Systematic whole-genome sequencing reveals an unexpected diversity among actinomycetoma pathogens and provides insights into their antibacterial susceptibilities

Mycetoma is a neglected tropical chronic granulomatous inflammatory disease of the skin and subcutaneous tissues. More than 70 species with a broad taxonomic diversity have been implicated as agents of mycetoma. Understanding the full range of causative organisms and their antibiotic sensitivity profiles are essential for the appropriate treatment of infections. The present study focuses on the analysis of full genome sequences and antibiotic inhibitory concentration profiles of actinomycetoma strains from patients seen at the Mycetoma Research Centre in Sudan with a view to developing rapid diagnostic tests. Seventeen pathogenic isolates obtained by surgical biopsies were sequenced using MinION and Illumina methods, and their antibiotic inhibitory concentration profiles determined. The results highlight an unexpected diversity of actinomycetoma causing pathogens, including three Streptomyces isolates assigned to species not previously associated with human actinomycetoma and one new Streptomyces species. Thus, current approaches for clinical and histopathological classification of mycetoma may need to be updated. The standard treatment for actinomycetoma is a combination of sulfamethoxazole/trimethoprim and amoxicillin/clavulanic acid. Most tested isolates had a high IC (inhibitory concentration) to sulfamethoxazole/trimethoprim or to amoxicillin alone. However, the addition of the β-lactamase inhibitor clavulanic acid to amoxicillin increased susceptibility, particularly for Streptomyces somaliensis and Streptomyces sudanensis. Actinomadura madurae isolates appear to have a particularly high IC under laboratory conditions, suggesting that alternative agents, such as amikacin, could be considered for more effective treatment. The results obtained will inform future diagnostic methods for the identification of actinomycetoma and treatment.     

Molecular Identification of Nanoplanktonic Protists Based on Small Subunit Ribosomal RNA Gene Sequences for Ecological Studies1

Nanoplanktonic protists are comprised of a diverse assemblage of species which are responsible for a variety of trophic processes in marine and freshwater ecosystems. Current methods for identifying small protists by electron microscopy do not readily permit both identification and enumeration of nanoplanktonic protists in field samples. Thus, one major goal in the application of molecular approaches in protistan ecology has been the detection and quantification of individual species in natural water samples. Sequences of small subunit ribosomal RNA (SSU rRNA) genes have proven to be useful towards achieving this goal. Comparison of sequences from clone libraries of protistan SSU rRNA genes amplified from natural assemblages of protists by the polymerase chain reaction (PCR) can be used to examine protistan diversity. Furthermore, oligonucleotide probes complementary to short sequence regions unique to species of small protists can be designed by comparative analysis of rRNA gene sequences. These probes may be used to either detect the RNA of particular species of protists in total nucleic acid extracts immobilized on membranes, or the presence of target species in water samples via in situ hybridization of whole cells. Oligonucleotide probes may also serve as primers for the selective amplification of target sequences from total population DNA by PCR. Thus, molecular sequence information is becoming increasingly useful for identifying and enumerating protists, and for studying their spatial and temporal distribution in nature. Knowledge of protistan species composition, abundance and variability in an environment can ultimately be used to relate community structure to various aspects of community function and biogeochemical activity.     

Identification of Heterotrophic Nanoflagellates by Restriction Fragment Length Polymorphism Analysis of Small Subunit Ribosomal DNA

Thirty clones derived from twenty isolates of heterotrophic nanoflagellates originating from a variety of marine and freshwater environments were examined by restriction fragment length polymorphism analysis of small subunit ribosomal RNA genes amplified by the polymerase chain reaction (riboprinting). The data were compared with light and electron microscopical identification of the isolates. On morphological criteria, sixteen of the thirty clones belonged to the genus Paraphysomonas De Saedeleer, seven to the genus Spumella Cienkowski, four to the genus Pteridomonas Penard and three to the genus Cafeteria Fenchel and Patterson. Among these taxa, eleven ribotypes were detected by analysis with the restriction enzymes Hinf I, Hae III, Sau3A I, and Msp I. Differentiation of nanoflagellate taxa by the riboprinting method supported taxonomic classification based on morphology at the generic and species level. The utility of the method for discriminating the 'naked' flagellates and for confirming the identity of polymorphic forms among species of Paraphysomonas is demonstrated.     

Die Kultur der Erdflechte Endocarpon pusillum im Labor

Zusammenfassung Kulturversuche mit der Erdflechte Endocarpon pusillum im Labor bestätigen die oft angezweifelten Beobachtungen und Ergebnisse von Stahl (1877). Eine reproduzierbare Kultur dieser Flechte im Labor von Spore zu Spore ist möglich. Bereits nach zwei Monaten läßt sich eine Differenzierung in Rinde, Algenschicht, Mark und Rhizinen erkennen; nach 5-6 Monaten sind reife, keimfähige Ascosporen vorhanden.Beobachtungen bei der Sporengewinnung zeigten, daß die Ascosporen bis zu einer Höhe von 5 cm ausgeschleudert werden. Der Sporenabschuß kann dabei mehrere Tage anhalten und zu dichten Sporenaussaaten führen.Die Thallusentwicklung (Keimung der Ascosporen, Umwandlung der Hymenialgonidien in Thallusgonidien, Bildung von Thallusinitialen, Prothalli und Primärthalli) und die Bildung reifer Perithecien mit keimfähigen Sporen in Laborkulturen wird beschrieben und abgebildet.Die Frage der speziellen Eignung schnellwüchsiger kleiner Erdflechten für die Laborkultur und das Problem der Flechtensynthese wird diskutiert.

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The culture of the lichen Endocarpon pusillum in the laboratory

Summary Culture experiments with the lichen Endocarpon pusillum corroborate the often doubted observations and results of Stahl (1877). It is possible to cultivate this lichen in the laboratory from spore to spore. After two months a differentiation into cortex, algae-layer, medulla and rhizines is developed and after 5–6 months the formation of mature and germinable ascospores is finished.Observations of the spore dispersal showed that the ascospores are ejected up to 5 cm. The spore dispersal can endure for some days and in this way produces a compact dissemination.The thallus development (germination of ascospores, transformation of hymenialalgae into thallus-algae, development of thallus initials, prothalli and primary thalli) and the formation of mature perithecia with germinable ascospores in laboratory cultures is described and illustrated.The opinion that such quickly growing and developing epigaeic lichens are especially suitable for laboratory cultures is emphasized and the problem of lichensynthesis is discussed in relation to the results of the experiments.

     

A comparison of balloon injury models of endovascular lesions in rat arteries

Background

Balloon injury (BI) of the rat carotid artery (CCA) is widely used to study intimal hyperplasia (IH) and decrease in lumen diameter (LD), but CCA's small diameter impedes the evaluation of endovascular therapies. Therefore, we validated BI in the aorta (AA) and iliac artery (CIA) to compare it with CCA.

Methods

Rats underwent BI or a sham procedure (control). Light microscopic evaluation was performed either directly or at 1, 2, 3, 4 and 16 weeks follow-up. The area of IH and the change in LD (LD at 16 weeks minus LD post BI) were compared.

Results

In the BI-groups the area of IH increased to 0.14 ± 0.08 mm² (CCA), 0.14 ± 0.03 mm² (CIA) and 0.12 ± 0.04 mm² (AA) at 16 weeks (NS). The LD decreased with 0.49 ± 0.07 mm (CCA), compared to 0.22 ± 0.07 mm (CIA) and 0.07 ± 0.10 mm (AA) at 16 weeks (p < 0.05). The constrictive vascular remodelling (CVR = wall circumference loss combined with a decrease in LD) was -0.17 ± 0.05 mm in CIA but absent in CCA and AA. No IH, no decrease in LD and no CVR was seen in the control groups.

Conclusions

BI resulted in: (1.) a decrease in LD in CCA due to IH, (2.) a decrease in LD in CIA due to IH and CVR, (3.) no change in LD in AA, (4.) Comparable IH development in all arteries, (5.) CCA has no vasa vasorum compared to CIA and AA, (6.) The CIA model combines good access for 2 F endovascular catheters with a decrease in LD due to IH and CVR after BI.     

Non-contiguous finished genome sequence of Staphylococcus capitis CR01 (pulsetype NRCS-A)

Staphylococcus capitis is a coagulase-negative staphylococcus (CoNS) commonly found in the human microflora. Recently, a clonal population of Staphylococcus capitis (denominated NRCS-A) was found to be a major cause of late-onset sepsis (LOS) in several neonatal intensive care units in France. Here, we report the complete genome sequence and annotation of the prototype Staphylococcus capitis NCRS-A strain CR01. The 2,504,472 bp long genome (1 chromosome and no plasmids) exhibits a G+C content of 32.81%, and contains 2,468 protein-coding and 59 tRNA genes and 4 rRNA genes.