Elina Jääskeläinen

In vitro assay for human toxicity of cereulide, the emetic mitochondrial toxin produced by food poisoning Bacillus cereus

The in vitro boar spermatozoon test was compared with the LC ion trap MS analysis for measuring the cereulide content of a pasta dish, implemented in serious emetic food poisoning caused by Bacillus cereus. Both assays showed that the poisonous food contained approximately 1.6 microg of cereulide g(-1) implying the toxic dose in human as < or =8 microg kg(-1) body weight. The threshold concentration of cereulide provoking visible mitochondrial damage in boar sperm exposed in vitro was 2 ng of cereulide ml(-1) of extended boar sperm. The same threshold value was found for cereulide extracted from the food and from the cultured bacteria. This shows that other constituents of the food did not enhance or mask the effects of cereulide. Exposure of four human cell lines (HeLa, Caco-2, Calu-3 and Paju) to cereulide showed that the threshold concentration for the loss of mitochondrial membrane potential in human cells was similar to that observed in boar sperm. Human cells and boar sperm were equally sensitive to cereulide. The results show that boar spermatozoan assay is useful for detecting cereulide concentrations toxic to humans. Spermatozoa in commercially available extended fresh boar and cryopreserved bull semen were compared, boar sperms were 100 times more sensitive to cereulide than bull sperms.

Family portrait of Bacillus cereus and Bacillus weihenstephanensis cereulide-producing strains

Two thousand Bacillus cereus sensu lato isolates from food and environmental matrices were screened by PCR for the presence of cereulide-producing strains. This survey identified 73 potential emetic strains, most of which originated from non-random food and clinical samplings. None of the 460 Bacillus thuringiensis, Bacillus mycoides and Bacillus pseudomycoides strains were PCR-positive for the cereulide genetic determinants. The chromosomal and extrachromosomal gene pool diversity of a subset of 30 cereulide-producing strains was then assessed using multilocus sequence typing, large plasmid gel electrophoresis and Southern blot hybridization. The strain toxicity on boar sperm and cereulide production were also analysed. The most striking observation was the identification of two distinct clusters of cereulide-producing strains, with members of the second group (cluster II) identified as psychrotolerant B. weihenstephanensis able to grow at 8°C. Moreover, the location of the cereulide genetic determinants was shown to vary depending on the strain, indicating a probable genomic mobility.

Potential of Bacillus cereus for Producing an Emetic Toxin, Cereulide, in Bakery Products: Quantitative Analysis by Chemical and Biological Methods

A method for the direct quantitative analysis of cereulide, the emetic toxin of Bacillus cereus, in bakery products was developed. The analysis was based on robotized extraction followed by quantitation of cereulide by liquid chromatography-mass spectrometry and an assay of toxicity by the boar sperm motility inhibition test. The bioassay and the chemical assay gave comparable results, demonstrating that the extracted cereulide was in a biologically active form. Cereulide was formed when cereulide-producing B. cereus strains were present at > or = 10(6) CFU/g in products with water activity values of > 0.953 and pHs of > 5.6. Rice-containing pastries accumulated high contents of cereulide (0.3 to 5.5 microg/g [wet weight]) when stored at nonrefrigeration temperatures (21 to 23 degrees C). Cereulide was not formed in products stored at refrigeration temperatures (4 to 8 degrees C). Cereulide is not inactivated by heating during food processing. Therefore, direct analysis of this toxin in food is preferable to cultivating methods for assessing the risk of food poisoning by emetic B. cereus.

Influence of type of food on the kinetics and overall production of Bacillus cereus emetic toxin

Potato puree and penne pasta were inoculated with cereulide producing B. cereus 5964a and B. cereus NS117. Static incubation at 28 degrees C proved these two foods to be a better substrate for higher cereulide production (4,080 ng/g in puree and 3,200 ng/g in penne were produced by B. cereus 5964a during 48 h of incubation) compared with boiled rice (2,000 ng/g). This difference occurred despite B. cereus counts of more than 10(8) CFU/g in all three products. Aeration of cultures had a negative effect on cereulide production, causing concentrations more than 10-fold lower than in some statically incubated samples. Cereulide production remained undetectable in shaken milk, whereas it reached 1,140 ng/ml in statically incubated milk. At 12 and 22 degrees C, presence of background flora was also a determinative factor. A total B. cereus count of more than 106 CFU/ml did not necessarily lead to uniform cereulide production and was also dependent on the B. cereus strain involved. In this study, we confirm that a number of factors play a crucial role in the determination of the extent to which, if at all, cereulide will be produced. Among those, type of the food, temperature, pH, and whether additional aeration (via incubation on an orbital shaker) is induced had an important role. An important effect was also induced by the cereulide-producing strain involved.

Significance of Heme-Based Respiration in Meat Spoilage Caused by Leuconostoc gasicomitatum

ABSTRACT Leuconostoc gasicomitatum is a psychrotrophic lactic acid bacterium (LAB) which causes spoilage in cold-stored modified-atmosphere-packaged (MAP) meat products. In addition to the fermentative metabolism, L. gasicomitatum is able to respire when exogenous heme and oxygen are available. In this study, we investigated the respiration effects on growth rate, biomass, gene expression, and volatile organic compound (VOC) production in laboratory media and pork loin. The meat samples were evaluated by a sensory panel every second or third day for 29 days. We observed that functional respiration increased the growth (rate and yield) of L. gasicomitatum in laboratory media with added heme and in situ meat with endogenous heme. Respiration increased enormously (up to 2,600-fold) the accumulation of acetoin and diacetyl, which are buttery off-odor compounds in meat. Our transcriptome analyses showed that the gene expression patterns were quite similar, irrespective of whether respiration was turned off by excluding heme from the medium or mutating the cydB gene, which is essential in the respiratory chain. The respiration-based growth of L. gasicomitatum in meat was obtained in terms of population development and subsequent development of sensory characteristics. Respiration is thus a key factor explaining why L. gasicomitatum is so well adapted in high-oxygen packed meat.

Development of spoilage bacterial community and volatile compounds in chilled beef under vacuum or high oxygen atmospheres.

Research into microbial community development and metabolism is essential to understand meat spoilage. Recent years have seen the emergence of powerful molecular techniques that are being used alongside conventional microbiology approaches. This enables more accurate studies on meat spoilage. The aim of this study was to investigate the influence of packaging (under vacuum and in high oxygen atmosphere) on the development of microbial communities and metabolic activities at 6 °C by using culture-dependent (cultivation, ribotyping) and culture-independent (amplicon sequencing) methods. At the beginning of shelf life, the microbial community mostly consisted of Carnobacterium and Lactobacillus. After two weeks of storage, Lactococcus and Lactobacillus were the dominant genera under vacuum and Leuconostoc in high oxygen meat packages. This indicates that oxygen favoured the genus Leuconostoc comprising only heterofermentative species and hence potential producers of undesirable compounds. Also the number of volatile compounds, such as diacetyl, 1-octen-3-ol and hexanoic acids, was higher in high oxygen packages than under vacuum packages. The beef in high oxygen atmosphere packaging was detected as spoiled in sensory evaluation over 10 days earlier than beef under vacuum packaging. Leuconostoc gelidum, Lactococcus piscium, Lactobacillus sakei and Lactobacillus algidus were the most common species of bacteria. The results obtained from identification of the isolates using ribotyping and amplicon sequencing correlated, except for L. algidus, which was detected in both types of packaging by amplicon sequencing, but only in vacuum packaged samples using the culture-based technique. This indicates that L. algidus grew, but was not cultivable in high oxygen beef using the Nordic Committee on Food Analysis standard method.

Genome Sequence and Transcriptome Analysis of Meat-Spoilage-Associated Lactic Acid Bacterium Lactococcus piscium MKFS47

ABSTRACT Lactococcus piscium is a psychrotrophic lactic acid bacterium and is known to be one of the predominant species within spoilage microbial communities in cold-stored packaged foods, particularly in meat products. Its presence in such products has been associated with the formation of buttery and sour off-odors. Nevertheless, the spoilage potential of L. piscium varies dramatically depending on the strain and growth conditions. Additional knowledge about the genome is required to explain such variation, understand its phylogeny, and study gene functions. Here, we present the complete and annotated genomic sequence of L. piscium MKFS47, combined with a time course analysis of the glucose catabolism-based transcriptome. In addition, a comparative analysis of gene contents was done for L. piscium MKFS47 and 29 other lactococci, revealing three distinct clades within the genus. The genome of L. piscium MKFS47 consists of one chromosome, carrying 2,289 genes, and two plasmids. A wide range of carbohydrates was predicted to be fermented, and growth on glycerol was observed. Both carbohydrate and glycerol catabolic pathways were significantly upregulated in the course of time as a result of glucose exhaustion. At the same time, differential expression of the pyruvate utilization pathways, implicated in the formation of spoilage substances, switched the metabolism toward a heterofermentative mode. In agreement with data from previous inoculation studies, L. piscium MKFS47 was identified as an efficient producer of buttery-odor compounds under aerobic conditions. Finally, genes and pathways that may contribute to increased survival in meat environments were considered.

Production of Buttery-Odor Compounds and Transcriptome Response in Leuconostoc gelidum subsp. gasicomitatum LMG18811T during Growth on Various Carbon Sources

ABSTRACT Leuconostoc gelidum subsp. gasicomitatum is a common spoilage bacterium in meat products packaged under oxygen-containing modified atmospheres. Buttery off-odors related to diacetyl/acetoin formation are frequently associated with the spoilage of these products. A whole-genome microarray study, together with gas chromatography (GC)-mass spectrometry (MS) analyses of the pathway end products, was performed to investigate the transcriptome response of L. gelidum subsp. gasicomitatum LMG18811T growing on semidefined media containing glucose, ribose, or inosine, which are essential carbon sources in meat. Generally, the gene expression patterns with ribose and inosine were quite similar, indicating that catabolism of ribose and nucleosides is closely linked. Diacetyl/acetoin concentrations as high as 110 or 470 μM were measured when growth was based on inosine or ribose, respectively. The gene expression results for pyruvate metabolism (upregulation of α-acetolactate synthase, downregulation of l-lactate dehydrogenase and pyruvate dehydrogenase) were as expected when diacetyl and acetoin were the end products. No diacetyl production (<7.5 μM) was detected with the glucose-containing medium, even though the cell counts of LMG18811T was 6 or 10 times higher than that on inosine or ribose, respectively. Although glucose was the most effective carbon source for the growth of L. gelidum subsp. gasicomitatum, utilization of inosine and ribose resulted in the production of the unwanted buttery-odor compounds. These results increase our understanding of which compounds are likely to enhance the formation of buttery odors during meat spoilage caused by L. gelidum subsp. gasicomitatum.

Lactobacillus oligofermentans glucose, ribose and xylose transcriptomes show higher similarity between glucose and xylose catabolism-induced responses in the early exponential growth phase

BackgroundLactobacillus oligofermentans has been mostly isolated from cold-stored packaged meat products in connection with their spoilage, but its precise role in meat spoilage is unknown. It belongs to the L. vaccinostercus group of obligate heterofermentative lactobacilli that generally ferment pentoses (e.g. xylose and ribose) more efficiently than hexoses (e.g. glucose). However, more efficient hexose utilization can be induced. The regulation mechanisms of the carbohydrate catabolism in such bacteria have been scarcely studied. To address this question, we provided the complete genome sequence of L. oligofermentans LMG 22743T and generated time course transcriptomes during its growth on glucose, ribose and xylose.ResultsThe genome was manually annotated and its main functional features were examined. L. oligofermentans was confirmed to be able to efficiently utilize several hexoses and maltose, which is, presumably, induced by its repeated cultivation with glucose in vitro. Unexpectedly, in the beginning of the exponential growth phase, glucose- and xylose-induced transcriptome responses were more similar, whereas toward the end of the growth phase xylose and ribose transcriptomes became more alike. The promoter regions of genes simultaneously upregulated both on glucose and xylose in comparison with ribose (particularly, hexose and xylose utilization genes) were found to be enriched in the CcpA- binding site. Transcriptionally, no glucose-induced carbon catabolite repression was detected. The catabolism of glucose, which requires initial oxidation, led to significant overexpression of the NAD(P)H re-oxidation genes, the upstream regions of which were found to contain a motif, which was highly similar to a Rex repressor binding site.ConclusionsThis paper presents the second complete genome and the first study of carbohydrate catabolism-dependent transcriptome response for a member of the L. vaccinostercus group. The transcriptomic changes detected in L. oligofermentans for growth with different carbohydrates differ significantly from those of facultative heterofermentative lactobacilli. The mechanism of CcpA regulation, putatively contributing to the observed similarities between glucose- and xylose-induced transcriptome responses and the absence of stringent carbon catabolite control, requires further studies. Finally, the cell redox balance maintenance, in terms of the NAD(P)+/NAD(P)H ratio, was predicted to be regulated by the Rex transcriptional regulator, supporting the previously made inference of Rex-regulons for members of the Lactobacillaceae family.

Retention of Bacillus cereus and its toxin, cereulide, in cellulosic fibres.

Abstract Bacillus cereus is the only pathogen that is occasionally found in paper and paper products, but there is no information on its prevalence. The aim of this work was to obtain data for a risk assessment of B. cereus in cellulosic fibre-based products. Handsheets were formed using laboratory papermaking equipment from stocks admixed with B. cereus. Then the distribution of B. cereus and its heat-stable toxin, cereulide, between the fibre web and the wire filtrate was measured. The handsheets retained 5% of the vegetative cells and spores of B. cereus and 10–15% of the cereulide. Transfer of cereulide into food or drink through contact with paper was investigated using ethanol and hot and cold water as food and drink simulants. Less than 0.2% of the cereulide from handsheets was recovered from hot and cold water, as measured by LC/MS and the boar sperm bioassay. Total immersion in 95% v/v ethanol leached nearly all cereulide present in the paper. The results obtained with the bioassay were equivalent to those obtained by LC/MS for the leachates, indicating that cereulide retained its toxicity through the handsheet-making process. The results indicate that cereulide in pulps is probably also present in paper products made from them, but the concentration appears to be too low to be relevant in terms of toxicity.