Optimization of disinfection procedures in food supply facilities of the German Armed Forces in view to the B. cereus prevalence of surfaces and food

Titelblatt, Inhaltsverzeichnis, Danksagung, Lebenslauf 1\. Einleitung 2\. Literaturýbersicht 3\. Eigene Untersuchungen 4\. Ergebnisse 5\. Diskussion 6\. Schlussfolgerungen 7\. Zusammenfassung 8\. Summary 9\. Anhang LiteraturverzeichnisRoutinemäßig durchgeführte epidemiologische Untersuchungen bei gehäuft auftretenden gastrointenstinalen Symptomatiken im Bereich der Bundeswehr liefern wertvolle Daten über die Prävalenz B. cereus-bedingter Erkrankungen. B. cereus dominiert bei weiterhin deutlich steigender Bedeutung als Verursacher lebensmittelbedingter Erkrankungen in der Bundeswehr. Die in den Jahren 1999 und 2000 routinemäßig durchgeführten mikrobiologischen Oberflächenuntersuchungen in 183 Verpflegungseinrichtungen der ehemaligen Wehrbereiche III und IV (diese umfassen die Bundesländer Nordrhein-Westfalen, Hessen, Rheinland-Pfalz und das Saarland) sind im Hinblick auf die B. cereus- Prävalenz auf Oberflächen und Bedarfsgegenständen ausgewertet worden. Weitere Untersuchungen sollten die sporozide Wirkung von Wofasteril® E400 / alcapur® und dem standardmäßig in Bundeswehrküchen verwendeten Desinfektionsmittel Tego 2000® vergleichend darstellen. Der qualitative Suspensionstest nach DVG-Methodik, bei dem unterschiedliche Desinfektionsmittelkonzentrationen auf eine Sporensuspension mit und ohne Eiweißbelastung einwirkten, wurde anhand einer durch Vermehrung überlebender Keime bedingten Trübung in einer Nährbouillon ausgewertet. So wurde ermittelt, inwieweit eine Wirksamkeit im Sinne der Abtötung aller eingesetzten Sporen (1,0 x 107 KbE) unter bestimmten Konzentrations-Zeit-Bedingungen vorliegt. Beim quantitativen Suspensionstest wurde die sporozide Wirkung durch die Reduktion der Keimzahl infolge der Einwirkung des Desinfektionsmittels nachgewiesen. Auch hier wirkten verschiedene Konzentrationen der Desinfektionsmittel in einem Reaktionsgefäß auf Sporen von B. cereus ein. Mit Sporensuspension beimpfte Keimträger aus V2A-Stahl und Polyethylen, welche in Küchen üblicherweise zu desinfizierende Materialien darstellen, sind ebenfalls einer quantitativen Desinfektionsmittelprüfung mit unterschiedlichen Konzentrationen der zu prüfenden Präparate unterzogen worden. Im Feldversuch sind insgesamt 700 Oberflächenproben von Bedarfsgegenständen in sieben Verpflegungseinrichtungen vor bzw. nach der Desinfektion auf das Vorhandensein von B. cereus untersucht worden. In den Verpflegungseinrichtungen der Bundeswehr zeigten die routinemäßigen Untersuchungen aus den Jahren 1999 und 2000, dass die Belastung der Oberflächen mit B. cereus mit 14 % positiver Proben (n=4412) hoch ist. In diesen Einrichtungen werden Amphotenside zur Desinfektion verwendet. Bei den durchgeführten Suspensionstests zeigte Tego 2000® keine sporozide Wirkung auf einen B. cereus-Teststamm. Das alternativ getestete Desinfektionsverfahren auf Peressigsäurebasis (Wofasteril® E400/ alcapur®) wirkte selbst unter Eiweißbelastung sporozid. Weitergehende praxisnahe Untersuchungen mit Keimträgern aus V2A-Stahl bzw. PE ergaben, dass Wofasteril® E400 / alcapur® bei einer Konzentration von 0,25% und einer Einwirkzeit von 30 min bei 20°C die Zahl der eingesetzten B. cereus-Sporen um etwa 5 lg KbE- Stufen zu reduzieren vermochte. Bei Verwendung von Tego 2000® in der für den belasteten Bereich empfohlenen Konzentration von 1% konnte im Keimträgertest keine Reduktion der Sporenzahl festgestellt werden, womit die Ergebnisse der Suspensionstests im praxisnahen Ansatz bestätigt wurden. Im Feldversuch konnte ein signifikanter Unterschied zwischen der sporoziden Wirkung von Tego 2000® und der von Wofasteril E400® / alcapur® belegt werden. Die Verwendung von Wofasteril® E400 / alcapur® unter Praxisbedingungen führte zu einer deutlich stärkeren Reduktion der B. cereus-Prävalenz als die vergleichsweise geprüfte Anwendung von Tego 2000®. Anhand des ermittelten Wertes für die Relative Effektivität wird deutlich, dass die sporozide Wirkung von Wofasteril® E400 / alcapur® unter Berücksichtigung der tatsächlichen Ausgangsprävalenz von B. cereus auf Oberflächen und Bedarfsgegenständen als 6,25-mal effektiver zu bewerten ist als die von Tego 2000®. Die ermittelte B. cereus-Prävalenz auf den Oberflächen der Verpflegungseinrichtungen im Zusammenhang mit der beschriebenen Häufigkeit B. cereus-bedingter Erkrankungen in der Gemeinschaftsverpflegung der Bundeswehr deutet darauf hin, dass dem Risiko der Kontamination von Lebensmitteln über belastete Flächen eine große Bedeutung zukommt. Es lag nahe, diese hohe Prävalenz durch die Auswahl eines geeigneten Desinfektionsmittels zu reduzieren. Die Durchführung von Desinfektionsmittelprüfungen und die Bewertung ihrer Ergebnisse sind aufgrund fehlender Normen für die Prüfung chemischer Desinfektionsmittel auf ihre sporozide Wirkung erschwert. In Anlehnung an bestehende Normen für die Wirksamkeitsprüfung von Desinfektionsmitteln auf vegetative Keime wurden Methoden modifiziert und ein Reduktionsfaktor von mindestens 3 lg KbE im quantitativen Suspensionstest sowie im Keimträgertest als Beleg für eine vorhandene sporozide Wirkung gefordert. Da die Resultate von Laboruntersuchungen aufgrund unterschiedlichster Einflussfaktoren unter Feldbedingungen nicht unbedingt auf die Praxis übertragbar sind, wurde zur Sporozidie-Prüfung der Desinfektionsmittel ein Feldversuch im vorgesehenen Anwendungsbereich durchgeführt. Trotz fehlender sporozider Wirkung des in Bundeswehrküchen üblicherweise eingesetzten Desinfektionsmittels sind bei Anwendungempfehlungen andere Auswahlkriterien wie Unbedenklichkeit für Mensch und Umwelt und die Aspekte der Arbeitssicherheit einzubeziehen. Eine ausschließliche Verwendung von Wofasteril® E400 / alcapur® in Verpflegungs-einrichtungen wird deshalb nicht grundsätzlich befürwortet. Vielmehr sollte die gute sporozide Wirkung von Peressigsäure gezielt genutzt werden, v.a. im Falle behördlich angeordneter Entseuchungen im Zusammenhang mit einer B. cereus-bedingten Gruppenerkrankung, zumal die Anwendung von Wofasteril® gemäß der "Liste der vom Bundesgesundheitsamt geprüften und anerkannten Desinfektionsmittel und -verfahren" des RKI für diesen Zweck zulässig ist und das Desinfektionsmittel in der DVG-Liste geführt wird. Darüber hinaus ist der gezielte Einsatz von Wofasteril® E400 / alcapur® sinnvoll, wenn bei mikrobiologischen Eigenkontrollen eine erhöhte B. cereus- Prävalenz in einer Verpflegungseinrichtungen festgestellt wurde. Führt die einmalige Anwendung von Wofasteril® E400 / alcapur® nur vorübergehend zum Erfolg, sollte der ausschließliche Einsatz von Wofasteril® E400 / alcapur® empfohlen werden. Im Rahmen von Bundeswehreinsätzen ist die ebenfalls ausschließliche Verwendung von Wofasteril® E400 / alcapur® angezeigt.Routine epidemiologic investigations of frequently occurring gastrointestinal diseases among German army personnel provides valuable data on the prevalence of B. cereus-associated diseases. B. cereus is the aprimary cause of foodborne diseases in the Bundeswehr and its importance continues to grow. Routine microbiological investigations on surfaces performed in the years 1999 and 2000 in 183 food supply facilities of the former military districts III and IV have been evaluated with a view to the B. cereus prevalence on surfaces of kitchen and cooking equipment. Further investigations were to provide a comparison between the sporocoidal effect of Wofasteril® E400 / alcapur® and Tego 2000® which is used as a standard disinfectant in Bundeswehr kitchens. The qualitative suspension test according to the DVG method, in which different disinfectant concentrations act upon a spore suspension with and without protein load, was evaluated on the basis of growth of surviving bacteria in a nutrient broth. Thus it was determined to which extent all spores used (1.0 x 107 KbE) are destroyed under certain concentration / time conditions. In the quantitative suspension test the sporocoidal effect was demonstrated by the reduction of bacteria due to the effect of the disinfectant. Various concentrations of the disinfectants acted upon spores of B. cereus in a reaction vessel, too. Spore carriers of stainless steel and polyethylene (PE) vaccinated with spore suspension, which are materials that normally have to be disinfected in kitchens, were also subjected to a quantitative disinfectant test with various concentrations of the preparations to be tested. In a field trial a total of 700 surface samples of daily used items in seven food supply facilities were tested for the presence of B. cereus prior to and after disinfection. Routine investigations in Bundeswehr food supply facilities in the years 1999 and 2000 show that the contamination of surfaces with B. cereus is at a high value of 14 % (n=4412). In these facilities amphotensides are used for disinfection. In the suspension tests performed Tego 2000® did not have any sporocoidal effect on a B. cereus test strain. The alternatively tested disinfection method on peracetic acid basis (Wofasteril® E400 / alcapur®) had a sporocoidal effect even under conditions of high protein load. Further reaching practical tests with spore carriers of stainless steel and PE showed that Wofasteril® E400 / alcapur®, in a concentration of 0.25 % and after a time of exposure of 30 min at 20°, could reduce the number of the B. cereus spores used by about log 5 KbE levels. When using Tego 2000® in the concentration of 1 % recommended for the contaminated area, no reduction of the number of spore carriers could be determined in the spore carrier test; thus the results of the suspension tests could be confirmed in a practial test. In the field trial a significant difference in the sporocoidal effect of Tego 2000® and Wofasteril® E400 / alcapur® could be demonstrated. The use of Wofasteril® E400 / alcapur® under practical conditions resulted in a significantly greater reduction of the B. cereus prevalence than that of Tego 2000® which was tested for comparison. The value determined for relative efficiency clearly shows that the sporocoidal effect of Wofasteril® E400 / alcapur®, under consideration of the actual starting prevalence of B. cereus on surfaces of daily used items, has to be rated 6.25 times greater than that of Tego 2000®. The B. cereus prevalence determined on surfaces of the food supply facilities in connection with the described frequency of B. cereus based diseases in the communal food service of the Bundeswehr indicates that the risk of food contamination via contaminated surfaces is highly significant. It suggests itself to reduce this high prevalence by the selection of a suitable disinfectant. The conduct of disinfectant tests and the evaluation of their results is made more difficult due to the lack of standards for the testing of chemical disinfectants for their sporocoidal effect. Analogously to existing standards for the efficiency test of disinfectants for non-sporeforming bacteria, methods were modified and a reduction factor of at least lg 3 KbE in the quantitative suspension test as well as in the spore carrier test was required as a proof of sporocidal efficiency. Since, due to different influencing factors, the results of laboratory tests can not necessarily be transferred to practical field conditions, the disinfectants were subjected to a field trial to test the sporocidal effect in the planned area of application. Despite the lack of a sporocidal effect of the disinfectant normally used in Bundeswehr kitchens, recommendations for application need to include other selection criteria like harmlessness for humans and the environment as well as ascpects of occupational safety. The exclusive use of Wofasteril® E400 / alcapur® in food supply facilities is therefore not generally recommended. The good sporocidal effect of peracetic acid should rather be used specifically, especially in case of officially ordered decontaminations in connection with a B. cereus based epidemic, particularly since the use of Wofasteril® is permissible for that purpose according to the "List of Disinfectants and Disinfection Methods Tested and Approved by the Federal Health Office" of the RKI and the disinfectant is included in the DVG list. Moreover, the specific use of Wofasteril® E400 / alcapur® is reasonable if an increased B. cereus prevalence was determined in a food supply facility during microbiological own checks. If the one-time use of Wofasteril® E400 / alcapur® only leads to termporary success, the exclusive use of Tego 2000® and Wofasteril® E400 / alcapur® should be recommended. During Bundeswehr missions abroad the exclusive use of Wofasteril® E400 /alcapur® is also advisable

Plant diversity and community history shift colonization success from early- to mid-successional species

Aims Functional traits are supposed to play an important role in determining the colonization success of new species into established communities. Short-term experimental studies have documented higher resistance of more diverse grasslands against colonization by new species. However, little is known about which traits colonizers should have to successfully invade diverse plant communities in the longer term and how community history may modify the resistance of diverse communities against colonization. Methods In a grassland biodiversity experiment (Jena Experiment) established with different species richness (SR; 1, 2, 4, 8 and 16) and functional group (FG) number and composition (1 to 4; legumes, grasses, small herbs, tall herbs), we studied colonization of naturally dispersed species in split-plots (i) with different duration of weeding (never weeded, weeded for 3 or 6 years and then un-weeded for 1 year) and (ii) with different duration of colonization (7 years, 4 years and 1 year after cessation of weeding). Important Findings Resistance against colonization by new species declined with increased duration of weeding (on average 13, 17 and 22 colonizer species in 1-, 4- and 7-year-old communities, respectively). Communities established at low diversity accumulated more colonizer species with a longer duration of weeding than more diverse communities. Duration of colonization had only small effects on the number of colonizer species. Colonizers with early successional traits, i.e. annual life cycle, reproduction by seeds, small seeds, long-lived seeds and an earlier start of a longer flowering period, were favoured in species-poor newly established experimental plant communities (short duration of weeding) and early after cessation of weeding (short duration of colonization). A change from early- to mid-successional traits, i.e. taller growth, perennial life cycle, vegetative reproduction, characterized colonization at increased plant diversity and in communities with legumes or without grasses. Legume absence/grass presence and increased duration of weeding led to a shift in colonizer strategies from rapid nutrient uptake and cycling (higher specific leaf area) to nutrient retention and symbiotic N2 fixation. Our study shows that non-random trait spectra of naturally dispersed colonizers encompass trade-offs between different functions (reproduction, persistence, growth) reflected in a change from early- to mid-successional traits at increasing plant diversity, with a longer duration of weeding and a longer time of colonizatio

Ex-vivo Clonally Expanded B Lymphocytes Infiltrating Colorectal Carcinoma Are of Mature Immunophenotype and Produce Functional IgG

Background: Tumor infiltrating B cells (TiBc) have not yet been investigated in detail. This may at least in part be due to technical difficulties. Here we describe a straightforward and reproducible method to isolate and culture TiBc from primary colorectal carcinomas (CRC). Methods/Results: TiBc cultures were generated by Epstein-Barr virus (EBV) immortalization. With this method, monoclonal TiBc cultures were obtained for 14/19 CRCs. As assessed by flow cytometry and ELISA, TiBc showed an activated immunophenotype (CD23 +, CD80 +) and produced immunoglobulin (Ig; IgG secretion in 55 % of the cultures). In functional in vitro analysis, most of the IgGs specifically bound to allogeneic CRC target cells. These data suggest that TiBc are antigenexperienced and thus may exhibit functionality in situ. Additionally, mini-cultures generated from 12 further CRCs revealed TiBc outgrowth exclusively in the presence of EBV. Conclusion: In summary, this simple method provides a cellular tool and our data set the stage for analysing the bivalent role of TiBc; being antigen-presenting cells on the one hand and tumor-specific antibody producers on the other. Additionally, the generation of long-term TiBc cultures and their monoclonal Ig may serve to identify novel tumor-specifi

13C, 15N Resonance Assignment of Parts of the HET-s Prion Protein in its Amyloid Form

The partial 15N and 13C solid-state NMR resonance assignment of the HET-s prion protein fragment 218-289 in its amyloid form is presented. It is based on experiments measured at MAS frequencies in the range of 20-40kHz using exclusively adiabatic polarization-transfer schemes. The resonance assignment within each residue is based on two-dimensional 13C--13C correlation spectra utilizing the DREAM mixing scheme. The sequential linking of the assigned residues used a set of two- and three-dimensional 15N--13C correlation experiments. Almost all cross peaks visible in the spectra are assigned, but only resonances from 43 of the 78 amino-acid residues could be detected. The missing residues are thought to be highly disordered and/or highly dynamic giving rise to broad resonance lines that escaped detection in the experiments applied. The line widths of the observed resonances are narrow and comparable to line widths observed in micro-crystalline samples. The 43 assigned residues are located in two fragments of about 20 residue

Differential effects of plant diversity on functional trait variation of grass species

Background and Aims Functional trait differences and trait adjustment in response to influences of the biotic environment could reflect niche partitioning among species. In this study, we tested how variation in above-ground plant traits, chosen as indicators for light and nitrogen acquisition and use, differs among taxonomically closely related species (Poaceae) to assess their potential for niche segregation at increasing plant diversity. Methods Traits of 12 grass species were measured in experimental grasslands (Jena Experiment) of varying species richness (from 1 to 60) and presence of particular functional groups (grasses, legumes, tall herbs and small herbs). Key Results Grass species increased shoot and leaf length, investment into supporting tissue (stem mass fraction) and specific leaf area as well as reduced foliar δ13C values with increasing species richness, indicating higher efforts for light acquisition. These species-richness effects could in part be explained by a higher probability of legume presence in more diverse communities. Leaf nitrogen concentrations increased and biomas s : N ratios in shoots decreased when grasses grew with legumes, indicating an improved nitrogen nutrition. Foliar δ15N values of grasses decreased when growing with legumes suggesting the use of depleted legume-derived N, while decreasing δ15N values with increasing species richness indicated a shift in the uptake of different N sources. However, efforts to optimize light and nitrogen acquisition by plastic adjustment of traits in response to species richness and legume presence, varied significantly among grass species. It was possible to show further that trait adjustment of grass species increased niche segregation in more diverse plant communities but that complementarity through niche separation may differ between light and nutrient acquisition. Conclusions The results suggest that even among closely related species such as grasses different strategies are used to cope with neighbours. This lack in redundancy in turn may facilitate complementary resource use and coexistenc

Cryopreservation of human colorectal carcinomas prior to xenografting

<p>Abstract</p> <p>Background</p> <p>Molecular heterogeneity of colorectal carcinoma (CRC) is well recognized, forming the rationale for molecular tests required before administration of some of the novel targeted therapies that now are rapidly entering the clinics. For clinical research at least, but possibly even for future individualized tumor treatment on a routine basis, propagation of patients' CRC tissue may be highly desirable for detailed molecular, biochemical or functional analyses. However, complex logistics requiring close liaison between surgery, pathology, laboratory researchers and animal care facilities are a major drawback in this. We here describe and evaluate a very simple cryopreservation procedure for colorectal carcinoma tissue prior to xenografting that will considerably reduce this logistic complexity.</p> <p>Methods</p> <p>Fourty-eight CRC collected ad hoc were xenografted subcutaneously into immunodeficient mice either fresh from surgery (N = 23) or after cryopreservation (N = 31; up to 643 days).</p> <p>Results</p> <p>Take rates after cryopreservation were satisfactory (71%) though somewhat lower than with tumor tissues fresh from surgery (74%), but this difference was not statistically significant. Re-transplantation of cryopreserved established xenografts (N = 11) was always successful. Of note, in this series, all of the major molecular types of CRC were xenografted successfully, even after cryopreservation.</p> <p>Conclusions</p> <p>Our procedure facilitates collection, long-time storage and propagation of clinical CRC specimens (even from different centres) for (pre)clinical studies of novel therapies or for basic research.</p

Back seat driving: hindlimb corticospinal neurons assume forelimb control following ischaemic stroke

Whereas large injuries to the brain lead to considerable irreversible functional impairments, smaller strokes or traumatic lesions are often associated with good recovery. This recovery occurs spontaneously, and there is ample evidence from preclinical studies to suggest that adjacent undamaged areas (also known as peri-infarct regions) of the cortex ‘take over' control of the disrupted functions. In rodents, sprouting of axons and dendrites has been observed in this region following stroke, while reduced inhibition from horizontal or callosal connections, or plastic changes in subcortical connections, could also occur. The exact mechanisms underlying functional recovery after small- to medium-sized strokes remain undetermined but are of utmost importance for understanding the human situation and for designing effective treatments and rehabilitation strategies. In the present study, we selectively destroyed large parts of the forelimb motor and premotor cortex of adult rats with an ischaemic injury. A behavioural test requiring highly skilled, cortically controlled forelimb movements showed that some animals recovered well from this lesion whereas others did not. To investigate the reasons behind these differences, we used anterograde and retrograde tracing techniques and intracortical microstimulation. Retrograde tracing from the cervical spinal cord showed a correlation between the number of cervically projecting corticospinal neurons present in the hindlimb sensory-motor cortex and good behavioural recovery. Anterograde tracing from the hindlimb sensory-motor cortex also showed a positive correlation between the degree of functional recovery and the sprouting of neurons from this region into the cervical spinal cord. Finally, intracortical microstimulation confirmed the positive correlation between rewiring of the hindlimb sensory-motor cortex and the degree of forelimb motor recovery. In conclusion, these experiments suggest that following stroke to the forelimb motor cortex, cells in the hindlimb sensory-motor area reorganize and become functionally connected to the cervical spinal cord. These new connections, probably in collaboration with surviving forelimb neurons and more complex indirect connections via the brainstem, play an important role for the recovery of cortically controlled behaviours like skilled forelimb reachin

Plant diversity and community history shift colonization success from early- to mid-successional species

Aims Functional traits are supposed to play an important role in determining the colonization success of new species into established communities. Short-term experimental studies have documented higher resistance of more diverse grasslands against colonization by new species. However, little is known about which traits colonizers should have to successfully invade diverse plant communities in the longer term and how community history may modify the resistance of diverse communities against colonization. Methods In a grassland biodiversity experiment (Jena Experiment) established with different species richness (SR; 1, 2, 4, 8 and 16) and functional group (FG) number and composition (1 to 4; legumes, grasses, small herbs, tall herbs), we studied colonization of naturally dispersed species in split-plots (i) with different duration of weeding (never weeded, weeded for 3 or 6 years and then un-weeded for 1 year) and (ii) with different duration of colonization (7 years, 4 years and 1 year after cessation of weeding). Important Findings Resistance against colonization by new species declined with increased duration of weeding (on average 13, 17 and 22 colonizer species in 1-, 4- and 7-year-old communities, respectively). Communities established at low diversity accumulated more colonizer species with a longer duration of weeding than more diverse communities. Duration of colonization had only small effects on the number of colonizer species. Colonizers with early successional traits, i.e. annual life cycle, reproduction by seeds, small seeds, long-lived seeds and an earlier start of a longer flowering period, were favoured in species-poor newly established experimental plant communities (short duration of weeding) and early after cessation of weeding (short duration of colonization). A change from early- to mid-successional traits, i.e. taller growth, perennial life cycle, vegetative reproduction, characterized colonization at increased plant diversity and in communities with legumes or without grasses. Legume absence/grass presence and increased duration of weeding led to a shift in colonizer strategies from rapid nutrient uptake and cycling (higher specific leaf area) to nutrient retention and symbiotic N2 fixation. Our study shows that non-random trait spectra of naturally dispersed colonizers encompass trade-offs between different functions (reproduction, persistence, growth) reflected in a change from early- to mid-successional traits at increasing plant diversity, with a longer duration of weeding and a longer time of colonization