Ribotyping of Pseudomonas aeruginosa Strains Isolated from Surgical Intensive Care Patients

To elucidate the sources of Pseudomonas aeruginosa on a surgical intensive care unit, rDNA restriction fragment length polymorphism analysis (ribotyping) was applied to analyze strains isolated during a 4-month prospective study. Samples included 1635 from 153 patients, 2463 from 97 staff members, and 581 from the environment. Only 18 patients were colonized. Isolation from their animate and inanimate environment was very low, with 3 and 2 samples, respectively, being positive. Samples from tap water were negative. Ribotyping could easily distinguish 16 different digest patterns with identical follow-up isolates of the same patient. Horizontal transmission occurred only twice. The discriminatory power of ribosomal DNA in differentiating strains was dependent on the restriction enzymes used; among eight different enzymes, PvuII was the most sensitive, producing 15 different patterns. Ribotyping showed high sensitivity in typing P. aeruginosa isolates and confirmed that colonization occurs from endogenous rather than from exogenous source

Comparison of the virulence of exopolysaccharide-producing Prevotella intermedia to exopolysaccharide non-producing periodontopathic organisms

<p>Abstract</p> <p>Background</p> <p>Evidence in the literature suggests that exopolysaccharides (EPS) produced by bacterial cells are essential for the expression of virulence in these organisms. Secreted EPSs form the framework in which microbial biofilms are built.</p> <p>Methods</p> <p>This study evaluates the role of EPS in <it>Prevotella intermedia </it>for the expression of virulence. This evaluation was accomplished by comparing EPS-producing <it>P. intermedia </it>strains 17 and OD1-16 with non-producing <it>P. intermedia </it>ATCC 25611 and <it>Porphyromonas gingivalis </it>strains ATCC 33277, 381 and W83 for their ability to induce abscess formation in mice and evade phagocytosis.</p> <p>Results</p> <p>EPS-producing <it>P. intermedia </it>strains 17 and OD1-16 induced highly noticeable abscess lesions in mice at 10⁷colony-forming units (CFU). In comparison, <it>P. intermedia </it>ATCC 25611 and <it>P. gingivalis </it>ATCC 33277, 381 and W83, which all lacked the ability to produce viscous materials, required 100-fold more bacteria (10⁹CFU) in order to induce detectable abscess lesions in mice. Regarding antiphagocytic activity, <it>P. intermedia </it>strains 17 and OD1-16 were rarely internalized by human polymorphonuclear leukocytes, but other strains were readily engulfed and detected in the phagosomes of these phagocytes.</p> <p>Conclusions</p> <p>These results demonstrate that the production of EPS by <it>P. intermedia </it>strains 17 and OD1-16 could contribute to the pathogenicity of this organism by conferring their ability to evade the host's innate defence response.</p

Large-Scale Screening of a Targeted Enterococcus faecalis Mutant Library Identifies Envelope Fitness Factors

Spread of antibiotic resistance among bacteria responsible for nosocomial and community-acquired infections urges for novel therapeutic or prophylactic targets and for innovative pathogen-specific antibacterial compounds. Major challenges are posed by opportunistic pathogens belonging to the low GC% Gram-positive bacteria. Among those, Enterococcus faecalis is a leading cause of hospital-acquired infections associated with life-threatening issues and increased hospital costs. To better understand the molecular properties of enterococci that may be required for virulence, and that may explain the emergence of these bacteria in nosocomial infections, we performed the first large-scale functional analysis of E. faecalis V583, the first vancomycin-resistant isolate from a human bloodstream infection. E. faecalis V583 is within the high-risk clonal complex 2 group, which comprises mostly isolates derived from hospital infections worldwide. We conducted broad-range screenings of candidate genes likely involved in host adaptation (e.g., colonization and/or virulence). For this purpose, a library was constructed of targeted insertion mutations in 177 genes encoding putative surface or stress-response factors. Individual mutants were subsequently tested for their i) resistance to oxidative stress, ii) antibiotic resistance, iii) resistance to opsonophagocytosis, iv) adherence to the human colon carcinoma Caco-2 epithelial cells and v) virulence in a surrogate insect model. Our results identified a number of factors that are involved in the interaction between enterococci and their host environments. Their predicted functions highlight the importance of cell envelope glycopolymers in E. faecalis host adaptation. This study provides a valuable genetic database for understanding the steps leading E. faecalis to opportunistic virulence

Comparative Opsonic and Protective Activities of Staphylococcus aureus Conjugate Vaccines Containing Native or Deacetylated Staphylococcal Poly-N-Acetyl-β-(1-6)-Glucosamine

Staphylococcus aureus and Staphylococcus epidermidis both synthesize the surface polysaccharide poly-N-acetyl-β-(1-6)-glucosamine (PNAG), which is produced in vitro with a high level (>90%) of the amino groups substituted by acetate. Here, we examined the role of the acetate substituents of PNAG in generating opsonic and protective antibodies. PNAG and a deacetylated form of the antigen (dPNAG; 15% acetylation) were conjugated to the carrier protein diphtheria toxoid (DT) and used to immunize animals. Mice responded in a dose-dependent fashion to both conjugate vaccines, with maximum antibody titers observed at the highest dose and 4 weeks after the last of three weekly immunizations. PNAG-DT and dPNAG-DT vaccines were also very immunogenic in rabbits. Antibodies raised to the conjugate vaccines in rabbits mediated the opsonic killing of various staphylococcal strains, but the specificity of the opsonic killing was primarily to dPNAG, as this antigen inhibited the killing of S. aureus strains by both PNAG- and dPNAG-specific antibodies. Passive immunization of mice with anti-dPNAG-DT rabbit sera showed significant levels of clearance of S. aureus from the blood (54 to 91%) compared to control mice immunized with normal rabbit sera, whereas PNAG-specific antibodies were ineffective at clearing S. aureus. Passive immunization of mice with a goat antiserum raised to the dPNAG-DT vaccine protected against a lethal dose of three different S. aureus strains. Overall, these data show that immunization of animals with a conjugate vaccine of dPNAG elicit antibodies that mediated opsonic killing and protected against S. aureus infection, including capsular polysaccharide types 5 and 8 and an untypable strain

Ribotyping of Pseudomonas aeruginosa Strains Isolated from Surgical Intensive Care Patients

To elucidate the sources of Pseudomonas aeruginosa on a surgical intensive care unit, rDNA restriction fragment length polymorphism analysis (ribotyping) was applied to analyze strains isolated during a 4-month prospective study. Samples included 1635 from 153 patients, 2463 from 97 staff members, and 581 from the environment. Only 18 patients were colonized. Isolation from their animate and inanimate environment was very low, with 3 and 2 samples, respectively, being positive. Samples from tap water were negative. Ribotyping could easily distinguish 16 different digest patterns with identical follow-up isolates of the same patient. Horizontal transmission occurred only twice. The discriminatory power of ribosomal DNA in differentiating strains was dependent on the restriction enzymes used; among eight different enzymes, PvuII was the most sensitive, producing 15 different patterns. Ribotyping showed high sensitivity in typing P. aeruginosa isolates and confirmed that colonization occurs from endogenous rather than from exogenous source

Immunochemical Properties of the Staphylococcal Poly-N-Acetylglucosamine Surface Polysaccharide

Staphylococcus aureus and Staphylococcus epidermidis often elaborate adherent biofilms, which contain the capsular polysaccharide-adhesin (PS/A) that mediates the initial cell adherence to biomaterials. Biofilm cells produce another antigen, termed polysaccharide intercellular adhesin (PIA), which is composed of a ∼28 kDa soluble linear β(1-6)-linked N-acetylglucosamine. We developed a new method to purify PS/A from S. aureus MN8m, a strain hyperproducing PS/A. Using multiple analytical techniques, we determined that the chemical structure of PS/A is also β(1-6)-N-acetylglucosamine (PNAG). We were unable to find N-succinylglucosamine residues in any of our preparations in contrast to previously reported findings (D. McKenney, K. Pouliot, Y. Wang, V. Murthy, M. Ulrich, G. Doring, J. C. Lee, D. A Goldmann, and G. B. Pier, Science 284:1523-1527, 1999). PNAG was produced with a wide range of molecular masses that could be divided into three major fractions with average molecular masses of 460 kDa (PNAG-I), 100 kDa (PNAG-II), and 21 kDa (PNAG-III). The purified antigens were not soluble at neutral pH unless first dissolved in 5 M HCl and then neutralized with 5 M NaOH. PNAG-I was very immunogenic in rabbits, but the responses of individual animals were variable. Immunization of mice with various doses (100, 50, or 10 μg) of PNAG-I, -II, and -III demonstrated that only PNAG-I was able to elicit an immunoglobulin G (IgG) immune response with the highest titers obtained with 100-μg dose. When we purified a small fraction of PNAG with a molecular mass of ∼780 kDa (PNAG-780) from PNAG-I, significantly higher IgG titers than those in mice immunized with the same doses of PNAG-I were obtained, suggesting the importance of the molecular mass of PNAG in the antibody response. These results further clarify the chemical structure of PS/A and help to differentiate it from PIA on the basis of immunogenicity, molecular size, and solubility