Difficult catheter access to target the carotid is common during acute endovascular treatment of stroke patients and is associated with a worse clinical outcome. If transfemoral access appears difficult, alternative access such as direct carotid puncture could be explored.

Escherichia coli represents a major cause of morbidity and mortality worldwide. The treatment of E. coli infections is now threatened by the emergence of antimicrobial resistance. The dissemination of resistance is associated with genetic mobile elements, such as plasmids, that may also carry virulence determinants. A proficient pathogen should be virulent, resistant to antibiotics, and epidemic. However, the interplay between resistance and virulence is poorly understood. This review aims to critically discuss the association and linked transmission of both resistance and virulence traits in strains from extraintestinal infections in E. coli, and intestinal pathotypes. Despite the numerous controversies on this topic, findings from research published to date indicate that there is a link between resistance and virulence, as illustrated by the successful E. coli ST131 epidemic clone. Perhaps the most commonly accepted view is that resistance to quinolones is linked to a loss of virulence factors. However, the low virulent phylogenetic groups might be more prone to acquire resistance to quinolones. Specific characteristics of the E. coli genome that have yet to be identified may contribute to such genetic linkages. Research based on bacterial populations is sorely needed to help understand the molecular mechanisms underlying the association between resistance and virulence, that, in turn, may help manage the future disseminations of infectious diseases in their entirety.

Of the 181 unduplicated Escherichia coli strains isolated in nine different hospitals in three Portuguese regions, 119 were extended-spectrum ␤-lactamase (ESBL)-CTX-M producers and were selected for phenotype and genotype characterization. CTX-M producer strains were prevalent among community-acquired infections (56%), urinary tract infections (76%), and patients >60 years old (76%). In MIC tests, all strains were resistant to cefotaxime, 92% were resistant to ceftazidime, 93% were resistant to quinolones, 89% were resistant to aminoglycoside, and 26% were resistant to trimethoprim-sulfamethoxazole; all strains were sensitive to carbapenems, and 92% of the strains had a multidrug resistance phenotype. Molecular methods identified 109 isolates harboring a bla CTX-M-15 gene, 1 harboring the bla CTX-M-32 gene (first identification in the country), and 9 harboring the bla CTX-M-14 gene. All isolates presented the ISEcp1 element upstream from the bla CTX-M genes; one presented the IS903 element (downstream of bla CTX-M-14 gene), and none had the IS26 element; 85% carried bla TEM-1B , and 84% also carried a bla OXA-30 . Genetic relatedness analysis based on pulsed-field gel electrophoresis defined five clusters and indicated that 76% of all isolates (from cluster IV) corresponded to a single epidemic strain. Of the 47 strains from one hospital, 41 belonged to cluster IV and were disseminated in three main wards. CTX-M-producing E. coli strains are currently a problem in Portugal, with CTX-M-15 particularly common. This study suggests that the horizontal transfer of bla CTX-M genes, mediated by plasmids and/or mobile elements, contributes to the dissemination of CTX-M enzymes to community and hospital environments. The use of extended-spectrum cephalosporins, quinolones, and aminoglycosides is compromised, leaving carbapenems as the therapeutic option for severe infections caused by ESBL producers.

Machado Joseph Disease (MJD) is the most frequent autosomal dominantly inherited cerebellar ataxia caused by the over-repetition of a CAG trinucleotide in the ATXN3 gene. This expansion translates into a polyglutamine tract within the ataxin-3 protein that confers a toxic gain-of-function to the mutant protein ataxin-3, contributing to protein misfolding and intracellular accumulation of aggregates and neuronal degeneration. Autophagy impairment has been shown to be one of the mechanisms that contribute for the MJD phenotype. Here we investigated whether this phenotype was present in patient-derived fibroblasts, a common somatic cell type used in the derivation of induced pluripotent stem cells and subsequent differentiation into neurons, for in vitro disease modeling. We generated and studied adult dermal fibroblasts from 5 MJD patients and 4 healthy individuals and we found that early passage MJD fibroblasts exhibited autophagy impairment with an underlying mechanism of decreased autophagosome production. The overexpression of beclin-1 on MJD fibroblasts reverted partially autophagy impairment by increasing the autophagic flux but failed to increase the levels of autophagosome production. Overall, our results provide a well-characterized MJD fibroblast resource for neurodegenerative disease research and contribute for the understanding of mutant ataxin-3 biology and its molecular consequences.

The new ␤-lactamase SHV-72 was isolated from clinical Klebsiella pneumoniae INSRA1229, which exhibited the unusual association of resistance to the amoxicillin-clavulanic acid combination (MIC, 64 g/ml) and susceptibility to cephalosporins, aztreonam, and imipenem. SHV-72 (pI 7.6) harbored the three amino acid substitutions Ile8Phe, Ala146Val, and Lys234Arg. SHV-72 had high catalytic efficiency against penicillins (k cat /K m , 35 to 287 M ؊1 ⅐ s ؊1 ) and no activity against oxyimino ␤-lactams. The concentration of clavulanic acid necessary to inhibit the enzyme activity by 50% was 10-fold higher for SHV-72 than for SHV-1. Moleculardynamics simulation suggested that the Lys234Arg substitution in SHV-72 stabilized an atypical conformation of the Ser130 side chain, which moved the O␥ atom of Ser130 around 3.5 Å away from the key O␥ atom of the reactive serine (Ser70). This movement may therefore decrease the susceptibility to clavulanic acid by preventing cross-linking between Ser130 and Ser70.The most common resistance mechanism in bacteria against ␤-lactam antibiotics is the production of ␤-lactamases (EC 3.5.2.6), which hydrolyze and inactivate ␤-lactams. ␤-Lactamases are divided into four major classes (A to D) on the basis of their primary sequence (1). While class B is composed of metalloenzymes that necessitate the presence of zinc cations for activity, classes A, C, and D are serine hydrolases (1, 7). Class A enzymes comprise several enzyme families, including the clinically relevant enzymes TEM and SHV (26).TEM and SHV enzymes initially had preferential activity against penicillins, as in the case of enzymes SHV-1 and TEM-1. Oxyimino ␤-lactams that are resistant to their hydrolytic activity and ␤-lactam inhibitors, such as clavulanic acid and tazobactam, have been developed to get around the activities of these enzymes (6,9,26). Nevertheless, the presence of point mutations in TEM and SHV enzymes has expanded the substrate spectrum to include oxyimino ␤-lactams and/or has conferred resistance to the inhibitors (3, 6, 9, 26).More than 28 inhibitor-resistant TEM enzymes have been detected. They harbor amino acid substitutions at positions 69, 130, 165, 182, 244, 275, and/or 276 that confer resistance to inhibitors (9, 15). Only three natural inhibitor-resistant SHVs (IRS) have been reported (http://www.lahey.org/studies). It has been proposed that substitutions at positions 69, 130, and 187 are involved in their resistance to inhibitors (10,14,31). IRS enzymes have also been constructed in vitro by site saturation mutagenesis at position 244 (37).In this study, we performed a phenotypic, molecular, and biochemical characterization of the new IRS-type ␤-lactamase SHV-72 from a clinical K. pneumoniae strain and investigated by molecular-dynamics simulations (MDSs) the role of the Lys234Arg substitution in its resistance to clavulanic acid. MATERIALS AND METHODSBacterial strains and plasmid. Klebsiella pneumoniae INSRA1229 was isolated from sputum of an 80-year-old male in an internal medicine service of a ge...