A rapid and simple method for constructing stable mutants of Acinetobacter baumannii

<p>Abstract</p> <p>Background</p> <p><it>Acinetobacter baumannii </it>is a multidrug-resistant bacterium responsible for nosocomial infections in hospitals worldwide. Study of mutant phenotypes is fundamental for understanding gene function. The methodologies developed to inactivate <it>A. baumannii </it>genes are complicated and time-consuming; sometimes result in unstable mutants, and do not enable construction of double (or more) gene knockout mutant strains of <it>A. baumannii</it>.</p> <p>Results</p> <p>We describe here a rapid and simple method of obtaining <it>A. baumannii </it>mutants by gene replacement via double crossover recombination, by use of a PCR product that carries an antibiotic resistance cassette flanked by regions homologous to the target locus. To demonstrate the reproducibility of the approach, we produced mutants of three different chromosomal genes (<it>omp33</it>, <it>oxyR</it>, and <it>soxR</it>) by this method. In addition, we disrupted one of these genes (<it>omp33</it>) by integration of a plasmid into the chromosome by single crossover recombination, the most widely used method of obtaining <it>A. baumannii </it>mutants. Comparison of the different techniques revealed absolute stability when the gene was replaced by a double recombination event, whereas up to 40% of the population reverted to wild-type when the plasmid was disrupting the target gene after 10 passages in broth without selective pressure. Moreover, we demonstrate that the combination of both gene disruption and gene replacement techniques is an easy and useful procedure for obtaining double gene knockout mutants in <it>A. baumannii</it>.</p> <p>Conclusions</p> <p>This study provides a rapid and simple method of obtaining stable mutants of <it>A. baumannii </it>free of foreign plasmidic DNA, which does not require cloning steps, and enables construction of multiple gene knockout mutants.</p

Optimization of transgenesis conditions for the generation of CXCL2-luciferase reporter mice line

Background: Transgenesis by microinjection has been widely used for the generation of different mouse models. Different variables of the procedure may critically affect the efficiency of the process. A DNA construction that carries the CXCL2 promoter gene and firefly luciferase has been used to optimize aspects of the procedure. Three different concentrations (0.5, 1.0 and 4.0 ng/μl) of the DNA construction to microinject a total of 1981 zygotes has been tested. Intact/injected embryos, pregnancy and birth rate, survival of pups 7 days after birth, number of transgenic pups and overall transgenic efficiency was registered and analyzed by Z test of proportions for each group. Results: A total of seven transgenic founders were detected for the three DNA concentrations used, 1 in 46 alive pups in the 0.5 ng/μl group, 5 in 38 alive pups in the 1 ng/μl group and 1 in 21 alive pups in the 4 ng/μl group (p < 0.1). The overall transgenic efficiency was higher for the 1 ng/μl concentration, with a transgenic rate of 13.2%. Conclusions: In conclusion, we have selected the best operative conditions to maximize the transgenesis efficiency. Furthermore, the transgenic lines developed could be used as a reporter model of innate immunity activation with many different applications in the fields of immunology, cancer and neurodegenerative diseases.Facultad de Ciencias Exacta

Optimization of transgenesis conditions for the generation of CXCL2-luciferase reporter mice line

Background: Transgenesis by microinjection has been widely used for the generation of different mouse models. Different variables of the procedure may critically affect the efficiency of the process. A DNA construction that carries the CXCL2 promoter gene and firefly luciferase has been used to optimize aspects of the procedure. Three different concentrations (0.5, 1.0 and 4.0 ng/μl) of the DNA construction to microinject a total of 1981 zygotes has been tested. Intact/injected embryos, pregnancy and birth rate, survival of pups 7 days after birth, number of transgenic pups and overall transgenic efficiency was registered and analyzed by Z test of proportions for each group. Results: A total of seven transgenic founders were detected for the three DNA concentrations used, 1 in 46 alive pups in the 0.5 ng/μl group, 5 in 38 alive pups in the 1 ng/μl group and 1 in 21 alive pups in the 4 ng/μl group (p < 0.1). The overall transgenic efficiency was higher for the 1 ng/μl concentration, with a transgenic rate of 13.2%. Conclusions: In conclusion, we have selected the best operative conditions to maximize the transgenesis efficiency. Furthermore, the transgenic lines developed could be used as a reporter model of innate immunity activation with many different applications in the fields of immunology, cancer and neurodegenerative diseases.Facultad de Ciencias Exacta

Practical guide for moisture associated skin damages

A presente guía intégrase dentro da Colección de Guías Prácticas de Feridas do Servizo Galego de Saúde (Sergas), de acordo coas estratexias e liñas de acción promovidas a través do Programa "Úlceras Fóra" que coordina a Subdirección Xeral de Ordenación Asistencial e Innovación Organizativa. Esta guía confórmase como unha síntese das mellores intervencións e prácticas preventivas ou terapéuticas disponibles para o coidado das persoas con lesións cutáneas asociadas á humidade. segundo a práctica clínica baseada na evidencia científicas mási actualA presente guía intégrase dentro da Colección de Guías Prácticas de Feridas do Servizo Galego de Saúde (Sergas), de acordo coas estratexias e liñas de acción promovidas a través do Programa "Úlceras Fóra" que coordina a Subdirección Xeral de Ordenación Asistencial e Innovación Organizativa. Esta guía confórmase como unha síntese das mellores intervencións e prácticas preventivas ou terapéuticas disponibles para o coidado das persoas con lesións cutáneas asociadas á humidade. segundo a práctica clínica baseada na evidencia científicas mási actua

Use of Technology-Based Interventions in the Treatment of Patients with Overweight and Obesity: A Systematic Review

Introduction: Obesity is one of the most important health problems worldwide. The prevalence of obesity has increased dramatically in the last decades and is now recognized as a global epidemic. Given the dramatic consequences of obesity, new intervention approaches based on the potential of technologies have been developed. Methods: We conducted a systematic review of studies using PubMed, ScienceDirect, Cochrane Library, and MedLine databases to assess how different types of technologies may play an important role on weight loss in obese patients. Results: Forty-seven studies using different types of technologies including smartphones, app, websites, virtual reality and personal digital assistant were included in the review. About half of interventions (47%) found a significant effect of the technology-based interventions for weight lost in obese patients. The provision of feedback could also be effective as a complement to interventions carried out using technology to promote weight loss. Conclusions: The use of technologies can be effective to increase weight loss in patients with obesity improving treatment adherence through self-monitoring.TIN2017-89069-R funded by the Ministry of Economy, Industry and Competitiveness and the European Regional Development Fund (FEDER)

Effectiveness of a Two-Year Multicomponent Intervention for the Treatment of Overweight and Obesity in Older People

This study aimed to assess the effectiveness of a two-year intervention based on the Mediterranean diet for the treatment of overweight and obesity in a sample of 51 older people from the Mediterranean city of Alicante (Spain). We also examined the effects of the intervention on psychological well-being. The participants were randomly assigned to the experimental and control groups. The experimental group received group nutritional education sessions, an individualized dietary–nutritional treatment based on a Mediterranean diet, and a physical activity program; the control group received Mediterranean nutritional education in a written format. The experimental group showed a greater loss in weight (p = 0.017) and percentage of fat mass (p = 0.049), and a greater reduction in body mass index (BMI) (p = 0.014) and waist circumference (p = 0.010). Both groups improved their depression scores using the PHQ-9; however, no significant improvement was seen in adherence to the Mediterranean diet (PREDIMED) and anxiety level (GAD-7). These results suggest that a two-year intervention based on the Mediterranean diet allows an older population with overweight or obesity to achieve greater weight loss and a greater decrease in BMI, waist circumference, and fat mass percentage. In relation to psychological well-being, depression levels improved at the end of said intervention.This research was funded by the Office of the Vice President of Research of the University of Alicante (GRE19-20)

Optimization of transgenesis conditions for the generation of CXCL2-luciferase reporter mice line

Background: Transgenesis by microinjection has been widely used for the generation of different mouse models. Different variables of the procedure may critically affect the efficiency of the process. A DNA construction that carries the CXCL2 promoter gene and firefly luciferase has been used to optimize aspects of the procedure. Three different concentrations (0.5, 1.0 and 4.0 ng/μl) of the DNA construction to microinject a total of 1981 zygotes has been tested. Intact/injected embryos, pregnancy and birth rate, survival of pups 7 days after birth, number of transgenic pups and overall transgenic efficiency was registered and analyzed by Z test of proportions for each group. Results: A total of seven transgenic founders were detected for the three DNA concentrations used, 1 in 46 alive pups in the 0.5 ng/μl group, 5 in 38 alive pups in the 1 ng/μl group and 1 in 21 alive pups in the 4 ng/μl group (p < 0.1). The overall transgenic efficiency was higher for the 1 ng/μl concentration, with a transgenic rate of 13.2%. Conclusions: In conclusion, we have selected the best operative conditions to maximize the transgenesis efficiency. Furthermore, the transgenic lines developed could be used as a reporter model of innate immunity activation with many different applications in the fields of immunology, cancer and neurodegenerative diseases.Facultad de Ciencias Exacta

In-Depth Analysis of the Role of the Acinetobactin Cluster in the Virulence of Acinetobacter baumannii

[Abstract] Acinetobacter baumannii is a multidrug-resistant pathogen that represents a serious threat to global health. A. baumannii possesses a wide range of virulence factors that contribute to the bacterial pathogenicity. Among them, the siderophore acinetobactin is one of the most important, being essential for the development of the infection. In this study we performed an in-depth analysis of the acinetobactin cluster in the strain A. baumannii ATCC 17978. For this purpose, nineteen individual isogenic mutant strains were generated, and further phenotypical analysis were performed. Individual mutants lacking the biosynthetic genes entA, basG, basC, basD, and basB showed a significant loss in virulence, due to the disruption in the acinetobactin production. Similarly, the gene bauA, coding for the acinetobactin receptor, was also found to be crucial for the bacterial pathogenesis. In addition, the analysis of the ΔbasJ/ΔfbsB double mutant strain demonstrated the high level of genetic redundancy between siderophores where the role of specific genes of the acinetobactin cluster can be fulfilled by their fimsbactin redundant genes. Overall, this study highlights the essential role of entA, basG, basC, basD, basB and bauA in the pathogenicity of A. baumannii and provides potential therapeutic targets for the design of new antivirulence agents against this microorganism.This work was funded by Projects PI15/00860 awarded to GB and PI17/01482 to AB and MP, all within in the National Plan for Scientific Research, Development and Technological Innovation 2013–2016 and funded by the ISCIII – General Subdirection of Assessment and Promotion of the Research-European Regional Development Fund (FEDER) “A way of making Europe.” The study was also funded by project IN607A 2016/22 (GAIN- Agencia Gallega de Innovación – Consellería de Economía, Emprego e Industria) awarded to GB. This work was also supported by Planes Nacionales de I + D + i 2008–2011/2013–2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/006) co-financed by European Development Regional Fund “A way to achieve Europe” and operative program Intelligent Growth 2014–2020. This work was also supported by Grant RTI2018-093634-B-C22 (AEI/FEDER, EU) from the State Agency for Research (AEI) of Spain, co-funded by the FEDER Programme from the European Union and Xunta de Galicia for the support of Grant ED431E 2018/03 for CICA-INIBIC strategic and the initiative “Seed Projects 2019–2020.” JV-U was financially supported by the ISCIII project FI18/00315, LÁ-F by the ISCIII project PI14/00059 and the IN606B-2018/011, MM-G was financially supported by the Grant Clara Roy (SEIMC, Spanish Society of Clinical Microbiology and Infectious Diseases), KC-P by IN607A 2016/22 and AECC (Asociación Española Contra el Cáncer) predoctoral fellowship and LA by Xunta de Galicia co-funded with the European Social Fund (FSE) of the European Union (ED481A-2019/081)Xunta de Galicia; IN607A 2016/22Xunta de Galicia; ED431E 2018/03Xunta de Galicia; IN606B-2018/011Xunta de Galicia; IN607A 2016/22Xunta de Galicia; ED481A-2019/08

Contribution of Efflux Pumps, Porins, and B-Lactamases to Multidrug Resistance in Clinical Isolates of Acinetobacter baumannii

Weinvestigated the mechanisms of resistance to carbapenems, aminoglycosides, glycylcyclines, tetracyclines, and quinolones in 90 multiresistant clinical strains of Acinetobacter baumannii isolated from two genetically unrelated A. baumannii clones: clone PFGEROC- 1 (53 strains producing the OXA-58B-lactamase enzyme and 18 strains with the OXA-24B-lactamase) and clone PFGE-HUI-1 (19 strains susceptible to carbapenems).Weused real-time reverse transcriptase PCR to correlate antimicrobial resistance (MICs) with expression of genes encoding chromosomalB-lactamases (AmpC and OXA-51), porins (OmpA, CarO, Omp33, Dcap-like, OprB, Omp25, OprC, OprD, and OmpW), and proteins integral to six efflux systems (AdeABC, AdeIJK, AdeFGH, CraA, AbeM, and AmvA). Overexpression of the AdeABC system (level of expression relative to that by A. baumannii ATCC 17978, 30- to 45-fold) was significantly associated with resistance to tigecycline, minocycline, and gentamicin and other biological functions. However, hyperexpression of the AdeIJK efflux pump (level of expression relative to that by A. baumannii ATCC 17978, 8- to 10-fold) was significantly associated only with resistance to tigecycline and minocycline (to which the TetB efflux system also contributed). TetB and TetA(39) efflux pumps were detected in clinical strains and were associated with resistance to tetracyclines and doxycycline. The absence of the AdeABC system and the lack of expression of other mechanisms suggest that tigecycline-resistant strains of the PFGE-HUI-1 clone may be associated with a novel resistance-nodulation-cell efflux pump (decreased MICs in the presence of the inhibitor Phe-ArgB-naphthylamide dihydrochloride) and the TetA(39) system

Assessment and integrated management of moisture associated skin damages (masd): consensus review

Objetivo: Determinar qué tipos de LESCAH hay, como identificarlas y valorarlas; así como saber cuál es la mejor práctica clínica y de cuidados para prevenirlas y tratarlas. Método: Estudio observacional descriptivo de revisión bibliográfica. Lectura crítica por medio de la técnica de grupo nominal y revisión de consenso por panel de expertos Resultados: Las LESCAH son el resultado de un modelo multicausal provocado por 3 factores determinantes (exceso de humedad, irritación física e irritación química sobre la piel). Se identificaron 6 tipos de LESCAH, siendo la Escala Visual del Eritema (EVE) y la SubEscala de la Humedad de Braden (SHB) las más utilizadas para su gradación y valoración del riesgo. Las recomendaciones sobre su prevención y tratamiento se dirigen hacia un correcto cuidado e higiene de la piel, el uso de cremas barrera, la formación del profesional y la educación sanitaria del paciente y familiares. Conclusiones: Debemos empezar a tomar conciencia de las LESCAH (diferenciándolas de otros tipos de lesiones), a fin de estandarizar procesos asistenciales y de cuidado, en busca de visibilizar el problema y su abordaje valoración y manejo integral.Objective: Determine what types of MASD there are, as identify and appraise them; as well as to know what the best clinical practice is and care to prevent and treat it. Methods: Descriptive observational study of bibliographic review. Critical reading through the nominal group technique and review of consensus by expert panel. Results: The MASD are the result of a multicausal model caused by 3 determinants (excess moisture, physical irritation and chemical irritation on the skin). We identified 6 types of MASD, being the Visual Scale of erythema and the subscale of the humidity of Braden the most used for its gradation and assessment of risk. Recommendations on its prevention and treatment are directed toward a proper care and hygiene of the skin, the use of barrier creams, professional training and health education of the patient and family. Conclusions: We must start to take conscience of the MASD (opposed to other type of injury), in order to standardize processes and care, search of increase the visibility of the problem and its valuation approach and integrated management.Enfermerí