Empiric antibiotic therapy in urinary tract infection in patients with risk factors for antibiotic resistance in a German emergency department

Background: The aim of this study was to identify clinical risk factors for antimicrobial resistances and multidrug resistance (MDR) in urinary tract infections (UTI) in an emergency department in order to improve empirical therapy. Methods: UTI cases from an emergency department (ED) during January 2013 and June 2015 were analyzed. Differences between patients with and without resistances towards Ciprofloxacin, Piperacillin with Tazobactam (Pip/taz), Gentamicin, Cefuroxime, Cefpodoxime and Ceftazidime were analyzed with Fisher’s exact tests. Results were used to identify risk factors with logistic regression modelling. Susceptibility rates were analyzed in relation to risk factors. Results: One hundred thirty-seven of four hundred sixty-nine patients who met the criteria of UTI had a positive urine culture. An MDR pathogen was found in 36.5% of these. Overall susceptibility was less than 85% for standard antimicrobial agents. Logistic regression identified residence in nursing homes, male gender, hospitalization within the last 30 days, renal transplantation, antibiotic treatment within the last 30 days, indwelling urinary catheter and recurrent UTI as risk factors for MDR or any of these resistances. For patients with no risk factors Ciprofloxacin had 90%, Pip/taz 88%, Gentamicin 95%, Cefuroxime 98%, Cefpodoxime 98% and Ceftazidime 100% susceptibility. For patients with 1 risk factor Ciprofloxacin had 80%, Pip/taz 80%, Gentamicin 88%, Cefuroxime 78%, Cefpodoxime 78% and Ceftazidime 83% susceptibility. For 2 or more risk factors Ciprofloxacin drops its susceptibility to 52%, Cefuroxime to 54% and Cefpodoxime to 61%. Pip/taz, Gentamicin and Ceftazidime remain at 75% and 77%, respectively. Conclusions: We identified several risk factors for resistances and MDR in UTI. Susceptibility towards antimicrobials depends on these risk factors. With no risk factor cephalosporins seem to be the best choice for empiric therapy, but in patients with risk factors the beta-lactam penicillin Piperacillin with Tazobactam is an equal or better choice compared to fluoroquinolones, cephalosporins or gentamicin. This study highlights the importance of monitoring local resistance rates and its risk factors in order to improve empiric therapy in a local environment

Transforming growth factor-β-induced upregulation of transforming growth factor-β receptor expression in pancreatic regeneration

AbstractThe transforming growth factor-β (TGFβ) signaling pathway is one important player in the regulation of extracellular matrix turnover and cell proliferation in epithelial regeneration. We used cerulein-induced pancreatitis in rats as a model to investigate the regulation of TGFβ receptor type I and type II expression on protein and messenger RNA level during regeneration. In the regenerating pancreas, mRNA levels of TGFβ receptor I and II were significantly increased with a maximum after 2 days. On protein level, expression of TGFβ receptor II was significantly increased after 3–5 days. This elevated expression could be inhibited by neutralizing the endogenous biological activity of TGFβ1 with a specific antibody. In cultured pancreatic epithelial cells, TGFβ1 reduced cell proliferation as measured by [3H]thymidine incorporation. Furthermore the transcript levels of TGFβ1 as well as mRNA and protein concentrations of type I and type II receptor increased during TGFβ stimulation in vitro. These results indicate that epithelial pancreatic cells contribute to the enhanced TGFβ1 synthesis during pancreatic regeneration by an autocrine mechanism. TGFβ1, furthermore, upregulates the expression of its own receptors during the regenerative process, thereby contributing to the increase of the TGFβ-induced cellular responses

Caveolin-1 protects B6129 mice against Helicobacter pylori gastritis.

Caveolin-1 (Cav1) is a scaffold protein and pathogen receptor in the mucosa of the gastrointestinal tract. Chronic infection of gastric epithelial cells by Helicobacter pylori (H. pylori) is a major risk factor for human gastric cancer (GC) where Cav1 is frequently down-regulated. However, the function of Cav1 in H. pylori infection and pathogenesis of GC remained unknown. We show here that Cav1-deficient mice, infected for 11 months with the CagA-delivery deficient H. pylori strain SS1, developed more severe gastritis and tissue damage, including loss of parietal cells and foveolar hyperplasia, and displayed lower colonisation of the gastric mucosa than wild-type B6129 littermates. Cav1-null mice showed enhanced infiltration of macrophages and B-cells and secretion of chemokines (RANTES) but had reduced levels of CD25+ regulatory T-cells. Cav1-deficient human GC cells (AGS), infected with the CagA-delivery proficient H. pylori strain G27, were more sensitive to CagA-related cytoskeletal stress morphologies ("humming bird") compared to AGS cells stably transfected with Cav1 (AGS/Cav1). Infection of AGS/Cav1 cells triggered the recruitment of p120 RhoGTPase-activating protein/deleted in liver cancer-1 (p120RhoGAP/DLC1) to Cav1 and counteracted CagA-induced cytoskeletal rearrangements. In human GC cell lines (MKN45, N87) and mouse stomach tissue, H. pylori down-regulated endogenous expression of Cav1 independently of CagA. Mechanistically, H. pylori activated sterol-responsive element-binding protein-1 (SREBP1) to repress transcription of the human Cav1 gene from sterol-responsive elements (SREs) in the proximal Cav1 promoter. These data suggested a protective role of Cav1 against H. pylori-induced inflammation and tissue damage. We propose that H. pylori exploits down-regulation of Cav1 to subvert the host's immune response and to promote signalling of its virulence factors in host cells

A Key Role for E-cadherin in Intestinal Homeostasis and Paneth Cell Maturation

E-cadherin is a major component of adherens junctions. Impaired expression of E-cadherin in the small intestine and colon has been linked to a disturbed intestinal homeostasis and barrier function. Down-regulation of E-cadherin is associated with the pathogenesis of infections with enteropathogenic bacteria and Crohn's disease. To genetically clarify the function of E-cadherin in intestinal homeostasis and maintenance of the epithelial defense line, the Cdh1 gene was conditionally inactivated in the mouse intestinal epithelium. Inactivation of the Cdh1 gene in the small intestine and colon resulted in bloody diarrhea associated with enhanced apoptosis and cell shedding, causing life-threatening disease within 6 days. Loss of E-cadherin led cells migrate faster along the crypt-villus axis and perturbed cellular differentiation. Maturation and positioning of goblet cells and Paneth cells, the main cell lineage of the intestinal innate immune system, was severely disturbed. The expression of anti-bacterial cryptidins was reduced and mice showed a deficiency in clearing enteropathogenic bacteria from the intestinal lumen. These results highlight the central function of E-cadherin in the maintenance of two components of the intestinal epithelial defense: E-cadherin is required for the proper function of the intestinal epithelial lining by providing mechanical integrity and is a prerequisite for the proper maturation of Paneth and goblet cells

Fractionation of the Epithelial Apical Junctional Complex: Reassessment of Protein Distributions in Different Substructures

The epithelial apical junctional complex (AJC) is an important regulator of cell structure and function. The AJC is compartmentalized into substructures comprising the tight and adherens junctions, and other membrane complexes containing the membrane proteins nectin, junctional adhesion molecule, and crumbs. In addition, many peripheral membrane proteins localize to the AJC. Studies of isolated proteins indicate a complex map of potential binding partners in which there is extensive overlap in the interactions between proteins in different AJC substructures. As an alternative to a direct search for specific protein-protein interactions, we sought to separate membrane substructures of the AJC in iodixanol density gradients and define their protein constituents. Results show that the AJC can be fractured into membrane substructures that contain specific membrane and peripheral membrane proteins. The composition of each substructure reveals a more limited overlap in common proteins than predicted from the inventory of potential interactions; some of the overlapping proteins may be involved in stepwise recruitment and assembly of AJC substructures

Concerted Action of Sphingomyelinase and Non-Hemolytic Enterotoxin in Pathogenic <i>Bacillus cereus</i>

<div><p><i>Bacillus cereus</i> causes food poisoning and serious non-gastrointestinal-tract infections. Non-hemolytic enterotoxin (Nhe), which is present in most <i>B. cereus</i> strains, is considered to be one of the main virulence factors. However, a <i>B. cereus</i> Δ<i>nheBC</i> mutant strain lacking Nhe is still cytotoxic to intestinal epithelial cells. In a screen for additional cytotoxic factors using an <i>in vitro</i> model for polarized colon epithelial cells we identified <i>B. cereus</i> sphingomyelinase (SMase) as a strong inducer of epithelial cell death. Using single and double deletion mutants of <i>sph</i>, the gene encoding for SMase, and <i>nheBC</i> in <i>B. cereus</i> we demonstrated that SMase is an important factor for <i>B. cereus</i> cytotoxicity <i>in vitro</i> and pathogenicity <i>in vivo.</i> SMase substantially complemented Nhe induced cytotoxicity <i>in vitro</i>. In addition, SMase but not Nhe contributed significantly to the mortality rate of larvae <i>in vivo</i> in the insect model <i>Galleria mellonella</i>. Our study suggests that the role of <i>B. cereus</i> SMase as a secreted virulence factor for <i>in vivo</i> pathogenesis has been underestimated and that Nhe and SMase complement each other significantly to cause full <i>B. cereus</i> virulence hence disease formation.</p></div

<i>Sph</i> deletion effected <i>B. cereus</i> virulence <i>in vitro</i>.

<p><b>A</b>. Cytotoxic effect of sterile <i>B. cereus</i> supernatants (1∶4 diluted) on IEC. Intact monolayer (green), cell rounding ≤50% (yellow), cell rounding >50% (orange) and 95–100% cell detachment (red) are indicated. <b>B</b>. Cytotoxic effects of <i>B. cereus</i> supernatant on IEC were analyzed using flow cytometry. Ptk6 cells were treated with various dilutions of bacterial supernatant of <i>B. cereus</i> NVH 0075-95 WT (black line), Δ<i>nheBC</i> mutant (grey line), Δ<i>sph</i> mutant (black dashed line) and Δ<i>nheBC</i>Δ<i>sph</i> mutant (grey dashed line). Samples were stained with Propidium iodide (PI) for dead epithelial cells and cytotoxicity is expressed in % of PI positive cells as determined by flow cytometric analysis. Cytotoxicity of the Δ<i>sph</i> mutant was strongly reduced at a dilution of 1∶8 compared to WT (a, <i>P</i><0.05). <i>Sph</i> deletion in addition to Nhe inactivation significantly reduced cytotoxicity compared to Nhe inactivation alone (b, <i>P</i><0.05). Data plotted represent mean values ± SEM (n = 3). <b>C</b>. Cooperative cytotoxic interaction of SMase and Nhe. Addition of various concentrations of recombinant SMase (0.05, 0.1 and 0.2 U/ml) to diluted (1∶16) bacterial supernatants caused significantly higher cytotoxicity against Ptk6 cells when subtoxic Nhe concentrations were present (Δ<i>sph</i> supernatant, black line) compared to supernatant without Nhe (Δ<i>nheBC</i>Δ<i>sph</i> supernatant, grey line) (*<i>P</i><0.05). Addition of anti-NheB (1E11) antibody (10 µg/well) neutralizes Nhe activity (Δ<i>sph</i> supernatant+α-NheB, black dotted line). Cytotoxicity is expressed in % of PI positive cells and data represent mean values ± SEM (n ≥3). <b>D</b>. CAMP-like test on sheep blood agar demonstrated complementation of extracellular hemolytic activity between <i>B. cereus</i> NVH 0075-95 Δ<i>sph and</i> Δ<i>nheBC</i>. Beta-hemolytic activity appeared as cleared zone around the colonies.</p

Cytotoxic effects of various <i>B. cereus</i> strains on intestinal epithelial cells (IEC).

<p>Ptk6 cells were treated with 14 different <i>B. cereus</i> strains and two isogenic mutants, morphological changes were monitored over time using light microscopy. <b>A.</b> At an MOI of 1 all strains tested caused epithelial cell rounding (yellow) and detachment (red) within 2–4 h after infection except for the <i>plcR</i> deletion mutant. Intact monolayer (green). <b>B.</b> Representative images of Ptk6 cell monolayers. Bar, 20 µm.</p

Identification of cytotoxic protein from <i>B. cereus</i> supernatant.

<p><b>A.</b> Bacterial supernatant of <i>B. cereus</i> NVH 0075-95 Δ<i>nheBC</i> was separated on a Superdex-75 10/300 GL gel filtration column. Chromatogram of fractionated bacterial proteins is shown (fraction1–24). Protein fractions were tested on Ptk6 cells for cytotoxicity as described in Experimental procedures. Protein fractions obtained from gel filtration were analyzed by SDS-PAGE. <b>B.</b> Gel filtration fractions 12–14 transferred cytotoxicity to Ptk6 cells and contained two distinct proteins migrating at 34 kDa and 25 kDa (red asterisks). <b>C.</b> Comparing total extracellular proteins of WT and mutant <i>B. cereus</i> strains, two potential cytotoxic proteins (red asterisks) were absent in the supernatant of avirulent Δ<i>plcR</i> strain.</p