The probiotic Lacticaseibacillus rhamnosus GG supplementation reduces Salmonella load and modulates growth, intestinal morphology, gut microbiota, and immune responses in chickens

Abstract

Salmonella, a leading cause of foodborne illnesses, is primarily transmitted to humans through the consumption of contaminated poultry products. The increasing resistance of Salmonella to antibiotics and lack of cross-protection by vaccines necessitate new control strategies in poultry production systems. This study assessed the efficacy of probiotics against Salmonella Typhimurium (ST) and Salmonella Enteritidis (SE). Lactobacillus acidophilus (LA), Lacticaseibacillus rhamnosus GG (LGG), and Bifidobacterium animalis subsp. lactis (Bb12) showed inhibition of ST and SE in agar well diffusion assay, with stable inhibitory properties. In co-culture assay, both LGG and Bb12 completely suppressed ST and SE growth. Liquid chromatography-with tandem mass spectrometry (LC-MS/MS) analysis of the LGG and Bb12 cell-free culture supernatant identified novel bioactive peptides with anti-Salmonella properties. Administering LGG in drinking water of chickens raised on built-up litter floor in experimental conditions significantly reduced the ST load (5.95 logs and 3.74 on 7 days post-infection [dpi] and 14 dpi, respectively). Gut microbiota analysis revealed increased abundance of several beneficial genera such as Butyricicoccus, Erysipelatoclostridium, Flavonifractor, and Bacillus in LGG-treated groups. Histomorphometry analysis demonstrated increased villus height (VH) and VH by crypt depth ratio in the ileum of the LGG-treated group on 14 dpi. These results highlight LGG as a promising probiotic for controlling Salmonella in chickens and reducing transmission to humans. The beneficial properties of LGG are attributed to the production of antimicrobial peptides, microbiota modulation, and enhanced intestinal integrity.IMPORTANCESalmonella is the leading cause of foodborne illnesses in the United States and worldwide. It is primarily transmitted through contaminated poultry and poultry products (eggs and poultry meat). Increasing resistance of Salmonella to antibiotics and lack of cross-protection by vaccines necessitate new control strategies to reduce Salmonella in poultry production system and minimize human infections. Probiotics, which are live beneficial microorganisms when administered in an optimum amount, have been increasingly used in recent years as alternatives to antibiotics to promote health. Our study showed that LGG exhibited superior probiotics properties and significantly reduced Salmonella load in chickens. Thus, LGG supplementation is a promising approach to prevent Salmonella infection and enhance performance of poultry thereby enhance food safety, proper antibiotic stewardship and public health.

Keywords: LGG; Salmonella; chickens; foodborne pathogens; probiotics.

Fig 1

Growth and persistence of ST and SE in mixed media (mixture of MRS and LB at a ratio of 1:1) and co-cultured with LA, LGG, Bb12, and Lbrev. Each probiotic (100 µL of 10⁸ CFU/mL) was individually co-cultured with ST and SE (100 µL of ~5 × 10⁷ CFU/mL) and incubated anaerobically for 24 h at 37°C. ST and SE were enumerated at 0, 6, 12, and 24 h in co-culture assay.

Fig 2

Inhibition of ST and SE by probiotics secreted products in a trans-well migration assay. Salmonella was added into a microcentrifuge tube containing 0.22 µm filter, and LGG and BB12 were added above the filter. Salmonella grown below and above the filter was used as control.

Fig 3

Effect of probiotic CFS to minimize invasion of ST (A) and SE (B) in HT-29 cells. Polarized HT-29 cells were infected with Salmonella enterica subsp. enterica serotype Typhimurium (A) and Salmonella enterica subsp. enterica serotype Enteritidis (B) and treated for 4 h with 12.5% and 25% CFS to determine the effect of probiotics on the invasion of Salmonella.

Fig 4

Impact of oral administration of probiotic against ST colonization in chicken. Birds were treated with LGG for 16 days and orally challenged with 10⁴ CFU nalidixic acid-resistant (Nal^r) ST on day 7. The five groups included were NC (neither infected nor treated), LS (infected with Salmonella and treated with LGG), BS (infected with Salmonella and treated with Bb12), LB_S (infected with Salmonella and treated with a mixture of LGG and Bb12 at the ratio of 1:1), and PC (infected with Salmonella). (A) Log CFU of ST colonization in cecum, (B) percent positive for ST in spleen and liver, and (C) body weight 10 days post-infection. N = 10/group; horizontal line: mean.

Fig 5

Efficacy of LGG supplemented in drinking water against ST colonization in chickens. Birds were treated with LGG for 13 days and orally challenged with 10⁴ CFU Nal^r ST on day 7. The four groups included were NC, LGG (not infected but treated with LGG), LS, and PC. (A) Log CFU of ST colonization in cecum, (B) percent positive for ST in spleen and liver, and (C) body weight 8 days post-infection, N = 15/group.

Fig 6

Efficacy of LGG supplemented in drinking water of ST infected chickens raised on the floor. Birds were treated with LGG for 13 days and orally challenged with 10⁴ CFU Nal^r ST on day 7. The five groups included were NC, LGG, LS, CS (infected with Salmonella and treated with commercial probiotics), and PC. Two necropsies were performed on 7 dpi (first necropsy) and 14 dpi (second necropsy). (A) Log CFU of ST colonization in cecum, (B) percent positive for ST in liver and spleen, and (C) body weight of chickens on 7 and 14 dpi, N = 20 birds/group.

Fig 7

Alpha diversity and beta diversity in cecal microbiota of different groups from chicken trial 3. Influence of LGG in the alpha diversity metrics Chao1, Observed, Shannon and Simpson on 7 dpi (A) and 14 dpi (B). (C) Principal component analysis using Bray-Curtis method of beta diversity on day 7 and 14 dpi.

Fig 8

Relative abundance of microbes at two different time points and three different levels: at phylum level on 7 dpi (A) and 14 dpi (B), at order level on 7 dpi (C) and 14 dpi (D), at genus level on 7 dpi (E) and 14 dpi (F). Bar graph showing the relative abundance of Lacticasebacillus in different groups (G).

Fig 9

VH and crypt depth (CD) of Ileum (A–F) and Jejunum (G–L). Salmonella infection reduced the VH and CD of the ileum, whereas inclusion of LGG significantly increased the VH.

Fig 10

LC-MS/MS profiling of different organic acids in the CFS of probiotic cultured alone and co-cultured with ST. As control, MRS media alone was used as control for monoculture study, and Salmonella grown in co-culture media as co-culture study.

Fig 11

Antimicrobial activity of LGG/Bb12-derived novel peptides against ST. ST (10⁵ CFU/mL) was incubated with PN-1, PN-2, PN-3, PN-4, and PN-5 peptides at 12 mM, and optical density (OD₆₀₀) was recorded using Tecan, an automated microplate reader, every 30 min. ST inhibition by PN-1, PN-2, PN-3, PN-4, and PN-5 was computed as inhibition percentage.