Comparative stability studies of poly(2-methyl-2-oxazoline) and poly(ethylene glycol) brush coatings

Non-fouling surfaces that resist non-specific adsorption of proteins, bacteria, and higher organisms are of particular interest in diverse applications ranging from marine coatings to diagnostic devices and biomedical implants. Poly(ethylene glycol) (PEG) is the most frequently used polymer to impart surfaces with such non-fouling properties. Nevertheless, limitations in PEG stability have stimulated research on alternative polymers that are potentially more stable than PEG. Among them, we previously investigated poly(2-methyl-2-oxazoline) (PMOXA), a peptidomimetic polymer, and found that PMOXA shows excellent anti-fouling properties. Here, we compare the stability of films self-assembled from graft copolymers exposing a dense brush layer of PEG and PMOXA side chains, respectively, in physiological and oxidative media. Before media exposure both film types prevented the adsorption of full serum proteins to below the detection limit of optical waveguide in situ measurements. Before and after media exposure for up to 2 weeks, the total film thickness, chemical composition, and total adsorbed mass of the films were quantified using variable angle spectroscopic ellipsometry (VASE), X-ray photoelectron spectroscopy (XPS), and optical waveguide lightmode spectroscopy (OWLS), respectively. We found (i) that PMOXA graft copolymer films were significantly more stable than PEG graft copolymer films and kept their protein-repellent properties under all investigated conditions and (ii) that film degradation was due to side chain degradation rather than due to copolymer desorption.

An attenuated Salmonella enterica serovar Typhimurium strain lacking the ZnuABC transporter induces protection in a mouse intestinal model of Salmonella infection

Salmonella enterica serovar Typhimurium has long been recognised as a zoonotic pathogen of economic significance in animals and humans. Attempts to protect humans and livestock may be based on immunization with vaccines aimed to induce a protective response. We recently demonstrated that the oral administration of a Salmonella enterica serovar Typhimurium strain unable to synthesize the zinc transporter ZnuABC is able to protect mice against systemic salmonellosis induced by a virulent homologous challenge. This finding suggested that this mutant strain could represent an interesting candidate vaccine for mucosal delivery. In this study, the protective effect of this Salmonella strain was tested in a streptomycin-pretreated mouse model of salmonellosis that is distinguished by the capability of evoking typhlitis and colitis. The here reported results demonstrate that mice immunized with Salmonella enterica serovar Typhimurium (S. Typhimurium) SA186 survive to the intestinal challenge and, compared to control mice, show a reduced number of virulent bacteria in the gut, with milder signs of inflammation. This study demonstrates that the oral administration a of S. Typhimurium strain lacking ZnuABC is able to elicit an effective immune response which protects mice against intestinal S. Typhimurium infection. These results, collectively, suggest that the streptomycin-pretreated mouse model of S. typhimurium infection can represent a valuable tool to screen S. typhimurium attenuated mutant strains and potentially help to assess their protective efficacy as potential live vaccines.

Streptococcus spp. and related bacteria: Their identification and their pathogenic potential for chronic mastitis - A molecular approach

Streptococcus spp. and related bacteria form a large group of organisms which are associated with bovine intramammary Infections (IMI). Some of them are the well-known mastitis pathogens Streptococcus uberis and Streptococcus agalactiae. In addition, there are a considerable number of these gram-positive, catalase-negative cocci (PNC) with unclear mastitic pathogenicity such as Aerococcus viridans which make the conventional diagnostics of PNC difficult. One diagnostic, API 20 Strep (API, Biomerieux) is recommended which, as a phenotypic assay, involves a series of miniaturized biochemical tests. Recently, preference is given to genotypic identification methods. In particular, sequencing of the 16S rRNA gene allows highly reproducible and accurate identification of bacteria and permits discovery of novel, clinically relevant bacteria. As a consequence, the aim of the present study was to compare identification of IMI-associated PNC by the API method as well as by sequencing of their 16S rRNA gene (16S). Furthermore, the correlation of these bacteria to bovine chronic mastitis and their phylogeny was investigated. 102 PNC isolated from single quarter milk samples were identified by API and 16S sequencing. Considering Streptococcus uberis, Streptococcus dysgalactiae subsp. dysgalactiae and Streptococcus agalactiae, both methods generated fully concordant results. In contrast, a very high disconcordance was observed for most of the other PNC, in particular Enterococcus spp., Aerococcus viridans and the viridans streptococci were shown as apathogenic. Lactococcus garvieae was found to be an opportunistic pathogen causing IMI during late lactation. In addition, PNC isolated from milk were frequently observed together with other bacteria, in particular with Staphylococcus spp. In these cases, the levels of somatic cell counts (SCC) were determined by the specific PNC present in the sample. Considering PNC phylogeny based on 16S sequencing, 3 major clusters were observed. They included all the common mastitis pathogens (cluster I), the Lactococcus spp., Enterococcus spp. and Aerococcus spp. (cluster II) and all the viridans streptococci (cluster III).

Isolated Rosai-Dorfman disease of intracranial meninges

Rosai-Dorfman disease (RDD) is a non-neoplastic proliferative histiocytic disorder that primarily affects lymph nodes (sinus histiocytosis with massive lymphadenopathy). Primary RDD of the central nervous system is most uncommon. We report on a 35-year-old man with isolated RDD of the meninges overlying the left cerebral hemisphere. Presenting signs and symptoms included severe progressive ipsilateral headaches of 4 months duration, as well as laboratory evidence of mild non-specific systemic inflammatory reaction. On magnetic resonance imaging, the lesion was seen as a contrast-enhancing, plaque-like thickening of the dura mater over the left convexity,without impinging on adjacent bone or cerebral parenchyma. Meningeal biopsy revealed a mixed mononuclear infiltrate dominated by CD68(+), S100(+), CD1a(-) non-Langerhans type histiocytes on a background of fibrosis. Bacteria, in particular mycobacteria, and fungi were excluded with special stains. Extensive clinical workup, encompassing computed tomography of thoracal and abdominal organs, bone marrow biopsy, and bronchoalveolar lavage failed to reveal any extracranial involvement. Laboratory tests for autoimmunity, including C- and P-antineutrophil cytoplasmic antibodies, antinuclear antibody, and serum rheumatoid factor, were negative. Methylprednisolone therapy induced complete remission of symptoms, with the neuroradiologic status remaining unchanged on follow-up after 2 months. We discuss the complex clinicopathologic differential diagnosis and therapeutic issues of this rare condition. While the correct diagnosis of central nervous system RDD is unlikely to be established without invasive procedures (biopsy), a conservative therapeutic approach may be considered a legitimate option.

Proposed minimal standards for the description of genera, species and subspecies of the Pasteurellaceae

Principles and guidelines are presented to ensure a solid scientific standard of papers dealing with the taxonomy of taxa of Pasteurellaceae Pohl 1981. The classification of the Pasteurellaceae is in principle based on a polyphasic approach. DNA sequencing of certain genes is very important for defining the borders of a taxon. However, the characteristics that are common to all members of the taxon and which might be helpful for separating it from related taxa must also be identified. Descriptions have to be based on as many strains as possible (inclusion of at least five strains is highly desirable), representing different sources with respect to geography and ecology, to allow proper characterization both phenotypically and genotypically, to establish the extent of diversity of the cluster to be named. A genus must be monophyletic based on 16S rRNA gene sequence-based phylogenetic analysis. Only in very rare cases is it acceptable that monophyly can not be achieved by 16S rRNA gene sequence comparison. Recently, the monophyly of genera has been confirmed by sequence comparison of housekeeping genes. In principle, a new genus should be recognized by a distinct phenotype, and characters that separate the new genus from its neighbours should be given clearly. Due to the overall importance of accurate classification of species, at least two genotypic methods are needed to show coherence and for separation at the species level. The main criterion for the classification of a novel species is that it forms a monophyletic group based on 16S rRNA gene sequence-based phylogenetic analysis. However, some groups might also include closely related species. In these cases, more sensitive tools for genetic recognition of species should be applied, such as DNA-DNA hybridizations. The comparison of housekeeping gene sequences has recently been used for genotypic definition of species. In order to separate species, phenotypic characters must also be identified to recognize them, and at least two phenotypic differences from existing species should be identified if possible. We recommend the use of the subspecies category only for subgroups associated with disease or similar biological characteristics. At the subspecies level, the genotypic groups must always be nested within the boundaries of an existing species. Phenotypic cohesion must be documented at the subspecies level and separation between subspecies and related species must be fully documented, as well as association with particular disease and host. An overview of methods previously used to characterize isolates of the Pasteurellaceae has been given. Genotypic and phenotypic methods are separated in relation to tests for investigating diversity and cohesion and to separate taxa at the level of genus as well as species and subspecies.

The association of the composite IL-1 genotype with periodontitis progression and/or treatment outcomes: a systematic review

BACKGROUND: Genetically transmitted traits such as cytokine gene polymorphisms may accentuate the host inflammatory response to the bacterial challenge and influence susceptibility to periodontitis. OBJECTIVE: To systematically review the evidence of an association between the interleukin-1 (IL-1) composite genotype, i.e. presence of the allele 2 in the gene clusters IL-1A-889 and in IL-1B +3953, and periodontitis progression and/or treatment outcomes. Material and Methods: Based on the focused question, a search was conducted for longitudinal clinical trials comparing progression of periodontitis and/or treatment outcomes in IL-1 genotype-positive (carrying allele 2) and IL-1 genotype-negative (not carrying allele 2) subjects. A search in the National Library of Medicine computerized bibliographic database MEDLINE and a manual search were performed. Selection of publications, extraction of data and validity assessment were made independently by two reviewers. RESULTS: The search provided 122 titles of which 11 longitudinal publications were included. The heterogeneity of the data prevented the performance of a meta-analysis. While findings from some publications rejected a possible role of IL-1 composite genotype on progression of periodontitis after various therapies, other reported a prognostic value for disease progression of the positive IL-1 genotype status. When assessed on a multivariate risk assessment model, several publications concluded that the assessment of the IL-1 composite genotype in conjunction with other covariates (e.g. smoking and presence of specific bacteria) may provide additional information on disease progression. The small sample size of the available publications, however, requires caution in the interpretation of the results. CONCLUSION: Based on these findings, (i) there is insufficient evidence to establish if a positive IL-1 genotype status contributes to progression of periodontitis and/or treatment outcomes. Therefore, (ii) results obtained with commercially available tests should be interpreted with caution.

Essential role of choline for pneumococcal virulence in an experimental model of meningitis

Objectives: The goal of the present study was to elucidate the contribution of the newly recognized virulence factor choline to the pathogenesis of Streptococcus pneumoniae in an animal model of meningitis. Results: The choline containing strain D39Cho(-) and its isogenic choline-free derivative D39Cho(-)licA64 -each expressing the capsule polysaccharide 2 - were introduced intracisternally at an inoculum size of 10(3) CFU into 11 days old Wistar rats. During the first 8 h post infection both strains multiplied and stimulated a similar immune response that involved expression of high levels of proinflammatory cytokines, the matrix metalloproteinase 9 (MMP-9), IL-10, and the influx of white blood cells into the CSF. Virtually identical immune response was also elicited by intracisternal inoculation of 10(7) CFU equivalents of either choline-containing or choline-free cell walls. At sampling times past 8 h strain D39Cho(-) continued to replicate accompanied by an intense inflammatory response and strong granulocytic pleiocytosis. Animals infected with D39Cho(-) died within 20 h and histopathology revealed brain damage in the cerebral cortex and hippocampus. In contrast, the initial immune response generated by the choline-free strain D39Cho(-)licA64 began to decline after the first 8 h accompanied by elimination of the bacteria from the CSF in parallel with a strong WBC response peaking at 8 h after infection. All animals survived and there was no evidence for brain damage. Conclusion: Choline in the cell wall is essential for pneumococci to remain highly virulent and survive within the host and establish pneumococcal meningitis.

Brain edema and increased intracranial pressure in the pathophysiology of bacterial meningitis

A number of advances in our understanding of the pathophysiology of bacterial meningitis have been made in recent years. In vivo studies have shown that bacterial cell wall fragments and endotoxins are highly active components, independent of the presence of viable bacteria in the subarachnoid space. Their presence in the cerebrospinal fluid is associated with the induction of inflammation and with the development of brain edema and increased intracranial pressure. Antimicrobial therapy may cause an additional increase of harmful bacterial products in the cerebrospinal fluid and thereby potentiate these pathophysiological alterations. These changes may contribute to the development of brain damage during meningitis. Some promising experimental work has been directed toward counteracting the above phenomena with non-steroidal or steroidal anti-inflammatory agents as well as with monoclonal antibodies. Although considerable advances have been made, further research needs to be done in these areas to improve the prognosis of bacterial meningitis.

Phylogeny and prediction of genetic similarity of Cronobacter and related taxa by multilocus sequence analysis (MLSA)

Multilocus sequence analysis (MLSA) based on recN, rpoA and thdF genes was done on more than 30 species of the family Enterobacteriaceae with a focus on Cronobacter and the related genus Enterobacter. The sequences provide valuable data for phylogenetic, taxonomic and diagnostic purposes. Phylogenetic analysis showed that the genus Cronobacter forms a homogenous cluster related to recently described species of Enterobacter, but distant to other species of this genus. Combining sequence information on all three genes is highly representative for the species' %GC-content used as taxonomic marker. Sequence similarity of the three genes and even of recN alone can be used to extrapolate genetic similarities between species of Enterobacteriaceae. Finally, the rpoA gene sequence, which is the easiest one to determine, provides a powerful diagnostic tool to identify and differentiate species of this family. The comparative analysis gives important insights into the phylogeny and genetic relatedness of the family Enterobacteriaceae and will serve as a basis for further studies and clarifications on the taxonomy of this large and heterogeneous family.

Mycoplasma leachii sp. nov. as a new species designation for Mycoplasma sp. bovine group 7 of Leach, and reclassification of Mycoplasma mycoides subsp. mycoides LC as a serovar of Mycoplasma mycoides subsp. capri

The Mycoplasma mycoides cluster consists of six pathogenic mycoplasmas causing disease in ruminants, which share many genotypic and phenotypic traits. The M. mycoides cluster comprises five recognized taxa: Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC), M. mycoides subsp. mycoides Large Colony (MmmLC), M. mycoides subsp. capri (Mmc), Mycoplasma capricolum subsp. capricolum (Mcc) and M. capricolum subsp. capripneumoniae (Mccp). The group of strains known as Mycoplasma sp. bovine group 7 of Leach (MBG7) has remained unassigned, due to conflicting data obtained by different classification methods. In the present paper, all available data, including recent phylogenetic analyses, have been reviewed, resulting in a proposal for an emended taxonomy of this cluster: (i) the MBG7 strains, although related phylogenetically to M. capricolum, hold sufficient characteristic traits to be assigned as a separate species, i.e. Mycoplasma leachii sp. nov. (type strain, PG50(T) = N29(T) = NCTC 10133(T) = DSM 21131(T)); (ii) MmmLC and Mmc, which can only be distinguished by serological methods and are related more distantly to MmmSC, should be combined into a single subspecies, i.e. Mycoplasma mycoides subsp. capri, leaving M. mycoides subsp. mycoides (MmmSC) as the exclusive designation for the agent of contagious bovine pleuropneumonia. A taxonomic description of M. leachii sp. nov. and emended descriptions of M. mycoides subsp. mycoides and M. mycoides subsp. capri are presented. As a result of these emendments, the M. mycoides cluster will hereafter be composed of five taxa comprising three subclusters, which correspond to the M. mycoides subspecies, the M. capricolum subspecies and the novel species M. leachii.

Genetic relationships of Aeromonas strains inferred from 16S rRNA, gyrB and rpoB gene sequences

Genetic relationships among bacterial strains belonging to the genus Aeromonas were inferred from 16S rRNA, gyrB and rpoB gene sequences. Twenty-eight type or collection strains of the recognized species or subspecies and 33 Aeromonas strains isolated from human and animal specimens as well as from environmental samples were included in the study. As reported previously, the 16S rRNA gene sequence is highly conserved within the genus Aeromonas, having only limited resolution for this very tight group of species. Analysis of a 1.1 kb gyrB sequence confirmed that this gene has high resolving power, with maximal interspecies divergence of 15.2 %. Similar results were obtained by sequencing only 517 bp of the rpoB gene, which showed maximal interspecies divergence of 13 %. The topologies of the gyrB- and rpoB-derived trees were similar. The results confirm the close relationship of species within the genus Aeromonas and show that a phylogenetic approach including several genes is suitable for improving the complicated taxonomy of the genus.

Phylogeny of the family Pasteurellaceae based on rpoB sequences

Sequences of the gene encoding the beta-subunit of the RNA polymerase (rpoB) were used to delineate the phylogeny of the family Pasteurellaceae. A total of 72 strains, including the type strains of the major described species as well as selected field isolates, were included in the study. Selection of universal rpoB-derived primers for the family allowed straightforward amplification and sequencing of a 560 bp fragment of the rpoB gene. In parallel, 16S rDNA was sequenced from all strains. The phylogenetic tree obtained with the rpoB sequences reflected the major branches of the tree obtained with the 16S rDNA, especially at the genus level. Only a few discrepancies between the trees were observed. In certain cases the rpoB phylogeny was in better agreement with DNA-DNA hybridization studies than the phylogeny derived from 16S rDNA. The rpoB gene is strongly conserved within the various species of the family of Pasteurellaceae. Hence, rpoB gene sequence analysis in conjunction with 16S rDNA sequencing is a valuable tool for phylogenetic studies of the Pasteurellaceae and may also prove useful for reorganizing the current taxonomy of this bacterial family.

Phylogenetic analysis of Pasteurella multocida subspecies and molecular identification of feline P. multocida subsp. septica by 16S rRNA gene sequencing

Pasteurella multocida is commonly found in the oral cavity of cats and dogs. In humans it is known as an opportunistic pathogen after bites from these animals. Phenotypic identification of P. multocida based on biochemical reactions is often limited and usually only done on a species level, even though 3 subspecies are described. For molecular taxonomy and diagnostic purposes a phylogenetic analysis of the three subspecies of P. multocida based on their 16S rRNA (rrs) gene sequence was therefore carried out. We found P. multocida subsp. septica on a distinguished branch on the phylogenetic tree of Pasteurellaceae, due to a 1.5% divergence of its rrs gene compared to the two other, more closely related subspecies multocida and gallicida. This phylogenetic divergence can be used for the identification of P. multocida subsp. septica by rrs gene determination since they form a phylogenetically well isolated and defined group as shown with a set of feline isolates. Comparison to routine phenotypic identification shows the advantage of the sequence-based identification over conventional methods. It is therefore helpful for future unambiguous identification and molecular taxonomy of P. multocida as well as for epidemiological investigations.

Induction of lucid dreams: A systematic review of evidence

In lucid dreams the dreamer is aware of dreaming and often able to influence the ongoing dream content. Lucid dreaming is a learnable skill and a variety of techniques is suggested for lucid dreaming induction. This systematic review evaluated the evidence for the effectiveness of induction techniques. A comprehensive literature search was carried out in biomedical databases and specific resources. Thirty-five studies were included in the analysis (11 sleep laboratory and 24 field studies), of which 26 employed cognitive techniques, 11 external stimulation and one drug application. The methodological quality of the included studies was relatively low. None of the induction techniques were verified to induce lucid dreams reliably and consistently, although some of them look promising. On the basis of the reviewed studies, a taxonomy of lucid dream induction methods is presented. Several methodological issues are discussed and further directions for future studies are proposed.

Antigens of the type-three secretion system of Aeromonas salmonicida subsp. salmonicida prevent protective immunity in rainbow trout

Aeromonas salmonicida subsp. salmonicida is the etiologic agent of furunculosis, a frequent and significant disease of fisheries worldwide. The disease is largely controlled by commercial oil adjuvanted vaccines containing bacterins. However, the mechanisms leading to a protective immune response remain poorly understood. The type-three secretion system (T3SS) plays a central role in virulence of A. salmonicida subsp. salmonicida and thus may have an influence on the immune response of the host. The aim of this study was to evaluate the role of the T3SS antigens in mounting a protective immune response against furunculosis. Rainbow trout were intraperitoneally vaccinated in two independent experiments with bacterins prepared from a wild-type A. salmonicida strain and an isogenic strain carrying a deletion in the T3SS (ΔascV). Fish were challenged with the wt strain eight weeks after vaccination. In both trials, the survival rate of trout vaccinated with the ΔascV strain was significantly higher (23-28%) in comparison to the group vaccinated with the wt strain. High-throughput proteomics analysis of whole bacteria showed the ascV deletion in the mutant strain resulted in lower expression of all the components of the T3SS, several of which have a potential immunosuppressive activity. In a third experiment, fish were vaccinated with recombinant AcrV (homologous to the protective antigen LcrV of Yersinia) or S-layer protein VapA (control). AcrV vaccinated fish were not protected against a challenge while fish vaccinated with VapA were partially protected. The presence of T3SS proteins in the vaccine preparations decreased the level of protection against A. salmonicida infection and that AcrV was not a protective antigen. These results challenge the hypothesis that mounting specific antibodies against T3SS proteins should bring better protection to fish and demonstrate that further investigations are needed to better understand the mechanisms underlying effective immune responses against A. salmonicida infection.