Real-time monitoring of fungal inhibition and morphological changes.

Mold growth constitutes a problem in many food and clinical environments and there is therefore focus on studying antifungal activity. Methods for determining growth inhibition by measuring colony growth or biomass are, however, time-taking and rapid methods for evaluation of antifungal effects are needed. Propionic acid and diacetyl are antifungal compounds produced by a range of dairy-associated bacteria. Their activity against Penicillium spp. was monitored real-time using an optical detection system with tilted focus plane to assess growth and morphological changes of Penicillium spp. by image recording inside a 96 well microplate. Images were used for generation of growth curves by using a segmentation and extraction of surface areas (SESA) algorithm and for quantifying morphology changes. Using image analysis growth could be detected within 15 h compared with more than 30 h when using standard optical density measurements. Induced morphological changes of fungi could furthermore be visualized and quantified using morphological descriptors such as circularity, branch points, perimeter and area of spores and growing hyphae. Propionic acid inhibited two out of two Penicillium spp. while morphological changes were strain dependent at the concentrations tested. Diacetyl inhibited six out of six Penicillium spp. strains and increased spore size and number of germination sites in two out of six of the strains prior to germination.

Contribution of volatiles to the antifungal effect of Lactobacillus paracasei in defined medium and yogurt.

Lactic acid bacteria with antifungal properties can be used to control spoilage of food and feed. Previously, most of the identified metabolites have been isolated from cell-free fermentate of lactic acid bacteria with methods suboptimal for detecting possible contribution from volatiles to the antifungal activity. The role of volatile compounds in the antifungal activity of Lactobacillus paracasei DGCC 2132 in a chemically defined interaction medium (CDIM) and yogurt was therefore investigated with a sampling technique minimizing volatile loss. Diacetyl was identified as the major volatile produced by L. paracasei DGCC 2132 in CDIM. When the strain was added to a yogurt medium diacetyl as well as other volatiles also increased but the metabolome was more complex. Removal of L. paracasei DGCC 2132 cells from CDIM fermentate resulted in loss of both volatiles, including diacetyl, and the antifungal activity towards two strains of Penicillium spp. When adding diacetyl to CDIM or yogurt without L. paracasei DGCC 2132, marked inhibition was observed. Besides diacetyl, the antifungal properties of acetoin were examined, but no antifungal activity was observed. Overall, the results demonstrate the contribution of diacetyl in the antifungal effect of L. paracasei DGCC 2132 and indicate that the importance of volatiles may have been previously underestimated.

Growth inhibition of Listeria monocytogenes by a nonbacteriocinogenic Carnobacterium piscicola.

AIMS: This study elucidates the mechanisms by which a nonbacteriocinogenic Carnobacterium piscicola inhibits growth of Listeria monocytogenes. METHODS AND RESULTS: Listeria monocytogenes was exposed to live cultures of a bacteriocin-negative variant of C. piscicola A9b in co-culture, in a diffusion chamber system, and to a cell-free supernatant. Suppression of maximum cell density (0-3.5 log units) of L. monocytogenes was proportional to initial levels of C. pisciola (10(3)-10(7) CFU ml(-1)). Cell-to-cell contact was not required to cause inhibition. The cell-free C. piscicola supernatant caused a decrease in L. monocytogenes maximum cell density, which was abolished by glucose addition but not by amino acid, vitamin or mineral addition. The fermentate also gave rise to a longer lag phase and a reduction in growth rate. These effects were independent of glucose and may have been caused by acetate production by C. piscicola. 2D gel-electrophoretic patterns of L. monocytogenes exposed to C. piscicola or to L. monocytogenes fermentate did not differ. Treatment with C. piscicola fermentate resulted in down-regulation (twofold) of genes involved in purine- or pyrimidine metabolism, and up-regulation (twofold) of genes from the regulon for vitamin B12 biosynthesis and propanediol and ethanolamine utilization. CONCLUSIONS: A nonbacteriocinogenic C. piscicola reduced growth of L. monocytogenes partly by glucose depletion. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the mechanism of microbial interaction enhances prediction of growth in mixed communities as well as use of bioprotective principles for food preservation.

Differential inactivation of Listeria monocytogenes by D- and L-lactic acid.

AIMS: To determine inactivation of Listeria monocytogenes by the two lactic acid isomers. METHODS AND RESULTS: The survival of four strains with varying sensitivity to acid was determined following treatment with L- or D-lactic acid at 100 mmol l(-1) (pH 3.7) or HCl at pH 3.37. There was some, but not complete, similarity in the relative sensitivity of the four strains to the two types of acid. All strains were most sensitive to D-lactic acid, which gave 0.6-2.2 log units greater reduction than L-lactic acid midway in the inactivation curves. Even very low concentrations of the two isomers had an immediate effect on pH(i) which was identical for the two isomers. CONCLUSIONS: The results show that L. monocytogenes is more sensitive to D- than to L-lactic acid; however, this difference is less than the strain variation in L-lactic acid sensitivity. SIGNIFICANCE AND IMPACT OF THE STUDY: This work has implications for the application of lactic acid for food preservation as well as for the understanding of the antibacterial mechanisms of weak organic acids.

Image analysis method for evaluation of specific and non-specific hand contamination.

AIMS: To evaluate a quantifying image analysis method for assessing the degree of hand contamination and efficacy of hand washing procedures. METHODS AND RESULTS: Two types of experimental design were used. In one, different concentrations of pure cultures of Escherichia coli, Listeria innocua and Pseudomonas flourescens were applied to hands. In the other, hands were contaminated by handling various raw foods. Imprints of the contaminated palms were made on 24.5 x 24.5 cm agar plates using appropriate agars. After incubation, digital photographs of the plates were analysed using image analysis. In pure culture studies with selective agars, levels from 1 to 10(6) CFU cm(-2) palm could be monitored. For aerobic, mesophilic organisms from raw chicken, levels from 10(3) to 10(6) CFU cm(-2) palm were correlated linearly to image analysis data. CONCLUSIONS: The image analysis of palm imprints made on agar plates was suitable for assessing the degree of contamination from foods on the palms. Sensitivity and specificity depended on the agar used and the type of contamination encountered. SIGNIFICANCE AND IMPACT OF THE STUDY: Data capture by the image analysis method is simple and can be partly automated. Sampling time is short for the person to be tested, which makes it an attractive method for assessing hand hygiene status in larger field trials.

Frequency of bacteriocin resistance development and associated fitness costs in Listeria monocytogenes.

Bacteriocin-producing starter cultures have been suggested as natural food preservatives; however, development of resistance in the target organism is a major concern. We investigated the development of resistance in Listeria monocytogenes to the two major bacteriocins pediocin PA-1 and nisin A, with a focus on the variations between strains and the influence of environmental conditions. While considerable strain-specific variations in the frequency of resistance development and associated fitness costs were observed, the influence of environmental stress seemed to be bacteriocin specific. Pediocin resistance frequencies were determined for 20 strains and were in most cases ca. 10(-6). However, two strains with intermediate pediocin sensitivity had 100-fold-higher pediocin resistance frequencies. Nisin resistance frequencies (14 strains) were in the range of 10(-7) to 10(-2). Strains with intermediate nisin sensitivity were among those with the highest frequencies. Environmental stress in the form of low temperature (10 degrees C), reduced pH (5.5), or the presence of NaCl (6.5%) did not influence the frequency of pediocin resistance development; in contrast, the nisin resistance frequency was considerably reduced (<5 x 10(-8)). Pediocin resistance in all spontaneous mutants was very stable, but the stability of nisin resistance varied. Pediocin-resistant mutants had fitness costs in the form of reduction down to 44% of the maximum specific growth rate of the wild-type strain. Nisin-resistant mutants had fewer and less-pronounced growth rate reductions. The fitness costs were not increased upon applying environmental stress (5 degrees C, 6.5% NaCl, or pH 5.5), indicating that the bacteriocin-resistant mutants were not more stress sensitive than the wild-type strains. In a saveloy-type meat model at 5 degrees C, however, the growth differences seemed to be negligible. The applicational perspectives of the results are discussed.

Structure and expression of Xenopus tropicalis BMP-2 and BMP-4 genes.

The Xenopus tropicalis BMP-2 and BMP-4 genes have been cloned and sequenced. A comparison with the corresponding genes from X. laevis reveals that the BMP-4 genes are conserved at a higher extent than the BMP-2 genes. This is especially evident for the intron sequences, but is also reflected by the exon sequences. While the amino acids of X. tropicalis and X. laevis BMP-4 proteins diverge at about 4%, those of BMP-2 proteins diverge at about 7%. By reverse transcriptase polymerase chain reaction analyses and whole mount in situ hybridizations, we demonstrate the temporal and spatial expression patterns of X. tropicalis BMP-2 and BMP-4 genes. BMP-2 is found to be expressed maternally, and later in development, in migrating heart progenitor cells as well as in the final heart, within the pineal gland and the olfactory placodes. BMP-4 is zygotically activated within the ventral marginal zone and later found in the eye, the otic vesicle, the heart and within blood islands. Although the overall patterns are very similar to those found in X. laevis, there is some distinct difference which might result from the accelerated development in X. tropicalis.

Spontaneous nisin-resistant Listeria monocytogenes mutants with increased expression of a putative penicillin-binding protein and their sensitivity to various antibiotics.

A concern regarding the use of bacteriocins, as for example the lantibiotic nisin, for biopreservation of certain food products is the possibility of resistance development and potential cross-resistance to antibiotics in the target organism. The genetic basis for nisin resistance development is as yet unknown. We analyzed changes in gene expression following nisin resistance development in Listeria monocytogenes 412 by restriction fragment differential display. The mutant had increased expression of a protein with strong homology to the glycosyltransferase domain of high-molecular-weight penicillin-binding proteins (PBPs), a histidine protein kinase, a protein of unknown function, and ClpB (putative functions from homology). The three former proteins had increased expression in a total of six out of 10 independent mutants originating from five different wild-type strains, indicating a prevalent nisin resistance mechanism under the employed isolation conditions. Increased expression of the putative PBP may affect the cell wall composition and thereby alter the sensitivity to cell wall-targeting compounds. The mutants had an isolate-specific increase in sensitivity to different beta-lactams and a slight decrease in sensitivity to another lantibiotic, mersacidin. A model incorporating these observations is proposed based on current knowledge of nisin's mode of action.

Factors influencing resuscitation and growth of heat injured Listeria monocytogenes 13-249 in sous vide cooked beef.

The growth of Listeria monocytogenes 13-249 in vacuum-packed, minced beef was investigated as a function of degree of heat injury (including no injury i.e. uncooked beef), growth phase (logarithmic and late stationary phase), pH (5.6 and 6.2), and storage temperature (3, 10 and 20 degrees C) during a storage period of 30 days. Late logarithmic and late stationary phase cultures of L. monocytogenes 13-249 showed similar growth in refrigerated, vacuum-packed, raw minced beef with a high pH (6.2). In normal pH (5.6) beef there was no growth at 3 degrees C while growth at 10 and 20 degrees C was only observed for logarithmic phase cultures. Heat injured late stationary phase cultures with 95-99.9% injured cells in the surviving population (as measured by differential plating on enriched vs. selective media after sous vide cooking) did not grow or repair sublethal injuries in sous vide cooked beef at 3 degrees C while repair and growth took place at 10 as well as at 20 degrees C. In logarithmic phase cultures heat injury occurred very rapidly and > or = 99.9% heat injury was observed in all trials in spite of much lower pasteurization values and fewer log10 reductions compared with late stationary phase cultures. Regardless of growth phase, all cultures where a high degree of heat injury (> or = 99.9%) was observed, did not subsequently grow in the beef product at 3 or 10 degrees C within 30 days. Growth of heat injured cultures preexposed to heat shock (46 degrees C, 30 min) or slowly rising temperatures (0.3 degrees C min(-1)) before heat injury was also investigated. Heat shocked or heat adapted cultures generally responded in the same manner as non-stressed cultures (no growth at 3 degrees C) except that a longer lag phase was observed in beef processed at slowly rising temperatures and in normal pH beef at 10 degrees C. Although processing at slowly rising temperatures may slightly increase the survival of L. monocytogenes 13-249 in cooked beef, there seem to be no indication of an increase in subsequent growth potential of the surviving cells.

c-Jun (AP-1) activates BMP-4 transcription in Xenopus embryos.

Zygotic expression of the BMP-4 gene in Xenopus embryos is regulated by an auto-regulatory loop. Since AP-1 is known as a mediator of auto-regulatory loops both in the case of the Drosophila dpp and the mammalian TGF-beta genes, we have analysed the potential of Xenopus c-Jun (AP-1) as a mediator of BMP-4 expression during Xenopus development. RNA injection experiments revealed that both heteromeric c-Fos/c-Jun and homodimeric c-Jun/c-Jun strongly activate BMP-4 transcription, whereas BMP signaling was found to activate the Xenopus c-Jun gene only at a rather low extent. In addition, the lack of zygotic c-Jun transcripts until the end of gastrulation should exclude a role of AP-1 in the activation and the early expression of BMP-4 during gastrulation in vivo. However, at later stages of Xenopus development, we find a spatial overlap of c-Jun and BMP-4 transcripts which suggests that AP-1 might serve as an additional activatory component for the auto-regulation of BMP-4. Promoter/reporter and gel mobility shift assays demonstrate multiple responsive sites for AP-1 in the 5' flanking region and two in the second intron of the BMP-4 gene. We further demonstrate that AP-1 acts independently of Xvent-2 which has recently been shown to mediate the early expression of BMP-4 in gastrula stage embryos.

The homeodomain transcription factor Xvent-2 mediates autocatalytic regulation of BMP-4 expression in Xenopus embryos.

Like other genes of the transforming growth factor-beta family, the BMP-4 gene is regulated by an autocatalytic loop. In Xenopus embryos this loop can be ectopically induced by injection of BMP-2 RNA. However, cycloheximide treatment subsequent to BMP-2 overexpression revealed that BMP signaling is not direct but requires additional factor(s). As putative mediator we have identified Xvent-2 which is activated by BMP-2/4 signaling and, in turn, activates BMP-4 transcription. Using promoter/reporter assays we have delineated Xvent-2 responsive elements within the BMP-4 gene. We further demonstrate that Xvent-2 which has recently been characterized as a transcriptional repressor can also act, context dependent, as an activator binding two copies of a 5'-CTAATT-3' motif in the second intron of the BMP-4 gene. Replacement of Xvent-2 target sites within the goosecoid (gsc) promoter by the BMP-4 enhancer converts Xvent-2 caused repression of gsc to strong activation. This switch is obviously due to adjacent nucleotides probably binding a transcriptional co-activator interacting with Xvent-2. A model is presented describing the mechanism of BMP-4 gene activation in Xenopus embryos at the early gastrula stage.

Growth condition-related response of Listeria monocytogenes 412 to bacteriocin inactivation.

Bacteriocin inactivation of Listeria monocytogenes 412 was studied as a function of growth phase. Cells were treated with nisin (300 IU ml-1) or pediocin (320 or 2560 AU ml-1) for 20 min at 30 degrees C. Inactivation with nisin or the low concentration of pediocin was growth phase dependent, with exponentially growing cells being more susceptible than stationary cells. No effect of growth phase was observed for the high pediocin concentration. Pediocin inactivation (320 AU ml-1) of L. monocytogenes 412 exposed to osmotic (6.5% NaCl) or low-temperature (5 degrees C) stress was investigated. Pediocin failed to inactivate osmotically stressed cultures and was unable to inhibit cold-stressed cells to the same degree as unstressed cells.

Variations in tolerance of Listeria monocytogenes to nisin, pediocin PA-1 and bavaricin A.

A collection of 381 Listeria monocytogenes strains was examined for strain variations in nisin and pediocin sensitivity at three different concentrations on Tryptic Soya Agar. Two of the strains were able to grow on agar plates containing 500 IU nisin ml-1. These strains were characterized as having an enhanced nisin tolerance. Twenty strains had normal growth on agar plates containing 1600 AU pediocin ml-1 and higher. These strains were characterized as pediocin-resistant. Another 34 strains, characterized as having enhanced tolerance, exhibited inhibited growth at 1600 AU pediocin ml-1. Bavaricin sensitivity, examined for 22 strains of L. monocytogenes, correlated with the pediocin sensitivity of the strains. It is therefore assumed that there is accordance between the pediocin and bavaricin sensitivity of the remaining strains. Cross-resistance between nisin and pediocin/bavaricin was not found.

Structural and functional analysis of the BMP-4 promoter in early embryos of Xenopus laevis.

The Xenopus laevis BMP-4 gene shows an evolutionary conserved structure containing two coding exons and a leader exon. The transcripts which are detected after zygotic activation of the gene in ventral mesoderm of late blastula stage embryos do either contain the leader exon or begin within the first intron. Luciferase reporter/promoter studies revealed multiple elements being required for the activation and for the spatial control of transcription. These elements are located within the upstream region and within the second intron and they interact with a most proximal located basal promoter being indispensable for transcriptional activation. The auto-activatory capacity of BMP-4 is mediated by several enhancer elements being responsive not only to BMP-4 but also to BMP-2 signaling. BMP-2 might thus function as a natural activator of the BMP-4 gene in the early embryo. Since reporter activity obtained with distinct BMP-2/4 responsive promoter deletion mutants is simultaneously inhibited by the dominant negative BMP receptor as well as by chordin, we suggest that down-regulation of the BMP-4 gene by chordin results from an interference with the auto-regulatory loop at the level of protein-protein interactions.

Thermal inactivation of Listeria monocytogenes during rapid and slow heating in sous vide cooked beef.

Heating at slowly rising temperatures is suspected to enhance thermotolerance in Listeria monocytogenes and, since anaerobic environments have been shown to facilitate resuscitation of heat-injured cells of this micro-organism, concern may arise about the possibility of L. monocytogenes surviving in minimally preserved products. The effect of rapid ( > 10 degrees C min-1) and slow (0.3 and 0.6 degrees C min-1) heating on survival of L. monocytogenes in sous vide cooked beef was therefore examined at mild processing temperatures of 56 degrees, 60 degrees and 64 degrees C. No statistically significant difference (P = 0.70) was observed between the tested heating regimes. Since the average pH of beef was low (5.6), and little or no effect was observed, a pH-dependency of heat shock-induced thermotolerance in L. monocytogenes is suggested to account for this result.

Effect of pre- and post-heat shock temperature on the persistence of thermotolerance and heat shock-induced proteins in Listeria monocytogenes.

The effect of incubation temperature, before and after a heat shock, on thermotolerance of Listeria monocytogenes at 58 degrees C was investigated. Exposing cells grown at 10 degrees C and 30 degrees C to a heat shock resulted in similar rises in thermotolerance while the increase was significantly higher when cells were grown at 4 degrees C prior to the heat shock. Cells held at 4 degrees C and 10 degrees C after heat shock maintained heat shock-induced thermotolerance for longer than cells held at 30 degrees C. The growth temperature prior to inactivation had negligible effect on the persistence of heat shock-induced thermotolerance. Concurrent with measurements of thermotolerance were measurements of the levels of heat shock-induced proteins. Major proteins showing increased synthesis upon the heat shock had approximate molecular weights of 84, 74, 63, 25 and 19 kDa. There was little correlation between the loss of thermotolerance after the heat shock and the levels of these proteins. Thermotolerance of heat shocked and non-heat shocked cells was described by traditional log-linear kinetics and a model describing a sigmoidal death curve (logistic model). Employing log-linear kinetics resulted in a poor fit to a major part of the data whereas a good fit was achieved by the use of a logistic model.

Emerging pathogens: Aeromonas spp.

Aeromonas spp. are Gram-negative rods of the family Vibrionaceae. They are normal water inhabitants and are part of the regular flora of poiquilotherm and homeotherm animals. They can be isolated from many foodstuffs (green vegetables, raw milk, ice cream, meat and seafood). Mesophilic Aeromonas spp. have been classified following the AeroKey II system (Altwegg et al., 1990; Carnahan et al., 1991). The major human diseases caused by Aeromonas spp. can be classified in two major groups: septicemia (mainly by strains of A. veronii subsp. sobria and A. hydrophila), and gastroenteritis (any mesophilic Aeromonas spp. but principally A. hydrophila and A. veronii). Most epidemiological studies have shown Aeromonas spp. in stools to be more often associated with diarrhea than with the carrier state; an association with the consumption of untreated water was also conspicuous. Acute self-limited diarrhea is more frequent in young children, in older patients chronic enterocolitis may also be observed. Fever, vomiting, and fecal leukocytes or erythrocytes (colitis) may be present (Janda, 1991). The main putative virulence factors are: exotoxins, endotoxin (LPS), presence of S-layers, fimbriae or adhesins and the capacity to form capsules.

Bone morphogenetic protein 2 in the early development of Xenopus laevis.

The temporal and spatial transcription patterns of the Xenopus laevis Bone morphogenetic protein 2 (BMP-2) gene have been investigated. Unlike the closely related BMP-4 gene, the BMP-2 gene is strongly transcribed during oogenesis. Besides some enrichment within the animal half, maternal BMP-2 transcripts are ubiquitously distributed in the early cleavage stage embryos but rapidly decline during gastrulation. Zygotic transcription of this gene starts during early neurulation and transcripts are subsequently localized to neural crest cells, olfactory placodes, pineal body and heart anlage. Microinjection of BMP-2 RNA into the two dorsal blastomeres of 4-cell stage embryos leads to ventralization of developing embryos. This coincides with a decrease of transcripts from dorsal marker genes (beta-tubulin, alpha-actin) but not from ventral marker genes (alpha-globin). BMP-2 overexpression inhibits transcription of the early response gene XFD-1, a fork head/HNF-3 related transcription factor expressed in the dorsal lip, but stimulates transcription of the posterior/ventral marker gene Xhox3, a member of the helix-turn-helix family. Activin A incubated animal caps from BMP-2 RNA injected embryos show transcription of ventral but an inhibition of dorsal marker genes; thus, BMP-2 overrides the dorsalizing activity of activin A. The results demonstrate that BMP-2 overexpression exerts very similar effects as have previously been described for BMP-4, and they suggest that BMP-2 may act already as a maternal factor in ventral mesoderm formation.

Growth and survival of Yersinia enterocolitica, Salmonella and Bacillus cereus in Brie stored at 4, 8 and 20 degrees C.

The potential of stabilised Brie to support growth of the food-borne pathogens, Yersinia enterocolitica serotypes 0:3 and 0:9, Salmonella typhimurium, S. dublin (both dairy isolates), S. thompson, and Bacillus cereus (3 dairy isolates), after contamination on opening the cheese package was evaluated. Growth kinetics of the different pathogens was determined in relation to inoculum size and storage temperature (4 degrees C, 8 degrees C and 20 degrees C). Only Y. enterocolitica was found to grow on the surfaces (outer and exposed) of Brie at all three storage temperatures. Growth of this pathogen during refrigerated storage must be avoided to ensure safety. The numbers of B. cereus and Salmonella increased at 20 degrees C but declined at a slow rate during storage at 4 degrees C and 8 degrees C. However, survival of these two pathogens for extended periods at abuse temperatures could pose a health hazard. Predictions from a predictive modelling program (MFS model) and a modelling database (Food Micromodel) were compared to observed growth values in Brie. Although accurate in the case of B. cereus at 20 degrees C, predicted generation times were in general found to be considerably shorter than the observed values, i.e., overall they were 'fail safe'. Predicted lag times, however, were generally longer compared to observed values in the case of Y. enterocolitica and at low inocula for Salmonella, and would 'fail dangerous' if used for predictive purposes.

Activin A induced expression of a fork head related gene in posterior chordamesoderm (notochord) of Xenopus laevis embryos.

A gene family encoding the fork head/HNF-3 domain has been identified in the South African clawed frog, Xenopus laevis. Screening of genomic DNA and gastrula stage derived cDNA libraries with a DNA fragment encoding the Drosophila fork head domain led to the isolation of a number of different clones encoding this motif. While one of the Xenopus fork head sequences, XFD-3, represents the Xenopus counterpart to rat HNF-3 beta, all other sequences encode novel types of fork head related proteins. Here we report on XFD-1, a DNA binding protein which can bind to the HNF-3 alpha target sequence. Analysis of temporal and spatial expression revealed that the gene is activated at blastula stage and that transcripts are localized in a rather thin stripe of cells invaginating the dorsal blastopore lip (organizer) during gastrulation. XFD-1 mRNA is localized within the notochord and, by the end of neurulation, is no longer detectable. In the animal cap assay the gene is activated by incubation with the vegetalizing factor (activin A) but not with bFGF.