Identifying More Epidemic Clones during a Hospital Outbreak of Multidrug-Resistant Acinetobacter baumannii

Infections caused by multidrug-resistant bacteria are a major concern in hospitals. Current infection-control practices legitimately focus on hygiene and appropriate use of antibiotics. However, little is known about the intrinsic abilities of some bacterial strains to cause outbreaks. They can be measured at a population level by the pathogen’s transmission rate, i.e. the rate at which the pathogen is transmitted from colonized hosts to susceptible hosts, or its reproduction number, counting the number of secondary cases per infected/colonized host. We collected data covering a 20-month surveillance period for carriage of multidrug-resistant Acinetobacter baumannii (MDRAB) in a surgery ward. All isolates were subjected to molecular fingerprinting, and a cluster analysis of profiles was performed to identify clonal groups. We then applied stochastic transmission models to infer transmission rates of MDRAB and each MDRAB clone. Molecular fingerprinting indicated that 3 clonal complexes spread in the ward. A first model, not accounting for different clones, quantified the level of in-ward cross-transmission, with an estimated transmission rate of 0.03/day (95% credible interval [0.012–0.049]) and a single-admission reproduction number of 0.61 [0.30–1.02]. The second model, accounting for different clones, suggested an enhanced transmissibility of clone 3 (transmission rate 0.047/day [0.018–0.091], with a single-admission reproduction number of 0.81 [0.30–1.56]). Clones 1 and 2 had comparable transmission rates (respectively, 0.016 [0.001–0.045], 0.014 [0.001–0.045]). The method used is broadly applicable to other nosocomial pathogens, as long as surveillance data and genotyping information are available. Building on these results, more epidemic clones could be identified, and could lead to follow-up studies dissecting the functional basis for variation in transmissibility of MDRAB lineages.     

Chitosomes lack concanavalin-A-binding sites

Whether intact or dissociated with digitonin, chitosomes isolated from the fungusMucor rouxii lack the ability to bind concanavalin A. The absence of external or internal concanavalin A-binding sites distinguishes the chitosome membrane no only from plasma membrane but also from membranes of other organelles (endoplasmic reticulum, mitochondrion, vacuole). This differential binding ability was used to partially separate chitosomal chitin synthetase from major membranes in a crude cell-free extract ofM. rouxii.     

Characterization of asymmetric fluorogenic phosphonates as probes for developing organophosphorus hydrolases with broader stereoselectivity

Organophosphorus hydrolases (OPH) such as mammalian plama paraoxonase (PON1) detoxify asymmetric toxic organophosphorus (OP) nerve agents by preferentially hydrolyzing the less toxic P(+) optical isomer. In order to develop new OPHs with broader stereoselectivity we have prepared a series of asymmetric fluorogenic organophosphonates (Flu-OPs). Such Flu-OPs may serve as molecular probes for screening large libraries of OP hydrolases during directed evolution. Flu-OPs were prepared as methylphosphonates (MPs) diesters containing either ethyl (E), isopropyl (I), cyclohexyl (C) or pinacolyl (P) groups that are structural congeners of the nerve agents VX, sarin, cyclosarin and soman, respectively. The second ester bond was formed with fluorescent moieties that are either 3-cyano-4-methyl-7-hydroxy coumarin (MeCyC) or 1,3-dichloro-7-hydroxy 9,9-dimethyl-9H-acridin-2-one (DDAO). To further characterize the Flu-OPs as surrogates of their respective nerve agents, we have studied the reactivation of Flu-OP-inhibited AChE using 2-PAM and toxogonin (TOX). AChE was 90–95% inhibited by all Flu-OPs (0.36–0.9 (M) and then was reactivated by either 2-PAM or TOX. TOX caused a more rapid reactivation than 2-PAM with the following rank order; EMP > IMP > CMP. TOX was also shown to be a better reactivator than 2-PAM for AChE inhibited by the nerve agents VX and cyclosarin. PMP-AChE could not be reactivated by either TOX or 2-PAM, similarly to aging of PMP-AChE formed by inhibition with soman.Racemic CMP-MeCyC was used for screening two new PON1 variants from a neutral library of PON1. These multiple mutation variants include replacement of active site amino acid residues. Neither mutation in these new variants appeared in PON1 variants previously discovered by directed evolution using symmetric Flu-OP. Detoxification rate of cylcosarin by these new PON1 variants was rather slow indicating the need to further screen PON1 clones using optically active Flu-OPs. Therefore, we have separated enzymatically the P(−) enantiomer of CMP-MeCyC and determined its 98% purity using chiral HPLC.     

Effects of temperature on the respiration rates and the kinetics of citrate synthase in two species of Idotea (Isopoda, Crustacea)

The two species of isopods, Idotea baltica (Pallas) and Idotea emarginata (Fabricius), co-occur frequently near Helgoland, North Sea, occupying different ecological niches. Respiration rates and kinetic properties of citrate synthase (CS) were compared in these species in order to identify possible mechanisms of temperature adaptation. Specimens were acclimated to 5 and 15 degrees C prior to further investigations. Respiration rates were measured under normoxic conditions at 5, 10 and 15 degrees C. CS was partly purified chromatographically and influences of temperature, pH, substrate saturation and ATP-concentration on enzyme activity were examined. In both species, rising temperatures led to linearly increasing oxygen consumption, with estimated Q10 values between 3.2 and 4.2. Only I. baltica showed an effect of short term acclimation: warm adapted animals had always higher respiration rates than cold adapted ones. In I. emarginata, the acclimation temperature had no effect on oxygen consumption. Furthermore, its CS slightly indicates higher affinity to oxaloacetic acid when specimens were adapted to 15 degrees C compared to those maintained at 5 degrees C. Any effect of the experimental temperature on CS in I. baltica was negligible. The results are discussed in view of the different habitats occupied by the species compared.     

Social Environment and Feeding State Influence Movement Decisions in a Web-building Spider

It is well recognized that feeding rate has a major influence on the amount of movement between microhabitats for many animals. However, the role of other extrinsic and intrinsic factors, and particularly how these factors may interact, is not well understood. This three-part study examines the movement decisions of a web-building spider, Latrodectus hesperus , by assessing microhabitat tenacity in established spiders and by testing how the presence of conspecific neighbours and the combined influence of individual feeding state (determined by prior feeding experience) and neighbour presence influence microhabitat residence time in unestablished spiders. The results show that naturally established spiders did not leave their microhabitats readily, emphasizing the importance of choosing a profitable location. Unestablished spiders stayed longer in microhabitats occupied by conspecifics than in unoccupied ones, and there was practically no cannibalism even though neighbours shared webs. Furthermore, feeding state and neighbour presence showed an interactive effect on microhabitat residence time. When spiders were housed alone, microhabitat residence time increased with feeding state. However, in the presence of conspecifics, spiders had a low propensity to move, regardless of feeding state. Together, these results demonstrate the combined importance of grouping dynamics and feeding state in shaping movement decisions.     

High rate of genetic recombination in murine leukemia virus: implications for influencing proviral ploidy

A significant difference in the recombination rates between human immunodeficiency virus type 1 (HIV-1) and the gammaretroviruses was previously reported, with the former being 10 to 100 times more recombinogenic. It is possible that preferential copackaging of homodimers in the case of gammaretroviruses, like murine leukemia virus (MLV), led to the underestimation of their rates of recombination. To reexamine the recombination rates for MLV, experiments were performed to control for nonrandom copackaging of viral RNA, and it was found that MLV and HIV-1 exhibit similar crossover rates. The implications for control of proviral ploidy and preferential recombination during minus-strand DNA synthesis are discussed.     

Deconvoluting post-transplant immunity: cell subset-specific mapping reveals pathways for activation and expansion of memory T, monocytes and B cells

A major challenge for the field of transplantation is the lack of understanding of genomic and molecular drivers of early post-transplant immunity. The early immune response creates a complex milieu that determines the course of ensuing immune events and the ultimate outcome of the transplant. The objective of the current study was to mechanistically deconvolute the early immune response by purifying and profiling the constituent cell subsets of the peripheral blood. We employed genome-wide profiling of whole blood and purified CD4, CD8, B cells and monocytes in tandem with high-throughput laser-scanning cytometry in 10 kidney transplants sampled serially pre-transplant, 1, 2, 4, 8 and 12 weeks. Cytometry confirmed early cell subset depletion by antibody induction and immunosuppression. Multiple markers revealed the activation and proliferative expansion of CD45RO(+)CD62L(-) effector memory CD4/CD8 T cells as well as progressive activation of monocytes and B cells. Next, we mechanistically deconvoluted early post-transplant immunity by serial monitoring of whole blood using DNA microarrays. Parallel analysis of cell subset-specific gene expression revealed a unique spectrum of time-dependent changes and functional pathways. Gene expression profiling results were validated with 157 different probesets matching all 65 antigens detected by cytometry. Thus, serial blood cell monitoring reflects the profound changes in blood cell composition and immune activation early post-transplant. Each cell subset reveals distinct pathways and functional programs. These changes illuminate a complex, early phase of immunity and inflammation that includes activation and proliferative expansion of the memory effector and regulatory cells that may determine the phenotype and outcome of the kidney transplant.     

Small mammals in high altitude phytophysiognomies in southeastern Brazil: are heterogeneous habitats more diverse?

The Chapada das Perdizes is considered a region of high biodiversity, but there are few studies that describe the small mammals in the region, mainly in higher portions of the area. The primary objective of this study was to estimate the richness and abundance of terrestrial small mammals within three different vegetation types in the high altitude region of southern Minas Gerais and to evaluate the relationship between the community composition of terrestrial small mammals and the heterogeneity of phytophysiognomies. Pitfall and cage trap grids were arranged in natural field, semi-deciduous and cloud forests. A total of 26 species was recorded. The compositions of the three phytophysiognomies were significantly different. The expansive study area is located in a region of tension between the Cerrado and Atlantic Forests and should be preserved due to high diversity and ecological importance.     

In vivo bioimaging as a novel strategy to detect doxorubicin-induced damage to gonadal blood vessels

Introduction

Chemotherapy may induce deleterious effects in normal tissues, leading to organ damage. Direct vascular injury is the least characterized side effect. Our aim was to establish a real-time, in vivo molecular imaging platform for evaluating the potential vascular toxicity of doxorubicin in mice.

Methods

Mice gonads served as reference organs. Mouse ovarian or testicular blood volume and femoral arterial blood flow were measured in real-time during and after doxorubicin (8 mg/kg intravenously) or paclitaxel (1.2 mg/kg) administration. Ovarian blood volume was imaged by ultrasound biomicroscopy (Vevo2100) with microbubbles as a contrast agent whereas testicular blood volume and blood flow as well as femoral arterial blood flow was imaged by pulse wave Doppler ultrasound. Visualization of ovarian and femoral microvasculature was obtained by fluorescence optical imaging system, equipped with a confocal fiber microscope (Cell-viZio).

Results

Using microbubbles as a contrast agent revealed a 33% (P<0.01) decrease in ovarian blood volume already 3 minutes after doxorubicin injection. Doppler ultrasound depicted the same phenomenon in testicular blood volume and blood flow. The femoral arterial blood flow was impaired in the same fashion. Cell-viZio imaging depicted a pattern of vessels'' injury at around the same time after doxorubicin injection: the wall of the blood vessels became irregular and the fluorescence signal displayed in the small vessels was gradually diminished. Paclitaxel had no vascular effect.

Conclusion

We have established a platform of innovative high-resolution molecular imaging, suitable for in vivo imaging of vessels'' characteristics, arterial blood flow and organs blood volume that enable prolonged real-time detection of chemotherapy-induced effects in the same individuals. The acute reduction in gonadal and femoral blood flow and the impairment of the blood vessels wall may represent an acute universal doxorubicin-related vascular toxicity, an initial event in organ injury.