Capillary electrophoresis study of outer membrane proteins of Pseudomonas strains upon antibiotic treatment.

Nosocomial wound infections by Pseudomonas aeruginosa strains have increasing importance in orthopaedic surgery. Outer membrane protein composition and cell-surface hydrophobicity of the bacteria have strong influence on adhesion to living tissues or artificial medical devices. Outer membrane proteins of five Pseudomonas strains (KT 2, KT 7, KT 25, KT 28, KT 39) isolated from orthopaedic patients' wounds and one standard strain NIH Hungary 170000 isolated from pus were examined. The capillary electrophoretic patterns of the outer membrane proteins were characteristic to each bacterial strains possessing different relative ratios of major and minor proteins. Antibiotic treatment of bacteria with three antibiotics, cefotaximum, amoxicillinum/clavulinic acid and amikacinum (applied often in prophylaxis and treatment of patients) induced changes in the electrophoretic profiles showing that outer membrane protein composition was altered significantly. The most pronounced alterations in the electrophoretic patterns after antibiotic treatment were obtained in the cases of the strains KT 2, KT 7 and KT 28. The amikacinum administration strongly decreased the relative percentage of the 38800 rel. mol. mass protein in KT 2 (from 20 to 6%). while the relative amount of the same protein increased significantly in KT 7 and KT 28 after cefotaximum treatment (from 2 to 16% and from 12 to 28%, respectively). Decrease in cell-surface hydrophobicity was also observed by salt aggregation test. The results obtained can be used to determine the therapeutic concentrations of antibiotics to induce changes in the adhesion properties of bacteria.

Effect of iron restriction on outer membrane protein composition of Pseudomonas strains studied by conventional and microchip electrophoresis.

In this study the outer membrane protein (OMP) composition of six Pseudomonas aeruginosa strains had been analyzed by conventional CE and microchip electrophoresis. Bacterial OMPs are important virulence factors and play a significant role in the pathogenesis of infectious diseases. Changes in their composition might refer to the altered pathogenic properties and antibiotic sensitivity of a certain strain. Pathogenic bacteria invading the human host have to multiplicate under iron-restricted conditions that induce changes in the OMP composition. High-molecular-weight OMPs have to be expressed, which serve as receptors for the iron-siderophore complexes. OMP patterns of bacteria obtained by the two different methods in this study were similar, all major proteins could be detected by both techniques, and the molecular weights showed good correlations, although direct comparison of the peak areas is not straightforward due to the different detection methods (UV and LIF). Changes in OMP composition under iron restriction could be detected, and appearance of a 92 kDa protein in all six P. aeruginosa strains and a 94 kDa protein in the KT 2 strain could be demonstrated. Besides that up- and downregulation of certain proteins could be also detected. The increased separation speed, picoliters of sample consumption, baseline separation achieved more frequently by this method--especially in the high-molecular-weight region--showed the advantages of microchip electrophoresis in the analysis of clinical samples.

Effect of antibiotic treatment on bacterial attachment to a DePuy Enduron orthopedic implant.

BACKGROUND: The increasing incidence of bacterial infections in orthopedic surgery might be related to the increasing application of artificial devices. In most cases, bacteria multiply on the surface of implants in biofilms. Poor penetration of antibiotics, frequent necessity of prosthesis removal, chronic processes and financial costs emphasize the significance of preventive measures. METHOD: Adhesion of bacterial strains (two Staphylococcus aureus, two coagulase-negative staphylococci and two Pseudomonas aeruginosa strains isolated from orthopedic patients' wounds) to the surface of a polyethylene cup was investigated using an ultrasonic method. Results were compared to the adhesive ability of three Hungarian standard strains. The effect of antibiotic treatment (cefuroxime, cefotaxime, amoxicillin with clavulanic acid and amikacin) has been examined. RESULTS: The staphylococcal strains showed significantly higher adhesive ability than Pseudomonas strains. Antibiotic treatment significantly reduced the attachment of bacteria. The higher the concentration of the antibiotics, the higher was the decrease in bacterial adhesion. CONCLUSIONS: Antibiotic prophylaxis was proven to be effective against bacterial adhesion, and, if applied at the proper time at the highest tolerable dose, it might prevent the formation of biofilms.

Comparison of antibody repertoires against Staphylococcus aureus in healthy individuals and in acutely infected patients.

The management of staphylococcal diseases is increasingly difficult with present medical approaches. Preventive and therapeutic vaccination is considered to be a promising alternative; however, little is known about immune correlates of protection and disease susceptibility. To better understand the immune recognition of Staphylococcus aureus by the human host, we studied the antistaphylococcal humoral responses in healthy people in comparison to those of patients with invasive diseases. In a series of enzyme-linked immunosorbent assay analyses performed using 19 recombinant staphylococcal cell surface and secreted proteins, we measured a wide range of antibody levels, finding a pronounced heterogeneity among individuals in both donor groups. The analysis revealed marked differences in the antibody repertoires of healthy individuals with or without S. aureus carriage, as well as in those of patients in the acute phase of infection. Most importantly, we identified antigenic proteins for which specific antibodies were missing or underrepresented in infected patients. In contrast to the well-described transient nature of disease-induced antistaphylococcal immune response, it was demonstrated that high-titer antistaphylococcal antibodies are stable for years in healthy individuals. In addition, we provide evidence obtained on the basis of opsonophagocytic and neutralizing activity in vitro assays that circulating antistaphylococcal serum antibodies in healthy donors are functional. In light of these data we suggest that proper serological analysis comparing the preexisting antibody repertoires of hospitalized patients with different outcomes for nosocomial staphylococcal infections could be extremely useful for the evaluation of candidate vaccine antigens in addition to protection data generated with animal models.

Effect of antibiotics on cell surface hydrophobicity of bacteria causing orthopedic wound infections.

BACKGROUND: Despite antibiotic prophylaxis and treatment, the incidence of wound infections in orthopedic surgery is significant. Postoperative wound infection is a multifactorial process, which can be modified by several bacterial factors. Cell surface hydrophobicity of bacteria is a very important physicochemical feature, which has a great influence on the ability of bacteria to adhere to the surface of host cells or medical implants. METHODS: In this study, the hydrophobic properties of thirteen bacterial strains (coagulase-negative staphylococci, Staphylococcus aureus and Pseudomonas aeruginosa) isolated from patients with postoperative deep wound infections following orthopedic procedures were determined by the salt aggregation test. Results were compared to the hydrophobicity of three Hungarian standard bacterial strains. The modifying effect of four antibiotics (cefuroxime, cefotaxime, amoxicillin combined with clavulanic acid and amikacin)--applied most often in our Department for prophylaxis and treatment of patients--were analyzed. RESULTS: The cell surface hydrophobicity of certain strains showed considerable changes after antibiotic treatment. These alterations indicated the decrease in hydrophobicity. Supra-inhibitory concentrations (2x minimum inhibitory concentrations, MIC) of the antibiotics were able to induce more frequent alterations in hydrophobicity than sub-inhibitory (0.5x MIC) levels. CONCLUSIONS: Alterations in cell surface hydrophobicity caused by antibiotics can modify the adhesion process and thus the pathogenicity of bacterial strains. These changes should be taken into consideration in the management of proper antibiotic prophylaxis and in the treatment of orthopedic patients.