Whole-Genome Sequencing and Molecular Analysis of Ceftazidime-Avibactam-Resistant KPC-Producing Klebsiella pneumoniae from Intestinal Colonization in Elderly Patients.

Ceftazidime-avibactam (CAZ-AVI) is an active antibiotic combination of a ß-lactam-ß-lactamase inhibitor against carbapenemase-producing Enterobacterales. Reports of resistance to CAZ-AVI other than metallo-ß-lactamases have increased in recent years. The aim of this study was to analyze KPC-Klebsiella pneumoniae (KP) isolates resistant to CAZ-AVI from the intestinal carriage of hospitalized elderly patients in Italy, in February 2018-January 2020. Characterization of CAZ-AVI-resistant KP isolates, including MLST, resistome, virulome and plasmid content, was performed by WGS analysis. Out of six CAZ-AVI-resistant KP isolates, three belonged to ST101 and three to ST512; two isolates produced KPC-3 (both ST512), four had mutated KPC-3 (KPC-31, in ST101 and ST512, and KPC-46, both ST101). All CAZ-AVI-resistant KP isolates were multidrug-resistant and carried several resistance genes. The yersiniabactin ybt9 gene cluster was present in all ST101 isolates, while, in ST512 isolates, no virulence genes were detected. Several plasmids were detected: IncF was present in all isolates, as well as IncR and Col440 in ST101 and IncX3 in ST512 isolates. In conclusion, it is important to monitor the circulation of K. pneumoniae resistant to CAZ-AVI to prevent the spread of clones causing difficult-to-treat infections. The presence of mutated KPC-3 in high-risk K. pneumoniae clones resistant to CAZ-AVI in hospitalized patients deserves attention.

Illumination and gaze effects on face evaluation: The Bi-AGI database.

Face evaluation and first impression generation can be affected by multiple face elements such as invariant facial features, gaze direction and environmental context; however, the composite modulation of eye gaze and illumination on faces of different gender and ages has not been previously investigated. We aimed at testing how these different facial and contextual features affect ratings of social attributes. Thus, we created and validated the Bi-AGI Database, a freely available new set of male and female face stimuli varying in age across lifespan from 18 to 87 years, gaze direction and illumination conditions. Judgments on attractiveness, femininity-masculinity, dominance and trustworthiness were collected for each stimulus. Results evidence the interaction of the different variables in modulating social trait attribution, in particular illumination differently affects ratings across age, gaze and gender, with less impact on older adults and greater effect on young faces.

Whole Genome Sequencing and Molecular Analysis of Carbapenemase-Producing Escherichia coli from Intestinal Carriage in Elderly Inpatients.

The spread of carbapenemase-producing (CP) Enterobacterales is currently a worldwide concern, especially in the elderly. Twelve CP-E. coli isolated from rectal swabs of colonized inpatients aged ≥65 years from four hospitals in two Italian cities (Milan and Rome) were analyzed by whole genome sequencing (WGS) to obtain multi-locus sequence typing (MLST), identification of carbapenemase-encoding genes, resistome, plasmid content, and virulence genes. MLST analysis showed the presence of 10 unrelated lineages: ST410 (three isolates from three different hospitals in two cities) and ST12, ST38, ST69, ST95, ST131, ST189, ST648, ST1288, and ST1598 (one isolate each). Most isolates (9/12, 75%) contained a serine-ß-lactamase gene (5 blaKPC-3, 2 blaKPC-2, and 2 blaOXA-181), while three isolates harbored a metallo-ß-lactamase gene (two blaNDM-5 and one blaVIM-1). In most CP-E. coli, the presence of more than one plasmid was observed, with the predominance of IncF. Several virulence genes were detected. All isolates contained genes enhancing the bacterial fitness, such as gad and terC, and all isolates but one, fimH, encoding type 1 fimbriae. In conclusion, CP-E. coli clones colonizing elderly patients showed heterogeneous genetic backgrounds. We recommend strict surveillance to monitor and prevent the spread of successful, high-risk clones in healthcare settings.

Dynamics of carbapenemase-producing Enterobacterales intestinal colonisation in the elderly population after hospital discharge, Italy, 2018-2020.

Carbapenemase-producing Enterobacterales (CPE) represent a serious threat to public health worldwide. Elderly patients are at increased risk of colonisation/infection with CPE. This study aimed to evaluate the persistence of CPE colonisation and the genotypic characteristics of persistent strains in elderly people discharged from Italian hospitals. A longitudinal study was conducted in two Italian cities (March 2018 to September 2020) enrolling 137 patients aged ≥65 years with CPE intestinal colonisation at hospital discharge. CPE colonisation was evaluated after 4, 8 and 12 months. Competing risk analysis was used to explore the association between baseline characteristics and persistence at 4 months. For all isolates, carbapenemase typing and multilocus sequence typing were performed. Persistent isolates underwent whole-genome sequencing. Of 137 patients, 91% carried carbapenemase-producing Klebsiella pneumoniae (CP-KP) and 8.8% carried carbapenemase-producing Escherichia coli. Although a large number of patients were lost to follow-up owing to death or withdrawal, 28/65 patients (43.1%) remained colonised at Month 4; 16/42 (38.1%) and 5/28 (17.9%) were found colonised up to Months 8 and 12, respectively. Colonisation persistence was more frequent in patients with bacteraemia or complicated urinary tract infection while in hospital and in those staying in long-term care facilities (LTCFs). Clonal characteristics of CP-KP isolates did not appear to influence persistence. Isolates obtained from each persistent carrier were identical or highly related by SNP phylogenetic analysis. Identification of patients at higher risk of persistent intestinal carriage after hospital discharge can prompt control measures to limit the transmission of CPE in the community, especially in LTCF settings.

Differentiation of human telencephalic progenitor cells into MSNs by inducible expression of Gsx2 and Ebf1.

Medium spiny neurons (MSNs) are a key population in the basal ganglia network, and their degeneration causes a severe neurodegenerative disorder, Huntington's disease. Understanding how ventral neuroepithelial progenitors differentiate into MSNs is critical for regenerative medicine to develop specific differentiation protocols using human pluripotent stem cells. Studies performed in murine models have identified some transcriptional determinants, including GS Homeobox 2 (Gsx2) and Early B-cell factor 1 (Ebf1). Here, we have generated human embryonic stem (hES) cell lines inducible for these transcription factors, with the aims of (i) studying their biological role in human neural progenitors and (ii) incorporating TF conditional expression in a developmental-based protocol for generating MSNs from hES cells. Using this approach, we found that Gsx2 delays cell-cycle exit and reduces Pax6 expression, whereas Ebf1 promotes neuronal differentiation. Moreover, we found that Gsx2 and Ebf1 combined overexpression in hES cells achieves high yields of MSNs, expressing Darpp32 and Ctip2, in vitro as well in vivo after transplantation. We show that hES-derived striatal progenitors can be transplanted in animal models and can differentiate and integrate into the host, extending fibers over a long distance.

Molecular and functional definition of the developing human striatum.

The complexity of the human brain derives from the intricate interplay of molecular instructions during development. Here we systematically investigated gene expression changes in the prenatal human striatum and cerebral cortex during development from post-conception weeks 2 to 20. We identified tissue-specific gene coexpression networks, differentially expressed genes and a minimal set of bimodal genes, including those encoding transcription factors, that distinguished striatal from neocortical identities. Unexpected differences from mouse striatal development were discovered. We monitored 36 determinants at the protein level, revealing regional domains of expression and their refinement, during striatal development. We electrophysiologically profiled human striatal neurons differentiated in vitro and determined their refined molecular and functional properties. These results provide a resource and opportunity to gain global understanding of how transcriptional and functional processes converge to specify human striatal and neocortical neurons during development.

Differences in visceral fat and fat bacterial colonization between ulcerative colitis and Crohn's disease. An in vivo and in vitro study.

Crohn's disease (CD) is notably characterized by the expansion of visceral fat with small adipocytes expressing a high proportion of anti-inflammatory genes. Conversely, visceral fat depots in ulcerative colitis (UC) patients have never been characterized. Our study aims were a) to compare adipocyte morphology and gene expression profile and bacterial translocation in omental (OM) and mesenteric (MES) adipose tissue of patients with UC and CD, and b) to investigate the effect of bacterial infection on adipocyte proliferation in vitro. Specimens of OM and MES were collected from 11 UC and 11 CD patients, processed and examined by light microscopy. Gene expression profiles were evaluated in adipocytes isolated from visceral adipose tissue using microarray and RTqPCR validations. Bacteria within adipose tissue were immuno-detected by confocal scanning laser microscopy. Adipocytes were incubated with Enterococcus faecalis and cells counted after 24 h. Morphology and molecular profile of OM and MES revealed that UC adipose tissue is less inflamed than CD adipose tissue. Genes linked to inflammation, bacterial response, chemotaxis and angiogenesis were down-regulated in adipocytes from UC compared to CD, whereas genes related to metallothioneins, apoptosis pathways and growth factor binding were up-regulated. A dense perinuclear positivity for Enterococcus faecalis was detected in visceral adipocytes from CD, whereas positivity was weak in UC. In vitro bacterial infection was associated with a five-fold increase in the proliferation rate of OM preadipocytes. Compared to UC, visceral adipose tissue from CD is more inflamed and more colonized by intestinal bacteria, which increase adipocyte proliferation. The influence of bacteria stored within adipocytes on the clinical course of IBD warrants further investigations.

Granule cell ascending axon excitatory synapses onto Golgi cells implement a potent feedback circuit in the cerebellar granular layer.

The function of inhibitory interneurons within brain microcircuits depends critically on the nature and properties of their excitatory synaptic drive. Golgi cells (GoCs) of the cerebellum inhibit cerebellar granule cells (GrCs) and are driven both by feedforward mossy fiber (mf) and feedback GrC excitation. Here, we have characterized GrC inputs to GoCs in rats and mice. We show that, during sustained mf discharge, synapses from local GrCs contribute equivalent charge to GoCs as mf synapses, arguing for the importance of the feedback inhibition. Previous studies predicted that GrC-GoC synapses occur predominantly between parallel fibers (pfs) and apical GoC dendrites in the molecular layer (ML). By combining EM and Ca(2+) imaging, we now demonstrate the presence of functional synaptic contacts between ascending axons (aa) of GrCs and basolateral dendrites of GoCs in the granular layer (GL). Immunohistochemical quantification estimates these contacts to be ∼400 per GoC. Using Ca(2+) imaging to identify synaptic inputs, we show that EPSCs from aa and mf contacts in basolateral dendrites display similarly fast kinetics, whereas pf inputs in the ML exhibit markedly slower kinetics as they undergo strong filtering by apical dendrites. We estimate that approximately half of the local GrC contacts generate fast EPSCs, indicating their basolateral location in the GL. We conclude that GrCs, through their aa contacts onto proximal GoC dendrites, define a powerful feedback inhibitory circuit in the GL.

Neural stem cells engrafted in the adult brain fuse with endogenous neurons.

Neural stem cells (NSCs) have become promising tools for basic research and regenerative medicine. Intracerebral transplantation studies have suggested that these cells may be able to adopt neuronal phenotypes typical of their engraftment site and to establish appropriate connections in the recipient circuitries. Here, we examined the in vivo neurogenic competence of well-characterized NSC lines subjected to in vitro priming and subsequent implantation into the adult intact mouse brain. Upon implantation into the hippocampus and, less frequently, in the striatum and in the cerebral cortex, numerous green fluorescent protein (GFP)-tagged cells acquired differentiated features indistinguishable from resident neurons. Upon closer examination, however, we found that this outcome resulted from fusion of donor cells with local neuronal elements generating long-term persistent GFP(+) neuronal hybrids. This fusogenic behavior of NSCs was unexpected and also observed in coculture with E18 hippocampal immature neural cells, but not with microglia or astrocytes. Similar findings were consistently obtained with different NSC lines, mouse recipients, and donor cell-labeling methods. The frequent and cell type-specific fusion of donor NSCs with host neurons highlights a previously underestimated biological property of the nervous tissue that might prove profitable for basic and therapeutically oriented studies.

Computational reconstruction of pacemaking and intrinsic electroresponsiveness in cerebellar Golgi cells.

The Golgi cells have been recently shown to beat regularly in vitro (Forti et al., 2006. J. Physiol. 574, 711-729). Four main currents were shown to be involved, namely a persistent sodium current (I(Na-p)), an h current (I(h)), an SK-type calcium-dependent potassium current (I(K-AHP)), and a slow M-like potassium current (I(K-slow)). These ionic currents could take part, together with others, also to different aspects of neuronal excitability like responses to depolarizing and hyperpolarizing current injection. However, the ionic mechanisms and their interactions remained largely hypothetical. In this work, we have investigated the mechanisms of Golgi cell excitability by developing a computational model. The model predicts that pacemaking is sustained by subthreshold oscillations tightly coupled to spikes. I(Na-p) and I(K-slow) emerged as the critical determinants of oscillations. I(h) also played a role by setting the oscillatory mechanism into the appropriate membrane potential range. I(K-AHP), though taking part to the oscillation, appeared primarily involved in regulating the ISI following spikes. The combination with other currents, in particular a resurgent sodium current (I(Na-r)) and an A-current (I(K-A)), allowed a precise regulation of response frequency and delay. These results provide a coherent reconstruction of the ionic mechanisms determining Golgi cell intrinsic electroresponsiveness and suggests important implications for cerebellar signal processing, which will be fully developed in a companion paper (Solinas et al., 2008. Front. Neurosci. 2:4).

Fast-reset of pacemaking and theta-frequency resonance patterns in cerebellar golgi cells: simulations of their impact in vivo.

The Golgi cells are inhibitory interneurons of the cerebellar granular layer, which respond to afferent stimulation in vivo with a burst-pause sequence interrupting their irregular background low-frequency firing (Vos et al., 1999a. Eur. J. Neurosci. 11, 2621-2634). However, Golgi cells in vitro are regular pacemakers (Forti et al., 2006. J. Physiol. 574, 711-729), raising the question how their ionic mechanisms could impact on responses during physiological activity. Using patch-clamp recordings in cerebellar slices we show that the pacemaker cycle can be suddenly reset by spikes, making the cell highly sensitive to input variations. Moreover, the neuron resonates around the pacemaker frequency, making it specifically sensitive to patterned stimulation in the theta-frequency band. Computational analysis based on a model developed to reproduce Golgi cell pacemaking (Solinas et al., 2008Front. Neurosci., 2:2) predicted that phase-reset required spike-triggered activation of SK channels and that resonance was sustained by a slow voltage-dependent potassium current and amplified by a persistent sodium current. Adding balanced synaptic noise to mimic the irregular discharge observed in vivo, we found that pacemaking converts into spontaneous irregular discharge, that phase-reset plays an important role in generating the burst-pause pattern evoked by sensory stimulation, and that repetitive stimulation at theta-frequency enhances the time-precision of spike coding in the burst. These results suggest that Golgi cell intrinsic properties exert a profound impact on time-dependent signal processing in the cerebellar granular layer.

Ionic mechanisms of autorhythmic firing in rat cerebellar Golgi cells.

Although Golgi cells (GoCs), the main type of inhibitory interneuron in the cerebellar granular layer (GL), are thought to play a central role in cerebellar network function, their excitable properties have remained unexplored. GoCs fire rhythmically in vivo and in slices, but it was unclear whether this activity originated from pacemaker ionic mechanisms. We explored this issue in acute cerebellar slices from 3-week-old rats by combining loose cell-attached (LCA) and whole-cell (WC) recordings. GoCs displayed spontaneous firing at 1-10 Hz (room temperature) and 2-20 Hz (35-37 degrees C), which persisted in the presence of blockers of fast synaptic receptors and mGluR and GABAB receptors, thus behaving, in our conditions, as pacemaker neurons. ZD 7288 (20 microM), a potent hyperpolarization-activated current (Ih) blocker, slowed down pacemaker frequency. The role of subthreshold Na+ currents (INa,sub) could not be tested directly, but we observed a robust TTX-sensitive, non-inactivating Na+ current in the subthreshold voltage range. When studying repolarizing currents, we found that retigabine (5 microM), an activator of KCNQ K+ channels generating neuronal M-type K+ (IM) currents, reduced GoC excitability in the threshold region. The KCNQ channel antagonist XE991 (5 microM) did not modify firing, suggesting that GoC IM has low XE991 sensitivity. Spike repolarization was followed by an after-hyperpolarization (AHP) supported by apamin-sensitive Ca2+-dependent K+ currents (I(apa)). Block of I(apa) decreased pacemaker precision without altering average frequency. We propose that feed-forward depolarization is sustained by Ih and INa,sub, and that delayed repolarizing feedback involves an IM-like current whose properties remain to be characterized. The multiple ionic mechanisms shown here to contribute to GoC pacemaking should provide the substrate for fine regulation of firing frequency and precision, thus influencing the cyclic inhibition exerted by GoCs onto the cerebellar GL.