Longer resistance of some DNA traits from BT176 maize to gastric juice from gastrointestinal affected patients.

The presence of antibiotic resistance marker genes in genetically engineered plants is one of the most controversial issues related to Genetically Modified Organism (GMO)-containing food, raising concern about the possibility that these markers could increase the pool of antibiotic resistance genes. This study investigates the in vitro survival of genes bla and cryIA(b) of maize Bt176 in human gastric juice samples. Five samples of gastric juice were collected from patients affected by gastro-esophageal reflux or celiac disease and three additional samples were obtained by pH modification with NaHCO3. DNA was extracted from maize Bt176 and incubated with samples of gastric juices at different times. The survival of the target traits (bla gene, whole 1914 bp gene cry1A(b), and its 211 bp fragment) was determined using PCR. The stability of the target genes was an inverse function of their lengths in all the samples. Survival in samples from untreated subjects was below the normal physiological time of gastric digestion. On the contrary, survival time in samples from patients under anti-acid drug treatment or in samples whose pH was modified, resulted strongly increased. Our data indicate the possibility that in particular cases the survival time could be so delayed that, as a consequence, some traits of DNA could reach the intestine. In general, this aspect must be considered for vulnerable consumers (people suffering from gastrointestinal diseases related to altered digestive functionality, physiological problems or drug side-effects) in the risk analysis usually referred to healthy subjects.

Melaleuca alternifolia essential oil possesses potent anti-staphylococcal activity extended to strains resistant to antibiotics.

Melaleuca alternifolia Cheel essential oil (TTO) and its major component terpinen-4-ol were examined against a large number of clinical isolates of Staphylococcus aureus to establish their anti-staphylococcal activities. Classic and established procedures were used to study M.I.C., time-kill curves, synergism and mutational frequency. The anti-staphylococcal activity of terpinen-4-ol and TTO were superior to those of antibiotics belonging to the major families (all the tested drugs are for topical use or included in ointments, eye drops or used during surgery); terpinen 4-ol and TTO were active against strains resistant to mupirocin, fusidic acid, vancomycin, methicillin and linezolid. TTO and terpinen-4-ol were bactericidal as revealed by time-kill curves; the frequency of mutational frequency to TTO was < 2.9 x 10 9. The study demonstrates good anti-staphylococcal activity of TTO and terpinen-4-ol against a large number of S.aureus isolates and suggests the possible application of these agents for topical treatment of staphylococcal infections. This is the first extensive study on the anti-staphylococcal activity of TTO. The results suggest that this compound may have application as a topical agent for the control of superficial staphylococcal infections, including activity against organisms resistant to antibiotics which can be used, or are specific, for topical use.

Genetic variability among Blastoschizomyces capitatus isolates from different clinical sources.

Sixteen clinical isolates and nine ATCC reference strains of Blastoschizomyces capitatus were analysed genetically, examined for the cellobiose, arbutin and salicin assimilation and tested for the aspartyl-proteinase secretion. The restriction endonuclease analysis (REA) with HpaII and HinfI enzymes and the electrophoretic karyotype (EK) were investigated. Both the restriction enzymes revealed two groups (I, II) constituted by the same isolates: 17 isolates (68%) in group I and 8 (32%) in group II. The EK analysis revealed sixteen groups. These data prompts for a genetic variability of the isolates of Blastoschizomyces capitatus and their account in two distinct genetic groups as suggested by REA. This grouping was confirmed by examining the utilisation of cellobiose, arbutin and salicin. The tests for secretory aspartyl proteinase (Sap) were positive only for three isolates, suggesting a marginal role of this specific enzyme in pathogenesis for these isolates.

Slime production by clinical isolates of Blastoschizomyces capitatus from patients with hematological malignancies and catheter-related fungemia.

In order to expand the present knowledge of the pathogenic potential of Blastoschizomyces capitatus in central venous catheter (CVC)-related bloodstream infections, six strains of the organism recovered from three leukemic patients with CVC-related fungemia in different years were investigated. Isolates and control strains were tested for their genetic relatedness and for their ability to produce slime in glucose-containing solutions. DNA restriction enzyme analysis revealed that all strains of B. capitatus were identical, whereas slime production assays and examination of ex vivo material showed that they were able to produce large amounts of slime. Slime production may therefore play a relevant pathogenic role in cases of CVC-related fungemia caused by B. capitatus.

Identification, molecular biotyping and ultrastructural studies of bacterial communities isolated from two damaged frescoes of St Damian's Monastery in Assisi.

AIM: To investigate the composition of the microbial community in biodeterioration of two frescoes in St Damian's Monastery in Assisi. METHODS AND RESULTS: A total of 1292 colonies were isolated from the most deteriorated parts, analysed by microbiological, biomolecular and ultrastructural techniques, and taxonomically classified. Molecular biotyping of Staphylococcus cohnii colonies, one of the most prevalent bacterial species, showed a very restricted genome diversity while Bacillus licheniformis were very homogeneous by RFLP, tDNA-PCR and random-amplified polymorphic DNA. Electron microscopy confirmed heterogeneity of the bacterial population in the different sampling areas. CONCLUSIONS: Several of the identified species are widespread in the soil or saprophytes of human skin. Although unable to demonstrate that they are involved in biodeterioration, they may represent trophic elements contributing to fungi-related chromatic alterations when adequate environmental conditions occur. Deterioration may in part be prevented or controlled by adequate air filtering or conditioning of the room.

Antimycotic activity of Melaleuca alternifolia essential oil and its major components.

AIMS: The aim of this study was to analyse the antimycotic properties of Melaleuca alternifolia essential oil (tea tree oil, TTO) and its principal components and to compare them with the activity of 5-fluorocytosine and amphotericin B. METHODS AND RESULTS: The screening for the antimycotic activity was performed by serial twofold dilutions in Roswell Park Memorial Institute medium with the inclusion of Tween-80 (0.5%). TTO and terpinen-4-olo were the most active compounds. CONCLUSIONS: The majority of the organisms were sensitive to the essential oil, with TTO and terpinen-4-olo being the most active oils showing antifungal activity at minimum inhibitory concentration values lower than other drugs. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides a sample large enough to determine the antifungal properties of TTO and terpinen-4-olo and suggests further studies for a possible therapeutic use.

Onychomycosis caused by Blastoschizomyces capitatus.

Blastoschizomyces capitatus was cultured from the nail of a healthy patient with onychomycosis. The identity of the isolate was initially established by standard methods and ultrastructural analysis and was verified by molecular probing. Strains ATCC 200929, ATCC 62963, and ATCC 62964 served as reference strains for these analyses. To our knowledge, this is the first case of nail infection secondary to paronychia caused by this organism reported in the English literature.

Correlation of SfiI macrorestriction endonuclease fingerprint analysis of Candida parapsilosis isolates with source of isolation.

SfiI macrorestriction digests from whole chromosome DNA preparations of 46 isolates of Candida parapsilosis from vaginal (20 isolates), blood (23 isolates) and soil (three isolates) sources were examined by CHEF-MAPPER pulsed-field electrophoresis. The isolates were grouped into nine macrorestriction endonuclease fingerprint (MEF) classes according to the number or size of the macrorestriction fragments, or both. The electrophoretic karyotype (EK) was also examined and found to contain 18 karyotypic classes (named A-R). A comparison between SfiI MEF and EK demonstrated that the former correlated much better than the latter with the source of C. parapsilosis isolates. Five SfiI classes (I-V) contained only vaginal isolates (or vaginal and three soil isolates, class I), and the blood isolates were distributed between four classes (VI-IX). This relationship was less evident with the EK classes as several of these were composed of both vaginal and blood isolates (B, G, L and M). The three soil isolates were in class A which also included one vaginal isolate. We conclude that SfiI macrorestriction endonuclease patterns seem to be useful in discriminating among C. parapsilosis isolates, with apparent association with source of isolation.

Biotype diversity of Candida parapsilosis and its relationship to the clinical source and experimental pathogenicity.

Environmental, vaginal, and blood isolates of Candida parapsilosis were biotyped by karyotype analysis in pulsed-field gel electrophoresis. Morphotype and resistotype were also determined as was aspartyl proteinase secretion and pathogenicity in a systemic mouse infection model. Overall, the karyotype patterns consisted of 6-9 chromosome bands (> 3.0-0.6 Mb) with limited clustering, since most isolates had unique chromosome profiles. Major clusters C. parapsilosis, differing by source of isolation and in experimental pathogenicity, could be discriminated by morphoresistotyping. The morphotypes of isolates from subjects with candidemia ranged from one that caused elevated mortality in the normal mouse to those that were totally avirulent in the neutropenic animal. Among clinical isolates, secretion of aspartyl proteinase was higher in vaginitis than in candidemia isolates and did not correlate with the experimental pathogenicity. These results emphasize the biotype diversity of C. parapsilosis and have potentially important epidemiologic and pathologic implications.

Filamentous growth and elevated vaginopathic potential of a nongerminative variant of Candida albicans expressing low virulence in systemic infection.

The vaginopathic potential and the intravaginal morphology of a nongerminative variant of Candida albicans, strain CA-2, were studied in a rat vaginitis model. Although it expressed low virulence in systemic infections, strain CA-2 was capable of causing a vaginal infection of the same duration and extent as that obtained in rats challenged with the germ-tube-forming strain C. albicans 3153 from the stock collection or with a fresh clinical isolate of C. albicans from a case of human vaginitis. During the experimental infection, the CA-2 cells did not maintain their yeast morphology but gave rise to single enlarged-elongated elements (1 to 2 days) which grew predominantly as coarse, short, pseudomycelium-like filaments (2 to 3 days) and then as long threads (7 days). These latter filaments were ultimately indistinguishable from the hyphal filaments formed by the germ-tube-forming strains, which, however, initially developed in the vagina by typical germ tube formation. This peculiar morphological development of strain CA-2 was not observed in organs of systemically infected mice, where, in contrast to strain 3153 which formed typical hyphae, strain CA-2 maintained a typical pattern of yeast growth. Vaginal isolates of strain CA-2 taken at different days of infection were found to be identical to the challenging CA-2 cells, in terms of biochemical characteristics, inability to form germ tubes in any medium at 37 degrees C in vitro, echinocandin resistance, DNA biotype, and low virulence in systemic infections in mice. Thus, experimental vaginitis by strain CA-2 is associated with a peculiar filamentous growth in the vagina, through an apparently novel morphological development bypassing classical germ tube formation but ultimately leading to ordinary hyphae. The elevated vaginopathic potential of strain CA-2, in contrast to its low virulence in systemic infection, also suggests that different Candida virulence factors (and host responses) come into play in local and disseminated candidal infections.

Fluconazole treatment of catheter-related right-sided endocarditis caused by Candida albicans and associated with endophthalmitis and folliculitis.

An unusual case of catheter-related right-sided endocarditis, endophthalmitis, and extensive folliculitis, apparently caused by a single DNA biotype of Candida albicans, was successfully treated with a 6-month course of fluconazole plus two intravitreous doses of amphotericin B. The patient was a 21-year-old man who underwent colectomy for diffuse polyposis and developed the clinical syndrome just described following total parenteral nutrition for the treatment of purulent anal fistulas. Fluconazole was initially administered at a daily dose of 200 mg, with 600 mg daily given after 4 weeks. Clinical improvement resulted, with no relapse during 14 months of follow-up. Sequential measurements by an enzyme-linked immunosorbent inhibition assay demonstrated that levels of circulating mannoprotein antigen of C. albicans fell from 75 ng/mL to less than 1 ng/mL after the institution of fluconazole therapy. These observations seem to confirm previous reports on the efficacy of fluconazole as sole therapy for candidal endocarditis and suggest a role for serological studies in clinical monitoring of severe candidal infections.

DNA fingerprinting and electrophoretic karyotype of environmental and clinical isolates of Candida parapsilosis.

The endonuclease restriction pattern (DNA fingerprinting) and the electrophoretic karyotype of 16 Candida parapsilosis isolates from environmental and clinical sources were investigated. DNA from both whole cells and separated mitochondria was digested with enzymes, including EcoRI, BamHI, KpnI, BglII, HpaII, PvuII, and HindIII. Regardless of their source and pathogenic properties, all isolates showed a uniform, reproducible, and overlapping whole-cell DNA fingerprinting with each endonuclease digest. Mitochondrial DNA fragments were, in all cases, major contributors to the total cellular DNA restriction pattern. In contrast, the electrophoretic karyotype generated by rotating field gel electrophoresis (RFGE) or contour clamped homogeneous field electrophoresis (CHEF) showed a remarkable polymorphism among the isolates. This polymorphism concerned the smaller molecular size section of the karyotype (range, 1.8 to 0.7 Mb), where at least two to five chromosomal bands could be consistently detected by both RFGE and CHEF. Larger (greater than or equal to 3.0 to 1.9 Mb) chromosome-sized DNA bands (four in CHEF and three in RFGE) were quite distinct and common to all isolates. Thus, seven karyotype classes could be defined, on the basis of both the number and size of putative chromosomes. The three categories of isolates (soil, vaginal, and hematological) were not randomly distributed among the seven classes. In particular, the four hematological isolates had a karyotype pattern which was clearly distinct from that shown by the three environmental isolates, and of the nine vaginal isolates only one shared a class with isolates from another source (soil). Although tentative, the classification was totally consistent with the independent and reproducible results obtained by the two pulse-field electrophoretic techniques employed. It is suggested that the electrophoretic analysis of the karyotype might be particularly useful for epidemiological and pathogenicity studies on biotypes of C. parapsilosis.