In Vivo Expression of Chicken Gut Anaerobes Identifies Carbohydrate- or Amino Acid-Utilising, Motile or Type VI Secretion System-Expressing Bacteria.

Complex gut microbiota increases chickens' resistance to enteric pathogens. However, the principles of this phenomenon are not understood in detail. One of the possibilities for how to decipher the role of gut microbiota in chickens' resistance to enteric pathogens is to systematically characterise the gene expression of individual gut microbiota members colonising the chicken caecum. To reach this aim, newly hatched chicks were inoculated with bacterial species whose whole genomic sequence was known. Total protein purified from the chicken caecum was analysed by mass spectrometry, and the obtained spectra were searched against strain-specific protein databases generated from known genomic sequences. Campylobacter jejuni, Phascolarctobacterium sp. and Sutterella massiliensis did not utilise carbohydrates when colonising the chicken caecum. On the other hand, Bacteroides, Mediterranea, Marseilla, Megamonas, Megasphaera, Bifidobacterium, Blautia, Escherichia coli and Succinatimonas fermented carbohydrates. C. jejuni was the only motile bacterium, and Bacteroides mediterraneensis expressed the type VI secretion system. Classification of in vivo expression is key for understanding the role of individual species in complex microbial populations colonising the intestinal tract. Knowledge of the expression of motility, the type VI secretion system, and preference for carbohydrate or amino acid fermentation is important for the selection of bacteria for defined competitive exclusion products.

Contact with adult hens affects the composition of skin and respiratory tract microbiota in newly hatched chicks.

Chickens in commercial production are hatched in hatcheries without any contact with their parents and colonization of their skin and respiratory tract is therefore dependent on environmental sources only. However, since chickens evolved to be hatched in nests, in this study we evaluated the importance of contact between hens and chicks for the development of chicken skin and tracheal microbiota. Sequencing of PCR amplified V3/V4 variable regions of the 16S rRNA gene showed that contact with adult hens decreased the abundance of E. coli, Proteus mirabilis and Clostridium perfringens both in skin and the trachea, and Acinetobacter johnsonii and Cutibacterium acnes in skin microbiota only. These species were replaced by Lactobacillus gallinarum, Lactobacillus aviarius, Limosilactobacillus reuteri, and Streptococcus pasterianus in the skin and tracheal microbiota of contact chicks. Lactobacilli can be therefore investigated for their probiotic effect in respiratory tract in the future. Skin and respiratory microbiota of contact chickens was also enriched for Phascolarctobacterium, Succinatimonas, Flavonifractor, Blautia, and [Ruminococcus] torque though, since these are strict anaerobes from the intestinal tract, it is likely that only DNA from nonviable cells was detected for these taxa.

Colonization of chickens with competitive exclusion products results in extensive differences in metabolite composition in cecal digesta.

The concept of competitive exclusion is well established in poultry and different products are used to suppress the multiplication of enteric pathogens in the chicken intestinal tract. While the effect has been repeatedly confirmed, the specific principles of competitive exclusion are less clear. The aim of the study was to compare metabolites in the cecal digesta of differently colonized chickens. Metabolites in the cecal contents of chickens treated with a commercial competitive exclusion product or with an experimental product consisting of 23 gut anaerobes or in control untreated chickens were determined by mass spectrometry. Extensive differences in metabolite composition among the digesta of all 3 groups of chickens were recorded. Out of 1,706 detected compounds, 495 and 279 were differently abundant in the chicks treated with a commercial or experimental competitive exclusion product in comparison to the control group, respectively. Soyasaponins, betaine, carnitine, glutamate, tyramine, phenylacetaldehyde, or 3-methyladenine were more abundant in the digesta of control chicks while 4-oxododecanedioic acid, nucleotides, dipeptides, amino acids (except for glutamate), and vitamins were enriched in the digesta of chickens colonized by competitive exclusion products. Metabolites enriched in the digesta of control chicks can be classified as of plant feed origin released in the digesta by degradative activities of the chicken. Some of these molecules disappeared from the digesta of chicks colonized by complex microbiota due to them being metabolized. Instead, nucleotides, amino acids, and vitamins increased in the digesta of colonized chicks as a consequence of the additional digestive potential brought to the cecum by microbiota from competitive exclusion products. It is therefore possible to affect metabolite profiles in the chicken cecum by its colonization with selected bacterial species.

Succession, Replacement, and Modification of Chicken Litter Microbiota.

Chickens are in constant interaction with their environment, e.g., bedding and litter, and their microbiota. However, how litter microbiota develops over time and whether bedding and litter microbiota may affect the cecal microbiota is not clear. We addressed these questions using sequencing of V3/V4 variable region of 16S rRNA genes of cecal, bedding, and litter samples from broiler breeder chicken flocks for 4 months of production. Cecal, bedding, and litter samples were populated by microbiota of distinct composition. The microbiota in the bedding material did not expand in the litter. Similarly, major species from litter microbiota did not expand in the cecum. Only cecal microbiota was found in the litter forming approximately 20% of total litter microbiota. A time-dependent development of litter microbiota was observed. Escherichia coli, Staphylococcus saprophyticus, and Weissella jogaejeotgali were characteristic of fresh litter during the first month of production. Corynebacterium casei, Lactobacillus gasseri, and Lactobacillus salivarius dominated in a 2-month-old litter, Brevibacterium, Brachybacterium, and Sphingobacterium were characteristic for 3-month-old litter, and Salinococcus, Dietzia, Yaniella, and Staphylococcus lentus were common in a 4-month-old litter. Although the development was likely determined by physicochemical conditions in the litter, it might be interesting to test some of these species for active modification of litter to improve the chicken environment and welfare. IMPORTANCE Despite intimate contact, the composition of bedding, litter, and cecal microbiota differs considerably. Species characteristic for litter microbiota at different time points of chicken production were identified thus opening the possibility for active manipulation of litter microbiota.

Host Species Adaptation of Obligate Gut Anaerobes Is Dependent on Their Environmental Survival.

The gut microbiota of warm-blooded vertebrates consists of bacterial species belonging to two main phyla; Firmicutes and Bacteroidetes. However, does it mean that the same bacterial species are found in humans and chickens? Here we show that the ability to survive in an aerobic environment is central for host species adaptation. Known bacterial species commonly found in humans, pigs, chickens and Antarctic gentoo penguins are those capable of extended survival under aerobic conditions, i.e., either spore-forming, aerotolerant or facultatively anaerobic bacteria. Such bacteria are ubiquitously distributed in the environment, which acts as the source of infection with similar probability in humans, pigs, chickens, penguins and likely any other warm-blooded omnivorous hosts. On the other hand, gut anaerobes with no specific adaptation for survival in an aerobic environment exhibit host adaptation. This is associated with their vertical transmission from mothers to offspring and long-term colonisation after administration of a single dose. This knowledge influences the design of next-generation probiotics. The origin of aerotolerant or spore-forming probiotic strains may not be that important. On the other hand, if Bacteroidetes and other host-adapted species are used as future probiotics, host preference should be considered.

Probiotic Lactobacilli Do Not Protect Chickens against Salmonella Enteritidis Infection by Competitive Exclusion in the Intestinal Tract but in Feed, Outside the Chicken Host.

Lactobacilli are commonly used as probiotics in poultry to improve production parameters and to increase chicken resistance to enteric infections. However, lactobacilli do not efficiently colonise the chicken intestinal tract, and also, their anti-infection effect in vivo is sometimes questionable. In this study, we therefore evaluated the potential of a mixture of four Lactobacillus species (L. salivarius, L. reuteri, L. ingluviei and L. alvi) for the protection of chickens against Salmonella Enteritidis infection. Whenever the chickens were inoculated by lactobacilli and S. Enteritidis separately, there was no protective effect of lactobacilli. This means that when lactobacilli and S. Enteritidis are exposed to each other as late as in the crop of chickens, lactobacilli did not influence chicken resistance to S. Enteritidis at all. The only positive effect was recorded when the mixture of lactobacilli and S. Enteritidis was used for the inoculation of feed and the feed was anaerobically fermented for 1 to 5 days. In this case, chickens fed such a diet remained S. Enteritidis negative. In vitro experiments showed that the protective effect was caused by acidification of feed down to pH 4.6 due to lactobacilli fermentation and was associated with S. Enteritidis inactivation. The probiotic effect of lactobacilli was thus expressed in the feed, outside the chicken host.

Eggshell and Feed Microbiota Do Not Represent Major Sources of Gut Anaerobes for Chickens in Commercial Production.

In this study, we addressed the origin of chicken gut microbiota in commercial production by a comparison of eggshell and feed microbiota with caecal microbiota of 7-day-old chickens, using microbiota analysis by 16S rRNA sequencing. In addition, we tested at which timepoint during prenatal or neonatal development it is possible to successfully administer probiotics. We found that eggshell microbiota was a combination of environmental and adult hen gut microbiota but was completely different from caecal microbiota of 7-day-old chicks. Similarly, we observed that the composition of feed microbiota was different from caecal microbiota. Neither eggshell nor feed acted as an important source of gut microbiota for the chickens in commercial production. Following the experimental administration of potential probiotics, we found that chickens can be colonised only when already hatched and active. Spraying of eggs with gut anaerobes during egg incubation or hatching itself did not result in effective chicken colonisation. Such conclusions should be considered when selecting and administering probiotics to chickens in hatcheries. Eggshells, feed or drinking water do not act as major sources of gut microbiota. Newly hatched chickens must be colonised from additional sources, such as air dust with spores of Clostridiales. The natural colonisation starts only when chickens are already hatched, as spraying of eggs or even chickens at the very beginning of the hatching process did not result in efficient colonisation.

Different Bacteroides Species Colonise Human and Chicken Intestinal Tract.

Bacteroidaceae are common gut microbiota members in all warm-blooded animals. However, if Bacteroidaceae are to be used as probiotics, the species selected for different hosts should reflect the natural distribution. In this study, we therefore evaluated host adaptation of bacterial species belonging to the family Bacteroidaceae. B. dorei, B. uniformis, B. xylanisolvens, B. ovatus, B. clarus, B. thetaiotaomicron and B. vulgatus represented human-adapted species while B. gallinaceum, B. caecigallinarum, B. mediterraneensis, B. caecicola, M. massiliensis, B. plebeius and B. coprocola were commonly detected in chicken but not human gut microbiota. There were 29 genes which were present in all human-adapted Bacteroides but absent from the genomes of all chicken isolates, and these included genes required for the pentose cycle and glutamate or histidine metabolism. These genes were expressed during an in vitro competitive assay, in which human-adapted Bacteroides species overgrew the chicken-adapted isolates. Not a single gene specific for the chicken-adapted species was found. Instead, chicken-adapted species exhibited signs of frequent horizontal gene transfer, of KUP, linA and sugE genes in particular. The differences in host adaptation should be considered when the new generation of probiotics for humans or chickens is designed.

Environmental Impact on Differential Composition of Gut Microbiota in Indoor Chickens in Commercial Production and Outdoor, Backyard Chickens.

In this study, we compared the caecal microbiota composition of egg-laying hens from commercial production that are kept indoors throughout their whole life with microbiota of hens kept outdoors. The microbiota of outdoor hens consisted of lower numbers of bacterial species than the microbiota of indoor hens. At the phylum level, microbiota of outdoor hens was enriched for Bacteroidetes (62.41 ± 4.47% of total microbiota in outdoor hens and 52.01 ± 6.27% in indoor hens) and Proteobacteria (9.33 ± 4.99% in outdoor and 5.47 ± 2.24% in indoor hens). On the other hand, Firmicutes were more abundant in the microbiota of indoor hens (33.28 ± 5.11% in indoor and 20.66 ± 4.41% in outdoor hens). Horizontally transferrable antibiotic resistance genes tetO, tet(32), tet(44), and tetW were also less abundant in the microbiota of outdoor hens than indoor hens. A comparison of the microbiota composition at the genus and species levels pointed toward isolates specifically adapted to the two extreme environments. However, genera and species recorded as being similarly abundant in the microbiota of indoor and outdoor hens are equally as noteworthy because these represent microbiota members that are highly adapted to chickens, irrespective of their genetics, feed composition, and living environment.

Gut Anaerobes Capable of Chicken Caecum Colonisation.

Chicks in commercial production are highly sensitive to enteric infections and their resistance can be increased by administration of complex adult microbiota. However, it is not known which adult microbiota members are capable of colonising the caecum of newly hatched chicks. In this study, we therefore orally inoculated chicks with pure cultures of 76 different bacterial isolates originating from chicken caecum on day 1 of life and determined their ability to colonise seven days later. The caecum of newly hatched chickens could be colonised by bacteria belonging to phyla Bacteroidetes, Proteobacteria, Synergistetes, or Verrucomicrobia, and isolates from class Negativicutes (phylum Firmicutes). On the other hand, we did not record colonisation with isolates from phyla Actinobacteria and Firmicutes (except for Negativicutes), including isolates from families Lachnospiraceae, Ruminococcaceae, Erysipelotrichaceae, and Lactobacillaceae. Representatives of genera commonly used in probiotics such as Lactobacillus, Enterococcus, or Bacillus therefore did not colonise the chicken intestinal tract after a single dose administration. Following challenge with Salmonella enterica serovar Enteritidis, the best protecting isolates increased the chicken's resistance to S. Enteritidis only tenfold, which, however, means that none of the tested individual bacterial isolates on their own efficiently protected chicks against S. Enteritidis.

Contact with adult hen affects development of caecal microbiota in newly hatched chicks.

Chickens in commercial production are hatched in a clean hatchery environment in the absence of any contact with adult hens. However, Gallus gallus evolved to be hatched in a nest in contact with an adult hen which may act as a donor of gut microbiota. In this study, we therefore addressed the issue of microbiota development in newly hatched chickens with or without contact with an adult hen. We found that a mere 24-hour-long contact between a hen and newly hatched chickens was long enough for transfer of hen gut microbiota to chickens. Hens were efficient donors of Bacteroidetes and Actinobacteria. However, except for genus Faecalibacterium and bacterial species belonging to class Negativicutes, hens did not act as an important source of Gram-positive Firmicutes. Though common to the chicken intestinal tract, Lactobacilli and isolates from families Erysipelotrichaceae, Lachnospiraceae and Ruminococcaceae therefore originated from environmental sources instead of from the hens. These observation may have considerable consequences for the evidence-based design of the new generation of probiotics for poultry.

Protein expression in the liver and blood serum in chickens in response to Salmonella Enteritidis infection.

Events occurring in the chicken caecum following Salmonella Enteritidis infection are relatively well-described. However, mechanisms of the immune response and defence beyond the intestinal tract are less well-described. In this study, we therefore determined changes in protein abundance in the liver and blood serum in response to S. Enteritidis infection using the unbiased approach of shotgun proteomics. Complement and coagulation cascades, TNF signalling, antigen processing and presentation was activated in the liver following infection with S. Enteritidis. Chicken proteins that decreased in the liver were involved in glycolysis, the citrate cycle, oxidative phosphorylation and fatty acid metabolism. No functional category was significantly activated or suppressed in the serum. Concerning individual proteins, VNN1, SAA, AVD, SERPINA3, SERPINB10, AGT, MRP126 or CP increased in abundance both in the liver and serum. MT4, MT3, PTGDS, GLRX and TGM4, though highly inducible in the liver, did not increase in the serum. PIGR, SERPINF2 and IGJ increased in the serum but not in the liver. SERPINA4, apoAIV, CLEC3B, SERPINF1, HRG, AHSG and ALB decreased both in the liver and serum. Avidin-like LOC431660, THRSP, GATM, GGACT, ACOX1, ALDOB or FABP7 decreased in the liver but not in the serum. Finally, CKM, CKB, PLTP, COMP, IGFALS, AMY1A or SERPIND1 decreased in the serum after S. Enteritidis infection but not in the liver. Differently abundant proteins characterise the chicken's response to infection and can be also used as markers of chicken health status.

Differential protein expression in chicken macrophages and heterophils in vivo following infection with Salmonella Enteritidis.

In this study we compared the proteomes of macrophages and heterophils isolated from the spleen 4 days after intravenous infection of chickens with Salmonella Enteritidis. Heterophils were characterized by expression of MMP9, MRP126, LECT2, CATHL1, CATHL2, CATHL3, LYG2, LYZ and RSFR. Macrophages specifically expressed receptor proteins, e.g. MRC1L, LRP1, LGALS1, LRPAP1 and a DMBT1L. Following infection, heterophils decreased ALB and FN1, and released MMP9 to enable their translocation to the site of infection. In addition, the endoplasmic reticulum proteins increased in heterophils which resulted in the release of granular proteins. Since transcription of genes encoding granular proteins did not decrease, these genes remained continuously transcribed and translated even after initial degranulation. Macrophages increased amounts of fatty acid elongation pathway proteins, lysosomal and phagosomal proteins. Macrophages were less responsive to acute infection than heterophils and an increase in proteins like CATHL1, CATHL2, RSFR, LECT2 and GAL1 in the absence of any change in their expression at RNA level could even be explained by capturing these proteins from the external environment into which these could have been released by heterophils.

Different roles of CD4, CD8 and γδ T-lymphocytes in naive and vaccinated chickens during Salmonella Enteritidis infection.

Lymphocytes represent the key antigen-specific leukocyte subpopulation. Despite their importance in mounting an immune response, an unbiased description of proteins expressed by chicken lymphocytes has not been presented. In this study, we therefore intravenously infected chickens with Salmonella Enteritidis, sorted CD4, CD8 and γδ T-lymphocytes from the spleen by flow cytometry and determined the proteome of each population by LC-MS/MS. CD4 T-lymphocyte characteristic proteins included ubiquitin SUMO-like domain and BAR domain containing proteins. CD8 T-lymphocyte specific proteins were characterized by purine ribonucleoside triphosphate binding and were involved in cell differentiation, cell activation and regulation of programmed cell death. γδ T-lymphocyte specific proteins exhibited enrichment of small GTPase of Rab type and GTP binding. Following infection, inducible proteins in CD4 lymphocytes included ribosomal proteins and downregulated proteins localized to the lysosome. CD8 T-lymphocytes induced MCM complex proteins, proteins required for DNA replication and machinery for protein processing in the endoplasmic reticulum. Proteins inducible in γδ T-lymphocytes belonged to immune system response, oxidative phosphorylation and the spliceosome. In this study, we predicted the likely events in lymphocyte response to systemic bacterial infection and identified proteins which can be used as markers specific for each lymphocyte subpopulation.

Immune protection of chickens conferred by a vaccine consisting of attenuated strains of Salmonella Enteritidis, Typhimurium and Infantis.

The colonization of poultry with different Salmonella enterica serovars poses an issue throughout the world. In this study we therefore tested the efficacy of a vaccine consisting of attenuated strains of Salmonella enterica serovars Enteritidis, Typhimurium and Infantis against challenge with the same serovars and with S. Agona, Dublin and Hadar. We tested oral and aerosol administration of the vaccine, with or without co-administration of cecal microbiota from adult hens. The protective effect was determined by bacterial counts of the challenge strains up to week 18 of life and by characterizing the immune response using real-time PCR specific for 16 different genes. We have shown that a vaccine consisting of attenuated S. Enteritidis, S. Typhimurium and S. Infantis protected chickens against challenge with the wild type strains of the same serovars and partially protected chickens also against challenge with isolates belonging to serovars Dublin or Hadar. Aerosol vaccination was more effective at inducing systemic immunity whilst oral vaccination stimulated a local immune response in the gut. Co-administration of cecal microbiota increased the protectiveness in the intestinal tract but slightly decreased the systemic immune response. Adjusting the vaccine composition and changing the administration route therefore affects vaccine efficacy.

Transient and Prolonged Response of Chicken Cecum Mucosa to Colonization with Different Gut Microbiota.

In this study we determined protein and gene expression in the caeca of newly hatched chickens inoculated with cecal contents sourced from hens of different ages. Over 250 proteins exhibited modified expression levels in response to microbiota inoculation. The most significant inductions were observed for ISG12-2, OASL, ES1, LYG2, DMBT1-L, CDD, ANGPTL6, B2M, CUZD1, IgM and Ig lambda chain. Of these, ISG12-2, ES1 and both immunoglobulins were expressed at lower levels in germ-free chickens compared to conventional chickens. In contrast, CELA2A, BRT-2, ALDH1A1, ADH1C, AKR1B1L, HEXB, ALDH2, ALDOB, CALB1 and TTR were expressed at lower levels following inoculation of microbiota. When chicks were given microbiota preparations from different age donors, the recipients mounted differential responses to the inoculation which also differed from the response profile in naturally colonised birds. For example, B2M, CUZD1 and CELA2A responded differently to the inoculation with microbiota of 4- or 40-week-old hens. The increased or decreased gene expression could be recorded 6 weeks after the inoculation of newly hatched chickens. To characterise the proteins that may directly interact with the microbiota we characterised chicken proteins that co-purified with the microbiota and identified a range of host proteins including CDD, ANGPTL6, DMBT1-L, MEP1A and Ig lambda. We propose that induction of ISG12-2 results in reduced apoptosis of host cells exposed to the colonizing commensal microbiota and that CDD, ANGPTL6, DMBT1-L, MEP1A and Ig lambda reduce contact of luminal microbiota with the gut epithelium thereby reducing the inflammatory response.

Composition of Gut Microbiota Influences Resistance of Newly Hatched Chickens to Salmonella Enteritidis Infection.

Since poultry is a very common source of non-typhoid Salmonella for humans, different interventions aimed at decreasing the prevalence of Salmonella in chickens are understood as an effective measure for decreasing the incidence of human salmonellosis. One such intervention is the use of probiotic or competitive exclusion products. In this study we tested whether microbiota from donor hens of different age will equally protect chickens against Salmonella Enteritidis infection. Newly hatched chickens were therefore orally inoculated with cecal extracts from 1-, 3-, 16-, 28-, and 42-week-old donors and 7 days later, the chickens were infected with S. Enteritidis. The experiment was terminated 4 days later. In the second experiment, groups of newly hatched chickens were inoculated with cecal extracts of 35-week-old hens either on day 1 of life followed by S. Enteritidis infection on day 2 or were infected with S. Enteritidis infection on day 1 followed by therapeutic administration of the cecal extract on day 2 or were inoculated on day 1 of life with a mixture of the cecal extract and S. Enteritidis. This experiment was terminated when the chickens were 5 days old. Both Salmonella culture and chicken gene expression confirmed that inoculation of newly hatched chickens with microbiota from 3-week-old or older chickens protected them against S. Enteritidis challenge. On the other hand, microbiota from 1-week-old donors failed to protect chickens against S. Enteritidis challenge. Microbiota from 35-week-old hens protected chickens even 24 h after administration. However, simultaneous or therapeutic microbiota administration failed to protect chickens against S. Enteritidis infection. Gut microbiota can be used as a preventive measure against S. Enteritidis infection but its composition and early administration is critical for its efficacy.

Important Metabolic Pathways and Biological Processes Expressed by Chicken Cecal Microbiota.

The gut microbiota plays important roles in its host. However, how each microbiota member contributes to the behavior of the whole population is not known. In this study, we therefore determined protein expression in the cecal microbiota in chickens of selected ages and in 7-day-old chickens inoculated with different cecal extracts on the day of hatching. Campylobacter, Helicobacter, Mucispirillum, and Megamonas overgrew in the ceca of 7-day-old chickens inoculated with cecal extracts from donor hens. Firmicutes were characterized by ABC and phosphotransferase system (PTS) transporters, extensive acyl coenzyme A (acyl-CoA) metabolism, and expression of l-fucose isomerase. Anaerostipes, Anaerotruncus, Pseudoflavonifractor, Dorea, Blautia, and Subdoligranulum expressed spore proteins. Firmicutes (Faecalibacterium, Butyrivibrio, Megasphaera, Subdoligranulum, Oscillibacter, Anaerostipes, and Anaerotruncus) expressed enzymes required for butyrate production. Megamonas, Phascolarctobacterium, and Blautia (exceptions from the phylum Firmicutes) and all Bacteroidetes expressed enzymes for propionate production pathways. Representatives of Bacteroidetes also expressed xylose isomerase, enzymes required for polysaccharide degradation, and ExbBD, TonB, and outer membrane receptors likely to be involved in oligosaccharide transport. Based on our data, Anaerostipes, Anaerotruncus, and Subdoligranulum might be optimal probiotic strains, since these represent spore-forming butyrate producers. However, certain care should be taken during microbiota transplantation because the microbiota may behave differently in the intestinal tract of a recipient depending on how well the existing communities are established.

Characterization of Antibiotic Resistance Gene Abundance and Microbiota Composition in Feces of Organic and Conventional Pigs from Four EU Countries.

One of the recent trends in animal production is the revival of interest in organic farming. The increased consumer interest in organic animal farming is mainly due to concerns about animal welfare and the use of antibiotics in conventional farming. On the other hand, providing animals with a more natural lifestyle implies their increased exposure to environmental sources of different microorganisms including pathogens. To address these concerns, we determined the abundance of antibiotic resistance and diversity within fecal microbiota in pigs kept under conventional and organic farming systems in Sweden, Denmark, France and Italy. The abundance of sul1, sul2, strA, tet(A), tet(B) and cat antibiotic resistance genes was determined in 468 samples by real-time PCR and the fecal microbiota diversity was characterized in 48 selected samples by pyrosequencing of V3/V4 regions of 16S rRNA. Contrary to our expectations, there were no extensive differences between the abundance of tested antibiotic resistance genes in microbiota originating from organic or conventionally housed pigs within individual countries. There were also no differences in the microbiota composition of organic and conventional pigs. The only significant difference was the difference in the abundance of antibiotic resistance genes in the samples from different countries. Fecal microbiota in the samples originating from southern European countries (Italy, France) exhibited significantly higher antibiotic resistance gene abundance than those from northern parts of Europe (Denmark, Sweden). Therefore, the geographical location of the herd influenced the antibiotic resistance in the fecal microbiota more than farm's status as organic or conventional.

Succession and replacement of bacterial populations in the caecum of egg laying hens over their whole life.

In this study we characterised the development of caecal microbiota in egg laying hens over their commercial production lifespan, from the day of hatching until 60 weeks of age. Using pyrosequencing of V3/V4 variable regions of 16S rRNA genes for microbiota characterisation, we were able to define 4 different stages of caecal microbiota development. The first stage lasted for the first week of life and was characterised by a high prevalence of Enterobacteriaceae (phylum Proteobacteria). The second stage lasted from week 2 to week 4 and was characterised by nearly an absolute dominance of Lachnospiraceae and Ruminococcaceae (both phylum Firmicutes). The third stage lasted from month 2 to month 6 and was characterised by the succession of Firmicutes at the expense of Bacteroidetes. The fourth stage was typical for adult hens in full egg production aged 7 months or more and was characterised by a constant ratio of Bacteroidetes and Firmicutes formed by equal numbers of the representatives of both phyla.