Characterization of brain transduction capability of a BBB-penetrant AAV vector in mice, rats and macaques reveals differences in expression profiles.

Different screening methods are being developed to generate adeno-associated viral vectors (AAV) with the ability to bypass the blood-brain barrier (BBB) upon intravenous administration. Recently, the AAV9P31 stood out as the most efficient version among a library of peptide-displaying capsids selected in C57BL/6 mice using RNA-driven biopanning. In this work we have characterized in detail its biodistribution in different mouse strains (C57BL/6 and Balb/c), as well as in Sprague Dawley rats and non-human primates (Macaca fascicularis). Using GFP and NanoLuc reporter genes, we confirmed homogeneous infection and transgene expression across the CNS of mice injected intravenously with AAV9P31. A more restricted pattern was observed upon either intracerebroventricular or intraparenchymal injection. Following intravenous delivery, region- and cell-specific differential patterns of transduction were observed in the mouse brain, including a preferential transduction of astrocytes and neurons in the cerebral cortex and striatum, whereas neurons were the only transduced cell type in subcortical locations across the hippocampus, thalamus, hypothalamus, mesencephalon, brainstem and cerebellum. Furthermore, transduced microglial cells were never found in any CNS location. Peripheral organs transduced upon intravenous administration included lung, liver, peritoneum, heart and skeletal muscle. However, a comparable performance of AAV9P31 to bypass the BBB in rats and macaques was not observed, although a more limited neuronal transduction was found in the brainstem of rats upon intravenous delivery. Finally, intracerebroventricular delivery in macaques resulted in neuronal transduction in cortical, subcortical structures and cerebellum following a patchy pattern. In conclusion, the widespread CNS transduction obtained in mice upon intravenous delivery of AAV9P31 represents a powerful tool for modeling a wide variety of neurological disorders as well as an appealing choice for the evaluation of gene therapy-based therapeutics.

The FICI paradigm: Correcting flaws in antimicrobial in vitro synergy screens at their inception.

Antibiotics have become the corner stone of modern medicine. However, our society is currently facing one of the greatest challenges of its time: the emergence of antimicrobial resistance. It is estimated that if no new therapies are implemented by 2050, 10 million people will die worldwide every year as a result of infections caused by bacteria resistant to current antibiotics; new antimicrobials are thus urgently needed. However, drug development is a tedious and very costly endeavor of hundreds of millions that can take up to 15-20 years from the bench discovery to the bedside. Under this scenario, drug repurposing, which consists in identifying new uses for old, clinically approved drugs, has gathered momentum within the pharmaceutical industry. Because most of these drugs have safety and toxicity information packages available, clinical evaluation could be done in a much shorter period than standard timelines. Synergistic combinations of these clinically approved drugs could also be a promising approach to identify novel antimicrobial therapies that might provide rational choices of available drugs to shorten treatment, increase efficacy, reduce toxicity, prevent resistance and treat infections caused by drug-resistant strains. However, although simple in its conception, translating results from in vitro synergy screens into in vivo efficacy or the clinical practice has proven to be a paramount challenge. In this Commentary, we will discuss common flaws at the inception of synergy research programs, with a special focus on the use of the Fractional Inhibitory Concentration Index (FICI), and evaluate potential interventions that can be made at different developmental pre-clinical stages in order to improve the odds of translation from in vitro studies.

Detection of carbapenemases and other mechanisms of enzymatic resistance to Beta-lactams in Enterobacteriaceae with diminished susceptibility to carbapenems in a tertiary care hospital / Detección de carbapenemasas y otros mecanismos de resistencia enzimáticos a Beta-lactámicos en enterobacterias con sensibilidad disminuida a carbapenems en un hospital de tercer nivel

INTRODUCTION: Our objective was to characterize the enzymatic Beta-lactam resistance in clinical Enterobacteriaceae isolates with diminished susceptibility to carbapenems from 2013 to 2014 at Hospital Universitario Miguel Servet. Material/methods: A total of 63 clinical isolates were analyzed for the presence of carbapenemases (KPC, OXA-48 and MBL), ESBLs and AmpC enzymes by combined disk methods and PCR detection of carbapenemase-encoding and beta-lactamase-encoding genes. RESULTS: Fifteen isolates had a phenotypic test compatible with carbapenemase production; two of these were confirmed by PCR as OXA-48 producers. ESBL detection was positive in 27 isolates (43%); plasmid-mediated AmpC was detected in nine isolates (14.2%) and derepressed AmpC β-lactamase was present in 18 isolates (28%). CONCLUSION: During the study period, the decreased susceptibility to carbapenems in Enterobacteriaceae in our area was not due to true carbapenemases but rather to Beta-lactamase activity (82.5% were ESBL or AmpC producers), probably in combination with decreased permeability of the outer membrane

INTRODUCCIÓN: Nuestro objetivo fue caracterizar la resistencia enzimática a Beta-lactámicos en aislados clínicos de Enterobacteriaceae con sensibilidad disminuida a carbapenems desde 2013 a 2014 en el Hospital Universitario Miguel Servet. Material/métodos: Se analizaron un total de 63 aislados clínicos para presencia de carbapenemasas (KPC, OXA-48 y MBL), BLEE y AmpC por método de discos combinados y detección de genes codificantes de carbapenemasas y betalactamasas por PCR. RESULTADOS: Quince aislados tuvieron un test fenotípico compatible con producción de carbapenemasas; dos de ellos confirmados por PCR como productores de OXA-48. La detección BLEE fue positiva en 27 aislados (43%); se detectó AmpC plasmídica en 9 aislados (14,2%) y se observó β-lactamasa AmpC desreprimida en 18 aislados (28%). CONCLUSIÓN: Durante el período de estudio, la sensibilidad disminuida a carbapenems en Enterobacteriaceae en nuestra área no se debió a verdaderas carbapenemasas sino a actividad Beta-lactamasa (82,5% eran productores de BLEE o AmpC) probablemente en combinación con permeabilidad disminuida de la membrana externa

Detection of carbapenemases and other mechanisms of enzymatic resistance to ß-lactams in Enterobacteriaceae with diminished susceptibility to carbapenems in a tertiary care hospital.

INTRODUCTION: Our objective was to characterize the enzymatic ß-lactam resistance in clinical Enterobacteriaceae isolates with diminished susceptibility to carbapenems from 2013 to 2014 at Hospital Universitario Miguel Servet. MATERIAL/METHODS: A total of 63 clinical isolates were analyzed for the presence of carbapenemases (KPC, OXA-48 and MBL), ESBLs and AmpC enzymes by combined disk methods and PCR detection of carbapenemase-encoding and beta-lactamase-encoding genes. RESULTS: Fifteen isolates had a phenotypic test compatible with carbapenemase production; two of these were confirmed by PCR as OXA-48 producers. ESBL detection was positive in 27 isolates (43%); plasmid-mediated AmpC was detected in nine isolates (14.2%) and derepressed AmpC ß-lactamase was present in 18 isolates (28%). CONCLUSION: During the study period, the decreased susceptibility to carbapenems in Enterobacteriaceae in our area was not due to true carbapenemases but rather to ß-lactamase activity (82.5% were ESBL or AmpC producers), probably in combination with decreased permeability of the outer membrane.

Towards the early detection of ß-lactamase-producing Enterobacteriaceae by MALDI-TOF MS analysis.

Objectives: Development of a novel MALDI-TOF MS-based method for the rapid detection of ESBL-producing Enterobacteriaceae. Methods: The method was evaluated in terms of sensitivity, specificity and turnaround time regarding the antibiotic used (cefotaxime, ceftazidime, ceftriaxone, cefpodoxime or cefepime) and the performance of the automated MBT STAR-BL software (Bruker Daltonik GmbH, Germany) relative to qualitative interpretation of spectra for detecting ß-lactamase resistance by MALDI-TOF MS (Bruker Daltonik) in a collection of 11 isogenic Escherichia coli control strains expressing different ß-lactamases. Finally, for clinical validation, ß-lactamase activity was determined under previously evaluated conditions in 100 clinical isolates previously characterized by PCR and sequencing. Results: Clinical validation of the assay showed 100% sensitivity and specificity for detecting ß-lactam resistance in 30 min by measuring hydrolysis of ceftriaxone (0.50 mg/mL) with the automated MBT STAR-BL software. Regarding the antibiotics evaluated, ceftriaxone yielded 70% more positive results than cefotaxime, 80% more than ceftazidime and 20% more than cefpodoxime, with 100% specificity. Cefepime revealed 100% sensitivity, but only 27% specificity. For the same incubation time, the automated software yielded on average 41% more positive results in relation to detecting resistance than qualitative interpretation of spectra. Conclusions: Our clinical validation of the method proved it to be highly reliable, simple to perform and time saving, transforming ß-lactam resistance detection by MALDI-TOF MS into a ready-to-use technique in clinical laboratories.