Bacteriemias por Escherichia coli: análisis clínico-epidemiológico, de factores de patogenicidad y mecanismos de resistencia a betalactámicos/inhibidores de betalactamasas

Las infecciones por bacterias multirresistentes son un serio problema a nivel mundial. La aparición constante de mecanismos de resistencia a los antibióticos junto con el limitado desarrollo de nuevos compuestos con actividad antibacteriana en los últimos años plantea la necesidad de abordar este problema de forma urgente. Esta Tesis Doctoral se ha centrado en el estudio de las infecciones por E. coli, en especial la bacteriemia y la infección intraabdominal, tanto desde el punto de vista del paciente como del microorganismo. Por un lado se analizaron las características clínicas y microbiológicas, así como factores de riesgo asociados con la mortalidad de los pacientes con bacteriemia por E. coli en comparación con pacientes con bacteriemia por K. pneumoniae. Este primer análisis mostró una serie de diferencias importantes entre uno y otro microorganismo. En la misma línea, se analizaron las características clínicas y microbiológicas de pacientes con infección intraabdominal por E. coli y su relación con la mortalidad, así como los factores de riesgo asociados al desarrollo de bacteriemia en este tipo de infección. Entre otros, la infección del tracto biliar parece jugar un papel importante, por lo que se decidió analizar la relación filogenética, el resistoma y el viruloma de un conjunto de aislados clínicos de E. coli causantes de infección biliar, con el objetivo de conocer si existen factores bacterianos que promuevan este tipo de infección. Aunque no existió relación filogenética entre los aislados, se encontraron numerosos factores de virulencia que podrían estar implicados en el desarrollo de infecciones del tracto biliar. Por otro lado, el siguiente objetivo fue estudiar la expresión de un factor de virulencia concreto como es la proteína de membrana externa A (OmpA) en E. coli, y su asociación con la mortalidad de los pacientes con bacteriemia por E. coli. Se analizó una amplia cohorte de aislados clínicos procedentes de pacientes con bacteriemia, en los que se determinó la expresión de OmpA y se encontró que la sobreexpresión de esta proteína es un factor de riesgo independiente para la mortalidad de los pacientes con bacteriemia por E. coli. Estos resultados sugieren que OmpA tiene un papel importante en la patogénesis de este microorganismo. Por último, y centrándonos en el tratamiento antibiótico de este tipo de infecciones, se estudió la existencia de un patrón de resistencia a los betalactámicos con inhibidores de betalactamasas (BL/IBL) en E. coli. Este patrón dio lugar a la descripción de un nuevo fenómeno denominado “resistencia de espectro extendido a los BL/IBL” o ESRI, el cual está favorecido por la presencia de concentraciones sub-óptimas de piperacilina/tazobactam, un antibiótico ampliamente utilizado para el tratamiento empírico de las infecciones graves por E. coli. Asimismo, y en relación con este objetivo, se llevó a cabo el diseño, desarrollo y validación de un sistema de detección rápida tanto de resistencia a piperacilina/tazobactam como de ESRI en E. coli. La descripción de este nuevo fenómeno de resistencia antibiótica, junto con la posibilidad de su detección anticipada, suponen una contribución innovadora a la par que prometedora en la lucha contra la resistencia a los antibióticos. En conjunto, los datos derivados de esta Tesis Doctoral dan cuenta de la importancia del abordaje multifocal de este tipo de infecciones, las cuales suponen un problema de gran magnitud, con elevados costes tanto para el paciente como para el sistema sanitario

De Pitágoras, habas y malaria

What does Pythagoras have to do with fava beans? And with malaria? Surely many of you are wondering this after reading the title. I’m going to tell you why and I hope not to disappoint you  ¿Qué tendrá que ver Pitágoras con las habas? ¿Y con la malaria? Seguramente muchos de vosotros os lo estaréis preguntando después de leer el título. Os voy a contar el porqué y espero no defraudaros

A selective culture medium for screening linezolid-resistant gram-positive bacteria

The SuperLinezolid medium was developed for screening resistance to linezolid (LZD) in Gram-positive bacteria (Staphylococcus spp., Enterococcus spp.). It was evaluated using LZD-susceptible (n = 20) and LZD-resistant (n = 17) Gram-positive isolates. The sensitivity was found to be 82% at 24 h (3 out of 17 isolates being missed), and reached 100% at 48 h. At 48 h, a single LZD-susceptible isolate grew (specificity 95%). By testing stools spiked with LZD-resistant Gram-positive strains, an excellent performance of the medium was observed, with a lowest detection limit ranging from 101 to 102 CFU/ml. Overall, this medium is accurate for detection of LZD-resistant Gram-positive isolates after 24 h of culture

In vitro effect of ceftazidime-avibactam pressure on ceftazidime-avibactam resistance in KPC-producing Klebsiella pneumoniae clinical isolates.

Motivation: Infections caused by KPC-producing Klebsiella pneumoniae represent a challenge due to the limited available treatement choices. In this context, ceftazidime-avibactam (CAZ-AVI) is postulated as an alternative treatment effective against class A beta-lactamases such as KPC [1]. But, recent data reported the failure of CAZ-AVI treatment of infections by KPC-producing K. pneumoniae due to the development of CAZ-AVI resistance [2]. However, little is known concernig the CAZ-AVI resistance developement by CAZ-AVI selective pressure. Here, we aimed to determinate in vitro whether the exposure of KPC-producing K. pneumoniae clinical isolates to CAZ-AVI subinhibitory concentrations could lead the selection of CAZ-AVI resistant isolates. Methods: Seventeen KPC-producing K. pneumoniae clinical isolates (7 KPC-2, 9 KPC-3 and 1 KPC-11) were analyzed. Minimum inhibitory concentrations (MICs) of CAZ-AVI were determined by broth microdilution using a fixed AVI cocentration of 4 mg/L [3]. Moreover, these isolates were further exposed to increasing concentrations of CAZ and fixed 4 mg/L of AVI, from a sub-MIC up to 256/4 mg/L of CAZ-AVI (or the concentration able to kill the bacterial isolate) at 37ºC with shaking during 24h. New MICs to CAZ-AVI were determined in each condition and after 15 days without CAZ-AVI pressure. Therefore, in order to demonstrated that blaKPC gene is responsible for acquisition of CAZ-AVI resistance in KPC-producing K. pneumoniae, blaKPC-2 and blaKPC-3 were cloned into a reference K. pneumoniae CECT 997 strain. Resistance or susceptibility were determined according to EUCAST criteria [3]. Results: All (17/17, 100%) KPC-producing K. pneumoniae isolates were able to grow at high concentrations of CAZ-AVI (≥64/4 mg/L), increasing their resistance to CAZ-AVI ≥8-fold. Likewise, fifteen of the 17 (88.2%) resistant isolates maintained the acquired CAZ-AVI resistance 15 days after without CAZ-AVI pressure. In addition, the CECT 997 mutants with blaKPC-2 or blaKPC-3 were able to grow up to 256/4 mg/L of CAZ-AVI, displaying and maintaining CAZ-AVI MIC shift from <0.01/4 mg/L (susceptible) to 512/4 mg/L (resistant). Conclusions: These data suggest that exposure of KPC-producing K. pneumoniae to subinhibitory CAZ-AVI concentrations could lead to the selection of CAZ-AVI resistance and this resistance is stable over the time

Phylogeny, Resistome, and Virulome of Escherichia coli Causing Biliary Tract Infections

Escherichia coli is the most frequent Gram-negative bacilli involved in intra-abdominal infections. However, despite high mortality rates associated with biliary tract infections due to E. coli, there is no study focusing on this pathogen. In this study, we have characterized a group of 15 E. coli isolates obtained from 12 patients with biliary tract infections. Demographic and clinical data of the patients were recovered. Phylogeny, resistome, and virulome analysis through whole genome sequencing and biofilm formation were investigated. Among the 15 E. coli isolates, no predominant sequence type (ST) was identified, although 3 of them belonged to unknown STs (20%). Resistance to ampicillin, amoxicillin/clavulanic acid, cotrimoxazole, and quinolones was more present in these isolates; whereas, third and fourth generation cephalosporins, carbapenems, amikacin, tigecycline, and colistin were highly active. Moreover, high diversity of virulence factors has been found, with sfa, fimH, and gad the most frequently detected genes. Interestingly, 26.6% of the E. coli isolates were high biofilm-producers. Altogether, our data characterized for the first time E. coli isolates associated with biliary tract infections in terms of genomic relationship, resistome, and virulome.España, Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades (CP15/00132)España, Plan Nacional de I+D+i 2013-2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spanish Network for Research in Infectious Diseases (RD16/0016/0009

Antibacterial activity of colloidal silver against Gram-negative and Gram-positive bacteria.

Motivation: Treatment of multidrug-resistant (MDR) bacteria represent a challenge for clinicians and public health authorities. Due to the emergence of resistance to a wide variety of antibiotics new alternative therapies are needed. Silver has been used to treat bacterial infections since antiquity due to its known antimicrobial properties [1]. The objective of this project was to study in vitro the activity of colloidal silver against Gram-negative and Gram-positive bacteria.Methods: Gram-negative bacteria [Acinetobacter baumannii (n=44), Pseudomonas aeruginosa (n=25) and Escherichia coli (n=79)] and Gram-positive bacteria [Staphylococcus aureus (n=34), Syaphylococcus epidermidis (n=14) and Enterococcus spp. (n=15)] were used. All strains were grown in a Mueller-Hinton Broth (MHB) at 37°C for 20-24 h. Minimal Inhibitory Concentration (MIC) was determined for all strains by using microdilution assay. To monitore the antibacterial activity, time-kill curve assays were performed on MHB at colloidal silver concentrations of 0.5x, 1x and 2x MIC with starting inoculum of 1x10^6 colony-forming units (cfu)/mL. Reactive Oxygen Species (ROS) production was measured at 6, 20 and 24 hours at colloidal silver concentrations of 0.25x, 0.5x and 1x MIC.Results:  Colloidal silver MIC range was from 4-8 mg/L for both Gram-negative and Gram-positive bacteria. Colloidal silver showed bactericidal activity against Gram-negative bacteria. However, it showed bacteriostatic activity against Gram-positive bacteria. For A. baumannii (Ab11 and ATCC 17978 strains), P. aeruginosa (Pa238 and Pa01 strains), and E. coli (mcr-1 positive strain) colloidal silver was bactericidal at 1x, and 2x MIC at 24h. However, at 24h, E. coli (ATCC 25922 strain) showed a regrowth at 0.5x, 1x and 2x MIC. Incubation of bacterial strains with colloidal silver led to a significant increase in ROS production at 24h in Gram-negative bacteria.Conclusions: Colloidal silver showed in vitro activity against these kind of pathogens, especially against Gram-negative bacteria. These results suggest that colloidal silver could be a new alternative for treatment of infections caused by MDR pathogens

Oligodendrocyte metabolism throughout its differentiation: immunocytochemistry study and its impact in remyelination

Introduction: Oligodendrocytes (OL) role in demyelinating pathologies such as multiple sclerosis and other neurodegenerative diseases is only recently being subject of extensive research. While the genetic and molecular aspects have been thoroughly studied, their metabolism was overshadowed. In order to develop new therapies to promote remyelination of already damaged axons, we need to accurately describe how OL metabolism affects axon myelination and trophic support (1). The objective of this study is to obtain cytological evidence of the extent of both glycolytic metabolism and oxidative phosphorylation by immunocytochemistry throughout the development of OL. Methods: Oligodendroglia cells from post-natal mice cortices were obtained and cultured. A wide assortment of differentiation-stage-specific cell surface antigens, a glycolytic and an oxidative phosphorylation marker were combined in several immunofluorescences to study both metabolic pathways in each step of differentiation. Results: After analysing them under confocal microscopy and imaging software, we observed a constant upregulation of glycolytic metabolism throughout differentiation, while oxidative phosphorylation seemed to increase with differentiation to then decrease when oligodendrocytes achieved their final maturation stage. Conclusions: Therefore, oxidative phosphorylation may be crucial in the differentiation of precursors and glycolysis would thus be the preferred metabolic pathway for fully matured OL. [1] Rosko L. et al. Neuroscientist. 2019;25(4):334–43.Supported by UMA and IBIMA and funding from two ongoing projects: - ‘Modulation of oligodendrocyte metabolism via blood vessel remodelling as target to promote remyelination’ (funding by NEURATRIS). - ‘Blood vessel remodelling modulates remyelination by oligodendrocyte metabolic reprogramming’ (funding by Arsep Foundation). Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Prevalence and Risk Factors for Multidrug-Resistant Organisms Colonization in Long-Term Care Facilities Around the World: A Review

Elderly people confined to chronic care facilities face an increased risk of acquiring infections by multidrug-resistant organisms (MDROs). This review presents the current knowledge of the prevalence and risk factors for colonization by MDROs in long-term care facilities (LTCF), thereby providing a useful reference to establish objectives for implementing successful antimicrobial stewardship programs (ASPs). We searched in PubMed and Scopus for studies examining the prevalence of MDROs and/or risk factors for the acquisition of MDROs in LTCF. One hundred and thirty-four studies published from 1987 to 2020 were included. The prevalence of MDROs in LTCF varies between the different continents, where Asia reported the highest prevalence of extended-spectrum ß-lactamase (ESBL) Enterobacterales (71.6%), carbapenem resistant (CR) Enterobacterales (6.9%) and methicillin-resistant Staphylococcus aureus (MRSA) (25.6%) and North America the highest prevalence to MDR Pseudomonas aeruginosa (5.4%), MDR Acinetobacter baumannii (15.0%), vancomycin-resistant Enterococcus spp. (VRE) (4.0%), and Clostridioides difficile (26.1%). Furthermore, MDRO prevalence has experienced changes over time, with increases in MDR P. aeruginosa and extended spectrum ß-lactamase producing Enterobacterales observed starting in 2015 and decreases of CR Enterobacterales, MDR A. baumannii, VRE, MRSA and C. difficile. Several risk factors have been found, such as male sex, chronic wounds, the use of medical devices, and previous antibiotic use. The last of these aspects represents one of the most important modifiable factors for reducing colonization with MDROs through implementing ASPs in LTCF.The study was funded by the Instituto de Salud Carlos III, the Spanish Ministry of Economy, Industry, and Competitiveness (grant number: PI17-02195) and was partially funded by the European Development Regional Fund “A way to achieve Europe”. A.R.V. and A.B.G.G. are supported by the Subprograma Río Hortega, Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spain. A.R.V. grant number: CM18/00122. A.B.G.G. grant number: CM19/00029. C.M.G. and J.C.C.R. are supported by the Instituto de Salud Carlos III (grant number: PI17-02195) and co-financed by European Development Regional Fund ‘A way to achieve Europe’ ERDF, Spanish Network for the Research in Infectious Diseases (REIPI RD16/0016/0009).G.P. is supported by the Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/0001)- co-financed by European Development Regional Fund “A way to achieve Europe”, Operative program Intelligent Growth 2014–2020. M.E.P.I. is a postdoctoral researcher belonging to the program “Nicolás Monardes” (C1-0038-2019), Servicio Andaluz de Salud, Junta de Andalucía, Spain. J.M.C. received funding for research from Plan Nacional de I+D+i 2013–2016 and Instituto de Salud Carlos III, Subdireccion General de Redes y Centros de InvestigacionCooperativa, Ministry of Economy, Industry and Competitiveness, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/0001, RD16/0016/0009), co-financed by the European Development Regional Fund “A way to achieve Europe”.Ye

Gastrointestinal cancer: Relationship between histology and microbiota

Este trabajo fue presentado como comunicación tipo póster en el citado congreso.Objectives: Review of the published literature concerning the relationship between microbiome and gastrointestinal cancer. Methods: Present work is focused on systematic research in the most prominent biomedical databases finds relevant works in Pubmed and the library’s catalog of the University of Málaga (Jábega) of published journals in the last 5 years. Results: In this work, the mechanisms used by the microbiome to damage gastrointestinal epithelial cells and cause cancer are explained. Some of them are the dysbiosis, destruction of the mucosal barrier, chronic inflammation, damage caused by metabolites produced in the digestion and the direct attack of certain toxins to the cell’s DNA. These mechanisms adjust the immune response, by activation or inhibition using different cytokines. There is also a deeper look into several microorganisms and how they cause gastrointestinal cancer using toxins or virulence factors to activate them. Conclusions: The evidence found so far about the microbiota and gastrointestinal cancer is enough to assume the relationship between them, although there is much left to research. With these findings, it can be expected that in a near future certain microorganisms could be used for screening purposes, due to their increase in early stages of the tumor genesis and also, in a preventive way to try to eradicate them, even avoid cancer. Studies on the microbiota are hardly beginning, and results appear to be promising.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Dendritic cell deficiencies persist seven months after SARS-CoV-2 infection

Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV)-2 infection induces an exacerbated inflammation driven by innate immunity components. Dendritic cells (DCs) play a key role in the defense against viral infections, for instance plasmacytoid DCs (pDCs), have the capacity to produce vast amounts of interferon-alpha (IFN-α). In COVID-19 there is a deficit in DC numbers and IFN-α production, which has been associated with disease severity. In this work, we described that in addition to the DC deficiency, several DC activation and homing markers were altered in acute COVID-19 patients, which were associated with multiple inflammatory markers. Remarkably, previously hospitalized and nonhospitalized patients remained with decreased numbers of CD1c+ myeloid DCs and pDCs seven months after SARS-CoV-2 infection. Moreover, the expression of DC markers such as CD86 and CD4 were only restored in previously nonhospitalized patients, while no restoration of integrin β7 and indoleamine 2,3-dyoxigenase (IDO) levels were observed. These findings contribute to a better understanding of the immunological sequelae of COVID-19