RESUMEN
Escherichia coli represents a classical intestinal gram-negative commensal. Despite this commensalism, different E. coli strains can mediate disparate immunogenic properties in a given host. Symbiotic E. coli strains such as E. coli Nissle 1917 (EcN) are attributed beneficial properties, e.g., promotion of intestinal homeostasis. Therefore, we aimed to identify molecular features derived from symbiotic bacteria that might help to develop innovative therapeutic alternatives for the treatment of intestinal immune disorders. This study was performed using the dextran sodium sulphate (DSS)-induced colitis mouse model, which is routinely used to evaluate potential therapeutics for the treatment of Inflammatory Bowel Diseases (IBDs). We focused on the analysis of flagellin structures of different E. coli strains. EcN flagellin was found to harbor a substantially longer hypervariable region (HVR) compared to other commensal E. coli strains, and this longer HVR mediated symbiotic properties through stronger activation of Toll-like receptor (TLR)5, thereby resulting in interleukin (IL)-22-mediated protection of mice against DSS-induced colitis. Furthermore, using bone-marrow-chimeric mice (BMCM), CD11c+ cells of the colonic lamina propria (LP) were identified as the main mediators of these flagellin-induced symbiotic effects. We propose flagellin from symbiotic E. coli strains as a potential therapeutic to restore intestinal immune homeostasis, e.g., for the treatment of IBD patients.
Asunto(s)
Escherichia coli/metabolismo , Flagelina/genética , Simbiosis/genética , Animales , Colitis/inducido químicamente , Colitis/inmunología , Modelos Animales de Enfermedad , Escherichia coli/genética , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/genética , Femenino , Flagelina/metabolismo , Mucosa Intestinal , Intestinos , Masculino , Ratones , Ratones Endogámicos C57BL , Transducción de Señal/inmunología , Simbiosis/fisiología , Receptor Toll-Like 5/metabolismoRESUMEN
BACKGROUND: Asymptomatic C. difficile colonization is believed to predispose to subsequent C. difficile infection (CDI). While emerging insights into the role of the commensal microbiota in mediating colonization resistance against C. difficile have associated CDI with specific microbial components, corresponding prospectively collected data on colonization with C. difficile are largely unavailable. METHODS: C. difficile status was assessed by GDH EIA and real-time PCR targeting the toxin A (tcdA) and B (tcdB) genes. 16S V3 and V4 gene sequencing results from fecal samples of patients tested positive for C. difficile were analyzed by assessing alpha and beta diversity, LefSe, and the Piphillin functional inference approach to estimate functional capacity. RESULTS: 1506 patients were recruited into a prospective observational study (DRKS00005335) upon admission into one of five academic hospitals. 936 of them provided fecal samples on admission and at discharge and were thus available for longitudinal analysis. Upon hospital admission, 5.5% (83/1506) and 3.7% (56/1506) of patients were colonized with toxigenic (TCD) and non-toxigenic C. difficile (NTCD), respectively. During hospitalization, 1.7% (16/936) acquired TCD. Risk factors for acquisition of TCD included pre-existing lung diseases, lower GI endoscopy and antibiotics. Species protecting against hospital-related C. difficile acquisition included Gemmiger spp., Odoribacter splanchnicus, Ruminococcus bromii and other Ruminococcus spp. Metagenomic pathway analysis identified steroid biosynthesis as the most underrepresented metabolic pathway in patients who later acquire C. difficile colonization. CONCLUSIONS: Gemmiger spp., Odoribacter splanchnicus, Ruminococcus bromii and other Ruminococci were associated with a decreased risk of C. difficile acquisition. CLINICAL TRIALS REGISTRATION: DRKS00005335.
Asunto(s)
Toxinas Bacterianas , Clostridioides difficile , Infecciones por Clostridium , Microbiota , Toxinas Bacterianas/genética , Bacteroidetes , Clostridioides , Clostridioides difficile/genética , Infecciones por Clostridium/epidemiología , Heces , Humanos , Estudios Prospectivos , Factores de Riesgo , RuminococcusRESUMEN
With the aim to identify potential new targets to restore antimicrobial susceptibility of multidrug-resistant (MDR) Pseudomonas aeruginosa isolates, we generated a high-density transposon (Tn) insertion mutant library in an MDR P. aeruginosa bloodstream isolate (isolate ID40). The depletion of Tn insertion mutants upon exposure to cefepime or meropenem was measured in order to determine the common resistome for these clinically important antipseudomonal ß-lactam antibiotics. The approach was validated by clean deletions of genes involved in peptidoglycan synthesis/recycling, such as the genes for the lytic transglycosylase MltG, the murein (Mur) endopeptidase MepM1, the MurNAc/GlcNAc kinase AmgK, and the uncharacterized protein YgfB, all of which were identified in our screen as playing a decisive role in survival after treatment with cefepime or meropenem. We found that the antibiotic resistance of P. aeruginosa can be overcome by targeting usually nonessential genes that turn essential in the presence of therapeutic concentrations of antibiotics. For all validated genes, we demonstrated that their deletion leads to the reduction of ampC expression, resulting in a significant decrease in ß-lactamase activity, and consequently, these mutants partly or completely lost resistance against cephalosporins, carbapenems, and acylaminopenicillins. In summary, the determined resistome may comprise promising targets for the development of drugs that may be used to restore sensitivity to existing antibiotics, specifically in MDR strains of P. aeruginosa.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/genética , Elementos Transponibles de ADN , Farmacorresistencia Bacteriana Múltiple/genética , Pseudomonas aeruginosa/genética , Resistencia betalactámica/genética , Proteínas Bacterianas/metabolismo , Cefepima/farmacología , Endopeptidasas/deficiencia , Endopeptidasas/genética , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Glicosiltransferasas/deficiencia , Glicosiltransferasas/genética , Humanos , Meropenem/farmacología , Pruebas de Sensibilidad Microbiana , Mutagénesis , Fosfotransferasas (Aceptor de Grupo Alcohol)/deficiencia , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/aislamiento & purificación , beta-Lactamasas/genética , beta-Lactamasas/metabolismoRESUMEN
Table 2 in the originally published article is not correct and is a duplicate of Table 3. The error happened during typesetting. The correct Table 2 is shown below.
RESUMEN
Fast identification of pathogens directly from positive blood cultures is of highest importance to supply an adequate therapy of bloodstream infections (BSI). There are several platforms providing molecular-based identification, detection of antimicrobial resistance genes, or even a full antimicrobial susceptibility testing (AST). Two of such test systems allowing rapid diagnostics were assessed in this study: The Biofire FilmArray® and the Genmark ePlex®, both fully automated test system with a minimum of hands-on time. Overall 137 BSI episodes were included in our study and compared to conventional culture-based reference methods. The FilmArray® is using one catridge including a panel for the most common bacterial and fungal BSI pathogens as well as selected resistance markers. The ePlex® offers three different cartridges for detection of Gram-positives, Gram-negatives, and fungi resulting in a broader panel including also rare pathogens, putative contaminants, and more genetic resistance markers. The FilmArray® and ePlex® were evaluated for all 137 BSI episodes with FilmArray® detecting 119 and ePlex® detecting 128 of these. For targets on the respective panel of the system, the FilmArray® generated a sensitivity of 98.9% with 100% specificity on Gram-positive isolates. The ePlex® system generated a sensitivity of 94.7% and a specificity of 90.7% on Gram-positive isolates. In each case, the two systems performed with 100% sensitivity and specificity for the detection of Gram-negative specimens covered by each panel. In summary, both evaluated test systems showed a satisfying overall performance for fast pathogen identification and are beneficial tools for accelerating blood culture diagnostics of sepsis patients.
Asunto(s)
Algoritmos , Técnicas de Diagnóstico Molecular/métodos , Sepsis/diagnóstico , Bacterias/genética , Bacterias/aislamiento & purificación , Cultivo de Sangre , Pruebas Diagnósticas de Rutina , Farmacorresistencia Microbiana/genética , Hongos/genética , Hongos/aislamiento & purificación , Humanos , Técnicas de Diagnóstico Molecular/normas , Sensibilidad y EspecificidadRESUMEN
Generated by gram-negative bacteria, lipopolysaccharides (LPSs) are one of the most abundant and potent immunomodulatory substances present in the intestinal lumen. Interaction of agonistic LPS with the host myeloid-differentiation-2/Toll-like receptor 4 (MD-2/TLR4) receptor complex results in nuclear factor κB (NF-κB) activation, followed by the robust induction of pro-inflammatory immune responses. Here we have isolated LPS from a common gut commensal, Bacteroides vulgatus mpk (BVMPK), which provides only weak agonistic activity. This weak agonistic activity leads to the amelioration of inflammatory immune responses in a mouse model for experimental colitis, and it was in sharp contrast to strong agonists and antagonists. In this context, the administration of BVMPK LPS into mice with severe intestinal inflammation re-established intestinal immune homeostasis within only 2 weeks, resulting in the clearance of all symptoms of inflammation. These inflammation-reducing properties of weak agonistic LPS are grounded in the induction of a special type of endotoxin tolerance via the MD-2/TLR4 receptor complex axis in intestinal lamina propria CD11c+ cells. Thus, weak agonistic LPS represents a promising agent to treat diseases involving pathological overactivation of the intestinal immune system, e.g., in inflammatory bowel diseases.
Asunto(s)
Homeostasis/inmunología , Inmunidad Mucosa , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Lipopolisacáridos/inmunología , Animales , Biomarcadores , Antígeno CD11c/metabolismo , Colitis/etiología , Colitis/metabolismo , Colitis/patología , Modelos Animales de Enfermedad , Microbioma Gastrointestinal/inmunología , Homeostasis/efectos de los fármacos , Humanos , Enfermedades Inflamatorias del Intestino/diagnóstico por imagen , Enfermedades Inflamatorias del Intestino/etiología , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/patología , Mucosa Intestinal/efectos de los fármacos , Lípido A/inmunología , Lipopolisacáridos/farmacología , Ratones , Ratones Noqueados , Tomografía de Emisión de PositronesRESUMEN
BACKGROUND: The selection pressure exercised by antibiotic drugs is an important consideration for the wise stewardship of antimicrobial treatment programs. Treatment decisions are currently based on crude assumptions, and there is an urgent need to develop a more quantitative knowledge base that can enable predictions of the impact of individual antibiotics on the human gut microbiome and resistome. RESULTS: Using shotgun metagenomics, we quantified changes in the gut microbiome in two cohorts of hematological patients receiving prophylactic antibiotics; one cohort was treated with ciprofloxacin in a hospital in Tübingen and the other with cotrimoxazole in a hospital in Cologne. Analyzing this rich longitudinal dataset, we found that gut microbiome diversity was reduced in both treatment cohorts to a similar extent, while effects on the gut resistome differed. We observed a sharp increase in the relative abundance of sulfonamide antibiotic resistance genes (ARGs) by 148.1% per cumulative defined daily dose of cotrimoxazole in the Cologne cohort, but not in the Tübingen cohort treated with ciprofloxacin. Through multivariate modeling, we found that factors such as individual baseline microbiome, resistome, and plasmid diversity; liver/kidney function; and concurrent medication, especially virostatic agents, influence resistome alterations. Strikingly, we observed different effects on the plasmidome in the two treatment groups. There was a substantial increase in the abundance of ARG-carrying plasmids in the cohort treated with cotrimoxazole, but not in the cohort treated with ciprofloxacin, indicating that cotrimoxazole might contribute more efficiently to the spread of resistance. CONCLUSIONS: Our study represents a step forward in developing the capability to predict the effect of individual antimicrobials on the human microbiome and resistome. Our results indicate that to achieve this, integration of the individual baseline microbiome, resistome, and mobilome status as well as additional individual patient factors will be required. Such personalized predictions may in the future increase patient safety and reduce the spread of resistance. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02058888 . Registered February 10 2014.
Asunto(s)
Antibacterianos/efectos adversos , Ciprofloxacina/efectos adversos , Farmacorresistencia Microbiana , Microbioma Gastrointestinal/efectos de los fármacos , Plásmidos/efectos de los fármacos , Combinación Trimetoprim y Sulfametoxazol/efectos adversos , Antibacterianos/uso terapéutico , Ciprofloxacina/uso terapéutico , Estudios de Cohortes , Farmacorresistencia Microbiana/efectos de los fármacos , Farmacorresistencia Microbiana/genética , Genes Bacterianos/efectos de los fármacos , Alemania , Humanos , Estudios Longitudinales , Metagenómica/métodos , Combinación Trimetoprim y Sulfametoxazol/uso terapéuticoRESUMEN
Vancomycin-resistant Enterococcus faecium (VREfm) is a frequent cause of nosocomial outbreaks. In the second half of 2015, a sharp increase in the incidence of VREfm was observed at our university medical center. Next-generation sequencing (NGS) was used to analyze the first isolates of VREfm recovered from patients between 2010 and 2016 (n = 773) in order to decipher epidemiological change, outbreak dynamics, and possible transmission routes. VREfm isolates were analyzed using whole-genome sequencing followed by sequence type extraction and phylogenetic analysis. We examined epidemiological data, room occupancy data, and patient transferals and calculated an intensity score for patient-to-patient contact. Phylogenetic analysis revealed the presence of 38 NGS clusters and 110 single clones. The increase of VREfm was caused mainly by the expansion of two newly introduced NGS clusters, comprising VanB-type strains determined by multilocus sequence typing (MLST) as sequence type 80 (ST80) and ST117. By combining phylogenetic information with epidemiological data, intrahospital transmission could be demonstrated, however to a lesser extent than initially expected based solely on epidemiological data. The outbreak clones were continuously imported from other hospitals, suggesting a change in the epidemiological situation at a regional scale. By tracking intrahospital patient transferals, two major axes could be identified that contributed to the spread of VREfm within the hospital. NGS-based outbreak analysis revealed a dramatic change in the local and regional epidemiology of VREfm, emphasizing the role of health care networks in the spread of VREfm.
Asunto(s)
Antibacterianos/farmacología , Vancomicina/farmacología , Enterococcus faecium/efectos de los fármacos , Enterococcus faecium/genética , Genoma Bacteriano/genética , Alemania , Humanos , Pruebas de Sensibilidad Microbiana , Tipificación de Secuencias Multilocus , Enterococos Resistentes a la Vancomicina/efectos de los fármacos , Enterococos Resistentes a la Vancomicina/genéticaRESUMEN
Type III secretion systems (T3SS) play a crucial role for virulence in many Gram-negative bacteria. After tight bacterial contact to host cells, the T3SS injects effector proteins into the host cells, which leads to cell invasion, tissue destruction and/or immune evasion. Over the last decade several attempts were made to characterize the host-cell interactions which precede and determine effector protein injection during infection. The development of the TEM-ß-lactamase reporter was an important breakthrough to achieve this goal. By this means it was demonstrated that during infection with many Gram-negative pathogens such as Salmonella, Pseudomonas or Yersinia the main targets of T3SS are leukocytes of the myeloid lineage such as neutrophils, macrophages or dendritic cells. This is due to the recruitment of these cells to the site of infection, but also due to the specific interplay between bacterial and host cells. Comprehensive studies on Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis effector translocation show that adhesins such as Invasin (Inv), Yersinia adhesin A (YadA) and attachment and invasion locus (Ail) are critical for effector translocation. Here, mainly the complex interaction of YadA and Ail with various host cell receptor repertoires on leukocytes and the modulatory effects of serum factors direct effector translocation predominantly towards myeloid cells. The current understanding suggests that mostly protein based interactions between bacteria and host determine host cell specific effector translocation during Yersinia infection. However, for Shigella dysenteriae infection it was shown that glycan-glycan interactions can also play a critical role for the adhesion preceding effector translocation. In addition, the Shigella infection model revealed that the activation status of cells is a further criterium directing effector translocation into a distinct cell population. In this review the current understanding of the complex and species-specific interaction between bacteria and host cells leading to type III secretion is discussed.
Asunto(s)
Adhesión Bacteriana , Interacciones Microbiota-Huesped , Transporte de Proteínas , Sistemas de Secreción Tipo III/metabolismo , Adhesinas Bacterianas/metabolismo , Animales , Proteínas de la Membrana Bacteriana Externa/metabolismo , Humanos , Shigella/inmunología , Shigella/patogenicidad , Virulencia/inmunología , Factores de Virulencia/metabolismo , Yersinia/inmunología , Yersinia/patogenicidadRESUMEN
Yersinia enterocolitica is an enteropathogenic bacterium that causes gastrointestinal disorders, as well as extraintestinal manifestations. To subvert the host's immune response, Y. enterocolitica uses a type III secretion system consisting of an injectisome and effector proteins, called Yersinia outer proteins (Yops), that modulate activation, signaling, and survival of immune cells. In this article, we show that galectin-1 (Gal-1), an immunoregulatory lectin widely expressed in mucosal tissues, contributes to Y. enterocolitica pathogenicity by undermining protective antibacterial responses. We found higher expression of Gal-1 in the spleen and Peyer's patches of mice infected orogastrically with Y. enterocolitica serotype O:8 compared with noninfected hosts. This effect was prevented when mice were infected with Y. enterocolitica lacking YopP or YopH, two critical effectors involved in bacterial immune evasion. Consistent with a regulatory role for this lectin during Y. enterocolitica pathogenesis, mice lacking Gal-1 showed increased weight and survival, lower bacterial load, and attenuated intestinal pathology compared with wild-type mice. These protective effects involved modulation of NF-κB activation, TNF production, and NO synthesis in mucosal tissue and macrophages, as well as systemic dysregulation of IL-17 and IFN-γ responses. In vivo neutralization of these proinflammatory cytokines impaired bacterial clearance and eliminated host protection conferred by Gal-1 deficiency. Finally, supplementation of recombinant Gal-1 in mice lacking Gal-1 or treatment of wild-type mice with a neutralizing anti-Gal-1 mAb confirmed the immune inhibitory role of this endogenous lectin during Y. enterocolitica infection. Thus, targeting Gal-1-glycan interactions may contribute to reinforce antibacterial responses by reprogramming innate and adaptive immune mechanisms.
Asunto(s)
Galectina 1/metabolismo , Interacciones Huésped-Patógeno , Yersiniosis/inmunología , Yersinia enterocolitica/inmunología , Animales , Carga Bacteriana , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Galectina 1/antagonistas & inhibidores , Galectina 1/genética , Galectina 1/inmunología , Interferón gamma/sangre , Interferón gamma/inmunología , Interleucina-17/sangre , Interleucina-17/inmunología , Intestinos/inmunología , Intestinos/microbiología , Intestinos/patología , Ratones , FN-kappa B/metabolismo , Óxido Nítrico/biosíntesis , Ganglios Linfáticos Agregados/inmunología , Ganglios Linfáticos Agregados/microbiología , Ganglios Linfáticos Agregados/patología , Proteínas Tirosina Fosfatasas/deficiencia , Proteínas Tirosina Fosfatasas/genética , Bazo/inmunología , Bazo/microbiología , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Klebsiella pneumoniae and related species are frequent causes of nosocomial infections and outbreaks. Therefore, quick and reliable strain typing is crucial for the detection of transmission routes in the hospital. The aim of this study was to evaluate Fourier transform infrared spectroscopy (FTIR) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) as rapid methods for typing clinical Klebsiella isolates in comparison to whole-genome sequencing (WGS), which was considered the gold standard for typing and identification. Here, 68 clinical Klebsiella strains were analyzed by WGS, FTIR, and MALDI-TOF MS. FTIR showed high discriminatory power in comparison to the WGS reference, whereas MALDI-TOF MS exhibited a low ability to type the isolates. MALDI-TOF mass spectra were further analyzed for peaks that showed high specificity for different Klebsiella species. Phylogenetic analysis revealed that the Klebsiella isolates comprised three different species: K. pneumoniae, K. variicola, and K. quasipneumoniae Genome analysis showed that MALDI-TOF MS can be used to distinguish K. pneumoniae from K. variicola due to shifts of certain mass peaks. The peaks were tentatively identified as three ribosomal proteins (S15p, L28p, L31p) and one stress response protein (YjbJ), which exhibit amino acid differences between the two species. Overall, FTIR has high discriminatory power to recognize the clonal relationship of isolates, thus representing a valuable tool for rapid outbreak analysis and for the detection of transmission events due to fast turnaround times and low costs per sample. Furthermore, specific amino acid substitutions allow the discrimination of K. pneumoniae and K. variicola by MALDI-TOF MS.
Asunto(s)
Técnicas de Tipificación Bacteriana/métodos , Infecciones por Klebsiella/microbiología , Klebsiella/clasificación , Klebsiella/aislamiento & purificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectroscopía Infrarroja por Transformada de Fourier , Técnicas de Tipificación Bacteriana/normas , Análisis por Conglomerados , Costos y Análisis de Costo , Genoma Bacteriano/genética , Humanos , Klebsiella/química , Klebsiella/genética , Infecciones por Klebsiella/diagnóstico , Polimorfismo de Nucleótido Simple/genética , Sensibilidad y Especificidad , Análisis de Secuencia de ADN , Factores de TiempoAsunto(s)
Diabetes Mellitus/genética , Ambiente , Predisposición Genética a la Enfermedad/genética , Hipersensibilidad/genética , Enfermedades Inflamatorias del Intestino/genética , Animales , Enfermedad Crónica , Congresos como Asunto , Diabetes Mellitus/inmunología , Diabetes Mellitus/terapia , Estudios de Asociación Genética , Humanos , Hipersensibilidad/inmunología , Hipersensibilidad/terapia , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/terapia , Metagenoma/inmunologíaRESUMEN
The metallo-beta-lactamase GIM-1 has been found in various bacterial host species nearly exclusively in western Germany. However, not much is known about the epidemiology of GIM-1-positive Serratia marcescens Here we report on a surprisingly protracted regional dissemination. In-hospital transmission was investigated by using conventional epidemiological tools to identify spatiotemporal links. Strain typing was performed using pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing (WGS). Bayesian phylogeny was used to infer the time axis of the observed occurrence. Thirteen S. marcescens strains from 10 patients from 6 different German hospitals were investigated. Suspected in-hospital transmissions were confirmed by molecular typing at a higher resolution by WGS than by PFGE. A detailed sequence analysis demonstrated the spread of one predominant strain variant but also provided evidence for transfer of the blaGIM-1 gene cassette between different strains. A Bayesian phylogenetic analysis showed that the most recent common ancestor of the identified clonal cluster could be dated back to April 1993 (95% highest posterior density interval, January 1973 to March 2003) and that this strain might have already harbored the blaGIM-1 at that time and, therewith, years before the first detection of this resistance gene in clinical specimens. This study shows a long-standing clonal and plasmid-mediated expansion of GIM-1-producing S. marcescens that might have gone unnoticed in the absence of a standardized and effective molecular screening for carbapenemases. The systematic and early detection of resistance is thus highly advisable, especially for the prevention of potentially long-term dissemination that may progress beyond control.
Asunto(s)
Infección Hospitalaria/transmisión , Genoma Bacteriano , Filogenia , Infecciones por Serratia/transmisión , Serratia marcescens/genética , Resistencia betalactámica/genética , beta-Lactamasas/genética , Antibacterianos/farmacología , Técnicas de Tipificación Bacteriana , Teorema de Bayes , Células Clonales , Infección Hospitalaria/tratamiento farmacológico , Infección Hospitalaria/epidemiología , Infección Hospitalaria/microbiología , Electroforesis en Gel de Campo Pulsado , Expresión Génica , Genotipo , Alemania , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Pruebas de Sensibilidad Microbiana , Epidemiología Molecular , Plásmidos/química , Plásmidos/metabolismo , Infecciones por Serratia/tratamiento farmacológico , Infecciones por Serratia/epidemiología , Infecciones por Serratia/microbiología , Serratia marcescens/clasificación , Serratia marcescens/efectos de los fármacos , Serratia marcescens/crecimiento & desarrollo , beta-Lactamasas/metabolismoRESUMEN
Bloodstream infections (BSI) are an important cause of morbidity and mortality. Increasing rates of antimicrobial-resistant pathogens limit treatment options, prompting an empirical use of broad-range antibiotics. Fast and reliable diagnostic tools are needed to provide adequate therapy in a timely manner and to enable a de-escalation of treatment. The Accelerate Pheno system (Accelerate Diagnostics, USA) is a fully automated test system that performs both identification and antimicrobial susceptibility testing (AST) directly from positive blood cultures within approximately 7 h. In total, 115 episodes of BSI with Gram-negative bacteria were included in our study and compared to conventional culture-based methods. The Accelerate Pheno system correctly identified 88.7% (102 of 115) of all BSI episodes and 97.1% (102 of 105) of isolates that are covered by the system's identification panel. The Accelerate Pheno system generated an AST result for 91.3% (95 of 104) samples in which the Accelerate Pheno system identified a Gram-negative pathogen. The overall category agreement between the Accelerate Pheno system and culture-based AST was 96.4%, the rates for minor discrepancies 1.4%, major discrepancies 2.3%, and very major discrepancies 1.0%. Of note, ceftriaxone, piperacillin-tazobactam, and carbapenem resistance was correctly detected in blood culture specimens with extended-spectrum beta-lactamase-producing Escherichia coli (n = 7) and multidrug-resistant Pseudomonas aeruginosa (n = 3) strains. The utilization of the Accelerate Pheno system reduced the time to result for identification by 27.49 h (P < 0.0001) and for AST by 40.39 h (P < 0.0001) compared to culture-based methods in our laboratory setting. In conclusion, the Accelerate Pheno system provided fast, reliable results while significantly improving turnaround time in blood culture diagnostics of Gram-negative BSI.
Asunto(s)
Técnicas Bacteriológicas/métodos , Sangre/microbiología , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/aislamiento & purificación , Infecciones por Bacterias Gramnegativas/diagnóstico , Sepsis/diagnóstico , Automatización de Laboratorios/métodos , Bacterias Gramnegativas/clasificación , Humanos , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
Autotransporter proteins comprise a large family of virulence factors that consist of a ß-barrel translocation unit and an extracellular effector or passenger domain. The ß-barrel anchors the protein to the outer membrane of Gram-negative bacteria and facilitates the transport of the passenger domain onto the cell surface. By inserting an epitope tag into the N terminus of the passenger domain of the inverse autotransporter intimin, we generated a mutant defective in autotransport. Using this stalled mutant, we could show that (i) at the time point of stalling, the ß-barrel appears folded; (ii) the stalled autotransporter is associated with BamA and SurA; (iii) the stalled intimin is decorated with large amounts of SurA; (iv) the stalled autotransporter is not degraded by periplasmic proteases; and (v) inverse autotransporter passenger domains are translocated by a hairpin mechanism. Our results suggest a function for the BAM complex not only in insertion and folding of the ß-barrel but also for passenger translocation.
Asunto(s)
Adhesinas Bacterianas/fisiología , Proteínas de Escherichia coli/fisiología , Escherichia coli/metabolismo , Adhesinas Bacterianas/química , Transporte Biológico , Membrana Celular/metabolismo , Clonación Molecular , Reactivos de Enlaces Cruzados/química , Epítopos/química , Proteínas de Escherichia coli/química , Células HeLa , Humanos , Microscopía Fluorescente , Chaperonas Moleculares/química , Mutagénesis Sitio-Dirigida , Mutación , Péptido Hidrolasas/química , Unión Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Propiedades de SuperficieRESUMEN
Yersinia enterocolitica evades the immune response by injecting Yersinia outer proteins (Yops) into the cytosol of host cells. YopH is a tyrosine phosphatase critical for Yersinia virulence. However, the mucosal immune mechanisms subverted by YopH during in vivo orogastric infection with Y. enterocolitica remain elusive. The results of this study revealed neutrophil recruitment to Peyer's patches (PP) after infection with a YopH-deficient mutant strain (Y. enterocolitica ΔyopH). While the Y. enterocolitica wild-type (WT) strain in PP induced the major neutrophil chemoattractant CXCL1 mRNA and protein levels, infection with the Y. enterocolitica ΔyopH mutant strain exhibited a higher expression of the CXCL1 receptor, CXCR2, in blood neutrophils, leading to efficient neutrophil recruitment to the PP. In contrast, migration of neutrophils into PP was impaired upon infection with Y. enterocolitica WT strain. In vitro infection of blood neutrophils revealed the involvement of YopH in CXCR2 expression. Depletion of neutrophils during Y. enterocolitica ΔyopH infection raised the bacterial load in PP. Moreover, the clearance of WT Y. enterocolitica was improved when an equal mixture of Y. enterocolitica WT and Y. enterocolitica ΔyopH strains was used in infecting the mice. This study indicates that Y. enterocolitica prevents early neutrophil recruitment in the intestine and that the effector protein YopH plays an important role in the immune evasion mechanism. The findings highlight the potential use of the Y. enterocolitica YopH-deficient strain as an oral vaccine carrier.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Infiltración Neutrófila/fisiología , Neutrófilos/citología , Ganglios Linfáticos Agregados/citología , Yersiniosis/inmunología , Yersinia enterocolitica/patogenicidad , Animales , Carga Bacteriana , Proteínas de la Membrana Bacteriana Externa/genética , Quimiocinas CXC/metabolismo , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos C57BL , Neutrófilos/inmunología , Neutrófilos/metabolismo , Receptores de Quimiocina/metabolismo , Virulencia/fisiología , Yersiniosis/metabolismo , Yersiniosis/microbiología , Yersinia enterocolitica/inmunologíaRESUMEN
Intimin and Invasin are prototypical inverse (Type Ve) autotransporters and important virulence factors of enteropathogenic Escherichia coli and Yersinia spp. respectively. In addition to a C-terminal extracellular domain and a ß-barrel transmembrane domain, both proteins also contain a short N-terminal periplasmic domain that, in Intimin, includes a lysin motif (LysM), which is thought to mediate binding to peptidoglycan. We show that the periplasmic domain of Intimin does bind to peptidoglycan both in vitro and in vivo, but only under acidic conditions. We were able to determine a dissociation constant of 0.8 µM for this interaction, whereas the Invasin periplasmic domain, which lacks a LysM, bound only weakly in vitro and failed to bind peptidoglycan in vivo. We present the solution structure of the Intimin LysM, which has an additional α-helix conserved within inverse autotransporter LysMs but lacking in others. In contrast to previous reports, we demonstrate that the periplasmic domain of Intimin mediates dimerisation. We further show that dimerisation and peptidoglycan binding are general features of LysM-containing inverse autotransporters. Peptidoglycan binding by the periplasmic domain in the infection process may aid in resisting mechanical and chemical stress during transit through the gastrointestinal tract.
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Adhesinas Bacterianas/química , Adhesinas Bacterianas/metabolismo , Escherichia coli Enteropatógena/metabolismo , Peptidoglicano/metabolismo , Yersinia/metabolismo , Adhesinas Bacterianas/genética , Sitios de Unión , Biología Computacional/métodos , Dimerización , Escherichia coli Enteropatógena/química , Escherichia coli Enteropatógena/genética , Concentración de Iones de Hidrógeno , Modelos Moleculares , Multimerización de Proteína , Estructura Secundaria de Proteína , Factores de Virulencia/química , Factores de Virulencia/metabolismo , Yersinia/química , Yersinia/genéticaRESUMEN
DCs are professional APCs playing a crucial role in the initiation of T-cell responses to combat infection. However, systemic bacterial infection with various pathogens leads to DC-depletion in humans and mice. The mechanisms of pathogen-induced DC-depletion remain poorly understood. Previously, we showed that mice infected with Yersinia enterocolitica (Ye) had impaired de novo DC-development, one reason for DC-depletion. Here, we extend these studies to gain insight into the molecular mechanisms of DC-depletion and the impact of different bacteria on DC-development. We show that the number of bone marrow (BM) hematopoietic progenitors committed to the DC lineage is reduced following systemic infection with different Gram-positive and Gram-negative bacteria. This is associated with a TLR4- and IFN-γ-signaling dependent increase of committed monocyte progenitors in the BM and mature monocytes in the spleen upon Ye-infection. Adoptive transfer experiments revealed that infection-induced monopoiesis occurs at the expense of DC-development. Our data provide evidence for a general response of hematopoietic progenitors upon systemic bacterial infections to enhance monocyte production, thereby increasing the availability of innate immune cells for pathogen control, whereas impaired DC-development leads to DC-depletion, possibly driving transient immunosuppression in bacterial sepsis.
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Diferenciación Celular/inmunología , Células Dendríticas/inmunología , Inmunidad Innata , Mielopoyesis/inmunología , Yersiniosis/inmunología , Yersinia enterocolitica/inmunología , Animales , Células Dendríticas/patología , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/patología , Interferón gamma/inmunología , Ratones , Ratones Noqueados , Transducción de Señal/inmunología , Receptor Toll-Like 4/inmunología , Yersiniosis/patologíaRESUMEN
Nasal colonization is a major risk factor for S. aureus infections. The mechanisms responsible for colonization are still not well understood and involve several factors on the host and the bacterial side. One key factor is the cell wall teichoic acid (WTA) of S. aureus, which governs direct interactions with nasal epithelial surfaces. We report here the first receptor for the cell wall glycopolymer WTA on nasal epithelial cells. In several assay systems this type F-scavenger receptor, termed SREC-I, bound WTA in a charge dependent manner and mediated adhesion to nasal epithelial cells in vitro. The impact of WTA and SREC-I interaction on epithelial adhesion was especially pronounced under shear stress, which resembles the conditions found in the nasal cavity. Most importantly, we demonstrate here a key role of the WTA-receptor interaction in a cotton rat model of nasal colonization. When we inhibited WTA mediated adhesion with a SREC-I antibody, nasal colonization in the animal model was strongly reduced at the early onset of colonization. More importantly, colonization stayed low over an extended period of 6 days. Therefore we propose targeting of this glycopolymer-receptor interaction as a novel strategy to prevent or control S. aureus nasal colonization.
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Adhesión Bacteriana/genética , Células Epiteliales/microbiología , Cavidad Nasal/microbiología , Receptores Depuradores de Clase F/fisiología , Staphylococcus aureus/crecimiento & desarrollo , Staphylococcus aureus/fisiología , Ácidos Teicoicos/metabolismo , Animales , Células CHO , Pared Celular/metabolismo , Células Cultivadas , Cricetinae , Cricetulus , Interacciones Huésped-Patógeno/genética , Humanos , Ratas , Receptores Depuradores de Clase F/metabolismo , Sigmodontinae , Infecciones Estafilocócicas/genética , Infecciones Estafilocócicas/metabolismo , Infecciones Estafilocócicas/microbiologíaRESUMEN
Lipopolysaccharides (LPS) of Gram negative bacteria are one of the most potent stimulators of the host innate immune system and LPS recognition is essential for the host organism to clear infections of invading bacterial pathogens. Here we review on the latest research on how LPS is sensed by host cells and how distinct LPS structures differentially modulate the strength of the host immune response. Much is known about host immunological reactions towards pathogens via recognition of their LPS, as well as strategies of pathogens to modulate their LPS structure in order to evade the immune system. However, less is known about differential sensing of lipopolysaccharides of commensal bacteria in the intestine and how this contributes to manifestation or destruction of the intestinal homeostasis. LPS sensing is necessary to fight pathogens. However, sensing of LPS of gut commensal bacteria can simultaneously be disadvantageous for the genetically predisposed host, since this might lead to damage of the intestinal homeostasis and therefore to chronic intestinal inflammation. However, less immunogenic LPS could also serve as therapeutics to antagonize an overreacting innate immune system. Therefore, commensal gut bacteria-derived LPS could prevent from uncontrolled intestinal immune response in the intestine which makes LPS an attractive therapeutical approach to treat e.g. IBD.