Aims
The aim of this study was to elucidate the prevalence of lichenysin production in Bacillus licheniformis and to see whether this feature was restricted to certain genotypes. Secondly, we wanted ...to see whether cytotoxicity reflected the measured levels of lichenysin.
Methods and Results
Fifty‐three genotyped strains of B. licheniformis, representing a wide variety of sources, were included. lchAA gene fragments were detected in all strains by polymerase chain reaction (PCR). All 53 strains produced lichenysins with four molecular masses as confirmed by LC‐MS/MS (liquid chromatography–tandem mass spectrometry) analysis. The amounts of lichenysin varied more than two orders of magnitude between strains and were irrespective of genotype. Finally, there was a strong association between lichenysin concentrations and toxicity towards boar spermatozoa, erythrocytes and Vero cells.
Conclusions
Lichenysin synthesis was universal among the 53 B. licheniformis strains examined. The quantities varied considerably between strains, but were not specifically associated with genotype. Cytotoxicity was evident at lichenysin concentrations above 10 μg ml−1, which is in accordance with previous studies.
Significance and Impact of Study
This study might be of interest to those working on B. licheniformis for commercial use as well as for authorities who make risk assessments of B. licheniformis when used as a food and feed additive.
Chitosans, polysaccharides obtained from the exoskeleton of crustaceans, have been shown to exert antibacterial activity in vitro and their use as a food preservative is of growing interest. However, ...beyond a consensus that chitosan appears to disrupt the bacterial cell membrane, published data are inconsistent on the chemical characteristics that confer the antibacterial activity of chitosan. While most authors agree that the net charge density of the polymer (reflected in the fraction of positively charged amino groups at the C-2 position of the glucosamine unit) is an important factor in antibacterial activity, conflicting data have been reported on the effect of molecular weight and on the susceptibility among different bacterial species to chitosan. Therefore, we prepared batches of water-soluble hydrochloride salts of chitosans with weight average molecular weights (M
w) of 2–224
kDa and degree of acetylation of 0.16 and 0.48. Their antibacterial activity was evaluated using tube inhibition assays and membrane integrity assays (
N-Phenyl-1-naphthylamine fluorescence and potassium release) against
Bacillus cereus,
Escherichia coli,
Salmonella Typhimurium and three lipopolysaccharide mutants of
E. coli and
S. Typhimurium. Chitosans with lower degree of acetylation (F
A
=
0.16) were more active than the more acetylated chitosans (F
A
=
0.48). No trends in antibacterial action related to increasing or decreasing M
w were observed although one of the chitosans (M
w 28.4
kDa, F
A
=
0.16) was more active than the other chitosans, inhibiting growth and permeabilizing the membrane of all the test strains included. The test strains varied in their susceptibility to the different chitosans with wild type
S. Typhimurium more resistant than the wild type
E. coli. Salmonellae lipopolysaccharide mutants were more susceptible than the matched wild type strain. Our results show that the chitosan preparation details are critically important in identifying the antibacterial features that target different test organisms.
► Antibacterial activity of chitosans varied with molecular weight, charge and organism. ► Chitosans with low degree of acetylation (0.16) were more active than 0.48-chitosans. ► Chitosan induced prompt bacterial membrane permeabilization. ► Impaired LPS structure increased susceptibility to chitosan for salmonellae. ► Our results show the importance of an accurate description of the test compounds.
A cytotoxin (CytK) has been isolated from a Bacillus cereus strain that caused a severe food poisoning outbreak killing three people. A protein of 34 kDa was highly cytotoxic, and the addition of ...other secreted proteins gave no synergistic effect. CytK was also necrotic and haemolytic. No known B. cereus enterotoxins were produced by this strain. A DNA sequence from 1.8 kb upstream to 0.2 kb downstream of the toxin gene was sequenced. The deduced amino acid sequence of the toxin showed similarity to Staphylococcus aureus leucocidins, γ‐haemolysin and α‐haemolysin, Clostridium perfringensβ‐toxin and B. cereus haemolysin II, all belonging to a family of β‐barrel channel‐forming toxins. There was no sequence similarity between CytK and enterotoxins of B. cereus. The upstream sequence contained a partial sequence of a putative histidine kinase gene. A recognition site for PlcR, which regulates the transcription of enterotoxins HBL and Nhe of B. cereus, was found in the promoter region of the toxin. This new cytotoxin may be responsible for a disease that is similar to, although not as severe as, the necrotic enteritis caused by the β‐toxin of C. perfringens type C.
In cooked-chilled and pasteurized vegetable products, initial numbers of Bacillus cereus were below 10 cfu g-1. Before the appearance of spoilage, numbers reached 6-8 log cfu g-1 at 20 degrees C and ...4-6 log cfu g-1 at 10 degrees C. Bacillus cereus was not detected in samples stored at 4 degrees C. Ten percent of strains isolated from the products were able to grow at 5 degrees C and 63% at 10 degrees C. Bacillus cereus strains unable to degrade starch, a feature linked to the production of emetic toxin, did not grow at 10 degrees C and had a higher heat resistance at 90 degrees C. Using immunochemical assays, enterotoxin was detected in the culture supernatant fluid of 97.5% of the strains. All culture supernatant fluids were cytotoxic but important variations in the level of activity were found. Psychrotrophic isolates of B. cereus were unable to grow in courgette broth at 7 degrees C whereas they grew in a rich laboratory medium. At 10 degrees C, these isolates grew in both media but lag time in courgette broth was 20-fold longer than in the rich laboratory medium.
To identify the phenolic compounds in the leaves of Sphagnum papillosum and examine their antibacterial activity at pH appropriate for the undissociated forms. Bacterial counts of overnight cultures ...showed that whilst growth of Staphylococcus aureus 50084 was impaired in the presence of milled leaves, the phenol-free fraction of holocellulose of S. papillosum had no bacteriostatic effect. Liquid chromatography-mass spectrometry analysis of an acetone-methanol extract of the leaves detected eight phenolic compounds. Antibacterial activity of the four dominating phenols specific to Sphagnum leaves, when assessed in vitro as minimal inhibitory concentrations (MICs), were generally >2·5 mg ml⁻¹. MIC values of the Sphagnum-specific compound 'sphagnum acid' p-hydroxy-β-(carboxymethyl)-cinnamic acid were >5 mg ml⁻¹. No synergistic or antagonistic effects of the four dominating phenols were detected in plate assays. Sphagnum-derived phenolics exhibit antibacterial activity in vitro only at concentrations far in excess of those found in the leaves. We have both identified the phenolic compounds in S. papillosum and assessed their antibacterial activity. Our data indicate that phenolic compounds in isolation are not potent antibacterial agents and we question their potency against food-borne pathogens.
Investigate if the antibacterial effect of sphagnan, a pectin-like carbohydrate polymer extracted from Sphagnum moss, can be accounted for by its ability to lower the pH. Antibacterial activity of ...sphagnan was assessed and compared to that of three other acids. Sphagnan in its acid form was able to inhibit growth of various food poisoning and spoilage bacteria on low-buffering solid growth medium, whereas sphagnan in its sodium form at neutral pH had no antibacterial activity. At similar acidic pH, sphagnan had comparable antibacterial activity to that of hydrochloric acid and a control rhamnogalacturonan pectin in its acid form. Sphagnan in its acid form is a weak macromolecular acid that can inhibit bacterial growth by lowering the pH of environments with a low buffering capacity. It has previously been suggested that sphagnan is an antimicrobial polysaccharide in the leaves of Sphagnum moss with a broad range of potential practical applications. Our results now show that sphagnan in its acid form can indeed inhibit bacterial growth, but only of acid-sensitive species. These findings represent increased knowledge towards our understanding on how sphagnan or Sphagnum moss might be used in practical applications.
Bacillus subtilis and the closely related species
Bacillus pumilus and
Bacillus licheniformis have periodically been suggested to play a role in the aetiology of food poisoning despite the fact that ...the organisms do not possess the genes associated with enteropathogenicity in
Bacillus cereus. We show here that
Bacillus mojavensis, an organism closely related to
B. subtilis, is able to produce toxic components which identify as a complex of three different surfactin analogues. These cyclic lipopeptides were soluble in methanol, heat stable after treatment in a boiling water bath for 10 min, resistant to enzymatic degradation by pepsin, trypsin, endoprotease V8 and proteinase K and formed pores in planar lipid bilayers. They were cytotoxic when tested in a series of commonly used laboratory cytotoxicity assays, namely, lactate dehydrogenase release, haemolysis, inhibition of both protein synthesis in Vero cells and motility in boar sperm. We show that such in vitro markers of enterotoxicity are due entirely to production of cyclic lipopeptides since deletion of
sfp, a gene essential for surfactin synthesis which abolished the cytotoxicity to Vero cells, boar sperm motility and haemolytic activity. Thus, the relevance of cyclic lipopeptides as food poisoning toxins needs to be evaluated in assays other than the cell cytotoxicity assays in common use.
Clostridium perfringens type A food poisoning is one of the more common in the industrialised world. This bacterium is also responsible for the rare but severe food borne necrotic enteritis.
C. ...perfringens enterotoxin (CPE) has been shown to be the virulence factor responsible for causing the symptoms of
C. perfringens type A food poisoning. CPE is a single polypeptide chain with a molecular weight of 3.5 kDa that binds to receptors on the target epithelial cells. Through a unique four-step membrane action it finally causes a breakdown in normal plasma membrane permeability properties. Genetic studies of
cpe have shown that
cpe can be either chromosomal or plasmid-borne and that only a small minority of the global
C. perfringens population is
cpe positive. CPE expression appears to be transcriptionally regulated during sporulation, at least in part, by regulatory factors that are common to all
C. perfringens isolates.
Three enterotoxic components have been isolated from a strain of Bacillus cereus which was involved in a large food poisoning outbreak in Norway in 1995. The components were purified by ...chromatography on three different columns. Three proteins of 39, 45 and 105 kDa, respectively, were found to be necessary for maximum cytotoxicity. The amino acid N-terminal sequences of the 39 and 45 kDa proteins were determined. The 45 kDa component was the same protein as the main antigen detected in the Bacillus Diarrhoeal Enterotoxin Visual Immunoassay (Tecra). The 39 kDa protein showed some similarity to the L1 protein of haemolysin BL from B. cereus. Furthermore, the three toxic components were all recognised by a polyclonal antiserum reported to detect enterotoxin from B. cereus. The proteins were different from the B- and L2-components of haemolysin BL, previously suggested to be a primary virulence factor, and had no detectable haemolytic activity.
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