Rapid and Specific Detection,Molecular Epidemiology,and Experimental Virulence of the O16 Subgroup within Escherichia coli Sequence Type 131

Escherichia coli sequence type 131 (ST131), a widely disseminated multidrug-resistant extraintestinal pathogen, typically exhibits serotype O25b:H4. However, certain ST131 isolates exhibit serotype O16:H5 and derive from a phylogenetic clade that is distinct from the classic O25b:H4 ST131 clade. Both clades are assigned to ST131 by the Achtman multilocus sequence typing (MLST) system and a screening PCR assay that targets ST131-specific sequence polymorphisms in the mdh and gyrB genes. However, they are classified as separate STs by the Pasteur Institute MLST system, and an ST131 PCR method that targets the O25b rfb region and an ST131-specific polymorphism in pabB detects only the O25b-associated clade. Here, we describe a novel PCR-based method that allows for rapid and specific detection of the O16-associated ST131 clade. The clade members uniformly contained allele 41 of fimH (type 1 fimbrial adhesin) and a narrow range of alleles of gyrA and parC (fluoroquinolone target genes). The virulence genotypes of the clade members resembled those of classic O25b:H4 ST131 isolates; representative isolates were variably lethal in a mouse subcutaneous sepsis model. Several pulsotypes spanned multiple sources (adults, children, pets, and human fecal samples) and locales. An analysis of recent clinical E. coli collections showed that the O16 ST131 clade is globally distributed, accounts for 1 to 5% of E. coli isolates overall, and, when compared with other ST131 isolates, it is associated with resistance to ampicillin, gentamicin, and trimethoprim-sulfamethoxazole and with susceptibility to fluoroquinolones and extended-spectrum cephalosporins. Attention to this O16-associated ST131 clade, which is facilitated by our novel PCR-based assay, is warranted in future epidemiological studies of ST131 and, conceivably, in clinical applications.     

Developmental maturation of innate immune cell function correlates with susceptibility to central nervous system autoimmunity

MS is an inflammatory CNS disorder, which typically occurs in early adulthood and rarely in children. Here we tested whether functional maturation of innate immune cells may determine susceptibility to CNS autoimmune disease in EAE. Two‐week‐old mice were resistant to active EAE, which causes fulminant paralysis in adult mice; this resistance was associated with an impaired development of Th1 and Th17 cells. Resistant, young mice had higher frequencies of myeloid‐derived suppressor cells and plasma‐cytoid DCs. Furthermore, myeloid APCs and B cells from young mice expressed lower levels of MHC class II and CD40, produced decreased amounts of proinflammatory cytokines, and released enhanced levels of anti‐inflammatory IL‐10. When used as APCs, splenocytes from 2‐week‐old mice failed to differentiate naive T cells into Th1 and Th17 cells irrespective of the T‐cell donor's age, and promoted development of Treg cells and Th2 cells instead. Adoptive transfer of adult APCs restored the ability of 2‐week‐old mice to generate encephalitogenic T cells and develop EAE. Collectively, these findings indicate that the innate immune compartment functionally matures during development, which may be a prerequisite for development of T‐cell‐mediated CNS autoimmune disease.     

Somatostatin Neurons of the Bed Nucleus of Stria Terminalis Enhance Associative Fear Memory Consolidation in Mice

Excessive fear learning and generalized, extinction-resistant fear memories are core symptoms of anxiety and trauma-related disorders. Despite significant evidence from clinical studies reporting hyperactivity of the bed nucleus of stria terminalis (BNST) under these conditions, the role of BNST in fear learning and expression is still not clarified. Here, we tested how BNST modulates fear learning in male mice using a chemogenetic approach. Activation of GABAergic neurons of BNST during fear conditioning or memory consolidation resulted in enhanced cue-related fear recall. Importantly, BNST activation had no acute impact on fear expression during conditioning or recalls, but it enhanced cue-related fear recall subsequently, potentially via altered activity of downstream regions. Enhanced fear memory consolidation could be replicated by selectively activating somatostatin (SOM), but not corticotropin-releasing factor (CRF), neurons of the BNST, which was accompanied by increased fear generalization. Our findings suggest the significant modulation of fear memory strength by specific circuits of the BNST.SIGNIFICANCE STATEMENT The bed nucleus of stria terminalis (BNST) mediates different defensive behaviors, and its connections implicate its integrative modulatory role in fear memory formation; however, the involvement of BNST in fear learning has yet to be elucidated in detail. Our data highlight that BNST stimulation enhances fear memory formation without direct effects on fear expression. Our study identified somatostatin (SOM) cells within the extended amygdala as specific neurons promoting fear memory formation. These data underline the importance of anxiety circuits in maladaptive fear memory formation, indicating elevated BNST activity as a potential vulnerability factor to anxiety and trauma-related disorders.     

Predictive Diagnostics for Escherichia coli Infections Based on the Clonal Association of Antimicrobial Resistance and Clinical Outcome

The ability to identify bacterial pathogens at the subspecies level in clinical diagnostics is currently limited. We investigated whether splitting Escherichia coli species into clonal groups (clonotypes) predicts antimicrobial susceptibility or clinical outcome. A total of 1,679 extraintestinal E. coli isolates (collected from 2010 to 2012) were collected from one German and 5 U.S. clinical microbiology laboratories. Clonotype identity was determined by fumC and fimH (CH) sequencing. The associations of clonotype with antimicrobial susceptibility and clinical variables were evaluated. CH typing divided the isolates into >200 CH clonotypes, with 93% of the isolates belonging to clonotypes with ≥2 isolates. Antimicrobial susceptibility varied substantially among clonotypes but was consistent across different locations. Clonotype-guided antimicrobial selection significantly reduced “drug-bug” mismatch compared to that which occurs with the use of conventional empirical therapy. With trimethoprim-sulfamethoxazole and fluoroquinolones, the drug-bug mismatch was predicted to decrease 62% and 78%, respectively. Recurrent or persistent urinary tract infection and clinical sepsis were significantly correlated with specific clonotypes, especially with CH40-30 (also known as H30), a recently described clonotype within sequence type 131 (ST131). We were able to clonotype directly from patient urine samples within 1 to 3 h of obtaining the specimen. In E. coli, subspecies-level identification by clonotyping can be used to significantly improve empirical predictions of antimicrobial susceptibility and clinical outcomes in a timely manner.     

Suppression of membrane microvesiculation — A possible anticoagulant and anti-tumor progression effect of heparin

Heparins (unfractionated and low molecular weight (LMWH) heparins) primarily used as anticoagulants, were found to be effective also in slowing down the development of some types of cancer. On the other hand, the number of microvesicles in the peripheral blood originating from the budding of cell membranes (mostly platelets) is increased in hypercoagulabile states as well as in cancer, indicating a possible common underlying mechanism. It was hypothesized that by mediating an attractive interaction between phospholipid membranes heparin suppresses microvesiculation and thereby acts as an anticoagulant and anti-tumor agent. In this work, the effect of LMWH nadroparin on phospholipid membranes was tested in vitro in a system of giant phospholipid vesicles (GPVs) created by electroformation and observed under the phase contrast microscope. Plasma of different blood donors containing different concentrations of nadroparin was added to the suspension of GPVs to induce adhesion between GPVs. The attractive interaction between membranes was assessed by measuring the average effective angle of contact between the adhered GPVs. It was found in healthy donors, in a donor with gastrointestinal cancer and in a donor with rheumatoid arthritis that adding therapeutic doses of nadroparin to the plasma samples enhanced adhesion of phospholipid membranes in a dose and time-dependent manner while nadroparin alone had no effect within the therapeutic concentration range. The results are in favor of the hypothesis that suppression of microvesiculation underlies both, the anticoagulant and the anti-tumor progression effect of heparin.     

The impact of preoperative micronutrient supplementation in lung surgery. A prospective randomized trial of oral supplementation of combined alpha-ketoglutaric acid and 5-hydroxymethylfurfural.

OBJECTIVE: Preoperative micronutrient supplementation in fast-track surgery programs have shown to reduce complications, shorten recovery, and thereby lower costs. In a prospective randomized study, the metabolic effects of a combination of alpha-ketoglutaric acid (alpha-KG) and 5-hydroxymethylfurfural (5-HMF) were evaluated concerning their impact on improvement of exercise capacity and reduction of oxidative stress in lung surgery. METHODS: Thirty-two consecutive patients admitted for lung resection due to NSCLC were randomized to the study protocol. All patients received preoperative nutritional guidelines according to general recommendations. In 16 (study group), a supplementation of 7.2g alpha-KG and 720 mg 5-HMF/day (SANOPAL) was administered from days 1 to 10. Spiroergometric evaluation was carried out at baseline and day 10 after micronutrient supplementation. Blood samples for the determination of oxidative stress, i.e. carbonyl proteins (CPs) and isoprostanes (IPs) were taken on at baseline, in the operating room just before resection treatment, and 25 min after single lung ventilation (SLV). RESULTS: Spiroergometric re-evaluation showed a significant increase of VO2max (p=0.0108) and Watt's (p=0.011) in favor of the study group. Determination of oxidative stress showed a significant reduction of CPs before (p=0.048) and after SLV (p=0.0001) for the study group compared to the control group. The same is true for IPs before (p=0.003) and after SLV (p=0.02). Hospitalization and intensive care unit (ICU) of the study group showed a significant reduction compared to the control group (p=0.03 and p=0.02, respectively). CONCLUSIONS: Simple oral supplementation using a combination of alpha-KG and 5-HMF of preoperative micronutrition may therefore be one further step in a multimodality approach of fast-track surgery programs also in lung surgery.     

Health is yearning--experiences of being conscious during ventilator treatment in a critical care unit.

The aim of this study was to investigate experiences of being conscious during ventilator treatment in the ICU from a patient perspective. Hermeneutic, phenomenological methods were used. Eight patients who had received ventilator treatment were interviewed. The time on a ventilator varied from 1 day to several months. Some patients had been more heavily sedated during the acute phase while some were only lightly sedated and others had no sedation at all. The motor activity assessment scale was used to rate the sedation level. The patients had been sufficiently conscious to communicate with the help of the alphabet board, by means of facial expression or by nodding or shaking their head. The results show that the experience of care by patients who were conscious during ventilator treatment was described under the headings of: memories, mastering the situation and individual consequences. Health in the ICU is associated with yearning. The patient undergoes different stages of yearning as part of his or her recovery process. The patient who is conscious during ventilator treatment views him/herself and his/her worth on the basis of the attitude and behaviour of the caregivers, where the value of caring consists of the holistic confirmation of individual suffering.     

Locally reduced levels of acidic FGF lead to decreased expression of 28‐kDa calbindin and contribute to the selective vulnerability of the neurons in the entorhinal cortex in Alzheimer's disease

Recent studies demonstrate that a disturbed calcium‐homeostasis leading to increased susceptibility to excitotoxic triggers plays a major role in the neurodegenerative process initiating in layer 2 of the entorhinal cortex (EC2) during Alzheimer's disease (AD). Thus, proteins binding free Ca ¹¹ (i.e. calbindin) and factors regulating these proteins are of great importance for the neuroprotective–neurotoxic balance in the affected brain regions. In the present combined human and in vitro study evidence is provided that altered levels of the acidic fibroblast growth factor (aFGF) and calbindin expression are concomitantly present in EC2 neurons and have interactive effects. A dramatic loss of aFGF‐ and calbindin‐labeled EC2 neurons was found. Further analysis of the surviving EC2 neurons revealed a strong immunoreactivity to calbindin and aFGF. In vitro experiments show that aFGF regulates calbindin expression, because treatment of differentiating neurons with recombinant aFGF increases calbindin expression in a time‐dependent fashion. The data imply that a reduced expression of aFGF in EC2 neurons of AD brains leads to lower levels of calbindin resulting in decreased neuroprotective capacity.     

Activity measurements with radionuclide calibrators in the Czech Republic.

Since radionuclide calibrators are used in nuclear medicine for the determination of the activity of radiopharmaceuticals administered to patients, it is important to ensure their long-term accuracy. Results of the checks performed in the Czech Republic during the year 2002 are given, together with a short overview of previous years. For the CMI 4pigamma ionization chamber, against which all the calibrators are checked, we calculated the response function and basic characteristics by a Monte Carlo method, using the MCNP4C code.