Chicken Gut Microbiota Research Articles

Experimental study of the impact of antimicrobial treatments on Campylobacter, Enterococcus and PCR-capillary electrophoresis single-strand conformation polymorphism profiles of the gut microbiota of chickens.

An experiment was conducted to compare the impact of antimicrobial treatments on the susceptibility of Campylobacter, Enterococcus faecium and Enterococcus faecalis, and on the diversity of broiler microbiota. Specific-pathogen-free chickens were first orally inoculated with strains of Campylobacter and Enterococcus faecium. Birds were then orally treated with recommended doses of oxytetracycline, sulfadimethoxine/trimethoprim, amoxicillin or enrofloxacin. Faecal samples were collected before, during and after antimicrobial treatment. The susceptibility of Campylobacter, Enterococcus faecium and Enterococcus faecalis strains isolated on supplemented or non-supplemented media was studied and PCR-capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) profiles of the gut microbiota were analysed. Enrofloxacin-resistant Campylobacter were selected in the enrofloxacin-treated group and showed the Thr86Ile mutation in the gyrA gene. Acquisition of the tetO gene in Campylobacter coli isolates was significantly more frequent in birds given oxytetracycline. No impact of amoxicillin treatment on the susceptibility of Campylobacter could be detected. Ampicillin- and sulfadimethoxine/trimethoprim-resistant Enterococcus faecium were selected in amoxicillin-treated broilers, but no selection of the inoculated vancomycin-resistant Enterococcus faecium could be detected, although it was also resistant to tetracycline and sulfadimethoxine/trimethoprim. PCR-CE-SSCP revealed significant variations in a few peaks in treated birds as compared with non-treated chickens. In conclusion, antimicrobial treatments perturbed chicken gut microbiota, and certain antimicrobial treatments selected or co-selected resistant strains of Campylobacter and Enterococcus.

Spatial heterogeneity and stability of bacterial community in the gastrointestinal tracts of broiler chickens

Bacterial communities in the different regions of gastrointestinal tract (GIT) of broiler chickens were analyzed by pyrosequencing approach to understand microbial composition and diversity. The DNA samples extracted from 7 different regions along the GIT were subjected to bacterial-community analysis by pyrosequencing of the V1–V3 region of 16S rRNA gene. Major bacterial phyla in the chicken-gut microbiota includedFirmicutes,Proteobacteria,Bacteroidetes,Actinobacteria, andAcidobacteria, butFirmicutes were mostly dominant (67.3 ± 16.1% of the total sequence reads identified). AmongFirmicutes,Lactobacillales, including the generaLactobacillus andEnterococcus, were the most dominant (51.8 ± 34.5% of the total sequence reads identified) from the crop to ileum. In contrast, in the cecum and large intestine, those genera were rarely detected, andClostridiales were dominant (55.9 ± 31.4%). Fast UniFrac analysis showed that microbial communities from the crop to jejunum of the same individual chicken were grouped together, and those from ileum, cecum, and large intestine were clustered in a more GIT-specific manner. The numbers of shared operational taxonomic units between the neighboring segments of GIT were low, ranging from 2.9 to 20.3%. However, the abundance of shared operational taxonomic units in each segment was relatively high, ranging from 61.7 to 85.0%, suggesting that substantial proportions of microbial communities were shared between each segment and its neighboring segments, comprising a core microbiota. Our results suggested that the microbial communities of 7 main segments in the chicken GIT were distinctive according to both individuals and the different segments of GIT, but their stability was maintained along the GIT.

Metaproteomics Analysis Reveals the Adaptation Process for the Chicken Gut Microbiota

The animal gastrointestinal tract houses a large microbial community, the gut microbiota, that confers many benefits to its host, such as protection from pathogens and provision of essential metabolites. Metagenomic approaches have defined the chicken fecal microbiota in other studies, but here, we wished to assess the correlation between the metagenome and the bacterial proteome in order to better understand the healthy chicken gut microbiota. Here, we performed high-throughput sequencing of 16S rRNA gene amplicons and metaproteomics analysis of fecal samples to determine microbial gut composition and protein expression. 16 rRNA gene sequencing analysis identified Clostridiales, Bacteroidaceae, and Lactobacillaceae species as the most abundant species in the gut. For metaproteomics analysis, peptides were generated by using the Fasp method and subsequently fractionated by strong anion exchanges. Metaproteomics analysis identified 3,673 proteins. Among the most frequently identified proteins, 380 proteins belonged to Lactobacillus spp., 155 belonged to Clostridium spp., and 66 belonged to Streptococcus spp. The most frequently identified proteins were heat shock chaperones, including 349 GroEL proteins, from many bacterial species, whereas the most abundant enzymes were pyruvate kinases, as judged by the number of peptides identified per protein (spectral counting). Gene ontology and KEGG pathway analyses revealed the functions and locations of the identified proteins. The findings of both metaproteomics and 16S rRNA sequencing analyses are discussed.

Ileal microbiota composition of broilers fed various commercial diet compositions

Microbiota plays a role in the release and absorption of nutrients from feed components, thereby affecting digesta composition and moisture content of the excreta. The objective of the current study was to determine the effects of 5 different diets varying in ingredients (medium-chain fatty acids, nonstarch polysaccharides, and starch) on the microbiota composition of ileal digesta of broiler chickens and excreta DM content. Each treatment was repeated 6 times in cages each containing 18 Ross 308 broilers, with growth performance measured from 0 to 34 d of age and excreta DM and ileal microbiota composition analyzed at 34 d of age. Microbiota composition was evaluated using a novel ribosomal RNA microarray technology containing 370 different probes covering various genera, groups of microbial species, and individual species of the chicken gut microbiota, of which 321 had a signal above the background threshold. Replacing part of the animal fat and soybean oil in the wheat-based diet with medium-chain fatty acids (MCFA; 0.3% C10 and 2.7% C12) improved feed efficiency compared with the other dietary treatments. This coincided with a suppression of gram-positive bacteria belonging to the phylum of the Firmicutes, including Lactobacillus species, and species belonging to the family of the Enterococcaceae and Micrococcaceae, whereas the gram-negative bacteria belonging to the family of the Enterobacteriaceae were promoted. None of the other diets used in the present study notably changed the ileal digesta bacteria composition. Excreta DM content was not affected by dietary treatment. The variation between individual birds per dietary treatment was more pronounced than variation caused by feed composition, with the exception of the digesta microbiota of the birds fed the MCFA diet. It is concluded that a diet with MCFA significantly changes the ileal microbiota composition, whereas the effect of the other diets on the composition of the microbiota and excreta DM content is small in broiler chickens.

Response of Intestinal Microbiota to Antibiotic Growth Promoters in Chickens

Antibiotic growth promoters (AGPs) have been used as feed additives to improve average daily weight gain and feed efficiency in food animals for more than five decades. However, use of AGPs is associated with the emergence of antibiotic-resistant human pathogens of animal origin, posing a significant threat to food safety and public health. Thus, development of novel alternatives to AGPs is important to mitigate antimicrobial resistance in foodborne pathogens. To achieve this goal, the mode of action of AGPs should be elucidated. In this study, the response of the chicken gut microbiota to AGPs was examined using two culture-independent approaches: phospholipid fatty acid (PLFA) biomarker analysis and 16S rDNA clone library sequencing. PLFA analysis showed that AGP tylosin treatment changed composition of the microbiota in various intestinal sites; however, total viable bacterial biomass in intestine was not affected by tylosin treatment. PLFA analysis also revealed an abundant viable fungal population in chicken microbiota. Eight 16S rDNA libraries (96 clones per library) were constructed using ileal samples from chickens that received either antibiotic-free or medicated feed. The 16S rDNA clone analysis of the growth-relevant samples showed the AGP treatment influenced the diversity of ileum microbiota in the chickens primarily in the Firmicutes division. In particular, Lactobacillus spp. populations in the ileum of AGP-treated chickens were significantly lower than those from chickens receiving antibiotic-free feed. Together, this study revealed novel features of the intestinal microbiota in response to AGP treatment and suggested approach to develop potential alternatives to AGPs for mitigation of antimicrobial resistance in foodborne pathogens.

A Bifidobacterium-based synbiotic product to reduce the transmission of C. jejuni along the poultry food chain

With the ban of dietary antimicrobial agents, the use of probiotics, prebiotics and synbiotics has attracted a great deal of attention in order to improve intestinal health and control food-borne pathogens, which is an important concern for the production of safe meat and meat products. Recently, Campylobacter jejuni has emerged as a leading bacterial cause of food-borne gastroenteritis in humans, and epidemiological evidences indicate poultry and poultry products as the main source of human infection. This work aimed at the development of a synbiotic mixture capable of modulating the gut microbiota of broiler chickens to obtain an increase of the beneficial bacteria (i.e. bifidobacteria, lactobacilli) and a competitive reduction of C. jejuni. The prebiotic compound used in the mixture was chosen after an in vivo trial: a fructooligosaccharide and a galactooligosaccharide were separately administered to broilers mixed with normal feed at a concentration of 0.5% and 3%, respectively. Quantitative PCR on DNA extracted from fecal samples revealed a significant (p<0.05) increase of Bifidobacterium spp. in broilers treated with the galactooligosaccharide, coupled to a decrease (p<0.05) of Campylobacter spp. The galactooligosaccharide was then combined with a probiotic Bifidobacterium strain (B. longum subsp. longum PCB133), possessing in vitro antimicrobial activity against C. jejuni. The strain was microencapsulated in a lipid matrix to ensure viability into the feed and resistance to stomach transit. Finally, the synbiotic mixture was administered to broiler chickens for 14days mixed with normal feed in order to have an intake of 109CFU of PCB133/day. Bifidobacterium spp., Lactobacillus spp., Campylobacter spp., B. longum subsp. longum and C. jejuni were quantified in fecal samples. PCB133 was recovered in feces of all animals. C. jejuni concentration in poultry feces was significantly (p<0.05) reduced in chickens administered with the synbiotic mixture. This study allowed to highlight the positive effect of the synbiotic approach for C. jejuni reduction in broiler chickens, which is of fundamental importance for the safety of poultry meat consumers.

Antimicrobials, Gut Microbiota and Immunity in Chickens

The use of antimicrobials will be soon removed due to an increase of occurrence of antibiotic-resistant bacteria or ionophore-resistant Eimeria species in poultry farms and consumers' preference on drug-free chicken meats or eggs. Although dietary antimicrobials contributed to the growth and health of the chickens, we do not fully understand their interrelationship among antimicrobials, gut microbiota, and host immunity in poultry. In this review, we explored the current understanding on the effects of antimicrobials on gut microbiota and immune systems of chickens. Based on the published literatures, it is clear that antibiotics and antibiotic ionophores, when used singly or in combination could influence gut microbiota. However, antimicrobial effect on gut microbiota varied depending on the samples (e.g., gut locations, digesta vs. mucosa) used and among the experiments. It was noted that the digesta vs. the mucosa is the preferred sample with the results of no change, increase, or decrease in gut microbiota community. In future, the mucosa-associated bacteria should be targeted as they are known to closely interact with the host immune system and pathogen control. Although limited, dietary antimicrobials are known to modulate humoral and cell-mediated immunities. Ironically, the evidence is increasing that dietary antimicrobials may play an important role in triggering enteric disease such as gangrenous dermatitis, a devastating disease in poultry industry. Future work should be done to unravel our understanding on the complex interaction of host-pathogen-microbiota-antimicrobials in poultry.

Immune Response of Chicken Gut to Natural Colonization by Gut Microflora and to Salmonella enterica Serovar Enteritidis Infection

In commercial poultry production, there is a lack of natural flora providers since chickens are hatched in the clean environment of a hatchery. Events occurring soon after hatching are therefore of particular importance, and that is why we were interested in the development of the gut microbial community, the immune response to natural microbial colonization, and the response to Salmonella enterica serovar Enteritidis infection as a function of chicken age. The complexity of chicken gut microbiota gradually increased from day 1 to day 19 of life and consisted of Proteobacteria and Firmicutes. For the first 3 days of life, chicken cecum was protected by increased expression of chicken β-defensins (i.e., gallinacins 1, 2, 4, and 6), expression of which dropped from day 4 of life. On the other hand, a transient increase in interleukin-8 (IL-8) and IL-17 expression could be observed in chicken cecum on day 4 of life, indicating physiological inflammation and maturation of the gut immune system. In agreement, the response of chickens infected with S. Enteritidis on days 1, 4, and 16 of life shifted from Th1 (characterized mainly by induction of gamma interferon [IFN-γ] and inducible nitric oxide synthase [iNOS]), observed in younger chickens, to Th17, observed in 16-day-old chickens (characterized mainly by IL-17 induction). Active modification of chicken gut microbiota in the future may accelerate or potentiate the maturation of the gut immune system and increase its resistance to infection with different pathogens.

Effect of potassium diformate on growth performance and gut microbiota in broiler chickens challenged with necrotic enteritis

1. The effect of potassium diformate (KDF) on mortality, growth performance, microbial populations, pH and short chain fatty acid concentrations in the intestinal tract of broiler chickens challenged with necrotic enteritis was investigated. 2. An experiment with 1050 Cobb male broiler chickens was conducted from 1 to 35 d of age. There were 7 treatment groups: (1) unchallenged negative control, (2) unchallenged KDF (4·50 g/kg feed), (3) challenged negative control, (4) challenged positive control (100 ppm monensin and 45 ppm Zn-bacitracin), and (5) to (7) challenged KDF (inclusion rate 2·25, 4·50 and 6·75 g/kg feed, respectively). 3. The necrotic enteritis challenge caused significant growth depression except in the challenged positive control group. Inclusion of KDF in feed had no significant effect on weight gain or feed conversion ratio, neither under challenged nor unchallenged conditions. Necrotic-enteritis-related mortalities were reduced in response to the positive control feed and KDF at 2·25 and 4·50 g/kg rates, but with no effect at the 6·75 g/kg rate. 4. There was no effect of KDF on Clostridium perfringens numbers in jejunum during the necrotic enteritis challenge (d 15). Post challenge (d 35), 4·5 g/kg KDF reduced the number of C. perfringens and Enterobacteria in jejunum compared with the negative control group. 5. Intestinal pH was not affected by adding KDF to the feed. The challenge reduced jejunum pH compared with the unchallenged treatment groups and challenged positive control group. 6. Chickens fed the KDF diets had detectable concentrations of formic acid in the jejunum. There was a tendency towards higher concentrations of acetic acid and lactic acid in the small intestine of unchallenged birds, whereas challenged birds had higher concentrations of butyric acid in the caeca. 7. It is concluded that KDF holds promise as an agent to control necrotic enteritis in broiler chickens and it may be possible to enhance the efficacy of KDF by manipulating dietary properties that may influence the dissociation kinetics of KDF in the gut.

Quantitative analysis of the intestinal bacterial community in one- to three-week-old commercially reared broiler chickens fed conventional or antibiotic-free vegetable-based diets

To explore the effect of drug-free poultry production on the intestinal microflora of broiler chickens, the bacterial community of this environment was quantitatively profiled in both conventionally reared birds and birds reared without antibiotic growth promotants (AGPs) on a vegetable-based diet. Quantitative, real-time PCR with group-specific 16S rDNA primer sets was used to enumerate the abundance of the following chicken gastrointestinal (GI) tract phylogenetic groups: the Clostridium leptum-Faecalibacterium prausnitzii subgroup (Clostridium genus cluster IV), the Clostridium coccoides - Eubacterium rectale subgroup (Clostridium cluster XIVa and XIVb), the Bacteroides group (including Prevotella and Porphyromonas), Bifidobacterium spp., the Enterobacteriaceae, the Lactobacillus group (including the genera Leuconostoc, Pediococcus, Aerococcus and Weissella), the Clostridium perfringens subgroup (Clostridium cluster I), Enterococcus spp., Veillonella spp., Atopobium spp., Campylobacter spp. and the domain Bacteria. A species-specific 5'-nuclease (Taqman) assay was also employed to specifically assess Cl. perfringens abundance. Ten birds were sampled from each of two commercial chicken houses, one in which feed was supplemented with AGPs and exogenous animal protein, and the other vegetable-based and drug-free, at 7, 14 and 21 days of age. The ileal community was dominated by two large populations, the lactobacilli and the Enterobacteriaceae, with those taxa much more numerous in drug-free vegetable-based diet fed birds than those conventionally reared at the 7- and 14-day time periods. The progressive changes in microflora in both the conventional and drug-free caeca were similar to each other, with the Enterobacteriaceae sequences dominating at day 7, but being replaced by obligate anaerobe signature sequences by day 14. Of note was the finding that all the day 14 and day 21 replicate caecal samples from the drug-free house were positive for Campylobacter spp. averaging >10(8) 16S rDNA gene copies per gram wet weight. Quantitative, real-time PCR indicates that the effects of drug-free rearing on the chicken GI tract microbial community are most pronounced in the ileal region, but AGPs may be important in controlling Campylobacter colonization of the caecum. A quantitative taxonomic understanding of the shifting microbial ecology of the broiler chicken gut microbiota is important in the light of AGP withdrawal. AGP withdrawal has occurred in response to concerns over the transfer of antimicrobial-resistant bacteria to humans via the food production chain.

Development and Assessment of a Real-Time PCR Assay for Rapid and Sensitive Detection of a Novel Thermotolerant Bacterium, Lactobacillus thermotolerans , in Chicken Feces

A new real-time PCR assay was successfully developed using a TaqMan fluorescence probe for specific detection and enumeration of a novel bacterium, Lactobacillus thermotolerans, in chicken feces. The specific primers and probe were designed based on the L. thermotolerans 16S rRNA gene sequences, and these sequences were compared to those of all available 16S rRNA genes in the GenBank database. The assay, targeting 16S rRNA gene, was evaluated using DNA from a pure culture of L. thermotolerans, DNA from the closely related bacteria Lactobacillus mucosae DSM 13345(T) and Lactobacillus fermentum JCM 1173(T), and DNA from other lactic acid bacteria in quantitative experiments. Serial dilutions of L. thermotolerans DNA were used as external standards for calibration. The minimum detection limit of this technique was 1.84 x 10(3) cells/ml of an L. thermotolerans pure culture. The assay was then applied to chicken feces in two different trials. In the first trial, the cell population was 10(4) cells/g feces on day 4 and 10(5) cells/g feces on days 11 to 18. However, cell populations of 10(6) to 10(7) cells/g feces were detected in the second trial. The total bacterial count, measured by 4',6-diamidino-2-phenylindole (DAPI) staining, was approximately 10(11) cells/g feces. These results suggest that in general, L. thermotolerans is a normal member of the chicken gut microbiota, although it is present at relatively low levels in the feces.