Development of azole resistance during fluconazole maintenance therapy for AIDS-associated cryptococcal disease.
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Biomedical subjects
Publications and source records attributed to R Füssle.
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We report an outbreak of sepsis from contaminated disinfectant in a neonatal and paediatric intensive-care unit. 28 infants were infected with Klebsiella oxytoca and basic measures to control the outbreak failed. The resistance of K. oxytoca against the disinfectant was probably mediated by capsule formation, visible as mucoid colonies.
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.
Depending on the aggressiveness of the pathogen and a patient's immunocompetence, fungal polypoid pansinusitis or allergic fungal sinusitis (AFS) may be a life-threatening disease. Apart from the clinical findings, its diagnosis is based on the demonstration of mucinous material with abundant eosinophils in the paranasal sinuses (indicating an allergic process), cultivation of the causative pathogen and immunocompetence of the patient. In a 20-year-old immigrant Sudanese woman, AFS due to Bipolaris (Drechslera) hawaiiensis was diagnosed. Because of intracranial extension, the disease had led to erosion of the cranial base and orbit with amaurosis on the right side and focal epilepsy. In addition to endonasal microsurgical pansinus operations, local irrigation therapy with amphothericin B was accompanied by systemic treatment with itraconazole after in vitro cultivation of the pathogen and determination of its sensitivities. Interdisciplinary management included a combination of endonasal surgery with debridement of infected tissues and wide drainage of the sinuses without removal of skull bone or the dural lesion in addition to specific antimycotic treatment. Injury to adjacent anatomical structures must be avoided in any case to prevent systemic or possibly lethal dissemination of infection.
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.
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.
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.
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.
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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.
Pulmonary infections are the most life-threatening infections in mechanically ventilated patients. Methods to avoid these infections by prophylactic systemic or local administration of antibiotics may promote resistance and selection of distinct groups of pathogens. In mechanically ventilated patients we studied the impact of early diagnosis and specific therapy on the prevention of pulmonary infections. Due to the very short interval between colonization and infection, daily microscopic and microbiologic examinations of tracheobronchial secretions proved to be essential for early and successful therapy and even prevention of pulmonary infections. PATIENTS AND METHODS. The present study comprised a total of 190 patients admitted to the surgical intensive care unit who required mechanical ventilation for a period of at least 48 h: 56 were admitted for multiple trauma; 38 had peritonitis; and the remainder had postoperative complications such as renal failure, cardiac problems, septicemia or pneumonia. Multitraumatized patients (16.5 days) and those with peritonitis (13.8 days) needed the most extensive ventilatory support. After admission antibiotic therapy was started with a second-generation cephalosporin or amoxicillin/clavulanic acid. Further antibiotic treatment was directed strictly against the isolated pathogens. Tracheobronchial secretions were monitored daily by microscopy and cultures. Microscopic evaluation was essential to discriminate between colonization and inflammation, and often indicated the infective agent. If infections were suspected provisional antibiograms were performed on the material. This procedure allowed a specific antibiotic treatment to be initiated 8-12 h later. In patients with pulmonary infections, additional bronchoscopic material was taken in order to correlate these findings with those gained from the tracheobronchial secretions. RESULTS. In 85% of cases massive colonization of the trachea with Pseudomonas aeruginosa, enterobacteria, Staphylococcus aureus or Streptococcus pneumoniae resulted in pulmonary infections 24-48 h later. The reduced virulence of Pseudomonas species (non-aeruginosa) and Acinetobacter species is reflected by an infection rate of 50% and an extended period of time to establish an infection (2-4 days). Only 30% of patients highly contaminated with Candida developed pulmonary infections after 3-6 days. A fair correlation (86%) was found between pathogens isolated in tracheobronchial secretions and bronchoscopic material. In the population studied, 68 patients (35.7%) developed pulmonary infections, 32 of them pneumonia (16.8%), and the others purulent tracheobronchitis with fever. Both groups were treated with antibiotics. Patients with multiple trauma, often accompanied by lung contusion, were most frequently affected. In 59 patients (87%) pulmonary infections were treated successfully by specific antibiotic therapy; 9 patients died so rapidly, that the pulmonary complication could not account for the fatal outcome. In 38 patients with massive contamination of the tracheobronchial system by enterobacteria, Pseudomonas aeruginosa or Staph. aureus, progression from colonization to infection was prevented by early administration of specific therapy. CONCLUSIONS. Because pulmonary infections in most cases arise very soon after pathogens have gained access to the tracheobronchial system daily monitoring of tracheobronchial secretions is required for early initiation of specific therapy.
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.
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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.
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 is produced by most strains of S. aureus and is considered a major pathogenic factor of these bacteria. The toxin is produced as a water-soluble molecule of MW 34000. Binding to a membrane target is accompanied by the formation of ring-structured hexamers with outer and inner diameters of 10 and 2-3 nm, respectively. The toxin rings carry lipid-binding surfaces that allow for insertion into and firm embedment within the membrane. Small transmembrane channels are thus generated that can induce a variety of pathological cellular changes. Large doses of toxin will generally cause cell lysis and death. However, sub-cytolytic toxin doses can also elicit major pathophysiological reactions. When introduced into the circulation of an isolated and perfused rabbit lung, the toxin causes steep rises in the pulmonary artery pressure, and lung edema results as a consequence of increases in vascular permeability occurring in parallel. These processes are the result of the activation of the arachidonic acid cascade by alpha-toxin in the lung. Studies using cultured endothelial cells as targets subsequently led to a hypothesis that would explain how membrane channel formation by a toxin could be linked to the observed arachidonic acid cascade activation. In essence, we propose that the toxin pores serve as non-physiological calcium channels, and that calcium influx triggers the observed reactions. It is probable that many other pathophysiological processes including inflammatory tissue reactions derive from such secondary effects of toxin action.(ABSTRACT TRUNCATED AT 250 WORDS)
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