Toxigenic Strains of Bacillus licheniformis Related to Food Poisoning

Abstract

Toxin-producing isolates of Bacillus licheniformis were obtained from foods involved in food poisoning incidents, from raw milk, and from industrially produced baby food. The toxin detection method, based on the inhibition of boar spermatozoan motility, has been shown previously to be a sensitive assay for the emetic toxin of Bacillus cereus, cereulide. Cell extracts of the toxigenic B. licheniformis isolates inhibited sperm motility, damaged cell membrane integrity, depleted cellular ATP, and swelled the acrosome, but no mitochondrial damage was observed. The responsible agent from the B. licheniformisisolates was partially purified. It showed physicochemical properties similar to those of cereulide, despite having very different biological activity. The toxic agent was nonproteinaceous; soluble in 50 and 100% methanol; and insensitive to heat, protease, and acid or alkali and of a molecular mass smaller than 10,000 g mol−1. The toxicB. licheniformis isolates inhibited growth ofCorynebacterium renale DSM 20688T, but not all inhibitory isolates were sperm toxic. The food poisoning-related isolates were beta-hemolytic, grew anaerobically and at 55°C but not at 10°C, and were nondistinguishable from the type strain of B. licheniformis, DSM 13T, by a broad spectrum of biochemical tests. Ribotyping revealed more diversity; the toxin producers were divided among four ribotypes when cut with PvuII and among six when cut withEcoRI, but many of the ribotypes also contained nontoxigenic isolates. When ribotyped with PvuII, most toxin-producing isolates shared bands at 2.8 ± 0.2, 4.9 ± 0.3, and 11.7 ± 0.5 or 13.1 ± 0.8 kb.

Toxic lactonic lipopeptide from food poisoning isolates of Bacillus licheniformis

“…In this study we show that sperm toxin produced by three food poisoning related isolates of B. licheniformis [3] was lichenysin A, known as a biosurfactant and an antibacterial agent produced by several B. licheniformis strains, BAS50, BNP29, ATCC 10716 and IM 1307 [6±10,22]. Lichenysin A is a cyclic heptalipopeptide having the sequence l-Gln, l-Leu, d-Leu, l-Val, l-Asp, d-Leu and l-Ile linked to 3-hydroxy fatty acid residue with amide (l-Gln) and lactone (l-Ile) bonds forming a cyclic structure.…”

“…The inhibition of the motility and the acrosome reaction of boar spermatozoa caused by toxins, damage to plasma membrane, swelling of mitochondria and loss of cellular ATP and NADH were analyzed as described elsewhere [3,19,21].…”

“…of Microbiology, GBF National Research Center for Biotechnology, Braunschweig, Germany). B. licheniformis strains 553/1 and 553/2 were isolated from an infant feed formula linked to fatal illness and B. licheniformis strain 575U/5 was isolated from an unused infant feed package of the same brand as 553/1 and 553/2 [3]. Cereulide was purified from methanol extract of Bacillus cereus strain F-4810/72 as described previously [18].…”

Technical Guidance on the assessment of the toxigenic potential of Bacillus species used in animal nutrition

“…To detect strains able to produce the lipopeptides, a new PCR screening approach has been developed using degenerated primers based on the intraoperon alignment of adenylation and thiolation nucleic acid domains of all enzymes implicated in the biosynthesis of each lipopeptide family (Tapi et al, 2010). All the Bacillus strains producing these lipopeptides are haemolytic, and a number of surveys have shown that about 5-6 % of Bacillus strains has the capacity to produce surfactins (From et al, 2005;Salkinoja-Salonen et al, 1999). It is know that these surfactins create pores in epithelial cells (From et al, 2007a;From et al, 2007b) and are toxic to sperm cells (Salkinoja-Salonen et al, 1999).…”

“…All the Bacillus strains producing these lipopeptides are haemolytic, and a number of surveys have shown that about 5-6 % of Bacillus strains has the capacity to produce surfactins (From et al, 2005;Salkinoja-Salonen et al, 1999). It is know that these surfactins create pores in epithelial cells (From et al, 2007a;From et al, 2007b) and are toxic to sperm cells (Salkinoja-Salonen et al, 1999).…”