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Shift in the chicken gut microbiota after Salmonella infection and probiotic treatment. A Changes in microbiome taxa after 6 h of treatment. B Changes in the microbiome taxa after 24 h of treatment. ControlN: chicken cecal microbiota (control group), Probiotics: chicken cecal microbiota supplemented with probiotics (probiotic control group), ControlP: chicken cecal microbiota infected with Salmonella Typhimurium. Probiotics + S: Salmonella-infected chicken cecal microbiota treated with probiotics
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In this study, we aimed to develop a protective probiotic coculture to inhibit the growth of Salmonella enterica serovar Typhimurium in the simulated chicken gut environment. Bacterial strains were isolated from the digestive mucosa of broilers and screened in vitro against Salmonella Typhimurium ATCC 14028. A biocompatibility coculture test was pe...
Citations
... Over the past two decades, many studies have demonstrated that lactic acid bacteria (LABs) are effective gut symbionts and probiotics in inhibiting enteric pathogenic microorganisms and stimulating the host immune system [30,42]. We recently reported the efficacy of novel bacteriocin-producing Ligilactobacillus strains isolated from healthy chicken intestinal mucosa in limiting the growth of enteric pathogens, such as Salmonella, Clostridium, and Campylobacter in vitro [42,47]. We observed a reduced relative abundance of Enterobacteriaceae (i.e., Salmonella) and increased gut microbiome production of SCFAs (acetic and propionic acids) ex vivo using the cell-free supernatant (CFS) of Ligilactobacillus strains, suggesting their potential to control Salmonella infections in poultry [47]. ...
... We recently reported the efficacy of novel bacteriocin-producing Ligilactobacillus strains isolated from healthy chicken intestinal mucosa in limiting the growth of enteric pathogens, such as Salmonella, Clostridium, and Campylobacter in vitro [42,47]. We observed a reduced relative abundance of Enterobacteriaceae (i.e., Salmonella) and increased gut microbiome production of SCFAs (acetic and propionic acids) ex vivo using the cell-free supernatant (CFS) of Ligilactobacillus strains, suggesting their potential to control Salmonella infections in poultry [47]. However, the probiotic-pathogen interplay and mechanism by which these strains exert their inhibitory activity remain largely unknown. ...
... The antimicrobial activity of EVs against S. Typhimurium and C. jejuni was tested in 96-well plates [47]. This antimicrobial assay is sensitive and can detect small differences in susceptibility to antibacterial components, making it useful for monitoring emerging resistance. ...
Bacterial intra-kingdom communication involves the secretion of outer membrane vesicles as signaling carriers to the target cells. However, limited research exists on extracellular vesicles (EVs) from Gram-positive gut bacteria, their interactions with enteric pathogens, and potential inhibitory effects. In this study, we characterized the structure, protein content, and inhibitory effects of EVs from three new potential probiotic gut symbionts, Ligilactobacillus salivarius UO.C109, Ligilactobacillus saerimneri UO.C121, and Ligilactobacillus salivarius UO.C249. EVs were isolated and characterized using three different methods (ultracentrifugation, density gradient purification, and size exclusion chromatography). The purity, dose-dependency, structure, and proteome profiles of the purified EVs were evaluated. Antibacterial and anti-virulence activities of EV subpopulations were assessed against Salmonella enterica serovar Typhimurium and Campylobacter jejuni. EVs from Lg. salivarius UO.C109 and Lg. saerimneri UO.C121 showed inhibitory activity against S. Typhimurium, whereas EVs from Lg. salivarius UO.C249 inhibited the growth of C. jejuni. Notably, purified F3 fraction exhibited the highest inhibitory activity and was enriched in lysin motif (LysM)-containing proteins, peptidoglycan hydrolases, peptidoglycan recognition proteins (PGRPs), and metallopeptidases, which have been shown to play a prominent role in antimicrobial activities against pathogens. F3 had the highest concentration (73.8%) in the 80–90 nm size compared to the other fractions. Gene expression analysis revealed that EVs from Lg. salivarius UO.C109 and Lg. saerimneri UO.C121 downregulated adhesion and invasion factors in S. Typhimurium. Likewise, EVs from Lg. salivarius UO.C249 reduced pathogenicity gene expression in C. jejuni. This study highlighted the potential of gut bacterial EVs as therapeutic agents against enteric pathogens.
... The tolerance test in simulated gastrointestinal fluids was modified from previously described methods (Saba et al., 2023). Given the fluctuating pH of gastric fluid, pH was adjusted to 2.0, 3.0, and 4.0, respectively. ...
In recent years, the use of fish-derived probiotics in aquaculture has become more widespread. However, research on Anguilla japonica-derived probiotics is still limited. To evaluate the potential of probiotics for disease control in eel aquaculture, isolates were obtained from the intestinal tract of healthy Anguilla japonica. These isolates were assessed for their adhesion properties, inhibition of pathogen adhesion, and hydrolytic enzyme production. Morphological characteristics and 16S rRNA sequence analysis were used for identification. Results showed that the AJQ03 strain adhered to the intestinal mucus and inhibited common pathogenic bacteria through adhesion inhibition, and further produced amylase, lipase, protease, and cellulase. Based on morphological characteristics and 16S rRNA sequencing, AJQ03 was identified as Bacillus subtilis. The strain demonstrated tolerance to various extreme conditions, as well as survival in simulated gastrointestinal fluids and superior growth in intestinal fluid compared to Luria-Bertani (LB) broth. In vitro safety tests showed that AJQ03 was not resistant to 32 antibiotics and exhibited γ hemolysis on blood plate. In vivo safety tests demonstrated a 100% survival rate for the fish, with stable organ indices, reduced bacterial loads in the liver and spleen, and complete bacterial clearance by day 7 without residue. Intestinal bacterial load results confirmed effective colonization by strain AJQ03. Analysis of the impact of AJQ03 on the gut microbiota of A. japonica revealed a significant increase in the relative abundance of Bacillus at the genus level, corroborating the colonization efficiency of AJQ03. Additionally, the relative abundances of Klebsiella, Pseudomonas, and Aeromonas were significantly lower compared to the controls, indicating that strain AJQ03 effectively reduced harmful bacteria and improved gut microbiota composition. This study confirms that B. subtilis AJQ03, isolated from the intestine of A. japonica, can serve as a probiotic candidate in A. japonica aquaculture.
... At the genus level, DON exposure significantly up-regulated the relative abundance of Helicobacter pylori, a type of Helicobacter, which was once considered to be associated positively with proinflammatory cytokines and varieties of diseases and was up-regulated to disrupt flora balance and promote intestinal inflammation [40,41]. In contrast, the relative abundances of Ligilactobacillus, Lachnospiraceae_NK4A136_group and Muribaculaceae were all down-regulated after DON exposure, with Ligilactobacillus being a probiotic showing potential in preventing various diseases such as treating obesity, alleviating constipation and resisting sepsis-associated acute liver injury [42][43][44]. Meanwhile, for Lachnospiraceae_NK4A136_group, a potential anti-inflammatory flora associated with some drugs for obesity and depression treatment, its vital role of anti-inflammatory effect was also weakened by DON [45][46][47]. ...
Deoxynivalenol (DON) is a mycotoxin produced by Fusarium fungi widespread in wheat, corn, barley and other grain crops, posing the potential for being toxic to human and animal health, especially in the small intestine, which is the primary target organ for defense against the invasion of toxins. This study firstly investigated DON contamination in a local area of a wheat production district in China. Subsequently, the mechanism of DON toxicity was analyzed through cellular molecular biology combining with intestinal flora and gene transcription analysis; the results indicated that DON exposure can decrease IPEC−J2 cell viability and antioxidant capacity, stimulate the secretion and expression of proinflammatory factors, destroy the gut microbiota and affect normal functions of the body. It is illustrated that DON could induce intestinal damage through structural damage, functional injury and even intestinal internal environment disturbance, and, also, these intestinal toxicity effects are intrinsically interrelated. This study may provide multifaceted information for the treatment of intestinal injury induced by DON.
... During the selection of probiotic candidates, tolerance to an acidic environment is a major factor affecting the survival of probiotic bacterial strains in the gastrointestinal tract (8). Our results indicated that the bacteriocinogenic strain Lg. salivarius UO.C249 exhibited a higher survival rate in the stomach and small intestine of chickens, indicating that it can survive under physiological pH conditions, in accordance with the study described by Miri et al. (62), making it useful as a probiotic candidate in chicken feed. In this study, we found a relative value of 54% for autoaggregation with Lg. salivarius UO.C249. ...
... Antimicrobial activity was evaluated against the target strain using 96-well flatbottomed plates (VWR, Monroeville, PA, USA), as described previously (62). A total of 100 µL from each CFS and its fraction 50 were added to MH broth and were used to perform twofold serial dilutions, which were then planted with 100 µL of 10 6 CFU/mL C. jejuni ATCC BAA1153. ...
... The counts of adherent and invading C. jejuni were normalized to 1.0 in control samples, whereas the counts in the presence of probiotic strains were reported as relative. The adhesion of probiotic candidates was expressed as the percentage of adhered bacteria to the total bacteria utilized in the experiment (62,72,73). ...
Campylobacter jejuni (C. jejuni) is one of the most common causes of foodborne infections worldwide and a major contributor to diarrheal diseases. This study aimed to explore the ability of commensal gut bacteria to control C. jejuni infection. Bacterial strains from the intestinal mucosa of broilers were screened in vitro against C. jejuni ATCC BAA1153. The cell-free supernatant (CFS) of Ligilactobacillus salivarius UO.C249 showed potent dose-dependent antimicrobial activity against the pathogen, likely due to the presence of bacteriocin-like moieties, as confirmed by protease treatment. Genome and exoproteome analyses revealed the presence of known bacteriocins, including Abp118. The genome of Lg. salivarius UO.C249 harbors a 1.8-Mb chromosome and a 203-kb megaplasmid. The strain was susceptible to several antibiotics and had a high survival rate in the simulated chicken gastrointestinal tract (GIT). Post-protease treatment revealed residual inhibitory activity, suggesting alternative antimicrobial mechanisms. Short-chain fatty acid (SCFA) quantification confirmed non-inhibitory levels of acetic (24.4 ± 1.2 mM), isovaleric (34 ± 1.0 µM), and butyric (32 ± 2.5 µM) acids. Interestingly, extracellular vesicles (EVs) isolated from the CFS of Lg. salivarius UO.C249 were found to inhibit C. jejuni ATCC BAA-1153. Proteome profiling of these EVs revealed the presence of unique proteins distinct from bacteriocins identified in CFS. The majority of the identified proteins in EVs are located in the membrane and play roles in transmembrane transport and peptidoglycan degradation, peptidase, proteolysis, and hydrolysis. These findings suggest that although bacteriocins are a primary antimicrobial mechanism, EV production also contributes to the inhibitory activity of Lg. salivarius UO.C249 against C. jejuni.
IMPORTANCE
Campylobacter jejuni (C. jejuni) is a major cause of gastroenteritis and a global public health concern. The increasing antibiotic resistance and lack of effective alternatives in livestock production pose serious challenges for controlling C. jejuni infections. Therefore, alternative strategies are needed to control this pathogen, especially in the poultry industry where it is prevalent and can be transmitted to humans through contaminated food products. In this study, Ligilactobacillus salivarius UO.C249 isolated from broiler intestinal mucosa inhibited C. jejuni and exhibited important probiotic features. Beyond bacteriocins, Lg. salivarius UO.C249 secretes antimicrobial extracellular vesicles (EVs) with a unique protein set distinct from bacteriocins that are involved in transmembrane transport and peptidoglycan degradation. Our findings suggest that beyond bacteriocins, EV production is also a distinct inhibitory signaling mechanism used by Lg. salivarius UO.C249 to control C. jejuni. These findings hold promise for the application of probiotic EVs for pathogen control.
Objective:
This study aimed to develop and evaluate the effectiveness of a water-soluble microencapsulation method for probiotic strains using gum Arabic (GA) and skim milk (SKM) over a three-month storage period following processing.
Methods:
Four strains of Pediococcus acidilactici (BYF26, BYF20, BF9, and BF14) that were typical lactic acid bacteria (LAB) isolated from the chicken gut were mixed with different ratios of GA and SKM as coating agents before spray drying at an inlet temperature 140°C. After processing, the survivability and probiotic qualities of the strains were assessed from two weeks to three months of storage at varied temperatures, and de-encapsulation was performed to confirm the soluble properties. Finally, the antibacterial activity of the probiotics was assessed under simulated gastrointestinal conditions.
Results:
As shown by scanning electron microscopy, spray-drying produced a spherical, white-yellow powder. The encapsulation efficacy (percent) was greatest for a coating containing a combination of 30% gum Arabic: 30% skim milk (w/v) (GA:SKM30) compared to lower concentrations of the two ingredients (p<0.05). Coating with GA:SKM30 (w/v) significantly enhanced (p<0.05) BYF26 survival under simulated gastrointestinal conditions (pH 2.5 to 3) and maintained higher survival rates compared to non-encapsulated cells under an artificial intestinal juices condition of pH 6. De-encapsulation tests indicated that the encapsulated powder dissolved in water while keeping viable cell counts within the effective range of 106 for 6 hours. In addition, following three months storage at 4°C, microencapsulation of BYF26 in GA:SKM30 maintained both the number of viable cells (p<0.05) and the preparation's antibacterial efficacy against pathogenic bacteria, specifically strains of Salmonella.
Conclusion:
Our prototype water-soluble probiotic microencapsulation GA:SKM30 effectively maintains LAB characteristics and survival rates, demonstrating its potential for use in preserving probiotic strains that can be used in chickens and potentially in other livestock.
