Insights into Broilers' Gut Microbiota Fed with Phosphorus, Calcium, and Phytase Supplemented Diets

Abstract

Phytase supplementation in broiler diets is a common practice to improve phosphorus (P) availability and to reduce P loss by excretion. An enhanced P availability, and its concomitant supplementation with calcium (Ca), can affect the structure of the microbial community in the digestive tract of broiler chickens. Here, we aim to distinguish the effects of mineral P, Ca, and phytase on the composition of microbial communities present in the content and the mucosa layer of the gastrointestinal tract (GIT) of broiler chickens.
Significant differences were observed between digesta and mucosa samples for the GIT sections studied (p = 0.001). The analyses of 56 individual birds showed a high microbial composition variability within the replicates of the same diet. The average similarity within replicates of digesta and mucosa samples across all diets ranged from 29 to 82% in crop, 19–49% in ileum, and 17–39% in caeca. Broilers fed with a diet only supplemented with Ca had the lowest body weight gain and feed conversion values while diets supplemented with P showed the best performance results. An effect of each diet on crop mucosa samples was observed, however, similar results were not obtained from digesta samples. Microbial communities colonizing the ileum mucosa samples were affected by P supplementation. Caeca-derived samples showed the highest microbial diversity when compared to the other GIT sections and the most prominent phylotypes were related to genus Faecalibacterium and Pseudoflavonifractor, known for their influence on gut health and as butyrate producers. Lower microbial diversity in crop digesta was linked to lower growth performance of birds fed with a diet only supplemented with Ca. Each diet affected microbial communities within individual sections, however, no diet showed a comprehensive effect across all GIT sections, which can primarily be attributed to the great variability among replicates. The substantial community differences between digesta and mucosa derived samples indicate that both habitats have to be considered when the influence of diet on the gut microbiota, broiler growth performance, and animal health is investigated.

Full text

ORIGINAL RESEARCH
published: 19 December 2016
doi: 10.3389/fmicb.2016.02033
Frontiers in Microbiology | www.frontiersin.org 1December 2016 | Volume 7 | Article 2033
Edited by:
Zhongtang Yu,
Ohio State University, USA
Reviewed by:
Jeffrey David Galley,
Baylor College of Medicine, USA
Robert J. Moore,
RMIT University, Australia
*Correspondence:
Amélia Camarinha-Silva
amelia.silva@uni-hohenheim.de
Specialty section:
This article was submitted to
Microbial Symbioses,
a section of the journal
Frontiers in Microbiology
Received: 01 September 2016
Accepted: 02 December 2016
Published: 19 December 2016
Citation:
Borda-Molina D, Vital M,
Sommerfeld V, Rodehutscord M and
Camarinha-Silva A (2016) Insights into
Broilers’ Gut Microbiota Fed with
Phosphorus, Calcium, and Phytase
Supplemented Diets.
Front. Microbiol. 7:2033.
doi: 10.3389/fmicb.2016.02033
Insights into Broilers’ Gut Microbiota
Fed with Phosphorus, Calcium, and
Phytase Supplemented Diets
Daniel Borda-Molina 1, Marius Vital 2, Vera Sommerfeld 1, Markus Rodehutscord 1and
Amélia Camarinha-Silva 1*
1Animal Nutrition Department, Institute of Animal Science, University of Hohenheim, Stuttgart, Germany, 2Microbial
Interactions and Processes Research Group, Helmholtz Centre for Infection Research, Braunschweig, Germany
Phytase supplementation in broiler diets is a common practice to improve phosphorus
(P) availability and to reduce P loss by excretion. An enhanced P availability, and its
concomitant supplementation with calcium (Ca), can affect the structure of the microbial
community in the digestive tract of broiler chickens. Here, we aim to distinguish the effects
of mineral P, Ca, and phytase on the composition of microbial communities present in
the content and the mucosa layer of the gastrointestinal tract (GIT) of broiler chickens.
Significant differences were observed between digesta and mucosa samples for the GIT
sections studied (p=0.001). The analyses of 56 individual birds showed a high microbial
composition variability within the replicates of the same diet. The average similarity within
replicates of digesta and mucosa samples across all diets ranged from 29 to 82% in
crop, 19–49% in ileum, and 17–39% in caeca. Broilers fed with a diet only supplemented
with Ca had the lowest body weight gain and feed conversion values while diets
supplemented with P showed the best performance results. An effect of each diet on crop
mucosa samples was observed, however, similar results were not obtained from digesta
samples. Microbial communities colonizing the ileum mucosa samples were affected by P
supplementation. Caeca-derived samples showed the highest microbial diversity when
compared to the other GIT sections and the most prominent phylotypes were related
to genus Faecalibacterium and Pseudoflavonifractor, known for their influence on gut
health and as butyrate producers. Lower microbial diversity in crop digesta was linked
to lower growth performance of birds fed with a diet only supplemented with Ca. Each
diet affected microbial communities within individual sections, however, no diet showed
a comprehensive effect across all GIT sections, which can primarily be attributed to
the great variability among replicates. The substantial community differences between
digesta and mucosa derived samples indicate that both habitats have to be considered
when the influence of diet on the gut microbiota, broiler growth performance, and animal
health is investigated.
Keywords: 16S sequencing, microbiota, chicken GIT, digesta, mucosa, phosphorus, calcium, phytase

Borda-Molina et al. Broiler Gastrointestinal Microbiota
INTRODUCTION
Broiler chickens are one of the most used farm animals due to
the efficient conversion of feed into body weight gain (Stanley
et al., 2014). Phosphorus (P) supply with the diet plays an
important role in skeletal system development and maintenance
of chickens. P is, however, a non-renewable resource that is
expected to be depleted in the next 100 years (Shastak and
Rodehutscord, 2013). Phytate, an organic source of P contained
in plant seeds and plant-based diets for broilers, is a principal
source of P for the animal, but it has the disadvantage of not
being easily accessible by broilers (Witzig et al., 2015; Zeller et al.,
2015). The P availability of plant-based diets can be improved by
supplementing the diets with phytase, an enzyme that increases
P digestibility and reduces P excretion (Witzig et al., 2015). In
consequence, the amount of calcium (Ca) and P required in
diet formulation can be reduced following release of these two
elements from phytate complexes (Zeller et al., 2015). Changes
in Ca and P supplementation affected the composition and
activity of the microbial community in the digestive tract of
broilers (Ptak et al., 2015). Because the microbes are involved
to a variable extent in enzymatic hydrolysis of nutrient fractions
in the digestive tract, it is necessary to understand the role of
the microbial community of the gut (Eeckhaut et al., 2011) and
its interaction with the host, in order improve the utilization of
nutrients such as phytate bound P by the bird.
The microbial community present in the broilers’
gastrointestinal tract (GIT) has more than 900 bacterial species
(Stanley et al., 2014). They play a crucial role in feed digestion,
breakdown of toxins, exclusion of pathogens, stimulation of
the immune system, and endocrine activity (Zhu et al., 2002).
Several studies have analyzed the microbiota from specific
sections of the GIT including the crop, ileum, and caeca (Sekelja
et al., 2012; Sergeant et al., 2014; Ptak et al., 2015; Witzig et al.,
2015), whereas only a few have focused on the whole GIT (Lu
et al., 2003; Sekelja et al., 2012). Nonetheless, it is now known
that they are highly connected and should influence up and
down-stream the different GIT sections (Stanley et al., 2014).
Most studies have focused on content of the GIT (digesta)
samples only (Sekelja et al., 2012; Walugembe et al., 2015;
Witzig et al., 2015), ignoring the mucosa communities, that are
the closest to the host epithelium (Collado and Sanz, 2007).
Epithelium attached microbial communities have biological
roles that should be characterized. A high bacterial diversity
was observed in the Pars non glandularis of the pig stomach
Abbreviations: P, Phosphorus; Ca, calcium; GIT, gastrointestinal tract; kg,
kilograms; FTU, phytase unit; CO2, carbon dioxide; N2, nitrogen; O2, oxygen;
cm, centimeters; ◦C, Celsius degrees; BW, body weight; FC, feed consumption;
PCR, polymerase chain reaction; bp, base pairs; OTU, operational taxonomic units;
RDP, ribosomal database project; ENA, European Nucleotide Archive; nMDS,
non-metric multidimensional scaling plots; PcoA, principal coordinate analysis;
SIMPER, similarity percentages analysis; PERMANOVA, permutational manova
H′, Shannon-weaver index of diversity; SCFA, short chain fatty acids; ANAE,
Anaeroplasmataceae; BURK, Burkholderiaceae; CARN, Carnobacteriaceae; CLOS
IV, Clostridiales incertae sedis IV; CLOS XI, Clostridiales incertae sedis XI; ERYS,
Erysipelotrichaceae; GRAC, Gracilibacteriaceae; LACH, Lachnospiraceae; LACT,
Lactobacillus; PEPT I, Peptococcaceae I; PEPT, Peptostreptococcaceae; PSEU,
Pseudomonadaceae; RUMI, Ruminococcaceae; STRE, Streptococcaceae.
(Mann et al., 2014) and previous reports in rats and humans
have found differences between the microbial counts in the
colonic mucosa and feces (Zoetendal et al., 2002; Haange et al.,
2012).
The crop, the section where feed is temporally stored
and fermentation activities initiate, is highly dominated by
Lactobacillus species (Stanley et al., 2014; Witzig et al., 2015).The
ileum, where nutrients are absorbed, is mainly colonized
by Lactobacillus species and also by partially characterized
bacteria with butyrate producing activities, such as Clostridium,
Streptococcus, and Enterococcus (Stanley et al., 2014). The
caeca, where complex substrates such as cellulose, other
polysaccharides, and phytate are fermented (Stanley et al.,
2014; Choi et al., 2015; Zeller et al., 2015) is the most
diverse section of the GIT and is highly dominated by
unknown microbes. The most abundant families in caeca are
Clostridiaceae, Bacteroidaceae, Lactobacillaceae, and butyrate
producers (Stanley et al., 2014).
Considering the low availability of P in plant-based diets,
and the effect of supplementing diets with phytase, Ca, and
P on chickens’ performance and phytate degradation in the
digestive tract, this study aims to investigate the influence of
these supplements, on the microbial communities of digesta
and mucosa samples of three sections of the GIT of broiler
chickens.
MATERIALS AND METHODS
Animal Sampling
The animal experiment was carried out in the Agricultural
Experiment Station of Hohenheim University, location
Lindenhöfe in Eningen (Germany). All procedures
regarding animal handling and treatments were approved
by the Regierungspräsidium Tübingen (approval number
HOH33|14TE).
A total of 1064 broiler chickens (unsexed, strain Ross 308)
were allocated to 56 floor pens. Animals were fed with a
commercial starter diet (Table S1) until day 14 of age. On day
15 each pen was randomly assigned to one of eight different
dietary treatments (seven pens per diet; Table 1). The diets were
mixed based on corn and soybean meal (Table S1) with the
supplementation of two levels of P (monosodium phosphate;
0 or 2 g P/kg), Ca (limestone; 0 or 3 g Ca/kg), and an
E. coli-derived 6-phytase QuantumTM Blue, AB Vista (0 or
1500 FTU/kg; Table 1). The experiment followed a 2 ×2×2
factorial arrangement of treatments. On day 26 one animal per
pen was euthanized by carbon dioxide asphyxiation following
anesthesia in a gas mixture (35% CO2, 35% N2, and 30%
O2;Zeller et al., 2015). The GIT was dissected immediately
after euthanization and crop, ileum (terminal two-thirds of
the section between Meckel’s diverticulum and 2 cm anterior
to the ileo-ceco-colonic junction) and the two caeca, were
opened longitudinally and digesta samples were collected with
a sterile spoon. The mucosa was washed with sterile phosphate-
buffered saline and scraped with a sterile glass slide. In some
cases, the amount of digesta contained in a certain section
was not sufficient, resulting in a total of 281 samples collected,
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Borda-Molina et al. Broiler Gastrointestinal Microbiota
TABLE 1 | Phosphorus (P), calcium (Ca), and phytase concentration in the eight dietary treatments.
Diets A B C D E F G H
P−P−P−P−P+P+P+P+
Ca−Ca−Ca+Ca+Ca−Ca−Ca+Ca+
Ph−Ph+Ph−Ph+Ph−Ph+Ph−Ph+
Total-P (g/kg) 4.1 4.1 4.1 4.1 6.9 6.9 6.9 6.9
Ca (g/kg) 6.2 6.2 10.4 10.4 6.2 6.2 10.4 10.4
Phytase (FTU/kg)a0 1500 0 1500 0 1500 0 1500
aThe calculated activity in the diet is based on enzyme supplements; intrinsic enzyme activity is not included. −, without supplementation; +, with supplementation.
which included 3–7 replicates per dietary treatment and sample
type (mucosa and digesta; Table S2A). Samples were stored at
−80◦C.
Broiler Performance Analysis
Information regarding final body weight (BW), feed
consumption (FC), BW gain and feed to gain ratio, was
obtained from day 15 to 26 and analyzed with MIXED procedure
of the software SAS (version 9.1.3, SAS Institute, Cary, NC). The
statistical model was yjjklm =µ+ri+Tj+βk+xl+(Tβ)jk +
(Tx)jl +(βx)kl +(Tβx)jkl +eijklm; where µ=general mean,
ri=effect of the block (random), Tj=effect of the P addition
(fixed), βk=effect of the Ca addition (fixed), xl=effect of the
phytase addition (fixed), (Tβ)jk, (Tx)il , (βx)kl are the two factor
interactions, (Tβx)jkl are the three factor interaction and eijklm =
random error of the observations. Statistical significance was
evaluated by one-way ANOVA. Differences between treatments
were tested with a multiple t-test (LSD). A significance level of p
<0.05 was considered.
DNA Extraction and Illumina Amplicon
Sequencing
DNA was extracted from 281 samples with FastDNATM SPIN Kit
for soil from MP Biomedicals (Solon, OH, USA), following the
instructions of the manufacturer’s protocol. DNA was quantified
in a NanoDrop 2000 spectrophotometer (Thermo Scientific,
Waltham, MA, USA) and stored at −20◦C.
Illumina library preparation with PCR amplification of the
V1-2 region of the 16S rRNA gene using PrimeSTAR HS DNA
Polymerase (Clontech Laboratories, Mountain View, CA, USA)
was performed according to Camarinha-Silva et al. (2014).
Amplicons were verified by agarose gel electrophoresis, purified
with Macherey-Nagel 96-well-plate (Macherey Nagel, Düren,
Germany) and quantified using a QuantiFluor R
dsDNA system
(Promega, Madison, USA). Equimolar ratios of amplicons (30
ng) were pooled followed by an ethanol precipitation in order
to remove any contaminants. Correct size of the PCR product
was obtained and purified with QIAquick gel extraction kit
(Qiagen, Hilden, Germany). Libraries were sequenced using
250 bp paired-end sequencing chemistry on an Illumina MiSeq
platform.
Bioinformatic processing of sequences was done according
to Camarinha-Silva et al. (2014) with some modifications. Raw
reads were assembled (Cole et al., 2014) and subsequently
aligned using MOTHUR (gotoh algorithm with the SILVA
reference database) prior to pre-clustering (diffs =2). Sequences
were clustered into operational taxonomic units (OTU) at
≥97% similarity. All OTUs with an average abundance lower
than 0.001% across all the samples and with sequence length
<250 bp were discarded from the analysis. Finally, 293,862
±1459 sequences were obtained per sample comprising
a total of 1796 OTUs that were taxonomically assigned
using the naïve Bayesian RDP classifier (Wang et al., 2007;
Table S3). OTUs were then manually evaluated against the
RDP database using Seqmatch function. Sequences are available
at the European Nucleotide Archive (ENA) under accession
number PRJEB14628 in http://www.ebi.ac.uk/ena/data/view/
PRJEB14628.
Multivariate Analysis
A multivariate dataset with the respective abundances of each
OTU on each sample was analyzed using PRIMER (version
7.0.9, PRIMER-E, Plymouth Marine Laboratory, Plymouth, UK;
Clarke and Warwick, 2001). Data was standardized and a sample
similarity matrix was created using Bray-Curtis coefficient (Bray
and Curtis, 1957). The community similarity structure was
depicted through non-metric multidimensional scaling plots
(nMDS) and shade plots were used to study species distributions
between the diets and each section (Clarke and Warwick, 2001).
Similarity percentages analysis (SIMPER) identified the species
contribution to the Bray-Curtis similarity among samples within
each diet (Clarke and Warwick, 2001). PERMANOVA routine
was used to study the significant differences and interactions
between factors [diet, type of sample (digesta or mucosa) and
GIT section], and differences between the diets were studied
based on the pair-wise tests using a permutation method under
a reduced model. Pielou’s evenness index and Shannon-weaver
index of diversity (H′) were used to calculate OTUs evenness and
diversity.
Differences in the abundance of OTUs of interest between
diets were evaluated using the unpaired Welch’s t-test that
can handle unequal variances, unequal sample sizes and
non-parametric data (Welch, 1947). OTUs abundances were
considered significantly different if p<0.05.
Correlations were estimated with Pearson correlation
coefficient (999 permutations) using PRISM 6 (GraphPad
Software, CA). Correlations were considered significantly
different if p<0.05.
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Borda-Molina et al. Broiler Gastrointestinal Microbiota
RESULTS AND DISCUSSION
Global Overview of Broiler Performance
and the Microbial Community in Crop,
Ileum, and Caeca
The growth performance of broiler chickens was significantly
affected by the levels of P, Ca, phytase, and their corresponding
interactions (Table 2). Final BW, FC, and BW gain increased in
diets that included P supplementation (E, F, G, and H) and in diet
B with only phytase supplementation (Tables 1, 2). The growth
performance of birds on these diets was significantly different
from the others. The lowest performance birds were those on diet
C, with only supplementation of Ca
Based on the taxonomic threshold defined by Yarza
et al. (2014), which takes into consideration a hierarchical
classification applied on both cultured and uncultured
microorganisms, 16S rRNA gene sequences were taxonomically
assigned with sequence identity of 82% to orders, 86.5% to
family, 94.5% to genera (Yarza et al., 2014), and 97% identity was
used for species identification (Konstantinidis and Tiedje, 2005).
A total of 1796 OTUs were classified into class (78.5%), order
(76.8%), family (63.4%), genera (22.8%), and species (4%). A total
of 3.8% of the sequences could only be assigned to the phylum
Firmicutes. This result confirmed previous findings, which
stated that gastrointestinal microbiota of the chicken remains
largely unexplored and <200 species are isolated from chicken
gastrointestinal tract (Stanley et al., 2014; Waite and Taylor,
2015). Next generation sequencing techniques have exposed
the hidden diversity of microorganisms, but its taxonomic
classification is difficult because of the time consuming effort to
isolate and biochemically characterize individual bacteria (Yarza
et al., 2014).
High variability in the microbial composition was observed
between individuals (3–7 birds) within each diet and section
(Table S2B). The average similarity of individuals in the studied
sections ranged in the crop digesta from 29 to 82% and crop
mucosa from 29 to 73%. In the ileum digesta the observed
similarity of individuals was between 19 and 49% and in the
ileum mucosa 25–47%. The caeca showed the lowest similarity
of individuals, namely 17–38% in digesta and 30–39% in mucosa
samples. The crop is dominated by Lactobacillus (Hagen et al.,
2005; Stanley et al., 2014; Witzig et al., 2015), explaining the
higher values of similarity and its simple structured microbiota
when compared to other sections of the GIT. In ileum and
caeca sections, the more diverse microbial communities are
responsible for phytate degrading activities (Palacios et al., 2008),
degrading complex organic substrates, and to the production
of short chain fatty acids (SCFA; Stanley et al., 2013b; Mann
et al., 2014; Choi et al., 2015). The average similarity decreased
in these sections, perhaps related to the presence of a higher
number of OTUs. Taking as an example diet H (with all
supplements) and diet A (without any supplement), a variation
in the relative abundance of predominant families was observed
between the replicates in each section (Figures S1A,B). The
variability between individuals has been previously reported in
two studies that characterized chicken caeca (Stanley et al., 2013b;
Sergeant et al., 2014) and in cattle feces (Durso et al., 2010).
Furthermore, human studies found inter-individual differences
in mucosa associated microbiota from colon and rectum samples
(Hong et al., 2011). These studies showed that, independently
of the core microbiota colonization, there is a great variation
in the relative abundance of the bacterial community between
individuals. A possible explanation is that shifts in microbial
composition are influenced by the initially colonizing microbiota,
diet, and immune system of the host (Donaldson et al., 2015).
Exploring the bacterial community structure of the 281
samples, regardless of the diet, a great distinction between crop,
ileum, and caeca was found to exist (p=0.001; Figure 1A
TABLE 2 | Broiler chickens performance data between day 15 and 26 for the eight dietary treatments.
Diets A B C D E F G H
P−P−P−P−P+P+P+P+
Ca−Ca−Ca+Ca+Ca−Ca−Ca+Ca+
Ph−Ph+Ph−Ph+Ph−Ph+Ph−Ph+
Final BW (g) 1433bc 1527a1202d1420c1510a1539a1492ab 1530a
FC (g/d) 117b121ab 96d112c124a123a119ab 122a
BW gain (g/d) 78b86a58c76b86a86a83a86a
F:G (g/g) 1.49b1.41d1.66a1.47bc 1.44cd 1.42d1.44cd 1.41d
p-value
Pooled SD P Ca Phy P*Ca P*phy Ca*phy P*Ca*phy
Final BW (g) 21.02 <0.0001 <0.0001 <0.0001 <0.0001 0.0003 0.0383 0.0756
FC (g/d) 1.26 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.0006 0.0526
BW gain (g/d) 1.31 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.0007 0.0406
F:G (g/g) 0.012 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.0178 0.0407
Final body weight (BW), feed consumption (FC), BW gain and feed to gain (F:G) ratio of broiler chickens. Data are given as treatment means with respective SD (standard deviation); n
=7 blocks, 16–18 animals per block, and means without common superscript resulted being significantly different (p <0.05).
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Borda-Molina et al. Broiler Gastrointestinal Microbiota
FIGURE 1 | Global bacterial community structure of 281 samples. Sequencing data was standardized prior to the use of Bray-Curtis similarity algorithm.
Non-metric multi-dimensional scaling (nMDS) plot illustrates: (A) crop, ileum and caeca samples, and (B) digesta and mucosa samples. The symbols represent a
unique sample comprising all OTUs and its abundance information. (C) Venn diagram of the OTUs common/unique to each type of sample in the crop, ileum, and
caeca. Overlapping areas show the OTUs shared between digesta and mucosa samples.
and Figure S2A). This confirms similar results from previous
studies (Stanley et al., 2014; Witzig et al., 2015). For the first
time, and in all three sections analyzed, a separation was observed
between digesta and mucosa samples (p=0.001; Figure 1B).
Additionally, PERMANOVA results using the total number of
OTUs indicated that two way interactions, diet ×section and
section ×type of sample, were significantly different (p<0.05),
showing that the type of community depends on the diet and
section studied and on the interactive effect of section and type
of sample.
Crop samples comprised 690 OTUs shared between digesta
and mucosa, a further 66 OTUs were specific to digesta and
583 OTUs to mucosa samples (Figure 1C). The diversity indices
showed on average the lowest Pielou’s evenness and Shannon
diversity for both digesta (0.33 and 1.47, respectively) and
mucosa (0.35 and 1.88, respectively), which is in accordance
with previous studies (Hagen et al., 2005; Witzig et al., 2015).
A similar diversity was observed in ileum digesta; however, an
increase in diversity was detected in the ileum mucosa (Pielou’s
evenness =0.49 and Shannon diversity =2.9). Specific OTUs
belonging to ileum digesta and mucosa samples were 64 and
490, respectively, while 1189 OTUs were observed in both
(Figure 1C). The higher microbial diversity could be attributed
to more suitable physicochemical conditions that allow a better
establishment of complex microbiota and influence their nutrient
availability (Stanley et al., 2014). Caecal digesta and mucosa
samples resulted in the highest OTUs evenness (0.68 and 0.73,
respectively) and diversity (4.15 and 4.6, respectively), when
compared with all other sections. In the caeca digesta and mucosa
1302 OTUs were detected. A total of 24 OTUs were only detected
in the digesta and 303 in the mucosa of caeca (Figure 1C).
Overall, mucosa samples shared more OTUs between the three
sections than digesta samples (Figure S2B). Several studies have
shown that this higher diversity in the caeca is due to the low
passage rate, pH, and the presence of small and soluble particles,
which enhance the role of the microorganisms in assimilation
of nutrients from food, in producing vitamins, and amino acids
(Zhu et al., 2002; Sergeant et al., 2014), and protecting the host
against pathogens (Stanley et al., 2014). Mucosa samples showed
higher species diversity than digesta in all GIT sections. Most
of the studies characterizing chicken microbiota have focused
on digesta of the different GIT sections (Deusch et al., 2015;
Waite and Taylor, 2015). The mucosa or mucous layer, which is
mainly composed by mucins and glycan, help the colonization
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Borda-Molina et al. Broiler Gastrointestinal Microbiota
of some groups of microorganisms in the gut (Donaldson et al.,
2015).
The majority of the microorganisms colonizing the three
GIT sections belonged to the phylum Firmicutes, as commonly
described in previous studies that characterized the microbial
communities of the chicken GIT (Stanley et al., 2013a;
Deusch et al., 2015). In the crop, the most abundant family
was Lactobacillaceae, which was previously reported as a
dominant group in that environment (Sekelja et al., 2012; Witzig
et al., 2015). Crop mucosa was additionally colonized with
Lachnospiraceae, Burkholderiaceae, Ruminococcaceae, and
Streptococcaceae (Figure 2). In the ileum, the dominance of
Lactobacillaceae family decreased in comparison to the crop,
showing 66% of abundance in digesta and 25% in the mucosa
samples. The percentage of this family in the luminal content is
in accordance to other broiler studies (Stanley et al., 2014; Witzig
et al., 2015). However, special attention should be given to the
lower abundance of Lactobacillaceae in the mucosa, which has
not been reported before (Figure 2). The caeca showed higher
family diversity in both digesta and mucosa samples, with similar
distribution of families Ruminococcacae, Lachnospiraceae,
Anaeroplasmataceae, Erysipelotrichaceae, Peptococcaceae, and
Lactobacillaceae (Figure 2).
Diet Effect in the Crop Microbial
Community
The composition of the microbial community of crop mucosa
was significantly affected by the diets (p=0.003). Such effect
was not found in digesta samples, highlighting the fact that both,
digesta and mucosa samples, should be studied in regard to diet
effects on gut homeostasis (Figure S3A). Pair-wise comparisons
showed that microbial communities of crop digesta of birds fed
with diet C were significantly distinct to those derived from other
diets (p<0.05), with the exception of diet D (Table S4). Lower
values of Shannon diversity were observed in diet C (Figure S4).
This reveals a diet effect in presence of only Ca supplementation,
which could be related to the lower growth and feed consumption
of birds obtained with diet C (Table 2). High dietary calcium
chelates part of the lipid fraction, which may reduce the energy
value of the diet (Driver et al., 2005). Additionally, Ca forms
insoluble complexes with phytate (Angel et al., 2002) and in
the lumen interacts with inorganic phosphorus resulting in Ca-
ortophosphate (Plumstead et al., 2008). Those complexes have
a negative impact on the birds’ performance due to the reduced
solubility and availability of the P (Hamdi et al., 2015). High Ca
diets have been associated with an increase of crop pH in chickens
(Shafey et al., 1991) and in an higher attachment of L. salivarius
to the GIT mucus of chickens when different Lactobacillus strains
were studied in vitro (Craven and Williams, 1998), however
in our study L. taiwanensis was the most abundant species in
mucosa samples (Figures 3C,D and Table S5).
The abundance of Lactobacillus had the greatest fluctuation
across all replicates when compared to other genera
(Figures 3A,B), indicating a high variability between individuals
at genus level. Lactobacillus was the most predominant genus
in crop digesta and mucosa (Figures 3A,B and Figure S3A).
FIGURE 2 | Family distribution of digesta and mucosa samples in the crop, ileum, and caeca. OTUs present in 281 samples were taxonomically assigned to
a family and families present in abundances higher than 1% plotted. Abbreviations in the graph represent each family: ANAE, Anaeroplasmataceae; BURK,
Burkholderiaceae; CARN, Carnobacteriaceae; CLOS IV, Clostridiales incertae sedis IV; CLOS XI, Clostridiales incertae sedis XI; ERYS, Erysipelotrichaceae; GRAC,
Gracilibacteriaceae; LACH, Lachnospiraceae; LACT, Lactobacillus; PEPT I, Peptococcaceae I; PEPT, Peptostreptococcacaea; PSEU, Pseudomonadaceae; RUMI,
Ruminococcaceae; STRE, Streptococcaceae, (Table S6).
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Borda-Molina et al. Broiler Gastrointestinal Microbiota
FIGURE 3 | Box-plots showing the relative abundance of the genus Lactobacillus in crop digesta (A) and mucosa (B) across eight dietary treatments
(Table 1). The box extends from the lower quartile (25%) to the higher quartile (75%). The line in the box is the median and the whiskers are the minimum and
maximum values. The column charts include the relative abundances (Mean, SEM) of the two main species of Lactobacillus,L. taiwanensis (OTU 1), and L. gallinarum
(OTU 2) detected in digesta (C) and mucosa (D) samples.
Bacteria belonging to this genus efficiently colonize the
squamous lining of the crop and decrease the pH due to the
production of organic acids (Abbas Hilmi et al., 2007). Its
presence in the gut has several advantages such as inhibition of
pathogens by colonization (Abbas Hilmi et al., 2007), production
of salt base hydrolase (BSH), and reduction of cholesterol
concentration (Ramasamy et al., 2009). L. taiwanensis was the
most dominant OTU in digesta and mucosa samples (OTU
1; Table S5). Birds fed with diet C showed a higher tendency
to be colonized more abundantly by this OTU (74%). This
result suggest that the presence of Ca favors this species. This
microorganism was previously observed in the GIT of chickens
fed with diets supplemented with monocalcium phosphate
(Witzig et al., 2015). OTU 1 was negatively correlated with
other species of Lactobacillus (p<0.003), and a negative
correlation between L. taiwanensis and L. crispatus has been
previously reported in the jejunum (Witzig et al., 2015). The
second most abundant OTU in crop digesta and mucosa was L.
gallinarum (OTU 2), a homofermentative lactic acid bacterium
(Hagen et al., 2005). Its abundance in crop mucosa was lower
in diet B supplemented with phytase when compared to diet
E, F and G (p<0.05). OTU 2 was found to be negatively
correlated with L. taiwanensis (p<0.001). The Lactobacillus
acidophilus complex, also studied in the crop (Hagen et al.,
2005), consists of L. amylovorus (OTU 9), L. crispatus (OTU 11),
L. mucosae (OTU 38), and L. vaginalis (OTU 25). Those OTUs
revealed a propensity to be detected in lower abundance in all
diets.
Diet Effects on the Microbial Community in
the Ileum
The ileum showed a higher diversity in the microbial
communities when compared to the crop. Digesta samples
belonging to diets C and H, that were both supplemented with
Ca, were significantly different from samples derived from
Ca-free diets E and F (p<0.05; Table S4). It is known that
higher doses of Ca in the diets can lead to an increase of the
pH (Ptak et al., 2015) and low precaecal P digestibility (Adeola
and Walk, 2013; Hamdi et al., 2015), which could possibly
influence the presence or absence of some OTUs. An effect of P
supplementation was observed in the microbial communities of
the ileum mucosa. Statistical differences were obtained between
diet A and F, G and H; B and F and G; diet C and F, G and H
(p<0.05; Table S4).
Lactobacillus, a genus widely present in crop, decreased in
abundance in the ileum for most diets analyzed. The exception
was for diets C and G, where it was detected at high abundances
(>83%) in digesta samples. With regards to the mucosa, this
genus was observed in higher abundance in diets F and G
(32–37%; Figures 4A,B and Figure S3B) when compared to the
other diets. Previous studies using mice and pigs have shown
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Borda-Molina et al. Broiler Gastrointestinal Microbiota
FIGURE 4 | Principal coordinate analysis (PCoA) ordination of the global bacterial community structure of ileum (A) digesta and (B) mucosa samples
across eight dietary treatments (A–H) (Table 1). Bubbles were superimposed to visualize the relative abundance of the most relevant genera, Lactobacillus and
Streptococcus and families, Peptostreptococcaceae, Burkholderiaceae, and Lachnospiraceae (slice scale 1–100% abundance).
that diets supplemented with P and Ca, like diet G, increases
Lactobacillus abundance (Ten Bruggencate et al., 2004; Metzler-
Zebeli et al., 2010). L. taiwanensis (OTU 1), highly abundant in
the crop, decreased its abundance in ileum digesta samples of
diets supplemented with Ca (C, G, and H; 27%), while in the
mucosa the highest percentage was observed on diet G (17%).
The second most abundant OTU was L. gallinarum (OTU 2),
which showed a tendency to be more abundant in diets A, C, and
F (27%) for digesta and 16% in mucosa samples of diet F.
Diets E and F in digesta, and F in mucosa, both with P
supplementation resulted in an increase of Streptococcus (44,
19, and 23%, respectively; Figures 4A,B). Lu et al. (2003)
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Borda-Molina et al. Broiler Gastrointestinal Microbiota
demonstrated that sequences of OTUs related to Streptococcus
were more prevalent in the ileum digesta than in the caeca (Lu
et al., 2003). In accordance with the study of Ptak et al. (2015),
Streptococcus abundance was reduced in diets supplemented with
Ca, P, and phytase (Ptak et al., 2015), represented in this study
by diet H. Streptococcus abundance was even lower in diet C,
with Ca supplementation only. OTUs assigned to uncultured
Clostridium XI tended to be detected in digesta in higher
abundances on diets D (18%) and E (23%) when compared to
other diets, which accounted for <14%. Likewise, in the mucosa,
colonization with this group mainly occurred with diet B (26%),
E (14%), H (13%), and D (12%), while other diets showed
abundances lower than 8%. In regards to ileum mucosa, OTUs
belonging to Burkholderiaceae accounted for more than 12% of
the total abundance in all dietary treatments, being detected in
higher abundance in diet A and C (30%). This bacterial group
showed moderate heritability in chickens, but it has not been
attributed any function (Meng et al., 2014). OTUs assigned to
Lachnospiraceae were commonly present in all treatments, with
relative abundance ranging from 2.4 to 5.9% (Figure 4B). This
family was reported to be associated with corn-based diets and is
mainly composed by anaerobes and some Clostridium members
(Munyaka et al., 2015).
Streptococcus alactolyticus (OTU 4) showed a tendency to be
present in higher abundance in digesta samples of diets E and
F with P addition (38 and 20%, respectively) and in mucosa
samples of diets F and D, with phytase supplementation (22 and
13%, respectively). This lactic acid bacteria has been found in
ileum samples of broilers fed with a commercial corn-soy diet (Lu
et al., 2003). An uncultured Clostridium XI (OTU 7) was found
with similar abundance in both digesta and mucosa samples,
with the highest values observed when fed diet B (33 and 26%,
respectively). Furthermore, diet B showed only 30% similarity
to other diets with OTU 7 responsible for the dissimilarity.
The closest relative sequence to OTU 7 was an uncultured
Clostridium XI previously isolated from ileum and caeca of
a conventional Ross 208 chickens grown under conditions of
organic farming (Bjerrum et al., 2006). Uncultured Ralstonia
(OTU 6),observed in the crop mucosa (<5%), showed a more
prominent increase of abundance in mucosa samples for diets
A and C (28 and 30%, respectively). Its abundance decreased in
diets supplemented with P. A trend was detected in the increase
of abundance of an OTU belonging to Clostridiaceae 1 (OTU 21)
in diet F digesta (15%) and diet H mucosa (30%); which have P
and phytase supplementation in common.
Diet Effect on the Microbial Community in
the Caeca
Caeca digesta and mucosa samples showed a more diverse
community at genus level than observed in the other sections
(Figure S3C). This fact was previously reported in chickens
under standard commercial conditions (Stanley et al., 2013a;
Sergeant et al., 2014; Mohd Shaufi et al., 2015) and in chickens
exposed to different supplementation of monocalcium phosphate
and phytase (Witzig et al., 2015). The highest OTU abundance
detected in both type of samples was 14% (OTU8). Pair-wise
comparison showed an effect of P in digesta samples of diet B
contrasted to E, F, G, and H, but also between diet C and E
(Table S4). This effect was also observed in the mucosa samples
of diet B compared to F, G and H; diet D with E, F, and
G; diet C with E and F, and lastly diet A and G. A high
proportion of microorganisms belonging to order Clostridiales
were detected in the caeca. This group is known to be an
indicator of healthy chickens, due to its main role in the
SCFA metabolism (Choi et al., 2015). SCFA have influence on
host physiology through regulatory, immunomodulatory, and
nutritional functions. They increase the growth of epithelial
cells, stimulate mineral absorption and inhibit the growth and
adherence of pathogenic microorganisms by decreasing the pH
(Walugembe et al., 2015).
OTUs belonging to Lachnospiraceae are known to degrade
complex polysaccharides to SCFA (Biddle et al., 2013). They
were more abundant in digesta samples of diets supplemented
with P (12–22%), while in the mucosa showed a similar
distribution within all diets (17–28%; Figure 5 and Figure S3C).
Ruminococcaceae is a common family reported in the chicken
caeca (Bjerrum et al., 2006; Mohd Shaufi et al., 2015) and it was
detected in both digesta (4–8%) and mucosa (3–13%) samples.
Both families have been associated with the maintenance of gut
health and have the enzymatic capability to degrade cellulose and
hemicellulose (Biddle et al., 2013). Erysipelotrichaceae showed an
abundance of 2% in the digesta samples of diets supplemented
with P, however in the mucosa a higher abundance was detected
(3–8%). In the caeca, protein sequences related to butyryl-
CoA production enzymes have been previously detected on this
family (Eeckhaut et al., 2011; De Maesschalck et al., 2014). One
group of OTUs, closely related to the family Anaeroplasmataceae,
were observed in all diets (Figure 5). This family has been
reported in the chicken gastrointestinal microbiome (Oakley
et al., 2014), but the exact role in chicken GIT remains unknown.
A species belonging to Anaeroplasmataceae was previously
described in rumen samples and related to bacteriolytic and non-
bacteriolytic activities (Robinson et al., 1975). This can explain
the negative correlation of OTU 8 (uncultured Anaeroplasma)
with other OTUs in digesta and mucosa samples such as
OTU 394 (uncultured Lachnospiraceae), OTU 116 (uncultured
Clostridium XIVa), OTU 390 (uncultured Ruminococcaceae),
and OTU 93 (uncultured Faecalibacterium;p<0.05).
The OTUs in digesta samples related to Lactobacillus were
more abundant when fed diet G (14.8%), with P and Ca
additions (Figure S3C), with L. gallinarum (OTU 2; 12%) and
L. taiwanensis (OTU 1; 2%) as the main colonizers. However, in
the other diets, these OTUs were present in abundances lower
than 2%. This is in accordance with a recent metagenomic
study on the chicken caeca that showed Lactobacillus in low
abundances (<4%; Mohd Shaufi et al., 2015). Diet E, with P
supplementation, showed a group of OTUs closely related to
Faecalibacterium in both type of samples. This genus is one
of the most prominent butyrate producers, providing energy
to the colonic mucosa and known to regulate gene expression,
inflammation, differentiation, and apoptosis in host cells (Luo
et al., 2013). Pseudoflavonifractor, detected in digesta and
mucosa, is a common caeca colonizer that has a protein from
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Borda-Molina et al. Broiler Gastrointestinal Microbiota
FIGURE 5 | Principal coordinate analysis (PCoA) ordination of the global bacterial community structure of caeca (A) digesta and (B) mucosa samples
across eight dietary treatments (A–H) (Table 1). Bubbles were superimposed to visualize the relative abundance of the most relevant genera, Faecalibacterium and
Pseudoflavonifractor and families, Lachnospiraceae, Ruminococcaceae, and Anaeroplasmataceae (slice scale 1–30% abundance).
class IV alcohol dehydrogenase that influences the final butyrate
production pathway (Polansky et al., 2015). Erysipelotrichaceae
incertae sedis previously reported in chicken caeca (Stanley et al.,
2012) was detected more consistently throughout the diets in
digesta samples and the same applied to Streptococcus in the
mucosa.
Supplementation of Ca in diet C enhanced the presence
of OTU 45 (5%) in caeca digesta. This OTU is related
to an uncultured Subdoligranulum sp. that was previously
found in the caeca of turkeys (Scupham, 2007) and is
capable of producing butyric acid. OTU 37, an uncultured
Ruminococcaceae, was detected in lower abundance (3%) in
diets without P supplementation (A to D) or P with phytase
supplementation (F) and has been previously detected in the
intestinal microbiota of preadolescent turkeys (Scupham, 2007).
In the caeca mucosa samples, an OTU with high similarity
to an uncultured Bacillales (OTU 23) was found. This OTU
was present in higher abundance on diet B (6.7%), with
phytase supplementation, when compared to diets A, E, and
F (4.5, 3.1, and 1.8%, respectively; p<0.05). Particularly,
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Borda-Molina et al. Broiler Gastrointestinal Microbiota
this OTU was negatively correlated with OTU 31, related to
an uncultured Lachnospiraceae, and OTU 91 related to an
uncultured Ruminococcaceae (p<0.05). Furthermore, OTU
4 identified as Streptococcus alactolyticus and highly abundant
in some ileum samples, decreased its abundance in the caeca
being 1.5% the highest value observed. This result contradicts a
previous study on broilers fed diets including peas and organic
acids where S. alactolyticus was a dominant species (Czerwiñski
et al., 2010).
In mucosa samples, the abundance of OTUs belonging to the
Clostridium XIVa and XIVb was higher than in digesta. The
first family comprises some microorganisms that are butyrate
producers while the second includes propionate producers and
therefore may be linked to beneficial effects in the GIT (De
Maesschalck et al., 2015). An uncultured Clostridium XIVb
(OTU 56) previously found in caeca of preadolescent turkeys
(Scupham, 2007), was present in birds fed diets B, C, D, E, and
F (2.5–3%). OTU 81, similar to uncultured Clostridium XIVb,
was positively correlated with OTU 56 (p<0.05) and was
previously reported to be present in the human ileum (Li et al.,
2012). OTU 87, an uncultured Clostridium XIVa found in human
feces (Turnbaugh et al., 2009), was more abundant on diet A and
F, without calcium supplementation when compared to diet D,
supplemented with Ca (p<0.05).
It is known that non-ruminant animals are not efficient in
utilizing phytate-P. In this study we have found, in the ileum and
caeca, OTUs related to the genus Clostridium, which have been
previously isolated and associated to the production of cysteine
phytase (Gruninger et al., 2009). Megasphaera elsdenii (OTU
111) and Mitsuokella spp. (OTU 1501), common members of
the rumen microbiota that have the ability to produce phytases
(Yanke et al., 1998), were also detected in the ileum and caeca
samples from birds on diets supplemented with Ca, P, or P with
phytase.
CONCLUSIONS
Diet supplementation with P, Ca, or phytase has an effect on
the microbial community that colonizes the GIT. However, a
consistent effect of diet on the microbiota harbored in the
different sections of the GIT was not observed. This was likely
due to the high variability between individuals. Lower microbial
diversity was associated with lower growth performance in
animals fed with a diet only supplemented with Ca. Diets
supplemented with P influenced the caeca microbiota and
positively affected the growth of the broilers. For a better
understanding of dietary effects on broiler performance, gut
function and balance, and the microbial community, digesta and
mucosa samples should be studied in separate as both showed
different microbial communities.
AUTHORS CONTRIBUTIONS
Conceived and designed the experiment: AC, VS,
MR. Performed the experiments: DB. Bioinformatics
analysis: MV. OTUs annotation: DB. Data analysis: DB,
AC. Performance data analysis: VS. Wrote the paper:
DB, AC. Article revision and final approval: MV, VS,
MR, AC.
FUNDING
This project has been funded in part by the Ministerium
für Wissenschaft, Forschung und Kunst Baden-Württemberg,
Stuttgart, Germany.
ACKNOWLEDGMENTS
The author would like to thank Maren Witzig for contributing
to the design of the experiment, Bruno Tilocca for animal
experiment set up and Silke Kahl for technical assistance.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: http://journal.frontiersin.org/article/10.3389/fmicb.
2016.02033/full#supplementary-material
Table S1 | Dietary composition of the commercial starter diet fed until day
14 and basal diet for the corresponding treatments with P, Ca and phytase
supplementation fed from days 15 to 26.
Table S2 | Description of the three GIT sections in regards to (A) number of
replicates per diet and type of sample and (B) average similarity of the replicates.
Table S3 | OTUs abundances across the eight dietary treatments and the
GIT sections.
Table S4 | Statistical differences between the sections and the type of
samples based on PERMANOVA results. Pairwise comparison results of the
diets that showed a significant difference.
Table S5 | Taxonomic assignment of the most relevant OTUs present in
the chicken gastrointestinal tract. The assignment was performed in the
Seqmatch function of the RDP database for type and non-type strain.
Table S6 | Percentages of the families present in crop, ileum and caeca for
digesta and mucosa.
Figure S1 | Shade plot showing the relative abundance of each family
present on each replicate of crop, ileum and caeca mucosa samples of (A)
diet H (Ca, P, and phytase supplementation) and (B) diet A (no supplementation).
The intensity of the color increases to black if the family was detected in higher
abundance, while white indicates family absence.
Figure S2 | (A) Non-metric multi-dimensional scaling (nMDS) plot to illustrates the
three GIT sections crop, ileum and caeca samples, splitted by the type of sample
digesta and mucosa. The symbols represent a unique sample comprising all
OTUs and its abundance information. (B) Venn diagrams of the OTUs
common/unique to the type of samples digesta and mucosa in the three GIT
sections: crop, ileum and caeca. Overlapping areas show the OTUs commonly
shared.
Figure S3 | Bar plots showing the relative abundance of the genus
detected in digesta and mucosa samples in the eight dietary treatments
(A) crop, (B) ileum, and (C) caeca.
Figure S4 | Diversity observed across the three GIT sections studied: crop,
ileum, and caeca and the two type of samples: digesta and mucosa, for
the eight dietary treatments. Values are calculated based on the Shannon
diversity index.
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Borda-Molina et al. Broiler Gastrointestinal Microbiota
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Conflict of Interest Statement: The authors declare that the research was
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