Reduzierung der Verbreitung von Antibiotika-Resistenzen durch gezielte Hygiene-Maßnahmen

In der vorliegenden Studie sollte der Zusammenhang zwischen einer erhöhten Zelldichte in Melkanlagen-Biofilmen und der vermehrten Verbreitung von Antibiotikaresistenzgenen über horizontalen Gentransfer überprüft werden. Hierfür wurden Tupferabstriche von verschiedenen Stellen innerhalb einer Melkanlage genommen und mit Hilfe einer Kombination aus kultivierungsgestützten und molekularbiologischen Verfahren untersucht. Zusätzlich wurden mit den gewonnenen antibiotikaresistenten Isolaten aus der Melkanlage Mating-Experimente durchgeführt, um die horizontale Übertragbarkeit der nachgewiesenen Resistenzgene in vitro zu überprüfen. Die Auswahl der Stellen zur Tupferprobenahme erfolgte anhand von bisherigen Arbeiten, in denen bereits Stellen mit vermehrt auftretender Biofilmbildung in der untersuchten Melkanlage erfasst wurden. Die getesteten Antibiotika wurden aufgrund der Häufigkeit ihres Einsatzes in dem untersuchten Milchviehhaltungsbetrieb ausgewählt. Während die kultivierungsgestützten Methoden in der Arbeitsgruppe bereits etabliert waren, wurden verschiedene RT-qPCR-Reaktionen für den molekularbiologischen Nachweis von Antibiotikaresistenzgenen neu etabliert. Insbesondere die PMA-Behandlung des Mastermixes vor dem Einsatz universeller Primer zum Nachweis der 16S rRNA-Gene erwies sich als geeignet zur Erzielung geringer Nachweisgrenzen durch Unterdrückung von Signalen in der Negativkontrolle. In der vorliegenden Studie wurde ein klarer Zusammenhang zwischen dem therapeutischen und präventiven Einsatz der getesteten Wirkstoffe zur Behandlung der Milchkühe und dem Auftreten entsprechender Resistenzen bestätigt. Gegen alle vier getesteten Antibiotika Cloxacillin, Ampicillin, Penicillin und Tetracyclin wurden Keimzahlen resistenter Mikroorganismen kulturell ermittelt. Der molekularbiologische Nachweis der Antibiotikaresistenzen gestaltete sich aufgrund der hohen Vielfalt entsprechender Resistenzgene und –gengruppen deutlich schwieriger. So wurden von den vier getesteten β-Lactamasegenen (blaZ, OXA-1, -2, und -10) lediglich OXA-2 Gene in den Tupfer-DNAExtrakten der Melkbecherablagen als am stärksten besiedelten Ort der Melkanlage detektiert. Im Gegensatz dazu wurden tetM-Gene in den DNA-Extrakten aller Tupferprobenahmeorte detektiert. Insgesamt konnte weder molekularbiologisch noch kulturell ein eindeutiger Zusammenhang zwischen der Besiedelungsdichte und der Häufigkeit des Auftretens von Antibiotikaresistenzen festgestellt werden. Folglich spielen vermutlich weitere Faktoren neben der Besiedelungsdichte eine Rolle für die Verbreitung von Resistenzgenen innerhalb von Biofilmen. Mit den zugewendeten Projektmitteln wurde eine zweijährige Doktorandenstelle finanziert. Zusätzlich wurden die Zuschüsse zur Anschaffung von Verbrauchsmaterial, insbesondere für die molekularbiologischen Verfahren, verwendet. Außerdem wurden die Kosten für die Teilnahme an einer internationalen Tagung in Valencia (Spanien) getragen, auf der die Ergebnisse des Projektes anhand einer Posterpräsentation vorgestellt wurden.The present study was conducted to test the interconnection between microbial cell density and appearance and horizontal transfer of antibiotic resistance genes in milking machine biofilms. Swab samples were taken from different parts of the milking machine and were investigated by a combination of cultivation and molecular methods including RT-qPCR detection of antibiotic resistance genes. Moreover, horizontal transferability of the detected antibiotic resistance genes was tested by in vitro mating experiments between resistant milking machine isolates and suitable sensitive recipients. The sampling sites were chosen according to earlier studies which revealed parts of the milking machine where biofilm formation takes place. The antibiotics tested were chosen according to their frequency of use on the investigated farm. While the cultivation techniques were already well established in the laboratory, the molecular methods, especially RT-qPCR reactions for the detection of 16S rRNA and antibiotic resistance genes, were newly established in the present study. This included PMA-treatment of mastermixes prior to use of universal 16S rRNA gene primers to exclude false positive signals from negative controls, which resulted in improved detection limits. The study showed a clear interconnection between the frequency of use of antibiotics in the investigated farm and the detection of antibiotic resistant biofilm inhabitants. Especially in the cultivation approach, bacterial counts were detected on agar media containing the four tested antibiotics cloxacillin, ampicillin, penicillin and tetracyclin. Cultivation counts were especially high on cloxacillin containing agar, an antibiotic used prophylactically at dry off. The molecular detection of antibiotic genes was difficult due to the high diversity of different resistance genes. While of the four tested β-lactamase genes (blaZ, OXA-1, -2, -10) only OXA-2 genes were detected only at the milking equipment retainers, tetM genes were detected in the DNA extracts of all swab samples. However, a clear relationship between biofilm cell density and frequency of appearance of antibiotic resistance genes could not be established. We concluded that additional factors must play a role in horizontal gene transfer frequency. The financial resources received were used to finance a two-year PhD-position, to purchase consumables, especially for the molecular methods, and for the participation on an international conference in Valencia (Spain) where the results of the study were presented in a poster presentation

Fungi under Modified Atmosphere—The Effects of CO2 Stress on Cell Membranes and Description of New Yeast Stenotrophomyces fumitolerans gen. nov., sp. nov.

High levels of carbon dioxide are known to inhibit the growth of microorganisms. A total of twenty strains of filamentous fungi and yeasts were isolated from habitats with enriched carbon dioxide concentration. Most strains were derived from modified atmosphere packed (MAP) food products or mofettes and were cultivated under an atmosphere of 20% CO₂ and 80% O₂. The influence of CO₂ on fungal cell membrane fatty acid profiles was examined in this study. Major changes were the increase in linolenic acid (C18:3 cis 9, 12, 15) and, additionally in most strains, linoleic acid (C18:2 cis 9, 12) with a maximum of 24.8%, at the expense of oleic (C18:1 cis 9), palmitic (C16:0), palmitoleic (C16:1 cis 9) and stearic acid (C18:0). The degree of fatty acid unsaturation increased for all of the strains in the study, which consequently led to lower melting temperatures of the cell membranes after incubation with elevated levels of CO₂, indicating fluidization of the membrane and a potential membrane malfunction. Growth was reduced in 18 out of 20 strains in laboratory experiments and a change in pigmentation was observed in several strains. Two of the isolated strains, strain WT5 and strain WR1, were found to represent a hitherto undescribed yeast for which the new genus and species Stenotrophomyces fumitolerans (MB# 849906) is proposed

Aclimatização de plantas micropropagadas de videira cv. Bordô (Vitis labrusca L.) em diferentes substratos

The substrate must promote the growth of the plants, being an important component during the acclimatization of micropropagated plants. The aim of this research was to evaluate the effects of different substrates during the acclimatization of fox grape cv. Bordô. The experiment was carried out at the greenhouse during February and March of 2009. Five substrates were tested: Plantmax® HT, Plantmax® HT: Coconut fiber (7,5:2,5 v/v), Plantmax® HT: Coconut fiber (1:1 v/v), Plantmax® HT: Coconut fiber (2,5:7,5 v/v) and Coconut fiber. The process of acclimatization consisted of three phases. After 20 days of in vitro culture, it was removed the PVC film (Polyvinyl chloride) that covered the flasks and after two days, flasks had lids partial opening for two hours at the growth room conditions. The plants were transferred for the different substrates and placed in a greenhouse with intermittent nebulization during 16 days proceeded by 20 days of daily manual irrigation.  The best substrate for acclimatization of fox grape cv. Bordô was Plantmax® HT, benefitting the formation of roots and aerial part of the plants. The coconut fiber must not be used alone as substrate or in larger proportions than 25% in the mixture with Plantmax® HT.O substrato deve promover o crescimento das plantas, sendo um componente importante durante a aclimatação das plantas micropropagadas. O objetivo desta pesquisa foi avaliar os efeitos de diferentes substratos durante a aclimatação da uva de raposa cv. Bordô. O experimento foi realizado em casa de vegetação nos meses de fevereiro e março de 2009. Cinco substratos foram testados: Plantmax® HT, Plantmax® HT: fibra de coco (7,5: 2,5 v / v), Plantmax® HT: fibra de coco ( 1: 1 v / v), Plantmax® HT: fibra de coco (2,5: 7,5 v / v) e fibra de coco. O processo de aclimatação consistiu em três fases. Após 20 dias de cultura in vitro, foi removido o filme de PVC (cloreto de polivinil) que cobria os frascos e após dois dias, os frascos tinham tampas abrindo parcialmente por duas horas nas condições da sala de crescimento. As plantas foram transferidas para os diferentes substratos e colocadas em casa de vegetação com nebulização intermitente durante 16 dias seguidos por 20 dias de irrigação manual diária. O melhor substrato para aclimatação da uva de raposa cv. O Bordô foi o Plantmax® HT, beneficiando a formação de raízes e parte aérea das plantas. A fibra de coco não deve ser usada sozinha como substrato ou em proporções maiores que 25% na mistura com Plantmax® HT

Answering Non-Monotonic Queries in Relational Data Exchange

Relational data exchange is the problem of translating relational data from a source schema into a target schema, according to a specification of the relationship between the source data and the target data. One of the basic issues is how to answer queries that are posed against target data. While consensus has been reached on the definitive semantics for monotonic queries, this issue turned out to be considerably more difficult for non-monotonic queries. Several semantics for non-monotonic queries have been proposed in the past few years. This article proposes a new semantics for non-monotonic queries, called the GCWA*-semantics. It is inspired by semantics from the area of deductive databases. We show that the GCWA*-semantics coincides with the standard open world semantics on monotonic queries, and we further explore the (data) complexity of evaluating non-monotonic queries under the GCWA*-semantics. In particular, we introduce a class of schema mappings for which universal queries can be evaluated under the GCWA*-semantics in polynomial time (data complexity) on the core of the universal solutions.Comment: 55 pages, 3 figure

Cultivation of a novel cold-adapted nitrite oxidizing betaproteobacterium from the Siberian Arctic

Permafrost-affected soils of the Siberian Arctic were investigated with regard to identification of nitrite oxidizing bacteria active at low temperature. Analysis of the fatty acid profiles of enrichment cultures grown at 4°C, 10°C and 17°C revealed a pattern that was different from that of known nitrite oxidizers but was similar to fatty acid profiles of Betaproteobacteria. Electron microscopy of two enrichment cultures grown at 10°C showed prevalent cells with a conspicuous ultrastructure. Sequence analysis of the 16S rRNA genes allocated the organisms to a so far uncultivated cluster of the Betaproteobacteria, with Gallionella ferruginea as next related taxonomically described organism. The results demonstrate that a novel genus of chemolithoautotrophic nitrite oxidizing bacteria is present in polygonal tundra soils and can be enriched at low temperatures up to 17°C. Cloned sequences with high sequence similarities were previously reported from mesophilic habitats like activated sludge and therefore an involvement of this taxon in nitrite oxidation in nonarctic habitats is suggested. The presented culture will provide an opportunity to correlate nitrification with nonidentified environmental clones in moderate habitats and give insights into mechanisms of cold adaptation. We propose provisional classification of the novel nitrite oxidizing bacterium as 'Candidatus Nitrotoga arctica'

Phenotypic and genomic assessment of the potential threat of human spaceflight-relevant Staphylococcus capitis isolates under stress conditions

Previous studies have reported that spaceflight specific conditions such as microgravity lead to changes in bacterial physiology and resistance behavior including increased expression of virulence factors, enhanced biofilm formation and decreased susceptibility to antibiotics. To assess if spaceflight induced physiological changes can manifest in human-associated bacteria, we compared three spaceflight relevant Staphylococcus capitis isolates (DSM 111179, ISS; DSM 31028, clean room; DSM 113836; artificial gravity bedrest study) with the type strain (DSM 20326T). We tested the three strains regarding growth, colony morphology, metabolism, fatty acid and polar lipid pattern, biofilm formation, susceptibility to antibiotics and survival in different stress conditions such as treatment with hydrogen peroxide, exposure to desiccation, and irradiation with X-rays and UV-C. Moreover, we sequenced, assembled, and analyzed the genomes of all four strains. Potential genetic determinants for phenotypic differences were investigated by comparative genomics. We found that all four strains show similar metabolic patterns and the same susceptibility to antibiotics. All four strains were considered resistant to fosfomycin. Physiological differences were mainly observed compared to the type strain and minor differences among the other three strains. The ISS isolate and the bedrest study isolate exhibit a strong delayed yellow pigmentation, which is absent in the other two strains. Pigments were extracted and analyzed by UV/Vis spectroscopy showing characteristic carotenoid spectra. The ISS isolate showed the highest growth rate as well as weighted average melting temperature (WAMT) of fatty acids (41.8°C) of all strains. The clean room isolate showed strongest biofilm formation and a high tolerance to desiccation. In general, all strains survived desiccation better in absence of oxygen. There were no differences among the strains regarding radiation tolerance. Phenotypic and genomic differences among the strains observed in this study are not inevitably indicating an increased virulence of the spaceflight isolate. However, the increased growth rate, higher WAMT and colony pigmentation of the spaceflight isolate are relevant phenotypes that require further research within the human spaceflight context. We conclude that combining genetic analysis with classical microbiological methods allows the detailed assessment of the potential threat of bacteria in highly regulated and extreme environments such as spaceflight environments

Diversity and Activity of Methanotrophic Bacteria in Different Upland Soils

Samples from diverse upland soils that oxidize atmospheric methane were characterized with regard to methane oxidation activity and the community composition of methanotrophic bacteria (MB). MB were identified on the basis of the detection and comparative sequence analysis of the pmoA gene, which encodes a subunit of particulate methane monooxygenase. MB commonly detected in soils were closely related to Methylocaldum spp., Methylosinus spp., Methylocystis spp., or the “forest sequence cluster” (USC α), which has previously been detected in upland soils and is related to pmoA sequences of type II MB (Alphaproteobacteria). As well, a novel group of sequences distantly related (<75% derived amino acid identity) to those of known type I MB (Gammaproteobacteria) was often detected. This novel “upland soil cluster γ” (USC γ) was significantly more likely to be detected in soils with pH values of greater than 6.0 than in more acidic soils. To identify active MB, four selected soils were incubated with (13)CH(4) at low mixing ratios (<50 ppm of volume), and extracted methylated phospholipid fatty acids (PLFAs) were analyzed by gas chromatography-online combustion isotope ratio mass spectrometry. Incorporation of (13)C into PLFAs characteristic for methanotrophic Gammaproteobacteria was observed in all soils in which USC γ sequences were detected, suggesting that the bacteria possessing these sequences were active methanotrophs. A pattern of labeled PLFAs typical for methanotrophic Alphaproteobacteria was obtained for a sample in which only USC α sequences were detected. The data indicate that different MB are present and active in different soils that oxidize atmospheric methane

Bacterial community composition of biofilms in milking machines of two dairy farms assessed by a combination of culture-dependent and -independent methods.

Dairy biofilms as a source of contamination of milk and its products are of great concern in the dairy industry. For a reliable risk assessment, knowledge about the microbial community composition of biofilms in the milking systems of dairy farms must be improved. In this work, swab samples of milking machine biofilms of two dairy farms were investigated by a combination of culture-dependent and -independent methods. Spots in the milking system with enhanced microbial colonization were identified by quantification on selective and non-selective media. In addition, stainless steel coupons were placed into the piping system of a milking machine, removed after several milking intervals, and investigated for colonization by cultivation and culture-independently. Isolates were differentiated and identified by a combination of chemotaxonomical methods and 16S rRNA sequencing. The culture-independent approach involved treatment of the samples with the viability dye propidium monoazide prior to direct DNA-extraction by enzymatic cell lysis and cloning to exclude bias from dead biomass. The milking equipment retainers and the outlet of the milk bulk tank were identified as highly colonized spots on both farms. A high bacterial diversity was detected covering the phyla Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria. Presence of biofilms was demonstrated on several materials including stainless steel and plastic, which are frequently used in milking machines, but also in dairy processing plants. Growth of mainly Gram-positive bacteria with high percentages of the phylum Actinobacteria was detected on the stainless steel coupons after exposition in the milking system for two to three days. Knowledge about the heterogenic microbial load on different parts of the milking machines and the stainless steel coupons will help to identify primary colonizers of the milking system, to assess the risk potential of biofilms for raw milk, to improve sanitation processes and to identify parts of the milking machine, which should be improved by hygienic design