A Stenotrophomonas maltophilia Multilocus Sequence Typing Scheme for Inferring Population Structure

Stenotrophomonas maltophilia is an opportunistic, highly resistant, and ubiquitous pathogen. Strains have been assigned to genogroups using amplified fragment length polymorphism. Hence, isolates of environmental and clinical origin predominate in different groups. A multilocus sequence typing (MLST) scheme was developed using a highly diverse selection of 70 strains of various ecological origins from seven countries on all continents including strains of the 10 previously defined genogroups. Sequence data were assigned to 54 sequence types (ST) based on seven loci. Indices of association for all isolates and clinical isolates of 2.498 and 2.562 indicated a significant linkage disequilibrium, as well as high congruence of tree topologies from different loci. Potential recombination events were detected in one-sixth of all ST. Calculation of the mean divergence between and within predicted clusters confirmed previously defined groups and revealed five additional groups. Consideration of the different ecological origins showed that 18 out of 31 respiratory tract isolates, including 12 out of 19 isolates from cystic fibrosis (CF) patients, belonged to genogroup 6. In contrast, 16 invasive strains isolated from blood cultures were distributed among nine different genogroups. Three genogroups contained isolates of strictly environmental origin that also featured high sequence distances to other genogroups, including the S. maltophilia type strain. On the basis of this MLST scheme, isolates can be assigned to the genogroups of this species in order to further scrutinize the population structure of this species and to unravel the uneven distribution of environmental and clinical isolates obtained from infected, colonized, or CF patients.Stenotrophomonas maltophilia is ubiquitous in nature. It has, for instance, been isolated from the rhizosphere of various plants and animals (14, 27, 37). Due to its tolerance against cadmium and its ability to degrade xenobiotic compounds, it has been proposed for remediation of contaminated ground (9, 39). Increasingly, it is being isolated from immunosuppressed individuals and intensive care and cystic fibrosis (CF) patients and has been shown to be resistant to many antimicrobial agents (16, 17, 69). However, the role of this opportunistic pathogen as an innocent bystander or causative agent often remains unclear (30), and little is known about its virulence factors (20, 33).Recently, novel Stenotrophomonas species were described: Stenotrophomonas nitritireducens sp. nov. (24), Stenotrophomonas acidaminiphila (3), Stenotrophomonas rhizophila (73), and Stenotrophomonas africana sp. nov. (21). However, the latter is a synonym of S. maltophilia (10).Using amplified fragment length polymorphism (AFLP) fingerprinting and DNA-DNA hybridizations, remarkable diversity has been shown to exist among S. maltophilia isolates recovered from both the environment and human clinical samples. This species can be subdivided into 10 AFLP genomic groups (35) that comprise to various extents both clinical and environmental isolates. Similarly, different genomic groups of the genus Stenotrophomonas can be distinguished using restriction fragment length polymorphisms (RFLP) in the gyrB gene (11). Surprisingly, 36 out of 40 isolates from CF patients are grouped in just two clusters. However, no such differences were seen in other investigations using pulsed-field gel electrophoresis (PFGE) after DraI digestion, molecular typing by BOX-PCR, or temperature-gradient gel electrophoresis of 16S rRNA PCR fragments (7). Later DNA sequence analyses of the 16S rRNA revealed three clusters, with grouping of the strains according to their sources of isolation and signature sequences in the region V1, which distinguishes clinical from environmental isolates (44).The objective of this study was to develop a multilocus sequence typing (MLST) scheme on the basis of a diverse strain collection comprising isolates from different ecological origins, continents, and DNA hybridization groups (35). We then employed this scheme to start initial analyses of the population structure of this species.(This study was conducted by S. Kaiser in partial fulfillment of the requirements for a diploma thesis in biology from the Faculty of Biology, University of Freiburg, Freiburg, Germany, 2007.)     

<Emphasis Type="Italic">Anchitherium aurelianense</Emphasis> (Equidae,Mammalia): a brachydont “dirty browser” in the community of herbivorous large mammals from Sandelzhausen (Miocene,Germany)

The Anchitheriinae are an extinct subfamily of Equidae that first appeared in North America near the base of the Miocene. Anchitheriinae are found in subtropical to warm temperate habitats and were long considered to be adapted to eating browse. In Europe the genus Anchitherium first occurred in the MN3 mammal zone and became extinct in MN9. The assemblage from Sandelzhausen (Early/Middle Miocene, boundary MN5) is one of the best dental samples known of Old World Anchitherium. The mesowear method was applied to reconstruct the dietary regime of A. aurelianense from Sandelzhausen. Hierarchical cluster statistics and principal component analysis was performed on mesowear variables. Thirty-five upper cheek teeth of A. aurelianense were analysed, and mesowear signatures compared with those of five ruminant species from Sandelzhausen. The extant analogue species indicate that A. aurelianense at Sandelzhausen had a diet similar in abrasiveness to that of extant mixed feeders and that the Sandelzhausen ruminants all occupied a browsing niche with little or no abrasives in their diet. Knowing now that brachydont anchitheres were able to cope with rather abrasive diets, it is assumed that hypsodont North American equids of the Cormohipparion clade, arriving in Europe successively with the extinction of anchitheres shortly after the “hipparion datum” in the MN9, introduced a new component of competition in their dietary niche. The extinction of the Anchitheriidae can now be better understood as response to this competition.      

Episodes in insect evolution

This article derives from a society-wide symposium organized by Timothy Bradley and Adriana Briscoe and presented at the 2009 annual meeting of the Society for Integrative and Comparative Biology in Boston, Massachusetts. David Grimaldi provided the opening presentation in which he outlined the major evolutionary events in the formation and subsequent diversification of the insect clade. This presentation was followed by speakers who detailed the evolutionary history of specific physiological and/or behavioral traits that have caused insects to be both ecologically successful and fascinating as subjects for biological study. These include a review of the evolutionary history of the insects, the origins of flight, osmoregulation, the evolution of tracheal systems, the evolution of color vision, circadian clocks, and the evolution of eusociality. These topics, as covered by the speakers, provide an overview of the pattern and timing of evolutionary diversification and specialization in the group of animals we know as insects.     

Antiretroviral therapy optimisation without genotype resistance testing: a perspective on treatment history based models

Background

Although genotypic resistance testing (GRT) is recommended to guide combination antiretroviral therapy (cART), funding and/or facilities to perform GRT may not be available in low to middle income countries. Since treatment history (TH) impacts response to subsequent therapy, we investigated a set of statistical learning models to optimise cART in the absence of GRT information.

Methods and Findings

The EuResist database was used to extract 8-week and 24-week treatment change episodes (TCE) with GRT and additional clinical, demographic and TH information. Random Forest (RF) classification was used to predict 8- and 24-week success, defined as undetectable HIV-1 RNA, comparing nested models including (i) GRT+TH and (ii) TH without GRT, using multiple cross-validation and area under the receiver operating characteristic curve (AUC). Virological success was achieved in 68.2% and 68.0% of TCE at 8- and 24-weeks (n = 2,831 and 2,579), respectively. RF (i) and (ii) showed comparable performances, with an average (st.dev.) AUC 0.77 (0.031) vs. 0.757 (0.035) at 8-weeks, 0.834 (0.027) vs. 0.821 (0.025) at 24-weeks. Sensitivity analyses, carried out on a data subset that included antiretroviral regimens commonly used in low to middle income countries, confirmed our findings. Training on subtype B and validation on non-B isolates resulted in a decline of performance for models (i) and (ii).

Conclusions

Treatment history-based RF prediction models are comparable to GRT-based for classification of virological outcome. These results may be relevant for therapy optimisation in areas where availability of GRT is limited. Further investigations are required in order to account for different demographics, subtypes and different therapy switching strategies.     

Disposable bioreactors: the current state-of-the-art and recommended applications in biotechnology

Disposable bioreactors have increasingly been incorporated into preclinical, clinical, and production-scale biotechnological facilities over the last few years. Driven by market needs, and, in particular, by the developers and manufacturers of drugs, vaccines, and further biologicals, there has been a trend toward the use of disposable seed bioreactors as well as production bioreactors. Numerous studies documenting their advantages in use have contributed to further new developments and have resulted in the availability of a multitude of disposable bioreactor types which differ in power input, design, instrumentation, and scale of the cultivation container. In this review, the term “disposable bioreactor” is defined, the benefits and constraints of disposable bioreactors are discussed, and critical phases and milestones in the development of disposable bioreactors are summarized. An overview of the disposable bioreactors that are currently commercially available is provided, and the domination of wave-mixed, orbitally shaken, and, in particular, stirred disposable bioreactors in animal cell-derived productions at cubic meter scale is reported. The growth of this type of reactor system is attributed to the recent availability of stirred disposable benchtop systems such as the Mobius CellReady 3 L Bioreactor. Analysis of the data from computational fluid dynamic simulation studies and first cultivation runs confirms that this novel bioreactor system is a viable alternative to traditional cell culture bioreactors at benchtop scale.     

Phospholipase A2 activity-dependent stimulation of Ca2+ entry by human parvovirus B19 capsid protein VP1

Recent reports demonstrated an association of human parvovirus B19 with inflammatory cardiomyopathy (iCMP), which is accompanied by endothelial dysfunction. As intracellular Ca(2+) activity is a key regulator of cell function and participates in mechanisms leading to endothelial dysfunction, the present experiments explored the effects of the B19 capsid proteins VP1 and VP2. A secreted phospholipase A2 (PLA2)-like activity has been located in the VP1 unique region of the B19 minor capsid protein. As PLA2 has recently been shown to activate the store-operated or capacitative Ca(2+) channel I(CRAC), we analyzed the impact of the viral PLA2 motif on Ca(2+) entry. We cloned the VP1 and VP2 genes isolated from a patient suffering from fatal B19 iCMP into eukaryotic expression vectors. We also generated a B19 replication-competent plasmid to demonstrate PLA2 activity under the control of the complete B19 genome. After the transfection of human endothelial cells (HMEC-1), cytosolic Ca(2+) activity was determined by utilizing Fura-2 fluorescence. VP1 and VP2 expression did not significantly modify basal cytosolic Ca(2+) activity or the decline of cytosolic Ca(2+) activity following the removal of extracellular Ca(2+). However, expression of VP1 and of the full-length B19 clone, but not of VP2, significantly accelerated the increase of cytosolic Ca(2+) activity following the readdition of extracellular Ca(2+) in the presence of thapsigargin, indicating an activation of I(CRAC.) The effect of VP1 was mimicked by the PLA2 product lysophosphatidylcholine and abolished by an inactivating mutation of the PLA2-encoding region of the VP1 gene. Our observations point to the activation of Ca(2+) entry by VP1 PLA2 activity, an effect likely participating in the pathophysiology of B19 infection.     

Co-management Policy Can Reduce Resilience in Traditionally Managed Marine Ecosystems

Best-practice environmental policy often suggests co-management of marine resources as a means of achieving sustainable development. Here we consider the impacts of superimposing co-management policy, in the form of territorial user rights for fishers over an existing traditional community-based natural-resource management system in Chile. We consider a broad definition of co-management that includes a spectrum of arrangements between governments and user groups described by different levels of devolution of power. We used participatory rural appraisal techniques and questionnaires to understand the mechanisms that underpin the traditional management system for the bull-kelp “cochayuyo” (Durvillaea antarctica). Traditional management was based on the allocation of informal access rights through a lottery system. This system was controlled by a complex web of traditional institutions that were shown to be successful in terms of equity and resilience. Using a similar approach, we analyzed the effects of superimposing a government-led co-management policy into this traditional system. Two major effects of the new policy were encountered. First, traditional institutions were weakened, which had negative effects on the levels of trust within the community and intensified conflict among users. Second, the management system’s adaptive capacity was reduced, thereby jeopardizing the ecosystem’s resilience. Our results suggest that the devolution of power to this kind of fisher community still has not reached the level required for fishers to legally address the local deficiencies of the Chilean co-management policy. Additionally, legal adjustments must be made to accommodate traditionally managed ecosystems that offer benefits comparable to those mandated under the formal policy. A fuller understanding of the interactions between co-management and traditional institutions can help us to identify ways to promote resilience and facilitate equal access by mitigating the potential negative effects of co-management policy and informing its future implementation.     

A novel esterase from Bacillus subtilis (RRL 1789): purification and characterization of the enzyme

An esterase (EC 3.1.1.1) produced by an isolated strain of Bacillus subtilis RRL 1789 exhibited moderate to high enantioselectivity in the kinetic resolution of several substrates like aryl carbinols, hydroxy esters, and halo esters. The enzyme named as B. subtilis esterase (BSE), was purified to >95% purity with a specific activity of 944 U/mg protein and 12% overall yield. The purified enzyme is approximately 52 kDa monomer, maximally activity at 37 degrees C and pH 8.0 and fairly stable up to 55 degrees C. The enzyme does not exhibit the phenomenon of interfacial activation with tributyrin and p-nitrophenyl butyrate beyond the saturation concentration. The enzyme showed preference for triacyglycerols and esters of p-nitrophenol with short chain fatty acid. Presence of Ca2+ ions increases the activity of enzyme by approximately 20% but its presence does not have any influence on the thermostability of the enzyme. The enzyme is not a metalloprotein and belongs to the family of serine proteases. The N-terminal amino acid sequence of BSE determined, as Met-Thr-Pro-Glu-Iso-Val-Thr-Thr-Glu-Tyr-Gly- revealed similarity with the N-terminal amino acid sequence of p-nitrobenzylesterase of B. subtilis.