FEMS immunology and medical microbiology最新文献

This study establishes the genetic relatedness among Brazilian Histoplasma capsulatum samples obtained from different sources. A PCR-based random amplified polymorphic DNA (RAPD) assay was used to delineate polymorphisms among isolates in geographically diverse regions in Brazil. RAPD fingerprints revealed distinct DNA profiles and provided a high level of discrimination among H. capsulatum strains from different locations. Cluster I was composed of H. capsulatum isolates from the northeast region. The majority of strains from southeast and south were categorized as major cluster II. The strain 84564 from Rio de Janeiro State showed no genetic correlation to any of the isolates from the same state. The RAPD patterns of H. capsulatum isolates from Goias (Cluster III) were unrelated to DNA fingerprints observed among the other H. capsulatum strains (48% similarity). This study is the first report that stratifies the clusters of H. capsulatum strains from Brazil by molecular typing and associates them with the geographical origin.

The detection of Pneumocystis DNA in clinical specimens by using PCR assays is leading to important advances in Pneumocystis pneumonia (PcP) clinical diagnosis, therapy and epidemiology. Highly sensitive and specific PCR tools improved the clinical diagnosis of PcP allowing an accurate, early diagnosis of Pneumocystis infection, which should lead to a decreased duration from onset of symptoms to treatment, a period with recognized impact on prognosis. This aspect has marked importance in HIV-negative immunocompromised patients, who develop often PcP with lower parasite rates than AIDS patients. The specific amplification of selected polymorphous sequences of Pneumocystis jirovecii genome, especially of internal transcribed spacer regions of the nuclear rRNA operon, has led to the identification of specific parasite genotypes which might be associated with PcP severity. Moreover, multi-locus genotyping revealed to be a useful tool to explore person-to-person transmission. Furthermore, PCR was recently used for detecting P. jirovecii dihydropteroate synthase gene mutations, which are apparently associated with sulfa drug resistance. PCR assays detected Pneumocystis-DNA in bronchoalveolar lavage fluid or biopsy specimens, but also in oropharyngeal washings obtained by rinsing of the mouth. This non-invasive procedure may reach 90%-sensitivity and has been used for monitoring the response to treatment in AIDS patients and for typing Pneumocystis isolates.

This review provides an overview of several molecular and cellular approaches that are likely to supply insights into the host–fungus interaction. Fungi present intra- and/or extracellular host–parasite interfaces, the parasitism phenomenon being dependent on complementary surface molecules. The entry of the pathogen into the host cell is initiated by the fungus adhering to the cell surface, which generates an uptake signal that may induce its cytoplasmatic internalization. Furthermore, microbial pathogens use a variety of their surface molecules to bind to host extracellular matrix (ECM) components to establish an effective infection. On the other hand, integrins mediate the tight adhesion of cells to the ECM at sites referred to as focal adhesions and also play a role in cell signaling. The phosphorylation process is an important mechanism of cell signaling and regulation; it has been implicated recently in defense strategies against a variety of pathogens that alter host-signaling pathways in order to facilitate their invasion and survival within host cells. The study of signal transduction pathways in virulent fungi is especially important in view of their putative role in the regulation of pathogenicity. This review discusses fungal adherence, changes in cytoskeletal organization and signal transduction in relation to host–fungus interaction.

Phloxine B (D&C red no. 28) is a color additive for food, drugs, and cosmetics. It has been previously shown to have anti-Staphylococcus aureus activities. In this work, the effect of Phloxine B on various gram-negative bacteria and other gram-positive bacteria including Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, Bacillus subtilis, Bacillus aureus, Salmonella, Escherichia coli and Shigella was studied, along with the mechanism of anti-microbial activity. In the presence of fluorescent light, the viable count for gram-positive bacteria, (Bacillus spp. and S. aureus) decreased in a dose and time dependent manner when incubated with Phloxine B. The viability of gram-positive bacteria was reduced by 99.99% in 40 min, while there was no effect on gram-negative bacteria (Salmonella choleraesuis, E. coli and Shigella flexneri). However, the use of ethylenediaminetetraacetic acid (EDTA) expands the spectrum of activity for Phloxine B to include gram-negative bacteria. EDTA increased membrane-permeability by releasing lipopolysaccharide. Overall, in an Agar diffusion test the light-dependent bactericidal activity of 1 μg of Phloxine B had a potency of 0.64 units of chloramphenicol and 0.5 units of tetracycline when tested on B. cereus, and had a potency of 0.7 units of chloramphenicol and 0.2 units of tetracycline when tested on S. aureus. The data suggest that the dye may have some potential anti-microbial applications.

Small-colony variants (SCVs) of Staphylococcus aureus exhibit characteristics of bacteria that can penetrate mammalian cells and remain intracellular and innocuous for indefinite periods. These properties make SCVs a convenient tool that can be used to identify new antibacterial agents having activity against intracellular, quiescent bacteria. Agents active against SCVs could be useful in the treatment of chronic staphylococcal infections such as bovine mastitis. An hemB deletion mutant of S. aureus Newbould, a bovine mastitis isolate, having a stable, genetically defined SCV phenotype, was used in a screening program to identify compounds active against intracellular, gram-positive bacteria. Out of more than 260,000 compounds screened, nine compounds having the desired properties were identified. The range of MICs against gram-positive bacteria was ⩽0.12–32 μg ml⁻¹. One of the compounds (no. 8) showed excellent activity against gram-positive (MICs ⩽0.12 μg ml⁻¹) and gram-negative (MICs ⩽0.12–4 μg ml⁻¹) bacteria. Each of the nine compounds demonstrated efficacy in a neutropenic mouse thigh infection model. Two compounds, including compound no. 8, reduced numbers of bacteria in a mouse mastitis model of infection. Application of a stepwise screening process has identified lead compounds that may be useful for treating persistent, intracellular infections.

To determine the significance of poultry and bovine as infectious sources of Campylobacter jejuni in Japan, the serotype distribution and pulsed-field gel electrophoresis (PFGE) patterns of poultry and bovine isolates were compared with those of isolates from patients with diarrhea in Akita (Japan). Serotypes O:2 and O:4-complex were common in human, poultry, and bovine isolates, and serotype O:23,36,53 was common in human and bovine isolates. SmaI PFGE patterns of isolates belonging to these serotypes were generated. Eight PFGE patterns were shared by poultry and human isolates and three patterns were shared by human and bovine isolates. Further analysis of the isolates having the same SmaI PFGE pattern by KpnI PFGE confirmed that four patterns and two patterns were still shared by poultry and human isolates, and bovine and human isolates, respectively. Thus, serotypic and genotypic data indicated a possible link between sporadic human campylobacteriosis and C. jejuni from retail poultry and bovine bile and feces, suggesting that bovine serves as an infectious source of C. jejuni in Japan, as is observed in other countries.

Microbial metabolites may influence the metabolic integrity of intestinal epithelial cells and induce mucosal immune responses. Therefore, we investigated the effects of the microbial metabolites butyrate, iso-valerate, and ammonium on Caco-2 cells and macrophages. Barrier functioning was determined by measuring transepithelial electrical resistance and basolateral recoveries of metabolites. The barrier function of Caco-2 cells remained intact after exposures. Basolateral recoveries ranged from 6.2% to 15.2%. Tumour necrosis factor-α and interleukin-10 were measured to determine immune reactions. The Caco-2 cells did not secrete both cytokines. Physiological concentrations of butyrate and iso-valerate stimulated the secretion of tumour necrosis factor-α and suppressed the secretion of interleukin-10 by macrophages that are not protected by an epithelial barrier. In contrast, ammonium concentrations as high as those produced by microbiotas of IBD patients suppressed the release of both cytokines when the barrier function is impaired.

Excess of iron promotes Mycobacterium tuberculosis infection, its replication and progression to clinical disease and death from tuberculosis. Chelation of iron may reduce M. tuberculosis replication, restore host defence mechanisms and it could constitute an application in the prevention and treatment strategies where both iron overload and tuberculosis are prevalent. We investigated the effect of iron and iron chelating agents, like desferrioxamine and silybin, individually and in combination with iron on mycobacterial number, viability in culture and after recovery from monocyte-macrophages, together with monocyte-macrophages viability and oxidative defence. Mycobacterial number and viability in culture were assessed using real-time quantitative PCR of H37Rv IS6110 DNA, 16S rRNA and 85B mRNA, whereas the microplate AlamarBlue^TM assay was used to detect viability in culture post-infection. Mitochondrial membrane potential and phosphatidyl serine exposure of monocyte-macrophages, detected using Mitotracker Red fluorescence and Annexin V binding, respectively, served as indicators of host cell viability. Superoxide generation served as marker of monocyte-macrophage effector functions. Extracellular H37Rv showed a significant increase in number and viability in presence of excess iron and, by large, a significant decrease in number and viability in presence of the iron chelating agents, silybin and desferrioxamine, compared to cultivation without supplementation. Intracellularly, excess iron increased H37Rv viability significantly but reduced monocyte-macrophages mitochondrial membrane potential and compromised superoxide production. Desferrioxamine had little influence on intracellular parameters, but consistently prevented effects of excess iron, while silybin significantly altered most intracellular parameters and mostly failed to prevent effects of excess iron. These findings suggest that chelation therapy should be considered in conditions of iron overload and that effective chelating agents like desferrioxamine, with limited intracellular access might need to be used in combination with lypophilic chelating agents.

Periodontitis is a multi-factorial chronic inflammatory and destructive disease of the tooth-supporting tissues. Quantitative anaerobic culture techniques have been used for microbial diagnosis of the different forms of the disease. The aim of this study was to compare real-time PCR with quantitative anaerobic culture for detection and quantification of 5 prominent periodontal pathogens. Real-time PCR assays with the 16s rRNA genes of Actinobacillus actinomycetemcomitans, Prevotella intermedia, Tannerella forsythensis, Peptostreptococcus micros and Fusobacterium spp. were developed. The PCR was validated on pure cultures of various bacterial strains. Subsequently, subgingival plaque samples from 259 adult patients with periodontitis were analyzed with quantitative anaerobic culture and real-time PCR. A standard curve for DNA quantification was created for each primer-probe set based on colony-forming units equivalents.

All bacterial species were correctly identified. The lower limits of detection by PCR varied between 1–50 colony-forming units equivalents depending on the species. No cross-reactivities with heterologous DNA of other bacterial species were observed. Real-time PCR results showed a high degree of agreement with anaerobic culture results. Real-time PCR is a reliable alternative for diagnostic quantitative anaerobic culture of subgingival plaque samples.