited a significantly greater richness (ANOVA, P = 0.034) and diversity (ANOVA, P = 0.048) of antibiotic resistance genes (ARGs) than the control pigs. Antibiotic administration significantly enriched the abundances of 41 ARGs, mainly from the tetracycline, betalactam and multidrug resistance classes. Compositional shifts in the bacterial communities were observed following 7 days of antibiotic adminstration, with the medicated pigs showing an increase in Escherichia (Proteobacteria) and Prevotella (Bacteroidetes) populations compared with the nonmedicated pigs. This might be explained by the potential of these taxa to carry ARGs that may be transferred to other susceptible bacteria in the densely populated gut environment. These findings will help in the optimization of therapeutic schemes involving antibiotic usage in swine production.

The intestinal microbiota of piglets fed with wheat bran variants as characterised by 16S rRNA next-generation amplicon sequencing.

The intestinal microbiota of piglets fed with a Control diet low in dietary fibre and modified wheat bran variants as an additional source of insoluble dietary fibre was characterised. In this context, variances in the microbiota of three different gut segments were assessed. Wheat bran was either included in its native form or modified by fermentation and extrusion before added at 150 g/kg to a basal diet for 48 piglets (12 animals per treatment). Total DNA was extracted from digesta samples from the jejunum, the end of the ileum and the colon ascendens. Samples were prepared accordingly for subsequent sequencing with the Illumina MiSeq. The obtained results revealed distinct location-specific differences in microbial composition. While Firmicutes were most predominant in all three gut segments, Bacteroidetes were additionally found in the colon at high abundance. The parameters of alpha and beta diversity analysis showed significant differences (p < 0.01) between the colon and the other two gut segments. Specialised bacterial groups like Prevotella and Ruminococcaceae were among the most predominant ones found in the colon, as they possess cellulolytic properties to degrade (at least partially) non-starch polysaccharides, while their abundance was negligible in the jejunum and the ileum. Conversely, the genera Lactobacillus, Bifidobacterium and Veillonella, for example, were among the most predominant groups in the jejunum and ileum, while in the colon they were hardly found. Although statistical taxonomical evaluation, following p-value correction, did not reveal pronounced differences in abundance related to bran modification, alpha and beta diversity analysis showed an influence regarding the various feeding strategies applied. Based on these findings, a more in-depth view on intestinal microbial composition within the gastrointestinal tract of young pigs fed with low- and high-fibre diets was generated.

Celeribacter persicus sp. nov., a polycyclic-aromatic-hydrocarbon-degrading bacterium isolated from mangrove soil.

A Gram-stain-negative, mesophilic bacterial strain, designated SBU1T, which degrades polycyclic aromatic hydrocarbons was isolated from the sediments of the mangrove forests of Nayband Bay in the Iranian Persian Gulf during a bioremediation experiment. The 16S rRNA gene sequence of strain SBU1T exhibited highest similarities with Celeribacter indicus P73T (98.52%) and Celeribacter neptunius H 14T (97.05%). Phylogenetic analysis, based on 16S rRNA gene sequences, demonstrated that strain SBU1T fell within a cluster consisting of the type strains of species of the genus Celeribacter and formed a stable clade with C. indicus P73T in trees generated with three algorithms. The fatty acid profile of strain SBU1T consisted of the major fatty acids C18:1ω7c/ω6c and C18:1ω7c 11-methyl. The major compounds in the polar lipid profile were one phosphatidylglycerol and four unidentified phospholipids. The quinone system exclusively comprised ubiquinone (Q-10). The DNA G+C content was 60.4 mol%. A combination of phylogenetic analysis, DNA-DNA hybridization estimation, average nucleotide identity results and differential phenotypic and chemotaxonomic characteristics demonstrated that strain SBU1T could be distinguished from its close relatives. Therefore, strain SBU1T is considered to represent a novel species of the genus Celeribacter for which the name Celeribacter persicus sp. nov. is proposed. The type strain is SBU1T (=MCCC 1A00672T=DSM 100434T).

Phylogenetic diversity and biological activity of culturable Actinobacteria isolated from freshwater fish gut microbiota.

The diversity of Actinobacteria isolated from the gut microbiota of two freshwater fish species namely Schizothorax zarudnyi and Schizocypris altidorsalis was investigated employing classical cultivation techniques, repetitive sequence-based PCR (rep-PCR), partial and full 16S rDNA sequencing followed by phylogenetic analysis. A total of 277 isolates were cultured by applying three different agar media. Based on rep-PCR profile analysis a subset of 33 strains was selected for further phylogenetic investigations, antimicrobial activity testing and diversity analysis of secondary-metabolite biosynthetic genes. The identification based on 16S rRNA gene sequencing revealed that the isolates belong to eight genera distributed among six families. At the family level, 72% of the 277 isolates belong to the family Streptomycetaceae. Among the non-streptomycetes group, the most dominant group could be allocated to the family of Pseudonocardiaceae followed by the members of Micromonosporaceae. Phylogenetic analysis clearly showed that many of the isolates in the genera Streptomyces, Saccharomonospora, Micromonospora, Nocardiopsis, Arthrobacter, Kocuria, Microbacterium and Agromyces formed a single and distinct cluster with the type strains. Notably, there is no report so far about the occurrence of these Actinobacteria in the microbiota of freshwater fish. Of the 33 isolates, all the strains exhibited antibacterial activity against a set of tested human and fish pathogenic bacteria. Then, to study their associated potential capacity to synthesize diverse bioactive natural products, diversity of genes associated with secondary-metabolite biosynthesis including PKS I, PKS II, NRPS, the enzyme PhzE of the phenazine pathways, the enzyme dTGD of 6-deoxyhexoses glycosylation pathway, the enzyme Halo of halogenation pathway and the enzyme CYP in polyene polyketide biosynthesis were investigated among the isolates. All the strains possess at least two types of the investigated biosynthetic genes, one-fourth of them harbours more than four. This study demonstrates the significant diversity of Actinobacteria in the fish gut microbiota and it's potential to produce biologically active compounds.

Influence of indoor air conditions on radon concentration in a detached house.

Radon is released from soil and building materials and can accumulate in residential buildings. Breathing radon and radon progeny for extended periods hazardous to health and can lead to lung cancer. Indoor air conditions and ventilation systems strongly influence indoor radon concentrations. This paper focuses on effects of air change rate, indoor temperature and relative humidity on indoor radon concentrations in a one family detached house in Stockholm, Sweden. In this study a heat recovery ventilation system unit was used to control the ventilation rate and a continuous radon monitor (CRM) was used to measure radon levels. FLUENT, a computational fluid dynamics (CFD) software package was used to simulate radon entry into the building and air change rate, indoor temperature and relative humidity effects using a numerical approach. The results from analytical solution, measurements and numerical simulations showed that air change rate, indoor temperature and moisture had significant effects on indoor radon concentration. Increasing air change rate reduces radon level and for a specific air change rate (in this work Ach = 0.5) there was a range of temperature and relative humidity that minimized radon levels. In this case study minimum radon levels were obtained at temperatures between 20 and 22 °C and a relative humidity of 50-60%.