Phenotypic antimicrobial resistance in Escherichia coli strains isolated from swine husbandries in North Western Germany - temporal patterns in samples from laboratory practice from 2006 to 2017.

BACKGROUND: Since 2011, antibiotic usage has decreased continuously in livestock in Germany. Whether this is accompanied by a reduction in bacterial antimicrobial resistance has not been proven so far. In this study 3054 Escherichia coli (E. coli) isolates from pigs which had suffered from disease on 2161 farms in North Western Germany were evaluated retrospectively from 2006 to 2017 for trends in their antimicrobial resistance pattern. Data were substantially related to the "pre-reduction period" and were therefore suggested as a basis for this task. Minimal inhibitory concentrations for selected antimicrobial substances were evaluated for E. coli strains isolated from different organs of diseased swine sampled for routine diagnostic. In total, 81% of E. coli were isolated from faeces or the gastrointestinal tract, 11% from the genito-urinary tract and 8% from other organs. Susceptibility testing and classification of isolates in accordance with clinical cut-offs followed the Clinical and Laboratory Standards Institute (CLSI). If no clinical cut-offs were available for the respective combination of species, substance and organ, other published clinical cut-offs were used. RESULTS: Differences in susceptibility patterns between isolates from the gastrointestinal and genito-urinary tract were found for most substances. Isolates from the genito-urinary tract were less frequently resistant to ampicillin, apramycin, colistin, neomycin, spectinomycin and tetracycline and more frequently resistant to enrofloxacin and florfenicol. A multifactorial logistic regression model revealed time-dependent decreases in frequency of resistant isolates for neomycin, spectinomycin and tetracycline. For colistin, the highest percentage of resistant isolates with 16.0% was found in 2015 followed by a decrease to the level of 2009-2010 in 2017. A decrease in frequencies of ampicillin-resistant isolates was dependent on the age-group and time period. Irrespective of the year, less than 15% E. coli isolates were resistant to apramycin, cephalosporins, colistin, enrofloxacin, florfenicol, gentamicin and neomycin. CONCLUSION: An overall time-dependent decrease in the percentage of resistant E. coli isolates was found for some substances. These data from diseased animals indicate an impact of a general reduction in antibiotic usage on development of bacterial antimicrobial resistance in the field and can support the decision-making of swine practitioners for treatment options in swine.

Vitamin D measurement standardization: The way out of the chaos.

Substantial variability is associated with laboratory measurement of serum total 25-hydroxyvitamin D [25(OH)D]. The resulting chaos impedes development of consensus 25(OH)D values to define stages of vitamin D status. As resolving this situation requires standardized measurement of 25(OH)D, the Vitamin D Standardization Program (VDSP) developed methodology to standardize 25(OH)D measurement to the gold standard reference measurement procedures of NIST, Ghent University and CDC. Importantly, VDSP developed protocols for standardizing 25(OH)D values from prior research based on availability of stored serum samples. The effect of such retrospective standardization on prevalence of "low" vitamin D status in national studies reported here for The Third National Health and Nutrition Examination Survey (NHANES III, 1988-1994) and the German Health Interview and Examination Survey for Children and Adolescents (KIGGS, 2003-2006) was such that in NHANES III 25(OH)D values were lower than original values while higher in KIGGS. In NHANES III the percentage with values below 30, 50 and 75 nmol/L increased from 4% to 6%, 22% to 31% and 55% to 71%, respectively. Whereas in KIGGS after standardization the percentage below 30, 50, and 70 nmol/L decreased from 28% to 13%, 64% to 47% and 87% to 85% respectively. Moreover, in a hypothetical example, depending on whether the 25(OH)D assay was positively or negatively biased by 12%, the 25(OH)D concentration which maximally suppressed PTH could vary from 20 to 35ng/mL. These examples underscore the challenges (perhaps impossibility) of developing vitamin D guidelines using unstandardized 25(OH)D data. Retrospective 25(OH)D standardization can be applied to old studies where stored serum samples exist. As a way forward, we suggest an international effort to identify key prior studies with stored samples for re-analysis and standardization initially to define the 25(OH)D level associated with vitamin D deficiency (rickets/osteomalacia). Subsequent work could focus on defining inadequacy. Finally, examples reported here highlight the importance of suspending publication of meta-analyses based on unstandardized 25(OH)D results.

Developing vitamin D dietary guidelines and the lack of 25-hydroxyvitamin D assay standardization: The ever-present past.

Unstandardized laboratory measurement of 25-hydroxyvitamin D (25(OH)D) confounds efforts to develop clinical and public health vitamin D guidelines. The Vitamin D Standardization Program (VDSP), an international collaborative effort, was founded in 2010 to correct this problem. Nearly all published vitamin D research is based on unstandardized laboratory 25(OH)D measurements. While it is impossible to standardize all old data, it may be possible to identify a small subset of prior studies critical to guidelines development. Once identified it may be possible to calibrate their 25(OH)D values to the NIST and Ghent University reference measurement procedures using VDSP methods thereby permitting future guidelines to be based on standardized results. We simulated the calibration of a small set of ten clinical trials of vitamin D supplementation on achieved 25(OH)D under minimal sun exposure. These studies were selected because they played a prominent role in setting the 2010 vitamin D dietary reference intakes (DRI). Using random-effects meta-regression analysis, Vitamin D External Quality Assessment (DEQAS) data on assay bias was used to simulate the potential bias due to the lack of assay standardization by calibrating the achieved 25(OH)D levels from those 10 studies to: (1) the largest negative, and (2) the largest positive bias from the DEQAS all laboratory trimmed mean (ALTM) for the appropriate assay and year of analysis. For a usual vitamin D intake of 600IU/day the difference in mean achieved 25(OH)D values for those two options was 20nmol/L. However, without re-calibration of 25(OH)D values it is impossible to know the degree to which any of the current guidelines may have been biased. This approach may help stimulate the search for and standardization of that small subset of key studies and, in the cases where standardization is impossible, to identify areas of urgently needed vitamin D research.

[Prediction of extubation failure in ELBW preterm infants]. / Prädiktion von Extubationsversagen bei ELBW-Frühgeborenen.

BACKGROUND: The accurately timed extubation of ventilated ELBW preterm infants is still a problem. With different data systems the attempt has been made to more accurately predict the successful extubation of these infants. However, there do not yet exist any satisfying solutions. PATIENTS/METHODS: We retrospectively analysed 66 ELBW preterm infants who were endotracheal intubated and ventilated within 24 h postnatal. Basic data, clinical and ventilation data immediately before planned extubation and in several intervals during the following 24 h, as well as outcome variables at discharge were interpreted. RESULTS: 51 patients were successfully extubated (EE-group), 15 (22.7%) failed extubation (reintubation within 48 h after extubation, EV-group). Immediately before extubation in the EE-group there was found a significantly higher inspiratory oxygen concentration (FiO2) in comparison to the EV-group (0.25 vs. 0.3; p=0.01). After the extubation attempt the inspiratory oxygen concentration stayed lower in the EE-group, whereas in the EV-group it rose remarkably (2 h after ext.: 0.26 vs. 0.4; p<0.001). Neither of the basic data showed any significant difference. The outcome analysis indicated a longer intensive care in the EV-group and a trend towards increased BPD and ROP. CONCLUSION: The study shows that for ELBW preterm infants the inspiratory oxygen concentration is especially important to predict a successful extubation. According to our data, the inspiratory oxygen demand before and immediately after extubation establishes the essential difference between successfully extubated and reintubated infants.

Measurement of the 1s-2s energy interval in muonium

The 1s-2s interval has been measured in the muonium (&mgr;(+)e(-)) atom by Doppler-free two-photon pulsed laser spectroscopy. The frequency separation of the states was determined to be 2 455 528 941.0(9.8) MHz, in good agreement with quantum electrodynamics. The result may be interpreted as a measurement of the muon-electron charge ratio as -1-1.1(2.1)x10(-9). We expect significantly higher accuracy at future high flux muon sources and from cw laser technology.

A systematic exploration of the influence of the protein stability on amyloid fibril formation in vitro.

There are a number of diseases in which normally soluble proteins associate into regular, insoluble amyloid fibrils. The development of in vitro model systems in which detailed structural, kinetic, and thermodynamic characterization are feasible is of critical importance to our understanding of the amyloid fibril phenomenon. The formation of amyloid fibrils by proteins that are not associated with disease has been recently described, suggesting that this may be a common property of many proteins and not only of the few proteins associated with amyloidoses. The B1 Ig-binding domain of protein G (beta1) is an extremely well-characterized model system. We have found that under certain experimental conditions, some variants of beta1 form fibrils with high reproducibility. By controlling the stability of the protein-either by mutations or by changing experimental conditions-we are able to modulate the ability of the protein to form fibrils. For all of the variants, we find that the key requirement for fibril formation is to choose conditions in which the population of intermediate conformations present during the unfolding transition is maximized.

Modulating protein folding rates in vivo and in vitro by side-chain interactions between the parallel beta strands of green fluorescent protein.

We have identified pairs of residues across the two parallel beta strands of green fluorescent protein that facilitate native strand register of the surface-exposed beta barrel. After constructing a suitable host environment around two guest residues, minimizing interactions of the guest residues with surrounding side-chains yet maintaining the wild-type protein structure and the chromophore environment, we introduced a library of cross-strand pairings by cassette mutagenesis. Colonies of Escherichia coli transformed with the library differ in intracellular fluorescence. Most of the fluorescent pairs have predominantly charged and polar guest site residues. The magnitude and the rate of fluorescence acquisition in vivo from transformed E. coli cells varies among the mutants despite comparable levels of protein expression. Spectroscopic measurements of purified mutants show that the native protein structure is maintained. Kinetic studies using purified protein with fully matured chromophores demonstrate that the mutants span a 10-fold range in folding rates with undetectable differences in unfolding rates. Thus, green fluorescent protein provides an ideal system for monitoring determinants of in vivo protein folding. Cross-strand pairings affect both protein stability and folding kinetics by favoring the formation of native strand register preferentially to non-native strand alignments.

Detection of gunshot residues in routine CTs.

The forensic assessment of non-fatal gunshot wounds often proves to be difficult as wounds have usually been cleaned and protected with a sterile bandage by the time of the examination. The aim of our investigation was to test the possible application of computed tomography (CT) for the forensic assessment. Doing so raised the questions whether gunshot residues in the soft tissues, detected by means of 3-dimensional CT, can be used as evidence of a close-range shot and whether conclusions can be drawn pertaining to the range of the shot or the type of bullet used based on the distribution of the radiologically detectable material? In this experimental study 39 shots were fired at fresh pig skin and it was possible to distinguish shots fired from distances of more than 10 cm and contact shots independent of the type of bullet. For unjacketed lead bullets, radiopaque material could be seen in the depth of the entrance would for firing distances up to 10 cm. In individual cases, CT data and the 3-D reconstruction could provide valuable information in the forensic assessment of patients with gunshot wounds.

Sidechain interactions in parallel beta sheets: the energetics of cross-strand pairings.

BACKGROUND: Both backbone hydrogen bonding and interactions between sidechains stabilize beta sheets. Cross-strand interactions are the closest contacts between the sidechains of a beta sheet. Here we investigate the energetics of cross-strand interactions using a variant of the B1 domain of immunoglobulin G (IgG) binding protein G (beta1) as our model system. RESULTS: Pairwise mutations of polar and nonpolar residues were made at a solvent-exposed site between the two central parallel beta strands of beta1. Both stabilizing and destabilizing interactions were measured. The greatest stabilizations were observed for charge-charge interactions. Our experimental study of sidechain interactions correlates with statistical preferences: residue pairs for which we measure stabilizing interaction energies occur together frequently, whereas destabilizing pairs are rarely observed together. CONCLUSIONS: Sidechain interactions modulate the stability of beta sheets. We propose that cross-strand sidechain interactions specify correct strand register and ordering through the energetic benefit of optimally arranged pairings.

Aromatic rescue of glycine in beta sheets.

BACKGROUND: Glycine is an intrinsically destabilizing residue in beta sheets. In natural proteins, however, this destabilization can be 'rescued' by specific cross-strand pairing with aromatic residues. Here, we present an experimental study of this effect. RESULTS: Protein variants containing glycine and aromatic residues positioned across beta strands in both antiparallel and parallel orientations were studied. The pairing of glycine and phenylalanine across antiparallel strands resulted in a synergistic increase in protein stability. Dramatic differences in stability were observed for the parallel beta-sheet mutants, which were dependent upon the type of site occupied by glycine as well as the type of aromatic residue with which it was cross-strand paired. CONCLUSIONS: Experimental results from a series of mutants suggest a thermodynamic benefit for glycine-aromatic pairing across antiparallel beta strands, consistent with the prevalence of such pairs in natural proteins. We also demonstrate the specificity of glycine-aromatic interactions across parallel beta strands, which defines strand register.

The ontogenetic transition of collagen deposition in rat skin.

Minimizing the morbidity of in utero surgery or, perhaps more important, capturing the unique characteristics of scarless remodeling, as is the fetal response to injury, demands better elucidation of the observed variations from adults in whom the normal progression of wound healing leads to fibrosis. Species-dependent fetal phlogistic responses and wound scar formation represent a temporal continuum before the onset of adult patterns. We have analyzed skin collagen synthesis and content in Sprague-Dawley rats as one possible factor in this evolution showing that the fetal characteristics of a high percentage of type III collagen relative to type I and low total collagen content are maintained as long as the first 10 to 15 days postpartum. Although extrapolation of such a crucial "golden period" to justify the delay of human surgical procedures while still capturing the benefits of the fetal milieu remains speculative, anecdotal observations of minimal scar formation lend some credibility for performing less invasive maneuvers in the neonate.

Assessing beliefs of older rural Americans about nutrition education: use of the focus group approach.

We used the focus group interview technique as a preliminary research step in developing a nutrition education intervention for rural seniors who, because of less than optimal eating habits and changing demographics, are an important target audience. Sixty-eight well, active, rural North Dakota seniors, 60 years or older, from communities of 2,500 or fewer people, participated in five focus groups conducted in late summer 1988. As a qualitative research approach, focus group interviews offer a means of obtaining in-depth information on a specific topic from representatives of a target audience in a discussion group atmosphere. Focus groups require careful preparation and structuring and should include a capable moderator, a prepared discussion guide, carefully recruited participants, and a comfortable setting. The process generated ideas that we are using to develop a health promotion nutrition intervention that will be a mailed-home approach, including use of incentives, social role models, cholesterol screening, and learning activities. The intervention relies on the interest and ability of seniors to make positive health changes. We conclude that the focus group approach is useful in developing nutrition education interventions.

Focus group interviews with seniors.

The focus group interview technique provides in-depth information on a specific topic in a group discussion setting and can be effectively used with older adults. Successful focus group interviews include careful question preparation and participant recruitment as well as securing a capable moderator and comfortable setting. The technique is used as a preliminary research step to elicit feelings, attitudes and perceptions about a topic. However, results should not be generalized without further quantitative research.

Analysis of collagen content in the fetal wound.

The absence of apparent scar formation following the creation of surgical wounds in utero appears to be a phenomenon peculiarly privileged as a sequela of fetal wound healing. Little information exists to explain this disparity from our knowledge of adult wound healing. Therefore, following creation of surgical wounds in fetal rats, at different intervals the healing wounds were harvested and analyzed for collagen content and types. The average proportion of type III collagen was elevated in normal (26.5%) as well as wounded fetal skin (33.8%) when compared with normal levels for the adult (15%). The total collagen content was markedly diminished in the fetal wound. Although embryonal collagen synthesis apparently does exist in fetal reparative processes, the relationship to the lack of gross scarring remains undetermined.

Type I and type III collagen content of healing wounds in fetal and adult rats.

Full-thickness, dermal wounds were surgically created on the dorsa of fetal rats on the 17th day of gestation. The granulation tissue which developed after 2 days (19 days of gestation) was harvested from six to nine animals and pooled and the collagen was extracted with 0.5 M acetic acid and acetic acid plus pepsin. The ratio of type III:type I collagen was estimated from densitometer scans of electrophoretically separated alpha-chains. Full-thickness (to fascia depth) wounds were also produced on the dorsa of adult rats and granulation tissue which had developed for different periods of time up to 30 days was excised. Relative proportions of type III and type I collagen were assessed in normal and granulation tissues taken from the adult rats. Both fetal and adult granulation tissues have elevated type III collagen content but normal fetal tissue has a much higher content of type III than does normal adult tissue.

Improved purification and some molecular and kinetic properties of sn-glycerol-3-phosphate dehydrogenase from Saccharomyces cerevisiae.

The purification procedure for isolating sn-glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) from Saccharomyces cerevisiae was improved by the introduction of an ion-exchange step. Enzyme yields were doubled and the specific activity was increased as compared to the original procedure. A new value of 42,000 was obtained for the molecular weight by several denaturing methods. By native gel chromatography the molecular weight appears to be 31,000 as reported earlier. Michaelis constants were found to be 0.37 mM with dihydroxyacetone phosphate as the variable substrate and 0.018 mM for NADH as the variable substrate.

Collagenolytic activity of Vibrio vulnificus: potential contribution to its invasiveness.

Vibrio vulnificus (lactose-positive vibrio) produced collagenase when grown in 2% synthetic sea salts supplemented with hydrolyzed casein. The addition of collagen or peptone to the medium increased the level of collagenase production. Collagenase activity was inhibited by EDTA but not by fetal calf serum.