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.

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.

Detoxification, Hydrogen Sulphide Metabolism and Wound Healing Are the Main Functions That Differentiate Caecum Protein Expression from Ileum of Week-Old Chicken.

Sections of chicken gut differ in many aspects, e.g., the passage of digesta (continuous vs. discontinuous), the concentration of oxygen, and the density of colonising microbiota. Using an unbiased LC-MS/MS protocol, we compared protein expression in 18 ileal and 57 caecal tissue samples that originated from 7-day old ISA brown chickens. We found that proteins specific to the ileum were either structural (e.g., 3 actin isoforms, villin, or myosin 1A), or those required for nutrient digestion (e.g., sucrose isomaltase, maltase-glucoamylase, peptidase D) and absorption (e.g., fatty acid-binding protein 2 and 6 or bile acid-CoA:amino acid N-acyltransferase). On the other hand, proteins characteristic of the caecum were involved in sensing and limiting the consequences of oxidative stress (e.g., thioredoxin, peroxiredoxin 6), cell adhesion, and motility associated with wound healing (e.g., fibronectin 1, desmoyokin). These mechanisms are coupled with the activation of mechanisms suppressing the inflammatory response (galectin 1). Rather prominent were also expressions of proteins linked to hydrogen sulphide metabolism in caecum represented by cystathionin beta synthase, selenium-binding protein 1, mercaptopyruvate sulphurtransferase, and thiosulphate sulphurtransferase. Higher mRNA expression of nuclear factor, erythroid 2-like 2, the main oxidative stress transcriptional factor in caecum, further supported our observations.

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.

Ecological Adaptations of Gut Microbiota Members and Their Consequences for Use as a New Generation of Probiotics.

In this review, we link ecological adaptations of different gut microbiota members with their potential for use as a new generation of probiotics. Gut microbiota members differ in their adaptations to survival in aerobic environments. Interestingly, there is an inverse relationship between aerobic survival and abundance or potential for prolonged colonization of the intestinal tract. Facultative anaerobes, aerotolerant Lactobacilli and endospore-forming Firmicutes exhibit high fluctuation, and if such bacteria are to be used as probiotics, they must be continuously administered to mimic their permanent supply from the environment. On the other hand, species not expressing any form of aerobic resistance, such as those from phylum Bacteroidetes, commonly represent host-adapted microbiota members characterized by vertical transmission from mothers to offspring, capable of long-term colonization following a single dose administration. To achieve maximal probiotic efficacy, the mode of their administration should thus reflect their natural ecology.

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.

Interleukin 4 inducible 1 gene (IL4I1) is induced in chicken phagocytes by Salmonella Enteritidis infection.

In attempt to identify genes that are induced in chickens by Salmonella Enteritidis we identified a new highly inducible gene, interleukin 4 induced 1 gene (IL4I1). IL4I1 reached its peak expression (458× induction) in the cecum of newly hatched chickens 4 days post-infection and remained upregulated for an additional 10 days. IL4I1 was expressed and induced in macrophages and granulocytes, both at the mRNA and protein level. IL4I1 was expressed and induced also in CD4 and γδ T-lymphocytes though at a 50-fold lower level than in phagocytes. Expression of IL4I1 was not detected in CD8 T lymphocytes or B lymphocytes. Mutation of IL4I1 in chicken HD11 macrophages did not affect their bactericidal capacity against S. Enteritidis but negatively affected their oxidative burst after PMA stimulation. We therefore propose that IL4I1 is not directly involved in bactericidal activity of phagocytes and, instead, it is likely involved in the control of inflammatory response and signaling to T and B lymphocytes.

Systematic Culturomics Shows that Half of Chicken Caecal Microbiota Members can be Grown in Vitro Except for Two Lineages of Clostridiales and a Single Lineage of Bacteroidetes.

Epidemiological data show that the composition of gut microbiota influences host health, disease status, and even behaviour. However, to confirm these epidemiological observations in controlled experiments, pure cultures of gut anaerobes must be obtained. Since the culture of gut anaerobes is not a simple task due to the large number of bacterial species colonising the intestinal tract, in this study we inoculated 174 different culture media with caecal content from adult hens, and compared the microbiota composition in the original caecal samples and in bacterial masses growing in vitro by 16S rRNA sequencing. In total, 42% of gut microbiota members could be grown in vitro and since there were some species which were not cultured but for which the culture conditions are known, it is likely that more than half of chicken gut microbiota can be grown in vitro. However, there were two lineages of Clostridiales and a single lineage of Bacteroidetes which were common in chicken caecal microbiota but resistant to culture. Of the most selective culture conditions, nutrient broths supplemented with mono- or di-saccharides, including those present in fruits, positively selected for Lactobacillaceae. The addition of bile salts selected for Veillonellaceae and YCFA (yeast casitone fatty acid agar) enriched for Desulfovibrionaceae. In addition, Erysipelotrichaceae were positively selected by colistin, trimethoprim, streptomycin and nalidixic acid. Culture conditions tested in this study can be used for the selective enrichment of desired bacterial species but also point towards the specific functions of individual gut microbiota members.

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.