The effect of Enterococcus faecium L6 and Lactobacillus plantarum L10 combination on broiler growth and protection against enterohemorrhagic Escherichia coli O157:H7 challenge

Abstract

Following the ban on antibiotic growth promoters in animal diet in China, probiotics have gained prominence as sustainable alternatives in poultry production. This study evaluated the effects of a novel probiotic combination, Enterococcus faecium (E. faecium) L6 and Lactobacillus plantarum (L. plantarum) L10 on both growth performance and protection against enterohemorrhagic Escherichia coli (EHEC) O157:H7 in broilers. A total of 120 one-day-old male white feather broilers were randomly allocated into 4 groups (n = 3 replicates of 10 chicks each): negative control group received basal diet, whereas the Lpro, Mpro, and Hpro groups received the same basal diet supplemented with the probiotics via drinking water at concentrations of 1, 2, and 4 g/L, respectively, from day 1 to 55. In a parallel experiment, 150 broilers were randomly assigned to 5 groups (n = 3 replicates of 10 chicks each) to evaluate protection against EHEC O157:H7 infection. The results demonstrated that probiotics supplementation improved growth performance and carcass yield, and significantly increased the immune organ indices (P < 0.05). Following EHEC O157:H7 challenge, probiotics treated broilers exhibited enhanced immune and antioxidant capacity, as evidenced by increased serum and ileum levels of immunoglobulin A (IgA), IgG, total antioxidant capacity (T-AOC), catalase (CAT), and glutathione peroxidase (GPx), along with reduced malondialdehyde (MDA) levels. In jejunal mucosa, mRNA expression of Occludin, Claudin-1, Claudin-4, ZO-1, and Mucin-2 was significantly increased. 16S rDNA sequencing of cecal contents revealed that probiotics administration enhanced microbial diversity and richness, and increased the relative abundances of Ruminococcus and Streptococcus. The Mpro group showed a marked proliferation of Lactobacillus. These findings demonstrate that combination of E. faecium L6 and L. plantarum L10 not only enhances broiler growth performance but also provides effective protection against EHEC O157:H7 infection, supporting its potency as a viable antibiotic alternative in broiler production.

Introduction

Poultry meat is a vital source of high-quality animal protein due to its relatively low calorie and cholesterol content, and global demand for poultry products continues to rise steadily (Karim et al., 2023). Nevertheless, industrial-scale poultry operations create conditions of heightened susceptibility to infectious disease, particularly from intestinal pathogens such as Escherichia coli (E. coli). Pathogenic E. coli can compromise the intestinal barrier, cause severe enteritis, and significantly hinder the development of the livestock industry (Guo et al., 2024). Among these pathogens, enterohemorrhagic E. coli (EHEC) O157:H7 is a major foodborne bacterium that poses a serious threat to poultry breeding industry. EHEC O157:H7 is characterized by its ability to induce hemorrhagic intestinal inflammation, disrupt epithelial transport and barrier function, and cause high morbidity and mortality rates in broilers (Liang et al., 2021; Guo et al., 2024). Usually, the antimicrobials are widely used for the prevention and treatment of EHEC O157:H7 infection, while the formidable biofilm-forming capacity of EHEC, which confers enhanced resistance to conventional antimicrobials, along with prevalent antibiotic residues in animal products, collectively threatens public health security (Ogbuewu et al., 2022; Jeon et al., 2025). Therefore, it is urging to find alternatives to reduce the incidence of bacterial infections, improve feed efficiency and economic benefit in broiler chickens since all antimicrobial growth promoters have been banned in China since 2020 according to the Announcement No. 194 of Ministry of Agriculture and Rural Affairs.

Probiotics have emerged as one of the most promising antibiotic alternatives (Tellez et al., 2012). These beneficial microorganisms can enhance growth performance, suppress specific enteric pathogens, and support intestinal and systemic health in both humans and animals (Zorriehzahra et al., 2016; Alagawany et al., 2018). Unlike antibiotics, probiotics exert their effects without leaving harmful residues or promoting antimicrobial resistance. They have been shown to enhance intestinal barrier function, boost immune responses, and increase antioxidant capacity, contributing to improved animal health and performance (Getahun et al., 2025).

Enterococcus faecium (E. faecium) and Lactobacillus plantarum (L. plantarum) are lactic acid bacteria belonging to the order Lactobacillales within the phylum Firmicutes, and they demonstrated significant improvements in growth performance and notable antimicrobial activity against bacterial infections, particularly the E. coli infection (Cao et al., 2024; Atan Cirpici and Kirkpinar, 2025; Cui et al., 2025; Tong et al., 2025). E. faecium L6, isolated from healthy grass carp in our prior research, possessed proven gastrointestinal tolerance, thermostability, and resistance to bile salts and acid. Furthermore, dietary supplementation with this strain significantly enhanced growth performance, immune function, and resistance to Aeromonas hydrophila in fish. (Sun et al., 2025). Our preliminary experiments indicated that E. faecium L6 exhibited a high survival rate and strong colonization capacity in the gastrointestinal tract of chickens. In parallel, L. plantarum L10 was isolated from the gut of healthy broilers and demonstrated potent antimicrobial activity, as evidenced in Supplementary Fig. S1 and S2. Given their complementary properties, this study aimed to investigate the effects of the combining E. faecium L6 L. plantarum L10 on growth performance in broiler chickens and infection resistance to EHEC O157:H7.

Materials and methods

Preparation of E. faecium L6 and L. plantarum L10 powder

A series of single-factor screening experiments were conducted to screen eight candidate cryoprotective agents, including sucrose, trehalose, maltodextrin, skim milk powder, sodium glutamate, mannitol, glycerol, and sodium L-ascorbate, for their efficacy in protecting bacterial viability during freeze-drying. Suspensions of E. faecium L6 and L. plantarum L10 in the optimized protective medium were lyophilized at a vacuum pressure of 0.37 mbar and a condenser temperature of -42°C. Post-lyophilization viability was quantified via standardized plate counting on DeMan, Rogosa and Sharpe (MRS) agar. Colony-forming units (CFUs) were adjusted according to the specific recovery rates of the protective matrix to ensure accurate quantification.

Broilers and experimental design

A total of 300 one-day-old male white feather broilers (Nanhai Xiaobai chicken) with uniform body weights was purchased from Yangling JuQin Commercial Hatchey (Yangling, Shaanxi, China). To evaluate the probiotic effect of E. faecium L6 and L. plantarum L10 combination, 120 broilers were randomly allocated into four groups (n=3 replicates/group, 10 chicks/replicate): negative control group (NC) fed basal diet; whereas probiotic supplementation groups (low-dose probiotics, Lpro; medium-dose probiotics, Mpro; high-dose probiotics, Hpro) received the same basal diet plus E. faecium L6 and L. plantarum L10 combination (1.275 × 10⁹ CFU/g) via drinking water at concentrations of 1, 2, and 4 g/L, respectively. The body weights were recorded on day 1, 14, 21, 28, 42, and 56 following a 12 h fasting period. On days 56, six broilers of each group were euthanized, the immune organs (thymus, spleen and bursa of Fabricius) were immediately excised and weighed to determine the organ indices. Cecal contents were aseptically collected for microbial diversity analysis.

To evaluate the protective effect of combined probiotics E. faecium L6 and L. plantarum L10 against EHEC O157:H7 infections, a total of 150 broilers were randomly divided into five groups (n=3 replicates/group, 10 chicks/replicate): negative control group (NC; basal diet); positive control group (PC; basal diet, EHEC infected); doxycycline group (Dox; infected, 2 g/L doxycycline for 7 days); Probiotic therapeutic group (ProT; infected, 2 g/L probiotics for 7 days post-infection); Probiotic preventive group (ProP; 2 g/L probiotics for 13 pre-infection). The doxycycline or probiotics (1.275 × 10⁹ CFU/g) were administered via drinking water. On day14, all broilers except the NC group were challenged with EHEC O157:H7 (5 × 10⁷ CFU/mL). The morbidity (%), mortality (%) and recovery rate (%) were subsequently monitored. On day 21, six broilers per group were euthanized aseptically. Broilers were considered recovered when their clinical symptoms had completely disappeared and no pathological changes were observed during autopsy on day 21. The recovery rate was defined as the number of recovered broilers divided by the total number of infected broilers. Tissue samples were either processed immediately for analysis, or flash-frozen in liquid nitrogen and stored at -80°C for subsequent studies.

Throughout the experimental period, the feed and water were offered ad libitum. The composition and nutrient profile of the basal diet is shown in Table 1. Room temperature was maintained at 35°C during the first week and gradually reduced to 24°C. All animal experiments were performed according to the protocols approved by the Animals Ethics Committee of Northwest A&F University (20250310).

Table 1.

Ingredient and nutrient composition of basal diet for broilers.

Ingredient (%)	Starter (day 1-22)	Grower (day 22-56)
Corn	55.0	62.0
Soybean meal	35.0	28.0
Fish meal	3.0	2.0
Soybean oil	3.0	4.5
Calcium carbonate	1.2	1.0
Calcium hydrogen phosphate	1.0	0.8
NaCl	0.3	0.25
Amino acidsa	0.3	0.2
Sodium bicarbonate	0.2	0.15
Vitamin premixb	0.5	0.5
Mineral premixc	0.5	0.5

^aThe amino acids was composed of the Lysine 80%, Methionine 15%, Tryptophan 5%.

^bThe vitamin premix was composed of the following per kg diet: Vit_A 12000 IU; Vit_D3, 3500 IU; Vit_E,40 IU; Vit_K3, 3.00 mg; Vit_B1, 3.00 mg; Vit_B2, 6.50 mg; Vit_B6, 2.60 mg; Vit_B12, 0.02 mg; D-Biotin, 0.22 mg; Nicotinic acid, 31.20 mg; Folic acid, 1.00 mg; Vit B5, 10.00 mg;

^cThe mineral premix was composed of the following per kg diet: Fe, 65.00 mg; Cu, 10.00 mg; Mn, 92.00 mg; Zn, 92.00 mg; I, 0.80 mg; Se, 0.40 mg.

Growth performance and carcass characteristics

At 1, 7, 28 and 55 days of age, broilers were fasted for 12 h, and then individually weighed. Growth performance parameters, including average daily feed intake (ADFI), average daily gain (ADG), and feed conversion ratio (FCR, ADF/ADG) were calculated. Additionally, carcass characteristics, including slaughter yield, breast muscle percentage, and leg muscle percentage were evaluated according to standard evisceration procedures.

Histological analysis

For the growth performance analysis, the mean villous height (VH) and crypt depth (CD) in duodenum of probiotic treated broilers were measured, and the VH:CD ratio was subsequently calculated. In the EHEC O157:H7 challenge experiment, duodenal and liver tissues were collected to assess histopathological changes. All tissue samples were fixed in 4% paraformaldehyde for 24 to 48 h, dehydrated with a graded ethanol series, embedded in paraffin, sectioned into 5 μm thickness, and stained with hematoxylin and eosin (H&E).

Determination of serum and intestinal tract biochemical indices

On day 21 post-infection with EHEC O157:H7, blood and ileum were collected from six randomly selected broilers per group. Antioxidant capacity, immunoglobulin concentrations, and inflammatory cytokine levels were determined using commercial assay kits according to manufacturer's instructions. T-AOC kit (AKAO012M, Boxbio, Beijing, China), Malondialdehyde (MDA) content assay kit (AKFA013M, Boxbio, Beijing, China), Catalase (CAT) activity assay kit (AKA0003-2M, Boxbio, Beijing, China), and Glutathione Peroxidase (GPx) activity assay kit (AKPR014M, Boxbio, Beijing, China). Immunoglobulin levels were assessed using chicken IgG and IgA enzyme-linked immunosorbent assay (ELISA) kits (F-4278-A, F4248-A, Shfksc, Shanghai, China). Inflammatory cytokines, including IL-2 and IL-6, were assessed using chicken-specific ELISA kits (YJ042736, YJ042757, Mlbio., Shanghai, China).

The mRNA expression levels of tight junction proteins (TJPs) related genes

To evaluate the protective efficacy of the E. faecium L6 and L. plantarum L10 combination against EHEC O157:H7-induced intestinal injury in broilers, we measured the mRNA expression of key tight junction protein (TJP)-related genes (Claudin-1, Claudin-4, Occludin, ZO-1, and Mucin-2) in the jejunum using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Total RNA was extracted from jejunum with the High Purity RNA Tissue Kit (Takara, Japan) followed by purity assessment (OD260/280) on a Nanodrop 2000c spectrophotometer (Thermo Scientific, USA). cDNA was subsequently synthesized using the Reverse Transcriptase M-MLV Kit (Takara, Japan) according to the manufacturer's instructions. qRT-PCR was performed on a 7500 Real Time PCR system (Applied Biosystem, USA) with β-actin as the internal control. Primer sequences for target genes are listed in Table S1. The thermal cycling conditions were 95°C for 30 s, followed by 40 cycles of 95°C for 10 s, 30 s under 60°C, and 72°C for 15 s. All reactions were run in triplicate, and gene expression was quantified using the 2^-ΔΔCt method.

Microbial diversity analysis

The cecal of microbiota composition was analyzed by 16S rRNA gene sequencing. Briefly, cecal content samples were aseptically collected under anaerobic conditions, immediately flash-frozen in liquid nitrogen, and stored at -80°C. Microbial DNA was subsequently extracted using the QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany) prior to sequencing. DNA quality and integrity were assessed via agarose gel electrophoresis and spectrophotometric analysis. The V3-V4 hypervariable region of the bacterial 16S rDNA gene was amplified by PCR using the following primers: forward 5′-GGTGCCAGCMGCCGCGG-3′, and reverse 5′-CCGTCAATTCMTTTRAGTTT-3′. PCR products were purified using MagicPure Size Selection Beads (TransGen Biotech. Co., Ltd., Beijing, China) and quantified using QuantiFluor^TM-ST dsDNA System (Promega, USA). Sequencing libraries were prepared with the TruSeq DNA LT Sample Preparation kit (Illumina Inc., San Diego, CA, USA), high-throughput sequencing was performed on an Illumina platform. Prior to analysis, sequences were clustered into 97% similarly operational taxonomic units (OTUs) using QIIME2 (v1.8.0). Representative sequences from each OUTs were taxonomic classified by alignment against reference databases. Alpha diversity indices were calculated with Mothur software (v1.30), while beta diversity analysis was conducted in QIIME2.

Statistical analysis

All statistical analyses were performed using SPSS software (version 26.0; IBM, Armonk, NY, USA). One-way analysis of variance (ANOVA) was used to evaluate differences among groups, followed by the least significant difference (LSD) post hoc test for multiple comparisons. Data are presented as means ± standard deviation (SD), and statistical significance was defined as P < 0.05.

Results

The preparation of E. faecium L6 and L. plantarum L10 combination powder

The optimum freeze-drying protective formulation was determined as 15% skim milk powder, 10% sucrose, and 1% glycerin. The probiotic strains were combined in a 1:1 (v/v) ratio and mixed with the protective agents at a 2:1 (v/v) ratio to the bacterial suspension (initial concentration: 1.5 × 10⁸ CFU/g). This formulation supported a survival rate of 85% after freeze-dryinge, and the final concentration is 1.275 × 10⁹ CFU/g.

Growth performance

As shown in Table 2, supplementation with E. faecium L6 and L. plantarum L10 combination improved the ADG and FCR compared to NC group. Notably, immune organs indices (bursa of Fabricius, thymus and spleen) were significantly higher in Mpro group than in NC group (P < 0.01), suggesting enhanced immune competence. Additionally, carcass traits, including dressing percentage, evisceration yield, pectoral muscle yield and leg muscle yield were all increased in the Mpro group compared to NC group. Duodenal histomorphology analysis revealed significantly greater VH and reduced CD in the probiotic-supplemented groups, leading to a significantly higher VH:CD ratio in both Mpro and Lpro groups compared to NC group (P < 0.01). These morphological improvements indicate an enhanced intestinal structure and improved nutrient absorption efficiency in broilers.

Table 2.

Effect of E. faecium L6 and L. plantarum L10 combination supplementation on growth performance and ileal mucosal structure of in broilers.

Items	Treatment				P-value
	NC	Lpro	Mpro	Hpro
d 56 Body weight (BW, g)	853.82±30.15	889.28±30.32	879.78±62.96	868.37±41.28	0.583
Average daily feed intake (ADFI, g/day)
Day 1-14	12.62	12.62	12.62	12.62
Day 15-28	35.36	35.36	35.36	35.36
Day 29-42	60.00	60.00	60.00	60.00
Day 43-56	90.59	90.59	90.59	90.59
Day 1-56	46.73	46.73	46.73	46.73
Average daily gain (ADG, g/day)
Day 1-14	6.475±0.20c	8.11±0.15a	7.79±0.12b	7.84±0.08b	< 0.001
Day 15-28	13.97±0.52	14.29±1.14	14.71±1.65	14.26±1.19	0.39
Day 29-42	18.08±1.12	17.99±1.34	11.89±9.86	17.35±2.81	0.67
Day 43-56	19.59±1.84	20.26±2.94	19.53±1.27	19.70±3.19	0.67
Day 1-56	14.53±0.54	15.16±0.54	14.99±1.13	14.79±0.73	0.21
Feed conversion ratio (FCR)
Day 1-14	1.95±0.06	1.56±0.03	1.62±0.03	1.61±0.02	< 0.001
Day 15-28	2.53±0.09	2.49±0.20	2.43±0.26	2.49±0.21	0.28
Day 29-42	3.33±0.21	3.35±0.22	3.56±1.02	3.53±0.54	0.53
Day 43-56	4.06±0.39	3.96±0.53	4.05±0.27	4.09±0.64	0.69
Day 1-56	3.22±0.12	3.09±0.11	3.13±0.23	3.17±0.15	0.20
d 56 Villus height, μm	1326.40±496.46ab	1172.70±220.53b	1551.300±206.8a	1079.10±135.49b	0.007
d 56 Crypt depth, μm	162.90±42.61a	88.50±22.00d	91.500±15.76bd	141.20±47.71ac	< 0.001
d 56 Villus height to crypt depth ratio (VH/CD)	8.06±1.525c	13.52±2.03b	17.397±3.69a	8.401±2.95c	< 0.001

NC, negative control; Lpro, low-dose probiotics; Mpro, medium-dose probiotics; Hpro, high-dose probiotics.

The results are presented as the mean ± standard deviation (SD), ^a,b,c,d Mean values within a row with no common superscript differ significantly (P < 0.05).

The therapeutic and preventive effects of E. faecium L6 and L. plantarum L10 combination against EHEC O157:H7 infection were summarized in Table 4. Following EHEC O157:H7 challenge, all broilers exhibited acute clinical symptoms, including lethargy, anorexia, diarrhea, and hyperpyrexia (≥ 41.5°C), with 100% incidence within 24 h. Mortality commenced within 12 hours post-infection in all infected groups. Bursa and thymus indices significantly declined in infected groups (P < 0.05). Following a seven-day treatment, both doxycycline and probiotics effectively alleviated clinical symptoms and reduced mortality from 48% (PC group) to 20%-25%. ProT achieved the highest recovery rate (32%-37%), outperforming the other treatments. Furthermore, ProT group exhibited the best growth recovery, with elevated ADG and reduced FCR relative to NC group. It is indicated that E. faecium L6 and L. plantarum L10 combination not only enhances resistance to EHEC O157:H7 but also effectively supports growth recovery post-infection.

Table 4.

Effect of E. faecium L6 and L. plantarum L10 combination on the growth performance and therapeutic efficacy for ETEC O157:H7-infected broilers.

Items	Treatment					P-value
	NC	PC	Dox	ProT	ProP
BW (g)	220.67±23.04	124.33±16.22	140.92±13.96	189.76±17.52	173.68±20.04	< 0.001
ADFI (g/day)	15.00	15.00	15.00	15.00	15.00
ADG (g/day)	7.04±0.13ad	4.16±0.40bd	5.11±0.06c	7.39±0.09ad	6.33±0.03d	< 0.001
FCR	2.52±0.04bc	2.82±0.27bd	2.95±0.09ad	2.40±0.03c	2.80±0.01bd	< 0.001
Spleen index	0.16±0.02b	0.20±0.08a	0.29±0.05a	0.26±0.05a	0.24±0.09a	0.008
Thymic index	0.65±0.16	0.49±0.07	0.49±0.07	0.61±0.23	0.54±0.21	0.23
Bursa Index	0.51±0.09a	0.18±0.07b	0.20±0.06b	0.21±0.11b	0.24±0.20b	< 0.001
Morbidity (%)	0	100.00	100.00	100.00	100.00
Mortality (%)	0	48.00±0.09	25.00±0.06	22.00±0.08	20.00±0.06	< 0.001
Recovery rate (%)	100.00	0	32.00±0.12	37.00±0.05	33.00±0.10	< 0.001

NC, negative control; PC, positive control; Dox, doxycycline; ProT, Probiotic therapeutic; ProP, Probiotic preventive. BW, body weight; ADFI, average daily feed intake; ADG, average daily gain; FCR, feed conversion ratio.

The results are presented as the mean ± standard deviation (SD), ^a,b,c,d Mean values within a row with no common superscript differ significantly (P < 0.05).

Statistical analysis showed that the ProT group achieved a significantly higher ADG than PC group (P < 0.05), while doxycycline treatment significantly reduced ADG (P < 0.05). Both probiotic groups (ProT and ProP) displayed superior ADG and significantly lower FCR relative to PC group (P < 0.05). Additionally, E. faecium L6 and L. plantarum L10 combination modestly improved immune organ indices, and significantly enhanced bursa of Fabricius and thymus indices compared to NC and PC groups (P < 0.05).

Anatomical observations and histopathological evaluation

Anatomical observations revealed marked pathological differences among the experimental groups (Fig. 1). The NC group exhibited normal tissue architecture across cardiac, hepatic, bursal, thymus, and intestinal tissues. In contrast, the PC group showed severe pathological alterations, including pericardial thickening with turbid myocardial tussue, hepatomegaly with multifocal necrotic lesions, and hemorrhagic congestion in both thymus and bursal tissues. The Dox group exhibited moderate pathological changes, characterized by cardiac fibrinous deposits with increased tissue firmness and petechial hemorrhages in hepatic, bursal, and thymus tissues. Notably, ProT and ProP groups exhibited only minor pathological changes, with minimal fibrinous exudate and rare hemorrhagic foci.

Fig. 1.

Pathological anatomical features of EHEC O157:H7-infected broilers. A. Effects of E. faecium L6 and L. plantarum L10 combination on heart, liver, bursal and thymus. B. Effects of E. faecium L6 and L. plantarum L10 combination on exterior morphology of small intestines.

Small intestinal morphology exhibited marked pathological differences across experimental groups. The PC group displayed severe intestinal edema along with extensive hemorrhagic lesions, whereas the Dox group exhibited only minor residual hemorrhagic spots. Importantly, both ProT and ProP groups maintained normal intestinal architecture, devoid of edema or visible hemorrhagic damage.

Histopathological analysis (Fig. 2. B, H&E staining) revealed severe lesions in the PC group, including villous atrophy, epithelial denudation, and dense neutrophilic infiltration in the small intestine, as well as nuclear pyknosis, extensive necrosis, and marked inflammatory infiltration in the hepatic parenchyma. The Dox group exhibited moderate villous damage and hepatic inflammation accompanied by vacuolar degeneration. In contrast, the ProT and ProP groups showed only mild villous abnormalities and limited hepatic inflammation, by fatty changes, primarily characterized by fatty changes, indicating substantial histological improvement relative to the PC group.

Fig. 2.

Histopathological images of E. faecium L6 and L. plantarum L10 combination on duodenal morphology of broilers (H&E staining, 200 ×) A. Histomorphology of duodenum in healthy broilers. B. Duodenal and liver tissues of EHEC O157:H7-infected broilers.

Immunoglobulin, inflammatory cytokines and antioxidant capacity

After 7 days of treatment, serum and intestinal levels of IgA and IgG in Dox, ProT and ProP groups increased by 1.26-2.38-fold increases compared to PC group following EHEC O157:H7 challenge (Fig. 3. A, P < 0.05). Furthermore, the anti-inflammatory cytokine IL-2 content increased by nearly 40%, while the pro-inflammatory factor IL-6 decreased by approximately 50% (Fig. 3. A, P < 0.05). No significant differences in these cytokine levels were observed among Dox, ProT and ProP groups (P > 0.05). Additionally, antioxidant enzyme activities, including the T-AOC, CAT and GPx were elevated by more than 3-fold in both serum and intestinal issues (Fig. 4), accompanied by a significant decline in the oxidative stress marker MDA.

Fig. 3.

The effects of probiotics treatment on the levels of IgA, IgG, IL-2 and IL-6 in serum (A) and ileum (B) of EHEC O157:H7 infected broilers (d 21) *P < 0.05, ^⁎⁎P < 0.01, ^⁎⁎⁎P < 0.001, ^⁎⁎⁎⁎P < 0.0001.

Fig. 4.

The effects of probiotics treatment on the levels of T-AOC, CAT, GPX, and MDAl in serum (A) and ileum (B) of EHEC O157:H7 infected broilers (d 21) *P < 0.05, ^⁎⁎P < 0.01, ^⁎⁎⁎P < 0.001, ^⁎⁎⁎⁎P < 0.0001.

The expression levels of tight junction proteins related genes

As shown in Fig. 5, EHEC O157:H7 infection significantly downregulated the mRNA expression levels of tight junction proteins Occludin, Claudin-1, Claudin-4, ZO-1, and Mucin-2 compared to the NC group (P < 0.01). These adverse effects were markedly reversed by both doxycycline and probiotics interventions. Notably, the ProP group showed significant upregulation of all five genes, whereas the ProT group showed the most pronounced increase in ZO-1 expression (Fig. 5). These results indicated that prophylactic administration of probiotics more effectively enhances intestinal barrier integrity than post-infection therapeutic intervention. Overall, the E. faecium L6 and L. plantarum L10 combination protected the small intestinal epithelium against EHEC O157:H7-induced tight junction disruption.

Fig. 5.

The effects of probiotics treatment on the relative mRNA expression levels of tight junction proteins (TJPs) related genes of EHEC O157:H7 infected broilers (d 21) *P < 0.05, ^⁎⁎P < 0.01, ^⁎⁎⁎P < 0.001, ^⁎⁎⁎⁎P < 0.0001.

Effects of E. faecium L6 and L. plantarum L10 combination on the intestinal microbiota composition

High-throughput 16S rRNA gene sequencing was utilized to evaluate the effects of E. faecium L6 and L. plantarum L10 combination on the gut microbiota of healthy and EHEC-infected broilers. Venn diagram analysis (Fig. 6. A) indicated that the MPro group exhibited a significantly higher number of unique microbial species compared to the other groups. Alpha-diversity indices (Shannon, Chao1, and ACE indices; Fig. 6. B-D) were significantly elevated in the MPro group relative to PC group (P < 0.05), suggesting that continuous probiotic supplementation enhances both microbial diversity and richness in the gut.

Fig. 6.

The effects of probiotics treatment on the gut microbial community profiling among different groups. (A) OTU venn plot. (B) Shannon index. (C) Chao 1 index. (D) ACE index.

Principal coordinates analysis (PCoA; Fig. 7. A) revealed distinct β-diversity clustering among the treatment groups. The ProP, ProT, and Mpro groups exhibited more dispersed distributions, whereas the NC and PC groups formed tighter clusters, reflecting pronounced differences in microbial community structure. At the phylum level (Fig. 7. B), Firmicutes and Bacteroidetes were dominated across all groups, with their relative abundance ratio remaining stable in the PC group following EHEC O157:H7 infection.

Fig. 7.

Probiotics affect gut microbiota composition. (A) PCoA analysis plot (B) Plot of relative abundance at phylum level (C) Plot of relative abundance at genus level.

Genus-level analysis (Fig. 7. C) indicated consistent enrichment of Ruminococcus and Streptococcus in all probiotic-treated groups, with a notable increase in Lactobacillus abundance in the Mpro group. LEfSe analyses identified 20 significantly differentially abundant families spanning five major taxa (Fig. 8. C). The PC group was characterized by an enrichment of Thermotogae, Thermoleophilia, and Spirochaetia, while the MPro group was dominated by Blastocatellia and Verrucomicrobiae. Linear discriminant analysis effect size (LDA-LEfSe) (Fig. 8) highlighted Firmicutes, specifically the classes Clostridia and Bacilli, and the order Lachnospirales were identified as key discriminative taxa in the MPro group, with Streptococcus and Clostridium as predominant genera. This patter aligned with the overall trends observed under probiotic intervention: Lactobacillus dominated the ProP group, whereas Bacteroidota characterized the ProT group.

Fig. 8.

Regulation of probiotics on intestinal flora. (A). LEfSe evolutionary branch diagram. (B) LDA value distribution histogram. (C). Species abundance clustering heatmap (family).

Discussion

As the implementation of the policy banning the use of antibiotics as antibacterial growth promoters in animal feed, probiotics have gained considerable attention as promising growth promoter and antibiotic alternatives. E. faecium L6 was isolated from the intestine of healthy grass carp, and exhibited beneficial biological properties in fish. We hypothesized that it might exert similar effects in poultry. This hypothesis prompted us to explore the efficacy of E. faecium L6 and L. plantarum L10 combination on broilers' growth performance and EHEC O157:H7 challenge. Our results showed that the E. faecium L6 and L. plantarum L10 combination moderately improved growth performance and carcass characteristics, while it significantly promoted the development of the immune organs in healthy broilers (Table 2, Table 3). The length of VH correlates positively with the absorptive function of the small intestine, whereas the length of CD reflects epithelial cell proliferation and regenerative capacity. A high VH:CD ratio indicates an expanded intestinal surface area conductive to nutrient digestion and absorption (Wang et al., 2021; Closs et al., 2025). In this study, medium-dose of probiotics significantly increased VH and VH:CD ratio, and enhanced the absorptive capacity of broilers. This aligns with previous reports showing that Lactobacillus salivarius and Lactobacillus bulgaricus improved villus morphology and growth performance in broilers (Shokryazdan et al., 2017; Xiang et al., 2022).

Table 3.

Effect of E. faecium L6 and L. plantarum L10 combination supplementation on immune organ indexes and carcass characteristics of broilers.

Items	Treatment				P-value
	NC	Lpro	Mpro	Hpro
Spleen index (d 56)	10.42±0.01b	18.68±0.01a	18.27±0.03a	18.26±0.03a	< 0.001
Thymic index (d 56)	42.89±0.08b	53.10±0.10ab	63.30±0.13a	58.42±0.07a	0.06
Bursa index (d 56)	39.90±0.03b	50.12±0.07ab	56.29±0.03a	56.17±0.13ab	0.03
Dressing rate (d 56)	76.66±0.07	80.63±0.07	81.29±0.11	82.71±0.07	0.35
Total evisceration rate (d 56)	52.35±0.07	58.93±0.05	57.77±0.09	58.52±0.05	0.22
Pectoral muscle rate (d 56)	19.39±0.03	17.53±0.01	20.46±0.04	17.91±0.03	0.25
Leg muscle rate (d 56)	10.54±0.02	9.96±0.01	10.69±0.01	10.99±0.02	0.27

NC, negative control; Lpro, low-dose probiotics; Mpro, medium-dose probiotics; Hpro, high-dose probiotics.

The results are presented as the mean ± standard deviation (SD), ^a,b,c,d Mean values within a row with no common superscript differ significantly (P < 0.05).

EHEC O157:H7 is characterized by a low infection dose and severe clinical manifestations, notably hemorrhagic intestinal inflammation. Chickens serve as one of its hosts, where EHEC infection can impair growth and increase mortality in broilers (Xiang et al., 2022). Probiotics are often administered in combination to enhance their collective efficacy against pathogenic infections (Closs et al., 2025). Synergistic interventions, including combination of L. plantarum and Lactobacillus acidophilus, have demonstrated efficacy in ameliorating Salmonella typhimurium infecton (Junaid et al., 2024). Our results showed that E. faecium L6 and L. plantarum L10 combination significantly attenuated intestinal hemorrhage and preserved normal mucosal morphology in infected broilers, indicating synergistic protective effects. Probiotic mechanisms likely involve lactic acid production and immune enhancement, consistent with previous findings (Onning et al., 2022). EHEC infection in broilers can triggers the expression of several proinflammatory and anti­inflammatory, and reduce the immunity. Our study indicated that EHEC O157:H7 infection significantly increased IL-6 levels, and reduced the anti-inflammatory cytokine IL-2, which were reversed by probiotic combination. IgA is critical for mucosal immunity by preventing pathogen adherence to epithelia surfaces, while IgG modulates systemic inflammation via pathogen neutralization (Corthesy, 2013). Consistent with our findings, Yu et al. reported that Bacillus coagulans and L. plantarum combination markedly enhanced IgA level in serum and jejunal mucosa (Yu et al., 2022). Notably, the E. faecium L6 and plantarum L10 combination effectively alleviated E. coli-induced immunosuppression. E. coli infection can induce significant oxidative stress in the host gut, leading to subsequent inflammation (Cui et al., 2025). Probiotics have been shown to mitigate oxidative damage by enhancing the expression of antioxidant enzymes such as SOD and CAT, thereby strengthening epithelial defense (Zhang et al., 2025). As expected, E. faecium L6 and plantarum L10 combination significantly enhanced the activity SOD, CAT, GSH-Px, with concomitant reduction of MDA production, indicating that the probiotic combination attenuated oxidative stress to protect against enhanced EHEC O157:H7 infected broilers.

The intestinal epithelial barrier serves as the frontline defense against bacterial translocation and harmful substance (Han et al., 2015; Zhao et al., 2021). Pathogenic bacterial infections can impair the integrity and function of the intestinal epithelial barrier by downregulating the ZO-1 and Occludin (Zha et al., 2025). Previous studies showed that EHEC O157:H7 infection downregulated ZO-1 expression, thereby compromising barrier function, while probiotic supplementation could enhance intestinal tight junctions and mitigate inflammatory damage (Wen and Duffy, 2017; Liu et al., 2024). Our findings corroborate these observation, demonstrating that both probiotics and doxycycline treatments significantly upregulated the mRNA expression of Claudin-1, Claudin-4, ZO-1, Occludin and Mucin-2. Notably, the ProP group exhibited the most pronounced effects, indicating that this probiotics combination effectively enhances intestinal barrier function and promotes repair in broilers.

The gut microbiota plays a critical role in modulating intestinal immunity and pathogen resistance in broilers, with the cecum serving as a key indicator of intestinal health (Chen et al., 2025). Our study showed that long-time probiotic administration enhanced microbial diversity and increased beneficial phyla such, aligning with reports that as Firmicutes and Actinobacteria, aligning with previous reports that probiotic promote microbiota maturation (Liu et al., 2023; Bumbie et al., 2024). The resulting microbiota was dominated by Firmicutes, Bacteroidetes, and Proteobacteria, a pattern typical in broilers (Gyawali et al., 2022). Notably, the enrichment of Firmicutes-associated taxa likely underlies the observed health benefits. Specifically, Lactobacillus spp. (e.g., L. plantarum AN1) and Clostridia contribute to anti-inflammatory responses and barrier function (Kuda et al., 2019; Han et al., 2020), while groups like Bacilli and Lachnospirales support nutrient digestion and immunity (Sun et al., 2023). Thus, our results indicated that Firmicutes enrichment mediated by probiotics contributes to improved growth and reduced intestinal disease in broilers.

The limitations of this study include the relatively small sample size, and lack of in-depth mechanisms insights into the action of E. faecium L6 and L. plantarum L10 combination. Additionally, the controlled laboratory conditions may not fully replicate infection dynamics in practical farming environments.

In conclusion, the E. faecium L6 and L. plantarum L10 combination in drink water effectively improved growth performance and carcass traits of broilers, and protects against EHEC O157:H7 infection. Its efficacy is mediated through enhanced nutrient utilization, immune function and antioxidant capacity, intestinal barrier integrity, and optimized gut microbiota. These results support the potential of this probiotic combination as a promising antibiotic alternative in poultry production.

CRediT authorship contribution statement

Weihua Zhang: Writing – original draft, Methodology, Conceptualization. Yu Sun: Methodology, Data curation. Dan Liu: Methodology. Yujun Gao: Data curation. Qiuyan Zhou: Writing – review & editing, Formal analysis. Ying Luo: Writing – review & editing, Formal analysis. Xinyu Huo: Writing – review & editing, Software, Formal analysis. Xiaoqiang Liu: Writing – review & editing, Supervision, Project administration, Funding acquisition.

Disclosures

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.