Cerebrospinal fluid proteome shows disrupted neuronal development in multiple sclerosis.

Despite intensive research, the aetiology of multiple sclerosis (MS) remains unknown. Cerebrospinal fluid proteomics has the potential to reveal mechanisms of MS pathogenesis, but analyses must account for disease heterogeneity. We previously reported explorative multivariate analysis by hierarchical clustering of proteomics data of MS patients and controls, which resulted in two groups of individuals. Grouping reflected increased levels of intrathecal inflammatory response proteins and decreased levels of proteins involved in neural development in one group relative to the other group. MS patients and controls were present in both groups. Here we reanalysed these data and we also reanalysed data from an independent cohort of patients diagnosed with clinically isolated syndrome (CIS), who have symptoms of MS without evidence of dissemination in space and/or time. Some, but not all, CIS patients had intrathecal inflammation. The analyses reported here identified a common protein signature of MS/CIS that was not linked to elevated intrathecal inflammation. The signature included low levels of complement proteins, semaphorin-7A, reelin, neural cell adhesion molecules, inter-alpha-trypsin inhibitor heavy chain H2, transforming growth factor beta 1, follistatin-related protein 1, malate dehydrogenase 1 cytoplasmic, plasma retinol-binding protein, biotinidase, and transferrin, all known to play roles in neural development. Low levels of these proteins suggest that MS/CIS patients suffer from abnormally low oxidative capacity that results in disrupted neural development from an early stage of the disease.

Growth Behavior of Listeria monocytogenes in a Traditional Norwegian Fermented Fish Product (Rakfisk), and Its Inhibition through Bacteriophage Addition.

Listeria monocytogenes may persist in food production environments and cause listeriosis. In Norway, a product of concern is the traditional and popular fermented fish product "rakfisk", which is made from freshwater salmonid fish by mild-salting and brine maturation at low temperatures for several months. It is eaten without any heat treatment, and L. monocytogenes, therefore, poses a potential hazard. We investigated the effect of salt and temperature on the growth of L. monocytogenes in rakfisk during the 91 days of maturation. The amounts of organic acids produced during fermentation were too low to inhibit growth of L. monocytogenes. Temperature was clearly the most important parameter for controlling L. monocytogenes. At 7 °C, approximately 2 log growth was observed during the first 14 days of fermentation, and the level of L. monocytogenes thereafter remained constant. At 4 °C, only a little growth potential of the pathogen was recorded. We also investigated the effect of the anti-Listeria bacteriophage P100 on rakfisk with added L. monocytogenes. The phage was introduced to the L. monocytogenes-inoculated fish before fermentation, and an average of 0.9 log reduction was observed throughout the fermentation period. This is the first study of L. monocytogenes behavior in rakfisk and points to possible measures for increasing the product safety.

Large Plasmid Complement Resolved: Complete Genome Sequencing of Lactobacillus plantarum MF1298, a Candidate Probiotic Strain Associated with Unfavorable Effect.

Considerable attention has been given to the species Lactobacillus plantarum regarding its probiotic potential. L. plantarum strains have shown health benefits in several studies, and even nonstrain-specific claims are allowed in certain markets. L. plantarum strain MF1298 was considered a candidate probiotic, demonstrating in vitro probiotic properties and the ability to survive passage through the human intestinal tract. However, the strain showed an unfavorable effect on symptoms in subjects with irritable bowel syndrome in a clinical trial. The properties and the genome of this strain are thus of general interest. Obtaining the complete genome of strain MF1298 proved difficult due to its large plasmid complement. Here, we exploit a combination of sequencing approaches to obtain the complete chromosome and plasmid assemblies of MF1298. The Oxford Nanopore Technologies MinION long-read sequencer was particularly useful in resolving the unusually large number of plasmids in the strain, 14 in total. The complete genome sequence of 3,576,440 basepairs contains 3272 protein-encoding genes, of which 315 are located on plasmids. Few unique regions were found in comparison with other L. plantarum genomes. Notably, however, one of the plasmids contains genes related to vitamin B12 (cobalamin) turnover and genes encoding bacterial reverse transcriptases, features not previously reported for L. plantarum. The extensive plasmid information will be important for future studies with this strain.

Chicken fillets subjected to UV-C and pulsed UV light: Reduction of pathogenic and spoilage bacteria, and changes in sensory quality.

We have compared the efficacy of continuous ultraviolet (UV-C) (254 nm) and pulsed UV light in reducing the viability of Salmonella Enteritidis, Listeria monocytogenes, Staphylococcus aureus, enterohemorrhagic Escherichia coli, Pseudomonas spp., Brochothrix thermospacta, Carnobacterium divergens, and extended-spectrum ß-lactamase producing E. coli inoculated on chicken fillet surface. Fluences from 0.05 to 3.0 J/cm2 (10 mW/cm2, from 5 to 300 s) used for UV-C light resulted in average reductions from 1.1 to 2.8 log cfu/cm2. For pulsed UV light, fluences from 1.25 to 18.0 J/cm2 gave average reductions from 0.9 to 3.0 log cfu/cm2. A small change in the odor characterized as sunburnt and increased concentration of volatile compounds associated with burnt odor posed restrictions on the upper limit of UV treatment, however no sensory changes were observed after cooking the meat. Treatments under modified atmosphere conditions using a UV permeable top film gave similar or slightly lower bacterial reductions. PRACTICAL APPLICATIONS: Ultraviolet (UV) light may be used for decontaminating the surface of food products and reduce viability of pathogenic and spoilage bacteria. Exposure of raw chicken fillet surface to various doses of continuous UV-C or pulsed UV light proposed in the present work represent alternatives for microbiological improvement of this product. Chicken fillets can be treated in intact packages covered with UV permeable top film, thus avoiding recontamination of the meat. UV-C light treatment is a low cost strategy with low maintenance, whereas pulsed UV light involves more elaborate equipment, but treatment times are short and less space is required. Both methods can be helpful for producers to manage the safety and quality of chicken fillets.

Comparison of UV-C and Pulsed UV Light Treatments for Reduction of Salmonella, Listeria monocytogenes, and Enterohemorrhagic Escherichia coli on Eggs.

Ten percent of all strong-evidence foodborne outbreaks in the European Union are caused by Salmonella related to eggs and egg products. UV light may be used to decontaminate egg surfaces and reduce the risk of human salmonellosis infections. The efficiency of continuous UV-C (254 nm) and pulsed UV light for reducing the viability of Salmonella Enteritidis, Listeria monocytogenes, and enterohemorrhagic Escherichia coli on eggs was thoroughly compared. Bacterial cells were exposed to UV-C light at fluences from 0.05 to 3.0 J/cm2 (10 mW/cm2, for 5 to 300 s) and pulsed UV light at fluences from 1.25 to 18.0 J/cm2, resulting in reductions ranging from 1.6 to 3.8 log, depending on conditions used. Using UV-C light, it was possible to achieve higher reductions at lower fluences compared with pulsed UV light. When Salmonella was stacked on a small area or shielded in feces, the pulsed UV light seemed to have a higher penetration capacity and gave higher bacterial reductions. Microscopy imaging and attempts to contaminate the interior of the eggs with Salmonella through the eggshell demonstrated that the integrity of the eggshell was maintained after UV light treatments. Only minor sensory changes were reported by panelists when the highest UV doses were used. UV-C and pulsed UV light treatments appear to be useful decontamination technologies that can be implemented in continuous processing.

Survival of Five Strains of Shiga Toxigenic Escherichia coli in a Sausage Fermentation Model and Subsequent Sensitivity to Stress from Gastric Acid and Intestinal Fluid.

The ability of foodborne pathogens to exhibit adaptive responses to stressful conditions in foods may enhance their survival when passing through the gastrointestinal system. We aimed to determine whether Escherichia coli surviving stresses encountered during a model dry-fermented sausage (DFS) production process exhibit enhanced tolerance and survival in an in vitro gastrointestinal model. Salami sausage batters spiked with five E. coli isolates, including enterohaemorrhagic E. coli strains isolated from different DFS outbreaks, were fermented in a model DFS process (20°C, 21 days). Control batters spiked with the same strains were stored at 4°C for the same period. Samples from matured model sausages and controls were thereafter exposed to an in vitro digestion challenge. Gastric exposure (pH 3) resulted in considerably reduced survival of the E. coli strains that had undergone the model DFS process. This reduction continued after entering intestinal challenge (pH 8), but growth resumed after 120 min. When subjected to gastric challenge for 120 min, E. coli that had undergone the DFS process showed about 2.3 log10⁡ lower survival compared with those kept in sausage batter at 4°C. Our results indicated that E. coli strains surviving a model DFS process exhibited reduced tolerance to subsequent gastric challenge at low pH.

Effects of glucose availability in Lactobacillus sakei; metabolic change and regulation of the proteome and transcriptome.

Effects of glucose availability were investigated in Lactobacillus sakei strains 23K and LS25 cultivated in anaerobic, glucose-limited chemostats set at high (D = 0.357 h-1) and low (D = 0.045 h-1) dilution rates. We observed for both strains a shift from homolactic towards more mixed acid fermentation when comparing high to low growth rates. However, this change was more pronounced for LS25 than for 23K, where dominating products were lactate>formate>acetate≥ethanol at both conditions. A multivariate approach was used for analyzing proteome and transcriptome data from the bacterial cultures, where the predictive power of the omics data was used for identifying features that can explain the differences in the end-product profiles. We show that the different degree of response to the same energy restriction revealed interesting strain specific regulation. An elevated formate production level during slow growth, more for LS25 than for 23K, was clearly reflected in correlating pyruvate formate lyase expression. With stronger effect for LS25, differential expression of the Rex transcriptional regulator and NADH oxidase, a target of Rex, indicated that maintainance of the cell redox balance, in terms of the NADH/NAD+ ratio, may be a key process during the metabolic change. The results provide a better understanding of different strategies that cells may deploy in response to changes in substrate availability.

Effect of relevant environmental stresses on survival of enterohemorrhagic Escherichia coli in dry-fermented sausage.

Dry-fermented sausages (DFSs) have been linked to several serious foodborne outbreaks of enterohemorrhagic Escherichia coli (EHEC). The ability of pathogens to utilize adaptive responses to different stressful conditions intended to control their growth in foods, food preparation and production processes may enhance their survival. In certain cases, induced tolerance to one type of stress may lead to enhanced resistance to the applied stress as well as to other stresses. We exposed two EHEC strains, MF3582 of serotype O157:H- and MF5554 of serogroup O145, to different stresses commonly encountered during a production process. The two EHEC strains, previously shown to have different abilities to survive DFS production process conditions, were subjected to low temperatures (4°C and 12°C), 5% NaCl or 1% lactic acid for 6days prior to being added to sausage batters. Survival of EHEC was recorded in salami of two recipes, fermented at two temperatures (20°C and 30°C). The results showed that recipe type had the largest impact on EHEC reductions where Moderate recipe (MR) salami batters containing increased levels of NaCl, glucose and NaNO2 provided enhanced EHEC reductions in salami (2.6 log10) compared to Standard recipe (SR) salami (1.7 log10). Effects of pre-exposure stresses were dependent both on strain and recipe. While acid adaptation of MF5554 provided enhanced log10 reductions from 2.0 to 3.0 in MR sausages, adaptation to a combination of acid and salt stress showed the opposite effect in SR sausages with reductions of only 1.1 log10 as compared to the average of 1.8 log10 for the other SR sausages. Otherwise, the salt and acid adaptation single stresses had relatively small effects on EHEC survival through the DFS production process and subsequent storage and freeze/thaw treatments. Growing cells and cells frozen in batter survived poorly in MR sausages with an average reduction of 3.4 and 3.2 log10, respectively. The reductions of EHEC after storage of DFS increased with higher temperature and storage time. Up to 3.7 log10 additional reduction was obtained when MF3582 was stored for 2months at 20°C. In conclusion, adaptation of EHEC to acid, salt and low temperatures prior to being introduced in a DFS production process has limited, but strain dependent effects on EHEC reductions. Producers should avoid conditions leading to acid and salt adapted cells that can contaminate the sausage batter. Recipe parameters had the largest impact on EHEC reductions while storage at 20°C is effective for enhanced reductions in finished products.

Genome Sequence of Lactobacillus sakei subsp. sakei LS25, a Commercial Starter Culture Strain for Fermented Sausage.

Lactobacillus sakei is a lactic acid bacterium associated primarily with fermented meat and fish. Here, we present the draft genome sequence of L. sakei subsp. sakei strain LS25, a commercial starter culture strain for fermented sausage.

Global transcriptome response in Lactobacillus sakei during growth on ribose.

BACKGROUND: Lactobacillus sakei is valuable in the fermentation of meat products and exhibits properties that allow for better preservation of meat and fish. On these substrates, glucose and ribose are the main carbon sources available for growth. We used a whole-genome microarray based on the genome sequence of L. sakei strain 23K to investigate the global transcriptome response of three L. sakei strains when grown on ribose compared with glucose. RESULTS: The function of the common regulated genes was mostly related to carbohydrate metabolism and transport. Decreased transcription of genes encoding enzymes involved in glucose metabolism and the L-lactate dehydrogenase was observed, but most of the genes showing differential expression were up-regulated. Especially transcription of genes directly involved in ribose catabolism, the phosphoketolase pathway, and in alternative fates of pyruvate increased. Interestingly, the methylglyoxal synthase gene, which encodes an enzyme unique for L. sakei among lactobacilli, was up-regulated. Ribose catabolism seems closely linked with catabolism of nucleosides. The deoxyribonucleoside synthesis operon transcriptional regulator gene was strongly up-regulated, as well as two gene clusters involved in nucleoside catabolism. One of the clusters included a ribokinase gene. Moreover, hprK encoding the HPr kinase/phosphatase, which plays a major role in the regulation of carbon metabolism and sugar transport, was up-regulated, as were genes encoding the general PTS enzyme I and the mannose-specific enzyme II complex (EIIman). Putative catabolite-responsive element (cre) sites were found in proximity to the promoter of several genes and operons affected by the change of carbon source. This could indicate regulation by a catabolite control protein A (CcpA)-mediated carbon catabolite repression (CCR) mechanism, possibly with the EIIman being indirectly involved. CONCLUSIONS: Our data shows that the ribose uptake and catabolic machinery in L. sakei is highly regulated at the transcription level. A global regulation mechanism seems to permit a fine tuning of the expression of enzymes that control efficient exploitation of available carbon sources.

Comparative genomics of Lactobacillus sakei with emphasis on strains from meat.

Lactobacillus sakei is a lactic acid bacterium important in food microbiology mainly due to its ability to ferment and preserve meat. The genome sequence of L. sakei strain 23K has revealed specialized metabolic capacities that reflect the bacterium's adaption to meat products, and that differentiate it from other LAB. An extensive genomic diversity analysis was conducted to elucidate the core features of the species, and to provide a better comprehension of niche adaptation of the organism. Here, we describe the genomic comparison of 18 strains of L. sakei originating mainly from processed meat against the 23K strain by comparative genome hybridization. Pulsed field gel electrophoresis was used to estimate the genome sizes of the strains, which varied from 1.880 to 2.175 Mb, and the 23K genome was among the smallest. Consequently, a large part of the genome of this strain belongs to a common gene pool invariant in this species. The majority of genes important in adaption to meat products, the ability to flexibly use meat components, and robustness during meat processing and storage were conserved, such as genes involved in nucleoside scavenging, catabolism of arginine, and the ability to cope with changing redox and oxygen levels, which is indicative of the role these genes play in niche specialization within the L. sakei species. Moreover, an additional set of sequenced L. sakei genes beyond the 23K genome was present on the microarray used, and it was demonstrated that all the strains carry remnants of or complete bacteriocin operons. The genomic divergence corresponded mainly to five regions in the 23K genome, which showed features consistent with horizontal gene transfer. Carbohydrate-fermentation profiles of the strains were evaluated in light of the CGH data, and for most substrates, the genotypes were consistent with the phenotypes. We have demonstrated a highly conserved organization of the L. sakei genomes investigated, and the 23K strain is a suitable model organism to study core features of the L. sakei species.

Primary metabolism in Lactobacillus sakei food isolates by proteomic analysis.

BACKGROUND: Lactobacillus sakei is an important food-associated lactic acid bacterium commonly used as starter culture for industrial meat fermentation, and with great potential as a biopreservative in meat and fish products. Understanding the metabolic mechanisms underlying the growth performance of a strain to be used for food fermentations is important for obtaining high-quality and safe products. Proteomic analysis was used to study the primary metabolism in ten food isolates after growth on glucose and ribose, the main sugars available for L. sakei in meat and fish. RESULTS: Proteins, the expression of which varied depending on the carbon source were identified, such as a ribokinase and a D-ribose pyranase directly involved in ribose catabolism, and enzymes involved in the phosphoketolase and glycolytic pathways. Expression of enzymes involved in pyruvate and glycerol/glycerolipid metabolism were also affected by the change of carbon source. Interestingly, a commercial starter culture and a protective culture strain down-regulated the glycolytic pathway more efficiently than the rest of the strains when grown on ribose. The overall two-dimensional gel electrophoresis (2-DE) protein expression pattern was similar for the different strains, though distinct differences were seen between the two subspecies (sakei and carnosus), and a variation of about 20% in the number of spots in the 2-DE gels was observed between strains. A strain isolated from fermented fish showed a higher expression of stress related proteins growing on both carbon sources. CONCLUSIONS: It is obvious from the data obtained in this study that the proteomic approach efficiently identifies differentially expressed proteins caused by the change of carbon source. Despite the basic similarity in the strains metabolic routes when they ferment glucose and ribose, there were also interesting differences. From the application point of view, an understanding of regulatory mechanisms, actions of catabolic enzymes and proteins, and preference of carbon source is of great importance.

Diversity of Lactobacillus sakei strains investigated by phenotypic and genotypic methods.

The diversity of 10 strains of Lactobacillus sakei, a commercially important species of lactobacilli, was characterized by studying food isolates. Growth characteristics varied among the strains when examined after growth in a complex medium and a defined medium with either glucose or ribose. A commercial starter culture strain showed the fastest growth rates and high biomass formation on all media, while two of the strains hardly grew on ribose. Based on acidification properties in a meat model, some of the strains had the ability to compete with the indigenous microbiota of the meat batter in addition to being fast acid producers. Carbohydrate-fermentation abilities revealed a relatively wide variation, clustering the strains into two phenotypic groups. The isolates were analyzed using different genetic fingerprinting techniques, demonstrating a distinction between two genetic groups, a grouping consistent with previous studies dealing with L. sakei strains. Comparative genome hybridization (CGH) was introduced for clustering the strains and the same division into two genetic groups was observed. Chromosomal sizes of the strains were estimated by pulsed field gel electrophoresis (PFGE) and were found to vary from 1884 to 2175 kb. The genetic groups did not correlate with the clustering obtained with carbohydrate-fermenting abilities or with chromosomal sizes.

Cloning and characterization of the executioner caspases 3, 6, 7 and Hsp70 in hyperthermic Atlantic salmon (Salmo salar) embryos.

Hyperthermia during embryogenesis has been reported to induce deformities in Atlantic salmon (Salmo salar). To examine the involvement of executioner caspases in hyperthermia-induced cell-death in a poikilotherm vertebrate species, five genes encoding caspase-3,-6, and -7 were cloned from Atlantic salmon, and the expression was studied in thermal stressed salmon embryos. The salmon genome contained two genetically distinct variants of both salmon caspase-3 and caspase-6 that is likely the result of two independent chromosome or genome duplications. Whereas only partial caspase-3A encoding sequences were isolated, the full-length caspase-3B cDNA encodes the inactive proenzyme of 279 amino acids (aa) consisting of an N-terminal prodomain and the large and the small subunit. The salmon caspase-6A and caspase-6B proenzymes include an additional linker region between the two subunits. The deduced salmon caspase-7 consists of only 245 aa and lacks the prodomain and part of the large subunit similar to the predicted caspase-7 of the puffer fish Tetraodon sp.. Increased apoptotic activity as evidenced by cleavage of nuclear DNA was demonstrated in salmon embryos incubated at 18-20 degrees C for 84 h after acclimatization at 8 degrees C. Hyperthermia-induced activation of the executioner caspases was indicated by the increased mRNA levels of caspase-3B, caspase-6A/B and caspase-7 after 54 h heat exposure as quantified by real-time RT-PCR. The 2-2.5 fold increase in the mRNA expression of the heat shock protein Hsp70 gene coincided with the peak mRNA values of the executioner caspases. Whole-mount in situ hybridization of the salmon embryo identified caspase-7 mRNA in the lens exclusively, while caspase-3B and caspase-6A/B were expressed in multiple tissues of exposed and control embryos. Interestingly, cardiac expression of caspase-6A/B was only identified in heat stressed embryos. Altogether, these results shed light on evolutionary aspects of the executioner caspases in vertebrates and their expression in salmon embryos exposed to hyperthermia. In particular, the heat sensitive caspase-6 expression in the embryonic heart is of interest since cardiac malformations are an emergent problem in salmon aquaculture.