Mannitol Does Not Enhance Tobramycin Killing of Pseudomonas aeruginosa in a Cystic Fibrosis Model System of Biofilm Formation

Cystic Fibrosis (CF) is a human genetic disease that results in the accumulation of thick, sticky mucus in the airways, which results in chronic, life-long bacterial biofilm infections that are difficult to clear with antibiotics. Pseudomonas aeruginosa lung infection is correlated with worsening lung disease and P. aeruginosa transitions to an antibiotic tolerant state during chronic infections. Tobramycin is an aminoglycoside currently used to combat lung infections in individuals with CF. While tobramycin is effective at eradicating P. aeruginosa in the airways of young patients, it is unable to completely clear the chronic P. aeruginosa infections in older patients. A recent report showed that co-addition of tobramycin and mannitol enhanced killing of P. aeruginosa grown in vitro as a biofilm on an abiotic surface. Here we employed a model system of bacterial biofilms formed on the surface of CF-derived airway cells to determine if mannitol would enhance the antibacterial activity of tobramycin against P. aeruginosa grown on a more clinically relevant surface. Using this model system, which allows the growth of robust biofilms with high-level antibiotic tolerance analogous to in vivo biofilms, we were unable to find evidence for enhanced antibacterial activity of tobramycin with the addition of mannitol, supporting the observation that this type of co-treatment failed to reduce the P. aeruginosa bacterial load in a clinical setting.     

Homogentisic acid induces morphological and mechanical aberration of ochronotic cartilage in alkaptonuria

Alkaptonuria (AKU) is a disease caused by a deficient homogentisate 1,2-dioxygenase activity leading to systemic accumulation of homogentisic acid (HGA), that forms a melanin-like polymer that progressively deposits onto connective tissues causing a pigmentation called “ochronosis” and tissue degeneration. The effects of AKU and ochronotic pigment on the biomechanical properties of articular cartilage need further investigation. To this aim, AKU cartilage was studied using thermal (thermogravimetry and differential scanning calorimetry) and rheological analysis. We found that AKU cartilage had a doubled mesopore radius compared to healthy cartilage. Since the mesoporous structure is the main responsible for maintaining a correct hydrostatic pressure and tissue homoeostasis, drastic changes of thermal and rheological parameters were found in AKU. In particular, AKU tissue lost its capability to enhance chondrocytes metabolism (decreased heat capacity) and hence the production of proteoglycans. A drastic increase in stiffness and decrease in dissipative and lubricant role ensued in AKU cartilage. Multiphoton and scanning electron microscopies revealed destruction of cell–matrix microstructure and disruption of the superficial layer. Such observations on AKU specimens were confirmed in HGA-treated healthy cartilage, indicating that HGA is the toxic responsible of morphological and mechanical alterations of cartilage in AKU.     

A complex species complex: The controversial role of ecology and biogeography in the evolutionary history of Syllis gracilis Grube, 1840 (Annelida,Syllidae)

The cryptic diversity in the polychaete Syllis gracilis Grube, 1840, in the Mediterranean Sea was examined with an integrative morpho-molecular approach. Individuals of S. gracilis were collected at eleven Mediterranean localities to provide an insight into the role of brackish environments in inducing cryptic speciation. The examination of morphological features combined with a molecular genetic analysis based on a partial sequence of the 16S rRNA gene highlighted discrepancies between morphological and molecular diversity. Morphological data allowed to identify a morphotype with short appendages occurring in coralline algae communities and another one with long appendages observed in brackish-water environments and Sabellaria reefs. Multivariate analyses showed that sampling localities were the greatest source of morphological divergence, suggesting that phenotypic plasticity may play a role in local adaptations of S. gracilis populations. Molecular data showed the occurrence of four divergent lineages not corresponding to morphological clusters. Different species delimitation tests gave conflicting results, retrieving, however, at least four separated entities. Some lineages occurred in sympatry and were equally distributed in marine and brackish-water environments, excluding a biogeographic or ecological explanation of the observed pattern and suggesting instead ancient separation between lineages and secondary contact. The co-occurrence of different lineages hindered the identification of the lineage corresponding to S. gracilis sensu stricto. The discrepancy between morphological and molecular diversity suggests that different environmental and biogeographic features may interact in a complex and unpredictable way in shaping diversity patterns. An integrative approach is needed to provide a satisfactory insight on evolutionary processes in marine invertebrates.     

Electrophoretic analysis of endonuclease-generated fragments of k-DNA, of esterase isoenzymes, and of surface proteins as aids for species identification of insect trypanosomatids

In order to verify the applicability of biochemical methods for species identification of Trypanosomatidae, 13 species of monoxenic trypanosomatids plus the heteroxenous Trypanosoma cruzi were comparatively analyzed by three different biochemical methods. Insect trypanosomatids examined were: Crithidia acanthocephali, C. fasciculata (three varieties), C. luciliae luciliae, C. luciliae thermophila, C. deanei, C. oncopelti, Herpetomonas muscarum muscarum, H. megaseliae, H. samuelpessoai, H. mariadeanei, Leptomonas seymouri, L. collosoma, L. samueli, and Blastocrithidia culicis. Also included in the survey were aposymbiotic strains of C. deanei and C. oncopelti. Methods used were: electrophoretic profiling of endonuclease-generated fragments of k-DNA, esterase isoenzymes profiling, and polyacrylamide-gel electrophoresis (SDS-PAGE) of radioiodinated cell surface proteins. Interspecific but not intraspecific differences were detected by all three methods among the 13 monoxenic species examined. Thus, it is concluded that these methods can be successfully used, in addition to classical criteria, for species identification of insect trypanosomatids.     

GarA is an essential regulator of metabolism in Mycobacterium tuberculosis

Alpha‐ketoglutarate is a key metabolic intermediate at the crossroads of carbon and nitrogen metabolism, whose fate is tightly regulated. In mycobacteria the protein GarA regulates the tricarboxylic acid cycle and glutamate synthesis by direct binding and regulation of three enzymes that use α‐ketoglutarate. GarA, in turn, is thought to be regulated via phosphorylation by protein kinase G and other kinases. We have investigated the requirement for GarA for metabolic regulation during growth in vitro and in macrophages. GarA was found to be essential to Mycobacterium tuberculosis, but dispensable in non‐pathogenic Mycobacterium smegmatis. Disruption of garA caused a distinctive, nutrient‐dependent phenotype, fitting with its proposed role in regulating glutamate metabolism. The data underline the importance of the TCA cycle and the balance with glutamate synthesis in M. tuberculosis and reveal vulnerability to disruption of these pathways.     

Selective enzymatic hydrolysis of aromatic diesters using esterase from Brevibacterium imperiale B222

Summary A practical procedure has been developed for the chemoselective microbial hydrolysis of aromatic dicarboxylic esters to give the corresponding monoesters, using cellular lysate and whole cell of Brevibacterium imperiale B222. The produced monoesters can be transformed into hydroxyacids, useful intermediates in the synthesis of polyesters.