Restrictions of in-feed antibiotics use in poultry has pushed research toward finding appropriate alternatives such as Direct-Fed Microbials (DFM). In this study, previously tested
Bacillus
isolates (
B
.
subtilis
and
B
.
amyloliquefaciens
) were used to evaluate their therapeutic and prophylactic effects against
Salmonella enterica
serovar Enteritidis (
S
. Enteritidis) in broiler chickens. For this purpose, initial antibacterial activity of
Bacillus
-DFM (10
4
spores/g or 10
6
spores/g) against
S
. Enteritidis colonization in crop, proventriculus and intestine was investigated using an
in vitro
digestive model. Furthermore, to evaluate therapeutic and prophylactic effects of
Bacillus
-DFM (10
4
spores/g) against
S
. Enteritidis colonization, altogether 60 (
n
= 30/group) and 30 (
n
= 15/group) 1-day-old broiler chickens were randomly allocated to either DFM or control group (without
Bacillus
-DFM), respectively. Chickens were orally gavaged with 10
4
cfu of
S
. Enteritidis per chicken at 1-day old, and cecal tonsils (CT) and crop were collected 3 and 10 days later during the therapeutic study, whereas they were orally gavaged with 10
7
cfu of
S
. Enteritidis per chicken at 6-day-old, and CT and crop were collected 24 h later from two independent trials during the prophylactic study. Serum superoxide dismutase (SOD), FITC-d and intestinal IgA levels were reported for both chicken studies, in addition cecal microbiota analysis was performed during the therapeutic study. DFM significantly reduced
S
. Enteritidis concentration in the intestine compartment, and in both proventriculus and intestine compartments as compared to the control when used at 10
4
spores/g and 10
6
spores/g, respectively (
p
< 0.05). DFM significantly reduced FITC-d and IgA as well as SOD and IgA levels (
p
< 0.05) compared to the control in therapeutic and prophylactic studies, respectively. Interestingly, in the therapeutic study, there were significant differences in bacterial community structure and predicted metabolic pathways between DFM and control. Likewise, phylum
Actinobacteria
and the genera
Bifidobacterium, Roseburia, Proteus
, and cc_115 were decreased, while the genus
Streptococcus
was enriched significantly in the DFM group as compared to the control (MetagenomeSeq,
p
< 0.05). Thus, the overall results suggest that the
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