Fitness of Enterohemorrhagic Escherichia coli (EHEC)/Enteroaggregative E. coli O104:H4 in Comparison to That of EHEC O157: Survival Studies in Food and In Vitro.

In 2011, one of the world's largest outbreaks of hemolytic-uremic syndrome (HUS) occurred, caused by a rare Escherichia coli serotype, O104:H4, that shared the virulence profiles of Shiga toxin-producing E. coli (STEC)/enterohemorrhagic E. coli (EHEC) and enteroaggregative E. coli (EAEC). The persistence and fitness factors of the highly virulent EHEC/EAEC O104:H4 strain, grown either in food or in vitro, were compared with those of E. coli O157 outbreak-associated strains. The log reduction rates of the different EHEC strains during the maturation of fermented sausages were not significantly different. Both the O157:NM and O104:H4 serotypes could be shown by qualitative enrichment to be present after 60 days of sausage storage. Moreover, the EHEC/EAEC O104:H4 strain appeared to be more viable than E. coli O157:H7 under conditions of decreased pH and in the presence of sodium nitrite. Analysis of specific EHEC strains in experiments with an EHEC inoculation cocktail showed a dominance of EHEC/EAEC O104:H4, which could be isolated from fermented sausages for 60 days. Inhibitory activities of EHEC/EAEC O104:H4 toward several E. coli strains, including serotype O157 strains, could be determined. Our study suggests that EHEC/EAEC O104:H4 is well adapted to the multiple adverse conditions occurring in fermented raw sausages. Therefore, it is strongly recommended that STEC strain cocktails composed of several serotypes, instead of E. coli O157:H7 alone, be used in food risk assessments. The enhanced persistence of EHEC/EAEC O104:H4 as a result of its robustness, as well as the production of bacteriocins, may account for its extraordinary virulence potential. IMPORTANCE: In 2011, a severe outbreak caused by an EHEC/EAEC serovar O104:H4 strain led to many HUS sequelae. In this study, the persistence of the O104:H4 strain was compared with those of other outbreak-relevant STEC strains under conditions of fermented raw sausage production. Both O157:NM and O104:H4 strains could survive longer during the production of fermented sausages than E. coli O157:H7 strains. E. coli O104:H4 was also shown to be well adapted to the multiple adverse conditions encountered in fermented sausages, and the secretion of a bacteriocin may explain the competitive advantage of this strain in an EHEC strain cocktail. Consequently, this study strongly suggests that enhanced survival and persistence, and the presumptive production of a bacteriocin, may explain the increased virulence of the O104:H4 outbreak strain. Furthermore, this strain appears to be capable of surviving in a meat product, suggesting that meat should not be excluded as a source of potential E. coli O104:H4 infection.

Spoilage of vacuum-packed beef by the yeast Kazachstania psychrophila.

A survey of the psychrotolerant yeast microbiota of vacuum-packed beef was conducted between 2010 and 2012. Chilled vacuum-packed beef (n = 50) sampled from 15 different producers was found to have a mean psychrotolerant yeast count of 3.76 log cfu per cm(2). During this assessment, a recently described yeast named Kazachstania psychrophila was shown to be associated with this product. In order to gain basic knowledge about the spoilage potential of K. psychrophila in vacuum-packed beef, challenge studies were performed and the survival of three different K. psychrophila strains was analyzed during storage of artificially contaminated beef. Beef samples were inoculated with the yeasts at a contamination level of 2 log cfu per cm(2). Survival and growth of K. psychrophila strains was monitored on malt extract agar at regular intervals over 84 days. Kazachstania levels rapidly increased about 5 log units within 16 days under chill conditions (4 °C). Gas bubbles were observed after 16 days, while discoloration and production of off-flavors became evident after 42 days in inoculated samples. This study demonstrates for the first time, that the psychrotolerant yeast K. psychrophila is a dominant spoilage microorganism of vacuum-packed beef products stored at low temperatures, causing sensory defects which result in reduced shelf life, and consequently in considerable economic losses.

Contribution of the NO-detoxifying enzymes HmpA, NorV and NrfA to nitrosative stress protection of Salmonella Typhimurium in raw sausages.

The antimicrobial action of the curing agent sodium nitrite (NaNO2) in raw sausage fermentation is thought to mainly depend on the release of cytotoxic nitric oxide (NO) at acidic pH. Salmonella Typhimurium is capable of detoxifying NO via the flavohemoglobin HmpA, the flavorubredoxin NorV and the periplasmic cytochrome C nitrite reductase NrfA. In this study, the contribution of these systems to nitrosative stress tolerance in raw sausages was investigated. In vitro growth assays of the S. Typhimurium 14028 deletion mutants ΔhmpA, ΔnorV and ΔnrfA revealed a growth defect of ΔhmpA in the presence of acidified NaNO2. Transcriptional analysis of the genes hmpA, norV and nrfA in the wild-type showed a 41-fold increase in hmpA transcript levels in the presence of 150 mg/l acidified NaNO2, whereas transcription of norV and nrfA was not enhanced. However, challenge assays performed with short-ripened spreadable sausages produced with 0 or 150 mg/kg NaNO2 failed to reveal a phenotype for any of the mutants compared to the wild-type. Hence, none of the NO detoxification systems HmpA, NorV and NrfA is solely responsible for nitrosative stress tolerance of S. Typhimurium in raw sausages. Whether these systems act cooperatively, or if there are other yet undescribed mechanisms involved is currently unknown.

Kazachstania psychrophila sp. nov., a novel psychrophilic yeast isolated from vacuum-packed beef.

Five novel ascosporogenous yeast strains (H382, H396, H409, H433(T) and H441) were found through a survey of vacuum-packed beef microbiota. Sequence analysis of ITS domain and LSU rRNA genes showed that the new strains represent a distinct lineage within the genus Kazachstania, closely related to Kazachstania lodderae (97.0 % identity) and Kazachstania ichnusensis (96.1 % identity). The main difference of strains H382, H396, H409, H433(T) and H441 to strains of known Kazachstania species is the maximum growth temperature, which is below 20 °C for the new strains, whereas related species grow at 25 °C. Furthermore, the strains differed from known Kazachstania species in assimilation and fermentation patterns of carbon sources. Based on these characteristics, the five strains are considered to represent a novel species of the genus Kazachstania for which the name Kazachstania psychrophila sp. nov. is proposed. The type strain is H433(T) (DSM 26230(T)=CBS 12689(T)). The Mycobank number of the type strain is MB 803980.

Dried Vegetables as Potential Clean-Label Phosphate Substitutes in Cooked Sausage Meat.

While phosphates are key additives in sausage production, their use conflicts with consumer preferences for "natural" foods. In this study, we investigated the potential of using vegetables as "clean-label" phosphate substitutes and their effects on water holding capacity, consumer acceptance, color, softness, and tenderness. Six freeze-dried vegetables with a pH above 6.0 were added to sausage meat on a laboratory scale. Adding 1.6% freeze-dried Brussels sprouts or Red Kuri squash resulted in a similar weight gain (7.0%) as the positive control of 0.6% commercial phosphate additive. Higher vegetable concentrations (2.2-4.0%) caused a significant increase in weight (p ≤ 0.05, 10.4-18.4% weight gain). Similar stress was needed to compress sausages containing 1.6/4.0% Brussels sprouts (14.2/11.2 kPa) and the positive control (13.2 kPa). Indentation tests also led to similar softness results for the sausages prepared with 1.6/4.0% Brussels sprouts (15.5 kPa/16.6 kPa) and the positive control (16.5 kPa). A force of 1.25 N was needed to shear the positive control, while 1.60 N/1.30 N was needed for the samples (1.6/4% Brussels sprouts). In summary, the present study indicates that freeze-dried vegetables have the potential to effectively replace phosphate in meat products.

Draft Genome Sequences of Three Lactic Acid Bacterial Strains Investigated for B Vitamin Biosynthesis.

The draft genome sequences of three lactic acid bacteria, namely, Limosilactobacillus reuteri 92071, Lactiplantibacillus plantarum 92117-i3, and Limosilactobacillus fermentum 92072, and the presence of genes involved in the biosynthesis of B vitamins were determined. Limosilactobacillus reuteri 92071 showed complete gene clusters for vitamin B12 biosynthesis, with a GC content of 38.52 mol%.

Shiga toxin-producing Escherichia coli serogroups in food and patients, Germany.

We compared 61 Shiga toxin-producing Escherichia coli (STEC) serogroups from 448 food isolates with 71 STEC serogroups from 1,447 isolates from patients in Germany. Two thirds (41/61), representing 72% of food isolates, were also found in patients. Serogroups typically isolated from patients with hemolytic uremic syndrome were rarely found in food.

Persistence and reduction of Shiga toxin-producing Escherichia coli serotype O26:H11 in different types of raw fermented sausages.

Fermented sausages have been identified as source of several outbreaks of Shiga toxin-producing Escherichia coli (STEC). Illnesses linked to non-O157 STEC serotypes appear to be on the rise worldwide, and serogroup O26 is the second most reported in Europe after O157. However, data on the behavior of serogroup O26 in food are rare, so that the aim of this study was to investigate the survival of STEC O26:H11 in different types of fermented sausages ("Teewurst", fast-ripened and long-fermented salami). Challenge studies were performed with an inoculation cocktail which consisted of three STEC O26:H11 strains isolated from human, cattle and food sources. In the short-ripened spreadable sausage type "Teewurst" STEC counts decreased by only 0.5 log10 within 28days. In contrast, STEC reductions from 2.2 to 2.6 log10 units were observed in the different salami products, while the most pronounced decrease of 1.0 log10 unit within one day was detected in fast-ripened sausages with glucono delta-lactone (GdL). Moreover, numbers of the food-associated E. coli O26:H11 strain were significantly higher (p<0.001) than those of the human and cattle STEC O26:H11 strains in all types of fermented sausages. Approximately 60% of all STEC isolates from GdL salami shared the genotypic virulence profile of the food-associated E. coli O26:H11 strain. In summary, hurdles of acidification and drying during salami ripening resulted in reductions of STEC O26:H11 counts. However, our results also indicate that STEC O26:H11 can persist in the environment of "Teewurst" and might therefore pose a risk to public health.

Biochemical evidence for the proteolytic degradation of infectious prion protein PrPsc in hamster brain homogenates by foodborne bacteria.

PrP(Sc) is a general term to describe the infectious agent causing transmissible spongiform encephalopathy (TSE), and the protease-resistant form of cellular PrP(C). In this study, we have identified several protease-secreting bacteria able to degrade PrP(Sc) under more or less native conditions (30 degrees C, pH 8), focusing on strains isolated mainly from cheese. One hundred and ninty-nine protease-secreting isolates belonging to the Actinomycetales and Bacillales were screened for the expression of PrP(Sc) degrading activity by a Western blot procedure. Only 6 strains belonging to the following species were found to exhibit such an activity: Arthrobacter nicotianae, Bacillus licheniformis, Brachybacterium conglomeratum, Brachybacterium tyrofermentans and Staphylococcus sciuri and Serratia spp. As revealed by a general protease assay based on dye-labeled Azocoll substrate, the PrP(Sc) degrading activity was not directly correlated to the total level of secreted proteolytic activity of these organisms. This indicates that specific proteases are required for the degradation of PrP(Sc). Our study also suggests the potential use of such starter bacteria or their proteases for application in PrP(Sc) degradation and decontamination under native conditions.

Acidified nitrite inhibits proliferation of Listeria monocytogenes - Transcriptional analysis of a preservation method.

Sodium nitrite (NaNO2) is added as a preservative during raw meat processing such as raw sausage production to inhibit growth of pathogenic bacteria. In the present study it was shown in challenge assays that the addition of sodium nitrite indeed inhibited growth and survival of Listeria monocytogenes in short-ripened spreadable raw sausages. Furthermore, in vitro growth analyses were performed, which took into account combinations of various parameters of the raw sausage ripening process like temperature, oxygen availability, pH, NaCl concentration, and absence or presence of NaNO2. Data based on 300 growth conditions revealed that the inhibitory effect of nitrite was most prominent in combination with acidification, a combination that is also achieved during short-ripened spreadable raw sausage production. At pH6.0 and below, L. monocytogenes was unable to replicate in the presence of 200mg/l NaNO2. During the adaptation of L. monocytogenes to acidified nitrite stress (pH6.0, 200mg/l NaNO2) in comparison to acid exposure only (pH6.0, 0mg/l NaNO2), a massive transcriptional adaptation was observed using microarray analyses. In total, 202 genes were up-regulated and 204 genes were down-regulated. In accordance with growth inhibition, a down-regulation of genes encoding for proteins which are involved in central cellular processes, like cell wall/membrane/envelope biogenesis, translation and ribosomal structure and biogenesis, transcription, and replication, recombination and repair, was observed. Among the up-regulated genes the most prominent group belonged to poorly characterized genes. A considerable fraction of the up-regulated genes has been shown previously to be up-regulated intracellularly in macrophages, after exposure to acid shock or to be part of the SigB regulon. These data indicate that the adaptation to acidified nitrite partly overlaps with the adaptation to stress conditions being present during host colonization.

[Salmonella spp. prevalence and contamination risk factors in broiler and broiler meat of Gallus gallus in Germany and the European Union]. / Prävalenz und Kontaminationsrisikofaktoren für Salmonella spp. in Hähnchen und Hähnchenfleisch in Deutschland und der Europäischen Union.

In order to reduce the prevalence of the Salmonella enterica serovars Typhimurium and Enteritidis as a main causative agent of human salmonellosis originating from poultry flocks and products, the EU regulations 2160/2003 and 2073/2005 and the German Hühner-Salmonellen-Verordnung were established ten years ago. A literature review shows that this aim could be reached to a large extend in many areas of the food production chain, e.g. in breeding and husbandry facilities in most EU member states including Germany. Nevertheless some exceptions exist, and there are other S. enterica serovars which have a human pathogenic potential comparable to S. Typhimurium and S. Enteritidis. Furthermore recent publications show, that especially processes in transport and slaughter of poultry can prevent successful husbandry sanitation measures. Especially in these areas a reasonable potential for hygiene improvements still exists. Based on the prevalence data obtained between 1996 and 2011 this review summarizes recent knowledge concerning possible risks of Salmonella cross contamination and suggests potential starting points for their mitigation.

The controversial nature of the Weissella genus: technological and functional aspects versus whole genome analysis-based pathogenic potential for their application in food and health.

Despite the use of several Weissella (W.) strains for biotechnological and probiotic purposes, certain species of this genus were found to act as opportunistic pathogens, while strains of W. ceti were recognized to be pathogenic for farmed rainbow trout. Herein, we investigated the pathogenic potential of weissellas based on in silico analyses of the 13 whole genome sequences available to date in the NCBI database. Our screening allowed us to find several virulence determinants such as collagen adhesins, aggregation substances, mucus-binding proteins, and hemolysins in some species. Moreover, we detected several antibiotic resistance-encoding genes, whose presence could increase the potential pathogenicity of some strains, but should not be regarded as an excluding trait for beneficial weissellas, as long as these genes are not present on mobile genetic elements. Thus, selection of weissellas intended to be used as starters or for biotechnological or probiotic purposes should be investigated regarding their safety aspects on a strain to strain basis, preferably also by genome sequencing, since nucleotide sequence heterogeneity in virulence and antibiotic resistance genes makes PCR-based screening unreliable for safety assessments. In this sense, the application of W. confusa and W. cibaria strains as starter cultures or as probiotics should be approached with caution, by carefully selecting strains that lack pathogenic potential.

Identification of H2O2 as a major antimicrobial component in coffee.

Coffee shows distinct antimicrobial activity against several bacterial genera. The present study investigated molecular mechanisms and active ingredients mediating the antimicrobial effect of coffee. Depending on concentration, roasted, but not raw coffee brew inhibited the growth of Escherichia coli and Listeria innocua. Several coffee ingredients with known antibacterial properties were tested for their contribution to the observed effect. In natural concentration, caffeine, ferulic acid and a mixture of all test compounds showed very weak, but significant activity, whereas trigonelline, 5-(hydroxymethyl)furfural, chlorogenic acid, nicotinic acid, caffeic acid, and methylglyoxal were not active. Antimicrobial activity, however, was completely abolished by addition of catalase indicating that H(2)O(2) is a major antimicrobial coffee component. In accordance with this assumption, bacterial counts during 16 h of incubation were inversely related to the H(2)O(2) concentration in the incubation solution. Pure H(2)O(2) showed slightly weaker activity. The H(2)O(2) dependent antimicrobial activity of coffee could be mimicked by a reaction mixture of d-ribose and l-lysine (30 min 120 °C) indicating that H(2)O(2) is generated in the coffee brew by Maillard reaction products. Identification of H(2)O(2) as major antimicrobial coffee component is important to evaluate the application of coffee or coffee extracts as natural preservatives.

Degradation of scrapie associated prion protein (PrPSc) by the gastrointestinal microbiota of cattle.

A food-borne origin of the transmission of bovine spongiform encephalopathy (BSE) to cattle is commonly assumed. However, the fate of infectious prion protein during polygastric digestion remains unclear. It is unknown at present, whether infectious prion proteins, considered to be very stable, are degraded or inactivated by microbial processes in the gastrointestinal tract of cattle. In this study, rumen and colon contents from healthy cattle, taken immediately after slaughter, were used to assess the ability of these microbial consortia to degrade PrP(Sc). Therefore, the consortia were incubated with brain homogenates of scrapie (strain 263K) infected hamsters under physiological anaerobic conditions at 37 degrees C. Within 20 h, PrP(Sc) was digested both with ruminal and colonic microbiota up to immunochemically undetectable levels. Especially polymyxin resistant (mainly gram-positive) bacteria expressed PrP(Sc) degrading activity. These data demonstrate the ability of bovine gastrointestinal microbiota to degrade PrP(Sc) during digestion.

[Occurrence of Salmonella spp. and shigatoxin-producing escherichia coli (STEC) in horse faeces and horse meat products]. / Vorkommen von Salmonella spp. und Shigatoxin-bildenden Escherichia coli (STEC) in Pferdefaeces und Pferdefleischprodukten.

In order to assess the relevance of horses as a possible reservoir of Salmonella and Shigatoxin-producing Escherichia coli (STEC), 400 samples of horse faeces and 100 samples of horse meat products were examined by PCR-screening methods. Salmonella enterica was not found in any of the samples. One faeces-sample and one horse meat product were proved to be STEC positive. The STEC-strain from faecal origin belonged to the serotype 0113:H21 and had the stx 2c gene and the enterohemolysin gene. The STEC-strain isolated from a horse meat product had the serotype O87:H16 and the stx 2d gene. The results indicate a very low risk for human to get a Salmonella- or EHEC- infection from horses in Germany.