Comparison of yeast-derived commercial feed additives on Salmonella Enteritidis survival and microbiota populations in rooster cecal in vitro incubations

Costello M, Rubinelli P, Brown J, Olson E, Dittoe D, Park SH, et al. (2023) Comparison of yeast-derived commercial feed additives on Salmonella Enteritidis survival and microbiota populations in rooster cecal in vitro incubations. PLoS ONE 18(12): e0295657. https://doi.org/10.1371/journal.pone.0295657

About

Food safety is a central concern in the poultry industry, with pathogens such as Salmonella causing outbreaks associated with meat and eggs. Salmonella causes 1.35 million cases of infection every year in the United States. Because poultry production is almost entirely vertically integrated, focusing on intervention strategies that control Salmonella infection from hatching egg production to slaughter reduces the risk of salmonellosis. Roosters are an integral part of breeding systems in commercial flocks. According to breeding recommendations, the ratio of roosters to hens is typically between 1:8 and 1:10. Natural breeding can be cost-friendly and feasible compared to other systems, though it introduces another route of exposure as GIT pathogens such as Salmonella can be spread through mating. With higher demands for poultry products, finding methods beyond antibiotics to reduce resistance and improve product quality is critical. Yeast postbiotics are dead or inactivated cells that contain key metabolites that may aid the growth of beneficial bacteria and inhibit Salmonella. If effective, yeast-derived products offer an alternative to antibiotics for small and large poultry producers while reducing the risk of foodborne illness. This study tested the effectiveness of various yeast-derived products on reducing S. Enteritidis in rooster ceca in vitro and to characterize changes to the rooster cecal microbiome.

Approach

Commercially available yeast-derived products were tested for their ability to inhibit the growth of an intestinal pathogen relevant to human health, specifically the strain S. Enteritidis in vitro. A poultry isolate of S. Enteritidis type 13A strain was obtained from the USDA National Veterinary Services Laboratory. Different commercial yeast-derived products were screened via an in vitro cecal incubation system. A total of 3 Single-Comb White Leghorn roosters (53 weeks of age) were fed the same wheat-based commercial-type laying hen diet consisting of 17.0% crude protein, 5.0% fat, and 4.1% fiber with 2,750 kcal ME/kg. Ceca (including contents) were collected, frozen at -20°C, and shipped on dry ice for analysis. An initial inoculum of approximately 1 x 107 CFU/mL of a nalidixic acid–resistant (NAR) marker strain, SE13A, of S. Enteritidis was placed in an incubator at 37°C for 48h. The cecal bacteria isolated from the roosters were pre-incubated for 24 hours with feed and their respective treatments, and S. Enteritidis was added after 24 hours. The treatments were cecal contents only, the basal diet and cecal contents, and various treatments with both the basal diet and cecal contents: Immunowall, Hilyses, Citristim, the control diet with Maxi-Gen Plus, and the control diet with Original XPC. Each control and treatment were tested on three individual ceca from the three roosters. Cultures were plated at each time point to determine the presence of S. Enteritidis 13A. If it was not found, the cultures were inoculated into TT enrichment broth to further confirm the absence of S. Enteritidis 13A.

Analysis of Results

Pathogenic bacteria from the GIT, such as Campylobacter, Clostridium perfringens, and Salmonella, are common and often naturally occurring in poultry semen. Since Salmonella is a GIT pathogen, dietary interventions offer the most promising solution. This study tests yeast-derived products and their effectiveness against S. Enteritidis in vitro to reduce Salmonella in the rooster GIT. Because of this potential transmission relationship, employing cecal contents in an in vitro system to screen the effects of different treatments on mitigating an S. Enteritidis in the presence of rooster cecal microbiota is a practical initial step to determine if feed additives such as yeast-based products would sufficiently restrict proliferation of S. Enteritidis beyond the GIT. A limitation of testing these products in vitro was the absence of host immune response data, which would have offered more insight into the apparent Salmonella reductions and microbial taxonomy as a function of the host response. In the current study, each of the five yeast-derived products, when introduced to the rooster cecal microbiota, inhibited S. Enteritidis SE13A. Citristim, Hilyses, MaxiGen, and XPC were inhibited to a comparable extent by 48 hours post-Salmonella inoculation. At 48 hours post-inoculation, Immunowall appeared to be somewhat more effective. At 48 hours, Hilyses and Immunowall had one and two ceca, respectively, with no detectable Salmonella. Microbiome 16S rDNA sequencing was conducted on the rooster in vitro cecal incubations to determine the level of diversity response within each yeast product incubation and comparisons among the different yeast products. There was a significant effect of the yeast product on cecal microbiota. This would indicate that each yeast product was equally supportive of a relatively diverse cecal microbial population. It would appear that each yeast product supported somewhat distinct microbial populations when compared with each other.

Application

The results indicate that each yeast-derived product tested can rapidly reduce S. Enteritidis populations by several orders of magnitude within 48 hours of Salmonella inoculation in rooster cecal in vitro cultures. A unique finding of this study is that Immunowall appeared to be somewhat more effective than the other yeast products, reducing S. Enteritidis almost -four orders of magnitude compared to the control cultures without yeast product. While these results are reflective of a rooster cecal response to various yeast-derived products, they may carry additional applications to the laying hen industry. To better understand the effects of these yeast-derived products on different aspects of the poultry industry, in vivo studies are necessary for both laying hens and roosters.

Abstract

Yeast-derived products have become more of an interest in the poultry industry as of late because of their use in modulating the gastrointestinal tract (GIT) microbiome to both improve production parameters and prevent infection. This study aimed to evaluate the effects of various yeast-derived products on Salmonella enterica inoculation in un in vitro rooster cecal incubations and associated effects on the cecal microbiome. Cecal contents were obtained from 53-wk old White Leghorn H & N Nick Chick roosters (n = 3) fed a wheat-based, commercial-type basal diet. Cecal contents were diluted 1:3000 in anaerobic dilution solution (ADS) in an anaerobic chamber, with 20 mL aliquoted to each serum bottle. There were three controls (n = 3): basal diet only, diluted cecal contents only, and basal diet and diluted cecal contents; and five treatments containing the basal diet and diluted cecal contents (n = 3): Citristim® (ADM), ImmunoWall® (ICC), Maxi-Gen Plus® (CBS Bio Platforms), Hilyses® (ICC), and Original XPC® (Diamond V). All treatments were applied at a rate of 2.5 kg/tonne or less. All groups were inoculated with a nalidixic acid-resistant strain of Salmonella Enteritidis at 10^7 CFU/mL and incubated at 37 deg C. Samples were collected at 0, 24, and 48 h for S. Enteritidis enumeration and 16S rDNA microbial sequencing. Salmonella data were log-transformed and analyzed in a two-way ANOVA with means separated using Tukey’s HSD (P≤0.05). Genomic DNA was extracted, and resulting libraries were prepared and sequenced using an Illumina MiSeq. Sequencing data were analyzed in QIIME2 (2021.4) with diversity metrics (alpha and beta), and an analysis of the composition of microbiomes (ANCOM) was performed. Main effects were considered significant at P≤0.05, with pairwise differences considered significant at Q≤0.05. There was an interaction of treatment and time on the enumeration of Salmonella where treatments of Citristim, Immunowall, Hilyses, and XPC reduced Salmonella by 1 log CFU/mL compared to the controls. At 48 h, each yeast product treatment reduced Salmonella by 3 log CFU/mL compared to the controls. There was no main effect of treatment on the alpha diversity metrics, richness, or evenness (P > 0.05). Treatment affected the beta diversity, abundance, and phylogenetic differences, but there were no pairwise differences (P>0.05, Q>0.05). Using ANCOM at the genus level, the taxa Synergistes, Alloprevotella, Sutterella, and Megasphaera abundance were significantly different (W = 154,147,145,140, respectively). These results demonstrate the potential of these yeast-derived products to reduce foodborne pathogens, such as Salmonella Enteriditis, in vitro, without negatively disrupting the cecal microbiome.