Continuous feeding of antimicrobial growth promoters to commercial swine during the growing/finishing phase does not modify faecal community erythromycin resistance or community structure

Aims: To investigate the effect of continuous feeding of antimicrobial growth promoters (tylosin or virginiamycin) on the swine faecal community. Methods and Results: The study consisted of two separate on‐farm feeding trials. Swine were fed rations containing tylosin (44 or 88 mg kg^?1 of feed) or virginiamycin (11 or 22 mg kg^?1 of feed) continuously over the growing/finishing phases. The temporal impact of continuous antimicrobial feeding on the faecal community was assessed and compared to nondosed control animals through anaerobic cultivation, the analysis of community 16S rRNA gene libraries and faecal volatile fatty acid content. Feeding either antimicrobial had no detectable effect on the faecal community. Conclusions: Erythromycin methylase genes encoding resistance to the macrolide–lincosamide–streptogramin B (MLS_B) antimicrobials are present at a high level within the faecal community of intensively raised swine. Continuous antimicrobial feeding over the entire growing/finishing phase had no effect on community erm‐methylase gene copy numbers or faecal community structure. Significance and Impact of the Study: Antimicrobial growth promoters are believed to function by altering gut bacterial communities. However, widespread MLS_B resistance within the faecal community of intensively raised swine likely negates any potential effects that these antimicrobials might have on altering the faecal community. These findings suggest that if AGP‐mediated alterations to gut communities are an important mechanism for growth promotion, it is unlikely that these would be associated with the colonic community.     

Evaluation of Two Simple Assay Methods for Detecting Antibiotic Residues in Chicken and Pig Muscle

Using 2 paper disc assay procedures, Bacillus cereus and Sarcina lutea were challenged with extracts from chicken and pig muscle paste to which various antibiotics had been added. The first method could detect tetracycline, chlortetracycline, oxytetracycline, penicillin, erythromycin, tylosin and virginiamycin at or <1 μg/g of muscle paste, whilst the second method was necessary to detect the remaining 5 antibiotics at this level. Using the first assay procedure, penicillin, zinc bacitracin and the 3 tetracycline compounds could be detected below FAO/WHO maximum permissible levels for meat for human consumption, whilst the second method was necessary to meet requirements for streptomycin, erythromycin and tylosin. Spiramycin could not be detected by either method at the levels permitted by FAO/WHO. The minimum levels of detection obtained for flavomycin, virginiamycin and monensin were 0.2, 0.02 and 0.5–1.0 μg/g, respectively.     

Residual concentrations of antimicrobial growth promoters in poultry litter favour plasmid conjugation among Escherichia coli

Considering that plasmid conjugation is a major driver for the dissemination of antimicrobial resistance in bacteria, this study aimed to investigate the effects of residual concentrations of antimicrobial growth promoters (AGPs) in poultry litter on the frequencies of IncFII-FIB plasmid conjugation among Escherichia coli organisms. A 2 × 5 factorial trial was performed in vitro, using two types of litter materials (sugarcane bagasse and wood shavings) and five treatments of litter: non-treated (CON), herbal alkaloid sanguinarine (SANG), AGPs monensin (MON), lincomycin (LCM) and virginiamycin (VIR). E. coli H2332 and E. coli J62 were used as donor and recipient strains, respectively. The presence of residues of monensin, lincomycin and virginiamycin increased the frequency of plasmid conjugation among E. coli in both types of litter materials. On the contrary, sanguinarine significantly reduced the frequency of conjugation among E. coli in sugarcane bagasse litter. The conjugation frequencies were significantly higher in wood shavings compared with sugarcane bagasse only in the presence of AGPs. Considering that the presence of AGPs in the litter can increase the conjugation of IncFII-FIB plasmids carrying antimicrobial resistance genes, the real impact of this phenomenon on the dissemination of antimicrobial resistant bacteria in the poultry production chain must be investigated.     

Effects of feed additives on the development on the ileal bacterial community of the broiler chicken

Intensifying concerns about the use of antimicrobials in meat and poultry production has enhanced interest in the application of prebiotics, probiotics and enzymes to enhance growth and prevent disease in food animals. Growth-promoting antibiotics enhance growth of animals by reducing the load of bacteria in the intestine, by reducing colonization by intestinal pathogens or by enhancing the growth and/or metabolism of beneficial bacteria in the intestine. Recently, molecular ecology, utilizing DNA-sequence heterogeneity of the 16S rRNA gene, has revealed a surprising diversity of uncharacterized bacteria inhabiting this ecosystem. We used this approach to determine the effect of growth-promoting antibiotics on the development and composition of the ileal bacterial community. Pairwise comparisons, correspondence analysis and community diversity indices revealed significant differences among the treatments (bacitracin/virginiamycin or monensin) and controls. Antibiotics reduced the diversity of the ileal bacterial community and induced communities rich in Clostridia throughout the life of the broiler chicken. These results indicate that some bacterial species, such as lactobacilli, were suppressed and also suggest that many intestinal Clostridia may be non-pathogenic. Future studies should focus on characterizing the important bacterial species needed to stabilize the intestinal microbiota and identifying those commensals that stimulate and enhance development of intestinal function.     

Characterization of intestinal microbiota and response to dietary virginiamycin supplementation in the broiler chicken

The inclusion of antibiotic growth promoters, such as virginiamycin, at subtherapeutic levels in poultry feeds has a positive effect on health and growth characteristics, possibly due to beneficial effects on the host gastrointestinal microbiota. To improve our understanding of the chicken gastrointestinal microbiota and the effect of virginiamycin on its composition, we characterized the bacteria found in five different gastrointestinal tract locations (duodenal loop, mid-jejunum, proximal ileum, ileocecal junction, and cecum) in 47-day-old chickens that were fed diets excluding or including virginiamycin throughout the production cycle. Ten libraries (five gastrointestinal tract locations from two groups of birds) of approximately 555-bp chaperonin 60 PCR products were prepared, and 10,932 cloned sequences were analyzed. A total of 370 distinct cpn60 sequences were identified, which ranged in frequency of recovery from 1 to 2,872. The small intestinal libraries were dominated by sequences from the Lactobacillales (90% of sequences), while the cecum libraries were more diverse and included members of the Clostridiales (68%), Lactobacillales (25%), and Bacteroidetes (6%). To assess the effects of virginiamycin on the gastrointestinal microbiota, 15 bacterial targets were enumerated using quantitative, real-time PCR. Virginiamycin was associated with increased abundance of many of the targets in the proximal gastrointestinal tract (duodenal loop to proximal ileum), with fewer targets affected in the distal regions (ileocecal junction and cecum). These findings provide improved profiling of the composition of the chicken intestinal microbiota and indicate that microbial responses to virginiamycin are most significant in the proximal small intestine.     

Characterization of Intestinal Microbiota and Response to Dietary Virginiamycin Supplementation in the Broiler Chicken

The inclusion of antibiotic growth promoters, such as virginiamycin, at subtherapeutic levels in poultry feeds has a positive effect on health and growth characteristics, possibly due to beneficial effects on the host gastrointestinal microbiota. To improve our understanding of the chicken gastrointestinal microbiota and the effect of virginiamycin on its composition, we characterized the bacteria found in five different gastrointestinal tract locations (duodenal loop, mid-jejunum, proximal ileum, ileocecal junction, and cecum) in 47-day-old chickens that were fed diets excluding or including virginiamycin throughout the production cycle. Ten libraries (five gastrointestinal tract locations from two groups of birds) of approximately 555-bp chaperonin 60 PCR products were prepared, and 10,932 cloned sequences were analyzed. A total of 370 distinct cpn60 sequences were identified, which ranged in frequency of recovery from 1 to 2,872. The small intestinal libraries were dominated by sequences from the Lactobacillales (90% of sequences), while the cecum libraries were more diverse and included members of the Clostridiales (68%), Lactobacillales (25%), and Bacteroidetes (6%). To assess the effects of virginiamycin on the gastrointestinal microbiota, 15 bacterial targets were enumerated using quantitative, real-time PCR. Virginiamycin was associated with increased abundance of many of the targets in the proximal gastrointestinal tract (duodenal loop to proximal ileum), with fewer targets affected in the distal regions (ileocecal junction and cecum). These findings provide improved profiling of the composition of the chicken intestinal microbiota and indicate that microbial responses to virginiamycin are most significant in the proximal small intestine.     

Comparative metagenomics reveals host specific metavirulomes and horizontal gene transfer elements in the chicken cecum microbiome

Background

The complex microbiome of the ceca of chickens plays an important role in nutrient utilization, growth and well-being of these animals. Since we have a very limited understanding of the capabilities of most species present in the cecum, we investigated the role of the microbiome by comparative analyses of both the microbial community structure and functional gene content using random sample pyrosequencing. The overall goal of this study was to characterize the chicken cecal microbiome using a pathogen-free chicken and one that had been challenged with Campylobacter jejuni.

Methodology/Principal Findings

Comparative metagenomic pyrosequencing was used to generate 55,364,266 bases of random sampled pyrosequence data from two chicken cecal samples. SSU rDNA gene tags and environmental gene tags (EGTs) were identified using SEED subsystems-based annotations. The distribution of phylotypes and EGTs detected within each cecal sample were primarily from the Firmicutes, Bacteroidetes and Proteobacteria, consistent with previous SSU rDNA libraries of the chicken cecum. Carbohydrate metabolism and virulence genes are major components of the EGT content of both of these microbiomes. A comparison of the twelve major pathways in the SEED Virulence Subsystem (metavirulome) represented in the chicken cecum, mouse cecum and human fecal microbiomes showed that the metavirulomes differed between these microbiomes and the metavirulomes clustered by host environment. The chicken cecum microbiomes had the broadest range of EGTs within the SEED Conjugative Transposon Subsystem, however the mouse cecum microbiomes showed a greater abundance of EGTs in this subsystem. Gene assemblies (32 contigs) from one microbiome sample were predominately from the Bacteroidetes, and seven of these showed sequence similarity to transposases, whereas the remaining sequences were most similar to those from catabolic gene families.

Conclusion/Significance

This analysis has demonstrated that mobile DNA elements are a major functional component of cecal microbiomes, thus contributing to horizontal gene transfer and functional microbiome evolution. Moreover, the metavirulomes of these microbiomes appear to associate by host environment. These data have implications for defining core and variable microbiome content in a host species. Furthermore, this suggests that the evolution of host specific metavirulomes is a contributing factor in disease resistance to zoonotic pathogens.     

Metagenomic abundance estimation and diagnostic testing on species level

One goal of sequencing-based metagenomic community analysis is the quantitative taxonomic assessment of microbial community compositions. In particular, relative quantification of taxons is of high relevance for metagenomic diagnostics or microbial community comparison. However, the majority of existing approaches quantify at low resolution (e.g. at phylum level), rely on the existence of special genes (e.g. 16S), or have severe problems discerning species with highly similar genome sequences. Yet, problems as metagenomic diagnostics require accurate quantification on species level. We developed Genome Abundance Similarity Correction (GASiC), a method to estimate true genome abundances via read alignment by considering reference genome similarities in a non-negative LASSO approach. We demonstrate GASiC’s superior performance over existing methods on simulated benchmark data as well as on real data. In addition, we present applications to datasets of both bacterial DNA and viral RNA source. We further discuss our approach as an alternative to PCR-based DNA quantification.     

Understanding microbial community diversity metrics derived from metagenomes: performance evaluation using simulated data sets

Metagenomics holds the promise of greatly advancing the study of diversity in natural communities, but novel theoretical and methodological approaches must first be developed and adjusted for these data sets. We evaluated widely used macroecological metrics of taxonomic diversity on a simulated set of metagenomic samples, using phylogenetically meaningful protein-coding genes as ecological proxies. To our knowledge, this is the first approach of this kind to evaluate taxonomic diversity metrics derived from metagenomic data sets. We demonstrate that abundance matrices derived from protein-coding marker genes reproduce more faithfully the structure of the original community than those derived from SSU-rRNA gene. We also found that the most commonly used diversity metrics are biased estimators of community structure and differ significantly from their corresponding real parameters and that these biases are most likely caused by insufficient sampling and differences in community phylogenetic composition. Our results suggest that the ranking of samples using multidimensional metrics makes a good qualitative alternative for contrasting community structure and that these comparisons can be greatly improved with the incorporation of metrics for both community structure and phylogenetic diversity. These findings will help to achieve a standardized framework for community diversity comparisons derived from metagenomic data sets.     

Cecal microbiome divergence of broiler chickens by sex and body weight

The divergence of gut bacterial community on broiler chickens has been reported as potentially possible keys to enhancing nutrient absorption, immune systems, and increasing poultry health and performance. Thus, we compared cecal bacterial communities and functional predictions by sex and body weight regarding the association between cecal microbiota and chicken growth performance. In this study, a total of 12 male and 12 female 1-day-old broiler chickens were raised for 35 days in 2 separate cages. Chickens were divided into 3 subgroups depending on body weight (low, medium, and high) by each sex. We compared chicken cecal microbiota compositions and its predictive functions by sex and body weight difference. We found that bacterial 16S rRNA genes were classified as 3 major phyla (Bacteroidetes, Firmicutes, and Proteobacteria), accounting for > 98% of the total bacterial community. The profiling of different bacterial taxa and predictive metagenome functions derived from 16S rRNA genes were performed over chicken sex and bodyweight. Male chickens were related to the enrichment of Bacteroides while female chickens were to the enrichment of Clostridium and Shigella. Male chickens with high body weight were associated with the enrichment of Faecalibacterium and Shuttleworthia. Carbohydrate and lipid metabolisms were suggested as candidate functions for weight gain in the males. This suggests that the variation of cecal bacterial communities and their functions by sex and body weight may be associated with the differences in the growth potentials of broiler chickens.     

Effects of two probiotic Lactobacillus strains on jejunal and cecal microbiota of broiler chicken under acute heat stress condition as revealed by molecular analysis of 16S rRNA genes

We examined the effects of probiotic Lactobacillus strains of Lactobacillus agilis JCM 1048 and Lactobacillus salivarius subsp. salicinius JCM 1230 on jejunal and cecal microbiota of broiler chicken under heat stress condition using terminal restriction fragment length polymorphism (T-RFLP) analysis. The jejunal bacterial community was limited to a few bacterial groups, mostly Lactobacillus spp. A relatively abundant and higher prevalence of Lactobacillus spp. were observed in the jejunal and cecal microbiota of the probiotic chickens compared with those of the control chickens under heat stress condition. In general, the probiotic strains did not significantly affect the abundance of L. agilis and L. salivarius in chicken intestine but clearly contributed to increasing their prevalence in the probiotic chickens. The probiotic Lactobacillus strains enriched the diversity of Lactobacillus flora in chicken jejunum and cecum by increasing the abundance and prevalence of Lactobacillus spp. inhabiting the intestine. The richness of Lactobacillus species tended to be similar among the jejunal and cecal microbiota. The bacterial community of cecum was complex and age-dependent. The major components of the cecal microbiota were clostridia and lactobacilli. The Clostridium subcluster XIVa was the most predominant group in chicken cecum. Probiotic Lactobacillus strains restored the microbial balance and maintained the natural stability of indigenous bacterial microbiota following heat stress-induced changes.     

Effect of Antibiotic Growth Promoters and Anticoccidials on Growth of Clostridium perfringens in the Caeca and on Performance of Broiler Chickens

The effects of the growth promoters avoparcin and avilamycin and the ionophore anticoccidials maduramicin, narasin and monensin on the growth of Clostridium perfringens (Cp) in the ceaca and on performance of broiler chickens were tested in 2 experiments. The supplements were fed as single feed additives or in some combinations. No clinical signs or lesions caused by coccidia were observed in any of the studies. All supplements had an antibacterial effect on Cp and improved growth rate significantly. Carcass yield of birds fed growth promoters avilamycin or avoparcin was significantly higher compared with birds fed anticoccidials. These data indicate that, what concerns bird performance, during good hygienic conditions supplementation with antibiotic growth promoters may not be necessary when the diet is supplemented with an anticoccidial with antibacterial effects.     

Comparative metagenomic,phylogenetic and physiological analyses of soil microbial communities across nitrogen gradients

Terrestrial ecosystems are receiving elevated inputs of nitrogen (N) from anthropogenic sources and understanding how these increases in N availability affect soil microbial communities is critical for predicting the associated effects on belowground ecosystems. We used a suite of approaches to analyze the structure and functional characteristics of soil microbial communities from replicated plots in two long-term N fertilization experiments located in contrasting systems. Pyrosequencing-based analyses of 16S rRNA genes revealed no significant effects of N fertilization on bacterial diversity, but significant effects on community composition at both sites; copiotrophic taxa (including members of the Proteobacteria and Bacteroidetes phyla) typically increased in relative abundance in the high N plots, with oligotrophic taxa (mainly Acidobacteria) exhibiting the opposite pattern. Consistent with the phylogenetic shifts under N fertilization, shotgun metagenomic sequencing revealed increases in the relative abundances of genes associated with DNA/RNA replication, electron transport and protein metabolism, increases that could be resolved even with the shallow shotgun metagenomic sequencing conducted here (average of 75 000 reads per sample). We also observed shifts in the catabolic capabilities of the communities across the N gradients that were significantly correlated with the phylogenetic and metagenomic responses, indicating possible linkages between the structure and functioning of soil microbial communities. Overall, our results suggest that N fertilization may, directly or indirectly, induce a shift in the predominant microbial life-history strategies, favoring a more active, copiotrophic microbial community, a pattern that parallels the often observed replacement of K-selected with r-selected plant species with elevated N.     

Sensitivity and Resistance to Growth Promoting Agents in Animal Lactobacilli

The minimal inhibitory concentrations of 11 growth promoting agents were determined by agar-dilution method against 113 strains of lactobacilli isolated from caeca of pigs, cattle and poultry. Only Lactobacillus acidophilus strains were susceptible to avoparcin and all strains were resistant to copper sulphate. Resistance was noted in cattle and poultry strains against bacitracin, in pig strains against virginiamycin, and in pig and poultry strains against nitrovin. Resistance against carbadox, flavomycin, lincomycin, oleandomycin, spiramycin and tylosin was noted in strains from all three host species.     

Screening of promoters from metagenomic DNA and their use for the construction of expression vectors

This study was focused on the screening of valuable genetic resources, such as promoters from metagenome, and describes a promoter trapping system with a bidirectional probe concept, which can select promoters or operons from various biological resources including metagenomic DNA. A pair of reporters, GFP and DsRed, facing the opposite direction without promoters, is an effective system that can function regardless of the direction of inserted promoters. The feasibility of this system was tested for the isolation of constitutively expressed promoters in E. coli from a soil metagenome, resulting in a potential pool of various promoters for practical application. The analyses of structural organization of the trapped genes demonstrated that constitutively expressible promoters in E. coli were broadly distributed within the metagenome, and suggested that some promoters were useful for the construction of expression vectors. Based on these observations, three constitutive promoters were employed in the expression vector system and their potentials for practical application were evaluated in terms of expression level, protein solubility, and effects on host growth.     

Metagenomic analysis reveals a functional signature for biomass degradation by cecal microbiota in the leaf-eating flying squirrel (Petaurista alborufus lena)

ABSTRACT: BACKGROUND: Animals co-evolve with their gut microbiota; the latter can perform complex metabolic reactions that cannot be done independently by the host. Although the importance of gut microbiota has been well demonstrated, there is a paucity of research regarding its role in foliage-foraging mammals with a specialized digestive system. RESULTS: In this study, a 16S rRNA gene survey and metagenomic sequencing were used to characterize genetic diversity and functional capability of cecal microbiota of the folivorous flying squirrel (Petaurista alborufus lena). Phylogenetic compositions of the cecal microbiota derived from 3 flying squirrels were dominated by Firmicutes. Based on end-sequences of fosmid clones from 1 flying squirrel, we inferred that microbial metabolism greatly contributed to intestinal functions, including degradation of carbohydrates, metabolism of proteins, and synthesis of vitamins. Moreover, 33 polysaccharide-degrading enzymes and 2 large genomic fragments containing a series of carbohydrate-associated genes were identified. CONCLUSIONS: Cecal microbiota of the leaf-eating flying squirrel have great metabolic potential for converting diverse plant materials into absorbable nutrients. The present study should serve as the basis for future investigations, using metagenomic approaches to elucidate the intricate mechanisms and interactions between host and gut microbiota of the flying squirrel digestive system, as well as other mammals with similar adaptations.     

Bacterial diversity of metagenomic and PCR libraries from the Delaware River

To determine whether metagenomic libraries sample adequately the dominant bacteria in aquatic environments, we examined the phylogenetic make-up of a large insert metagenomic library constructed with bacterial DNA from the Delaware River, a polymerase chain reaction (PCR) library of 16S rRNA genes, and community structure determined by fluorescence in situ hybridization (FISH). The composition of the libraries and community structure determined by FISH differed for the major bacterial groups in the river, which included Actinobacteria, beta-proteobacteria and Cytophaga-like bacteria. Beta-proteobacteria were underrepresented in the metagenomic library compared with the PCR library and FISH, while Cytophaga-like bacteria were more abundant in the metagenomic library than in the PCR library and in the actual community according to FISH. The Delaware River libraries contained bacteria belonging to several widespread freshwater clusters, including clusters of Polynucleobacter necessarius, Rhodoferax sp. Bal47 and LD28 beta-proteobacteria, the ACK-m1 and STA2-30 clusters of Actinobacteria, and the PRD01a001B Cytophaga-like bacteria cluster. Coverage of bacteria with > 97% sequence identity was 65% and 50% for the metagenomic and PCR libraries respectively. Rarefaction analysis of replicate PCR libraries and of a library constructed with re-conditioned amplicons indicated that heteroduplex formation did not substantially impact the composition of the PCR library. This study suggests that although it may miss some bacterial groups, the metagenomic approach can sample other groups (e.g. Cytophaga-like bacteria) that are potentially underrepresented by other culture-independent approaches.     

Virulence-associated and antibiotic resistance genes of microbial populations in cattle feces analyzed using a metagenomic approach

The bovine fecal microbiota impacts human food safety as well as animal health. Although the bacteria of cattle feces have been well characterized using culture-based and culture-independent methods, techniques have been lacking to correlate total community composition with community function. We used high throughput sequencing of total DNA extracted from fecal material to characterize general community composition and examine the repertoire of microbial genes present in beef cattle feces, including genes associated with antibiotic resistance and bacterial virulence. Results suggest that traditional 16S sequencing using “universal” primers to generate full-length sequence may under represent Acitinobacteria and Proteobacteria. Over eight percent (8.4%) of the sequences from our beef cattle fecal pool sample could be categorized as virulence genes, including a suite of genes associated with resistance to antibiotic and toxic compounds (RATC). This is a higher proportion of virulence genes found in Sargasso sea, chicken cecum, and cow rumen samples, but comparable to the proportion found in Antarctic marine derived lake, human fecal, and farm soil samples. The quantitative nature of metagenomic data, combined with the large number of RATC classes represented in samples from widely different habitats indicates that metagenomic data can be used to track relative amounts of antibiotic resistance genes in individual animals over time. Consequently, these data can be used to generate sample-specific and temporal antibiotic resistance gene profiles to facilitate an understanding of the ecology of the microbial communities in each habitat as well as the epidemiology of antibiotic resistant gene transport between and among habitats.     

Acquired Resistance to Macrolide–Lincosamide–Streptogramin Antibiotics in Lactic Acid Bacteria of Food Origin

Antibiotic resistance is a growing problem in clinical settings as well as in food industry. Lactic acid bacteria (LAB) commercially used as starter cultures and probiotic supplements are considered as reservoirs of several antibiotic resistance genes. Macrolide–lincosamide–streptogramin (MLS) antibiotics have a proven record of excellence in clinical settings. However, the intensive use of tylosin, lincomysin and virginamycin antibiotics of this group as growth promoters in animal husbandry and poultry has resulted in development of resistance in LAB of animal origin. Among the three different mechanisms of MLS resistance, the most commonly observed in LAB are the methylase and efflux mediated resistance. This review summarizes the updated information on MLS resistance genes detected and how resistance to these antibiotics poses a threat when present in food grade LAB.     

Metagenomic microbial community profiling using unique clade-specific marker genes

Metagenomic shotgun sequencing data can identify microbes populating a microbial community and their proportions, but existing taxonomic profiling methods are inefficient for increasingly large data sets. We present an approach that uses clade-specific marker genes to unambiguously assign reads to microbial clades more accurately and >50× faster than current approaches. We validated our metagenomic phylogenetic analysis tool, MetaPhlAn, on terabases of short reads and provide the largest metagenomic profiling to date of the human gut. It can be accessed at http://huttenhower.sph.harvard.edu/metaphlan/.