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. licheniformis isolates 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 toxic B. licheniformis isolates inhibited growth of Corynebacterium renale DSM 20688(T), but not all inhibitory isolates were sperm toxic. The food poisoning-related isolates were beta-hemolytic, grew anaerobically and at 55 degrees C but not at 10 degrees C, and were nondistinguishable from the type strain of B. licheniformis, DSM 13(T), 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 with EcoRI, 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.

FIG. 1

Ribotyping of 25 isolates and strains of B. licheniformis of different origins, with PvuII (A) or EcoRI (B) and hybridization with labeled whole ribosomal operon of E. coli. The patterns obtained from B. amyloliquefaciens DSM 7^T, B. cereus DSM 31^T, and B. subtilis ATCC 6051^T are also shown. Strains indicated as being of the same ribotype exhibited patterns with a similarity value of >0.95. (A) Lane A, DSM 13^T, 553/2 (toxic), 575E/P (toxic), and TSP29a; lane B, 553/1 (toxic), F287/91 (toxic), F231/97 (toxic), Hulta 53/97, and Hulta 54/97 (toxic); lane E, F2943/92 (toxic); lane H, 575U/5 (toxic), F2667/94, F5520/96 (toxic), Hulta 52/97 (toxic), and TSP19; lanes C, D, F, G, I, J, and K, no toxic isolates; lanes L, M, and N, reference strains B. amyloliquefaciens DSM 7^T, B. subtilis ATCC 6051^T, and B. cereus DSM 31^T, respectively. (B) Lane A, DSM 13^T, 553/1 (toxic), 575U/5 (toxic), F281/91 (toxic), F5734/93, TSP29a, and Hulta 52/97 (toxic); lane C, F9229/95, Hulta 53/97, and Hulta 54/97 (toxic); lane D, F2943/92 (toxic), F2896/95, and F231/97 (toxic); lane E, 575E/P (toxic); lane F, 553/2 (toxic); lane I, F5520/96 (toxic); lanes B, G, H, and J, no toxic isolates; lanes K, L, and M, reference strains B. amyloliquefaciens DSM 7^T, B. subtilis ATCC 6051^T, and B. cereus DSM 31^T, respectively.

FIG. 2

Fluorescence micrographs of boar spermatozoa stained for determination of viability after being exposed to cell extracts of different B. licheniformis strains. (A) Sperm cells (5 × 10⁶) in 1 ml of extended (with commercial extender) boar semen were exposed to cell extract from 4 mg (wet weight) of B. licheniformis DSM 13^T cells. Over 80% of the spermatozoa showed intact cell membranes. Similar results were obtained with spermatozoa exposed to the negative control (staining green). (B) Effect of extract prepared from 2 mg (wet weight) of cells of B. licheniformis 553/2. Fifty percent of the sperm cells lost integrity in the cell membrane (staining orange). (C) Sperm cells were exposed to extract prepared from 4 mg (wet weight) of the same isolate as in panel B. Seventy percent of sperm cells lost cell membrane integrity (red). Magnification in all panels, ×2,000 (i.e., sperm head dimensions are 2 to 3 by 3 to 5 μm).

FIG. 3

Light micrographs of Giemsa stained boar spermatozoa exposed to cell extracts of B. cereus and toxic and nontoxic strains of B. licheniformis. (A and B) Extended boar semen (1 ml) exposed to extract from 4 mg (wet weight) of cells of the emetic B. cereus strain 4810/72 (A) or the (nontoxic) B. licheniformis type strain DSM 13^T (B). Over 90% of the exposed sperm cells showed heavily staining dark intact acrosomes, similar to spermatozoa exposed to negative control extract (data not shown). (C) Extended boar semen (2 ml) exposed to B. licheniformis 553/2 (extract from 4 mg [wet weight] of cells). Over 50% of the cells showed lightly staining fused acrosomes or swollen and disrupted acrosomes. Magnification in all panels, ×2,000 (i.e., sperm head dimensions are 2 to 3 by 3 to 5 μm).

FIG. 4

Thin cross sections of the middle segments of boar spermatozoa exposed for 4 days to cell extracts of B. licheniformis 553/2 and F287/91, the B. licheniformis type strain DSM 13^T, and an emetic toxin producer strain, B. cereus 4810/72. (A and B) Sperm cells exposed to cell extracts prepared from 4 mg (wet weight) of isolates 553/2 and F287/91. These sperm cells had lost motility and ATP and showed damaged cell plasma membrane, but the mitochondria were intact. (C) Spermatozoon exposed to extract of the type strain B. licheniformis DSM 13^T (nontoxic). These cells displayed normal motility and cellular ATP content after exposure, and the figure shows an intact plasma membrane. (D) Sperm cell exposed to cell extract from 2 mg (wet weight) of the emetic strain B. cereus 4810/72 (12) ml⁻¹. These cells have a morphologically intact plasma membrane and normal ATP content, but over 90% of the cells exposed showed no motility, and their mitochondria were swollen with a disrupted outer membrane. Bars, 200 nm.