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Background: Routine urine culture testing is not recommended for uncomplicated urinary tract infections (UTIs). As a result, the antibiotic resistance patterns or the organisms causing UTIs are not adequately reflected in routine data. We studied the sensitivity of Escherichia coli (E. coli) to trimethoprim (TMP) and to cotrimoxazole (i.e., trimethoprim/sulfamethoxazole, TMP/SMX) in community-acquired UTI and compared the findings with the resistance data of the Antimicrobial Resistance Surveillance System (ARS). Methods: General practitioners and internists in private practice prospectively recruited all of their adult patients with symptoms of a urinary tract infection from May 2015 to February 2016. Urine specimens from all patients were tested (including urine culture testing and antibiotic susceptibility) and infections were defined as uncomplicated or complicated UTIs. Results: 1245 participants from 58 medical practices were enrolled in the study. Pathogenic organisms were found in the urine of 877 patients, of whom 74.5% had E. coli infections. Among the E.-coli-positive UTIs, 52.4% were classified as uncomplicated and 47.6% as complicated. The prevalence of E. coli that was resistant to TMP and to TMP/SMX in uncomplicated UTIs was 15.2% and 13.0%, respectively, compared to 25.3% and 24.4%, respectively, from all UTIs in ARS in 2015. Study participants who had previously taken antibiotics had the highest prevalence of E. coli resistance (30.9%), followed by those who had two or more UTIs within the past six months (28.9%). Conclusion: E. coli with resistance to TMP was significantly less prevalent among the study patients with uncomplicated UTIs than in the routine data of the ARS. Accordingly, TMP should still be considered as an option for the treatment of uncomplicated UTIs. TMP/SMX is considered the agent of second choice because of its side effects. Surveillance systems based on routine data do not yield a representative sample for the evaluation of the resistance situation in patients with uncomplicated UTIs.

Inflammation is a hallmark of microbial infection in mammals and is the result of a pathogen-induced release of inflammatory effectors, In humans a variety of germ-line encoded receptors, so-called pattern-recognition receptors, respond to conserved signatures on invading pathogens, which results in the transcriptional activation of pro-inflammatory responses. Inflammation is often detrimental to the host and leads to tissue damage and/or systemic dysfunctions. Thus, specific inhibitors of these pathways are desirable for medical interventions. Herein we report on the synthesis and use of some chromium-containing compounds (arene-Cr(CO) 3 complexes) with a core structure related to anti-inflammatory diterpenes produced by the sea whip Pseudopterogorgia elisabethae. By using cell-based reporter assays we identified complexes with a potent inhibitory activity on tumour necrosis factor (TNF), Toll-like receptor (TLR), and nucleotide binding domain, leucine-rich repeat-containing receptor (NLR) pathways. Moreover, we found one complex to be a specific inhibitor of inflammatory responses mediated by the NLR protein NOD2, a pivotal innate immune receptor involved in bacterial recognition. Synthesis and characterisation of a set of derivatives of this substance revealed structural requirements for NOD2 specificity. Taken together, our studies suggest this type of arene-Cr(CO) 3 complex as a potential lead for the development of antiphlogistica and pharmacologically relevant NOD2 inhibitors.

Capsular polysaccharides of the human physiologic bacterial flora are immunogenic components that first encounter the human immune system during initial colonization and at the time that the immune system is developing and maturing. As opposed to common negatively charged polysaccharides, the biologic activities of certain commensal bacterial polysaccharides are unique in their ability to stimulate CD4+ T cells in vivo and in vitro. They direct the development of the systemic cellular immune response by correcting CD4+ T-cell deficiencies and TH1/TH2 imbalances toward a TH1 immune response. Responses to the polysaccharides are conferred by CD4+ T cells, not B cells or other T cells (14, 18, 28, 31-33). Examples of such bacteria are the ubiquitous anaerobic member of the gut flora Bacteroides fragilis, Staphylococcus aureus as a temporary member of the skin and mucosal flora, and Streptococcus pneumoniae of the upper respiratory tract flora. CD4+ T-cell activation induced by these polysaccharides depends on their unique electrical charge: each repeating unit has a minimum of one positive and one negative charge, leading to their common three-dimensional configuration characterized by a right-handed helix with repeating negatively charged grooves, with the positive charges being on the outer surface of the lateral boundaries (5, 14, 31, 36). Presentation of the so-called zwitterionic polysaccharide (ZPS) from S. pneumoniae serotype 1 (Sp1) by major histocompatibility complex (MHC) class II molecules requires its retrograde transport from lysosomes to the cell surface within tubules as a ZPS-MHC class II complex and also requires the DM molecule (12, 22). DM is known to catalyze and edit the exchange of the self-peptide CLIP with processed antigen in MHC class II compartments. The requirement of DM for Sp1 presentation via MHC class II suggests presentation within the antigen binding groove, which is supported by recent studies demonstrating that binding of the ZPS PS A1 from B. fragilis to MHC class II molecules can be competed by peptides known to be presented in the antigen binding cleft (7). For protein-derived T-cell antigens, it is well established that their presence in the MHC class II binding groove leads to the recognition of their antigenic epitopes by the T-cell receptor (TCR), within the CDR3 antigen binding domain of the β-chain variable (BV) region, and to subsequent T-cell activation and oligoclonal T-cell proliferation. However, for ZPS, besides the requirement of engaging the MHC class II molecule with the αβ TCR for ZPS-induced CD4+ T-cell activation (6, 29), little is known about the clonality of the T-cell response. So far, attempts to generate ZPS-specific T-cell clones have been unsuccessful. In rats, only two ZPS-cross-reactive T-cell hybridomas have been established (23). The ZPS-specific hybridomas responded to a variety of other ZPS but not to nonzwitterionic polysaccharides, indicating cross-reactivity between different ZPS. In the present study, we tested the hypothesis that ZPS induce host memory responses in the mature host immune system. We further studied the clonality of the immune response. Using the ZPS Sp1 in an experimental mouse model of cellular immunity, besides TH1-polarized cells, we found predominantly CD4+ T cells of a TH17-polarized phenotype. They were characterized by their CD44high CD62Llow CD25− phenotype as memory CD4+ T cells. In an experimental mouse model of humoral immunity, subcutaneous immunization with Sp1 resulted in significant Sp1-specific immunoglobulin G (IgG) titers, predominantly of the IgG1 subclass. In vivo and in vitro stimulation of murine CD4+ T cells with Sp1 resulted in oligoclonal T-cell expansion within numerous TCR BV families.

We describe the recovery and identification of a bacterium that represents a new species of the genus Cupriavidus from cultures of respiratory tract specimens from two patients with cystic fibrosis (CF). The elucidation of the role of this species in CF lung disease will require an evaluation of a greater number of cases.

Aim: To investigate the prevalence and genetic background of Escherichia coli collected from different patient populations in the Euroregion Meuse–Rhine. Materials & methods: Susceptibility testing was performed on 1651 E. coli isolates with broth microdilution. Their genetic background was determined using pulsed-field gel electrophoresis and multilocus sequence typing. Results: The prevalence of resistance varied significantly between the populations. Approximately 10% of the E. coli isolates were multidrug-resistant and/or a β-lactamase producer. The most prevalent extended-spectrum β-lactamase type was CTX-M-15 and ST131 was the most prevalent multilocus sequence typing type. Results from pulsed-field gel electrophoresis of the ST131 isolates indicate the spread of these isolates in the Euroregion. Conclusion: E. coli ST131 was the most prevalent sequence type in our Euroregional study. It is essential to control the spread of these resistant strains (e.g., with infection-control policies, antibiotic stewardship programs and antibiotic resistance surveillance). In this way we could observe shifts in the prevalence of resistance of the E. coli population and act accordingly.

The detection of pathogen-derived molecules as foreign particles by adaptive immune cells triggers T and B lymphocytes to mount protective cellular and humoral responses, respectively. Recent immunological advances elucidated that proteins and some lipids are the principle biological molecules that induce protective T cell responses during microbial infections. Polysaccharides are important components of microbial pathogens and many vaccines. However, research concerning the activation of the adaptive immune system by polysaccharides gained interest only recently. Traditionally, polysaccharides were considered to be T cell-independent antigens that did not directly activate T cells or induce protective immune responses. Here, we review several recent advances in “carbohydrate immunobiology”. A group of bacterial polysaccharides that are known as “zwitterionic polysaccharides (ZPSs)” were recently identified as potent immune modulators. The immunomodulatory effect of ZPSs required antigen processing and presentation by antigen presenting cells, the activation of CD4 T cells and subpopulations of CD8 T cells and the modulation of host cytokine responses. In this review, we also discuss the potential use of these unique immunomodulatory ZPSs in new vaccination strategies against chronic inflammatory conditions, autoimmunity, infectious diseases, allergies and asthmatic conditions.

Endothelial progenitor cells (EPC) play a fundamental role in tissue regeneration and vascular repair. Current research suggests that EPC are more resistant to oxidative stress as compared to differentiated endothelial cells. Here we hypothesized that EPC not only possess the ability to protect themselves against oxidative stress but also confer this protection upon differentiated endothelial cells by release of paracrine factors. To test this hypothesis, HUVEC incubated with conditioned medium obtained from early EPC cultures (EPC-CM) were exposed to H2O2 to assess the accumulation of intracellular ROS, extent of apoptosis and endothelial cell functionality. Under oxidative stress conditions HUVEC treated with EPC-CM exhibited substantially lower levels of intracellular oxidative stress (0.2+/-0.02 vs. 0.4+/-0.03 relative fluorescence units, p0.05) compared to control medium. Moreover, the incubation with EPC-CM elevated the expression level of antioxidant enzymes in HUVEC (catalase: 2.6+/-0.4; copper/zinc superoxide dismutase (Cu/ZnSOD): 1.6+/-0.1; manganese superoxide dismutase (MnSOD): 1.4+/-0.1-fold increase compared to control, all p0.05). Furthermore, EPC-CM had the distinct potential to reverse the functional impairment of HUVEC as measured by their capability to form tubular structures in vitro. Finally, incubation of HUVEC with EPC-CM resulted in a significant reduction of apoptosis (0.34+/-0.01 vs. 1.52+/-0.12 relative fluorescence units, p0.01) accompanied by an increased expression ratio of the anti/pro-apoptotic factors Bcl-2/Bax to 2.9+/-0.7-fold (compared to control, p0.05). Most importantly, neutralization of selected cytokines such as VEGF, HGF, IL-8 and MMP-9 did not significantly reverse the cyto-protective effect of EPC-CM (p0.05), suggesting that soluble factors secreted by EPC, possibly via broad synergistic actions, exert strong cyto-protective properties on differentiated endothelium through modulation of intracellular antioxidant defensive mechanisms and pro-survival signals.

Dendritic cells (DCs) link the innate and adaptive immune system. Currently, murine DCs for cell biology investigations are developed from MHC class II-negative bone marrow (BM) precursor cells, non-depleted BM cells or BM monocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Here we demonstrate an isolation procedure of functionally intact myeloid CD11c(+) CD11b(+) DCs derived from murine CD34-positive precursors. DCs derived from CD34(+) cells show functional internalization, maturation, cytokine secretion, MHC-restricted antigen presentation, and MHCII retrograde transport of antigens from the lysosomes to the cell surface. In comparison to the established method, the advantages of this isolation procedure are a shorter cultivation period, a superior transfection efficiency, the yield of a purer and more homogeneous population of immature DCs, and less consumption of cell culture medium and GM-CSF. The new isolation procedure and the functional quality of CD34(+) cell-derived murine myeloid DCs make them ideally suited for immunology and cell biology studies.

Enterobacteriaceae causing community-acquired urinary tract infections were examined in selected outpatient clinics and hospitals in Belgium, Germany and Spain using EUCAST breakpoints for susceptibility. A total of 1190 isolates were collected. Escherichia coli isolates were resistant to amoxicillin-clavulanic acid (28.1%), ciprofloxacin (23.4%) and trimethoprim-sulfamethoxazole (21.4%) compared with fosfomycin and nitrofurantoin (each

Zwitterionic capsular polysaccharides (ZPS) of commensal bacteria are characterized by having both positive and negative charged substituents on each repeating unit of a highly repetitive structure that has an α-helix configuration. In this paper we look at the immune response of CD8+ T cells to ZPSs. Intraperitoneal application of the ZPS Sp1 from Streptococcus pneumoniae serotype 1 induces CD8+CD28− T cells in the spleen and peritoneal cavity of WT mice. However, chemically modified Sp1 (mSp1) without the positive charge and resembling common negatively charged polysaccharides fails to induce CD8+CD28− T lymphocytes. The Sp1-induced CD8+CD28− T lymphocytes are CD122lowCTLA-4+CD39+. They synthesize IL-10 and TGF-β. The Sp1-induced CD8+CD28− T cells exhibit immunosuppressive properties on CD4+ T cells in vivo and in vitro. Experimental approaches to elucidate the mechanism of CD8+ T cell activation by Sp1 demonstrate in a dimeric MHC class I-Ig model that Sp1 induces CD8+ T cell activation by enhancing crosslinking of TCR. The expansion of CD8+CD28− T cells is independent, of direct antigen-presenting cell/T cell contact and, to the specificity of the T cell receptor (TCR). In CD8+CD28− T cells, Sp1 enhances Zap-70 phosphorylation and increasingly involves NF-κB which ultimately results in protection versus apoptosis and cell death and promotes survival and accumulation of the CD8+CD28− population. This is the first description of a naturally occurring bacterial antigen that is able to induce suppressive CD8+CD28− T lymphocytes in vivo and in vitro. The underlying mechanism of CD8+ T cell activation appears to rely on enhanced TCR crosslinking. The data provides evidence that ZPS of commensal bacteria play an important role in peripheral tolerance mechanisms and the maintenance of the homeostasis of the immune system., Author Summary One of the most difficult challenges for the mammalian immune system is to protect its host from pathogens and cancer while at the same time avoiding a self-destructive or overwhelming immune response. In addition to so-called central tolerance induced in the thymus, the immune system relies on peripheral control mechanisms. One of the most important brakes of the peripheral tolerance system is constituted by so-called regulatory T lymphocytes. The predominately investigated regulatory T lymphocytes belong to the CD4+ subset but CD8+ regulatory T lymphocytes are now also believed to play a major role in controlling immune responses. Herein, we describe for the first time a natural occurring saccharide antigen from a commensal bacterium which induces the accumulation of a defined population of CD8+ regulatory T lymphocytes. These CD8+ regulatory lymphocytes suppress inflammatory immune responses in vivo and in in vitro assays. We also describe how the bacterial antigen induces the activation of CD8+ T cells. Our findings not only describe a novel mechanism of saccharide-mediated T cell activation but also provide evidence that commensal bacteria play an important role in the induction of peripheral tolerance and maintenance of the mammalian immune system.

To the Editor: Infections are the most frequent and serious wound complications in injection drug users (IDUs). Wound botulism is primarily caused by Clostridium botulinum (1) and was first observed in IDUs in New York in 1982 (2). It results from the introduction of C. botulinum spores into a wound and their multiplication, germination, in situ synthesis, and secretion of toxin under anaerobic conditions. Of 7 designated toxin types, neurotoxins A, B, E, and F result in human disease. During the 1990s, wound botulism cases among IDUs increased in the United States in conjunction with the use of black-tar heroin (3). Since 2000, wound botulism cases in IDUs have been reported in Europe (4). To our knowledge, molecular epidemiologic analyses have not been performed to confirm suspected outbreaks. Within a 6-week period in October and November 2005, 12 clinical cases were recognized in the metropolitan area of Cologne, Germany (5). Six patients were successfully treated at teaching hospitals of the University of Cologne. On admission, all socially nonrelated patients had signs of bilateral symmetric cranial neuropathies such as ptosis, diplopia, blurred vision, dysphagia, dysarthria associated with symmetrical descending weakness of the upper extremities, and no sensory deficiencies. Treatment of patients included administration of trivalent A, B, and E antitoxin; antimicrobial drugs such as penicillin G or mezlocillin with metronidazole; and surgical drainage of any existing abscesses. Patient 1, a 31-year-old female IDU, had multiple abscesses on both legs. Four days after her admission, wound botulism was suspected and antitoxin administered. Respiratory failure required mechanical ventilation for 11 weeks. Patient 2, a 51-year-old male IDU, had 1 large abscess on the left lower leg. Antitoxin was administered within 3 days of hospital admission. Mechanical ventilation was required for 5 weeks. Patient 3, a 25-year-old male IDU, had a large abscess on the left forearm. Patient 4, a 43-year-old man who used heroin intramuscularly, had an abscess of moderate size on the left forearm. Antitoxin was administered within 12 hours of admission to patients 3 and 4, and both patients required 2 weeks of respiratory support. Patient 5, a 32-year-old male IDU who was positive for hepatitis C virus, had purchased heroin from the same dealer as patient 2. Abscesses were absent. Antitoxin was administered within several hours of admission. Within 10 days, the patient recovered fully without need for mechanical ventilation. Patient 6, a 44-year-old male IDU, had several skin lesions at injection sites on his arms, but no abscesses. He received antitoxin treatment within several hours of admission and was discharged with minimal residual neck weakness after 7 days. Serum specimens were obtained from patients 1, 2, 5, and 6. Botulinum toxin detected by the mouse bioassay in serum of patients 1 and 2, but not of patients 5 and 6, was neutralized by polyvalent antitoxin (Novartis Behring, Marburg, Germany). Abscess specimens were available from patients 2, 3, and 4. Anaerobic cultures grew C. botulinum, which was identified by Gram stain, culture morphologic features, Rapid ID 32A (bioMerieux, Marcy l’Etoile, France), and 16S rDNA sequencing. All strains were susceptible to penicillin G and metronidazole, as determined by the E-test (AB Biodisk, Solna, Sweden). PCR assays performed for C. botulinum type A, B, E, and F neurotoxin genes (6,7) identified the single toxin B. Toxin B production was confirmed by the mouse bioassay. Pulsed-field gel electrophoresis (PFGE) after SmaI, SacII, and XhoI restriction (8) showed indistinguishable strains from patients 2, 3, and 4 (shown for SmaI in the Figure). Figure Fingerprint patterns obtained for Clostridium botulinum isolates following pulsed-field gel electrophoresis after SmaI restriction show identical strains. Lane 1, 100-bp ladder; lanes 2–4, abscess fluid isolates from patients 2, 3, and 4, respectively. ... To our knowledge, this is the first outbreak of wound botulism in IDUs that was confirmed by molecular epidemiologic typing. PFGE suggests a single-source exposure with C. botulinum type B in at least 3 IDUs; this implies that the heroin was obtained from a common source, where contamination with C. botulinum spores may have been introduced when mixed with adulterants or diluted with substances such as dextrose or dyed paper. Skin popping (subcutaneous and intramuscular injection), which may increase the odds of wound botulism by a factor >15 (9), was used by all patients for drug delivery. This study confirms previous observations that the duration of clinical symptoms before antitoxin administration affects the need for and duration of mechanical ventilation (10). Here, the time from hospital admission to antitoxin treatment ranged from several hours to 4 days and correlated with the mechanical ventilation interval ranging from 0 days to 11 weeks. In addition, the extent of abscesses, which ranged from no abscesses to multiple abscesses, seems to affect clinical outcome. As soon as an index case of wound botulism in IDUs is diagnosed, a coordinated public health case-management effort, including hospitals, outpatient clinics, and information centers for drug addicts, is mandatory to alert the medical community and the drug users to consider wound botulism if typical symptoms occur and to enable the prompt administration of antitoxin. Obtaining tissue samples or abscess fluid for culture and molecular epidemiologic studies of C. botulinum isolates is necessary to facilitate identification of the source of the contaminated heroin.

Bacterial capsular polysaccharides are virulence factors and are considered T cell–independent antigens. However, the capsular polysaccharide Sp1 from Streptococcus pneumoniae serotype 1 has been shown to activate CD4+ T cells in a major histocompatibility complex (MHC) class II–dependent manner. The mechanism of carbohydrate presentation to CD4+ T cells is unknown. We show in live murine dendritic cells (DCs) that Sp1 translocates from lysosomal compartments to the plasma membrane in MHCII-positive tubules. Sp1 cell surface presentation results in reduction of self-peptide presentation without alteration of the MHCII self peptide repertoire. In DM-deficient mice, retrograde transport of Sp1/MHCII complexes resulting in T cell–dependent immune responses to the polysaccharide in vitro and in vivo is significantly reduced. The results demonstrate the capacity of a bacterial capsular polysaccharide antigen to use DC tubules as a vehicle for its transport as an MHCII/saccharide complex to the cell surface for the induction of T cell activation. Furthermore, retrograde transport requires the functional role of DM in self peptide–carbohydrate exchange. These observations open new opportunities for the design of vaccines against microbial encapsulated pathogens., Author Summary Microorganisms are comprised of proteins, carbohydrates, lipids, and nucleic acids. Current immunologic paradigms state that activation of T lymphocytes required for humoral and cellular immune responses resulting in immunologic memory to the pathogens is solely brought about by proteinaceous antigens, processed and degraded to small peptides, loaded onto major histocompatibility complex (MHC) molecules, and transported as MHC/peptide complexes to the cell surface, where the MHC/peptide complex is recognized by the T cell antigen receptor. The findings of the present study elucidate the mechanism of MHC class II (MHCII)–dependent presentation of the bacterial capsular polysaccharide of Streptococcus pneumoniae serotype 1 (Sp1) that results in effective T cell activation. Sp1 is transported in MHCII-positive tubules from lysosomal compartments to the plasma membrane for presentation. In the absence of the DM molecule, known as an editor and catalyst of self and foreign peptide exchange, retrograde transport of carbohydrate/MHCII complexes resulting in dendritic cell engagement with T cells in vitro and T cell–dependent immune responses to the polysaccharide in vivo fail. The results suggest a fundamental shift in the immunologic paradigm, offering previously unrecognized opportunities for the design of new classes of vaccines against infectious diseases.