Competing microorganisms with exclusion effects against multidrug-resistant Salmonella Infantis in chicken litter supplemented with growth-promoting antimicrobials

Abstract

Background and Aim: The widespread use of antibiotic growth promoters (AGPs) in poultry production has been implicated in altering gut microbiota and promoting the excretion of multidrug-resistant (MDR) bacteria into the environment. Salmonella enterica serovar Infantis (Salmonella Infantis [S.I]), a prevalent zoonotic pathogen, has demonstrated increasing resistance in poultry systems. This study aimed to evaluate the efficacy of natural control microorganisms (NCM), Bacillus subtilis and Lactobacillus plantarum, in reducing the abundance of MDR S.I in fresh chicken litter from birds raised with or without AGP supplementation. It also examined how physicochemical properties and microbial dynamics influence pathogen persistence. Materials and Methods: Microcosms were constructed using litter from broilers raised under two dietary regimes (with and without avilamycin). Treatments included combinations of AGP, S.I, and NCM. Bacterial enumeration was performed using selective media, and whole-genome sequencing of S.I was conducted to characterize antimicrobial resistance and virulence genes. Physicochemical parameters (pH, humidity, temperature, and ammonia) were measured and correlated with microbial loads. Antagonistic activity of NCM strains was assessed using agar diffusion assays. Results: Genome analysis revealed that S.I carried multiple resistance genes (e.g., blaCTX-M-65, tet(A), and sul1) and efflux systems conferring MDR. In vitro assays showed strong antagonism by L. plantarum and moderate activity by B. subtilis. In microcosms, S.I counts significantly decreased in the presence of both AGP and NCM, indicating synergistic inhibition. Conversely, in the absence of AGP, NCM had a limited effect. Statistical analyses showed strong correlations between microbial groups and physicochemical variables, particularly during later production stages. Conclusion: The application of B. subtilis and L. plantarum in chicken litter significantly reduced S.I colonization under AGP supplementation, suggesting their potential as biocontrol agents. These findings support the development of integrated litter management strategies to mitigate zoonotic and resistant pathogen dissemination, particularly in AGP-using systems. However, the effectiveness of such interventions may vary across farms due to differences in microbial ecology and environmental conditions.

Keywords

• antimicrobial resistance
• bacillus subtilis
• competitive exclusion
• food safety
• lactobacillus plantarum
• poultry litter
• salmonella infantis