Phylogenetic Analysis of Mycobacterium tuberculosis Strains in Wales by Use of Core Genome Multilocus Sequence Typing To Analyze Whole-Genome Sequencing Data.

An inability to standardize the bioinformatic data produced by whole-genome sequencing (WGS) has been a barrier to its widespread use in tuberculosis phylogenetics. The aim of this study was to carry out a phylogenetic analysis of tuberculosis in Wales, United Kingdom, using Ridom SeqSphere software for core genome multilocus sequence typing (cgMLST) analysis of whole-genome sequencing data. The phylogenetics of tuberculosis in Wales have not previously been studied. Sixty-six Mycobacterium tuberculosis isolates (including 42 outbreak-associated isolates) from south Wales were sequenced using an Illumina platform. Isolates were assigned to principal genetic groups, single nucleotide polymorphism (SNP) cluster groups, lineages, and sublineages using SNP-calling protocols. WGS data were submitted to the Ridom SeqSphere software for cgMLST analysis and analyzed alongside 179 previously lineage-defined isolates. The data set was dominated by the Euro-American lineage, with the sublineage composition being dominated by T, X, and Haarlem family strains. The cgMLST analysis successfully assigned 58 isolates to major lineages, and the results were consistent with those obtained by traditional SNP mapping methods. In addition, the cgMLST scheme was used to resolve an outbreak of tuberculosis occurring in the region. This study supports the use of a cgMLST method for standardized phylogenetic assignment of tuberculosis isolates and for outbreak resolution and provides the first insight into Welsh tuberculosis phylogenetics, identifying the presence of the Haarlem sublineage commonly associated with virulent traits.

Comparison of bacterial identification by MALDI-TOF mass spectrometry and conventional diagnostic microbiology methods: agreement, speed and cost implications.

Identification of microbial pathogens still relies primarily on culture and phenotypic methods, which is labour-intensive and time-consuming. In this study, identification of bacteria with valid standard identification using BD Phoenix, API panels and other recommended procedures is compared to identification with matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry using the MALDI Biotyper (Bruker Daltonics) in the setting of a routine NHS diagnostic microbiology laboratory. In total, 928 bacterial isolates obtained from blood (n=463), wounds and pus (n=208), respiratory tract (n=100), faeces (n=86) and urines (n=71) were analysed. There were 721 (77.7%) isolates with a MALDI Biotyper score > or =2.0, indicating secure genus and probable species identification; and 149 (16.1%) isolates with a score > or =1.7 and <2.0 indicating probable genus identification. The isolates with scores of > or =2.0 and > or =1.7 comprised 31 and 33 genera and 65 and 67 species, respectively. Overall, 99.4% and 99.1% of organism identifications were in agreement between the MALDI Biotyper and conventional identification at the genus level, and 89.3% and 87.8% at species level when analysing organisms with MALDI Biotyper scores > or =2.0 and > or =1.7, respectively. With many but not all organisms, identification at the genus level is sufficient; however, MALDI Biotyper separation of 208 staphylococci into Staphylococcus aureus and coagulase-negative staphylococci was always correct when scores were > or =1.7. First results were obtained after 5-10 min and analysis of a full 96-well target plate was completed in approximately 90 min. Substantial savings of between pounds 1.79 and pounds 2.56 per isolate, depending on the cost model of acquisition of the MALDI Biotyper system and number of isolates tested, would be realised when all 928 isolates were identified using the MALDI Biotyper and disk-susceptibility testing when compared to the cost for 618 Phoenix ID panels and 158 API panels and disk-susceptibility tests only (i.e., not taking into account costs incurred for identification of the remaining 152 mixed isolates). Microbial identification by MALDI Biotyper offers a rare opportunity for significant cost-neutral or even cost-saving quality improvements in medical diagnostics.

Increased vascularity and spontaneous metastasis of breast cancer by hedgehog signaling mediated upregulation of cyr61.

The Hedgehog (Hh) pathway is well known for its involvement in angiogenesis and vasculogenesis during ontogeny. The ligand, Sonic Hh (SHH), has an important role in vascular formation during development. However, SHH expression is upregulated on tumor cells and can impact the tumor microenvironment. We have investigated the effects of autocrine as well as paracrine Hh signaling on tumor cells as well as on endothelial cells, respectively. Upon constitutive expression of SHH, breast cancer cells showed aggressive behavior and rapid xenograft growth characterized by highly angiogenic tumors that were spontaneously metastatic. In these cells, SHH caused activation of the Hh transcription factor, GLI1, leading to upregulated expression of the potent pro-angiogenic secreted molecule, CYR61 (cysteine-rich angiogenic inducer 61). Silencing of CYR61 from these SHH-expressing Hh activated cells blunted the malignant behavior of the tumor cells and resulted in reduced tumor vasculature and limited hematogenous metastases. Thus, CYR61 is a critical downstream contributor to the Hh influenced pro-angiogenic tumor microenvironment. We also observed concomitant upregulation of SHH and CYR61 transcripts in tumors from patients with advanced breast cancer, further ratifying the clinical relevance of our findings. In summary, we have defined a novel, VEGF-independent, clinically relevant, pro-angiogenic factor, CYR61, that is a transcriptional target of Hh-GLI signaling.

Rewritable glycochips.

We describe microarraying of carbohydrates for protein screening using either disulfide bridge or Schiff base imine immobilization chemistries on plasmachemical deposited functional nanolayers. The commonly observed issue of nonspecific background binding of proteins is overcome by spotting carbohydrates through a protein-resistant overlayer yielding spatially localized interaction with a reactive functional underlayer.

Weak effect of metal type and ica genes on staphylococcal infection of titanium and stainless steel implants.

Currently, ica is considered to be the major operon responsible for staphylococcal biofilm. The effect of biofilm on susceptibility to staphylococcal infection of different implant materials in vivo is unclear. The interaction of ica-positive (wild-type (WT)) and ica-negative (ica(-)) Staphylococcus aureus and Staphylococcus epidermidis strains with titanium and both smooth and rough stainless steel surfaces was studied by scanning electron microscopy in vitro and in a mouse tissue cage model during 2 weeks following perioperative or postoperative inoculation in vivo. In vitro, WT S. epidermidis adhered equally and more strongly than did WT S. aureus to all materials. Both WT strains, but not ica(-) strains, showed multilayered biofilm. In vivo, 300 CFUs of WT and ica(-)S. aureus led, in all metal cages, to an infection with a high level of planktonic CFUs and only 0.89% adherent CFUs after 8 days. In contrast, 10(6) CFUs of the WT and ica(-) strains were required for postoperative infection with S. epidermidis. In all metal types, planktonic numbers of S. epidermidis dropped to <100 WT, and adherent CFUs were low in WT-infected cages and absent in ica(-)-infected cages after 14 days. Perioperative S. epidermidis inoculation resulted in slower clearance than postoperative inoculation, and in titanium cages adherent WT bacteria survived in higher numbers than ica(-) bacteria. In conclusion, the metal played a minor role in susceptibility to and persistence of staphylococcal infection; the presence of ica genes had a strong effect on biofilm in vitro and a weak effect in vivo; and S. epidermidis was more pathogenic when introduced during implantation than after implantation.

Reduced medical infection related bacterial strains adhesion on bioactive RGD modified titanium surfaces: a first step toward cell selective surfaces.

Ideally, implants should inhibit nonspecific protein adsorption, bacterial adhesion, and at the same time, depending on the final application be selective toward cellular adhesion and spreading for all or only selected cell types. Poly(L-lysine)-grafted-poly(ethylene glycol) (PLL-g-PEG) polymers have been shown to adsorb from aqueous solution onto negatively charged metal oxide surfaces, reducing protein adsorption as well as fibroblast, osteoblast and epithelial cell adhesion significantly. PLL-g-PEG can be functionalized with bioligands such as RGD (Arg-Gly-Asp), which then restores host cell adhesion, but the surface remains resistant to nonspecific protein adsorption. Previously, it was also shown that both nonfunctionalized PLL-g-PEG and RGD-peptide functionalized PLL-g-PEG reduced the adhesion of Staphylococcus aureus to titanium (Ti) surfaces. The present study looked at the effect of other implant associated infection relevant bacteria, Staphylococcus epidermidis, Streptococcus mutans and Pseudomonas aeruginosa towards the same surface chemistries. The different surfaces were exposed to the bacteria for 1-24 h, and bacteria surface density was evaluated using scanning electron microscopy (SEM) and fluorescence light microscopy (FM). The adhesion of all bacteria strains tested was reduced on Ti surfaces coated with PLL-g-PEG compared to uncoated Ti surfaces even in the presence of RGD. The percentage reduction in bacterial adhesion over the 24-h culture time investigated was 88%-98%, depending on the bacteria type. Therefore, coating surfaces with PLL-g-PEG/PEG-RGD allows cells such as fibroblasts and osteoblasts to attach but not bacteria, resulting in a selective biointeractive pattern that may be useful on medical implants.

Mimicking a Stenocara beetle's back for microcondensation using plasmachemical patterned superhydrophobic-superhydrophilic surfaces.

A simple two-step plasmachemical methodology is outlined for the fabrication of microcondensor surfaces. This comprises the creation of a superhydrophobic background followed by pulsed plasma deposition of a hydrophilic polymer array. Microcondensation efficiency has been explored in terms of the chemical nature of the hydrophilic pixels and their dimensions. These results are compared to the hydrophilic-hydrophobic pattern present on the Stenocara beetle's back, which is used by the insect to collect water in the desert. Potential applications include fog harvesting, microfluidics, and biomolecule immobilization.

Biofilm formation in medical device-related infection.

Medical device-associated infections, most frequently caused by coagulase-negative staphylococci, especially Staphylococcus epidermidis, are of increasing importance in modern medicine. Regularly, antimicrobial therapy fails without removal of the implanted device. The most important factor in the pathogenesis of medical device-associated staphylococcal infections is the formation of adherent, multilayered bacterial biofilms. There is urgent need for an increased understanding of the functional factors involved in biofilm formation, the regulation of their expression, and the interaction of those potential virulence factors in device related infection with the host. Significant progress has been made in recent years which may ultimately lead to new rational approaches for better preventive, therapeutic, and diagnostic measures.

Bacteria and cell cytocompatibility studies on coated medical grade titanium surfaces.

Acute and chronic osteomyelitis caused by staphylococci can be difficult to treat by conventional means and often has marked consequences for the patient. Current methods of treatment involve the use of systemic antibiotics, the local implantation of nondegradable drug carriers, and surgical debridement. A possible solution that could prevent initial bacterial adhesion could be to modify the implant surface with an antimicrobial coating while maintaining biocompatibility to host cells. This study describes the cytocompatibility evaluation of different coatings (poly(D,L-lactide) (PDLLA), politerefate (PTF), calcium phosphate/anodic plasma-chemical treatment (CaP/APC), polyurethane (PU), and polyvinylpyrollidone (PVP) on titanium surfaces with and without chlorhexidine diacetate (CHA) to Staphylococcus aureus, Staphylococcus epidermidis, and hTERT human fibroblasts. Surface characterization of the coatings showed no significant variation in the roughness or hydrophobicity of the coated surfaces, except the CaP/APC surface that was porous yet the smoothest, and PVP, PVP+CHA, and CaP/APC+CHA that were more hydrophilic in nature than the others. On the surfaces without CHA, both staphylococcal strains and spread fibroblasts were observed, but on the CHA impregnated surfaces few bacteria and no intact fibroblasts were seen. Flow cytometry found fewer bacteria in the media and on the surfaces containing CHA in comparison to the surfaces without CHA. The release kinetics varied from slow (over 200 h) to burst release: PDLLA>PTF>PU>CaP/APC=PVP. This study showed that PDLLA and PTF have the best potential as coatings on implants for drug delivery, as they were cytocompatible to hTERT fibroblasts, eluted CHA effectively, and passed mechanical testing. The actual release kinetics of PDLLA and PTF are important, as the amount of CHA present should remain above the minimal inhibitory concentration value for a limited time before disappearing completely.

Biodegradable polyurethane cytocompatibility to fibroblasts and staphylococci.

Biodegradable polyurethanes have potential for use as implantable devices (orthopedic, maxillofacial, cardiovascular, wound dressing and plastic surgery) because of their controllable elasticity, and the possibility of changing their chemistry and structure. Studying bacterial and cell adhesion to polyurethanes helps to determine surface cytocompatibility and suitability for in vivo trials. Staphylococcus aureus, Staphylococcus epidermidis and hTERT human fibroblast cells were used to determine the cytocompatibility of experimental biodegradable polyurethanes (PUs) with different hydrophobic-to-hydrophilic (pho:phi) content ratios (100% pho, 70% pho, and 30% pho). Poly(L/DL-lactide) 70/30% (PLDL) and Thermanox were used as control surfaces. Surface characterization using noncontact profilometry, contact angles, and scanning electron microscopy (SEM) showed that the three PU surfaces, PLDL, and Thermanox have different properties. On the 100% PU and 30% PU surfaces, hTERT cells spread less in comparison to the 70% PU, PLDL, and Thermanox surfaces. The adsorption of fibronectin to the surfaces had no effect on the adhesion and spreading of hTERT cells when compared to the uncoated surfaces. The trend for S. aureus was the most adhered on the 70% PU and 30% PU, then Thermanox, followed by 100% PU and PLDL, respectively. The amount of S. epidermidis adhesion followed the trend of the most on 70% PU, then 100% PU, then 30% PU and PLDL, and the least on Thermanox. These results suggest that the 70% PU surface is cytocompatible to hTERT fibroblasts, while the 100% PU and 30% PU were not. All surfaces encouraged S. aureus and S. epidermidis colonization, particularly the 70% PU.

Staphylococcus aureus adhesion to different treated titanium surfaces.

Staphylococcus aureus is a major pathogen, associated with medical-device related infections. Converting biomaterial surfaces into non-interactive surfaces requires a specific surface/interface design. One approach is to polish the surface, and a second is to coat the surface with an antimicrobial or protein resistant coating. This study showed that polishing a titanium surface or coating titanium with various treatments that decreased the surface's coefficient of friction, had no significant effect on minimising S. aureus adhesion to these surfaces under static conditions in comparison to standard medical grade titanium. The cell promoting coating, TAST, was found to increase the S. aureus density on its surface as expected. The only coating that significantly decreased the density of adhering S. aureus was the titanium surface coated with sodium hyaluronate. Thus such a coating could have potential use as a coating for ostoesynthesis, orthopaedic or dental implants.

Staphylococcus aureus adhesion to titanium oxide surfaces coated with non-functionalized and peptide-functionalized poly(L-lysine)-grafted-poly(ethylene glycol) copolymers.

Implanted biomaterials are coated immediately with host plasma constituents, including extracellular matrix (ECM); this reaction may be undesirable in some cases. Poly(L-lysine)-grafted-poly(ethylene glycol) (PLL-g-PEG) has been shown to spontaneously adsorb from aqueous solution onto metal oxide surfaces, effectively reducing the degree of non-specific adsorption of blood and ECM proteins, and decreasing the adhesion of fibroblastic and osteoblastic cells to the coated surfaces. Cell adhesion through specific peptide-integrin receptors could be restored on surfaces coated with PLL-g-PEG functionalized with peptides of the RGD (Arg-Asp-Gly) type. To date, no study has examined the effect of surface modifications by PLL-g-PEG-based polymers on bacterial adhesion. The ability of Staphylococcus aureus to adhere to the ECM and plasma proteins deposited on biomaterials is a significant factor in the pathogenesis of medical-device-related infections. This study describes methods for visualizing and quantifying the adhesion of S. aureus to smooth and rough (chemically etched) titanium surfaces without and with monomolecular coatings of PLL-g-PEG, PLL-g-PEG/PEG-RGD and PLL-g-PEG/PEG-RDG. The different surfaces were exposed to S. aureus cultures for 1-24h and bacteria surface density was evaluated using scanning electron microscopy and fluorescence microscopy. Coating titanium surfaces with any of the three types of copolymers significantly decreased the adhesion of S. aureus to the surfaces by 89-93% for PLL-g-PEG, and 69% for PLL-g-PEG/PEG-RGD. Therefore, surfaces coated with PLL-g-PEG/PEG-RGD have the ability to attach cells such as fibroblasts and osteoblasts while showing reduced S. aureus adhesion, resulting in a selective biointeraction pattern that may be useful for applications in the area of osteosynthesis, orthopaedic and dental implantology.

An introduction to Staphylococcus aureus, and techniques for identifying and quantifying S. aureus adhesins in relation to adhesion to biomaterials: review.

The ability of Staphylococcus aureus to adhere to the extracellular matrix and plasma proteins deposited on biomaterials is a significant factor in the pathogenesis of orthopaedic-device related infections. S. aureus possesses many adhesion proteins on its surface, but it is not known how they interact with each other to form stable interactions with the substrate. A novel method was developed for extracting adhesins from the S. aureus cell wall, which could then be further analysed. The protocol involves using a FastPrep instrument to mechanically disrupt the cell walls resulting in native cell walls. Ionically and covalently bound proteins were then solubilised using sodium dodecyl sulphate (SDS) and lysostaphin, respectively. Western blot analysis of covalently bound proteins using anti-protein A and anti-clumping factor A sera showed that S. aureus produces most surface proteins in early growth, and less in post-exponential and stationary growth. Immuno-gold labelling of protein A, and clumping factor A was observed all over the bacteria and showed no distinct surface distribution pattern. However, this labelling showed expression of surface associated proteins varied in a growth-phase dependent and cell-density dependent manner.

Measurement of fibroblast and bacterial detachment from biomaterials using jet impingement.

Fibroblast and Staphylococcus aureus detachment strength from orthopaedic alloys and a tissue culture plastic (Thermanox) have been investigated with jet impingement. For S. aureus, unlike fibroblasts, detachment is caused more by pressure than shear. For these biomaterials, detachment strength is much higher for S. aureus than fibroblasts. Comparing materials under equivalent flow conditions, S. aureus attach to stainless steel and titanium with equal strength and more strongly than to Thermanox. For fibroblasts, detachment strength from all materials was similar. Fibroblast detachment strength from these biomaterials substantially decreases with time at equal flow rates and increases with flow rate at equal exposure times. Detachment strength is very similar for 3T3 and L929 fibroblasts on Thermanox for equivalent flow rate/time combinations, though enhanced adhesion of 3T3 cells was often noted for metals. Time effects are less evident for S. aureus. S. aureus adhesion to metals is more affected by flow rate than fibroblast adhesion.

Influence of parathyroid hormone, calcitonin, 1,25(OH)2 cholecalciferol, calcium, and the calcium ionophore A23187 on erythrocyte morphology and blood viscosity.

Parathyroid hormone and calcitonin, both endocrine modulators of calcium homeostasis, may influence blood rheology. Parathyroid hormone is known to reduce erythrocyte survival, leading to anemia. Calcitonin has been found to have some vascular effects. We have analyzed the Influence of parathyroid hormone (10(-7) to 10(-10) mol/L), calcitonin (10(-6) to 10(-12) mol/L), 1,25(OH)2 cholecalciferol (10(-7) to 10(-10) mol/L), additional calcium in plasma (+1 and 2 mmol/L), and the calcium lonophore A23187 (50 micromol/L) on erythrocyte morphology and blood viscosity at high shear rate (94 s(-1)) and low shear rate (0.1 s(-1)) in vitro. The loading of erythrocytes with calcium by the ionophore A23187 produced a marked echinocytic shape transformation, an increased blood viscosity at high shear rate caused by decreased deformability of these cells, and a decreased viscosity at low shear rate caused by decreased aggregation of echinocytes. In contrast, increasing plasma calcium concentrations, parathyroid hormone, calcitonin, and 1,25(OH)2 vitamin D3 had no effect on erythrocyte morphology and blood viscosity. We conclude that an increase in intraerythrocytic calcium leads to severe echinocytosis and altered blood viscosity. The endocrine modulators of calcium homeostasis--namely, parathyroid hormone, calcitonin, and 1,25(OH)2 vitamin D3--apparently do not influence intraerythrocytic calcium to a significant degree and have, therefore, no influence on cell morphology and blood viscosity.

Influence of nitrovasodilators and endothelin-1 on rheology of human blood in vitro.

1. The shear stress of flowing blood profoundly influences the release of endothelium-dependent vasodilative and constrictive factors. Conversely, the influence of these mediators such as nitric oxide (NO) or endothelin-1 (ET-1) on blood rheology remains elusive. In the present study the influence of nitrovasodilators and ET-1 on red blood cell (RBC) shape and whole blood viscosity were investigated. 2. Incubation of whole blood with sodium-nitroprusside (SNP, 10-5 - 10-2 M), glyceryl trinitrate (GTN, 0.0001 - 0.1 mg mL-1), S-nitroso-N-acetylpenicillamine (SNAP, 10-6 - 10-3 M), and the active metabolite of molsidomine (SIN-1, 10-6 - 10-3 M), but not molsidomine (10-6 - 10-3 M), resulted in significantly increased amounts of methaemoglobin, indicating a relevant interaction with RBCs. Treatment with SNP at 10-2 M induced a marked echinocytosis (morphological index: 2.23+/-0.98 vs -0.17+/-0.10; P<0.001) and increased blood viscosity (haematocrit 45%) at a high shear rate of 94.5 s-1 (6.46+/-0.60 vs 5.07+/-0.35 mPa.s; P<0.01) and a low shear rate of 0.1 s-1 (88.6+/-36.8 vs 42.1+/-11.7 mPa.s; P<0.01). Echinocytosis was probably due to cyanide accumulation. SIN-1 at 10-3 M slightly decreased high shear viscosity (4.88+/-0.28 vs 4. 95+/-0.30 mPa.s; P<0.05). SNAP at 10-3 M slightly increased both high (5.14+/-0.23 vs 5.05+/-0.24 mPa.s; P<0.01) and low shear (53.9+/-7.2 vs 51.2+/-5.9 mPa.s; P<0.05) viscosity. Molsidomine and GTN failed to influence whole blood viscosity. ET-1 (10-9 - 10-6 M) had no effect on RBC shape and viscosity. 3. We conclude that the most important modulators of vascular tone, NO and ET-1, do not affect RBC shape and blood viscosity, which is important from both a physiological and a pharmacological point of view.

Spinal and combined spinal epidural techniques for labor analgesia: clinical application in a small hospital.

Providing safe and effective analgesia to laboring parturients presents a challenge to anesthesia providers in small hospitals. The necessary time commitment and additional staff needed to provide coverage for the obstetrical area can strain resources. Offering the spinal opioid block as the first choice for labor analgesia and the combined spinal epidural block in selected cases permits a labor anesthesia service to address the needs of the community hospital. Sufentanil injected into cerebral spinal fluid provides effective analgesia for 124 minutes. Adding 2.5 mg of bupivacaine further increases effective analgesia time to 170 minutes. The combined spinal epidural block offers the advantages of spinal opioid analgesia but with the flexibility of having an epidural catheter in place. The epidural catheter can be dosed intermittently for parturients in whom labor is prolonged, who require surgical manipulation for vaginal delivery, or who require cesarean section for delivery. By offering both blocks to laboring parturients, the appropriate block can be applied in each situation.

Community recovery after storm damage: a case of facilitation in primary succession.

Manipulations of early arriving, fast-growing algal stands, which appeared soon after a severe storm denuded a Southern California marine reef habiatat, indicated that the dense cover protected newly settled kelp plants from excessive damage by grazing fishes. This is an example of refuge facilitation in primary succession after a major natural disturbance, a mechanism that may contribute substantially to the regeneration of a kelp forest.

An infraciliary network in statocyst hair cells.

Ultrastructural analysis of the statocyst, a primitive vestibular organ, of the nudibranch mollusc Hermissenda crassicornis, indicates that in addition to the basal foot, there is an infraciliary rootlet system between basal bodies of adjacent sensory cilia. These rootlets project perpendicularly from the basal bodies and parallel to the cell surface in an astral array. A polarity within the network also appears to exist; the array is longest and most extensive on the side of the basal body directed away from the cell centre, but the overall arrangement of the basal bodies indicates a multidirectional sensitivity for each of the 13 sensory cells. This rootlet system, in conjunction with the attachment system of the basal bodies to the cell membrane (button anchors), may serve an integrative function for the mechanical stimuli experienced by sensory cells and/or be involved with their transductive processes by maximizing the stress to, and membrane distortion of, the transductive site caused by weighting of the cilia. Evidence was also obtained for the intracellular synthesis of statoconia by the nonsensory supporting cells.