Correlation between Bacterial Cell Density and Abundance of Antibiotic Resistance on Milking Machine Surfaces Assessed by Cultivation and Direct qPCR Methods.

The relative abundance of antibiotic-resistant bacteria and antibiotic-resistance genes was surveyed for different parts of a milking machine. A cultivation approach based on swab samples showed a highly diverse microbiota, harboring resistances against cloxacillin, ampicillin, penicillin, and tetracycline. This approach demonstrated a substantial cloxacillin resistance of numerous taxa within milking machine microbiota coming along with regular use of cloxacillin for dry-off therapy of dairy cows. For the less abundant tetracycline-resistant bacteria we found a positive correlation between microbial cell density and relative abundance of tetracycline-resistant microorganisms (R2 = 0.73). This indicated an accelerated dispersion of resistant cells for sampling locations with high cell density. However, the direct quantification of the tetM gene from the swap samples by qPCR showed the reverse relation to bacterial density if normalized against the abundance of 16S rRNA genes (R2 = 0.88). The abundance of 16S rRNA genes was analyzed by qPCR combined with a propidium monoazide treatment, which eliminates 16S rRNA gene signals in negative controls.

Analysis of sugar crystal size in honey.

Honey consists typically of more than 80% sugars, predominantly fructose and glucose. Glucose-rich honey crystallizes more rapidly than honey with a high fructose content. However, the size of the sugar crystals is crucial for the mouth feel of crystallised honey. Honeys containing small crystals have a creamy consistency, which is preferred by most consumers. In contrast, large crystals cause a coarse mouth feel. Factors affecting crystal size are of vital interest for the production of high-quality honey and thus analysis of sugar crystal size in honey is crucial. Here we present a simple and efficient method for measuring the size of sugar crystals in honey. A honey drop is placed on a coverslip, which is centrifuged using a converted smoothie maker. This spreads the drop over the coverslip and separates the sugar crystals from each other. Subsequently, the size of the crystals can be conveniently measured by microscopy. Compared to squeezing the honey drop between slide and coverslip, this approach avoids the risk of breaking the crystals. Moreover, the method is highly reproducible as indicated by intra-day and inter-day standard deviations of 7 to 14% for crystal sizes. Simple method for preparation of honey for crystal size analysis by microscopy. Use of cheap, easily accessible equipment. High intra and inter-day reproducibility.

Polar lipid characterization and description of Chryseobacterium capnotolerans sp. nov., isolated from high CO2-containing atmosphere and emended descriptions of the genus Chryseobacterium, and the species C. balustinum, C. daecheongense, C. formosense, C. gleum, C. indologenes, C. joostei, C. scophthalmum and C. ureilyticum.

Modified atmosphere (MA) packaging plays an important role in improving food quality and safety. By using different gas mixtures and packaging materials the shelf life of fresh produce can significantly be increased. A Gram-negative-staining, rod-shaped, orange-pigmented strain DH-B6T, has been isolated from MA packed raw pork sausage (20% CO2, 80% O2). The strain produced biofilms and showed growth at high CO2 levels of up to 40%. Complete 16S rRNA gene and whole-genome sequences revealed that strain DH-B6T belongs to the genus Chryseobacterium, being closely related to strain Chryseobacterium indologenes DSM 16777T (98.4%), followed by Chryseobacterium gleum NCTC11432T (98.3%) and Chryseobacterium lactis KC1864T (98.2%). Average nucleotide identity value between DH-B6T and C. indologenes DSM 16777T was 81.1% and digital DNA-DNA hybridisation was 24.9%, respectively. The DNA G+C content was 35.51 mol%. Chemotaxonomical analysis revealed the presence of the rare glycine lipid cytolipin, the serine-glycine lipid flavolipin and the sulfonolipid sulfobacin A, as well as phosphatidylethanolamine, monohexosyldiacylglycerol and ornithine lipid, including the hydroxylated forms. Major fatty acids were iC15 : 0 (50.7%) and iC17 : 1 cis 9 (28.7%), followed by iC15 : 0 2-OH (7.0%) and iC17 : 0 3-OH (6.2%). The isolated strain contained MK-6 as the only respiratory quinone and flexirubin-like pigments were detected as the major pigments. Based on the phenotypic, chemotaxonomic and phylogenetic characteristics, the strain DH-B6T (=DSM 110542T=LMG 31915T) represents a novel species of the genus Chryseobacterium, for which the name Chryseobacterium capnotolerans sp. nov. is proposed. Emended descriptions of the genus Chryseobacterium and eight species of this genus based on polar lipid characterisation are also proposed.

Influence of Stirring Parameters on Creaminess of Spring Blossom Honey Measured by Crystal Size, Whiteness Index and Mouthfeel.

Spring blossom honey from regions with many rape fields tends to crystalize rapidly after harvesting. The crystallization process needs to be controlled by stirring in order to avoid the formation of coarse crystals and to ensure the creaminess of honey. The aim of this study was to investigate how various parameters of the stirring process influence the creaminess of spring blossom honey in order to give recommendations for beekeeping practices. The creaminess was quantified by measuring the crystal size by microscopic analysis, measuring the whiteness index by color analysis using CIE Lab and by sensory analysis. We investigated the influence of five stirring parameters, including the type of stirring device, honey pretreatment, stirring temperature (14 °C to room temperature), stirring interval (1 to 24 times) and stirring time (1-15 min) on the creaminess of honey. We found that the stirring temperature is the most important factor for honey creaminess. At the optimal temperature of 14 °C, other factors like seed honey, stirring time and stirring interval have only a neglectable effect. If the optimal temperature of 14 °C cannot be maintained, as it may happen in beekeepers' practice, sieving the honey with a mesh size of 200 µm before stirring, the addition of seed honey prepared with a kitchen food processor, and using a stirring screw and stirring several times per day is recommended.

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.

Proteome and Phosphoproteome Analysis in TNF Long Term-Exposed Primary Human Monocytes.

To better understand the inflammation-associated mechanisms modulating and terminating tumor necrosis factor (TNF-)induced signal transduction and the development of TNF tolerance, we analyzed both the proteome and the phosphoproteome in TNF long term-incubated (i.e., 48 h) primary human monocytes using liquid chromatography-mass spectrometry. Our analyses revealed the presence of a defined set of proteins characterized by reproducible changes in expression and phosphorylation patterns in long term TNF-treated samples. In total, 148 proteins and 569 phosphopeptides were significantly regulated (103 proteins increased, 45 proteins decreased; 377 peptides with increased and 192 peptides with decreased phosphorylation). A variety of these proteins are associated with the non-canonical nuclear factor κB (NF-κB) pathway (nuclear factor κB (NFKB) 2, v-rel reticuloendotheliosis viral oncogene homolog (REL) B, indolamin-2,3-dioxygenase (IDO), kynureninase (KYNU)) or involved in the negative regulation of the canonical NF-κB system. Within the phosphopeptides, binding motifs for specific kinases were identified. Glycogen synthase kinase (GSK) 3 proved to be a promising candidate, since it targets NF-κB inhibiting factors, such as CCAAT/enhancer binding protein (C/EBP) ß. Our experiments demonstrate that both proteome and phosphoproteome analysis can be effectively applied to study protein/phosphorylation patterns of primary monocytes. These results provide new regulatory candidates and evidence for a complex network of specific but synergistically acting/cooperating mechanisms enabling the affected cells to resist sustained TNF exposure and resulting in the resolution of inflammation.

Isolation and characterization of Corynebacterium spp. from bulk tank raw cow's milk of different dairy farms in Germany.

We detected Corynebacterium spp. in raw milk samples of three farms by means of a selective, tellurite-containing medium. The isolated strains were identified based on full 16S rRNA gene sequences and partial rpoB gene sequences as C. xerosis, C. variabile, C. lactis, C. callunae, C. confusum, C. glutamicum and C. crudilactis. The identification based on 16S rRNA and rpoB sequences was not reliable for isolates of C. xerosis. Chemotaxonomic markers of the isolates, fatty acids, acyl type of peptidoglycan, presence and length of mycolic acids, quinone patterns, and polar lipids, were in accord with the known characteristics of these species. Biochemical profiles, analyzed with the API Coryne system, were able to differentiate all groups, but were unable to identify the strains due to an inappropriate database for raw-milk associated corynebacteria. Most of the tested isolates showed a single-substance resistance against oxacillin, but three single isolates were classified as multidrug resistant.

Stenotrophomonas lactitubi sp. nov. and Stenotrophomonas indicatrix sp. nov., isolated from surfaces with food contact.

Five Gram-stain-negative, rod-shaped, none-spore-forming isolates were obtained from biofilms on different sites of a milking machine in Germany. Another strain with similar morphological characteristics was isolated from dirty dishes. Based on phylogenetic analysis of the 16S rRNA and gyrB genes, all isolates were assigned to the genus Stenotrophomonas, but were divided into three different groups. Chemotaxonomic characterization of the isolates led to the detection of iso-C15 : 0 and anteiso-C15 : 0 as the predominant cellular fatty acids, as well as small amounts of the hydroxyl fatty acids iso-C11 : 0 3-OH, C12 : 0 3-OH and iso-C13 : 0 3-OH. One group could be assigned to the species Stenotrophomonas maltophilia, while the genome sequences of two groups displayed average nucleotide identity values of less than 94 % between each other and the genome sequences of the next related type strains Stenotrophomonas maltophilia ATCC 13637T and Stenotrophomonas rhizophila DSM 14405T. Further phylogenetic, phenotypic and chemotaxonomic analyses enabled the differentiation of these strains from these closely related species. They are therefore considered to represent two novel species, for which the names Stenotrophomonaslactitubi and Stenotrophomonasindicatrix are proposed, with strains M15T (=DSM 104152T=LMG29943T) and WS40T (=DSM28278T=LMG29942T) as type strains.

Plate Osteosynthesis of Proximal Ulna Fractures-A Biomechanical Micromotion Analysis.

PURPOSE: Double plating has been promoted, in recent years, as an alternative treatment method for proximal ulna fractures. This study aimed to compare the biomechanical properties of double-plate osteosynthesis with posterior plate fixation using a novel investigational design utilizing a 3-dimensional camera system to analyze fracture micromotion. METHODS: Fourteen fresh-frozen specimens were available for this study. Mayo type IIA fractures of the olecranon were created and internal fixation was performed with either an angular stable posterior plate or angular stable double plates. Fracture micromotion was evaluated by means of digital image correlation with a 3-dimensional camera system before and after dynamic cyclic loading from 15° to 90° of elbow flexion with a pulling force of 25 N to 80 N. RESULTS: Micromotion of fragments was less pronounced in double-plate osteosynthesis when compared with single plates before and after cyclic loading. However, overall results were similar. Two of the single plates failed during cyclic loading but there were no failures in the double plates. CONCLUSIONS: This biomechanical analysis shows that single and double plating results in comparable stability of fixation. Although the double-plating technique tends to provide more stable fixation, relevant differences were not observed. CLINICAL RELEVANCE: Double plating potentially represents an efficient option for fixation of proximal ulna fractures. It could decrease the risk of soft tissue complications owing to their low profile and the superior soft tissue coverage.

Comparative analysis of bacterial community composition in bulk tank raw milk by culture-dependent and culture-independent methods using the viability dye propidium monoazide.

Microbial diversity of 3 raw milk samples after 72 h of storage at 4 °C in a bulk tank was analyzed by culture-dependent and -independent methods. The culture-dependent approach was based on the isolation of bacteria on complex and selective media, chemotaxonomic differentiation of isolates, and subsequent identification by 16S rRNA gene sequencing. The culture-independent approach included the treatment of raw milk with the dye propidium monoazide before direct DNA extraction by mechanic and enzymatic cell lysis approaches, and cloning and sequencing of the 16S rRNA genes. The selective detection of viable bacteria improved the comparability between bacterial compositions of raw milk based on culture-dependent and -independent methods, which was the major objective of this study. Several bacterial species of the phyla Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria were detected by the culture-dependent method, whereas mainly bacteria of the phylum Proteobacteria as well as low proportions of the phyla Bacteroidetes and Actinobacteria were detected by the culture-independent method. This led to the conclusion that the phylum Firmicutes was strongly discriminated by the culture-independent approach. Generally, species richness detected by the culture-dependent method was higher than that detected by the culture-independent method for all samples. However, few taxa could be detected solely by the direct DNA-based method. In conclusion, the combination of culture-dependent and -independent methods led to the detection of the highest bacterial diversity for the raw milk samples analyzed. It was shown that DNA extraction from raw milk as the essential step in culture-independent methods causes the discrimination of taxa by incomplete cell lysis. Treatment of raw milk with the viability dye propidium monoazide was optimized for the application in raw milk without former removal of milk ingredients and proved to be a suitable tool to ensure comparability of bacterial diversity depicted by both methods.