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Translation research strategy in infectious diseases, combining the results from basic research with patient-orientated research, aims to bridge the gap between laboratory findings and clinical infectious disease practice to improve disease management. In an era of increasing antimicrobial resistance, there are four main areas of clinical and scientific uncertainty that need to be urgently addressed by translational research: (i) early diagnosis of antibiotic-resistant infections and the appropriateness of empirical antibiotic therapy; (ii) the identification of reservoirs of antibiotic-resistant pathogens; (iii) the development of new antibiotics with lower propensities to evoke resistance; and (iv) the development of new non-antibiotic drugs to be used in the prevention of the spread of resistant bacterial strains. Strict European collaboration among major stakeholders is therefore essential. Appropriate educational tools to train a new generation of scientists with regard to a multifaceted approach to antimicrobial resistance research should be developed. Key areas include the support and implementation of European networks focused on translational research and related education activities, making potential therapeutics more attractive to investors and helping academic investigators to determine whether new molecules can be developed with clinical applicability., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Mutations in the nucleotide-binding oligomerization domain-containing protein 2 (NOD2) play an important role in the pathogenesis of Crohn's disease. NOD2 is an intracellular pattern recognition receptor (PRR) that senses bacterial peptidoglycan (PGN) structures, e.g., muramyl dipeptide (MDP). Here we focused on the effect of more-cross-linked, polymeric PGN fragments (PGNpol) in the activation of the innate immune system. In this study, the effect of combined NOD2 and Toll-like receptor 2 (TLR2) stimulation was examined compared to single stimulation of the NOD2 receptor alone. PGNpol species derived from a lipoprotein-containing Staphylococcus aureus strain (SA113) and a lipoprotein-deficient strain (SA113 Δlgt) were isolated. While PGNpol constitutes a combined NOD2 and TLR2 ligand, lipoprotein-deficient PGNpolΔlgt leads to activation of the immune system only via the NOD2 receptor. Murine bone marrow-derived dendritic cells (BMDCs), J774 cells, and Mono Mac 6 (MM6) cells were stimulated with these ligands. Cytokines (interleukin-6 [IL-6], IL-12p40, and tumor necrosis factor alpha [TNF-α]) as well as DC activation and maturation parameters were measured. Stimulation with PGNpolΔlgt did not lead to enhanced cytokine secretion or DC activation and maturation. However, stimulation with PGNpol led to strong cytokine secretion and subsequent DC maturation. These results were confirmed in MM6 and J774 cells. We showed that the NOD2-mediated activation of DCs with PGNpol was dependent on TLR2 costimulation. Therefore, signaling via both receptors leads to a more potent activation of the immune system than that with stimulation via each receptor alone., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Yersinia adhesin A (YadA) belongs to a class of bacterial adhesins that form trimeric structures. Their mature form contains a passenger domain and a C-terminal β-domain that anchors the protein in the outer membrane (OM). Little is known about how precursors of such proteins cross the periplasm and assemble into the OM. In the present study we took advantage of the evolutionary conservation in the biogenesis of β-barrel proteins between bacteria and mitochondria. We previously observed that upon expression in yeast cells, bacterial β-barrel proteins including the transmembrane domain of YadA assemble into the mitochondrial OM. In the current study we found that when expressed in yeast cells both the monomeric and trimeric forms of full-length YadA were detected in mitochondria but only the trimeric species was fully integrated into the OM. The oligomeric form was exposed on the surface of the organelle in its native conformation and maintained its capacity to adhere to host cells. The co-expression of YadA with a mitochondria-targeted form of the bacterial periplasmic chaperone Skp, but not with SurA or SecB, resulted in enhanced levels of both forms of YadA. Taken together, these results indicate that the proper assembly of trimeric autotransporter can occur also in a system lacking the lipoproteins of the BAM machinery and is specifically enhanced by the chaperone Skp., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

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.

Background & Aims: The intestinal microbiota is an important determinant of the mucosal response. In patients with inflammatory bowel diseases, the mucosal immune system has inappropriate interactions with the intestinal microbiota. We investigated how the composition of the intestinal microbiota affects its endotoxicity and development of colitis in mice., Methods: Germ-free C57BL/6J-Rag(1tm1Mom) (Rag1(-/-)) mice were colonized with 2 different types of complex intestinal microbiota. Colitis was induced in Rag1(-/-) mice by transfer of CD4(+)CD62L(+) T cells from C57BL/6J mice. Colonic tissues were collected and used for histologic analysis and cell isolation. Activation of lamina propria dendritic cells and T cells was analyzed by flow cytometry., Results: After transfer of CD4(+)CD62L(+) T cells, mice with intestinal Endo(lo) microbiota (a low proportion of Enterobacteriaceae, high proportion of Bacteroidetes, and low endotoxicity) maintained mucosal immune homeostasis, and mice with highly endotoxic Endo(hi) microbiota (a high proportion of Enterobacteriaceae and low proportion of Bacteroidetes) developed colitis. To determine whether the effects of Endo(hi) microbiota were related to the higher endotoxic activity of lipopolysaccharide (LPS), we compared LPS from Enterobacteriaceae with that of Bacteroidetes. Administration of Escherichia coli JM83 (wild-type LPS) to the mice exacerbated colitis, and Escherichia coli JM83 + htrBPG (mutated LPS, with lower endotoxicity, similar to that of Bacteroidetes) prevented development of colitis after transfer of the T cells to mice., Conclusions: The endotoxicity of LPS produced by the intestinal microbiota is a determinant of whether mice develop colitis after transfer of CD4(+)CD62L(+) T cells. This finding might aid the design of novel biologics or probiotics to treat inflammatory bowel disease., (Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Migration of dendritic cells (DCs) plays an important role in T-cell-mediated adaptive immune responses. Lipopolysaccharide (LPS) sensed by Toll-like receptor 4 (TLR4) serves as a signal for DC migration. We analyzed LPS-induced DC volume changes preceding the directed movement towards chemoattractants. Treatment with LPS resulted in rapid, prolonged cell swelling in wild-type (WT), but not in TLR4(-/-) bone marrow-derived (BM) DCs indicating that TLR4 signaling is essential for LPS-induced swelling. As a consequence, LPS-treatment enhanced the migratory activity along a chemokine (CCL21)-gradient in WT, but not in TLR4-deficient BMDCs suggesting that the LPS/TLR4-induced swelling response facilitates DC migration. Moreover, the role of calcium-activated potassium channels (K(Ca) 3.1) as putative regulators of immune cell volume regulation and migration was analyzed in LPS-challenged BMDCs. We found that the LPS-induced swelling of K(Ca) 3.1-deficient DCs was impaired when compared to WT DCs. Accordingly, the LPS-induced increase in [Ca(2+)](i) detected in WT DCs was reduced in K(Ca) 3.1-deficient DCs. Finally, directed migration of LPS-challenged K(Ca) 3.1-deficient DCs was low compared to WT DCs indicating that activation of K(Ca) 3.1 is involved in LPS-induced DC migration. These findings suggest that both TLR4 and K(Ca) 3.1 contribute to the migration of LPS-activated DCs as an important feature of the adaptive immune response., (© 2013 The Societies and Wiley Publishing Asia Pty Ltd.)

The incidence of tuberculosis (TB) and especially multidrug-resistant TB (MDR) continues to increase alarmingly worldwide, and reliable and fast diagnosis of MDR is essential for the adequate treatment of patients. In contrast to the standard culture methods, nucleid acid amplification tests (NAATs) provide information about presence of Mycobacterium tuberculosis complex (MTBC) DNA and a potential resistance pattern within hours. We analyzed specimens of 110 patients from Nigeria comparing culture-based drug susceptibility testing (DST) to NAAT assays detecting isoniazid (INH), rifampicin (RMP) (GenoType MTBDRplus), and ethambutol (EMB) (GenoType MTBDRsl) resistance. Compared to DST, the GenoType MTBDRplus and MTBDRsl showed a specificity of 100% (86.3-100) and a sensitivity of 86% (42.1-99.6%) for detection of INH and a specificity of 100% (86.3-100) and a sensitivity of 83% (35.9-99.6%) for detection of RMP, and a sensitivity 100% (47.8-100%) for EMB resistance. However, in two strains, the NAAT assays provided false susceptible results as the mutations causing resistance were in genomic regions not covered by the probes of the GenoType MTBDRplus assay. We show that, in combination to DST, application of the GenoType MTBDRplus and GenoType MTBDRsl assays might be a useful additional tool to allow a rapid and safe diagnosis of MDR and extensively drug-resistant (XDR) MTBC.

Objectives: The time to positivity (TTP), measured as the time span between the start of incubation and the alert signal from the blood culture device, has been described as useful tool of prognosis in patients suffering from blood stream infection with Staphylococcus aureus, Escherichia coli and Klebsiella pneumonia. The present study investigates the relationship between TTP and in-hospital mortality in patients with monomicrobial Pseudomonas aeruginosa blood stream infection (PA-BSI)., Methods: From 2006 until 2012 a retrospective cohort study was undertaken in 3 hospitals in the region surrounding Tübingen, Germany. Seventy-four patients with monomicrobial PA-BSI were studied. TTP and clinical parameters were determined and analyzed by receiver operating characteristic (ROC) analysis and Cox regression., Results: The in-hospital mortality of our clinical cohort was 33.78%. In multivariate Cox regression, a TTP ≤ 18 h proved to be independently associated with mortality (HR 3.83, P = 0.012) along with SAPS II score (HR 1.04, P = 0.006), cardiac disease (HR 0.33, P = 0.008) and appropriate definitive antimicrobial treatment (HR 0.21, P = 0.013)., Conclusions: TTP is an easy-to-measure laboratory tool for prognosis in patients with monomicrobial PA-BSI, providing useful information in addition to clinical parameters., (Copyright © 2013 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

Background: Blood stream infections (BSI) with Pseudomonas aeruginosa lead to poor clinical outcomes. The worldwide emergence and spread of metallo-β-lactamase (MBL) producing, often multidrug-resistant organisms may further aggravate this problem. Our study aimed to investigate the effect of MBL-producing P. aeruginosa (MBL-PA) and various other resistance phenotypes on clinical outcomes., Methods: A retrospective cohort study was conducted in three German hospitals. Medical files from 2006 until 2012 were studied, and a number of 113 patients with P. aeruginosa BSI were included. The presence of VIM, IMP and NDM genes was detected using molecular techniques. Genetic relatedness was assessed through multilocus sequence typing (MLST). The effect of resistance patterns or MBL production on clinical outcomes was investigated by using multivariate Cox regression models., Results: In-hospital mortality was significantly higher in patients with MBL-PA and multidrug-resistant P. aeruginosa. However, neither BSI with MBL-PA nor BSI with various resistance phenotypes of P. aeruginosa were independently associated with mortality or length of hospital stay. In multivariate models, the SAPS II score (HR 1.046), appropriate definitive treatment (HR range 0.25-0.26), and cardiovascular disease (HR range 0.44-0.46) were independent predictors of mortality. Concomitant infections were associated with an excess length of stay (HR < 1)., Conclusions: Medication with appropriate antimicrobial agents at any time during the course of infection remains the key for improving clinical outcomes in patients with P. aeruginosa BSI and should be combined with a strict implementation of routine infection control measures.

Type I IFN signaling amplifies the secretion of LPS-induced proinflammatory cytokines such as TNF-α or IL-6 and might thus contribute to the high mortality associated with Gram-negative septic shock in humans. The underlying molecular mechanism, however, is ill defined. In this study, we report the generation of mice deficient in IFN-induced protein with tetratricopeptide repeats 2 (Ifit2) and demonstrate that Ifit2 is a critical signaling intermediate for LPS-induced septic shock. Ifit2 expression was significantly upregulated in response to LPS challenge in an IFN-α receptor- and IFN regulatory factor (Irf)9-dependent manner. Also, LPS induced secretion of IL-6 and TNF-α by bone marrow-derived macrophages (BMDMs) was significantly enhanced in the presence of Ifit2. In accordance, Ifit2-deficient mice exhibited significantly reduced serum levels of IL-6 and TNF-α and reduced mortality in an endotoxin shock model. Investigation of the underlying signal transduction events revealed that Ifit2 upregulates Irf3 phosphorylation. In the absence of Irf3, reduced Ifn-β mRNA expression and Ifit2 protein expression after LPS stimulation was found. Also, Tnf-α and Il-6 secretion but not Tnf-α and Il-6 mRNA expression levels were reduced. Thus, IFN-stimulated Ifit2 via enhanced Irf3 phosphorylation upregulates the secretion of proinflammatory cytokines. It thereby amplifies LPS-induced cytokine production and critically influences the outcome of endotoxin shock.

In industrialized countries bacterial intestinal infections are commonly caused by enteropathogenic Enterobacteriaceae. The interaction of the microbiota with the host immune system determines the adequacy of an appropriate response against pathogens. In this study we addressed whether the probiotic Bifidobacterium adolescentis is protective during intestinal Yersinia enterocolitica infection. Female C57BL/6 mice were fed with B. adolescentis, infected with Yersinia enterocolitica, or B. adolescentis fed and subsequently infected with Yersinia enterocolitica. B. adolescentis fed and Yersinia infected mice were protected from Yersinia infection as indicated by a significantly reduced weight loss and splenic Yersinia load when compared to Yersinia infected mice. Moreover, protection from infection was associated with increased intestinal plasmacytoid dendritic cell and regulatory T-cell frequencies. Plasmacytoid dendritic cell function was investigated using depletion experiments by injecting B. adolescentis fed, Yersinia infected C57BL/6 mice with anti-mouse PDCA-1 antibody, to deplete plasmacytoid dendritic cells, or respective isotype control. The B. adolescentis-mediated protection from Yersinia dissemination to the spleen was abrogated after plasmacytoid dendritic cell depletion indicating a crucial function for pDC in control of intestinal Yersinia infection. We suggest that feeding of B. adolescentis modulates the intestinal immune system in terms of increased plasmacytoid dendritic cell and regulatory T-cell frequencies, which might account for the B. adolescentis-mediated protection from Yersinia enterocolitica infection.

The association between antimicrobial consumption and resistance in nonfermentative Gram-negative bacteria is well-known. Antimicrobial restriction, implemented in clinical routines by antibiotic stewardship programs (ASPs), is considered a means to reduce resistance rates. Whether and how antimicrobial restriction can accomplish this goal is still unknown though. This leads to an element of uncertainty when designing strategies for ASPs. From January 2002 until December 2011, an observational study was performed at the University Hospital Tübingen, Tübingen, Germany, to investigate the association between antimicrobial use and resistance rates in Pseudomonas aeruginosa. Transfer function models were used to determine such associations and to simulate antimicrobial restriction strategies. Various positive associations between antimicrobial consumption and resistance were observed in our setting. Surprisingly, impact estimations of different antimicrobial restriction strategies revealed relatively low intervention expenses to effectively attenuate the observed increase in resistance. For example, a simulated intervention of an annual 4% reduction in the use of meropenem over 3 years from 2009 until 2011 yielded a 62.5% attenuation (95% confidence interval, 15% to 110%) in the rising trend of multidrug-resistant Pseudomonas aeruginosa (three- and four-class-resistant P. aeruginosa [34MRGN-PA]). Time series analysis models derived from past data may be a tool to predict the outcome of antimicrobial restriction strategies, and could be used to design ASPs.

The intestinal microbiota is a complex community of microorganisms that colonizes the gastrointestinal tract. The composition of the intestinal microbiota and the number of microorganisms differ in dependency of the local environmental conditions. The intestinal microbiota has an important impact on the development of the intestinal architecture and function, it influences the development of the gut-associated immune system, and epithelial cell functions. One of the most important functions of the intestinal microbiota is the prevention of bacterial overgrowth and susceptibility to infection with enteropathogenic organisms. Additionally, the intestinale microbiota plays a crucial role in the development of the systemic immunity and has an important influence on the host nutrition and metabolism. However, in genetically predisposed hosts, the intestinal microbiota is involved in the pathophysiology of inflammatory bowel diseases and pouchitis. Additionally, recent studies suggest that there might be an inflammation triggering effect of the intestinal microbiota in necrotizing enterocolitis. Here, we give an overview of the intestinal microbiota and its variety of roles in health and disease.

Yersinia adhesin A (YadA) is a major virulence factor of Yersinia enterocolitica. YadA mediates host cell binding and autoaggregation and protects the pathogen from killing by the complement system. Previous studies demonstrated that YadA is the most important single factor mediating serum resistance of Y. enterocolitica, presumably by binding C4b binding protein (C4BP) and factor H, which are both complement inhibitors. Factor H acts as a cofactor for factor I-mediated cleavage of C3b into the inactive form iC3b and thus prevents formation of inflammatory effector compounds and the terminal complement complex. In this study, we challenged the current direct binding model of factor H to YadA and show that Y. enterocolitica YadA recruits C3b and iC3b directly, without the need of an active complement cascade or additional serum factors. Enhanced binding of C3b does not decrease survival of YadA-expressing Yersiniae because C3b becomes readily inactivated by factor H and factor I. Binding of factor H to YadA is greatly reduced in the absence of C3. Experiments using Yersinia lacking YadA or expressing YadA with reduced trimeric stability clearly demonstrate that both the presence and full trimeric stability of YadA are essential for complement resistance. A novel mechanism of factor H binding is presented in which YadA exploits recruitment of C3b or iC3b to attract large amounts of factor H. As a consequence, formation of the terminal complement complex is limited and bacterial survival is enhanced. These findings add a new aspect of how Y. enterocolitica effectively evades the host complement system.

Background: "Quorum sensing" (QS) is the phenomenon which allows single bacterial cells to measure the concentration of bacterial signal molecules. Two principle different QS systems are known, the Autoinducer 1 system (AI-1) for the intraspecies communication using different Acyl-homoserine lactones (AHL) and AI-2 for the interspecies communication. Aim of this study was to investigate QS of Escherichia coli Nissle 1917 (Mutaflor)., Results: While E. coli Nissle is producing AI-2 in a density dependent manner, no AI-1 was produced. To study the effect of AI-2 in the DSS (dextran sulphate sodium) induced mouse model of acute colitis, we silenced the corresponding gene luxS by intron insertion. The mutant bacterium E. coli Nissle::luxS was equally effective in colonizing the colon and the mutation turned out to be 100% stable during the course of the experiment. Isolating RNA from the colon mucosa and performing semiquantitative RT PCR, we were able to show that the expression of the pro-inflammatory cytokine IFN-y was suppressed in mice being infected with the E. coli Nissle wild type. Mice infected with the E. coli Nissle::luxS mutant showed a suppressed expression of IL-10 compared to uninfected mice, while the expression of the pro-inflammatory cytokines IL-6 and TNF-α was higher in these mice. The expression of mBD-1 was suppressed in mice being infected with the mutant in comparison to the mice not infected or infected with the wild type. No differences were seen in the histological examination of the colon sections in the different groups of mice., Conclusions: E. coli Nissle is producing AI-2 molecules, which are influencing the expression of cytokines in the mucosa of the colon in the DSS mice. However, if QS has a direct influence on the probiotic properties of E. coli Nissle remains to be elucidated.

The prevalence of infections with enterococci is increasing worldwide. However, little is known about the mechanisms which enable these opportunistic pathogens to cause infections of their host. Here we demonstrate that Enterococcus faecium in the presence of lysozyme induces necrosis in human and mouse cells after 4 h indicated by disrupted cellular membranes of epithelial (HeLa), myeloid (U937, J774A.1) and lymphoid (Jurkat J16, thymocytes), but not intestinal epithelial cells (CaCo-2, CMT-93). Using an appropriate mutant strain it was shown that the enterococcal surface-protein SgrA is involved in cell death induction in mouse cells (J774A.1, thymocytes). Microscopic analyses of epithelial cells 30 min post infection revealed that lysozyme increases adhesion of E. faecium to HeLa, but not CaCo-2 cells. At that time the phalloidin-FITC-stained cytoskeleton of infected cells was still intact, whereas 2 h post infection the F-actin network of HeLa, but not CaCo-2 cells was disrupted. Hence, the early, lysozyme-mediated increase of bacterial adherence plays an important role for cell death induction by E. faecium in HeLa cells. Moreover, bacterial extracellular hydrogen peroxide might contribute to necrosis induction, since the rate of propidium iodide-positive HeLa and J774A.1 cells was lowered after infection with a ROS-deficient E. faecium mutant., (Copyright © 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Background: DC are among the first antigen presenting cells encountering bacteria at mucosal surfaces, and play an important role in maintenance of regular homeostasis in the intestine. Upon stimulation DC undergo activation and maturation and as initiators of T cell responses they have the capacity to stimulate naïve T cells. However, stimulation of naïve murine DC with B. vulgatus or LPS at low concentration drives DC to a semimature (sm) state with low surface expression of activation-markers and a reduced capacity to activate T-cells. Additionally, semimature DC are nonresponsive to subsequent TLR stimulation in terms of maturation, TNF-α but not IL-6 production. Ligation of CD40 is an important mechanism in enhancing DC maturation, function and capacity to activate T-cells. We investigated whether the DC semimaturation can be overcome by CD40 ligation., Results: Upon CD40 ligation smDC secreted IL-12p40 but not the bioactive heterodimer IL-12p70. Additionally, CD40 ligation of smDC resulted in an increased production of IL-6 but not in an increased expression of CD40. Analysis of the phosphorylation pattern of MAP kinases showed that in smDC the p38 phosphorylation induced by CD40 ligation is inhibited. In contrast, phosphorylation of ERK upon CD40 ligation was independent of the DC maturation state., Conclusion: Our data show that the semimature differentiation state of DC can not be overcome by CD40 ligation. We suggest that the inability of CD40 ligation in overcoming DC semimaturation might contribute to the tolerogenic phenotype of semimature DC and at least partially account for maintenance of intestinal immune homeostasis.

Dendritic cells (DCs) as professional antigen-presenting cells play an important role in the initiation and modulation of the adaptive immune response. However, their role in the innate immune response against bacterial infections is not completely defined. Here we have analyzed the role of DCs and their impact on the innate anti-bacterial host defense in an experimental infection model of Yersinia enterocolitica (Ye). We used CD11c-diphtheria toxin (DT) mice to deplete DCs prior to severe infection with Ye. DC depletion significantly increased animal survival after Ye infection. The bacterial load in the spleen of DC-depleted mice was significantly lower than that of control mice throughout the infection. DC depletion was accompanied by an increase in the serum levels of CXCL1, G-CSF, IL-1α, and CCL2 and an increase in the numbers of splenic phagocytes. Functionally, splenocytes from DC-depleted mice exhibited an increased bacterial killing capacity compared to splenocytes from control mice. Cellular studies further showed that this was due to an increased production of reactive oxygen species (ROS) by neutrophils. Adoptive transfer of neutrophils from DC-depleted mice into control mice prior to Ye infection reduced the bacterial load to the level of Ye-infected DC-depleted mice, suggesting that the increased number of phagocytes with additional ROS production account for the decreased bacterial load. Furthermore, after incubation with serum from DC-depleted mice splenocytes from control mice increased their bacterial killing capacity, most likely due to enhanced ROS production by neutrophils, indicating that serum factors from DC-depleted mice account for this effect. In summary, we could show that DC depletion triggers phagocyte accumulation in the spleen and enhances their anti-bacterial killing capacity upon bacterial infection.

Urinary tract infections (UTI) are one of the most common infections in both hospitalised and ambulant patients. Rapid diagnostic of UTIs is necessary to provide early information about the presence of bacteria and the indication to administer an antibiotic therapy. Here we report on a study comparing 3 different rapid automated systems with the semiquantitative plate culture reference method in a university hospital with a highly complex patient population. In total, 2230 urine samples were consecutively tested using the UroQuick (Alifax), the BACSYS-40i (Sysmex), and the UF-1000i (Sysmex) system. In comparison to the results obtained by culture techniques, the automated systems showed a sensitivity of 73.0-80.9% and a specificity ranging between 61.8% and 92.8%. Additionally, sensitivity and specificity for the most common UTI-causing microorganisms were analysed and showed that sensitivity and specificity correlate with the colony forming units of microorganisms in the urine, with a sensitivity of nearly 90% for Gram-negative rods, typical for community acquired UTIs, but a very low sensitivity for Gram-positive bacteria and yeasts. This led us to the conclusion that the currently available automated systems might be rather helpful to analyse a typical UTI in an ambulant patient population but not for rapid diagnosis of UTIs in a complex population of hospitalized patients., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Glucocorticoid induced-leucine zipper (GILZ) has been shown to be induced in cells by different stimuli such as glucocorticoids, IL-10 or deprivation of IL-2. GILZ has anti-inflammatory properties and may be involved in signalling modulating apoptosis. Herein we demonstrate that wildtype Yersinia enterocolitica which carry the pYV plasmid upregulated GILZ mRNA levels and protein expression in epithelial cells. Infection of HeLa cells with different Yersinia mutant strains revealed that the protease activity of YopT, which cleaves the membrane-bound form of Rho GTPases was sufficient to induce GILZ expression. Similarly, Clostridium difficile toxin B, another bacterial inhibitor of Rho GTPases induced GILZ expression. YopT and toxin B both increased transcriptional activity of the GILZ promoter in HeLa cells. GILZ expression could not be linked to the inactivation of an individual Rho GTPase by these toxins. However, forced expression of RhoA and RhoB decreased basal GILZ promoter activity. Furthermore, MAPK activation proved necessary for profound GILZ induction by toxin B. Promoter studies and gel shift analyses defined binding of upstream stimulatory factor (USF) 1 and 2 to a canonical c-Myc binding site (E-box) in the GILZ promoter as a crucial step of its trans-activation. In addition we could show that USF-1 and USF-2 are essential for basal as well as toxin B induced GILZ expression. These findings define a novel way of GILZ promoter trans-activation mediated by bacterial toxins and differentiate it from those mediated by dexamethasone or deprivation of IL-2.

Small-colony variants (SCVs) of bacteria are slow-growing subpopulations which can cause latent or recurrent infections due to better intracellular survival compared to their wild-type counterparts. Atypical colony morphology and altered biochemical profile may lead to failure in identification of SCV strains. We here report for the first time the isolation of an Enterococcus faecium SCV phenotype. The case of a 65-year-old woman with acute myeloid leukaemia who developed symptoms of sepsis during induction chemotherapy is presented. E. faecium with normal and SCV phenotype was isolated from blood cultures. At the same time urine culture was positive with E. faecium suggesting that bacteraemia originated from the urinary tract. The SCV phenotype was characterized by atypical growth behaviour. Electron microscopic analyses revealed perturbation of the separation of daughter cells and the accumulation of cell wall material. Accordingly, the SCV variant showed a dysfunction or lack of spontaneous autolysis whereas the normal phenotype did not. In contrast to conventional identification systems based on biochemical characteristics, the E. faecium SCV was precisely identified by MALDI-TOF MS analysis implemented in our laboratory. Hence, the increasing use of MALDI-TOF MS analysis for the identification of bacteria might be an appropriate tool for the detection of SCV variants, the diagnosis of which is of importance for the clinical outcome and the antibiotic treatment., (Copyright © 2011. Published by Elsevier GmbH.)

Invasin and intimin are major virulence factors of enteropathogenic Yersiniae and Escherichia coli, mediating invasion into and intimate adherence to host cells, respectively. Several studies have hinted that extracellular portion of these homologous proteins might be exported via an autotransport mechanism, but rigorous experimental proof has been lacking. Here, we present a topology model for invasin and intimin, consistent with the hypothesis that the N-terminal β-barrel domain acts as a translocation pore to secrete the C-terminal passenger domain. We confirmed this topology model by inserting epitope tags into the loops of the β-barrel. We further show that obstructing the pore of β-barrel hinders the export of the passenger domain. As for classical autotransport, the biogenesis of invasin and intimin is dependent on the Bam complex and the periplasmic chaperone SurA, whereas the chaperone/protease DegP is involved in quality control. However, compared to classical autotransporters (Type Va secretion), the domain structure of intimin and invasin is inverted. We conclude that proteins of the intimin and invasin family constitute a novel group of autotransported proteins, and propose that this class of autotransporters be termed Type Ve secretion.

Background: Extended-spectrum β-lactamase (ESBL)-producing organisms are spreading worldwide in hospital and community settings., Methods: A total of 328 unduplicated ESBL-producing Enterobacteriaceae isolated in 2008 and 2009 at the University Hospital of Tübingen were analysed retrospectively., Results: Escherichia coli (n = 253) and Klebsiella spp. (n = 46) were the most frequent ESBL-producing species. The ESBL rates among E. coli and Klebsiella spp. increased from 3.8 and 2.1%, respectively, in 2008, to 5.2 and 2.4%, respectively, in 2009. Two E. coli and 3 Klebsiella pneumoniae ESBL producers were non-susceptible to ertapenem, most likely due to loss of porins. Antimicrobial susceptibility testing of selected, molecularly characterized ESBL producers revealed susceptibility to tigecycline among 97.9% (191/195) of the E. coli and 78.8% (26/33) of the K. pneumoniae isolates. PCR analysis and sequencing showed the presence of CTX-M-type enzymes in 91.3% of the E. coli and 87.9% of the K. pneumoniae isolates, whereby bla(CTX-M-15) was the most frequent ESBL gene both in E. coli (50.0%) and K. pneumoniae (51.5%). Only 7 single cases of potential patient-to-patient transmissions of E. coli strains were observed., Conclusions: Our data suggest that the increase in ESBL-producing E. coli and K. pneumoniae isolates at our hospital is mainly caused by growing import of Enterobacteriaceae harbouring CTX-M-type ESBLs., (Copyright © 2012 S. Karger AG, Basel.)

Over the last half century, a dramatic increase in the incidence of chronic inflammatory diseases, such as asthma, allergy, and irritable bowel syndrome, has rightfully led to concern about how the modern lifestyle might inappropriately trigger innate physiologic defense mechanisms. Health care research in the Western world is faced with a significant challenge if it is to meet the needs of its populations in the decades ahead. The tools with which we hope to advance our understanding of the intrinsic and extrinsic mechanisms of chronic inflammatory diseases must therefore be adequately exploited and further developed to identify treatment and prevention strategies. There is an urgent need to prioritize resources and identify the most efficient scientific and societal initiatives to be adopted within this area. In this context national collaboration within Europe and beyond to establish state-of-the-art practices with an interdisciplinary perspective and promote an efficient exchange of best practices is essential. Such an approach likely represents the most efficient manner in which strategies for amelioration of the increase of chronic inflammatory diseases in the Western world can be achieved. The present report is based on a Forward Look initiative conducted by the European Medical Research Councils under the European Science Foundation. Experts from industry and academia, as well as relevant interest organizations, have been consulted in the process of conducting this initiative and have, based on this work, developed a set of final recommendations that target academic research, science funders, and policy makers., (Copyright © 2011 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Background: Gastrointestinal infections have been proposed to predict subsequent irritable bowel syndrome (IBS) but large-scale infectious events are rare and long-term data are missing., Methods: We identified 576 individuals with a Salmonella or Campylobacter infection between 2000 and 2009 that were followed by a short postal questionnaire asking for the presence of current symptoms in 2010. In case of agreement (n = 90), an extended postinfectious (PI)-IBS questionnaire was mailed including the Hospital Anxiety Depression Scale and the Patient Health Questionnaire., Key Results: A total of 189 patients reported back (36%); 98 had a Salmonella and 91 had a Campylobacter infection, of which 56 reported persistent symptoms (9.7% of the initial sample). Fifty-one patients returned the PI-IBS questionnaire. Of 48 patients with complete data, 15 reported no or mild symptoms of abdominal pain or discomfort while 17 had moderate and 16 severe symptoms. Twenty-two met Rome IBS criteria, 14 (29%) reported GI symptoms before the infection. Patients with moderate and/or severe PI-IBS symptoms were significantly more often females, were more often infected by Salmonella than by Campylobacter, had more severe symptoms during the initial infection, and had more often GI symptoms prior to the infection. They reported higher anxiety, depression, and somatisation scores, but were not different with respect to acute stool habits., Conclusions & Inferences: Nearly 10% of patients with an intestinal bacterial infection report postinfectious symptoms up to 10 years after the infectious event. They represent a clinically important population with high psychiatric comorbidity and somatic symptom burden., (© 2011 Blackwell Publishing Ltd.)

β-barrel proteins are found in the outer membranes of eukaryotic organelles of endosymbiotic origin as well as in the outer membrane of Gram-negative bacteria. Precursors of mitochondrial β-barrel proteins are synthesized in the cytosol and have to be targeted to the organelle. Currently, the signal that assures their specific targeting to mitochondria is poorly defined. To characterize the structural features needed for specific mitochondrial targeting and to test whether a full β-barrel structure is required, we expressed in yeast cells the β-barrel domain of the trimeric autotransporter Yersinia adhesin A (YadA). Trimeric autotransporters are found only in prokaryotes, where they are anchored to the outer membrane by a single 12-stranded β-barrel structure to which each monomer is contributing four β-strands. Importantly, we found that YadA is solely localized to the mitochondrial outer membrane, where it exists in a native trimeric conformation. These findings demonstrate that, rather than a linear sequence or a complete β-barrel structure, four β-strands are sufficient for the mitochondria to recognize and assemble a β-barrel protein. Remarkably, the evolutionary origin of mitochondria from bacteria enables them to import and assemble even proteins belonging to a class that is absent in eukaryotes.

Our laboratory strain Yersinia enterocolitica strain WA-314 biogroup 1B serotype O:8 displayed a different adhesion behavior to host cells compared to other Yersinia strains. To investigate whether this is based on differences in the gene content of the large pYV virulence plasmid which contains the major Yersinia adhesin YadA, we set out to sequence pYV(WA-314). pYV(WA-314) is very similar to pYV127/90, with a notable difference in the length of the Type III secretion system component YscP, which determines the needle length of the system. We found that we could improve the annotation of proteins previously described as "hypothetical" in pYV127/90 and other pYV plasmids, and show that pYV plasmids contain several and seemingly redundant plasmid partitioning and stabilization systems, explaining why these plasmids are not easily lost in laboratory cultures of Yersinia strains., (Copyright © 2010 Elsevier Inc. All rights reserved.)

CD4(+) T cells are essential for the control of Yersinia enterocolitica (Ye) infection in mice. Ye can inhibit dendritic cell (DC) antigen uptake and degradation, maturation and subsequently T-cell activation in vitro. Here we investigated the effects of Ye infection on splenic DCs and T-cell proliferation in an experimental mouse infection model. We found that OVA-specific CD4(+) T cells had a reduced potential to proliferate when stimulated with OVA after infection with Ye compared to control mice. Additionally, proliferation of OVA-specific CD4(+) T cells was markedly reduced when cultured with splenic CD8α(+) DCs from Ye infected mice in the presence of OVA. In contrast, T-cell proliferation was not impaired in cultures with CD4(+) or CD4(-)CD8α(-) DCs isolated from Ye infected mice. However, OVA uptake and degradation as well as cytokine production were impaired in CD8α(+) DCs, but not in CD4(+) and CD4(-)CD8α(-) DCs after Ye infection. Pathogenicity factors (Yops) from Ye were most frequently injected into CD8α(+) DCs, resulting in less MHC class II and CD86 expression than on non-injected CD8α(+) DCs. Three days post infection with Ye the number of splenic CD8α(+) and CD4(+) DCs was reduced by 50% and 90%, respectively. The decreased number of DC subsets, which was dependent on TLR4 and TRIF signaling, was the result of a faster proliferation and suppressed de novo DC generation. Together, we show that Ye infection negatively regulates the stimulatory capacity of some but not all splenic DC subpopulations in vivo. This leads to differential antigen uptake and degradation, cytokine production, cell loss, and cell death rates in various DC subpopulations. The data suggest that these effects might be caused directly by injection of Yops into DCs and indirectly by affecting the homeostasis of CD4(+) and CD8α(+) DCs. These events may contribute to reduced T-cell proliferation and immune evasion of Ye.

The Bam complex is a highly conserved multiprotein machine essential for the assembly of β-barrel outer membrane proteins. It is composed of the essential outer membrane protein BamA and four outer membrane associated lipoproteins BamB-E. The Yersinia enterocolitica Adhesin A (YadA) is the prototype of trimeric auotransporter adhesins (TAAs), consisting of a head, stalk and a β-barrel membrane anchor. To investigate the role of BamA in biogenesis of TAAs, we expressed YadA in a BamA-depleted strain of Escherichia coli, which resulted in degradation of YadA. Yeast-two-hybrid experiments and immunofluorescence studies revealed that BamA and YadA interact directly and colocalize. As BamA recognizes the C-terminus of OMPs, we exchanged the nine most C-terminal amino acids of YadA. Substitution of the amino acids in position 1, 3 or 5 from the C-terminus with glycine resulted in DegP-dependent degradation of YadA. Despite degradation all YadA proteins assembled in the outer membrane. In summary we demonstrate that (i) BamA is essential for biogenesis of the TAA YadA, (ii) BamA interacts directly with YadA, (iii) the C-terminal amino acid motif of YadA is important for the BamA-dependent assembly and differs slightly compared with other OMPs, and (iv) BamA and YadA colocalize., (© 2010 Blackwell Publishing Ltd.)

Enterococci are commensal organisms in the alimentary tract. However, they can cause a variety of life-threatening infections, especially in nosocomial settings. We hypothesized that induction of cell death might enable these facultative pathogenic bacteria to evade the innate immune response and to cause infections of their host. We demonstrate that E. faecium when exposed to lysozyme induces cell death in macrophages in vitro and in vivo. Flow cytometric analyses of J774A.1 macrophages infected with E. faecium revealed loss of cell membrane integrity indicated by uptake of propidium iodide and decrease of the inner mitochondrial transmembrane potential DeltaPsi(m). Inhibition of caspases, treatment of macrophages with cytochalasin D, or rifampicin did not prevent cells from dying, suggesting cell death mechanisms that are independent of caspase activation, bacterial uptake, and intracellular bacterial replication. Characteristics of necrotic cell death were demonstrated by both lack of procaspase 3 activation and cell shrinkage, electron microscopy, and release of lactate dehydrogenase. Pretreatment of E. faecium with lysozyme and subsequently with broad spectrum protease considerably reduced cell death, suggesting that a bacterial surface protein is causative for cell death induction. Moreover, in a mouse peritonitis model we demonstrated that E. faecium induces cell death of peritoneal macrophages in vivo. Altogether, our results show that enterococci, under specific conditions such as exposure to lysozyme, induce necrotic cell death in macrophages, which might contribute to disseminated infections by these facultative pathogenic bacteria.

A young, previously healthy and immunocompetent patient was transferred to our hospital to recover a suspected Ascaris worm from his gall bladder. Although the diagnosis of Ascaris infection could not be confirmed, the patient suffered from cholecystitis. To our surprise, the respiratory situation of the patient deteriorated within 24 h under antibiotic therapy and he had to be transferred to the intensive care unit for mechanical respiration. Human cytomegalovirus (HCMV) was isolated directly from a bronchoalveolar lavage (BAL) sample, and Mycoplasma pneumoniae DNA was detected by PCR in an enrichment culture of the same BAL sample. Serology for HCMV and M. pneumoniae clearly supported a primary/post-primary infection for both agents (IgM detection, increase of IgG titres and, in the case of HCMV, a low avidity index of only 22 %). Therefore, we assumed that a rare HCMV and M. pneumoniae coinfection was the aetiology of the fulminant pneumonia. Under broad antibiotic and antiviral treatment, the situation of the patient improved only very slowly.

Probiotics are viable microorganisms that are increasingly used for treatment of a variety of diseases. Occasionally, however, probiotics may have adverse clinical effects, including septicemia. Here we examined the role of the intestinal microbiota and the adaptive immune system in preventing translocation of probiotics (e.g., Escherichia coli Nissle). We challenged C57BL/6J mice raised under germfree conditions (GF-raised C57BL/6J mice) and Rag1(-/-) mice raised under germfree conditions (GF-raised Rag1(-/-) mice) and under specific-pathogen-free conditions (SPF-raised Rag1(-/-) mice) with probiotic E. coli strain Nissle 1917, strain Nissle 1917 mutants, the commensal strain E. coli mpk, or Bacteroides vulgatus mpk. Additionally, we reconstituted Rag1(-/-) mice with CD4(+) T cells. E. coli translocation and dissemination and the mortality of mice were assessed. In GF-raised Rag1(-/-) mice, but not in SPF-raised Rag1(-/-) mice or GF-raised C57BL/6J mice, oral challenge with E. coli strain Nissle 1917, but not oral challenge with E. coli mpk, resulted in translocation and dissemination. The mortality rate was significantly higher for E. coli strain Nissle 1917-challenged GF-raised Rag1(-/-) mice (100%; P < 0.001) than for E. coli strain Nissle 1917-challenged SPF-raised Rag1(-/-) mice (0%) and GF-raised C57BL/6J mice (0%). Translocation of and mortality due to strain E. coli Nissle 1917 in GF-raised Rag1(-/-) mice were prevented when mice were reconstituted with T cells prior to strain E. coli Nissle 1917 challenge, but not when mice were reconstituted with T cells after E. coli strain Nissle 1917 challenge. Cocolonization experiments revealed that E. coli mpk could not prevent translocation of strain E. coli Nissle 1917. Moreover, we demonstrated that neither lipopolysaccharide structure nor flagella play a role in E. coli strain Nissle 1917 translocation and dissemination. Our results suggest that if both the microbiota and adaptive immunity are defective, translocation across the intestinal epithelium and dissemination of the probiotic E. coli strain Nissle 1917 may occur and have potentially severe adverse effects. Future work should define the possibly related molecular factors that promote probiotic functions, fitness, and facultative pathogenicity.

Yersinia adhesin A (YadA) is a trimeric autotransporter adhesin with multiple functions in host-pathogen interactions. The aim of this study was to dissect the virulence functions promoted by YadA in vitro and in vivo. To accomplish this, we generated Yersinia enterocolitica O:8 mutants expressing point mutations in YadA G389, a highly conserved residue in the membrane anchor of YadA, and analyzed their impact on YadA expression and virulence functions. We found that point mutations of YadA G389 led to impaired transport, stability, and surface display of YadA. YadA G389A and G389S mutants showed comparable YadA surface expression, autoagglutination, and adhesion to those of wild-type YadA but displayed reduced trimer stability and complement resistance in vitro and were 10- to 1,000-fold attenuated in experimental Y. enterocolitica infection in mice. The G389T, G389N, and G389H mutants lost trimer stability, exhibited strongly reduced surface display, autoagglutination, adhesion properties, and complement resistance, and were avirulent (>10,000-fold attenuation) in mice. Our data demonstrate that G389 is a critical residue of YadA, required for optimal trimer stability, transport, surface display, and serum resistance. We also show that stable trimeric YadA protein is essential for virulence of Y. enterocolitica.

DJ-1 is a neuroprotective gene mutated in recessive Parkinson's disease (PD). In addition to direct protective functions in neurons, DJ-1 regulates neuroinflammatory signaling in primary mouse brain astrocytes. To assess the influence of DJ-1 on innate immunity signaling in vivo, we have generated djr-1 knockout Caenorhabditis elegans. When grown on pathogenic gram-negative bacteria, djr-1 (-/-) worms showed stronger phosphorylation of p38 mitogen-activated protein kinase (PMK-1) and hyper-induction of PMK-1 target genes. Thus, PD-associated DJ-1 contributes to regulation of innate immunity.

Dendritic cells (DCs) are key players in activation of the adaptive immune system by their ability of antigen presentation to and priming of T cells. An increasing body of evidence suggests that DCs may also play an important role in induction of tolerance, predominantly by induction of regulatory T cells (T(reg)). More recently, data have been published on how Toll-like receptor (TLR) ligands and cytokines affect DC differentiation, and how DC subsets might be involved in immunoregulation and tolerance rather than in T cell activation. The most important features of tolerance-inducing DCs appear to be their maturation state and their cytokine secretion pattern. The following types of tolerance-inducing DCs have been reported: immature DCs (DCs(im)) or DCs in the steady state (DCs(st)), DCs(IL-10), semi-mature DCs(TNF-alpha), semi-mature DCs(IL-6). With this review article we would like to discuss the aforementioned types of tolerogenic DCs with a focus on semi-mature DCs(IL-6) and discuss their potential role in maintenance of (hepatic or intestinal) immune homeostasis and inflammatory diseases such as inflammatory bowel disease., (Copyright 2009 Elsevier GmbH. All rights reserved.)

Antigen uptake by dendritic cells is essential for the induction of antigen-specific T-cell responses. Here, we investigate the ability of dendritic cells from different mouse strains to endocytose antigens. The uptake of different fluorescently labelled soluble antigens by bone marrow-derived dendritic cells from BALB/c, C57BL/6 and C3H/HeN mice was analysed by flow cytometry. Using transferrin as a specific marker for clathrin-mediated endocytosis, we observed no significant differences of transferrin uptake by dendritic cells from BALB/c, C57BL/6 and C3H/HeN mice. Similar results were obtained by analysing macropinocytosis with lucifer yellow. In contrast, analysing the uptake of ovalbumin, which is predominantly mediated by clathrin-mediated endocytosis via the macrophage mannose receptor, we found that dendritic cells from C3H/HeN mice take up three- to fivefold more ovalbumin than dendritic cells from BALB/c or C57BL/6 mice. Blocking the uptake of ovalbumin via the macrophage mannose receptor by using mannan led to a comparable uptake of ovalbumin by dendritic cells from all three mouse strains. Consistently, dendritic cells from C3H/HeN mice displayed significantly increased expression of the macrophage mannose receptor compared to dendritic cells from BALB/c or C57BL/6 mice. In conclusion, receptors involved in antigen uptake such as the macrophage mannose receptor may be differentially expressed and may explain variations of T-cell responses after vaccination in different individuals.

Yersinia enterocolitica (Ye) evades the immune system of the host by injection of Yersinia outer proteins (Yops) via a type three secretion system into host cells. In this study, a reporter system comprising a YopE-beta-lactamase hybrid protein and a fluorescent staining sensitive to beta-lactamase cleavage was used to track Yop injection in cell culture and in an experimental Ye mouse infection model. Experiments with GD25, GD25-beta1A, and HeLa cells demonstrated that beta1-integrins and RhoGTPases play a role for Yop injection. As demonstrated by infection of splenocyte suspensions in vitro, injection of Yops appears to occur randomly into all types of leukocytes. In contrast, upon infection of mice, Yop injection was detected in 13% of F4/80(+), 11% of CD11c(+), 7% of CD49b(+), 5% of Gr1(+) cells, 2.3% of CD19(+), and 2.6% of CD3(+) cells. Taking the different abundance of these cell types in the spleen into account, the highest total number of Yop-injected cells represents B cells, particularly CD19(+)CD21(+)CD23(+) follicular B cells, followed by neutrophils, dendritic cells, and macrophages, suggesting a distinct cellular tropism of Ye. Yop-injected B cells displayed a significantly increased expression of CD69 compared to non-Yop-injected B cells, indicating activation of these cells by Ye. Infection of IFN-gammaR (receptor)- and TNFRp55-deficient mice resulted in increased numbers of Yop-injected spleen cells for yet unknown reasons. The YopE-beta-lactamase hybrid protein reporter system provides new insights into the modulation of host cell and immune responses by Ye Yops.

The spread of Gram-negative bacteria with plasmid-borne extended-spectrum beta-lactamases (ESBLs) has become a worldwide problem. This study analysed a total of 366 ESBL-producing Enterobacteriaceae strains isolated from non-selected patient specimens at the university hospital of Tübingen in the period January 2003 to December 2007. Although the overall ESBL rate was comparatively low (1.6 %), the percentages of ESBL-producing Enterobacter spp. and Escherichia coli increased from 0.8 and 0.5 %, respectively, in 2003 to 4.6 and 3.8 % in 2007. In particular, the emergence was observed of one carbapenem-resistant ESBL-producing E. coli isolate and five carbapenem-non-susceptible ESBL-positive Klebsiella pneumoniae isolates, in two of which carbapenem resistance development was documented in vivo under a meropenem-containing antibiotic regime. The possible underlying mechanism for this carbapenem resistance in three of the K. pneumoniae isolates was loss of the Klebsiella porin channel protein OmpK36 as shown by PCR analysis. The remaining two K. pneumoniae isolates exhibited increased expression of a tripartite AcrAB-TolC efflux pump as demonstrated by SDS-PAGE and mass spectrometry analysis of bacterial outer-membrane extracts, which, in addition to other unknown mechanisms, may contribute towards increasing the carbapenem MIC values further. Carbapenem-non-susceptible ESBL isolates may pose a new problem in the future due to possible outbreak situations and limited antibiotic treatment options. Therefore, a systematic exploration of intestinal colonization with ESBL isolates should be reconsidered, at least for haemato-oncological departments from where four of the five carbapenem-non-susceptible ESBL isolates originated.

Bartonella henselae is a slow growing, fastidious and facultative intracellular pathogen causing cat scratch disease and vasculoproliferative disorders. To date, knowledge about the pathogenicity of this human pathogenic bacterium is limited and, additionally, serodiagnosis still needs further improvement. Here, we investigated the proteome of B. henselae using 2-D SDS-PAGE and MALDI-TOF-MS. We provide a comprehensive 2-D proteome reference map of the whole cell lysate of B. henselae with 431 identified protein spots representing 191 different proteins of which 16 were formerly assigned as hypothetical proteins. To unravel immunoreactive antigens, we applied 2-D SDS-PAGE and subsequent immunoblotting using 33 sera of patients suffering from B. henselae infections. The analysis revealed 79 immunoreactive proteins of which 71 were identified. Setting a threshold of 20% seroreactivity, 11 proteins turned out to be immunodominant antigens potentially useful for an improved Bartonella-specific serodiagnosis. Therefore, we provide for the first time (i) a comprehensive 2-D proteome map of B. henselae for further proteome-based studies focussed on the pathogenicity of B. henselae and (ii) an integrated view into the humoral immune responses targeted against this newly emerged human pathogenic bacterium.

Oxygen-dependent antimicrobial activity of human polymorphonuclear leukocytes (PMNs) relies on the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to generate oxidants. As the oxidase transfers electrons from NADPH the membrane will depolarize and concomitantly terminate oxidase activity, unless there is charge translocation to compensate. Most experimental data implicate proton channels as the effectors of this charge compensation, although large-conductance Ca2+-activated K+ (BK) channels have been suggested to be essential for normal PMN antimicrobial activity. To test this latter notion, we directly assessed the role of BK channels in phagocyte function, including the NADPH oxidase. PMNs genetically lacking BK channels (BK(-/-)) had normal intracellular and extracellular NADPH oxidase activity in response to both receptor-independent and phagocytic challenges. Furthermore, NADPH oxidase activity of human PMNs and macrophages was normal after treatment with BK channel inhibitors. Although BK channel inhibitors suppressed endotoxin-mediated tumor necrosis factor-alpha secretion by bone marrow-derived macrophages (BMDMs), BMDMs of BK(-/-) and wild-type mice responded identically and exhibited the same ERK, PI3K/Akt, and nuclear factor-kappaB activation. Based on these data, we conclude that the BK channel is not required for NADPH oxidase activity in PMNs or macrophages or for endotoxin-triggered tumor necrosis factor-alpha release and signal transduction BMDMs.

Background: Interferon induced tetratricopeptide repeat protein 2 (IFIT-2, P54) belongs to the type I interferon response genes and is highly induced after stimulation with LPS. The biological function of this protein is so far unclear. Previous studies indicated that IFIT-2 binds to the initiation factor subunit eIF-3c, affects translation initiation and inhibits protein synthesis. The aim of the study was to further characterize the function of IFIT-2., Results: Stimulation of RAW264.7 macrophages with LPS or IFN-gamma leads to the expression of IFIT-2 in a type I interferon dependent manner. By using stably transfected RAW264.7 macrophages overexpressing IFIT-2 we found that IFIT-2 inhibits selectively LPS induced expression of TNF-alpha, IL-6, and MIP-2 but not of IFIT-1 or EGR-1. In IFIT-2 overexpressing cells TNF-alpha mRNA expression was lower after LPS stimulation due to reduced mRNA stability. Further experiments suggest that characteristics of the 3'UTR of transcripts discriminate whether IFIT-2 has a strong impact on protein expression or not., Conclusion: Our data suggest that IFIT-2 may affect selectively LPS induced protein expression probably by regulation at different posttranscriptional levels.

Background: Yersinia outer protein (Yop) H is a secreted virulence factor of Yersinia enterocolitica (Ye), which inhibits phagocytosis of Ye and contributes to the virulence of Ye in mice. The aim of this study was to address whether and how YopH affects the innate immune response to Ye in mice., Results: For this purpose, mice were infected with wild type Ye (pYV+) or a YopH-deficient Ye mutant strain (DeltayopH). CD11b+ cells were isolated from the infected spleen and subjected to gene expression analysis using microarrays. Despite the attenuation of DeltayopH in vivo, by variation of infection doses we were able to achieve conditions that allow comparison of gene expression in pYV+ and DeltayopH infection, using either comparable infection courses or splenic bacterial burden. Gene expression analysis provided evidence that expression levels of several immune response genes, including IFN-gamma and IL-6, are high after pYV+ infection but low after sublethal DeltayopH infection. In line with these findings, infection of IFN-gammaR-/- and IL-6-/- mice with pYV+ or DeltayopH revealed that these cytokines are not necessarily required for control of DeltayopH, but are essential for defense against infection with the more virulent pYV+. Consistently, IFN-gamma pretreatment of bone marrow derived macrophages (BMDM) strongly enhanced their ability in killing intracellular Ye bacteria., Conclusion: In conclusion, this data suggests that IFN-gamma-mediated effector mechanisms can partially compensate virulence exerted by YopH. These results shed new light on the protective role of IFN-gamma in Ye wild type infections.

During 2004 and at the start of 2005 a university hospital in Southwest Germany was affected by an extensive outbreak of vancomycin-resistant Enterococcus faecium (VRE). Although the outbreak was contained, linezolid-resistant enterococci emerged during and after the outbreak as the usage of linezolid became more common. Linezolid resistance was no longer limited to VRE. Nosocomial spread of linezolid-resistant but vancomycin-susceptible E. faecium was detected and these strains also emerged in patients without prior drug exposure. Linezolid should therefore be used with caution and the susceptibility of isolates monitored over time. Isolation precautions and screening of contacts should be considered to avoid spread of resistant isolates.

Background: IL-2 deficient (IL-2(-/-)) mice mono-colonized with E. coli mpk develop colitis whereas IL-2(-/-)-mice mono-colonized with B. vulgatus mpk do not and are even protected from E. coli mpk induced colitis., Methodology/principal Findings: We investigated if mono-colonization with E. coli mpk or B. vulgatus mpk differentially modulates distribution, activation and maturation of intestinal lamina propria (LP) dendritic cells (DC). LP DC in mice mono-colonized with protective B. vulgatus mpk or co-colonized with E. coli mpk/B. vulgatus mpk featured a semi-mature LP DC phenotype (CD40(lo)CD80(lo)MHC-II(hi)) whereas mono-colonization with colitogenic E. coli mpk induced LP DC activation and maturation prior to onset of colitis. Accordingly, chemokine receptor (CCR) 7 surface expression was more strikingly enhanced in mesenteric lymph node DC from E. coli mpk than B. vulgatus mpk mono- or co-colonized mice. Mature but not semi-mature LP DC promoted Th1 polarization. As B. vulgatus mpk promotes differentiation of semi-mature DC presumably by IL-6, mRNA and protein expression of IL-6 was investigated in LP DC. The data demonstrated that IL-6 mRNA and protein was increased in LP DC of B. vulgatus mpk as compared to E. coli mpk mono-colonized IL-2(-/-)-mice. The B. vulgatus mpk mediated suppression of CCR7 expression and DC migration was abolished in IL-6(-/-)-DC in vitro., Conclusions/significance: From this data we conclude that the B. vulgatus triggered IL-6 secretion by LP DC in absence of proinflammatory cytokines such as IL-12 or TNF-alpha induces a semi-mature LP DC phenotype, which might prevent T-cell activation and thereby the induction of colitis in IL-2(-/-)-mice. The data provide new evidence that IL-6 might act as an immune regulatory cytokine in the mucosa by targeting intestinal DC.

Yersinia enterocolitica (Ye) disrupts the ability of dendritic cells (DC) to prime CD4+ T cells suggesting that Ye may subvert uptake and/or processing of soluble antigens (Ag). To investigate this Ye-infected DC were loaded with fluorescently labelled ovalbumins as markers for Ag uptake and processing, and analysed by flow cytometry, fluorometry and microscopy. Wild type pYV+ as well as plasmidless pYV(-) bacteria inhibited Ag degradation in DC by 40% compared to non-infected cells. Microscopic analyses of pYV(-)-infected DC revealed that 40% of DC contained intracellular bacteria, and that DC without intracellular bacteria had degraded more Ag. When internalization of pYV(-) was blocked by cytochalasin D, Ag degradation was no longer inhibited indicating the competition between degradation of bacteria and ovalbumin. In contrast, cytochalasin D pre-treated DC infected with pYV+ inhibited Ag degradation by a mechanism dependent on the presence of virulence plasmid pYV encoding YopE, YopH, YopM, YopP, YopT and YopO. As no single Yop inhibited Ag degradation, interaction of multiple Yops might account for this effect, possibly by inhibiting Rho GTPases, because of a significant decrease of Ag degradation observed in DC incubated with toxin B of C. difficile. However, the contribution of other pYV-encoded factors cannot be excluded.

Together with animal experiments, organotypical cell cultures are important models for analyzing cellular interactions of the mucosal epithelium and pathogenic mechanisms in the gastrointestinal tract. Here, we introduce a three-dimensional culture model from the adult mouse colon for cell biological investigations in an in vivo-like environment. These explant cultures were cultured for up to 2 weeks and maintained typical characteristics of the intestinal mucosa, including a high-prismatic epithelium with specific epithelial cell-to-cell connections, a basal lamina and various connective tissue cell types, as analyzed with immunohistological and electron microscopic methods. The function of the epithelium was tested by treating the cultures with dexamethasone, which resulted in a strong upregulation of the serum- and glucocorticoid-inducible kinase 1 similar to that found in vivo. The culture system was investigated in infection experiments with the fungal pathogen Candida albicans. Wildtype but not Deltacph1/Deltaefg1-knockout Candida adhered to, penetrated and infiltrated the epithelial barrier. The results demonstrate the potential usefulness of this intestinal in vitro model for studying epithelial cell-cell interactions, cellular signaling and microbiological infections in a three-dimensional cell arrangement.

Cysteine-rich protein 61 (Cyr61/CCN1) and connective tissue growth factor (CTGF/CCN2) are members of the CCN (CYR61, CTGF, nephroblastoma overexpressed gene) family and exert pleiotropic functions such as regulation of adhesion, migration, extracellular matrix deposition, or cell differentiation, and play an important role in wound healing. This study focused on the nature of the so far unknown CTGF and CYR61 mRNA expression of epithelial cells after infection with bacteria. We demonstrate that infection of epithelial cells with attenuated Yersinia enterocolitica lacking the virulence plasmid pYV leads to the expression of CYR61 and CTGF. Virulent Y. enterocolitica bearing the pYV virulence plasmid suppressed the mRNA expression of these genes. Yersinia-mediated inhibition of CTGF and CYR61 mRNA expression is partially mediated by the cysteine protease YopT. Further characterization of the Yersinia factors, which trigger CTGF and CYR61 mRNA expression, demonstrated that these factors were secreted and could be enriched in lipid extracts. Beside Yersinia, several other bacteria such as Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis, or Staphylococcus aureus, as well as supernatants of these bacteria induced CTGF and CYR61 expression. Blocking experiments with the lysophosphatidic acid (LPA) receptor-specific inhibitor Ki16425 suggest a general involvement of LPA receptors in bacteria-triggered CTGF and CYR61 expression. These data suggest that LPA receptor-dependent expression of CTGF and CYR61 represents a common host response after interaction with bacteria.

Background & Aims: Hypoxia inducible factor-1 (HIF-1) is the key transcriptional regulator during adaptation to hypoxia. Recent studies provide evidence for HIF-1 activation during bacterial infections. However, molecular details of how bacteria activate HIF-1 remain unclear. Here, we pursued the role of bacterial siderophores in HIF-1 activation during infection with Enterobacteriaceae., Methods: In vivo, HIF-1 activation and HIF-1-dependent gene induction in Peyer's patches were analyzed after orogastric infection with Yersinia enterocolitica. The course of an orogastric Y enterocolitica infection was determined using mice with a deletion of HIF-1alpha in the intestine. In vitro, the mechanism of HIF-1 activation was analyzed in infections with Y enterocolitica, Salmonella enterica subsp enterica, and Enterobacter aerogenes., Results: Infection of mice with Y enterocolitica led to functional activation of HIF-1 in Peyer's patches. Because mice with deletion of HIF-1alpha in the intestinal epithelium showed a significantly higher susceptibility to orogastric Y enterocolitica infections, bacterial HIF-1 activation appears to represent a host defense mechanism. Additional studies with Y enterocolitica, S enterica subsp enterica, or E aerogenes, and, moreover, application of their siderophores (yersiniabactin, salmochelin, aerobactin) caused a robust, dose-dependent HIF-1 response in human epithelia and endothelia, independent of cellular hypoxia. HIF-1 activation occurs most likely because of inhibition of prolylhydroxylase activity and is abolished upon infection with siderophore uptake deficient bacteria., Conclusions: Taken together, this study reveals what we believe to be a previously unrecognized role of bacterial siderophores for hypoxia-independent activation of HIF-1 during infection with human pathogenic bacteria.