# Bacillus spp. Potentiate the Virulence and Intracellular Invasion of A. paragallinarum in Chickens

## TL;DR

This study shows that Bacillus bacteria can help Avibacterium paragallinarum grow and infect chickens more effectively, raising concerns about probiotic safety.

## Contribution

The study reveals that Bacillus metabolites enhance A. paragallinarum virulence and intracellular invasion in chickens.

## Key findings

- Bacillus spp. metabolites promote A. paragallinarum growth and infection in vitro and in vivo.
- Bacillus colonization increases the risk of A. paragallinarum infection in chicken respiratory tracts.
- B. cereus worsens rhinitis symptoms in chickens co-infected with A. paragallinarum.

## Abstract

Bacterial coinfection poses severe threats to poultry health. One common bacterium, Avibacterium paragallinarum, relies on extracellular growth factors acquired from other organisms or its surrounding environment and is more susceptible to coinfection. In this study, Bacillus promotes the growth and infection of Avibacterium paragallinarum in vitro and in vivo by metabolites, especially some Bacillus strains isolated from probiotics. This study highlights the necessity for enhanced safety assessments of probiotic Bacillus strains to evaluate their potential role in facilitating coinfections.

Coinfection poses severe threats to poultry health, particularly due to the complexity and resilience of multispecies interactions, increasing the difficulty of treatment. Haemophilus spp., a heterotrophic bacterium, heavily relies on extracellular growth factors acquired from other organisms or its surrounding environment. Although coinfections by Avibacterium paragallinarum and Bacillus have been reported, the underlying mechanism of the cooperative interaction remains poorly understood. In this study, we characterized the growth-promoting properties and nicotinamide adenine dinucleotide production of some Bacillus species, including probiotic Bacillus, to evaluate the feasibility of A. paragallinarum coinfection in vitro. Meanwhile, we determined the minimum inhibitory concentration (MIC) and population dynamics of cocultured Bacillus and A. paragallinarum to assess the effect of bacterial interactions on antibiotic efficacy. Additionally, we demonstrated that B. cereus aggravates rhinitis symptoms in chickens infected with A. paragallinarum. Our findings reveal that Bacillus spp.-derived metabolites sustain A. paragallinarum growth and enhance its survival, thereby highlighting the infection risks associated with Bacillus colonization in the respiratory tract.