Reduction of the nosocomial meticillin-resistant Staphylococcus aureus incidence density by a region-wide search and follow-strategy in forty German hospitals of the EUREGIO, 2009 to 2011.

Meticillin-resistant Staphylococcus aureus (MRSA) disseminates between hospitals serving one patient catchment area. Successful prevention and control requires concerted efforts and regional surveillance. Forty hospitals located in the German EUREGIO have established a network for combating MRSA. In 2007 they agreed upon a synchronised strategy for screening of risk patients and a standard for transmissionbased precautions (search and follow). The same year, the hospitals started synchronised MRSA prevention and annually reporting MRSA-data to the public health authorities. The median rate of screening cultures per 100 patients admitted increased from 4.38 in 2007 to 34.4 in 2011 (p<0.0001). Between 2007 and 2011, the overall incidence density of MRSA (0.87 MRSA cases/1,000 patient days vs 1.54; p<0.0001) increased significantly. In contrast, both the incidence density of nosocomial MRSA cases (0.13 nosocomial MRSA cases/1,000 patient days in 2009 vs 0.08 in 2011; p=0.0084) and the MRSA-days-associated nosocomial MRSA rate (5.51 nosocomial MRSA cases/1,000 MRSA days in 2009 vs 3.80 in 2011; p=0.0437) decreased significantly after the second year of the project. We documented adherence to the regional screening strategy resulting in improved detection of MRSA carriers at admission. Subsequently, after two years the nosocomial MRSA-incidence density was reduced. Regional surveillance data, annually provided as benchmarking to the regional hospitals and public health authorities, indicated successful prevention.

Genomic and functional analysis of Vibrio phage SIO-2 reveals novel insights into ecology and evolution of marine siphoviruses.

We report on a genomic and functional analysis of a novel marine siphovirus, the Vibrio phage SIO-2. This phage is lytic for related Vibrio species of great ecological interest including the broadly antagonistic bacterium Vibrio sp. SWAT3 as well as notable members of the Harveyi clade (V.harveyi ATTC BAA-1116 and V.campbellii ATCC 25920). Vibrio phage SIO-2 has a circularly permuted genome of 80598 bp, which displays unusual features. This genome is larger than that of most known siphoviruses and only 38 of the 116 predicted proteins had homologues in databases. Another divergence is manifest by the origin of core genes, most of which share robust similarities with unrelated viruses and bacteria spanning a wide range of phyla. These core genes are arranged in the same order as in most bacteriophages but they are unusually interspaced at two places with insertions of DNA comprising a high density of uncharacterized genes. The acquisition of these DNA inserts is associated with morphological variation of SIO-2 capsid, which assembles as a large (80 nm) shell with a novel T=12 symmetry. These atypical structural features confer on SIO-2 a remarkable stability to a variety of physical, chemical and environmental factors. Given this high level of functional and genomic novelty, SIO-2 emerges as a model of considerable interest in ecological and evolutionary studies.

Legionella pneumophila pneumonia in a pregnant woman treated with anti-TNF-α antibodies for Crohn's disease: a case report.

Anti-TNF-α antibodies are widely used. The indications for their usage are still increasing. With their emerging use, their infectious complications are seen more often. We describe the first case of a pneumonia with Legionella pneumophila in a pregnant women with Crohn's disease, during treatment with anti-TNF-α antibodies. She was treated with erythromycin and made a full recovery.

Jumbo bacteriophages.

There is currently a handful of genome sequences available for tailed bacteriophages with genomes of more than 200 kbp of DNA, designated here as giant or jumbo phages. The majority of the proteins predicted from the genome sequences of these phages have no matches in the current sequence databases, and the genomes themselves are diverse enough to preclude the sorts of detailed comparative analysis that has benefited study of the smaller phages, for which hundreds of genome sequences are available. However, it is informative to extrapolate the better known genome organizations and mechanisms of evolution seen in the smaller phages to the jumbo phages. In this way, we see that the jumbo phages encode the same functions as the smaller phages, supplemented with large numbers of mostly small genes of mostly undiscovered functions. A case can be made that the jumbo phages evolved from smaller tailed phages, possibly in a process mediated by the constraints imposed on genome size by capsid size.

Expanded nonhuman primate tregs exhibit a unique gene expression signature and potently downregulate alloimmune responses.

We have established two complementary strategies for purifying naturally occurring regulatory T cells (Tregs) from rhesus macaques in quantities that would be sufficient for use as an in vivo cellular therapeutic. The first strategy identified Tregs based on their being CD4+/CD25(bright). The second incorporated CD127, and purified Tregs based on their expression of CD4 and CD25 and their low expression of CD127. Using these purification strategies, we were able to purify as many as 1x10(6) Tregs from 120 cc of peripheral blood. Cultures of these cells with anti-CD3, anti-CD28 and IL-2 over 21 days yielded as much as a 450-fold expansion, ultimately producing as many as 4.7x10(8) Tregs. Expanded Treg cultures potently inhibited alloimmune proliferation as measured by a carboxyfluorescein succinimidyl ester- mixed lymphocyte reaction (CFSE-MLR) assay even at a 1:100 ratio with responder T cells. Furthermore, both responder-specific and third-party Tregs downregulated alloproliferation similarly. Both freshly isolated and cultured Tregs had gene expression signatures distinguishable from concurrently isolated bulk CD4+ T-cell populations, as measured by singleplex reverse transcriptase-polymerase chain reaction (RT-PCR) and gene array. Moreover, an overlapping yet distinct gene expression signature seen in freshly isolated compared to expanded Tregs identifies a subset of Treg genes likely to be functionally significant.

The role of atypical respiratory pathogens in exacerbations of chronic obstructive pulmonary disease.

The aetiology of acute exacerbations of chronic obstructive pulmonary disease (COPD) is heterogeneous and still under discussion. Serological studies have suggested that Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila may play a role in acute exacerbations of COPD. The presence of these atypical pathogens in sputum samples was investigated in patients with stable COPD and with acute exacerbations of COPD using real-time PCR. The present study was part of a randomised, double-blind, single-centre study and a total of 248 sputum samples from 104 COPD patients were included. In total, 122 samples obtained during stable disease (stable-state sputa) and 126 samples obtained during acute exacerbations of COPD (exacerbation sputa) were tested. Of the 122 stable-state sputa, all samples were negative for M. pneumoniae and C. pneumoniae DNA, whereas one sample was positive for Legionella non-pneumophila DNA. Of the 126 exacerbation sputa, all samples were negative for M. pneumoniae and C. pneumoniae DNA, whereas one sample was positive for Legionella non-pneumophila DNA. The possible relationship between the presence of atypical pathogens and the aetiology of acute exacerbations in chronic obstructive pulmonary disease was investigated in patients with stable disease and in those with acute exacerbations using real-time PCR. No indication was found of a role for Legionella spp., Chlamydia pneumoniae or Mycoplasma pneumoniae in stable, moderately severe chronic obstructive pulmonary disease and in its exacerbations.

Induction of chimerism in rhesus macaques through stem cell transplant and costimulation blockade-based immunosuppression.

A strategy for producing high-level hematopoietic chimerism after non-myeloablative conditioning has been established in the rhesus macaque. This strategy relies on hematopoietic stem cell transplantation after induction with a non-myeloablative dose of busulfan and blockade of the IL2-receptor in the setting of mTOR inhibition with sirolimus and combined CD28/CD154 costimulation blockade. Hematopoietic stem cells derived from bone marrow and leukopheresis products both were found to be successful in inducing high-level chimerism. Mean peripheral blood peak donor chimerism was 81% with a median chimerism duration of 145 days. Additional immune modulation strategies, such as pre-transplant CD8 depletion, donor-specific transfusion, recipient thymectomy or peritransplant deoxyspergualin treatment did not improve the level or durability of chimerism. Recipient immunologic assessment suggested that chimerism occurred amidst donor-specific down-regulation of alloreactive T cells, and the reappearance of vigorous T-mediated alloreactivity accompanied rejection of the transplants. Furthermore, viral reactivation constituted a significant transplant-related toxicity and may have negatively impacted the ability to achieve indefinite survival of transplanted stem cells. Nevertheless, this chimerism-induction regimen induced amongst the longest-lived stem cell chimerism reported to date for non-human primates and thus represents a platform upon which to evaluate emerging tolerance-induction strategies.

Crosslinking in viral capsids via tiling theory.

A vital part of a virus is its protein shell, called the viral capsid, that encapsulates and hence protects the viral genome. It has been shown in Twarock [2004. A tiling approach to vius capsids assembly explaining a structural puzzle in virology. J. Theor. Biol. 226, 477-482] that the surface structures of viruses with icosahedrally symmetric capsids can be modelled in terms of tilings that encode the locations of the protein subunits. This theory is extended here to multi-level tilings in order to model crosslinking structures. The new framework is demonstrated for the case of bacteriophage HK97, and it is shown, how the theory can be used in general to decide if crosslinking, and what type of crosslinking, is compatible from a mathematical point of view with the geometrical surface structure of a virus.

Reducing whole-body vibration and musculoskeletal injury with a new car seat design.

A new car seat design, which allows the back part of the seat (BPS) to lower down while a protruded cushion supports the lumbar spine, was quantitatively tested to determine its effectiveness and potentials in reducing whole-body vibration (WBV) and musculoskeletal disorders in automobile drivers. Nine subjects were tested to drive with the seat in: 1) the conventional seating arrangement (Normal posture); and 2) the new seating design (without BPS (WO-BPS) posture). By reducing contact between the seat and the ischial tuberosities (ITs), the new seating design reduced both contact pressure and amplitude of vibrations transmitted through the body. Root-mean-squared values for acceleration along the z-axis at the lumbar spine and ITs significantly decreased 31.6% (p < 0.01) and 19.8% (p < 0.05), respectively, by using the WO-BPS posture. At the same time, vibration dose values significantly decreased along the z-axis of the lumbar spine and ITs by 43.0% (p < 0.05) and 34.5% (p < 0.01). This reduction in WBV allows more sustained driving than permitted by conventional seating devices, by several hours, before sustaining unacceptable WBV levels. Such seating devices, implemented in large trucks and other high-vibration vehicles, may reduce the risk of WBV-related musculoskeletal disorders among drivers.

FEM model for evaluating buttock tissue response under sitting load.

Development of a model for evaluating tissue compression during sitting has been hampered by limitation of implementing the anatomical and mechanical parameters of the various layers of tissue. This study proposes a new method to setup and validate a finite element model for buttock tissue to evaluate the tissue response under external sitting load. Multi-layer tissue deformation was measured with ultrasound imaging under simulated sitting load, applied and monitored through an indenter which integrates ultrasound probe and force/pressure sensors. Subject-specific FE model can then be setup and validated. Pilot test of this model revealed that the highest peak stress was found in the vicinity of the ischial tuberosity and the greatest deformation seen in the muscle layer. The authors conclude that this new method allows accurate multi-layer tissue deformation be obtained under close to real sitting load, therefore, provides a better way to evaluate tissue response to external sitting load for seated individuals in a true and subject-specific fashion, which may provide helpful information to improve the sitting comfort and prevent pressure ulcers in wheelchair bound individuals.

Gene expression analysis in human renal allograft biopsy samples using high-density oligoarray technology.

BACKGROUND: High-density oligoarray technology is a novel method for screening the expression of thousands of genes in a small tissue sample. Oligoarray analysis of genes expressed during human renal allograft rejection has not been reported previously. METHODS: Seven human renal allograft biopsies with histologic evidence of acute cellular rejection and three renal allograft biopsies without evidence of rejection (control) were analyzed for the expression of 6800 human genes using high-density oligoarrays (GeneChip, Affymetrix, Santa Clara, CA). Quantitative expression of gene transcripts was determined and a comparison analysis between acute rejection and control biopsy samples was performed. Up-regulation of a specific gene transcript during acute rejection was considered to be significant if transcript abundance increased fourfold or more relative to control biopsy samples. RESULTS: Comparison analysis revealed that between 32 and 219 gene transcripts are up-regulated (>fourfold) during acute rejection. Of these transcripts, only four (human monokine induced by interferon-gamma, T-cell receptor active beta-chain protein, interleukin-2 stimulated phosphoprotein, and RING4 (a transporter involved in antigen presentation)) were consistently up-regulated in each acute rejection sample relative to at least two of three control biopsy samples. Six other genes were up-regulated in six of seven acute rejection samples. These were interferon-stimulated growth factor-3, complement factor 3, nicotinamide N-methyltransferase, macrophage inflammatory protein-3beta, myeloid differentiation protein, and CD18. Only two gene transcripts were down-regulated in five of seven acute rejection samples. Significant up-regulation of cytotoxic T-cell effector molecules, previously reported as markers of acute renal rejection in humans, was not detected. CONCLUSIONS: High-density oligoarray technology is useful for screening gene expression in transplanted tissues undergoing acute rejection. Because this method does not rely on a priori knowledge of which genes are involved in acute rejection, it is likely to yield novel insights into the mechanisms and diagnosis of rejection.

Muscle strength in knee varus and valgus.

PURPOSE: The purpose of this study was to investigate the lower-limb muscle strength in knee varus-valgus and its dependence on knee varus-valgus position. The hypothesis was that humans could differentially contract the medial and lateral muscles crossing the knee and generate significant moments in knee valgus-varus. METHODS: The subjects sat with the knee at full extension and secured from the medial, lateral, anterior, and posterior sides. Both hips were clamped from the lateral sides. The subjects adducted (abducted) the ipsilateral hip during the knee valgus (varus) maximal voluntary contraction with EMG signals recorded from muscles crossing the knee and knee joint moments measured using a six-axis force sensor. Frontal plane tibiofemoral movement was evaluated using a fluoroscope. RESULTS AND CONCLUSIONS: The subjects differentially contracted the medial and lateral muscles, and fluoroscope images showed the corresponding tibiofemoral movement. The subjects showed considerable strength in knee varus and valgus. The active knee varus strength increased significantly with increasing knee valgus angle, and the valgus strength was significantly higher when the knee was in varus position (P < 0.039). Active valgus muscle strength at 5 degrees knee varus was significantly higher than the active varus strength at 5 degrees valgus (P = 0.002). The passive resistance moment increased linearly with increasing knee valgus and varus angles, and it accounted for 28% and 35% of the total (active plus passive) moment at the 5 degrees varus and 5 degrees valgus, respectively. The significant varus-valgus muscle strength demonstrated in this study may play important roles in performing various functional tasks, maintaining joint stability, and preventing potential injuries, whether the major load and motion at the knee is in the frontal plane or not.

Virus maturation involving large subunit rotations and local refolding.

Large-scale conformational changes transform viral precursors into infectious virions. The structure of bacteriophage HK97 capsid, Head-II, was recently solved by crystallography, revealing a catenated cross-linked topology. We have visualized its precursor, Prohead-II, by cryoelectron microscopy and modeled the conformational change by appropriately adapting Head-II. Rigid-body rotations ( approximately 40 degrees) cause switching to an entirely different set of interactions; in addition, two motifs undergo refolding. These changes stabilize the capsid by increasing the surface area buried at interfaces and bringing the cross-link-forming residues, initially approximately 40 angstroms apart, close together. The inner surface of Prohead-II is negatively charged, suggesting that the transition is triggered electrostatically by DNA packaging.

Where are the pseudogenes in bacterial genomes?

Most bacterial genomes have very few pseudogenes; notable exceptions include the genomes of the intracellular parasites Rickettsia prowazekii and Mycobacterium leprae. As DNA can be introduced into microbial genomes in many ways, the compact nature of these genomes suggests that the rate of DNA influx is balanced by the rate of DNA deletion. We propose that the influx of dangerous genetic elements such as transposons and bacteriophages selects for the maintenance of relatively high deletion rates in most bacteria; the sheltered lifestyle of intracellular parasites removes this threat, leading to reduced deletion rates and larger pseudogene loads.

Genome organization and characterization of mycobacteriophage Bxb1.

Mycobacteriophage Bxb1 is a temperate phage of Mycobacterium smegmatis. The morphology of Bxb1 particles is similar to that of mycobacteriophages L5 and D29, although Bxb1 differs from these phages in other respects. First, it is heteroimmune with L5 and efficiently forms plaques on an L5 lysogen. Secondly, it has a different host range and fails to infect slow-growing mycobacteria, using a receptor system that is apparently different from that of L5 and D29. Thirdly, it is the first mycobacteriophage to be described that forms a large prominent halo around plaques on a lawn of M. smegmatis. The sequence of the Bxb1 genome shows that it possesses a similar overall organization to the genomes of L5 and D29 and shares weak but detectable DNA sequence similarity to these phages within the structural genes. However, Bxb1 uses a different system of integration and excision, a repressor with different specificity to that of L5 and encodes a large number of novel gene products including several with enzymatic functions that could degrade or modify the mycobacterial cell wall.

The origins and ongoing evolution of viruses.

Genome analyses of double strand DNA tailed bacteriophages argue that they evolve by recombinational reassortment of genes and by the acquisition of novel genes as simple genetic elements termed morons. These processes suggest a model for early virus evolution, wherein viruses can be regarded less as having derived from cells and more as being partners in their mutual co-evolution.

Topologically linked protein rings in the bacteriophage HK97 capsid.

The crystal structure of the double-stranded DNA bacteriophage HK97 mature empty capsid was determined at 3.6 angstrom resolution. The 660 angstrom diameter icosahedral particle contains 420 subunits with a new fold. The final capsid maturation step is an autocatalytic reaction that creates 420 isopeptide bonds between proteins. Each subunit is joined to two of its neighbors by ligation of the side-chain lysine 169 to asparagine 356. This generates 12 pentameric and 60 hexameric rings of covalently joined subunits that loop through each other, creating protein chainmail: topologically linked protein catenanes arranged with icosahedral symmetry. Catenanes have not been previously observed in proteins and provide a stabilization mechanism for the very thin HK97 capsid.

The LE1 bacteriophage replicates as a plasmid within Leptospira biflexa: construction of an L. biflexa-Escherichia coli shuttle vector.

We have discovered that LE1, one of the plaque-forming phages previously described as lytic for the Leptospira biflexa saprophytic spirochete (I. Saint Girons, D. Margarita, P. Amouriaux, and G. Baranton, Res. Microbiol. 141:1131-1138, 1990), was indeed temperate. LE1 was found to be unusual, as Southern blot analysis indicated that it is one of the few phages to replicate in the prophage state as a circular plasmid. The unavailability of such small endogenous replicons has hindered genetic experimentation in Leptospira. We have developed a shuttle vector with DNA derived from LE1. Random LE1 DNA fragments were cloned into a pGEM 7Zf(+) derivative devoid of most of the bla gene but carrying a kanamycin resistance marker from the gram-positive bacterium Enterococcus (Streptococcus) faecalis. These constructs were transformed into L. biflexa strain Patoc 1 by electroporation, giving rise to kanamycin-resistant transformants. A 2.2-kb fragment from LE1 was responsible for replication of the vector in L. biflexa. However, a larger region including an intact parA gene homologue was necessary for the stability of the shuttle vector. Direct repeats and AT-rich regions characterized the LE1 origin of replication. Our data indicate that the replicon derived from the LE1 leptophage, together with the kanamycin resistance gene, is a promising tool with which to develop the genetics of Leptospira species.