In vitro antagonistic activities of Lactobacillus spp. against Brachyspira hyodysenteriae and Brachyspira pilosicoli

doi:10.1016/j.vetmic.2009.03.020

Veterinary Microbiology

Volume 138, Issues 1–2, 2 July 2009, Pages 184-190

https://doi.org/10.1016/j.vetmic.2009.03.020 Get rights and content

Abstract

The sensitivity of Brachyspira hyodysenteriae and Brachyspira pilosicoli, respectively the causative agents of Swine Dysentery and Porcine Intestinal Spirochaetosis to two probiotic Lactobacillus strains, L. rhamnosus CNCM-I-3698 and L. farciminis CNCM-I-3699 was studied through viability, motility and coaggregation assays. The cell-free supernatant of these lactobacilli contains lactic acid, that is stressful for Brachyspira (leading to the formation of spherical bodies), and lethal. It was demonstrated for the first time the in vitro coaggregation properties of two probiotic Lactobacillus strains (active or heat-treated) with two pathogenic strains of Brachyspira, leading to (1) trapping of spirochaetal cells in a physical network as demonstrated by SEM; (2) inhibition of the motility of Brachyspira. Such in vitro studies should encourage in vivo studies in animal model to evaluate the potential of the use of probiotic lactobacilli through a feeding strategy for the prevention of B. hyodysenteriae and B. pilosicoli.

Introduction

Brachyspira hyodysenteriae and Brachyspira pilosicoli are important pathogenic intestinal spirochaetes that cause costly diarrhoeal diseases for the pig industry (Smith, 2005), respectively Swine Dysentery (SD) and Porcine Intestinal Spirochaetosis (PIS). Economical losses (poor gain weight performance, high medication and death) have been recognized worldwide for decades. Recently, B. pilosicoli was considered as a zoonotic agent (Hampson et al., 2006).

Antimicrobial agents, mainly tylosin and tiamulin are widely used for extended periods to control SD and PIS in affected herds. However, in the light of increasing multiple drug resistance (Karlsson et al., 2003), more consideration has been given to reduce the risk of infection by applying preventive measures, and the use of antibiotic treatment should be combined with hygienic, technical and nutritional management practices. A nutritional strategy is the use of probiotics—live microorganisms which when administered in adequate amounts confer a health benefit on the host (FAO/WHO, 2001). Supplementing animal feed with probiotic lactobacilli is well documented (reviewed by Bernardeau et al., 2006). Some probiotics contribute to improve herd performance (Torres-Rodriguez et al., 2007, Vasconcelos et al., 2008) and health management (Sargeant et al., 2007). Multiple mechanisms of action for the beneficial effect of probiotics have been postulated, including preventing growth of pathogenic bacteria (Tsai et al., 2008, Wagner et al., 2009), production of antimicrobial agents (Gillor et al., 2008), coaggregation with pathogens (Golowczyc et al., 2007) and stimulation of mucosal barrier function (Isolauri et al., 2008).

To our knowledge, neither in vitro nor in vivo interactions between Brachyspira and probiotic strains have been studied to date. Thus, the aim of this study was to investigate the in vitro antagonistic activities of the cells of two Lactobacillus strains, Lactobacillus rhamnosus CNCM-I-3698 and L. farciminis CNCM-I-3699, known as probiotics (Bernardeau, 2006) and their cell-free supernatants against B. pilosicoli and B. hyodysenteriae.

Section snippets

Bacterial strains and microbial cell suspensions

The two strains of lactobacilli, stored at −80 °C, were cultured for 24 hours in 9.0 ml of MRS broth (MRS, De Man Rogosa Sharpe, AES, Combourg, France) or milk (Lait G, Standa Industrie, France) in anaerobic conditions (GENbox anaer, Biomérieux, Marcy l’Etoile, France) at 37 °C prior to testing. The final Lactobacillus population was around 1 × 10⁹ c.f.u. ml⁻¹. Lactobacillus cells were separated from cell-free supernatants by centrifugation at 10,000 × g at 4 °C for 10 min, the pellet was washed three

Effect of cell-free Lactobacillus supernatant on viability and morphology of Brachyspira cells

Lactobacillus supernatant obtained from MRS was an acidic solution (pH 3.5), and was shown to contain 14.4 g of lactic acid/l (especially the L-form: 13.7 g/l as determined by D-Lactic acid/L-Lactic acid Enzymatic kit, Boehringer Mannheim). A highly significant (P < 0.001) inhibitory effect against B. hyodysenteriae 9828978 and B. pilosicoli ATCC-51139 was observed after 1 hour of contact time with Lactobacillus supernatant obtained from MRS since the initial bacterial count of 1 × 10⁹ c.f.u. ml⁻¹

Discussion

Here we demonstrated and illustrated the sensitivity of one strain of B. hyodysenteriae and of one strain of B. pilosicoli (two pathogenic agents of economical importance in the pig industry) to two probiotic Lactobacillus strains. Contact with cell-free Lactobacillus supernatant had a lethal effect on Brachyspira cells, which was characterized by broken cells walls. Sensitivity of Brachyspira to acidic compounds and disinfectants has already been shown by one of us (Corona-Barrera, 2002,

Acknowledgments

This study was supported by a grant from the Association Nationale de la Recherche Technique (France) and Sorbial, a French company. Authors wish to thank Atmane Asselate and Raouf Tareb for their technical support. Authors gratefully acknowledge Dr. Didier Goux from the Microscopy centre of the University of Caen (France) for his scientific and technical assistance getting the electron micrographs.

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Short communicationIn vitro antagonistic activities of Lactobacillus spp. against Brachyspira hyodysenteriae and Brachyspira pilosicoli

Abstract

Introduction

Section snippets

Bacterial strains and microbial cell suspensions

Effect of cell-free Lactobacillus supernatant on viability and morphology of Brachyspira cells

Discussion

Acknowledgments

J Food Prot

FEMS Microbiol Lett

Int J Food Microbiol

Biophys J

J Food Prot

Poult Sci

Anaerobe

Zentralbl Bakteriol

Beneficial lactobacilli in food and feed: long-term use, biodiversity and proposals for specific and realistic safety assessments

FEMS Microbiol Rev