Rapid and Accurate Identification of Nontuberculous Mycobacteria Directly from Positive Primary MGIT Cultures by MALDI-TOF MS

Over the last years, nontuberculous mycobacteria (NTM) have emerged as important human pathogens. Accurate and rapid mycobacterial species identification is needed to successfully diagnose, treat, and manage infections caused by NTM. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, MALDI-TOF MS, was demonstrated to effectively identify mycobacteria isolates subcultured from solid or liquid media rather than new positive cultures. The present study aims to develop a new extraction protocol to yield rapid and accurate identification of NTM from primary MGIT cultures by MALDI-TOF MS. A total of 60 positive MGIT broths were examined by the Bruker Biotyper system with Mycobacteria Library v. 2.0 (Bruker Daltonics GmbH & Co. KG., Bremen, Germany). The results were compared with those obtained by the molecular method, line probe assay GenoType Mycobacterium CM/AS/NTM-DR. All samples were concordantly identified by MALDI-TOF MS and the molecular test for all the tested mycobacteria. Fifty-seven (95%) MGIT positive cultures for NTM from clinical samples had a MALDI-TOF MS analysis score S ≥ 1.8. Although a small number of strains and a limited diversity of mycobacterial species were analysed, our results suggest that MALDI-TOF MS could represent a promising routine diagnostic tool for identifying mycobacterial species directly from primary liquid culture

Spotlight on the Compositional Quality of Probiotic Formulations Marketed Worldwide

On the worldwide market, a great number of probiotic formulations are available to consumers as drugs, dietary supplements, and functional foods. For exerting their beneficial effects on host health, these preparations should contain a sufficient amount of the indicated living microbes and be pathogen-free to be safe. Therefore, the contained microbial species and their amount until product expiry are required to be accurately reported on the labels. While commercial formulations licensed as drugs are subjected to rigorous quality controls, less stringent regulations are generally applied to preparations categorized as dietary supplements and functional foods. Many reports indicated that the content of several probiotic formulations does not always correspond to the label claims in terms of microbial identification, number of living organisms, and purity, highlighting the requirement for more stringent quality controls by manufacturers. The main focus of this review is to provide an in-depth overview of the microbiological quality of probiotic formulations commercialized worldwide. Many incongruences in the compositional quality of some probiotic formulations available on the worldwide market were highlighted. Even if manufacturers carry at least some of the responsibility for these inconsistencies, studies that analyze probiotic products should be conducted following recommended and up-to-date methodologies

Sperm ultrastructure and spermiogenesis in two Exogone species (Polychaeta, Syllidae, Exogoninae)

The spermatozoa of Exogone naidina and E. dispar are characterized by a prominent bell-shaped acrosome, a spheroidal nucleus, and a conventional flagellum. During spermiogenesis, the acrosomal vesicle undergoes conspicuous modifications leading to its final bell shape with a posterior opening. The subacrosomal material initially shows radiating filaments but in mature sperms it appears as a meshwork of electron-opaque material. The acrosomal axis is oblique with respect to the main longitudinal sperm axis. The chromatin is arranged in electronopaque strands in the early spermatids, then becomes amorphous, and is finally organized in filaments in mature sperms. Centrioles are orthogonally arranged beneath the nucleus and fibers radiate from the distal centriole to contact the plasma membrane and the single mitochondrion. The latter is located eccentrically on the side of the nucleus opposite the acrosome. A disk-shaped structure is evident beneath the distal centriole. The flagellar axoneme has a 9+2 microtubule pattern. A conspicuous glycocalyx surrounds the flagellar plasma membrane, and an electron-lucent space is present between these two structures at the distal tip of the flagellum. We compare the sperm morphology of these two species of Exogone with that described in other members of the subfamily Exogoninae. The fine structure of these two species supports the occurrence of an ent-aquasperm type within Exogoninae, in accordance with the brood strategy present within this subfamily. The mode of reproduction is of taxonomic importance for defining subfamilies within Syllidae, and is likely also of phylogenetic significance. Because epitoky is probably plesiomorphic, the ent-aquasperm type found in Exogoninae can be considered a derived feature within Syllidae

Survival and persistence of Bacillus clausii in the human gastrointestinal tract following oral administration as spore-based probiotic formulation

AIMS: This study aimed to investigate the fate of Bacillus clausii spores orally administered as lyophilized or liquid formulation to healthy volunteers. METHODS AND RESULTS: The study was a randomized, open-label, cross-over trial in which two commercial probiotic formulations containing spores of four antibiotic-resistant B. clausii strains (OC, NR, SIN, T) were given as a single dose administration. Faecal B. clausii units of each strain were counted on selective media and extrapolated for the total weight of evacuated faeces. RAPD-PCR typing was used to confirm B. clausii identification. Bacillus clausii was found alive in faeces for up to 12 days. In some volunteers, the recovered amount of OC, NR or SIN was higher than the number of administered spores. Bioequivalence among the two formulations was demonstrated. CONCLUSIONS: Bacillus clausii spores survive transit through the human gastrointestinal tract. They can undergo germination, outgrowth and multiplication as vegetative forms. Bacillus clausii strains can have different ability to survive in the intestinal environment. Bacillus clausii spores administered as liquid suspension or lyophilized form behave similarly in vivo. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes towards a better understanding of the behaviour of B. clausii spores as probiotics

Impact of polystyrene nanoparticles on marine diatom Skeletonema marinoi chain assemblages and consequences on their ecological role in marine ecosystems.

Marine diatoms have been identified among the most abundant taxa of microorganisms associated with plastic waste collected at sea. However, the impact of nano-sized plastic fragments (nanoplastics) at single cell and population level is almost unknown. We exposed the marine diatom Skeletonema marinoi to model polystyrene nanoparticles with carboxylic acid groups (PS-COOH NPs, 90 nm) for 15 days (1, 10, 50 mu g/mL). Growth, reactive oxygen species (ROS) production, and nano-bio-interactions were investigated. No effect on diatom growth was observed, however Dynamic light scattering (DLS) demonstrated the formation of large PS aggregates which were localized at the diatoms' fultoportula process (FPP), as shown by TEM images. Increase production of ROS and reduction in chain length were also observed upon PS NPs exposure (p < 0.005). The observed PS-diatom interaction could have serious consequences on diatoms ecological role on the biogeochemical cycle of carbon, by impairing the formation of fast-sinking aggregates responsible for atmospheric carbon fixation and sequestration in the ocean sea floor. S. marinoi exposure to PS NPs caused an increase of intracellular and extracellular oxidative stress, the reduction of diatom's chain length and the adhesion of PS NPs onto the algal surface

External gestation of Exogone naidina Öersted, 1845 (Polychaeta, Syllidae): Ventral attachment of eggs and embryos

The external gestation of sexually ripe females of the species Exogone naidina (Polychaeta, Syllidae) is described by means of SEM and TEM analysis. The eggs, embryos and juveniles are attached in close vicinity of each parapodial complex in a position immediately below each ventral cirrus and are connected to the female by a cup like structure. The formation of this adhesive disk is linked to secretory cells scattered between dermal cells of ripe female. This adhesive disk is present only in sexually mature animals and is considered as epitokous structure. The evolutive significance of ventral and dorsal attachment found within the Exogoninae is also discussed. © 2003 Elsevier Ltd. All rights reserved

Involvement of basic fibroblast growth factor in suramin-induced inhibition of V79/AP4 fibroblast cell proliferation.

The V79/AP4 Chinese hamster fibroblasts were densely stained with the anti-basic fibroblast growth factor (bFGF) antibody demonstrating an endogenous production of the peptide. The in vitro proliferation of these cells was stimulated by exogenous bFGF and the maximum growth (259% increase in 3H-thymidine incorporation into DNA) was reached with bFGF 10 ng ml-1. Inhibition of bFGF-mediated mitogenic pathway was obtained with a 15-mer antisense oligodeoxynucleotide targeted against bFGF mRNA and with suramin, a drug which blocks the biological activity of heparin-binding growth factors. bFGF antisense oligomer reduced the synthesis of DNA by 79.5 and 89.5% at 20 and 60 microM, respectively; this effect was reversed by the addition of exogenous bFGF to the culture medium. A short-term exposure to suramin 300 micrograms ml-1 produced a modest reduction in 3H-thymidine incorporation but suppressed the mitogenic effect of bFGF on V79/AP4 cells. In cells treated with suramin 300 micrograms ml-1 the drug concentration increased linearly over 3 days, reaching 13.15 micrograms mg-1 of protein; cell proliferation was inhibited in a dose-related manner as evaluated by the colony formation assay (IC50: 344.22 micrograms ml-1) and by the number of mitoses observed in culture. Furthermore, the drug induced ultrastructural alterations, consisting of perinuclear cisternae swelling, chromatin condensation, nucleolar segregation and cytoplasmic vacuolations. These findings demonstrated that the endogenous production of bFGF plays an important role in V79/AP4 fibroblasts proliferation, and the inhibition of bFGF-mediated mitogenic signalling with bFGF antisense oligomer or suramin is an effective mean of reducing cell growth

INVOLVEMENT OF BASIC FIBROBLAST GROWTH-FACTOR IN SURAMIN-INDUCED INHIBITION OF V79/AP4 FIBROBLAST CELL-PROLIFERATION

The V79/AP4 Chinese hamster fibroblasts were densely stained with the anti-basic fibroblast growth factor (bFGF) antibody demonstrating an endogenous production of the peptide. The in vitro proliferation of these cells was stimulated by exogenous bFGF and the maximum growth (259% increase in H-3-thymidine incorporation into DNA) was reached with bFGF 10 ng ml-1. Inhibition of bFGF-mediated mitogenic pathway was obtained with a 15-mer antisense oligodeoxynucleotide targeted against bFGF mRNA and with suramin, a drug which blocks the biological activity of heparin-binding growth factors. bFGF antisense oligomer reduced the synthesis of DNA by 79.5 and 89.5% at 20 and 60 muM, respectively; this effect was reversed by the addition of exogenous bFGF to the culture medium. A short-term exposure to suramin 300 mug ml-1 produced a modest reduction in H-3-thymidine incorporation but suppressed the mitogenic effect of bFGF on V79/AP4 cells. In cells treated with suramin 300 mug ml-1 the drug concentration increased linearly over 3 days, reaching 13.15 mug mg-1 of protein; cell proliferation was inhibited in a dose-related manner as evaluated by the colony formation assay (IC50: 344.22 mug ml-1) and by the number of mitoses observed in culture. Furthermore, the drug induced ultrastructural alterations, consisting of perinuclear cisternae swelling, chromatin condensation, nucleolar segregation and cytoplasmic vacuolations. These findings demonstrated that the endogenous production of bFGF plays an important role in V79/AP4 fibroblasts proliferation, and the inhibition of bFGF-mediated mitogenic signalling with bFGF antisense oligomer or suramin is an effective mean of reducing cell growth

Comparative distribution of azithromycin in lung tissue of patients given oral daily doses of 500 and 1000 mg

OBJECTIVES: The administration of antibacterial agents should be optimized on the basis of their distribution to enhance drug exposure and obtain bacterial eradication. This study examines the pharmacokinetics of azithromycin in plasma, lung tissue and bronchial washing in patients after oral administration of 500 mg versus 1000 mg once daily for 3 days. PATIENTS AND METHODS: Samples of plasma, lung tissue and bronchial washing were obtained from a cohort of 48 patients during open-chest surgery for lung resection up to 204 h after the last drug dose, and assayed for antibiotic concentrations. RESULTS: Azithromycin was widely distributed within the lower respiratory tract and sustained levels of the drug were detectable at the last sampling time in lung tissue. Doubling the dose of the antibiotic resulted in a proportional increase in lung area under the curve (AUC, 1245.4 versus 2514.2 h x mg/kg) and peak tissue concentration (Cmax, 8.93 +/- 2.05 versus 18.6 +/- 2.20 mg/kg). The pharmacodynamic parameter AUC/MIC for susceptible and intermediate strains of Streptococcus pneumoniae (MICs 0.5 and 2 mg/L, respectively) increased after administration of the 1000 mg schedule compared with 500 mg (AUC/MIC0.5 2414 versus 1144 and AUC/MIC2 2112 versus 814.1 h x mg/kg, respectively) in pulmonary tissue. CONCLUSIONS: Lung exposure to azithromycin is increased proportionally by doubling the dose, which results in a predictable pharmacokinetic behaviour of the drug in the lower respiratory tract