Use of microbiology tests in the era of increasing AMR rates- a multicentre hospital cohort study.

Background: Effective use of microbiology test results may positively influence patient outcomes and limit the use of broad-spectrum antibiotics. However, studies indicate that their potential is not fully utilized. We investigated microbiology test ordering practices and the use of test results for antibiotic decision-making in hospitals. Methods: A multicentre cohort study was conducted during five months in 2014 in Medical departments across three hospitals in Western Norway. Patients treated with antibiotics for sepsis, urinary tract infections, skin and soft tissue infections, lower respiratory tract infections or acute exacerbations of chronic obstructive pulmonary disease were included in the analysis. Primary outcome measures were degree of microbiology test ordering, compliance with microbiology testing recommendations in the national antibiotic guideline and proportion of microbiology test results used to inform antibiotic treatment. Data was obtained from electronic- and paper medical records and charts and laboratory information systems. Results: Of the 1731 patient admissions during the study period, mean compliance with microbiology testing recommendations in the antibiotic guideline was 89%, ranging from 81% in patients with acute exacerbations of chronic obstructive pulmonary disease to 95% in patients with sepsis. Substantial additional testing was performed beyond the recommendations with 298/606 (49%) of patients with lower respiratory tract infections having urine cultures and 42/194 (22%) of patients with urinary tract infections having respiratory tests. Microbiology test results from one of the hospitals showed that 18% (120/672) of patient admissions had applicable test results, but only half of them were used for therapy guidance, i.e. in total, 9% (63/672) of patient admissions had test results informing prescription of antibiotic therapy. Conclusions: This study showed that despite a large number of microbiology test orders, only a limited number of tests informed antibiotic treatment. To ensure that microbiology tests are used optimally, there is a need to review the utility of existing microbiology tests, test ordering practices and use of test results through a more targeted and overarching approach.

Structural and functional evolution of the translocator protein (18 kDa).

Translocator proteins (TSPO) are the products of a family of genes that is evolutionarily conserved from bacteria to humans and expressed in most mammalian tissues and cells. Human TSPO (18 kDa) is expressed at high levels in steroid synthesizing endocrine tissues where it localizes to mitochondria and functions in the first step of steroid formation, the transport of cholesterol into the mitochondria. TSPO expression is elevated in cancerous tissues and during tissue injury, which has lead to the hypothesis that TSPO has roles in apoptosis and the maintenance of mitochondrial integrity. We recently identified a new paralog of Tspo in both the human and mouse. This paralog arose from an ancient gene duplication event before the divergence of the classes aves and mammals, and appears to have specialized tissue-, cell-, and organelle-specific functions. Evidence from the study of TSPO homologs in mammals, bacteria, and plants supports the conclusion that the TSPO family of proteins regulates specialized functions related to oxygen-mediated metabolism. In this review, we provide a comprehensive overview of the divergent function and evolutionary origin of Tspo genes in Bacteria, Archaea, and Eukarya domains.

Characteristics of breast milk and serology of women donating breast milk to a milk bank.

OBJECTIVE: Breast milk is the most important nutrient to all newborn babies. If the mother's milk production is insufficient, it is important to provide donor breast milk without reduction of its immunologic and antimicrobial properties. Early use of breast milk to preterm infants has shown a reduced incidence of necrotising enterocolitis, a faster tolerance of enteral feeding, and a reduced need of parenteral nutrition. It is important to have milk from a CMV-IgG negative donor to VLBW infants considered immunocompromised. METHODS: Between January 1st and December 31st 2001, 69 women delivered 1.973 litres (mean 28.6 litres/woman/year). 73% had college education, were primipara, and with a mean age of 30.7 years. Those who smoked, used alcohol or any medications were refused as donors. They started to deliver approximately 7 weeks after having given birth and continued for a mean of 4 months. Each milk sample was tested for bacterial growth. Every donor was screened for HIV, CMV-IgG and hepatitis B/C before donating milk and thereafter every third month. RESULTS: 62.3% was CMV-IgG positive. Samples containing staphylococcus aureus, klebsialla-, enterobacter- and serratia-species or E. coli, and all samples containing > 10(4) cfu/ml were pasteurised. Overall, only 10.5% of the samples were pasteurised. CONCLUSION: It is possible and important to provide VLBW babies with fresh frozen unpasteurised CMV-IgG negative breast milk until their own mothers' milk production is sufficient.

Cloning and functional expression in Escherichia coli of a cDNA encoding cardenolide 16'-O-glucohydrolase from Digitalis lanata Ehrh.

A clone of cardenolide 16'-O-glucohydrolase cDNA (CGH I) was obtained from Digitalis lanata which encodes a protein of 642 amino acids (calculated molecular mass 73.2 kDa). The amino acid sequence derived from CGH I showed high homology to a widely distributed family of beta-glucohydrolases (glycosyl hydrolases family 1). The recombinant CGH I protein produced in Escherichia coli had CGH I activity. CGH I mRNA was detected in leaves, flowers, stems and fruits of D. lanata.

Antiproliferative effect of a polysaccharide fraction of a 20% methanolic extract of stinging nettle roots upon epithelial cells of the human prostate (LNCaP).

In Germany, plant extracts are often used in the treatment of early stages of benign prostate hyperplasia (BPH). The effects of different concentrations of the polysaccharide fraction of the 20% methanolic extract of stinging nettle roots (POLY-M) on the cellular proliferation of lymph node carcinoma of the prostate (LNCaP) cells were determined by measurement of the genomic DNA content of the samples. All concentrations of POLY-M showed an inhibitory effect on the growth of the LNCaP cells during 7 days except the two lowest concentrations. The reduced proliferation of POLY-M treated LNCaP cells was significantly (p < 0.05) different from the untreated control. The inhibition was time- and concentration-dependent with the maximum suppression (50%) on day 6 and at concentrations of 1.0E-9 and 1.0E-11 mg/ml. No cytotoxic effect of POLY-M on cell proliferation was observed. The in vitro results show for the first time an antiproliferative effect of Urtica compounds on human prostatic epithelium and confirm our previous in vivo findings.

Delta(5)-3beta-hydroxysteroid dehydrogenase from Digitalis lanata Ehrh. - a multifunctional enzyme in steroid metabolism?

Delta(5)-3beta-Etaydroxysteroid dehydrogenase (Delta(5)-3beta-HSD; EC 1.1.1.145), an enzyme converting pregn-5-ene-3beta-ol-20-one (pregnenolone) to pregn-5-ene-3,20-dione (isoprogesterone), was isolated from the soluble fraction of suspension-cultured cells of Digitalis lanata L. strain VIII. Starting with acetone dry powder the enzyme was purified in three steps using column chromatography on Fractogel-TSK DEAE, hydroxyapatite and Sephacryl G-200. Fractions with highest Delta(5)-3beta-HSD activity were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After in-situ digestion the resulting bands were sequenced N-terminally. The 29-kDa band yielded three fragments with high sequence homology to members of the superfamily of short-chain dehydrogenases/reductases. High similarity was found to microbial hydroxysteroid dehydrogenases. The band may therefore represent the Delta(5)-3beta-HSD. The purified enzyme was characterized with respect to kinetic parameters, substrate specificity and localization. The function of the enzyme in steroid metabolism is discussed.

[Nutritional content in the daily diet from selected nursing homes for the aged in State of Hessen Second Report--minerals]. / Nhrstoffgehalt in Tagesverpflegungen ausgewählter hessischer Altenheime Zweite Mitteilung--Mineralstoffe.

Daily diet from 20 nursing homes for the elderly in the German state of Hessen were collected over a period of seven days. After weighing and protocoling, the components of the meals were combined to one sample per day. The homogenized samples were analyzed for the minerals sodium, potassium, calcium, magnesium, phosphorus, iron, zinc, manganese, copper, nickel, and chromium. For evaluation of the nutrient value the mineral contents were compared to the recommendations of the German Association for Nutrition (Deutsche Gesellschaft für Ernährung) for nutrient intake of the elderly. Whereas mean content of sodium and of calculated sodium chloride per day drastically exceeded the recommendation, the magnesium and chromium recommendations were not reached by far. Regarding high nutrient density requirements with respect to a lower energy demand, only a few diets could reach the recommendations for calcium, magnesium, iron, and zinc density given for the elderly.

Cardenolide 16'-O-glucohydrolase from Digitalis lanata. Purification and characterization.

A three-step chromatographic procedure was developed for purification of cardenolide 16'-O-glucohydrolase (CGH) from Digitalis lanata Ehrh. leaves, including Phenyl-Sepharose hydrophobic interaction chromatography followed by SP-Sepharose cation exchange and Q-Sepharose anion-exchange chromatography. Starting with acetone dry powder the purification resulted in an 760-fold enrichment of CGH. Molecular weight, substrate specificity, pH optimum and temperature stability of CGH were determined. Antibodies against CGH were prepared in rabbits. The SDS gel electrophoresis of protein extracts from leaves of D. lanata and other D. species showed bands at 70 kDa and 36 kDa reacting with the antibodies. The 70-kDa protein is the main protein stained with CGH antibodies in freshly prepared extracts of D. lanata. It may represent undegraded CGH. The 36-kDa protein is enriched in aged CGH preparations. It is probably a degradation product. Proteins related to 70-kDa and 36-kDa bands also occur in crude protein preparations from leaves of D. heywoodii P. et M. Silva, D. mariana Boiss., D. purpurea L., and D. thapsi L. indicating that CGH is also present in these species. Purified CGH was digested with proteases V8 and Lys-C and the resulting fragments obtained were sequenced. One fragment had the typical amino-acid sequence of the catalytic center of family-1 glycosyl hydrolases (EC 3.2.1.x). Cardenolide 16'-O-glucohydrolase, like the other members of this enzyme family, appeared to have a glutamic acid residue directly involved in glycosidic bond cleavage as a nucleophile.

Purification and characterization of lanatoside 15'-O-acetylesterase from Digitalis lanata Ehrh.

Lanatoside 15'-O-acetylesterase (LAE) from in-vitro-cultivated cells of Digitalis lanata Ehrh. was isolated and partially sequenced. The enzyme was extracted with citrate buffer from acetone dry powder. It was purified in a two-step chromatographical procedure including Phenyl Sepharose hydrophobic interaction chromatography followed by CM Sepharose cation-exchange chromatography to more than 330 mumol.s-1.(g protein)-1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified protein showed a major band at 39 kDa. The protein was identified by correlation of band intensity on SDS-PAGE and enzyme activity of CM Sepharose column fractions. Size-exclusion chromatography on Sephacryl 200 revealed a single activity peak with an apparent molecular mass of about 85 kDa. Electrophoresis under nondenaturating conditions of purified LAE showed only one band with esterase activity. The intensity of this band was correlated with that of the 39-kDa band after SDS-PAGE. About 30% of the protein, including the N-terminus and several fragments obtained by Lys-C protease digestion, was sequenced. A fragment obtained by Lys-C digestion showed partial homology to other hydrolases and apoplasmic proteins. It included the probable location of an active-site histidine. The activity of LAE was high in non-morphogenic D. lanata cell strains selected for high activities in the chemical transformation of cardenolides, but rather low in the proembryogenic masses of the embryogenic cell strain VIII. It increased during the development of somatic embryos. The LAE activity in leaves of D. lanata plants was in the range 4-24 nmol.s-1.(g protein)-1.

Effects of bound monoclonal antibodies on the decay of the phototransformation intermediates I700(1,2) from native Avena phytochrome.

The kinetics of the microsecond phototransformation intermediates of 124 kDa Avena phytochrome (I700(1,2) were studied in the presence of bound monoclonal antibodies at various temperatures. A global analysis was applied to the decays at all wavelengths at each temperature in order to derive the rate constants and the decay-associated spectra of the three decay components. Monoclonal antibodies bound to specific epitopes altered the Arrhenius parameters of both I700(1,2) decay components. The strongest influence on these parameters was observed with OAT 8 (epitope between residues 624 and 686), which decreased by more than 50% the activation parameters of both components. This decrease is interpreted to result from an increased flexibility induced by this antibody in the ground state or in the transition state of bonds changing during the decay of both I700 transients. Thus, the OAT 8 epitope appears to be functionally important during the decay of the I700(1,2) intermediates. For the case of I700(1), bound OAT 23 and OAT 25 (epitopes between residues 1 and 66) reduced even further the relatively small flexibility of these bonds in the red light-absorbing form of phytochrome (Pr) without antibodies, as reflected by the high preexponential factors for its decay. This resulted also in higher activation energies for this decay in the presence of the antibodies. Thus, the amino-terminus should act as a rigid spacer of the chromophore cavity without affecting it during the microsecond transformation, because the Arrhenius parameters for these decays are similar to those for small phytochrome. The possible implications of the influence of the various antibodies on the bleaching remaining after the decay of I700(1,2) are discussed.

Fourier transform resonance Raman spectroscopy of phytochrome.

The Pr and Pfr forms of phytochrome in H2O and D2O have been studied by Fourier transform resonance Raman spectroscopy with near-infrared excitation (1064 nm). It is demonstrated that this technique is a powerful method for analyzing the chromophore structures of photosensitive pigments. The high spectral quality allows discussion of vibrational assignments based on an empirical approach using previously published data obtained from model compounds. The reduction in intensity of a high-frequency band assigned to the ring-C/D methine bridge vibration is an indication for the non-coplanarity of the ring D in Pfr. The high intensity of a C-H out-of-plane vibration also supports this hypothesis. In Pr, a broad peak at approximately 1100 cm-1 is assigned to an out-of-plane vibration of a strongly hydrogen-bonded pyrrole C=NH+ group. It is missing in Pfr, suggesting deprotonation of the corresponding ring during the transformation from Pr to Pfr.

Partial purification and initial characterization of phytochrome from the mossAtrichum undulatum P. Beauv. grown in the light.

The extraction and partial purification of phytochrome from light-grownAtrichum undulatum P. Beauv., a chlorophyllous moss, is described. Polyethyleneimine and salt fractionation followed by hydroxyapatite and Affi-gel-blue chromatography were used to separate phytochrome from chlorophyll, and to purify the pigment. All steps were performed in the presence of Triton X-100 which improved the yield by a factor of about three. The protein has a molecular weight some-what larger than that ofAvena phytochrome (124 kDa), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis. It cross-reacts with a monoclonal antibody against phytochrome from etiolated corn (Zea) and a polyclonal antibody against phytochrome from etiolated oat (Avena), and its photoreversibility is similar to that of phytochrome from greenAvena.