Heidrun Schmidt

Adhesiveness and invasiveness of staphylococcal species in a cell culture model

A model was established for the study of adhesiveness and invasiveness of staphylococcal species. Five collection strains from each of the species Staphylococcus aureus, S. epidermidis, and S. saprophyticus and 26 fresh isolates from patients with urinary tract infections were tested for adhesiveness and invasiveness in HEp‐2 cell cultures. All the strains of S. saprophyticus were able to invade the cells and localize intracellularly in the cultures, whereas the invasive potential among the strains of S. aureus and S. epidermidis was lower. The number of adhesive bacteria was also highest among the S. saprophyticus strains, whereas S. epidermidis was the least adhesive. The model may be suitable for further study of urinary tract infection strains.

Detection of different fimbriae-like structures on the surface of Staphylococcus saprophyticus

In relation to the ability of adhesiveness to HEp-2-cells the sizes of capsules on three strains of Staphylococcus saprophyticus were examined using an electron microscopic India ink technique. There was a good correlation between small amounts of capsular material and good adherence to the tissue culture cells. In contrast to this a very large capsule could be demonstrated on a strain which adhered only in a poor manner. Some indications of fimbriae-like structures could be confirmed by negative staining. Three different appendages: thick, short sticks, long, branched fimbriae and fine, thin filaments on the surface of S. saprophyticus were detected. The possible functions of these surface structures in the adherence process represented in a hypothetical cell model is discussed.

Adherence of staphylococci to various intraocular lenses with different levels of hydrophobicity

Background: A major goal in research on intraocular lenses (IOL) is the development of new polymers and modifications to reduce foreign-body reactions after implantation. This effect may be achieved by a reduction in the surface hydrophobicity of the polymers. To illustrate the influence of surface modifications on bacterial adhesiveness, the most often isolated organism in “low-grade” postoperative endophthalmitis, Staphylococcus epidermidis, was used. Materials and methods: For this reason three strains of this species, the type strain ATCC 14990 and two clinical isolates (8687, 6579 I) with different hydrophobic surface properties were studied. IOL, used in the experiments were either made of PMMA or silicone with modified surfaces (unpolished, polished, heparinized). The adhesiveness of H3-thymidin-labeled bacteria was calculated/mm2 of lens surface. Each experiment was performed in triplicate and repeated three times. Results: The hydrophobic-type strain showed stronger adherence to unpolished PMMA surface (8000 bacteria per mm2) compared to the polished (5200 bacteria/mm2). In contrast, the hydrophilic strain adhered with 2000 bacteria/mm2 to the unpolished and with 4200 bacteria/mm2 to the polished surface. Polishing PMMA lenses diminished the differences between the three strains. However, surface passivation of silicone lenses increased the adhesion rate of the hydrophilic strain up to 9600 bacteria/mm2. Treatment of PMMA lenses with heparin increased the adhesiveness of the hydrophilic strain and reduced the adhesion rate of the hydrophobic type strain to 250 bacteria/mm2. Conclusions: It was demonstrated that bacterial adherence to IOL also involves hydrophobic interactions. Obviously, however, that adherence reflects a complex of interactions between the two surfaces.Fragestellung: Ein Hauptziel in der Entwicklung von Intraokularlinsen (IOL) ist die Anwendung neuer Polymere und Modifikationen zur Reduzierung von Fremdkörperreaktionen. Dies kann durch eine Verminderung der Oberflächenhydrophobizität der Polymere erreicht werden. Inwieweit eine Oberflächenmodifizierung auch die bakterielle Haftung beeinträchtigt, soll mit dem am häufigsten bei Low-grade-Endophthalmitiden isolierten Keim Staphylococcus epidermidis untersucht werden. Material und Methode: Für die Untersuchungen wurden 3 Staphylococcus-epidermidis -Stämme, ATCC 14990 und 2 klinische Isolate (8687, 6579 I) mit unterschiedlicher Hydrophobizität und verschiedene IOL (PMMA, Silikon) mit unterschiedlichen Oberflächenmodifikationen (unpoliert, poliert, heparinisiert) ausgewählt. Die Haftung von H3-Thymidin-markierten Bakterien wurde auf mm2 Linsenoberfläche berechnet. Aufgrund bekannter Schwankungen bei Adhärenzuntersuchungen wurden pro Bakterienstamm und Versuch 3 Linsen parallel getestet und jeder Versuch insgesamt 3 mal an 3 verschiedenen Tagen durchgeführt, so daß pro Stamm 9 Einzelwerte erhalten wurden. Ergebnisse: Der hydrophobe Stamm zeigte eine stärkere Haftung an unpolierten (8000 Bakterien/mm2) als an polierten PMMA-Linsen (5200 Bakterien/mm2). Im Gegensatz dazu adhärierte der hydrophile Stamm mit 2000 Bakterien/mm2 an unpolierten und mit 4200 Bakterien/mm2 an polierten Oberflächen. Die Polierung von PMMA-Linsen reduzierte die Differenzen in der Haftung der 3 Staphylococcus-epidermidis -Stämme unterschiedlicher Hydrophobizität. Bei der Verwendung von Silikonlinsen stieg die Anzahl adhärierter Bakterien des hydrophilen Stamms auf 9600 Bakterien/mm2. Die Oberflächenmodifizierung von PMMA-Linsen mit Heparin verstärkte die Haftung des hydrophilen Stamms und führte zu einer Adhärenzreduzierung des hydrophoben Stamms auf 250 Bakterien/mm2. Schlußfolgerung: Es konnte gezeigt werden, daß in die bakterielle Adhärenz an IOL auch hydrophobe Wechselwirkungen integriert sind. Offensichtlich handelt es sich jedoch um ein komplexes Wechselspiel zwischen beiden Oberflächen.

Safety evaluation for a cell-based immune support system in an ex vivo rat model of gram-negative sepsis.

Granulocyte dysfunction is a central component of immunodeficiency in septic patients. Granulocyte transfusions appear to be pathophysiologically useful; however, they cause unwanted side‐effects in the lungs and other organs. This study evaluates the safety of an extracorporeal immune support system with granulocytic cells in a rat model of Gram‐negative sepsis. Three groups of male CD rats received either saline (control group, I), a dose of Escherichia coli O7:K1 lethal to 90% of the animals (LD90) (septic group, II), or an LD90 dose of E. coli that was incubated with the human promyelocytic leukemia cell line (HL‐60) (differentiated into the granulocytic direction) for 20 min prior to infusion (second septic group, III). The animals were observed for seven days. Pre‐treatment with HL‐60 cells resulted in no adverse effects in the group III animals. Significantly lower bacterial counts and endotoxin levels in the plasma were detected after 24 h as compared to group II (P < 0.05). Group III animals had better weight gain and more stable hemodynamics than group II animals (P < 0.01). Seven day survival was 0/8 in group II, 6/8 in group III, and 8/9 in group I (log–rank test: II–III: P < 0.001). The data suggest that extracorporeal use of granulocytes allows the therapeutic use of these cells while avoiding unwanted effects resulting from direct contact to internal organs.

A Phosphatase-Novobiocin-Mannose-Inhibition Test (PNMITest) for Routine Identification of the Coagulase-Negative Staphylococcal Urinary Tract Pathogens, S. aureus, S. epidermidis, and S. saprophyticus

Abstract A modified Kloos/Schlei fer -scheme proved to be useful in identifying coagulase-negative staphylococci isolated from urine. S. epidermidis (44,2%) and S. saprophyticus (21.5%) were the most frequent species. Analysis of patients confirmed both species as urinary pathogens. Using an abbreviated scheme of 6 characteristics, S. saprophyticus was misclassified in 19.5% of cases. A Phosphatase-Novobiocin-Mannose-Inhibition Test (PNMITest) together with a high NaCl concentration (10%) in combination with a coagulase test seems to be an acceptable compromise for routine identification of the three most important staphylococcal urinary tract pathogens, S. aureus , S. epidermidis , and S. saprophyticus . The technical and financial expenditure can be reduced considerably, because an extended identification has to be applied only to strains which cannot be identified by the PNMI-Test.