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1.
Artigo em Inglês | MEDLINE | ID: mdl-31818817

RESUMO

With the aim to identify potential new targets to restore antimicrobial susceptibility of multidrug-resistant (MDR) Pseudomonas aeruginosa (Pa), we generated a high-density transposon (Tn) insertion mutant library in a MDR Pa bloodstream 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 lytic transglycosylase MltG, the murein endopeptidase MepM1, the MurNAc/GlcNAc-kinase AmgK and the uncharacterized protein YgfB that all were identified in our screen as playing a decisive role for survival of treatment with cefepime or meropenem. We found that the antibiotic resistance of Pa can be overcome by targeting usually non-essential 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 of 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 developing drugs that could be used to restore the sensitivity towards existing antibiotics specifically in MDR strains of Pa.

2.
Front Microbiol ; 10: 2582, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31781074

RESUMO

Members of the Enterobacter (E.) cloacae complex have emerged as important pathogens frequently encountered in nosocomial infections. Several outbreaks with E. cloacae complex have been reported in recent years, especially in neonatal units. Fast and reliable strain typing methods are crucial for real-time surveillance and outbreak analysis to detect pathogen reservoirs and transmission routes. The aim of this study was to evaluate the performance of Fourier-transform infrared (FTIR) spectroscopy as a fast method for typing of clinical E. cloacae complex isolates, when whole genome sequencing (WGS) analysis was used as reference. First, the technique was used retrospectively on 24 first isolates of E. cloacae complex strains from neonatal patients and showed good concordance with SNP-based clustering [adjusted rand index (ARI) = 0.818] and with the sequence type (ST) (ARI = 0.801). 29 consecutive isolates from the same patients were shown by WGS analysis to almost always belong to the same SNP cluster as the first isolates, which was only inconsistently recognized by FTIR spectroscopy. Training of an artificial neural network (ANN) with all FTIR spectra from sequenced strains markedly improved the recognition of related and unrelated isolate spectra. In a second step, FTIR spectroscopy was applied on 14 strains during an outbreak with E. cloacae complex and provided fast typing results that were confirmed by WGS analysis. In conclusion, FTIR spectroscopy is a promising tool for strain typing of clinical E. cloacae complex strains. Discriminatory power can be improved by implementing an ANN for spectrum analysis. Due to its low costs and fast turnaround times, the method presents a valuable tool for real-time surveillance as well as outbreak analysis.

3.
BMC Biol ; 17(1): 76, 2019 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-31533707

RESUMO

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.

4.
Front Microbiol ; 10: 1742, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31440214

RESUMO

In this study, we aimed to elucidate a prolonged outbreak of extensively drug-resistant (XDR) Pseudomonas aeruginosa, at two adjacent hospitals over a time course of 4 years. Since all strains exhibited a similar antibiotic susceptibility pattern and carried the carbapenemase gene blaVIM, a monoclonal outbreak was assumed. To shed light on the intra-hospital evolution of these strains over time, whole genome sequence (WGS) analysis of 100 clinical and environmental outbreak strains was employed. Phylogenetic analysis of the core genome revealed the outbreak to be polyclonal, rather than monoclonal as initially suggested. The vast majority of strains fell into one of two major clusters, composed of 27 and 59 strains, and their accessory genome each revealed over 400 and 600 accessory genes, respectively, thus indicating an unexpectedly high structural diversity among phylogenetically clustered strains. Further analyses focused on the cluster with 59 strains, representing the hospital from which both clinical and environmental strains were available. Our investigation clearly shows both accumulation and loss of genes occur very frequently over time, as reflected by analysis of protein enrichment as well as functional enrichment. In addition, we investigated adaptation through single nucleotide polymorphisms (SNPs). Among the genes affected by SNPs, there are a multidrug efflux pump (mexZ) and a mercury detoxification operon (merR) with deleterious mutations, potentially leading to loss of repression with resistance against antibiotics and disinfectants. Our results not only confirm WGS to be a powerful tool for epidemiologic analyses, but also provide insights into molecular evolution during an XDR P. aeruginosa hospital outbreak. Genome mutation unveiled a striking genetic plasticity on an unexpectedly high level, mostly driven by horizontal gene transfer. Our study adds valuable information to the molecular understanding of "real-world" Intra-hospital P. aeruginosa evolution and is a step forward toward more personalized medicine in infection control.

5.
Mol Ther ; 27(11): 1974-1991, 2019 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-31416777

RESUMO

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.

6.
Int J Med Microbiol ; 309(5): 344-350, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31178419

RESUMO

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.


Assuntos
Aderência Bacteriana , Interações entre Hospedeiro e Microrganismos , Transporte Proteico , Sistemas de Secreção Tipo III/metabolismo , Adesinas Bacterianas/metabolismo , Animais , Proteínas da Membrana Bacteriana Externa/metabolismo , Humanos , Shigella/imunologia , Shigella/patogenicidade , Virulência/imunologia , Fatores de Virulência/metabolismo , Yersinia/imunologia , Yersinia/patogenicidade
7.
PLoS Biol ; 17(6): e3000334, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31206517

RESUMO

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.

8.
Sci Rep ; 9(1): 6406, 2019 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-30992476

RESUMO

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

9.
Front Microbiol ; 10: 100, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30846971

RESUMO

Pseudomonas aeruginosa is one of the main causative agents of nosocomial infections and the spread of multidrug-resistant strains is rising. Therefore, novel strategies for therapy are urgently required. The outer membrane composition of Gram-negative pathogens and especially of Pa restricts the efficacy of antibiotic entry into the cell and determines virulence. For efficient outer membrane protein biogenesis, the ß-barrel assembly machinery (BAM) complex in the outer membrane and periplasmic chaperones like Skp and SurA are crucial. Previous studies indicated that the importance of individual proteins involved in outer membrane protein biogenesis may vary between different Gram-negative species. In addition, since multidrug-resistant Pa strains pose a serious global threat, the interference with both virulence and antibiotic resistance by disturbing outer membrane protein biogenesis might be a new strategy to cope with this challenge. Therefore, deletion mutants of the non-essential BAM complex components bamB and bamC, of the skp homolog hlpA as well as a conditional mutant of surA were investigated. The most profound effects for both traits were associated with reduced levels of SurA, characterized by increased membrane permeability, enhanced sensitivity to antibiotic treatment and attenuation of virulence in a Galleria mellonella infection model. Strikingly, the depletion of SurA in a multidrug-resistant clinical bloodstream isolate re-sensitized the strain to antibiotic treatment. From our data we conclude that SurA of Pa serves as a promising target for developing a drug that shows antiinfective activity and re-sensitizes multidrug-resistant strains to antibiotics.

10.
Sci Rep ; 8(1): 13767, 2018 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-30213965

RESUMO

High throughput sequencing has been proposed as a one-stop solution for diagnostics and molecular typing directly from patient samples, allowing timely and appropriate implementation of measures for treatment, infection prevention and control. However, it is unclear how the variety of available methods impacts the end results. We applied shotgun metagenomics on diverse types of patient samples using three different methods to deplete human DNA prior to DNA extraction. Libraries were prepared and sequenced with Illumina chemistry. Data was analyzed using methods likely to be available in clinical microbiology laboratories using genomics. The results of microbial identification were compared to standard culture-based microbiological methods. On average, 75% of the reads corresponded to human DNA, being a major determinant in the analysis outcome. None of the kits was clearly superior suggesting that the initial ratio between host and microbial DNA or other sample characteristics were the major determinants of the proportion of microbial reads. Most pathogens identified by culture were also identified through metagenomics, but substantial differences were noted between the taxonomic classification tools. In two cases the high number of human reads resulted in insufficient sequencing depth of bacterial DNA for identification. In three samples, we could infer the probable multilocus sequence type of the most abundant species. The tools and databases used for taxonomic classification and antimicrobial resistance identification had a key impact on the results, recommending that efforts need to be aimed at standardization of the analysis methods if metagenomics is to be used routinely in clinical microbiology.


Assuntos
Bactérias/classificação , Bactérias/genética , DNA Bacteriano/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Metagenômica/métodos , Tipagem Molecular/métodos , Líquidos Corporais/microbiologia , Farmacorresistência Bacteriana/genética , Humanos , Testes de Sensibilidade Microbiana , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
11.
J Clin Microbiol ; 56(11)2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30135233

RESUMO

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.


Assuntos
Técnicas de Tipagem Bacteriana/métodos , Infecções por Klebsiella/microbiologia , Klebsiella/classificação , Klebsiella/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectroscopia de Infravermelho com Transformada de Fourier , Técnicas de Tipagem Bacteriana/normas , Análise por Conglomerados , Custos e Análise de Custo , Genoma Bacteriano/genética , Humanos , Klebsiella/química , Klebsiella/genética , Infecções por Klebsiella/diagnóstico , Polimorfismo de Nucleotídeo Único/genética , Sensibilidade e Especificidade , Análise de Sequência de DNA , Fatores de Tempo
13.
J Innate Immun ; 9(1): 33-51, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27798934

RESUMO

Complement resistance is an important virulence trait of Yersinia enterocolitica (Ye). The predominant virulence factor expressed by Ye is Yersinia adhesin A (YadA), which enables bacterial attachment to host cells and extracellular matrix and additionally allows the acquisition of soluble serum factors. The serum glycoprotein vitronectin (Vn) acts as an inhibitory regulator of the terminal complement complex by inhibiting the lytic pore formation. Here, we show YadA-mediated direct interaction of Ye with Vn and investigated the role of this Vn binding during mouse infection in vivo. Using different Yersinia strains, we identified a short stretch in the YadA head domain of Ye O:9 E40, similar to the 'uptake region' of Y. pseudotuberculosis YPIII YadA, as crucial for efficient Vn binding. Using recombinant fragments of Vn, we found the C-terminal part of Vn, including heparin-binding domain 3, to be responsible for binding to YadA. Moreover, we found that Vn bound to the bacterial surface is still functionally active and thus inhibits C5b-9 formation. In a mouse infection model, we demonstrate that Vn reduces complement-mediated killing of Ye O:9 E40 and, thus, improved bacterial survival. Taken together, these findings show that YadA-mediated Vn binding influences Ye pathogenesis.


Assuntos
Adesinas Bacterianas/metabolismo , Vitronectina/metabolismo , Yersiniose/imunologia , Yersinia enterocolitica/fisiologia , Animais , Bacteriólise , Proteínas do Sistema Complemento/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Imunomodulação , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Domínios Proteicos/genética , Especificidade da Espécie , Virulência , Vitronectina/genética , Yersinia enterocolitica/patogenicidade
15.
Artigo em Inglês | MEDLINE | ID: mdl-27956426

RESUMO

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.


Assuntos
Infecção Hospitalar/transmissão , Genoma Bacteriano , Filogenia , Infecções por Serratia/transmissão , Serratia marcescens/genética , Resistência beta-Lactâmica/genética , beta-Lactamases/genética , Antibacterianos/farmacologia , Técnicas de Tipagem Bacteriana , Teorema de Bayes , Células Clonais , Infecção Hospitalar/tratamento farmacológico , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , Eletroforese em Gel de Campo Pulsado , Expressão Gênica , Genótipo , Alemanha , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Plasmídeos/química , Plasmídeos/metabolismo , Infecções por Serratia/tratamento farmacológico , Infecções por Serratia/epidemiologia , Infecções por Serratia/microbiologia , Serratia marcescens/classificação , Serratia marcescens/efeitos dos fármacos , Serratia marcescens/crescimento & desenvolvimento , beta-Lactamases/metabolismo
16.
Sci Rep ; 6: 39053, 2016 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-27982054

RESUMO

ß-Barrel proteins are found in the outer membrane (OM) of Gram-negative bacteria, chloroplasts and mitochondria. The assembly of these proteins into the corresponding OM is facilitated by a dedicated protein complex that contains a central conserved ß-barrel protein termed BamA in bacteria and Tob55/Sam50 in mitochondria. BamA and Tob55 consist of a membrane-integral C-terminal domain that forms a ß-barrel pore and a soluble N-terminal portion comprised of one (in Tob55) or five (in BamA) polypeptide transport-associated (POTRA) domains. Currently the functional significance of this difference and whether the homology between BamA and Tob55 can allow them to replace each other are unclear. To address these issues we constructed hybrid Tob55/BamA proteins with differently configured N-terminal POTRA domains. We observed that constructs harboring a heterologous C-terminal domain could not functionally replace the bacterial BamA or the mitochondrial Tob55 demonstrating species-specific requirements. Interestingly, the various hybrid proteins in combination with the bacterial chaperones Skp or SurA supported to a variable extent the assembly of bacterial ß-barrel proteins into the mitochondrial OM. Collectively, our findings suggest that the membrane assembly of various ß-barrel proteins depends to a different extent on POTRA domains and periplasmic chaperones.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Escherichia coli/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Evolução Molecular , Mitocôndrias/genética , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência , Especificidade da Espécie
17.
PLoS One ; 11(10): e0164163, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27701464

RESUMO

Most frequently, gram-negative bacterial infections in humans are caused by Enterobacteriaceae and remain a major challenge in medical diagnostics. We non-invasively imaged moderate and severe systemic Yersinia enterocolitica infections in mice using the positron emission tomography (PET) tracer 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT), which is a marker of proliferation, and compared the in vivo results to the ex vivo biodistributions, bacterial loads, and histologies of the corresponding organs. Y. enterocolitica infection is detectable with histology using H&E staining and immunohistochemistry for Ki 67. [18F]FLT revealed only background uptake in the spleen, which is the main manifestation site of systemic Y. enterocolitica-infected mice. The uptake was independent of the infection dose. Antibody-based thymidine kinase 1 (Tk-1) staining confirmed the negative [18F]FLT-PET data. Histological alterations of spleen tissue, observed via Ki 67-antibody-based staining, can not be detected by [18F]FLT-PET in this model. Thus, the proliferation marker [18F]FLT is not a suitable tracer for the diagnosis of systemic Y. enterocolitica infection in the C57BL/6 animal model of yersiniosis.


Assuntos
Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/farmacocinética , Yersiniose/diagnóstico por imagem , Yersinia enterocolitica/fisiologia , Animais , Carga Bacteriana , Camundongos , Camundongos Endogâmicos C57BL , Traçadores Radioativos , Baço/metabolismo , Distribuição Tecidual
18.
Infect Immun ; 84(11): 3172-3181, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27550935

RESUMO

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.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Infiltração de Neutrófilos/fisiologia , Neutrófilos/citologia , Nódulos Linfáticos Agregados/citologia , Yersiniose/imunologia , Yersinia enterocolitica/patogenicidade , Animais , Carga Bacteriana , Proteínas da Membrana Bacteriana Externa/genética , Quimiocinas CXC/metabolismo , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Neutrófilos/metabolismo , Receptores de Quimiocinas/metabolismo , Virulência/fisiologia , Yersiniose/metabolismo , Yersiniose/microbiologia , Yersinia enterocolitica/imunologia
19.
J Autoimmun ; 75: 82-95, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27484364

RESUMO

Cathepsin S (CTSS) is a lysosomal protease whose activity regulation is important for MHC-II signaling and subsequent activation of CD4+ T cell mediated immune responses. Dysregulation of its enzymatic activity or enhanced secretion into extracellular environments is associated with the induction or progression of several autoimmune diseases. Here we demonstrate that commensal intestinal bacteria influence secretion rates and intracellular activity of host CTSS and that symbiotic bacteria, i.e. Bacteroides vulgatus mpk, may actively regulate this process and help to maintain physiological levels of CTSS activities in order to prevent from induction of pathological inflammation. The symbiont-controlled regulation of CTSS activity is mediated by anticipating reactive oxygen species induction in dendritic cells which, in turn, maintains cystatin C (CysC) monomer binding to CTSS. CysC monomers are potent endogenous CTSS inhibitors. This Bacteroides vulgatus caused and CysC dependent CTSS activity regulation is involved in the generation of tolerant intestinal dendritic cells contributing to prevention of T-cell mediated induction of colonic inflammation. Taken together, we demonstrate that symbionts of the intestinal microbiota regulate host CTSS activity and secretion and might therefore be an attractive approach to deal with CTSS associated autoimmune diseases.


Assuntos
Bactérias/imunologia , Catepsinas/imunologia , Microbioma Gastrointestinal/imunologia , Simbiose/imunologia , Animais , Bacteroides/imunologia , Bacteroides/fisiologia , Infecções por Bacteroides/imunologia , Infecções por Bacteroides/microbiologia , Benzopiranos/farmacologia , Western Blotting , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/microbiologia , Carbamatos/farmacologia , Catepsinas/antagonistas & inibidores , Catepsinas/genética , Células Cultivadas , Colite/imunologia , Colite/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Microbioma Gastrointestinal/fisiologia , Expressão Gênica/imunologia , Interações Hospedeiro-Patógeno/imunologia , Tolerância Imunológica/imunologia , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
20.
Int J Syst Evol Microbiol ; 66(8): 3005-3009, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27150727

RESUMO

A strain of obligately anaerobic, Gram-stain-negative and non-spore-forming rod-shaped bacterium was isolated from a human wound and characterized both phenotypically and genotypically. The strain was moderately saccharolytic and proteolytic. Phylogenetic analysis was based on full-length 16S rRNA gene sequence analysis and revealed the strain to represent a member of the genus Prevotella, but to be different from the described species, with the closest relationship to Prevotella bergensis and Prevotella multisaccharivorax. The genomic DNA G+C content was 43.2 mol%. The most abundant cellular long-chain fatty acids were 3-OH iso-C17 : 0, anteiso-C15 : 0 and iso-C15 : 0. In view of phenotypical and biochemical characteristics as well as gene sequencing, strain A1336T is considered to represent a novel species within the genus Prevotella, for which the name Prevotella colorans sp. nov. is proposed. The type strain is A1336T (=DSM 100333T =CCUG 67421T =CCOS 902T).


Assuntos
Filogenia , Prevotella/classificação , Ferimentos e Lesões/microbiologia , Idoso , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Feminino , Humanos , Pigmentação , Prevotella/genética , Prevotella/isolamento & purificação , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
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