Dual bacteriocin and extracellular vesicle-mediated inhibition of
Campylobacter jejuni
by the potential probiotic candidate
Ligilactobacillus salivarius
UO.C249
Authors: Chiba Mariem , Miri Saba , Yousuf Basit , Esmail Galal Ali , Leao Luana , Li Yingxi , Hincke Maxwell , Minic Zoran , Mottawea Walid , Hammami Riadh 
Published: July 2024

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.