On the mechanism of membrane damage by Staphylococcus aureus alpha-toxin.

Rabbit or human erythrocytes lysed with Staphylococcus aureus alpha-toxin were solubilized with Triton X-100, and the toxin was subsequently isolated by gel chromatography, sucrose density gradient centrifugation, and reincorporation into liposomes. In the presence of Triton X-100, the toxin exhibited a sedimentation coefficient of 11S and eluted at a position between those of IgG and alpha 2-macroglobulin in gel chromatography. A single polypeptide subunit of 34,000 mol wt was found in SDS PAGE. In the electron microscope, ring-shaped or cylindrical structures were observed, 8.5-10 nm in diameter, harboring central pits or channels 2-3 nm in diameter. An amphiphilic nature of these structures was evident from their capacity to bind lipid and detergent, aggregation in the absence of detergents, and low elutability from biological and artificial membranes through ionic manipulations. In contrast to the membrane-derived form of alpha-toxin, native toxin was a water-soluble, 34,000 mol wt, 3S molecule, devoid of an annular structure. Because studies on the release of radioactive markers from resealed erythrocyte ghosts indicated the presence of circumscribed lesions of approximately 3-nm effective diameter in toxin-treated membranes, the possibility is raised that native alpha-toxin oligomerizes on and in the membrane to form an amphiphilic annular complex that, through its partial embedment within the lipid bilayer, generates a discrete transmembrane channel.

Examination of computer assisted prescribing of an initial calculated antibiotic treatment.

The objective of this prospective clinical usage study was to examine the value of the rule based 'Therapeutic Assistant' integrated into an existing Patient Data Management System (PDMS) in helping to prescribe a initial antibiotic regime in accordance with the requirements of accepted guidelines. A prospective study comparing data before and after the introduction of the 'Therapeutic Assistant' was carried out. An adequate therapy resulted significantly more often after the introduction of the 'Therapeutic Assistant' [p<0.05]; however no difference between the regimes with and without the 'Therapeutic Assistant' in the period after its introduction could be established. Whether the 'Therapeutic Assistant' influenced the prescriptions made without it will have to be established in a further study.

Perioperative microbiologic monitoring of tracheal aspirates as a predictor of pulmonary complications after cardiac operations.

The value of preoperative and early postoperative microbiologic testing of tracheal aspirates as a prognostic indicator of the development of pneumonia was evaluated in a prospective study of 213 cardiac surgical patients. Tracheal aspirates were obtained immediately after intubation and after the patient's arrival at the intensive care unit. Diagnosis of pneumonia was accepted if at least three of the following criteria were fulfilled: leukocytosis > 15,000 cells/mm3, body temperature >38.5 degrees C, positive results of auscultation, positive results of radiography (new infiltrates that seemed to be consistent with pneumonia), and increased core-reactive protein for more than 2 days after operation. Potentially pathogenic microorganisms were found in 54 (25.4%) of the preoperative tracheal aspirates and in 27 (12.7%) of the early postoperative tracheal aspirates. Positive microbiologic findings correlated with pneumonia in the postoperative course in 24.1% (p < 0.001) if the preoperative culture results were positive, in 48.2% (p < 0.001) if the postoperative culture results were positive, and in 44.0% (p < 0.001) if both were positive. The risk of pneumonia was increased in male patients (p < 0.05) and in patients with chronic obstructive pulmonary disease (p < 0.05). Demographic variables, smoking, acute pulmonary symptoms, temperature, leukocyte count at the day of the operation, and data on the operation and the extracorporeal circulation were not significantly related to pneumonia in the early postoperative course. The risk of development of postoperative pneumonia is significantly higher among patients with colonization of the lower respiratory tract. Positive culture results in routine microbiologic monitoring of tracheal aspirates are predictive of pulmonary complications after cardiac operations.

Preoperative microbiologic screening and antibiotic prophylaxis in pulmonary resection operations.

BACKGROUND: Pulmonary resection is associated with considerable risk of infection, so antibiotic prophylaxis has become routine practice in pulmonary operations. We studied two standard flash antibiotic prophylaxis regimens and matched them to preoperatively acquired microorganisms. METHODS: In 120 patients scheduled for elective pulmonary resection, aspirates were taken separately from the left and the right lung using a double-lumen tube. Then the patients received either 1.5 g of sulbactam plus ampicillin (n = 60; group 1) or 2 g of cefazolin (n = 60; group 2) intravenously as a single-shot antibiotic prophylaxis according to a prospective randomized sequence. When bacteria were found in the aspirates, both antibiotics were tested for susceptibility. The patients were monitored for the first 3 postoperative days with regard to bronchopulmonary infections. RESULTS: Fifty-eight pathogens were isolated from the 120 patients. The cultured bacteria did not differ significantly between the two groups. In group 1 all found bacteria were susceptible to the used antibiotic prophylaxis, whereas in group 2 eight of the 25 found bacteria were not susceptible to antibiotic prophylaxis. Postoperatively, group 2 showed significantly more signs of bronchopulmonary infections than the group 1 and subsequently needed additional antibiotics more often. Intensive care unit stay was longer in patients of group 2 and costs were higher for these patients. CONCLUSIONS: Preoperative microbiologic examination could be helpful to evaluate efficacy of the antibiotic prophylaxis regimen. Sulbactam plus ampicillin was significantly more effective than cefazolin.

[Reduction of bacterial contamination during mechanical autotransfusion by uv irradiation--initial results]. / Reduktion von bakteriellen Kontaminationen während maschineller Autotransfusion durch UV-Bestrahlung--Erste Ergebnisse.

OBJECTIVE: To investigate the effects of UV-radiation of autologous blood on bacteria and red blood cells when using intraoperative sampling of autologous blood using a cell separator--an established method for reducing the need for donor blood during surgery--which is reported to have a bacterial contamination rate of 5-75%, due mainly to coagulase-negative staphylococci (CNS). METHODS: Cell-separator blood was diluted to a haematocrit level permitting transmission of 1% of the UV-radiation used in this study (lambda 254 nm, coat thickness 1 mm). CNS samples were irradiated for 2, 4, 10, 20 and 30 seconds. Free haemoglobin and methaemoglobin levels were measured, and erythrocytes examined microscopically at the end of the procedure. RESULTS: Blood samples had to be diluted to a haematocrit of 1% to permit transmission of 1% of the UV light. The optimal irradiation duration was 4 seconds, when bacteria were completely eliminated. Longer irradiation durations were associated with increasing levels of free haemoglobin and methaemoglobin, the levels of which at 4 seconds exposure were 12.5 mg/L and 15.5%, respectively. CONCLUSIONS: It is possible to prevent CNS contamination of cell-separator blood by irradiation with UV light. Prior to clinical application, however, the method will need to be modified to minimize side effects and increase its decontamination efficacy.

Diagnosis of fungal infections.

Fungal infections become more and more important due to their increasing incidence in immunocompromised patients. In these patients opportunistic fungi (Candida, Aspergillus, Cryptococcus) can cause life-threatening infections. Diagnosis of fungal infections is difficult. Clinical symptoms are uncharacteristic, and laboratory diagnosis is confronted with many problems and requires expert interpretation.

Incorporation of staphylococcal alpha-toxin in glutaraldehyde fixed erythrocytes.

The incorporation of staphylococcal alpha-toxin into glutaraldehyde fixed erythrocytes occurs in the same way as with native erythrocytes. Binding of alpha-toxin to the cells is accompanied by oligomerization of native 3 S toxin to the membrane-bound 11 S toxin hexamer, which is embedded into the lipid bilayer of the membrane. Antibodies against alpha-toxin, build up during an infection with S. aureus, can be determined in a passive hemagglutination test (IHT) using glutaraldehyde fixed and alpha-toxin treated erythrocytes. To test the validity of this IHT, antibodies to alpha-toxin were determined in 550 human sera of patients from hospitals of the University of Giessen suspected to suffer from staphylococcal infections and in 300 sera of healthy blood donors. The results were compared with the titres obtained by a convenient neutralisation test (ASTA). All sera with elevated titres in the ASTA test also showed high titres in the IHT. Because it is simple to perform and highly reproducible, the IHT seems to be a valuable test for detection of antibodies against staphylococcal alpha-toxin.

Alteration of the erythrocyte membrane during Newcastle disease virus-induced hemolysis.

In attempting to explain the mechanism whereby hemoglobin is released during NDV-induced hemolysis it was found that marker substances like albumin or myoglobin with diameters larger than 30 A are retained in the erythrocyte membrane, whereas low molecular weight material is released. This indicates that NDV-induced hemolysis does not result in the formation of membrane lesions but rather in the alteration of membrane permeability. A prerequisite for this process is the insertion of viral envelope components into the cell membrane. NDV-induced hemolysis can be enhanced by virus-specific antibodies and complement. In this case the release of marker substances is due to membrane lesions since large molecules also are released from the erythrocytes.

[Diseases caused by group G beta-hemolytic streptococci]. / Erkrankungen durch beta-hämolysierende Streptokokken der Gruppe G.

Group G beta-haemolytic streptococci were isolated from two patients, one with life-threatening septicaemia and meningitis, the other with puerperal sepsis and endometritis. The bacteria were isolated by blood culture and from cerebrospinal fluid in one patient, and from cervical swabs and urine in the other. In both treatment with a cephalosporin followed by penicillin G promptly led to recovery. These cases may signal an increasing incidence of severe infections caused by group G beta-haemolytic streptococci.

Correlation between toxin binding and hemolytic activity in membrane damage by staphylococcal alpha-toxin.

The binding of Staphylococcus aureus alpha-toxin to rabbit and human erythrocytes was studied by hemolytic assays and sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting. Hemolytic assays showed that toxin binding to 10% cell suspensions at neutral pH was very ineffective in the concentration range 3 X 10(-8) to 3 X 10(-7) M (1 to 10 micrograms/ml), and less than 5% of added toxin became cell bound. However, binding was augmented as toxin levels were raised, abruptly increasing to 50 to 60% at 2 X 10(-6) to 3 X 10(-6) M (60 to 100 micrograms/ml). When rabbit erythrocytes were lysed with 1 to 5 micrograms of toxin per ml, both monomeric and hexameric forms of the toxin could be detected on the membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting. In contrast, human erythrocytes treated with 1 to 6 micrograms of toxin per ml did not lyse, and membrane-bound toxin was not detectable. When toxin concentrations were raised to 30 to 100 micrograms/ml, human erythrocytes also lysed and toxin hexamers became membrane bound in comparable amounts as on rabbit cell membranes. Lowering the pH led to a marked increase in susceptibility of human, but not rabbit erythrocytes towards alpha-toxin. When human cells were lysed at pH 5.0 with 5 micrograms of toxin per ml, membrane-bound hexameric toxin became detectable. The demonstrated correlation between the presence of hexameric, cell-bound toxin and hemolytic activity supports the channel concept of toxin-mediated cytolysis. The results also show that toxin binding does not exhibit overall characteristics of a simple receptor-ligand interaction.

Staphylococcal alpha-toxin: oligomerization of hydrophilic monomers to form amphiphilic hexamers induced through contact with deoxycholate detergent micelles.

Native staphylococcus aureus alpha-toxin is secreted as a hydrophilic polypeptide chain of Mr 34,000. The presence of deoxycholate above the critical micellar concentration induced the toxin monomers to self-associate, forming ring or cylindrical oligomers. The oligomers were amphiphilic and bound detergent. In deoxycholate solution, the protein-detergent complexes exhibited a sedimentation coefficient of 10.4 S. A Mr of 238,700 was determined by ultracentrifugation analyses at sedimentation equilibrium. Because quantitative detergent-binding studies indicated a protein/detergent ratio of approximately 5:1 (wt/wt), the protein moiety in each protein-detergent complex was determined to be approximately Mr 200000, corresponding to a hexamer of the native molecule. The amphiphilic toxin hexamers were ultrastructurally indistinguishable from the cytolytic, annular toxin complexes that form on and in biological target membranes. They bound lipid and could be incorporated into artificial lecithin lipid vesicles. The transition of toxin protein molecules from a hydrophilic monomer to an amphiphilic oligomer through self-association has thus been shown to be inducible solely through contact of the native protein molecules with an appropriate amphiphilic substrate.

Development of resistance by Enterobacter cloacae during therapy of pulmonary infections in intensive care patients.

The emergence of resistance during therapy and the efficacy of different antibiotic therapy regimens were studied in 38 intensive care patients suffering from pulmonary infections caused by Enterobacter cloacae. Every three days a fresh isolate was obtained from each patient and tested in vitro for susceptibility to 16 antibiotics by determination of the minimal inhibitory concentrations. During therapy with cefotaxime and tobramycin the E. cloacae strains from 47% of the patients became resistant to cefotaxime within 6 days. In all cases resistance encompassed all other broad-spectrum penicillins and cephalosporins tested, as well as aztreonam. Development of resistance regularly led to persistence of bacteria. Resistance to tobramycin, ciprofloxacin or imipenem was not observed. Treatment of 25 patients with persisting E. cloacae infections was successful in 17 out of 18 patients treated with imipenem and in 6 out of 7 patients receiving ciprofloxacin.

Influence of the age of sheep red blood cells on virus-induced and hypotonic hemolysis.

The effect of red blood cell age on the susceptibility towards NDV- induced hemolysis has been investigated. It was found that young sheep red blood cells (SRBC), although more resistant to hypotonic hemolysis, are more susceptible towards viral hemolysis than older cells. This may be because young red blood cells have a higher content of neuraminic acid, which functions as a receptor for NDV.

[Candida infections in intensive care patients]. / Candida-Infektionen bei Intensivpatienten.

In 31 of 60 long-term ventilated intensive-care patients, a significant Candida antigen titer in serum was found that correlated with positive Candida cultures. In some cases the detection of the antigen was the first indication of Candida infection, while the isolation of the infectious agent required a culture period of 24-48 hours. In addition to routinely performed controls of Candida colonisation, determination of Candida antigen in the serum appears to be a valuable tool in the early diagnosis of candida infections.

Nosocomial infections in general surgery: surveillance report from a German university clinic.

At the general surgery clinics, University of Giessen, we developed our own system for surveillance of nosocomial infections according to the guidelines of the Centers of Disease Control. Atlanta, USA, and according to the results of the SENIC Project. We wanted to receive information about the overall infection rate, the procedure specific infection rate, site specific infection rate, distribution of nosocomial infections by pathogen and resistance pattern of antibiotics at the general surgery clinics. The overall infection rate of operations, classified as clean, clean--contaminated, and contaminated and dirty, was 13%. The surgical wound infection rate of 3% after clean operations was mainly caused by an elevated infection rate of 13% after clean operations of a prolonged duration and hyperthermic perfusion of the extremities in patients with melanoma. There is also a difference in nosocomial infection rates at the general surgery ward (11%) and at the intensive care unit (29%). At the intensive care unit candida and coagulase negative staphylococci are mainly isolated whereas Escherichia coli, Staphylococcus aureus and Enterococcus faecalis dominated the general surgery ward. Different operations show different distributions of isolates; operations on the pancreas are prone to have infections with coagulase negative staphylococci, candida and Pseudomonas aeruginosa. The antibiotic susceptibility tests for the most commonly used antibiotics revealed no resistance problems for E. coli, E. faecalis, and Staphylococcus aureus, common pathogens at the general surgery ward, but did for coagulase-negative staphylococci where we can consider only a few antibiotics like amikacin in obvious infections at the intensive care unit.

[Rapid and reliable detection of multiresistent Staphylococcus aureus (MRSA) by multiplex PCR]. / Schneller und zuverlässiger Nachweis multiresistenter Staphylococcus aureus (MRSA) durch Multiplex-PCR.

BACKGROUND AND OBJECTIVE: Staphylococci are widespread pathogens and are frequently associated with nosocomial infections. Many hospitals struggle with increasing amounts of methicillin-resistant Staphylococcus aureus (MRSA) which are "multiresistant" against all betalactam antibiotics. Often, applicable antibiotics for treatment are only glycopeptides like vancomycin and teicoplanin. In addition, MRSA infected patients require expensive intensive isolation measures and strict hygiene. To efficiently prevent dissemination of these pathogens rapid and reliable identification and a close collaboration between clinicians and microbiologists are required. The purpose of our study was to set up a rapid and reliable identification procedure for MRSA by the amplification of specific gene determinants by PCR in order to to efficiently support therapy and eradication of the pathogen. METHODS: 153 strains of staphylococci isolated from in-patients of the hospital of the Justus-Liebig University of Giessen were examined. The femB gene was used to differentiate between Staphylococcus aureus (S. aureus) and coagulase-negative staphylococci (CNS), a gene which allows the species-specific identification of methicillin-resistant (MRSA) and -susceptible S. aureus (MSSA). Additionally, MRSA harbor the mecA gene encoding methicillin-resistance, which is absent in MSSA strains. RESULTS: Using a multiplex PCR with femB and mecA gene-specific oligonucleotides MRSA strains were unequivocally detected within 3 hours. The femB gene was detected in all 102 strains of S. aureus but in none of the 51 CNS. The mecA determinant was detected in 12 S. aureus. Among these, 11 strains were phenotypically methicillin-resistant and one strain was susceptible. The methicillin-resistance of this particular mecA-positive/methicillin-susceptible strain (cryptic MRSA) was inducible by cultivation on agar plates supplemented with flucloxacillin. CONCLUSIONS: The described method specifically detects S. aureus and identifies phenotypical and cryptic MRSA. These cryptic MRSA are of particular relevance since they are undetectable using common phenotypically based detection methods. It is conceivable that the methicillin resistance of these strains is induced under antibiotic therapy with flucloxacillin and that the mec-encoded feature of methicillin-resistance can be transferred to previously methicillin-susceptible strains. Using the reliable detection of these strains by PCR, failure of flucloxacillin therapy is avoidable.

Function of the adrenal cortex during therapy with fluconazole in intensive care patients.

An impairment of cortisol synthesis can be assumed for the new antimycotic fluconazole based on its chemical structure (triazole derivative) and mechanism of action (inhibition of ergosterol synthesis). In healthy volunteers, however, no influence on steroid hormone production could be found. The present study was undertaken to clarify whether this is also true for critically ill, long-term patients in an intensive care unit. The basal cortisol and adrenocorticotropic hormone (ACTH) levels were determined by means of radioimmunoassay in 11 patients being treated with antimycotics at fixed times. Antimycotic treatment was carried out using either fluconazole (n = 6) or a combination of amphotericin B and flucytosine (n = 5) for 14 days. Seven days after cessation of the treatment the above-mentioned hormones were again determined. Patients with the same baseline criteria who did not require antimycotic treatment (n = 8) served as controls. During the entire study period adequate cortisol synthesis was found after ACTH stimulation in all three patient groups. They all presented with relatively raised basal cortisol levels (range 16.4-31.0 micrograms dl-1) and an increase in ACTH-stimulated cortisol synthesis from 31% (group ampho B/flucytosine) to 78% (group fluconazole). The basal ACTH values were always within the normal range (9.2-16.4 pg ml-1). Neither the basal ACTH levels nor the basal cortisol levels as well as the cortisol levels determined after the ACTH test showed adrenocortical suppression in the patients of all three groups. Thus, according to the present results clinically relevant impairment of cortisol synthesis after treatment with fluconazole can be excluded.