
==== Front
Poult Sci
Poult Sci
Poultry Science
0032-5791
1525-3171
Elsevier

S0032-5791(24)00786-7
10.1016/j.psj.2024.104207
104207
MICROBIOLOGY AND FOOD SAFETY
Protective effects of potential probiotics Lacticaseibacillus rhamnosus SN21-1 and Lactiplantibacillus plantarum SN21-2 against Salmonella typhimurium infection in broilers
Lou Haibo *
Wang Jian *
Wang Yaping *
Gao Yongdong †
Wang Wei weiwang@ecust.edu.cn
*1
⁎ State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China
† Agriculture Technology Extension Service Center of Shanghai, Shanghai 201103, China
1 Corresponding author: weiwang@ecust.edu.cn
11 8 2024
12 2024
11 8 2024
103 12 10420713 6 2024
7 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
This study aimed to explore the probiotic characteristics of Lacticaseibacillus rhamnosus SN21-1 and Lactiplantibacillus plantarum SN21-2 by genotype and phenotype analysis, assess their safety in vitro and in vivo, and investigate the effects of L. rhamnosus SN21-1 and L. plantarum SN21-2 on Salmonella typhimurium-infected broilers in an in vivo experiment. L. rhamnosus SN21-1 and L. plantarum SN21-2 showed antimicrobial activity against pathogens, including S. Typhimurium, resistance to simulated gastrointestinal digestive fluid, and adhesion to HT-29 cells. In addition, L. rhamnosus SN21-1 and L. plantarum SN21-2 showed no resistance to most common antimicrobial agents and no haemolysis in vitro. Whole-genome sequence analyses of L. rhamnosus SN21-1 and L. plantarum SN21-2 provided basic genomic information, functional genes underlying the probiotic characteristics, and evidence of safety. Furthermore, feeding with L. rhamnosus SN21-1 or L. plantarum SN21-2 for 28 d had no significant effect on the growth or blood biochemical parameters of the broilers, and hematoxylin-eosin staining revealed no liver, spleen, heart, or kidney damage. Additionally, L. rhamnosus SN21-1 or L. plantarum SN21-2 did not translocate to the blood, liver, spleen, heart, or kidney of the broilers. Moreover, L. rhamnosus SN21-1 and L. plantarum SN21-2 significantly reduced S. Typhimurium counts in the faeces and caecal contents of S. Typhimurium-infected broilers and reduced small intestinal bleeding in S. Typhimurium-infected broilers. Consequently, L. rhamnosus SN21-1 and L. plantarum SN21-2 have excellent probiotic characteristics and are safe for use as anti-S. typhimurium probiotics in broilers.

Key words

probiotic characteristic
safety
whole-genome sequencing
anti-S. typhimurium
broiler
==== Body
pmcINTRODUCTION

With the ever-growing demand for poultry and poultry products, the poultry industry has grown at a rate of 5% over the last 50 years (Yakubu et al., 2024). Bacterial infection in the poultry industry leads to vast economic losses and reduced animal welfare (Tan et al., 2023). Salmonella typhimurium, one of the most common zoonotic pathogens, poses significant risks to both animal health and global public health (Wang et al., 2023). Young broilers are susceptible to S. Typhimurium because this age group has an immature immune system (Alkie et al., 2019), and S. Typhimurium can lead to compromised growth performance, gut health, and immunity (Choi et al., 2022).

In the past few decades, antibiotics have been widely applied to control or limit bacterial infection and to improve growth performance in animals. However, the extensive use of antibiotics in the poultry industry has led to the emergence of antibiotic-resistant bacteria and antibiotic residues in poultry products. A study on the antimicrobial resistance of 196 Salmonella strains showed that 84.7% of the strains were resistant to at least 1 antimicrobial agent, and 66.8% of the strains were multidrug resistant (Zhang et al., 2021). Hence, finding alternative therapeutics and subtherapeutic agents for use in poultry production is urgently needed.

Probiotics are defined as “live microorganisms that when administered in adequate amounts confer a health benefit on the host” (Gibson et al., 2017). Probiotics are not only effective in treating a variety of animal diseases but also considered safe alternatives to antibiotics. Several studies have collectively demonstrated that probiotics can inhibit the growth of pathogens and have beneficial effects on intestinal morphology, intestinal microflora composition, antioxidative capacity, nutrient absorption, blood biochemical parameters, and immune function in broilers (Cirilo et al., 2023; Khan and Chousalkar, 2020; Wu et al., 2019). Considering the large demand for probiotics in poultry farming, screening new probiotic strains is an important area of research in the poultry industry (Kassa et al., 2023).

In this study, the probiotic characteristics, including antimicrobial activity, resistance to simulated gastrointestinal digestive fluid, adhesion to HT-29 cells and safety, of L. rhamnosus SN21-1 and L. plantarum SN21-2 were evaluated. Furthermore, the genomes of L. rhamnosus SN21-1 and L. plantarum SN21-2 were sequenced to better understand the mechanisms underlying their probiotic characteristics and safety. Finally, the effects of L. rhamnosus SN21-1 and L. plantarum SN21-2 on S. Typhimurium-infected broilers were investigated. This study aimed to screen novel probiotics with anti-S. Typhimurium activity and provide probiotic strain resources for the treatment of S. Typhimurium infection in broilers.

MATERIALS AND METHODS

The animal experimental protocols were approved by the East China University of Science and Technology Bioethics Committee (approval number: ECUST-2023-080). Ciprofloxacin hydrochloride (CIP-HCl) powder (Hefei dragon god Animal Pharmaceutical Co., Ltd, Anhui, China) was used as a positive control against S. Typhimurium infection in the animal experiment. Unless otherwise specified, all the consumables and reagents employed in the present study were acquired from Shanghai Titan Scientific Co., Ltd.

Bacterial Strains and Culture Conditions

L. rhamnosus SN21-1 and L. plantarum SN21-2 were isolated from the gastrointestinal tract of healthy broilers (Shanghai, China). Lacticaseibacillus rhamnosus GG (LGG), L. rhamnosus SN21-1, and L. plantarum SN21-2 were grown at 37°C in deMan, Rogosa, and Sharpe (MRS) media, and the pathogenic strains Salmonella typhimurium SL1344, Escherichia coli ATCC35150, and Staphylococcus aureus ATCC 6538P were grown at 37°C in 2× yeast extract tryptone (2×YT) media. S. Typhimurium SL1344 is resistant to streptomycin. For enumeration of S. Typhimurium SL1344, samples were cultured on Xylose Lysine Desoxycholate (XLD) agar containing 50 µg/mL streptomycin sulfate (Sangon Biotech Co., Ltd., Shanghai, China), which was referred to as XLD-strep.

Evaluation of the Probiotic Characteristics of L. Rhamnosus SN21-1 and L. Plantarum SN21-2

Antimicrobial Activity

Bacterial suspensions of S. aureus, E. coli, and S. Typhimurium were spread onto MRS plates. Sterilized paper discs with a diameter of 7 mm were positioned on the plates, after which 4 μL of the L. rhamnosus SN21-1 or L. plantarum SN21-2 bacterial suspension (∼109 CFU/mL) was impregnated into the sterilized paper discs. CIP paper discs with a diameter of 7 mm (5 μg/disc) and MRS broth-loaded paper discs were used as positive and negative controls, respectively.

Bacterial suspensions of S. aureus, E. coli, and S. Typhimurium were spread onto MRS plates, after which Oxford cups were positioned on the plates. Two hundred microlitres of cell-free supernatant of L. rhamnosus SN21-1 or L. plantarum SN21-2 cultured for 48 h was added to the Oxford cup. MRS broth and the adjusted cell-free supernatant of L. rhamnosus SN21-1 or L. plantarum SN21-2, the pH of which was adjusted to 7.0, were used as controls.

The inhibition zone diameters were determined after culture at 37°C for 48 h.

Resistance to Simulated Gastrointestinal Digestive Fluid

The resistance of L. rhamnosus SN21-1 and L. plantarum SN21-2 to simulated gastrointestinal digestive fluid was tested based on the method of Saelim et al. (2017).

Adhesion to HT-29 Cells

The adhesion ability of L. rhamnosus SN21-1 and L. plantarum SN21-2 to HT-29 cells was measured following our previous method (Lou et al., 2024).

Evaluation of the In Vitro Safety of L. Rhamnosus SN21-1 and L. Plantarum SN21-2

Haemolytic Activity

The haemolytic capacity of L. rhamnosus SN21-1 and L. plantarum SN21-2 was tested according to the method of Biswas, Soren, & Das Mohapatra (2024). S. aureus and LGG were used as β haemolysis control and γ haemolysis control, respectively.

Antimicrobial Susceptibility

The susceptibilities of L. rhamnosus SN21-1 and L. plantarum SN21-2 to common antimicrobial agents, namely, lincomycin (LIN, 2 μg/disc), ciprofloxacin (CIP, 5 μg/disc), ampicillin (AMP, 10 μg/disc), gentamicin (GEN, 10 μg/disc), erythromycin (ERY, 15 μg/disc), tetracycline (TET, 30 μg/disc), chloramphenicol (CHL, 30 μg/disc), and cefazolin (CEF, 30 μg/disc), were individually measured by the method of Das et al. (2016).

Whole-Genome Sequencing and Comparative Genomic Analyses of L. Rhamnosus SN21-1 and L. Plantarum SN21-2

Genome Sequencing and Gene Annotation

Whole-genome sequencing and gene annotation of L. rhamnosus SN21-1 and L. plantarum SN21-2 were performed according to our previous method (Lou et al., 2024).

Comparative Genomics Analysis

The online software Automated multilocus species tree (autoMLST) was used to construct the species phylogenetic trees of L. rhamnosus SN21-1 and L. plantarum SN21-2 (Alanjary et al., 2019). Based on the phylogenetic trees, the 5 strains with genome assembly levels reaching “chromosome” or “complete” that had the closest phylogenetic relationship with L. rhamnosus SN21-1 or L. plantarum SN21-2 were selected for comparative genomic analysis. FastANI (version 1.32) was used to calculate the average nucleotide identity (ANI) values. CD-HIT rapid clustering of similar proteins software (version v4.6.6) with a threshold of 50% pairwise identity and 0.7 length difference cutoff in amino acid was used to analyze the core genes and specific genes, and phylogenetic trees based on the core genes were generated by TreeBeST 1.9.2 using the neighbour-joining method to further analyze the evolutionary relationships among the strains.

Assessment of the Safety of L. Rhamnosus SN21-1 and L. Plantarum SN21-2 in Broilers

Animal Experiments

A total of 42 three-yellow broilers of 1 day old were raised under suitable conditions. After becoming acclimatized to the environment for 7 d, they were randomly allocated to 3 groups (control group, SN21-1 treatment group, and SN21-2 treatment group) with 14 broilers per group. L. rhamnosus SN21-1 and L. plantarum SN21-2 cells were collected and resuspended in 0.85% NaCl solution to 1×109 CFU/mL. During the experiment, the broilers from control group were treated by gavage with 0.85% NaCl solution, while the broilers from SN21-1 treatment group or SN21-2 treatment group were treated by gavage with L. rhamnosus SN21-1 or L. plantarum SN21-2 bacterial suspension. In all experimental groups, every broiler was fed 0.2 mL corresponding solution once every day for 28 d.

Growth Performance

The growth, behavior, health status, and mortality of the broilers were observed every day during the experiment. Mortality was recorded every day, and body weights were measured on d 1, 3, 7, 14, 21, and 28 of the experiment.

Blood Biochemical Indices

Three broilers per group were randomly selected for whole blood collection on d 28 of the experiment. Total protein (TP), aspartate aminotransferase (AST), glucose (GLU), total cholesterol (TC), and triglyceride (TG) in blood serum were measured by a fully automatic biochemical analyzer.

Histopathological Analysis

On d 28 of the experiment, the liver, spleen, heart and kidneys of 1 broiler per group were removed. The hematoxylin-eosin (H&E) staining was performed using our previous method (Lou et al., 2024).

Assessment of the Translocation Capacity of L. Rhamnosus SN21-1 and L. Plantarum SN21-2

On d 28 of the experiment, blood samples, livers, spleens, hearts and kidneys of 10 broilers per group were collected. Livers, spleens, hearts and kidneys were homogenized in 0.85% NaCl solution to a concentration of 1 g/mL, and then blood (3×100 μL) and tissue suspensions (3×100 μL) were added to solid MRS media. Colonies were counted after culture for 48 h.

Effects of L. Rhamnosus SN21-1 and L. Plantarum SN21-2 on S. Typhimurium-Infected Broilers

Animal Experiments

After acclimating to the environment for 7 d, 50 three-yellow broilers of 1 day old were randomly allocated into 5 groups (the control group, disease model group, SN21-1 treatment group, SN21-2 treatment group, and CIP treatment group). The broilers in the control group were treated by gavage with 0.2 mL of 0.85% NaCl solution once every day for 14 d. For the disease model group, the broilers were infected with 0.2 mL of S. Typhimurium bacterial suspension (1×109 CFU/mL) on the 8th d, and the broilers were treated by gavage with 0.2 mL of 0.85% NaCl solution once every day during the remaining experiment. The broilers in the SN21-1 treatment group or the SN21-2 treatment group were treated by gavage with 0.2 mL of L. rhamnosus SN21-1 or L. plantarum SN21-2 bacterial suspension (1×109 CFU/mL) once every day for 14 d, and the broilers were infected with 0.2 mL of S. Typhimurium bacterial suspension (1×109 CFU/mL) on the 8th d. For the CIP treatment group, the broilers were infected with 0.2 mL of S. Typhimurium bacterial suspension (1×109 CFU/mL) on the 8th d, and CIP-HCl powder was added to the drinking water to a concentration of 1 g/L from the 8th to the 10th d.

Growth Performance

The broilers were examined for growth, behaviour, health status, and death during the experiment. Mortality was recorded every d, and body weights were measured on the 1st, 8th, 10th, and 14th d of the experiment.

Enumeration of S. Typhimurium in Broiler Faeces

Broiler faeces were collected on the 8th, 10th, 12th, and 14th d of the experiment. The collected faeces were homogenized in 0.85% NaCl solution to a concentration of 0.1 g/mL and 10-fold serially diluted, after which 3×10 μL of each dilution was spotted on XLD-strep plates. The plates were cultured for 48 h before colonies were counted.

Exterior Morphological Observation of Small Intestines and Enumeration of S. Typhimurium in Caecal Contents of Broilers

Three broilers from each group were randomly selected and euthanized on the 14th d. The exterior morphology of the small intestines was observed. The caecal contents were collected and homogenized in 0.85% NaCl solution to a concentration of 0.1 g/mL and 10-fold serially diluted, and 3×10 μL of each dilution was spotted on XLD-strep plates. The plates were cultured for 48 h before colonies were counted.

Statistical Analysis

Each experiment in this study was carried out in triplicate. The means ± SDs were used to present the experimental data. The statistical significance was analyzed by the unpaired 2-tailed Student's t test or 1-way analysis of variance (ANOVA) following Tukey's post hoc test using GraphPad Prism (version 9). P < 0.05 indicated a statistically significant difference. The S. Typhimurium counts in the faeces and caecal contents of the broilers were converted to log (CFU+1) since some samples had no S. Typhimurium detected (zero count) and log (zero) did not exist.

RESULTS

Probiotic Properties of L. Rhamnosus SN21-1 and L. Plantarum SN21-2

Antimicrobial Activity

The inhibitory areas of CIP against S. aureus, E. coli, and S. Typhimurium were 34.22±1.09 mm, 57.89±1.05 mm, and 48.22±1.64 mm, respectively (Figure 1). The inhibitory areas of L. rhamnosus SN21-1 against S. aureus, E. coli, and S. Typhimurium were 15.11±0.93 mm, 31.67±1.58 mm, and 31.22±2.17 mm, respectively, and the inhibitory areas of L. plantarum SN21-2 against S. aureus, E. coli, and S. Typhimurium were 16.33±1.12 mm, 32.11±2.15 mm, and 31.78±2.44 mm, respectively (Figure 1).Figure 1 Zones of inhibitory activity of SN21-1 and SN21-2 against S. typhimurium (A), E. coli (B), and S. aureus (C). (D) Inhibition zone diameters of SN21-1 and SN21-2 against S. typhimurium, E. coli, and S. aureus. A-C: a-d represent CIP, MRS control, SN21-1, and SN21-2, respectively. D: Different letters indicate significant differences (P < 0.05).

Figure 1

Organic acids and bacteriocin are considered 2 key antimicrobial factors of LAB (Wu et al., 2023). Two bacteriocin clusters, garvieacin Q and carnocin CP52, were identified by BAGEL 4 in the L. rhamnosus SN21-1 genome. One bacteriocin cluster, plantaricin E/F, was identified by BAGEL 4 in the L. plantarum SN21-2 genome. Garvieacin Q can inhibit pathogenic Enterococcus faecalis, Bacillus coagulans, Lactococcus garvieae, Listeria ivanovii, Listeria monocytogenes, and Pediococcus pentosaceus (Tosukhowong et al., 2012). Carnocin CP52 exhibited inhibitory activity against Enterococcus and Listeria (Yang et al., 2023). Plantaricin E/F exhibits a comparatively narrow antimicrobial spectrum and is effective mainly against Lactobacilli species (Diep et al., 2009). Garvieacin Q, carnocin CP52, and plantaricin E/F did not show antibacterial activity against S. aureus, E. coli, or S. typhimurium.

The inhibitory area sizes of the cell-free culture supernatant (pH = 4.0) of L. rhamnosus SN21-1 against S. aureus, E. coli, and S. Typhimurium were 31.00±1.73 mm, 36.67±2.52 mm, and 38.67±2.31 mm, respectively, and the inhibitory area sizes of the cell-free culture supernatant (pH=4.0) of L. plantarum SN21-2 against S. aureus, E. coli, and S. Typhimurium were 20.33±2.08 mm, 23.67±0.58 mm, and 26.00±1.73 mm, respectively, while the adjusted cell-free culture supernatants (pH = 7.0) of L. rhamnosus SN21-1 and L. plantarum SN21-2 or MRS broth control could not inhibit these 3 pathogens (Figure 2). The results demonstrated that the antibacterial activity of L. rhamnosus SN21-1 and L. plantarum SN21-2 against these 3 pathogens was mainly due to organic acids.Figure 2 Antibacterial activity of cell-free culture supernatants of SN21-1 and SN21-2 against S. typhimurium, E. coli, and S. aureus. A-C represent the antibacterial activity of the cell-free culture supernatant of SN21-1 against S. typhimurium (A), E. coli (B), and S. aureus (C), respectively, and D-F represent the antibacterial activity of the cell-free culture supernatant of SN21-2 against S. typhimurium (D), E. coli (E), and S. aureus (F), respectively. a, Cell-free culture supernatant (pH = 4.0) of SN21-1 cultured for 48 h; b, adjusted cell-free culture supernatant of SN21-1 cultured for 48 h for which the pH was adjusted to 7.0; c, MRS broth; d, cell-free culture supernatant (pH = 4.0) of SN21-2 cultured for 48 h; e, adjusted cell-free culture supernatant of SN21-2 cultured for 48 h for which the pH was adjusted to 7.0.

Figure 2

Resistance to Simulated Gastrointestinal Digestive Fluid

L. rhamnosus SN21-1 displayed a 99.77%±0.85% survival rate after 2 h of incubation in simulated gastric fluid, a 52.43%±0.90% survival rate after 6 h of incubation in simulated intestinal fluid, and a final 52.31%±1.14% survival rate following exposure to simulated gastric fluid and simulated intestinal fluid, while L. plantarum SN21-2 displayed a 100.41%±0.44% survival rate after 2 h of incubation in simulated gastric fluid, a 73.51%±1.53% survival rate after 6 h of incubation in simulated intestinal fluid, and a final 73.81%±1.65% survival rate following exposure to simulated gastric fluid and simulated intestinal fluid (Table 1).Table 1 Resistances of SN21-1 and SN21-2 to the simulated gastrointestinal digestive fluid.

Table 1Strain	Simulated gastrointestinal digestive fluid resistance	Survival (%)	
	Simulated gastric fluid (log CFU/ml)	Survival (%)	Simulated intestinal fluid (log CFU/ml)	Survival (%)		
	0 h	2 h		8 h			
SN21-1	9.15±0.05	9.13±0.04	99.77±0.85a	4.78±0.10	52.43±0.90b	52.31±1.14b	
SN21-2	9.03±0.03	9.07±0.03	100.41±0.44a	6.66±0.14	73.51±1.53a	73.81±1.65a	
Note: Data are expressed as the means ± SD. Different letters in each column indicate significant differences (P < 0.05).

In the genome of L. rhamnosus SN21-1, F0F1-ATPase genes, ornithine decarboxylase gene, ATP-binding cassette (ABC) superfamily genes, and major facilitator superfamily (MFS) transporter genes were identified (Supplementary Table S1). In the genome of L. plantarum SN21-2, F0F1-ATPase genes, glutamate decarboxylase gene, choloylglycine hydrolase gene, ABC superfamily multidrug efflux pump genes, and MFS transporter genes were detected (Supplementary Table S2). DNA repair protein genes, universal stress protein genes, protease genes, and chaperone genes were identified in L. rhamnosus SN21-1 (Supplementary Table S1) and L. plantarum SN21-2 (Supplementary Table S2).

F0F1-ATPases contribute to maintaining cytoplasmic pH homeostasis by extruding excess H+ ions from cells when exposed to an acidic environment (Arnold et al., 2018). Amino acid decarboxylase helps maintain pH homeostasis by consuming intracellular protons (Wang et al., 2020). Ornithine decarboxylase catalyses the decarboxylation of ornithine. Ornithine decarboxylase contributes to resisting external acidic environments and maintaining normal bacterial growth (Romano et al., 2014). Glutamate decarboxylase catalyzes the decarboxylation of glutamate, so probiotics can exploit this property to remove intracellular protons in acidic environments (Feehily and Karatzas, 2013).

The ABC-type multidrug transport system and MFS transporter can expel various compounds from the cell, including bile salts. The ABC-type multidrug transport system and MFS transporter have been reported to be involved in bile tolerance in L. acidophilus NCFM (Pfeiler and Klaenhammer, 2009). BSH can hydrolyze conjugated bile salts and reduce their toxicity (Papadimitriou et al., 2015), which improves the bile salt resistance of probiotics (Bi et al., 2016). ABC-type multidrug transport system genes and MFS transporter genes were identified in both the L. rhamnosus SN21-1 and L. plantarum SN21-2 genomes, while the BSH gene was identified in only the L. rhamnosus SN21-1 genome. The difference in genotype between these 2 strains may explain why the survival rate of L. plantarum SN21-2 was greater than that of L. plantarum SN21-2 after 6 h of incubation in simulated intestinal fluid (Table 1).

Universal stress proteins help bacteria survive in stressful environments (Luo et al., 2023). In adverse environments, the timely repair of damaged proteins and DNA is crucial for the viability of probiotics. DNA repair proteins help detect and repair various types of DNA damage. Chaperones protect normal proteins and refold proteins that have been damaged or misfolded, while proteases breakdown irreversibly damaged proteins into their constituent amino acids, which are then recycled.

Taken together, these genes enable L. rhamnosus SN21-1 and L. plantarum SN21-2 to survive in simulated gastrointestinal conditions.

Adhesion to HT-29 Cells

The adhesion percentage of L. rhamnosus SN21-1 to HT-29 cells, 58.24%±1.98%, was greater than that of L. plantarum SN21-2, 49.00%±0.96% (Figure 3).Figure 3 Adhesion of SN21-1 and SN21-2 to HT-29 cells. Different letters indicate significant differences (P < 0.05).

Figure 3

In the genomes of L. rhamnosus SN21-1 and L. plantarum SN21-2, elongation factor Tu (EF-Tu), enolase (ENO), pyruvate dehydrogenase E1 component beta subunit (PDHB), sortase A, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and triosephosphate isomerase (TPI) genes were detected (Table S1, Table S2). In addition, fibronectin binding protein was detected in the genome of L. rhamnosus SN21-1 (Table S1). Fibronectin binding proteins bind to fibronectin, a glycoprotein present on the surface of animal intestinal epithelial cells and can be used as a substratum by bacteria for adhesion (Jayashree et al., 2018). PDHB improved the adhesion of L. plantarum to fibronectin (Vastano et al., 2014). Three moonlighting proteins, GAPDH, EF-Tu, and ENO, are involved in the adhesion of L. plantarum PO23 to intestinal epithelial cells (Liu et al., 2024). Sortase A is involved in the anchoring of adhesive proteins to the cell wall (Kumar et al., 2022), which contributes to the binding of adhesive proteins to host cells. TPI is involved in the adhesion of L. plantarum 423 to Caco-2 cells (Ramiah et al., 2008).

These genes enable L. rhamnosus SN21-1 and L. plantarum SN21-2 to adhere to HT-29 cells.

Assessment of the Safety of L. rhamnosus SN21-1 and L. plantarum SN21-2 In Vitro

Potential probiotic strains should be evaluated for antibiotic resistance to prevent the transfer of undesirable antibiotic resistance into the intestinal niche (Parveen Rani et al., 2016). No antimicrobial resistance genes were detected in either the L. rhamnosus SN21-1 genome or the L. plantarum SN21-2 genome through ResFinder 4.3.3. L. rhamnosus SN21-1 exhibited no resistance to any of the tested antimicrobial agents, and L. plantarum SN21-2 exhibited no resistance to any of the tested antimicrobial agents except lincomycin (Table 2). The ABC-type multidrug transport system was shown to expel antibiotics out of bacteria (Browning et al., 2018), so the resistance of L. plantarum SN21-2 to lincomycin may be attributed to the presence of an ABC-type multidrug transport system.Table 2 Sensitivity of SN21-1 and SN21-2 to antimicrobial agents.

Table 2Strains	GEN	TET	CIP	ERY	CHL	AMP	LIN	CEF	
SN21-1	14.56 ± 1.24 (I)	38.33 ± 1.66 (S)	31.67 ± 2.29 (S)	42.11 ± 2.37 (S)	37.11 ± 2.71 (S)	23.33 ± 1.50 (S)	24.56 ± 2.24 (S)	23.00 ± 2.00 (S)	
SN21-2	15.89 ± 1.27 (S)	20.67 ± 1.58 (S)	16.89 ± 1.17 (I)	32.44 ± 1.67 (S)	32.00 ± 1.94 (S)	32.67 ± 2.00 (S)	8.89 ± 0.78 (R)	29.11 ± 2.89 (S)	
Abbreviations: R, resistant; S, sensitive; I, intermediate.

UTP-glucose-1-phosphate uridylyltransferase, EF-Tu, response regulator ArlR, dTDP-glucose-4,6-dehydratase, and glucose-1-phosphate thymidylyltransferase genes were detected in the Virulence Factor Database (VFDB) in the genomes of L. rhamnosus SN21-1 and L. plantarum SN21-2 (Table S3). In addition, the ATP-dependent Clp protease gene and enolase gene were detected by VFDB in the genome of L. plantarum SN21-2 (Table S3). UTP-glucose-1-phosphate uridylyltransferase is involved in capsule and lipopolysaccharide biosynthesis and biofilm formation in several bacterial pathogens (Wu et al., 2021), but these functions contribute to the beneficial effects of probiotics. Glucose-1-phosphate thymidylyltransferase and dTDP-glucose 4,6-dehydratase are key to the synthesis of L-rhamnose. The EF-Tu is involved in the adherence of bacteria to host cells. The response regulator ArlR influences biofilm formation, agglutination, and autolysis in S. aureus (Zhou et al., 2024). ATP-dependent Clp protease plays a crucial role in responding to diverse environmental stresses in bacteria (Ni et al., 2021). Enolase plays a role both in glycolysis and in bacterial adhesion (Xie et al., 2023). Hence, no virulence genes exist in L. rhamnosus SN21-1 or L. plantarum SN21-2.

Haemolysis of pathogenic bacteria can cause anaemia and oedema in the infected host. LGG, L. rhamnosus SN21-1 and L. plantarum SN21-2 caused γ haemolysis (no haemolysis), while S. aureus caused β haemolysis (Figure 4).Figure 4 Haemolytic activity tests of SN21-1 (A) and SN21-2 (B). a-d represent LGG, S. aureus, SN21-1, and SN21-2, respectively.

Figure 4

Genomic Properties of L. Rhamnosus SN21-1 and L. Plantarum SN21-2

The genome profiles of L. rhamnosus SN21-1 and L. plantarum SN21-2 are shown in Figure 5 and Table 3. The complete genome sequence of L. rhamnosus SN21-1 consisted of a single circular chromosome of 2 962 769 bp with an average GC content of 46.80%, and the complete genome sequence of L. plantarum SN21-2 consisted of a single, circular chromosome of 3,199,580 bp with 44.66% average GC content. The whole-genome sequences of L. rhamnosus SN21-1 and L. plantarum SN21-2 have been deposited in GenBank under the BioProject number PRJNA1037306 for L. rhamnosus SN21-1 and the BioProject number PRJNA1037313 for L. plantarum SN21-2. The L. rhamnosus SN21-1 genome contained 2 832 genes, 59 tRNAs, 15 rRNAs, 7 sRNAs, 172 tandem repeats and 5 CRISPR sequences, and the L. plantarum SN21-2 genome contained 3 046 genes, 66 tRNAs, 16 rRNAs, 1 sRNA, 134 tandem repeats and 1 CRISPR sequence.Figure 5 Circular maps of the SN21-1 chromosome (A) and SN21-2 chromosome (B). From the outside to the center, the outermost circle is the identification of genome size; the second and third circles are coding sequences on positive and negative chains colored according to the functional classification of COG; the fourth and fifth circles are the ncRNAs on positive and negative chains; the sixth circle is the repeat sequences; and the seventh and eighth circles are GC content and GC skew value, respectively.

Figure 5

Table 3 Genomic attributes of SN21-1 and SN21-2.

Table 3Attributes	SN21-1	SN21-2	
Genome size	2,962,769	3,199,580	
GC content (%)	46.80%	44.66%	
Genes	2,832	3 046	
tRNA	59	66	
5s rRNA	5	6	
16s rRNA	5	5	
23s rRNA	5	5	
sRNA	7	1	
Plasmids	0	0	
Tandem repeats	172	134	
Prophages	0	0	
CRISPR	5	1	

Comparative Genomics Analysis

The autoMLST-constructed phylogenetic tree of L. rhamnosus SN21-1 showed that L. rhamnosus BFE5264, L. rhamnosus LRB, L. rhamnosus GG_GG, L. rhamnosus GG, and L. rhamnosus 4B15 had the closest evolutionary relationships with L. rhamnosus SN21-1 (Supplementary Figure S1A). The average nucleotide identity (ANI) threshold of 95–96% was used to define the same bacterial species (Yang et al., 2024). The 6 strains were confirmed to be the same species based on the ANI values among them (Supplementary Figure S2A). These 6 L. rhamnosus strains contained 2,013 core genes (Figure 6A). Unique gene numbers among the 6 L. rhamnosus strains varied from 0 (L. rhamnosus GG) to 177 (L. rhamnosus SN21-1) (Figure 6A). The phylogenetic tree generated based on sequence similarities of the 2,013 core genes showed that L. rhamnosus SN21-1 had the closest genetic relationship with L. rhamnosus GG (Figure 6B).Figure 6 Comparative genomic analyses of 6 L. rhamnosus strains or 6 L. plantarum strains. (A) The core genes and unique genes among the 6 L. rhamnosus strains. (B) Phylogenetic tree based on sequence similarities among 2013 core genes of 6 L. rhamnosus strains. (C) The core genes and unique genes among the 6 L. plantarum strains. (D) Phylogenetic tree based on sequence similarities among 2335 core genes of 6 L. plantarum strains.

Figure 6

The autoMLST-constructed phylogenetic tree of L. plantarum SN21-2 showed that L. plantarum DOMLa, L. plantarum JDM1, L. plantarum subsp. plantarum P-8, L. plantarum 16, and L. plantarum ZJ316 had the closest evolutionary relationships with L. plantarum SN21-2 (Supplementary Figure S1B). The 6 strains were confirmed to be the same species based on the ANI values among them (Supplementary Figure S2B). These 6 L. plantarum strains contained 2 335 core genes. Unique gene numbers in the 6 L. rhamnosus strains varied from 2 (L. plantarum DOMLa, L. plantarum JDM1) to 193 (L. plantarum ZJ316), with L. plantarum SN21-2 having 101 unique genes (Figure 6C). The phylogenetic tree generated based on sequence similarities of the 2,335 core genes showed that L. plantarum SN21-2 had the closest genetic relationship with L. plantarum JDM1 (Figure 6D).

Assessment of the Safety of L. Rhamnosus SN21-1 and L. Plantarum SN21-2 In Broilers

Growth Performance

The broilers in all treatment groups behaved normally. Treatment with L. rhamnosus SN21-1 or L. plantarum SN21-2 did not significantly influence the body weight of the broilers on d 1, 3, 7, 14, 21, or 28 of the experiment (Figure 7).Figure 7 Effects of SN21-1 and SN21-2 on the body weight of broilers on d 3, 7, 14, 21, and 28 of the experiment.

Figure 7

Detection of Blood Biochemical Indices

Treatment with L. rhamnosus SN21-1 or L. plantarum SN21-2 had no significant influence on the TP, AST, GLU, TC, or TG content of the broilers (Figure 8). AST is regarded as a highly sensitive indicator of hepatotoxicity in birds (Harr, 2002). The normal AST level indicated that L. rhamnosus SN21-1 and L. plantarum SN21-2 did not cause liver damage in broilers. Similarly, TP, GLU, TC, and TG concentrations showed that L. rhamnosus SN21-1 and L. plantarum SN21-2 did not significantly impact protein synthesis, glucose metabolism or lipid metabolism in broilers.Figure 8 Effects of SN21-1 and SN21-2 on blood biochemical indices of broilers.

Figure 8

Bacterial Translocation Detection and Histopathological Observation

In the L. rhamnosus SN21-1-treated group and L. plantarum SN21-2-treated group, no L. rhamnosus SN21-1 or L. plantarum SN21-2 were determined in the blood, liver, spleen, heart, or kidney (Table 4), and no liver, spleen, heart, or kidney damage was found (Figure 9).Table 4 Translocation of SN21-1 and SN21-2 to blood, liver, spleen, heart, or kidney in broilers.

Table 4Organs	Treatments	
NaCl	SN21-1	SN21-2	
Blood	0/10	0/10	0/10	
Heart	0/10	0/10	0/10	
Liver	0/10	0/10	0/10	
Spleen	0/10	0/10	0/10	
Kidney	0/10	0/10	0/10	

Figure 9 Histopathological observation of the liver, spleen, heart, and kidney in broilers fed SN21-1 and SN21-2.

Figure 9

Effects of L. Rhamnosus SN21-1 and L. Plantarum SN21-2 on S. Typhimurium-Infected Broilers

Growth Performance

During the experiment, there were no deaths or signs of disease visible to the naked eye for the broilers in any of the treatment groups. No significant differences in the body weights of the broilers were detected among the treatment groups on d 1, 8, 10, and 14 of the experiment (Figure 10).Figure 10 Effects of SN21-1 and SN21-2 on the body weight of S. Typhimurium-infected broilers on d 8, 10, and 14 of the experiment.

Figure 10

Enumeration of S. Typhimurium in the Faeces and Caecal Contents of Broilers

Treatments with L. rhamnosus SN21-1, L. plantarum SN21-2, or CIP decreased the S. Typhimurium counts in the faeces and caecal contents of the S. Typhimurium-infected broilers (Table 5). The faeces of the broilers in the CIP treatment group had the lowest S. Typhimurium counts during the 3 d of CIP treatment, while the S. Typhimurium counts in the faeces of the broilers in the CIP treatment group tended to increase after 3 d of CIP treatment. In the L. rhamnosus SN21-1 treatment group and L. plantarum SN21-2 treatment group, S. Typhimurium counts in the faeces of broilers continuously decreased. On the 14th d of the experiment, S. Typhimurium strains were not detected in the caecal contents of broilers from the L. plantarum SN21-2 treatment group, and fewer S. Typhimurium strains were detected in the caecal contents of broilers from the L. rhamnosus SN21-1 treatment group than in the caecal contents of broilers from the CIP treatment group.Table 5 S. Typhimurium counts in broilers of different treatment groups.

Table 5S. Typhimurium counts	Treatment	
Control group	Disease model group	SN21-1 treatment group	SN21-2 treatment group	CIP treatment group	
Faeces	D8	0.00±0.00d	7.39±0.01a	6.19±0.08b	5.68±0.17c	5.36±0.10c	
D10	0.00±0.00d	5.37±0.05a	4.06±0.03b	3.20±0.06c	0.00±0.00d	
D12	0.00±0.00d	4.02±0.06a	3.52±0.07b	0.00±0.00c	3.36±0.10b	
D14	0.00±0.00b	3.69±0.09a	0.00±0.00b	0.00±0.00b	3.56±0.07a	
Caecal contents	D14	0.00±0.00d	5.66±0.10a	3.50±0.17c	0.00±0.00d	3.88±0.03b	
Note: Data are expressed as the means ± SDs of the Log (CFU/g+1). Different letters in each row indicate significant differences (P < 0.05).

Exterior Morphological Observation of Small Intestines

As shown in Figure 11, L. rhamnosus SN21-1, L. plantarum SN21-2, and CIP reduced small intestinal bleeding caused by S. Typhimurium.Figure 11 Effects of SN21-1 and SN21-2 on exterior morphology of small intestines in S. Typhimurium-infected broilers. A-E represent the small intestines of broilers from control group, disease model group, SN21-1 treatment group, SN21-2 treatment group, and CIP treatment group, respectively.

Figure 11

DISCUSSION

Once potential probiotic strains have been screened and identified, they must undergo extensive screening studies to ensure their efficacy and safety. This study aimed to screen 2 novel probiotic strains, namely, L. rhamnosus SN21-1 and L. plantarum SN21-2, which have excellent probiotic properties and safety, and to explore their effects on S. Typhimurium-infected broilers.

The antibacterial activity of L. rhamnosus SN21-1 and L. plantarum SN21-2 against S. aureus, E. coli, and S. typhimurium was mainly attributed to organic acids. Tejero-Sariñena et al. (2012) reported that the inhibitory effect of 8 potential probiotic strains on S. Typhimurium, E. coli, Enterococcus faecalis, S. aureus and Clostridium difficile was due to the production of organic acids. Among 18 L. plantarum strains, Bu et al. (2022) reported that the inhibitory effect of 14 L. plantarum strains on Listeria monocytogenes was due to organic acids, while the inhibitory effect of another 4 L. plantarum strains against L. monocytogenes was due to bacteriocins (Bu et al., 2022). Hence, the antagonistic mechanism of LAB strains against pathogens is highly strain specific.

Acid and bile salts cause the death of bacteria by disrupting bacterial cell membranes, inducing DNA damage and modifying protein structure (Schumacher et al., 2023; Urdaneta & Casadesús, 2017). Probiotics must withstand the stress caused by acid and bile salts and survive in harsh gastrointestinal environments. Both L. rhamnosus SN21-1 and L. plantarum SN21-2 exhibited extremely high tolerances to simulated gastric juice, while they exhibited relatively weak tolerances to simulated intestinal fluid (Table 1). Ren et al. (2024) reported that the viable counts of 4 probiotics did not change significantly after the simulated gastric fluid test but decreased sharply after the simulated intestinal fluid test, which was consistent with our results.

The ability of probiotic strains to adhere to intestinal epithelial cells is a critical factor for their colonization (Jayashree et al., 2018). Bock et al. (2024) reported that L. plantarum KU210152 displayed 5.58% adhesion to HT-29 cells, which was lower than the adhesion percentages of L. rhamnosus SN21-1 and L. plantarum SN21-2 to HT-29 cells (58.24±1.98% and 49.00±0.96%, respectively).

The safety of probiotic strains is a crucial prerequisite for their application. No antimicrobial resistance genes or virulence genes were detected in the genomes of L. rhamnosus SN21-1 and L. plantarum SN21-2, and L. rhamnosus SN21-1 and L. plantarum SN21-2 did not exhibit resistance to the most common antimicrobial agents or haemolytic activity. Furthermore, L. rhamnosus SN21-1 and L. plantarum SN21-2 had no effect on the growth performance or blood biochemistry indices of broilers, and L. rhamnosus SN21-1 and L. plantarum SN21-2 had no translocation capacity in broilers and did not damage crucial organs. Consequently, both L. rhamnosus SN21-1 and L. plantarum SN21-2 are safe and can be used as broiler probiotics.

According to the phylogenetic tree based on the 2013 core genes of 6 L. rhamnosus strains, L. rhamnosus SN21-1 had the closest relationship with L. rhamnosus GG, which is widely marketed as a probiotic strain due to its ability to prevent diarrhoea, acute gastroenteritis, malnutrition, milk protein allergy, and respiratory diseases (Xavier-Santos et al., 2022). A total of 177 unique genes in the L. rhamnosus SN21-1 genome showed the specificity of L. rhamnosus SN21-1. According to the phylogenetic tree generated based on 2 335 core genes of the 6 L. plantarum strains, L. plantarum SN21-2 had the closest relationship with L. plantarum JDM1 (Figure 5C), which is a broadly used commercial probiotic strain in China (Zhang et al., 2012). A total of 101 unique genes in the L. plantarum SN21-2 genome also showed the specificity of L. plantarum SN21-2. The results of comparative genomics analysis show that L. rhamnosus SN21-1 and L. plantarum SN21-2 not only have extremely close evolutionary relationships with commercial probiotic strains but also exhibit unique genomes. This means that they may possess novel characteristics compared with other marketed probiotics.

In animal experiments, L. rhamnosus SN21-1 and L. plantarum SN21-2 significantly reduced S. Typhimurium counts in the faeces and caecal contents of S. Typhimurium-infected broilers and reduced small intestinal bleeding in S. Typhimurium-infected broilers. These results demonstrated that L. rhamnosus SN21-1 and L. plantarum SN21-2 had protective effects on S. Typhimurium-infected broilers.

In conclusion, we screened 2 novel broiler probiotic strains, L. rhamnosus SN21-1 and L. plantarum SN21-2, which possess antimicrobial activity against pathogens, resistance to simulated gastrointestinal fluid, and adhesion ability. The results of the safety assessment demonstrated that L. rhamnosus SN21-1 and L. plantarum SN21-2 were safe enough to be used as probiotic strains. The whole-genome sequence analysis of L. rhamnosus SN21-1 and L. plantarum SN21-2 provided molecular information on the factors underlying the probiotic properties and safety of L. rhamnosus SN21-1 and L. plantarum SN21-2. The 177 unique genes of L. rhamnosus SN21-1 and 101 unique genes of L. plantarum SN21-2 showed the specificity of these 2 strains. In the in vivo experiment, L. rhamnosus SN21-1 and L. plantarum SN21-2 significantly reduced S. Typhimurium counts in the faeces and caecal contents of S. Typhimurium-infected broilers and reduced small intestinal bleeding in S. Typhimurium-infected broilers. Briefly, this study identified 2 novel probiotic isolates for broilers, and these 2 probiotics represent a promising strategy for the treatment of S. Typhimurium-infected broilers.

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.

Appendix Supplementary materials

Image, application 1

ACKNOWLEDGMENTS

This research was supported by the Science and Technology Commission of Shanghai Municipality, China (Grant numbers: No. 23N51900200 ).

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.psj.2024.104207.
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