Maria Rasch

Food spoilage—interactions between food spoilage bacteria

Food spoilage is a complex process and excessive amounts of foods are lost due to microbial spoilage even with modern day preservation techniques. Despite the heterogeneity in raw materials and processing conditions, the microflora that develops during storage and in spoiling foods can be predicted based on knowledge of the origin of the food, the substrate base and a few central preservation parameters such as temperature, atmosphere, a(w) and pH. Based on such knowledge, more detailed sensory, chemical and microbiological analysis can be carried out on the individual products to determine the actual specific spoilage organism. Whilst the chemical and physical parameters are the main determining factors for selection of spoilage microorganisms, a level of refinement may be found in some products in which the interactive behavior of microorganisms may contribute to their growth and/or spoilage activity. This review gives three such examples. We describe the competitive advantage of Pseudomonas spp. due to the production of iron-chelating siderophores, the generation of substrates for spoilage reactions by one organism from another microorganism (so-called metabiosis) and the up-regulation of phenotypes potentially involved in spoilage through cell-to-cell communication. In particular, we report for the first time the widespread occurrence of N-acyl homoserine lactones (AHL) in stored and spoiling fresh foods and we discuss the potential implications for spoilage and food preservation.

An inhibitor of bacterial quorum sensing reduces mortalities caused by Vibriosis in rainbow trout (Oncorhynchus mykiss, Walbaum).

The fish pathogen Vibrio anguillarum produces quorum sensing signal molecules, N-acyl homoserine lactones (AHLs), which in several Gram-negative human and plant pathogenic bacteria regulate virulence factors. Expression of these factors can be blocked using specific quorum-sensing inhibitors (QSIs). The purpose of this study was to investigate the effect of a QSI, furanone C-30, on mortality of rainbow trout during challenge with V. anguillarum. Addition of 0.01 or 0.1 microM furanone C-30 to rainbow trout infected by cohabitation caused a significant reduction in accumulated mortality from 80-100% in challenge controls to 4-40% in treated groups. Furanone C-30 had no effect in an immersion challenge system, probably due to a very high water exchange and a rapid dilution of furanone C-30. Growth and survival of V. anguillarum were not affected by the concentrations of furanone C-30 used in the challenge experiments, thus avoiding selection for resistance. To elucidate the mechanism of disease control by furanone C-30, we determined its effect on the bacterial proteome, motility, and respiration. No effects were seen of furanone C-30 in any of these experiments. Although no cytotoxic effect on HeLa cells were observed, exposure to 1 microM (or higher) concentrations of furanone C-30 had detrimental effects on the rainbow trout. Our results indicate that QSIs can be used in non-antibiotic based control of fish diseases. However, they also underline the need for development of novel, less toxic QSI compounds and the need for understanding the exact mechanism(s) of action.

Involvement of bacterial quorum-sensing signals in spoilage of bean sprouts

ABSTRACT Bacterial communication signals, acylated homoserine lactones (AHLs), were extracted from samples of commercial bean sprouts undergoing soft-rot spoilage. Bean sprouts produced in the laboratory did not undergo soft-rot spoilage and did not contain AHLs or AHL-producing bacteria, although the bacterial population reached levels similar to those in the commercial sprouts, 108 to 109 CFU/g. AHL-producing bacteria (Enterobacteriaceae and pseudomonads) were isolated from commercial sprouts, and strains that were both proteolytic and pectinolytic were capable of causing soft-rot spoilage in bean sprouts. Thin-layer chromatography and liquid chromatography-high-resolution mass spectrometry revealed the presence of N-3-oxo-hexanoyl-l-homoserine lactone in spoiled bean sprouts and in extracts from pure cultures of bacteria. During normal spoilage, the pH of the sprouts increased due to proteolytic activity, and the higher pH probably facilitated the activity of pectate lyase. The AHL synthetase gene (I gene) from a spoilage Pectobacterium was cloned, sequenced, and inactivated in the parent strain. The predicted amino acid sequence showed 97% homology to HslI and CarI in Erwinia carotovora. Spoilage of laboratory bean sprouts inoculated with the AHL-negative mutant was delayed compared to sprouts inoculated with the wild type, and the AHL-negative mutant did not cause the pH to rise. Compared to the wild-type strain, the AHL-negative mutant had significantly reduced protease and pectinase activities and was negative in an iron chelation (siderophore) assay. This is the first study demonstrating AHL regulation of iron chelation in Enterobacteriaceae. The present study clearly demonstrates that the bacterial spoilage of some food products is influenced by quorum-sensing-regulated phenotypes, and understanding these processes may be useful in the development of novel food preservation additives that specifically block the quorum-sensing systems.

A comparative study on the use of flow cytometry and colony forming units for assessment of the antibacterial effect of bacteriocins

Flow cytometry was investigated as a rapid method to determine the antibacterial effect of the bacteriocins nisin, pediocin PA-1, and sakacin A on the indicator organisms Lactobacillus reuteri DSM 12246, Lactobacillus sakei NCFB 2714 and Lactobacillus sakei DSM 20017, respectively. Fluorescence intensities of the cells were measured by flow cytometry upon exposure to bacteriocins after staining with carboxyfluorescein diacetate (cFDA) and were compared to the number of colony forming units (CFU). The fluorescence index (FI) of the bacterial populations decreased when exposed to the bacteriocins. For the different bacteriocins the pattern of decreases in FI and colony forming units differed at equal bacteriostatic concentrations. FI was the most sensitive measure of bacteriocin activity, i.e. the decrease in FI was observed at lower bacteriocin concentrations than decrease in CFU. It was demonstrated that the decrease in FI was caused by rapid leakage of carboxyfluorescein from cells exposed to pediocin. Cells showing severe leakage after pediocin treatment could be detected as CFU when transferred to a rich medium. Such a repair was less pronounced for cells exposed to sakacin and very limited for cells exposed to nisin. The influence of temperature and NaCl in combination with pediocin on FI and CFU of Lactobacillus sakei NCFB 2714 was examined at conditions relevant to foods. At all temperatures (5, 10, 20 and 37 degrees C) and NaCl concentrations (0, 2 and 4% w/v) investigated the flow cytometric measurements were significantly more sensitive compared to CFU. Both methods showed that the inhibitory effect of pediocin increased with increasing temperatures and decreased with increasing NaCl concentrations.

The ¤influence of temperature, salt and pH on the inhibitory effect of reuterin on Escherichia coli

The inhibitory effect of reuterin on Escherichia coli K12 was investigated at different conditions of temperature (10-30 degrees C), pH (4.5-6.5), and NaCl (0.5-3% (w/v)). The maximum specific growth rate as a function of the different environmental conditions was modelled with a polynomial model. At increasing temperatures, there was an increasing effect of reuterin, whereas neither pH nor salt showed interactions with the effect of reuterin. The model was validated against new experimental data and proved to perform satisfactorily based on the bias and accuracy factors.

Quorum sensing signals are produced by Aeromonas salmonicida and quorum sensing inhibitors can reduce production of a potential virulence factor

Many pathogens control production of virulence factors by self-produced signals in a process called quorum sensing (QS). We demonstrate that acyl homoserine lactone (AHL) signals, which enable bacteria to express certain phenotypes in relation to cell density, are produced by a wide spectrum of Aeromonas salmonicida strains. All 31 typical strains were AHL producers as were 21 of 26 atypical strains, but on a strain population basis, production of virulence factors such as protease, lipase, A-layer or pigment did not correlate with the production and accumulation of AHLs in the growth medium. Pigment production was only observed in broth under highly aerated conditions. Quorum sensing inhibitors (QSIs) are compounds that specifically block QS systems without affecting bacterial growth and 2 such compounds, sulphur-containing AHL-analogues, reduced production of protease in a typical strain of Aeromonas salmonicida. The most efficient compound N-(heptylsulfanylacetyl)-L-homoserine lactone (HepS-AHL), reduced protease production by a factor of 10. Five extracellular proteases were detected on gelatin-containing sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels and 3 of these were completely down regulated by HepS-AHL. Hence, QSIs can curb virulence in some strains and could potentially be pursued as bacterial disease control measures in aquaculture.

Profiling acylated homoserine lactones in Yersinia ruckeri and influence of exogenous acyl homoserine lactones and known quorum‐sensing inhibitors on protease production

Aims:  To profile the quorum‐sensing (QS) signals in Yersinia ruckeri and to examine the possible regulatory link between QS signals and a typical QS‐regulated virulence phenotype, a protease.

Well‐known quorum sensing inhibitors do not affect bacterial quorum sensing‐regulated bean sprout spoilage

Aim:  To investigate the potential of quorum sensing inhibitors (QSI) as food preservative agents in a food product, where bacterial spoilage is controlled by quorum sensing (QS).

Characterisation and modelling of oscillatory behaviour related to reuterin production by Lactobacillus reuteri.

During reuterin production by Lactobacillus reuteri in a chemostat, the growth, substrate and metabolite concentrations showed oscillatory behaviour. The sensitivity of L. reuteri towards reuterin was shown to be a possible explanation of the oscillatory behaviour. A deterministic mathematical model consisting of four coupled differential equations describing the concentrations of biomass, glucose, glycerol and reuterin with time was developed. With a set of parameter values determined from batch experiments, the model was able to predict both oscillatory and steady state behaviour in a chemostat by changing the input variables. In a batch system, the model was able to give a satisfactory description of the glucose and glycerol concentrations but not of the biomass and reuterin concentrations. Mathematical modelling of the system was shown to be an effective and systematic approach in exploring a complex biological system.