Inhibitor of complement, Compstatin, prevents polymer-mediated Mac-1 up-regulation of human neutrophils independent of biomaterial type tested.

The inflammatory reaction after cell contact with polymer materials is primarily mediated by activated neutrophils and may, in some cases, lead to exhaustion of neutrophil cell function. A direct consequence of this can be impairment of local or even systemic host defense mechanisms, which in turn can result in foreign body infections. Neutrophil activation, as indicated by the up-regulation of the Mac-1 adhesion receptor, is a reliable parameter for estimating the inflammatory risk due to implanted biomaterials. Because at blood contact, biomaterials immediately acquire a material-specific layer of blood proteins on their surface, including fibrinogen, complement, and immunoglobulin G, it is generally believed that after biomaterial contact, neutrophil activation primarily occurs by interaction with this protein layer. In this study, using our recently established polymer bead in vitro assay, we investigated whether complement inhibition alone can reduce biomaterial-mediated neutrophil activation, independent of the type of polymer and, hence, also its surface chemistry. Complement inhibition was achieved by using Compstatin, a recently developed complement inhibitor that binds to the complement component C3 preventing C3 convertase formation. We revealed significantly reduced (p < or = 0.025) Mac-1 receptor expression levels after 45 min of blood contact with the following polymers (without and with Compstatin): 1. polyurethane, 98.3%, 13.6%; 2. polymethylmetacrylate, 88.5%, 11.0%; and poly-D,L-lactide, 71.8%, 8.4%. Although these three polymer types acquire material-specific protein layers because of their different surface chemistry, complement inhibition by Compstatin alone proved to be sufficient to reduce neutrophil activation after surface contact, thus reducing the risk of biomaterial-mediated inflammatory reaction.

Analyses of phagocytosis, evoked oxidative burst, and killing of black yeasts by human neutrophils: a tool for estimating their pathogenicity?

The pathogenicity of several dematiaceous yeasts that have, to date, rarely been isolated in humans remains unclear. Because professional phagocytes are prominent in lesions caused by dematiaceous fungi, we address this issue by comparing phagocytosis, evoked oxidative burst and killing by human neutrophils of different black yeasts in vitro. Whereas phagocytosis of all black yeasts tested and evoked oxidative burst yielded comparable results, in contrast, the degree of killing differed significantly after 5 h. Thereby, two groups could be identified; one in which strains are killed at high rates, for example, Hortaea werneckii (81 +/- 11.6%), Exophiala castellanii (96 +/- 8.6%), Phaeoannellomyces elegans (93 +/- 9.7%), Phaeococcomyces exophialae (87 +/- 8.7%), and the other in which strains are killed to a lesser degree, for example, Exophiala dermatitidis (ATCC 34100) (61 +/- 9.5%), E. dermatitidis (CBS 207.35) (66 +/- 7.5%), E. jeanselmei (50 +/- 10.5%), E. mesophila (63 +/- 11.6%), E. bergeri (63 +/- 9.1%), and E. spinifera (57 +/- 9.6%). Non-pigmented yeasts were killed at levels comparable with those at which the white mutant strain of E. dermatitidis (ATCC 44504) was killed (95 +/- 7.5%); the yeast strains tested were Candida albicans (DSM 11943) (95 +/- 4.0% killing) and Saccharomyces cerevisiae (DSM 1333) (95 +/- 10.3%). Comparison of killing rates with the observed pathogenicity of the melanized species suggests that low killing rates might indicate or even predict a high degree of invasiveness. Although previous experiments revealed that melanization conferred killing resistance on E. dermatitidis, the differences in killing rates of other dematious fungi suggest that melanization of the cell wall is in itself insufficient to confer virulence.

Simultaneous measurement of biopolymer-mediated Mac-1 up-regulation and adherence of neutrophils: a novel flow cytometric approach for predicting initial inflammatory interaction with foreign materials.

Implantation of any medical device normally causes an inflammatory cell interaction with the foreign material. In vitro cell activation of human neutrophils (Mac-1 upregulation) has been taken as one measure to assess the attributable risk of inflammation due to biopolymers before their clinical application. Mac-1 expression has generally been measured by flow cytometric assays, whereas quantification of neutrophil adhesion to the biopolymer surfaces has been performed by separate and time-consuming assays, e.g. microscopically by differential cell counting. However, due to an increasing number of surface-modified novel biopolymers entering clinical usage, effective testing of their inflammatory potential is now mandatory. To facilitate these analyses, we have developed a novel flow cytometric assay permitting simultaneous measurement of biopolymer-mediated neutrophil activation and adhesion. The biopolymers were used as beads (diameter 25+/-10 microm), and were demonstrated to be non-phagocytosable and non-fluorescent before being co-incubated with whole human blood (range of ratio granulocytes/beads from 5:1 to 1:1). Besides flow cytometric measurement of Mac-1 up-regulated neutrophils as fluorescing events, a fluorescence of the bead population indicates the adherence of activated neutrophils to the biopolymer surface.After establishing this assay, we evaluated it by comparing six different biopolymers. We observed high levels of Mac-1 expression (>70% of positive control) accompanied by increased adhesiveness (>60% of neutrophils) for polyurethane (PUR), polymethylmetacrylate (PMMA), and poly-DL-lactide (PDLLA) beads. Low Mac-1 expression levels (<10%) accompanied by a low percentage of adhering neutrophils (<10%) were observed for polyethylene (PE), polyisoprene (PI), and silicone (SI) beads.

Assessment of acid production by various human oral micro-organisms when palatinose or leucrose is utilized.

One promising way of reducing caries is by using sucrose substitutes in food, e.g., palatinose or leucrose. Previous experiments addressing cariogenic potential of sucrose substitutes have focused mainly on Streptococcus mutans. However, given the many other micro-organisms in the oral cavity, this study compared the acid production of 100 bacterial strains representing 44 different species, by batch fermentation in a test tube containing, as a sole carbohydrate source, glucose, sucrose, palatinose, or leucrose. Selected strains were further analyzed in a fermenter. Additionally, 30 yeast strains were tested by an auxanographic sugar assimilation test. Only Lactobacillus spp., Stomatococcus mucilaginosus, Leuconostoc mesenteroides, and Weissella paramesenteroides, and some of the yeasts studied-i.e., Candida albicans, C. tropicalis, C. parapsilosis, and Saccharomyces cerevisiae-utilized leucrose and/or palatinose well. Strikingly, Stomatococcus mucilaginosus produced water-insoluble polysaccharides by fermentation of leucrose and palatinose. In the fermenter, the respective sucrose substitutes were not only cleaved but also utilized. Thus, extracellular cleavage by autochthonous micro-organisms may produce cariogenic cleavage products (glucose, fructose) that can be used by other well-characterized cariogenic bacteria found in the oral flora. Therefore, the anticariogenic potential of sucrose substitutes in food might be limited.

Case report. Pathohistological findings in a clinical case of disseminated infection with Fusarium oxysporum.

Despite appropriate antimicrobial and antifungal therapy (amphotericin B), a disseminated infection with Fusarium oxysporum in a 75-year-old immunocompromised patient (acute myeloid leukaemia, minimal leucocyte count of 0.5 giga l-1) led rapidly to death. A similarly fatal course of an F. oxysporum infection has been reported in several cases. Fusarium oxysporum could be isolated shortly before death from blood cultures and from a swab taken from skin efflorescences. An autopsy revealed histopathologically typical fungal infiltrates in the mucosa of the pharynx, epiglottis, trachea, and oesophagus and in the parenchyma of the spleen, the lung and both kidneys. Because of the high risk of a fatal outcome of this infection, the clinician should aim at maximum diagnostic enforcement. We propose both analysis of blood cultures and immediate skin biopsy--with PAS-staining--of suspicious dermal efflorescences for microscopic examination. The treatment of choice is discussed controversially but a beneficial effect has been reported from granulocyte transfusion, subcutaneous administration of GM-CSF and concomitant treatment with amphotericin B.

Differentiation between Candida dubliniensis and Candida albicans by fatty acid methyl ester analysis using gas-liquid chromatography.

Candida dubliniensis is often found in mixed culture with C. albicans, but its recognition is hampered as the color of its colonies in primary culture on CHROMagar Candida varies. Furthermore, definite identification of C. dubliniensis is difficult to achieve, time-consuming, and expensive. Therefore, a method to discriminate between these two closely related yeast species by fatty acid methyl ester (FAME) analysis using gas-liquid chromatography (Sherlock Microbial Identification System [MIS]; MIDI, Inc., Newark, Del.) was developed. Although the chromatograms of these two species revealed no obvious differences when applying FAME analysis, a new library (CADLIB) was successfully created using Sherlock Library Generation Software (MIDI). The amount and frequency of FAME was analyzed using library training files (n = 10 for each species), preferentially those comprising reference strains. For testing the performance of the CADLIB, clinical isolates genetically assigned to the respective species (C. albicans, n = 32; C. dubliniensis, n = 28) were chromatographically analyzed. For each isolate tested, MIS computed a similarity index (SI) indicating a hierarchy of possible strain fits. When using the newly created library CADLIB, the SIs for C. albicans and C. dubliniensis ranged from 0.11 to 0.96 and 0.53 to 0. 93 (for all but one), respectively. Only three isolates of C. albicans (9.4%) were misidentified as C. dubliniensis, whereas all isolates of C. dubliniensis were correctly identified. Resulting differentiation accuracy was 90.6% for C. albicans and 100% for C. dubliniensis. Cluster analysis and principal component analysis of the resulting FAME profiles showed two clearly distinguishable clusters matching up with two assigned species for the strains tested. Thus, the created library proved to be well suited to discriminate between these two species.

Phagocytosis, oxidative burst, and killing of Candida dubliniensis and Candida albicans by human neutrophils.

Candida dubliniensis is a phylogenetically closely related species to Candida albicans. So far virtually nothing is known about the virulence factors of C. dubliniensis. Cell surface hydrophobicity (CSH) plays a critical role in adhesion of microorganisms to phagocytic cells; hydrophobic cells of C. albicans have been reported to be less sensitive to phagocytic killing than hydrophilic cells. C. dubliniensis displays CSH at 37 degrees C in contrast to C. albicans. To elucidate this issue, we determined levels of phagocytosis, oxidative burst and killing by human neutrophils of C. dubliniensis (n=10) compared to C. albicans (n=10) both cultured at 37 degrees C. Obtained test results revealed no statistically significant differences between these two yeast species for the level of phagocytosis (77.3 vs. 76.2% after 60 min), evoked oxidative burst (64.5 vs. 67.3% after 30 min) and killing (72.7 vs. 73.1% after 240 min). Therefore, human neutrophils can be considered to be equally efficient against these two yeast species.

Investigation of infectious organisms causing pericoronitis of the mandibular third molar.

PURPOSE: The purpose of the study was to identify the most frequently encountered pyogenic organisms involved in pericoronitis to permit more targeted antibiotic therapy. PATIENTS AND METHODS: Pericoronal pockets of mandibular third molars from 37 patients showing symptoms of acute, severe pericoronitis were sampled and subjected to microbiologic analysis, including primary evaluation by phase-contrast microscopy. To avoid overgrowth with faster-growing, less fastidious organisms, specimens were cultured on a wide variety of selective media (supporting growth of fastidious bacteria, protozoa, and fungi). RESULTS: Microscopic examination indicated spirochetes in 55% and fusiform bacteria in 84% of the samples. A total of 441 microorganisms were isolated and identified from the 37 cultured samples. Besides obligate anaerobic bacteria, including various Actinomyces and Prevotella species, a predominantly facultative anaerobic microflora was cultivated, that is, Streptococcus milleri group (78% of samples), Stomatococcus mucilaginosus (71%), and Rothia dentocariosa (57%). CONCLUSION: It was concluded that the Streptococci milleri group bacteria, well-known for their ability to cause suppurative infections, are most likely involved in the pathogenesis of acute severe pericoronitis of the lower third molar.

Discriminative power of fatty acid methyl ester (FAME) analysis using the microbial identification system (MIS) for Candida (Torulopsis) glabrata and Saccharomyces cerevisiae.

Candida (Torulopsis) glabrata is frequently isolated in cases of fungal infection and commonly shows acquired or innate fluconazole resistance. Saccharomyces cerevisiae, an emerging opportunistic yeast pathogen, causes serious systemic infections in immunocompromised, and vaginitis and superficial infections in immunocompetent patients. For both species reliable identification in the routine laboratory is mandatory, but species identification of strains, e.g. trehalose-negative C. glabrata, may be difficult. Therefore, gas-liquid chromatography (GLC) of whole cell fatty acid methyl ester (FAME) profiles, that is independent of assimilation profiles of strains and suitable for reliable and rapid identification of clinically important yeasts, was applied. However, frequent misidentification of C. glabrata as S. cerevisiae has been reported when using the Yeast Clinical Database of MIS. Accuracy of MIS identification may be strongly influenced by the amounts of cell mass analyzed. Therefore, the present study compared the MIS results of these two yeasts achieved with different cell masses. Primarily we optimized, especially with respect to cost-effectiveness, the recommended streaking technique yielding a maximal recovery of 90-130 mg of cell mass from one plate, enabling testing of poor growing strains of C. glabrata. For all C. glabrata strains tested (n = 10) the highest identification scores (SI [Similarity Index] range 0.525-0.963, median 0.832) were achieved with 30 to 45 mg of cell mass. Only 5 of 10 S. cerevisiae strains revealed good library comparisons (SI > or = 0.5) when using 30 mg of cell mass, whereas with 45 mg all strains but two revealed this SI-level. For S. cerevisiae a higher amount of cell mass processed (up to 90 mg) was correlated with better identification scores (SI range using 90 mg: 0.464-0.870, median, 0.737). Several passages prior to FAME analysis of C. glabrata strains on recommended media revealed narrowing of SI ranges, but differences in SI values were not statistically significant.

Black fungi: clinical and pathogenic approaches.

Data are presented on the clinically relevant black yeasts and their relatives, i.e., members of the Ascomycete order Chaetothyriales. In order to understand the pathology of these fungi it is essential to know their natural ecological niche. From a relatively low degree of molecular variability of the black yeast Exophiala dermatitidis, potential agent of brain infections in patients from East Asia, it is concluded that this species is an emerging pathogen, currently going through a process of active speciation. It is found to be an oligotrophic fungus in hot, moist environments, such as steambaths. Cladophialophora-, Fonsecaea- and Ramichloridium-like strains, known in humans as agents of chromoblastomycosis, are frequently found on rotten plant material, but the fungal molecular diversity in the environment is much higher than that on the human patient, so that it is difficult to trace the etiological agents of the disease with precision. This approach has been successful with Cladophialophora carrionii, of which cells resembling muriform cells, the tissue form of chromoblastomycosis, were found to occur in drying spines of cacti. Phagocytosis assays provide a method to distinguish between pathogens and non-pathogens, as the killing rates of strict saprobes proved to be consistently higher than of those species frequently known as agents of disease. The therapeutic possibilities for patients with chromoblastomycosis are reviewed.

Effect of melanin and carotenoids of Exophiala (Wangiella) dermatitidis on phagocytosis, oxidative burst, and killing by human neutrophils.

The black yeast Exophiala (Wangiella) dermatitidis is an increasingly recognized pathogen and a leading cause of severe pheohyphomycosis. Melanin is thought to contribute to the virulence of E. dermatitidis. Whereas the synthesis and the redox properties of melanin have been studied intensively, the influence of melanin and carotenoids on the phagocytosis, the oxidative burst, and the killing of E. dermatitidis by human neutrophils has not been studied. To study their effects on these phenomena, we applied a combination of flow cytometry and a colony-count-dependent method. Using E. dermatitidis wild-type strain 8565 and several melanin-deficient mutants that have been described previously, we demonstrate that melanin prevents this pathogen from being killed in the phagolysosome of the neutrophils. Melanin did not influence the phagocytosis or the oxidative burst of the neutrophils involved. The carotenoids torulene and torularhodine were not found to contribute to the prevention of killing. The ability of E. dermatitidis to block the effects of the neutrophil oxidative burst may critically impair the potential of the host to sufficiently eliminate this fungal pathogen and thus may play an important role in the pathogenesis of phaeohyphomycosis.

Rapid identification of Candida glabrata by using a dipstick to detect trehalase-generated glucose.

Candida glabrata is a yeast frequently isolated from human specimens. Based upon its well-known ability to rapidly hydrolyze trehalose, we have developed a novel and cost-effective test incubating one yeast colony emulsified in 50 microl of citrate buffer (0.1 M [pH 5. 0]) containing 4% (wt/vol) trehalose for 3 h at 37 degrees C. Trehalase-generated glucose is detected with a commercially available dipstick (range, 1.0 to 50 g/liter). For evaluation, consecutive clinical isolates and several reference strains of C. glabrata (n = 160), C. albicans (n = 120), and other yeast species with potential ability for utilization of trehalose (C. dubliniensis, n = 11; C. famata, n = 15; C. guilliermondii, n = 5; C. lusitaniae, n = 16; C. parapsilosis, n = 20; C. tropicalis, n = 34; C. viswanathii, n = 5; Pichia angusta, n = 2; C. zeylanoides, n = 2; Saccharomyces cerevisiae, n = 16; C. neoformans, n = 7) were tested. Identification of C. glabrata is achieved within 3 h, with a specificity of 99.1% and a sensitivity of 98.8% when grown on Sabouraud dextrose agar supplemented with 4% glucose.

Novel microtiter plate format for testing germ tube formation and proposal of a cost-effective scheme for yeast identification in a clinical laboratory.

The germ tube test is most widely used for presumptive identification of Candida albicans. Conventional testing is relatively time-consuming due to the hands-on time involved in preparing and viewing each isolate. In order to reduce work-load and costs we have developed a novel microtiter plate test format that offers several advantages: (i) use of removable strips of microtiter wells placed in lockwell frames, (ii) only one pipetting step for each isolate, (iii) direct micromorphological evaluation using an inverse microscope, (iv) use of a novel synthetic germ tube test medium, (v) reduction of the inoculum, permitting testing of minute colonies, (vi) thus, testing of different colonies in potentially mixed primary cultures of clinical specimens is encouraged and facilitated. Implementing this microtiter based germ tube test with simultaneous trehalase test for presumptive identification of Torulopsis (Candida) glabrata, we propose an identification scheme including this test format. This has been implemented in our routine laboratory permitting cost-effective presumptive identification of almost all clinically relevant yeast species.

[Methicillin-resistant Staphylococcus aureus (MRSA)--clinical implications]. / Methicillin-resistente Staphylococcus aureus (MRSA)--klinische Implikationen.

The isolation rate of MRSA from Staphylococcus aureus infections rose to 8.7% in German hospitals between 1990 and 1995. Patients undergoing surgical treatment or physiotherapy showed a threefold increase in the risk of being colonized or infected with MRSA. Surgical wound infection is the most frequent among MRSA-induced infections (28%). The main transmission path of MRSA inside a hospital is bacterial spread from one patient to another through contact with the hands of nursing staff. Therefore, the crucial strategy in avoiding the spread of bacteria is strict hygiene management through hand disinfection. The most widespread therapeutic regimen for simultaneous eradication of nasal colonization and treatment of infection on other body sites is mupirocin nasal ointment combined with parenteral vancomycin application. The non-indicated use of vancomycin, e.g., for perioperative prophylaxis or prevention of catheter-induced infections, should be avoided, especially after the appearance of vancomycin-intermediately sensitive S. aureus (VISA) strains that have been reported recently from Japan and the USA.