Phenotypic and Genotypic Characterization of Daptomycin-Resistant Methicillin-Resistant Staphylococcus aureus Strains: Relative Roles of mprF and dlt Operons

Development of in vivo daptomycin resistance (DAP-R) among Staphylococcus aureus clinical isolates, in association with clinical treatment failures, has become a major therapeutic problem. This issue is especially relevant to methicillin-resistant S. aureus (MRSA) strains in the context of invasive endovascular infections. In the current study, we used three well-characterized and clinically-derived DAP-susceptible (DAP-S) vs. resistant (DAP-R) MRSA strain-pairs to elucidate potential genotypic mechanisms of the DAP-R phenotype. In comparison to the DAP-S parental strains, DAP-R isolates demonstrated (i) altered expression of two key determinants of net positive surface charge, either during exponential or stationary growth phases (i.e., dysregulation of dltA and mprF), (ii) a significant increase in the D-alanylated wall teichoic acid (WTA) content in DAP-R strains, reflecting DltA gain-in-function; (iii) heightened elaboration of lysinylated-phosphatidylglyderol (L-PG) in DAP-R strains, reflecting MprF gain-in-function; (iv) increased cell membrane (CM) fluidity, and (v) significantly reduced susceptibility to prototypic cationic host defense peptides of platelet and leukocyte origins. In the tested DAP-R strains, genes conferring positive surface charge were dysregulated, and their functionality altered. However, there were no correlations between relative surface positive charge or cell wall thickness and the observed DAP-R phenotype. Thus, charge repulsion mechanisms via altered surface charge may not be sufficient to explain the DAP-R outcome. Instead, changes in the compositional or biophysical order of the DAP CM target of such DAP-R strains (i.e., increased fluidity) may be essential to this phenotype. Taken together, DAP-R in S. aureus appears to involve multi-factorial and strain-specific adaptive mechanisms.     

A study on the interactions of surfactin with phospholipid vesicles.

Surfactin, an acidic lipopeptide produced by various strains of Bacillus subtilis, behaves as a very powerful biosurfactant and posses several other interesting biological activities. By means of differential scanning calorimetry and X-ray diffraction the effect of surfactin on the phase transition properties of bilayers composed of different phospholipids, including lipids forming hexagonal-HII phases, has been studied. The interactions of surfactin with phosphatidylcholine and phosphatidylglycerol seem to be optimal in the case of myristoyl acyl chains, which have a similar length to the surfactin hydrocarbon tail. Data are shown that support formation of complexes of surfactin with phospholipids. The ionized form of surfactin seems to be more deeply inserted into negatively charged bilayers when Ca2+ is present, also supporting the formation of surfactin-Ca2+ complexes. In mixtures with dielaidoylphosphatidylethanolamine, a hexagonal-HII phase forming lipid, surfactin displays a bilayer stabilizing effect. Our results are compatible with the marked amphiphilic nature of surfactin and may contribute to explain some of its interesting biological actions; for instance the formation of ion-conducting pores in membranes.     

Risk factors associated with negative in-vivo diagnostic results in bovine tuberculosis-infected cattle in Spain

Background

Despite great effort and investment incurred over decades to control bovine tuberculosis (bTB), it is still one of the most important zoonotic diseases in many areas of the world. Test-and-slaughter strategies, the basis of most bTB eradication programs carried out worldwide, have demonstrated its usefulness in the control of the disease. However, in certain countries, eradication has not been achieved due in part to limitations of currently available diagnostic tests. In this study, results of in-vivo and post-mortem diagnostic tests performed on 3,614 animals from 152 bTB-infected cattle herds (beef, dairy, and bullfighting) detected in 2007–2010 in the region of Castilla y León, Spain, were analyzed to identify factors associated with positive bacteriological results in cattle that were non-reactors to the single intradermal tuberculin test, to the interferon-gamma (IFN-γ) assay, or to both tests applied in parallel (Test negative/Culture + animals, T-/C+). The association of individual factors (age, productive type, and number of herd-tests performed since the disclosure of the outbreak) with the bacteriology outcome (positive/negative) was analyzed using a mixed multivariate logistic regression model.

Results

The proportion of non-reactors with a positive post-mortem result ranged from 24.3% in the case of the SIT test to 12.9% (IFN-γ with 0.05 threshold) and 11.9% (95% CI 9.9-11.4%) using both tests in parallel. Older (>4.5 years) and bullfighting cattle were associated with increased odds of confirmed bTB infection by bacteriology, whereas dairy cattle showed a significantly lower risk. Ancillary use of IFN-γ assay reduced the proportion of T-/C + animals in high risk groups.

Conclusions

These results demonstrate the likelihood of positive bacteriological results in non-reactor cattle is influenced by individual epidemiological factors of tested animals. Increased surveillance on non-reactors with an increased probability of being false negative could be helpful to avoid bTB persistence, particularly in chronically infected herds. These findings may aid in the development of effective strategies for eradication of bTB in Spain.     

Phylogenetic relationships of three porcine mycoplasmas, Mycoplasma hyopneumoniae, Mycoplasma flocculare, and Mycoplasma hyorhinis, and complete 16S rRNA sequence of M. flocculare.

The nucleotide sequence of the 16S rRNA gene of Mycoplasma flocculare was determined and was compared with the sequence of a related porcine mycoplasma, Mycoplasma hyopneumoniae. While the overall level of DNA-DNA homology was approximately 11%, sequence alignment of the two 16S rRNA genes yielded a homology value of more than 95%, emphasizing the highly conserved nature of the 16S rRNA gene. Multiple sequence alignments with other mollicutes indicated that M. flocculare, M. hyopneumoniae, and Mycoplasma hyorhinis form a subcluster within the fermentans phylogroup, and this subcluster is distinct from the Mycoplasma pneumoniae phylogroup. Thus, the three mycoplasmas isolated from porcine respiratory systems exhibit phylogenetic similarities.     

Aminopeptidase N (CD13): Expression,Prognostic Impact,and Use as Therapeutic Target for Tissue Factor Induced Tumor Vascular Infarction in Soft Tissue Sarcoma

Aminopeptidase N (CD13) is expressed on tumor vasculature and tumor cells. It represents a candidate for targeted therapy, e.g., by truncated tissue factor (tTF)-NGR, binding to CD13, and causing tumor vascular thrombosis. We analyzed CD13 expression by immunohistochemistry in 97 patients with STS who were treated by wide resection and uniform chemo-radio-chemotherapy. Using a semiquantitative score with four intensity levels, CD13 was expressed by tumor vasculature, or tumor cells, or both (composite value, intensity scores 1-3) in 93.9% of the STS. In 49.5% tumor cells, in 48.5% vascular/perivascular cells, and in 58.8%, composite value showed strong intensity score 3 staining. Leiomyosarcoma and synovial sarcoma showed low expression; fibrosarcoma and undifferentiated pleomorphic sarcoma showed high expression. We found a significant prognostic impact of CD13, as high expression in tumor cells or vascular/perivascular cells correlated with better relapse-free survival and overall survival. CD13 retained prognostic significance in multivariable analyses. Systemic tTF-NGR resulted in significant growth reduction of CD13-positive human HT1080 sarcoma cell line xenografts. Our results recommend further investigation of tTF-NGR in STS patients. CD13 might be a suitable predictive biomarker for patient selection.     

Phenomics allows identification of genomic regions affecting maize stomatal conductance with conditional effects of water deficit and evaporative demand

Stomatal conductance is central for the trades‐off between hydraulics and photosynthesis. We aimed at deciphering its genetic control and that of its responses to evaporative demand and water deficit, a nearly impossible task with gas exchanges measurements. Whole‐plant stomatal conductance was estimated via inversion of the Penman–Monteith equation from data of transpiration and plant architecture collected in a phenotyping platform. We have analysed jointly 4 experiments with contrasting environmental conditions imposed to a panel of 254 maize hybrids. Estimated whole‐plant stomatal conductance closely correlated with gas‐exchange measurements and biomass accumulation rate. Sixteen robust quantitative trait loci (QTLs) were identified by genome wide association studies and co‐located with QTLs of transpiration and biomass. Light, vapour pressure deficit, or soil water potential largely accounted for the differences in allelic effects between experiments, thereby providing strong hypotheses for mechanisms of stomatal control and a way to select relevant candidate genes among the 1–19 genes harboured by QTLs. The combination of allelic effects, as affected by environmental conditions, accounted for the variability of stomatal conductance across a range of hybrids and environmental conditions. This approach may therefore contribute to genetic analysis and prediction of stomatal control in diverse environments.     

Carbon isotope discrimination in Quercus ilex resprouts after fire and tree-fell

Ecophysiological differences related to photosynthesis were compared in holm oak Quercus ilex leaves from undisturbed holm-oak vegetation, resprouts after fire and resprouts after tree-fell. No significant differences in any parameter measured were observed between the two kinds of resprout throughout the first growing season following disturbance. Resprouting leaves showed lower carbon isotope discrimination () and intercellular CO₂ concentration (p _i), and higher photosynthesis, leaf conductance and transpiration rates than leaves from undisturbed stands. Nitrogen, soluble protein content and ribulose bisphosphate carboxylase (RuBPCase) activity were 88%, 96% and 45% higher respectively, in both kinds of resprout. The results indicate that photosynthetic capacity, rather than stomatal conductance, is the limiting factor in photosynthesis in resprouts, Chlorophyll content and chlorophyll a/b ratio did not differ between resprouts and undisturbed leaves, indicating that the observed differences were not a result of differences in light environment during leaf development. Leaf mass per area (LMA), was 80% higher in the resprouts, and was negatively related (r=–0.86) to and positively related (r=0.87) to N content. Enhanced carbon assimilation after disturbances resulted in higher water use efficiency, as indicated by lower values in the resprouts. We conclude that the cause of defoliation was not relevant in the physiology of the resprouts, suggesting the importance of underground organs.     

Quantitation of peptide hormone in single cultured secretory neurons of the crab,Cardisoma carnifex

The content of crustacean hyperglycemic hormone (CHH) in single cultured neurons of the crab Cardisoma carnifex was determined by a sensitive enzyme-linked immunosorbent assay (ELISA), using purified CHH (1–50 pg) of the crab Carcinus maenas as standard. The somata were dissociated from the group of 150 peptidergic neurons that form the X-organ—sinus gland neuroendocrine system. As previously reported, the neurons show immediate regenerative outgrowth in defined culture conditions, and develop, generally, into one of two morphological types: cells that produce broad, lamelliform growth cones (veils), and others that are characterized by branching of neurites. In this study, all but one of 64 veiling cells taken after various times in culture up to 12 days contained CHH. They could be readily categorized as having high (>33 pg; mean 86±5, S.E., n=47) or low (33pg; mean 22±2.5; n=17) Carcinus CHH equivalents. Thus, CHH is associated with neurons showing veiling outgrowth, but veiling neurons with low CHH form a distinct, but not morphologically distinguishable group. They may contain an isoform of CHH with limited cross-reactivity. In 24 branching neurons assayed, Carcinus CHH equivalents averaged 7.2±2 pg. This figure includes 14 neurons in which CHH was undetectable, and one that had 40 pg of Carcinus CHH equivalents. There was no significant change of the hormone content in cells of either type during 6 days of culturing.