Comparative in vitro activity of oral antimicrobial agents against Enterobacteriaceae from patients with community-acquired urinary tract infections in three European countries.

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, <1.5%). Ceftibuten (MIC50/90 0.25/0.5 mg/L) and ceftriaxone activity (MIC50/90 ≤0.25 mg/L) was comparable. Ceftibuten (MIC90 ≤0.25 mg/L) was also active against Proteus mirabilis and Klebsiella spp. Extended-spectrum ß-lactamase phenotypes were 7.1% for E. coli, 5.6% for Klebsiella pneumoniae and 0.4% for P. mirabilis. Resistance was common among men and elderly women.

Evaluation of an infectious disease consultation programme in a German tertiary care hospital.

PURPOSE: To evaluate a newly implemented infectious disease (ID) consultation service in terms of patient care, outcome and antibiotic prescription and to describe factors influencing adherence to recommendations. METHODS: Data from consultations during the first 6 months of the ID consultation program were collected and evaluated. Consultation requests, diagnostic results, treatment outcomes and antibiotic recommendations were categorised. Diagnostic and therapeutic recommendations were assessed and rated for adherence and outcome. Statistical analysis was performed to identify factors influencing adherence and treatment outcome. RESULTS: A total of 251 consultations were assessed. In most cases, ID specialists were asked for further advice regarding a previously initiated anti-infective treatment (N = 131, 52 %). In 54 of 195 (28 %) first consultations, the ID specialist proposed a differential diagnosis that differed from that of the working diagnoses submitted with the consultation request, and which was subsequently confirmed in 80 % of these cases. Diagnostic and therapeutic recommendations were made in 190 (76 %) and 240 (96 %) of the consultations, respectively. A change in the current treatment was recommended in 66 % of consultations; 37 % of recommendations were cost-saving and 26 % were cost-neutral. Compliance with diagnostic and therapeutic recommendations was rated as good by pre-specified criteria in 65 and 86 % of consultations, respectively. Treatment outcome was correlated with adherence to diagnostic recommendations (P = 0.012). Twenty-nine patients (16 %) died during the same hospital stay. CONCLUSION: Infectious disease consultations may help to establish the correct diagnosis, resulting in the appropriate treatment being provided to a severely sick patient population. Treatment outcome was improved in cases of good diagnostic adherence to the recommendations of the ID specialist.

Rapid identification of bacteria in positive blood culture by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Blood culture is probably the most significant specimen used for the diagnosis of bacterial infections, especially for bloodstream infections. In the present study, we compared the resin-containing BD BACTEC™ Plus-Aerobic (Becton Dickinson), non-charcoal-containing BacT/Alert(®) SA (bioMérieux), and charcoal-containing BacT/Alert(®) FA (bioMérieux) blood culture bottles with direct identification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). A total of 103 bacterial isolates, from clinical blood cultures, representing the most frequent 13 genera and 24 species were examined. Bacteria were extracted from positive blood culture broth by density centrifugation and then subjected to identification by MALDI-TOF MS using two different volumes and chemical treatments. Overall, correct identification by MALDI-TOF MS was obtained for the BD BACTEC™ Plus-Aerobic, BacT/Alert(®) SA, and BacT/Alert(®) FA blood culture bottles in 72%, 45.6%, and 23%, respectively, for gram-negative bacteria in 86.6%, 69.2%, and 47.1%, respectively, and for gram-positive bacteria in 60.0%, 28.8%, and 5.4%, respectively. The lack of identification was observed mainly with viridans streptococci. Depending on the blood culture bottles used in routine diagnostic procedures and the protocol for bacterial preparation, the applied MALDI-TOF MS represents an efficient and rapid method for direct bacterial identification.

Immunochemical and biological characterization of three capsular polysaccharides from a single Bacteroides fragilis strain.

Although Bacteroides fragilis accounts for only 0.5% of the normal human colonic flora, it is the anaerobic species most frequently isolated from intra-abdominal and other infections with an intestinal source. The capsular polysaccharides of B. fragilis are part of a complex of surface polysaccharides and are the organism's most important virulence factors in the formation of intra-abdominal abscesses. Two capsular polysaccharides from strain NCTC 9343, PS A1 and PS B1, have been characterized structurally. Their most striking feature is a zwitterionic charge motif consisting of both positively and negatively charged substituent groups on each repeating unit. This zwitterionic motif is essential for abscess formation. In this study, we sought to elucidate structural features of the capsular polysaccharide complex of a commonly studied B. fragilis strain, 638R, that is distinct from strain 9343. We sought a more general picture of the species to establish basic structure-activity and structure-biosynthesis relationships among abscess-inducing polysaccharides. Strain 638R was found to have a capsular polysaccharide complex from which three distinct carbohydrates could be isolated by a complex purification procedure. Compositional and immunochemical studies demonstrated a zwitterionic charge motif common to all of the capsular polysaccharides that correlated with their ability to induce experimental intra-abdominal abscesses. Of interest is the range of net charges of the isolated polysaccharides-from positive (PS C2) to balanced (PS A2) to negative (PS 3). Relationships among structural components of the zwitterionic polysaccharides and their molecular biosynthesis loci were identified.

Structural basis of the abscess-modulating polysaccharide A2 from Bacteroides fragilis.

Zwitterionic capsular polysaccharides from pathogenic bacteria have peculiar immunological properties. They are capable of eliciting T-cell proliferation and modulating the course of abscess formation. To understand the molecular basis of this characteristic immune response, we are conducting detailed structure-function studies on these polysaccharides. We have identified, purified, and characterized an abscess-modulating polysaccharide, PS A2, from the clinical strain Bacteroides fragilis 638R. Here, we report the elucidation of both the chemical and three-dimensional structures of PS A2 by NMR spectroscopy, chemical methods, gas chromatography-mass spectrometry, and restrained molecular dynamics calculations. PS A2 consists of a pentasaccharide repeating unit containing mannoheptose, N-acetylmannosamine, 3-acetamido-3,6-dideoxyglucose, 2-amino-4-acetamido-2,4,6-trideoxygalactose, fucose, and 3-hydroxybutanoic acid. PS A2 is zwitterionic and carries one cationic free amine and one anionic carboxylate in each repeating unit. It forms an extended right-handed helix with two repeating units per turn and a pitch of 20 A. Positive and negative charges are exposed on the outer surface of the polymer in a regularly spaced pattern, which renders them easily accessible to other molecules. The helix is characterized by repeated large grooves whose lateral boundaries are occupied by the charges. The three-dimensional structure of PS A2 explicitly suggests mechanisms of interaction between zwitterionic polysaccharides and proteins.

Bacterial pathogens induce abscess formation by CD4(+) T-cell activation via the CD28-B7-2 costimulatory pathway.

Abscesses are a classic host response to infection by many pathogenic bacteria. The immunopathogenesis of this tissue response to infection has not been fully elucidated. Previous studies have suggested that T cells are involved in the pathologic process, but the role of these cells remains unclear. To delineate the mechanism by which T cells mediate abscess formation associated with intra-abdominal sepsis, the role of T-cell activation and the contribution of antigen-presenting cells via CD28-B7 costimulation were investigated. T cells activated in vitro by zwitterionic bacterial polysaccharides (Zps) known to induce abscess formation required CD28-B7 costimulation and, when adoptively transferred to the peritoneal cavity of naïve rats, promoted abscess formation. Blockade of T-cell activation via the CD28-B7 pathway in animals with CTLA4Ig prevented abscess formation following challenge with different bacterial pathogens, including Staphylococcus aureus, Bacteroides fragilis, and a combination of Enterococcus faecium and Bacteroides distasonis. In contrast, these animals had an increased abscess rate following in vivo T-cell activation via CD28 signaling. Abscess formation in vivo and T-cell activation in vitro required costimulation by B7-2 but not B7-1. These results demonstrate that abscess formation by pathogenic bacteria is under the control of a common effector mechanism that requires T-cell activation via the CD28-B7-2 pathway.

Bacteroides fragilis NCTC9343 produces at least three distinct capsular polysaccharides: cloning, characterization, and reassignment of polysaccharide B and C biosynthesis loci.

Bacteroides fragilis produces a capsular polysaccharide complex (CPC) that is directly involved in its ability to induce abscesses. Two distinct capsular polysaccharides, polysaccharide A (PS A) and PS B, have been shown to be synthesized by the prototype strain for the study of abscesses, NCTC9343. Both of these polysaccharides in purified form induce abscesses in animal models. In this study, we demonstrate that the CPC of NCTC9343 is composed of at least three distinct capsular polysaccharides: PS A, PS B, and PS C. A previously described locus contains genes whose products are involved in the biosynthesis of PS C rather than PS B as was originally suggested. The actual PS B biosynthesis locus was cloned, sequenced, and found to contain 22 genes in an operon-type structure. A mutant with a large chromosomal deletion of the PS B biosynthesis locus was created so that the contribution of PS B to the formation of abscesses could be assessed in a rodent model. Although purified PS B can induce abscesses, removal of this polysaccharide does not attenuate the organism's ability to induce abscesses.

Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization.

Animal studies and preliminary results in humans suggest that lower extremity and myocardial ischemia can be attenuated by treatment with angiogenic cytokines. The resident population of endothelial cells that is competent to respond to an available level of angiogenic growth factors, however, may potentially limit the extent to which cytokine supplementation enhances tissue neovascularization. Accordingly, we transplanted human endothelial progenitor cells (hEPCs) to athymic nude mice with hindlimb ischemia. Blood flow recovery and capillary density in the ischemic hindlimb were markedly improved, and the rate of limb loss was significantly reduced. Ex vivo expanded hEPCs may thus have utility as a "supply-side" strategy for therapeutic neovascularization.

Effect of molecular size on the ability of zwitterionic polysaccharides to stimulate cellular immunity.

The large-molecular-sized zwitterionic capsular polysaccharide of the anaerobe Bacteroides fragilis NCTC 9343, designated polysaccharide (PS) A, stimulates T cell proliferation in vitro and induces T cell-dependent protection against abscess formation in vivo. In the present study, we utilized a modification of a recently developed ozonolytic method for depolymerizing polysaccharides to examine the influence of the molecular size of PS A on cell-mediated immunity. Ozonolysis successfully depolymerized PS A into structurally intact fragments. PS A with average molecular sizes of 129.0 (native), 77.8, 46.9, and 17.1 kDa stimulated CD4+-cell proliferation in vitro to the same degree, whereas the 5.0-kDa fragment was much less stimulatory than the control 129.0-kDa PS A. Rats treated with 129.0-kDa, 46.9-kDa, and 17.1-kDa PS A molecules, but not those treated with the 5.0-kDa molecule, were protected against intraabdominal abscesses induced by challenge with viable B. fragilis. These results demonstrate that a zwitterionic polysaccharide as small as 22 repeating units (88 monosaccharides) elicits a T cell-dependent immune response. These findings clearly distinguish zwitterionic T cell-dependent polysaccharides from T cell-independent polysaccharides and give evidence of the existence of a novel mechanism for a polysaccharide-induced immune response.