Real-time quantitative PCR assay with Taqman(®) probe for rapid detection of MCR-1 plasmid-mediated colistin resistance.

Here we report the development of two rapid real-time quantitative PCR assays with TaqMan(®) probes to detect the MCR-1 plasmid-mediated colistin resistance gene from bacterial isolates and faecal samples from chickens. Specificity and sensitivity of the assay were 100% on bacterial isolates including 18 colistin-resistant isolates carrying the mcr-1 gene (six Klebsiella pneumoniae and 12 Escherichia coli) with a calibration curve that was linear from 10(1) to 10(8) DNA copies. Five out of 833 faecal samples from chickens from Algeria were positive, from which three E. coli strains were isolated and confirmed to harbour the mcr-1 gene by standard PCR and sequencing.

Noncontiguous finished genome sequence and description of Enterococcus massiliensis sp. nov.

Enterococcus massiliensis strain sp. nov. (= CSUR P1927 = DSM 100308) is a new species within the genus Enterococcus. This strain was first isolated from a fresh stool sample of a man during culturomics study of intestinal microflora. Enterococcus massiliensis is a Gram-positive cocci, facultative anaerobic and motile. E. massiliensis is negative for mannitol and positive for ß-galactosidase, contrary to E. gallinarum. The complete genome sequence is 2 712 841 bp in length with a GC content of 39.6% and contains 2617 protein-coding genes and 70 RNA genes, including nine rRNA genes.

Genome sequence and description of Actinomyces polynesiensis str. MS2 sp. nov. isolated from the human gut.

Actinomyces polynesiensis strain MS2 gen. nov., sp. nov. is a newly proposed genus within the family Actinomycetaceae, isolated from the stools of a healthy individual in Raiatea Island (French Polynesia, South Pacific). Actinomyces massiliensis is an anaerobic, Gram-positive organism. Here we describe the features of this organism, together with the complete genome sequence and annotation-2 943 271 bp with a 70.80% G+C content, assembled into 15 scaffolds and containing 2080 genes.

Noncontiguous finished genome sequence and description of Bacillus testis strain SIT10 sp. nov.

Bacillus testis strain SIT10 (= CSUR P1492 = DSMZ 101190) is the new type strain collected from stool from a 2-year-old boy from Senegal during a culturomics study. This Gram-positive bacterium is a facultative anaerobic rod and a member of the Bacillaceae family. We describe here the features of this bacterium, together with the complete genome sequence and annotation. The 3 987 349 bp long genome (one chromosome but no plasmid) with 42.8% GC content contains 4005 protein-coding and 171 sRNA genes, including 19 5S rRNA gene, 15 16S rRNA genes and ten 23S rRNA genes.

Evaluation of the PREVI® Isola automated seeder system compared to reference manual inoculation for antibiotic susceptibility testing by the disk diffusion method.

The disk diffusion (DD) method remains the most popular manual technique for antibiotic susceptibility testing (AST) in clinical microbiology laboratories. This is because of its simplicity, reproducibility, and limited cost compared to (automated) microdilution systems, which are usually less sensitive at detecting certain important mechanisms of resistance. Here, we evaluate the PREVI® Isola automated seeder system using a new protocol for spreading bacterial suspensions (eight deposits of calibrated inocula of bacteria, followed by two rounds of rotation) in comparison with manual DD reference testing on a large series of clinical and reference strains. The average time required for seeding one agar plate for DD with this new protocol was 51 s per plate, i.e., 70 agar plates/h. Reproducibility and repeatability was assessed on three reference and three randomly chosen clinical strains, as usually requested by the European Committee on Antimicrobial Susceptibility Testing (EUCAST), and was excellent compared to the manual method. The standard deviations of zones of growth inhibition showed no statistical discrimination. The correlation between the two methods, assessed using 294 clinical isolates and a panel of six antibiotics (n = 3,528 zones of growth inhibition measured), was excellent, with a correlation coefficient of 0.977. The new PREVI® Isola protocol adapted for DD had a sensitivity of 99 % and a specificity of 100 % compared to the manual technique for interpreting DD as recommended by the EUCAST.

[Functional outcome and quality of life after pancreaticoduodenectomy]. / Séquelles fonctionnelles et qualité de vie après duodénopancréatectomie céphalique.

AIM OF STUDY: Pancreaticoduodenectomy is a major surgical procedure whose physiological effects may weigh heavily on quality of life. The goal of this retrospective unicentric pilot study was to assess the the functional outcome after pancreaticoduodenectomy and its effect on the patient's quality of life. PATIENTS AND METHOD: Thirty patients free from tumor recurrence more than one year after pancreaticoduodenectomy responded to the GIQLI questionnaire (Gastro Intestinal Quality of Life Index) and to a specific questionnaire evaluating long-term functional outcome. RESULTS: The acceptability rate was 100%. The internal coherence of the GIQLI questionnaire was good (a Cronbach rate=0.85). The average total score of the GIQLI was 94 (IC-95%=[86-101]) compared to an ideal rate of 144. The quality of life was significantly impaired by steatorrhea, need for treatment of diarrhea, or need for enzymatic substitutive treatment. CONCLUSION: Compared to the reference for the normal population, patients post-pancreaticoduodenectomy have an average 25% decrease of quality of life scores (although more than 25% of patients experience a normal quality of life). The impairment of quality of life after pancreaticoduodenectomy appears to be related to the functional digestive consequences of the procedure. The GIQLI score could be used to assess the technical surgical variants.

Genetic analysis of a documented population bottleneck: introduced Bennett's wallabies (Macropus rufogriseus rufogriseus) in New Zealand.

Few bottlenecks of wild populations are sufficiently well-documented to constitute models for testing theories about the impact of bottlenecks on genetic variation, and subsequent population persistence. Relevant details of the Bennett's wallaby (Macropus rufogriseus rufogriseus) introduction into New Zealand were recorded (founder number, source and approximate bottleneck duration) and suggest this may provide a rare opportunity to examine the efficacy of tests designed to detect recent bottlenecks in wild populations. We first assessed the accuracy of historic accounts of the introduction using genetic diversity detected in mitochondrial DNA (mtDNA) and at five microsatellite loci. Phylogenetic analyses of mtDNA D-loop sequence haplotypes were consistent with the reported origin of the founders as Tasmania, rather than one of the Bass Strait islands in which Bennett's wallabies are also found. Microsatellite allele frequencies from the Tasmanian source population were then used to seed bottleneck simulations encompassing varying sizes and numbers of generations, in order to assess the severity of bottleneck consistent with diversity observed in the New Zealand population. The results suggested that the founder number was unlikely to have been as small as the three animals suggested by the account of the introduction. Nonetheless, the bottleneck was probably severe; in the range of three to five pairs of wallabies for one to three generations. It resulted in significantly reduced levels of allelic diversity and heterozygosity relative to the source population. This bottleneck is only detectable under the infinite allele model (IAM) and not under the stepwise mutation model (SMM) or the two-phase model (TPM), and possible explanations for this are discussed.

Nuclear import of insulin-like growth factor-binding protein-3 and -5 is mediated by the importin beta subunit.

Although insulin-like growth factor-binding protein (IGFBP)-3 and IGFBP-5 are known to modulate cell growth by reversibly sequestering extracellular insulin-like growth factors, several reports have suggested that IGFBP-3, and possibly also IGFBP-5, have important insulin-like growth factor-independent effects on cell growth. These effects may be related to the putative nuclear actions of IGFBP-3 and IGFBP-5, which we have recently shown are transported to the nuclei of T47D breast cancer cells. We now describe the mechanism for nuclear import of IGFBP-3 and IGFBP-5. In digitonin-permeabilized cells, where the nuclear envelope remained intact, nuclear translocation of wild-type IGFBP-3 appears to occur by a nuclear localization sequence (NLS)-dependent pathway mediated principally by the importin beta nuclear transport factor and requiring both ATP and GTP hydrolysis. Under identical conditions, an NLS mutant form of IGFBP-3, IGFBP-3[(228)KGRKR --> MDGEA], was unable to translocate to the nucleus. In cells where both the plasma membrane and nuclear envelope were permeabilized, wild-type IGFBP-3, but not the mutant form, accumulated in the nucleus, implying that the NLS was also involved in mediating binding to nuclear components. By fusing wild-type and mutant forms of NLS sequences (IGFBP-3 [215-232] and IGFBP-5 [201-218]) to the green fluorescent protein, we identified the critical residues of the NLS necessary and sufficient for nuclear accumulation. Using a Western ligand binding assay, wild-type IGFBP-3 and IGFBP-5, but not an NLS mutant form of IGFBP-3, were shown to be recognized by importin beta and the alpha/beta heterodimer but only poorly by importin alpha. Together these results suggest that the NLSs within the C-terminal domain of IGFBP-3 and IGFBP-5 are required for importin-beta-dependent nuclear uptake and probably also accumulation through mediating binding to nuclear components.

Induction of adjuvant arthritis in mice.

Adjuvant arthritis, induced by injections of Freund's complete adjuvant into the footpads of some rat strains, has been recognized as a useful animal model for many years. There has, however, been notable lack of success in reproducing this model in other species. We now describe the development of adjuvant arthritis in healthy strain mice approximately 2 months after injection of Freund's complete adjuvant. Although the clinical appearance of the mice and the joint histopathology closely resemble the adjuvant arthritis reported in the rat, we were unable to detect rheumatoid factor in sera from the affected animals. In parallel studies of T cell proliferation, affected animals responded to some mycobacterial antigens but not to the 65-kD heat shock protein of Mycobacterium tuberculosis, suggesting that some other epitope is important in the development of the disease.

Independent analysis of the 16/6 idiotype lupus model. A role for an environmental factor?

The recent description of a lupus-like disease in normal mice after injections of human mAb that bind DNA and carry the common Id 16/6 Id has excited much attention. In an effort to reproduce this model we have performed two experiments using eight human mAb three of which bear the 16/6 Id. Despite using an injection protocol very similar to that of the original authors and waiting for up to 1 yr we were unable to detect any autoantibodies or any evidence of renal disease. We suspect that our failure to reproduce the model may be due to differences in either or both the batches of CFA used, or in the animal house environments. It supports the view that a mosaic of effects is required to induce clinical expression of an autoimmune disease.

Autoantibody and idiotype profile of lung involvement in autoimmune rheumatic disease.

Several reports have linked the presence of certain serum autoantibodies with particular clinical manifestations of autoimmune disease. For example, the Jo-1 antibody is now established as a marker for fibrosing alveolitis in polymyositis. To investigate the possible association of further autoantibodies or idiotypes with fibrosing alveolitis in autoimmune rheumatic disease a panel of autoantibodies was measured in serum samples from 28 patients with systemic lupus erythematosus (SLE) (10 with and 18 without lung involvement), 21 patients with scleroderma (12 with fibrosing alveolitis and nine without), and 41 patients with 'lone' fibrosing alveolitis. Antibodies measured were IgM and IgG anti-dsDNA and anti-ssDNA antibody; IgG and IgM anticardiolipin antibody; anti-poly (ADP-ribose) antibody; antibodies to two common idiotypes of anti-DNA antibodies, designated 134 and 16/6; and IgM, IgG, and IgA isotypes of rheumatoid factor. None of these antibodies was specifically associated with lung involvement in SLE or scleroderma, but a trend was found towards an increase in all autoantibodies in association with lung disease in SLE, while the reverse trend was seen in scleroderma.

Disease specificity of antibodies to poly (ADP-ribose); their relationships to anti-DNA antibodies and to disease activity in lupus.

In this study we have measured the level of anti poly (ADP-ribose) antibodies in the sera of a number of patients with SLE and their relatives, patients with a wide variety of other autoimmune and infectious diseases, and a group of normal healthy controls. It was found that these antibodies were not disease specific but were present in nine out of thirteen groups tested in significant numbers. The levels of anti poly (ADP-ribose) antibodies and anti DNA antibodies in SLE patients bled serially were also measured. The level of these antibodies fluctuated in parallel in many of these patients, although the anti poly (ADP-ribose) antibodies reflected disease activity more accurately in some.

Analysis of autoantibody reactivity and common idiotype PR4 expression of myeloma proteins.

Sera from 75 patients with monoclonal gammopathies and with no clinical evidence of autoimmune disease have been screened for a wide range of autoreactivity including binding to DNA, cardiolipin, extractable nuclear antigen (ENA), rheumatoid factor activity and the presence of the common anti-DNA antibody idiotype PR4. The sera of 17/75 (23%) patients possessed autoreactivity: six were positive for anti-DNA activity, two had anticardiolipin activity and the PR4 ID was found in two sera (both of which possessed anti-DNA activity). Antibodies to ENA were found in one serum (anti-Ro) and anti-organ-specific antibodies in five. Using iso-electric focusing and immunoblotting we have shown that the PR4 ID and DNA binding activity are carried on the paraprotein and not on some other serum constituent. The IgG subclass distribution of 55 IgG paraproteins has also been investigated. The majority of IgG paraproteins belong to IgG1 subclass (55%), with the others, being IgG2 (4%), IgG3 (9%) and IgG4 (27%). In this study we have shown that sera from myeloma patients frequently possess autoreactivity, and that in many cases this can be attributed to the paraprotein.

Relation between lymphocytotoxic antibodies, anti-DNA antibodies and a common anti-DNA antibody idiotype PR4 in patients with systemic lupus erythematosus, their relatives and spouses.

Forty-two patients with systemic lupus erythematosus (SLE), 65 of their healthy relatives and 20 spouses were studied for the presence of lymphocytotoxic antibodies (LCA), anti-lymphocyte antibodies (ALA), antibodies to DNA and a common idiotype (Id) PR4. Seventy-one per cent of the patients had positive levels of LCA, and in 34% the PR4 Id was detected; normal levels were found in their families. Anti-PR4, an anti-Id, failed to block the lymphocytotoxic activity in those nine patients who both carried the Id and had LCA. This indicates that the Id was not present on LCA. There was no correlation between anti-DNA antibodies and LCA, suggesting that different mechanisms are involved in their expression.

A comparison of autoantibodies and common DNA antibody idiotypes in SLE patients and their spouses.

In order to determine whether environmental influence per se might influence autoantibody production, sera from the spouses of 20 SLE patients were examined. No antibodies to cardiolipin, poly (ADP-ribose), or ENA were detected and none had detectable rheumatoid factor. One weakly positive ANA reaction was noted, one had anti-DNA antibodies (by RIA and ELISA) and in two sera the common DNA antibody idiotype 16/6 was found. The idiotype was not, however, present on either anti-DNA or anti-K30 antibodies. Although long-term analyses are required, it is evident that sharing the same environment with patients who commonly express a wide range of autoantibodies and common idiotypes rarely leads to their expression in non-autoimmune subjects.

Serial studies of the IgG subclass and functional affinity of DNA antibodies in systemic lupus erythematosus.

The functional affinity and IgG subclass of antibodies to ss and dsDNA were measured by ELISA in five serial samples from 41 patients with systemic lupus erythematosus (SLE) who were divided into relatively homogeneous disease subgroups. Anti-dsDNA antibodies were restricted to IgG1 and IgG3 in renal disease and levels increased with disease severity. Functional affinity of IgG1 and IgG3 anti-dsDNA fell in patients with severe renal disease, suggesting that the high affinity antibody population lost from the serum was localizing in the kidneys. IgG2 anti-dsDNA were found in patients with joint and skin disease alone and in the thrombotic/spontaneous abortion subgroup. IgG2 antibody levels did not correlate with disease severity but did correlate with the presence of antibodies to Klebsiella K30 and may have represented a cross-reactive antibody population.