Evaluation of the Uro-Quick system for antibiotic susceptibility tests of strains collected from intensive care units

During the period January–June 2004, 525 pathogens isolated from intensive care units were examined with the new rapid Uro-Quick method for antibiotic susceptibility tests. The results were compared with those obtained by the reference NCCLS methods (disk diffusion or dilution). Antibiotic (in appropriate concentration) was introduced in a vial containing 2 ml of Mueller-Hin ton broth, then 0.5 ml of 5×10 or 106 cells/ml of the strain culture were added. After 3–6 h of incubation, depending on the microorganism studied, the instrument printed the results: no growth and a growth curve similar to that of the untreated control are representative of a susceptible and resistant strain respectively. The following drugs were tested: ciprofloxacin, ampicillin, aztreonam, co-clavulanate, piperacillin/tazobactam, ceftazidime, cefotaxime, cefuroxime, ceftriaxone, imipenem, amikacin, gentamicin, trimethoprim-sulfamethoxazole, clindamycin, erythromycin, linezolid, penicillin, tetracycline, vancomycin, oxacillin. Gram-negative strains tested were 252 and Gram-positive 273: agreement between the two methods ranged from 85.6% (piperacillin/tazobactam) to 98.5% (ciprofloxact) in Gram-negative pathogens, from 90 to 100% in Gram-positive, with the exception of erythromycin (84.2%) against enterococci. On the basis of the present findings the Uro-Quick system appears to be very useful for the rapid detection of antibiotic susceptibility in pathogens collected from intensive care units

A Novel Mutation in Lamin A/C Gene: Phenotype and Consequences on the Protein Structure and Flexibility

n/

Genetic loci linked to Type 1 Diabetes and Multiple Sclerosis families in Sardinia

<p>Abstract</p> <p>Background</p> <p>The Mediterranean island of Sardinia has a strikingly high incidence of the autoimmune disorders Type 1 Diabetes (T1D) and Multiple Sclerosis (MS). Furthermore, the two diseases tend to be co-inherited in the same individuals and in the same families. These observations suggest that some unknown autoimmunity variant with relevant effect size could be fairly common in this founder population and could be detected using linkage analysis.</p> <p>Methods</p> <p>To search for T1D and MS loci as well as any that predispose to both diseases, we performed a whole genome linkage scan, sequentially genotyping 593 microsatellite marker loci in 954 individuals distributed in 175 Sardinian families. In total, 413 patients were studied; 285 with T1D, 116 with MS and 12 with both disorders. Model-free linkage analysis was performed on the genotyped samples using the Kong and Cox logarithm of odds (LOD) score statistic.</p> <p>Results</p> <p>In T1D, aside from the HLA locus, we found four regions showing a lod-score ≥1; 1p31.1, 6q26, 10q21.2 and 22q11.22. In MS we found three regions showing a lod-score ≥1; 1q42.2, 18p11.21 and 20p12.3. In the combined T1D-MS scan for shared autoimmunity loci, four regions showed a LOD >1, including 6q26, 10q21.2, 20p12.3 and 22q11.22. When we typed more markers in these intervals we obtained suggestive evidence of linkage in the T1D scan at 10q21.2 (LOD = 2.1), in the MS scan at 1q42.2 (LOD = 2.5) and at 18p11.22 (LOD = 2.6). When all T1D and MS families were analysed jointly we obtained suggestive evidence in two regions: at 10q21.1 (LOD score = 2.3) and at 20p12.3 (LOD score = 2.5).</p> <p>Conclusion</p> <p>This suggestive evidence of linkage with T1D, MS and both diseases indicates critical chromosome intervals to be followed up in downstream association studies.</p

Epidemiological study of pathogens collected from blood for a period of a year (2008-2009)

n/

Large genotype-phenotype study in carriers of D4Z4 borderline alleles provides guidance for facioscapulohumeral muscular dystrophy diagnosis.

Facioscapulohumeral muscular dystrophy (FSHD) is a myopathy with prevalence of 1 in 20,000. Almost all patients affected by FSHD carry deletions of an integral number of tandem 3.3 kilobase repeats, termed D4Z4, located on chromosome 4q35. Assessment of size of D4Z4 alleles is commonly used for FSHD diagnosis. However, the extended molecular testing has expanded the spectrum of clinical phenotypes. In particular, D4Z4 alleles with 9-10 repeat have been found in healthy individuals, in subjects with FSHD or affected by other myopathies. These findings weakened the strict relationship between observed phenotypes and their underlying genotypes, complicating the interpretation of molecular findings for diagnosis and genetic counseling. In light of the wide clinical variability detected in carriers of D4Z4 alleles with 9-10 repeats, we applied a standardized methodology, the Comprehensive Clinical Evaluation Form (CCEF), to describe and characterize the phenotype of 244 individuals carrying D4Z4 alleles with 9-10 repeats (134 index cases and 110 relatives). The study shows that 54.5% of index cases display a classical FSHD phenotype with typical facial and scapular muscle weakness, whereas 20.1% present incomplete phenotype with facial weakness or scapular girdle weakness, 6.7% display minor signs such as winged scapula or hyperCKemia, without functional motor impairment, and 18.7% of index cases show more complex phenotypes with atypical clinical features. Family studies revealed that 70.9% of relatives carrying 9-10 D4Z4 reduced alleles has no motor impairment, whereas a few relatives (10.0%) display a classical FSHD phenotype. Importantly all relatives of index cases with no FSHD phenotype were healthy carriers. These data establish the low penetrance of D4Z4 alleles with 9-10 repeats. We recommend the use of CCEF for the standardized clinical assessment integrated by family studies and further molecular investigation for appropriate diagnosis and genetic counseling. Especially in presence of atypical phenotypes and/or sporadic cases with all healthy relatives is not possible to perform conclusive diagnosis of FSHD, but all these cases need further studies for a proper diagnosis, to search novel causative genetic defects or investigate environmental factors or co-morbidities that may trigger the pathogenic process. These evidences are also fundamental for the stratification of patients eligible for clinical trials. Our work reinforces the value of large genotype-phenotype studies to define criteria for clinical practice and genetic counseling in rare diseases

Dynorphin B is an agonist of nuclear opioid receptors coupling nuclear protein kinase C activation to the transcription of cardiogenic genes in GTR1 embryonic stem cells

The cardiac differentiation of embryonic stem (ES) cells was found to involve prodynorphin gene and dynorphin B expression and was associated with the interaction of secreted dynorphin B with cell surface opioid receptors coupled with protein kinase C (PKC) signaling and complex subcellular redistribution patterning of selected PKC isozymes. Here, confocal microscopy revealed the presence of immunoreactive dynorphin B–like material in GTR1 ES cells, suggesting that dynorphin peptides may also act intracellularly. Opioid binding sites were identified in ES cell nuclei, with a single dissociation constant in the low nanomolar range. A significant increase in Bmax for a k opioid receptor ligand was observed in nuclei isolated from ES-derived cardiomyocytes compared with nuclei from undifferentiated cells. Direct exposure of nuclei isolated from undifferentiated ES cells to dynorphin B or U-50,488H, a synthetic K opioid receptor agonist, time- and dose-dependently activated the transcription of GATA-4 and Nkx-2.5, 2 cardiac lineage–promoting genes. Nuclear exposure to dynorphin B also enhanced the rate of prodynorphin gene transcription. These responses were abolished in a stereospecific fashion by the incubation of isolated nuclei with selective opioid receptor antagonists. Nuclei isolated from undifferentiated cells were able to phosphorylate the acrylodan-labeled MARCKS peptide, a high-affinity fluorescent PKC substrate. Exposure of isolated nuclei to dynorphin B induced a remarkable increase in nuclear PKC activity, which was suppressed by opioid receptor antagonists. Nuclear treatment with PKC inhibitors abolished the capability of dynorphin B to prime the transcription of cardiogenic genes

Protein kinase C signaling transduces endorphin-primed cardiogenesis in GTR1 embryonic stem cells

The prodynorphin gene and its product, dynorphin B, have been found to promote cardiogenesis in embryonic cells by inducing the expression of GATA-4 and Nkx-2.5, two transcription factor–encoding genes essential for cardiogenesis. The molecular mechanism(s) underlying endorphin-induced cardiogenesis remain unknown. In the present study, we found that GTR1 embryonic stem (ES) cells expressed cell surface opioid receptors, as well as protein kinase C (PKC)-α, -ß1, -ß2, -δ, -ε, and -ζ. Cardiac differentiation was associated with a marked increase in the Bmax value for a selective opioid receptor ligand and complex subcellular redistribution of selected PKC isozymes. PKC-α, -ß1, -ß2, -δ, and -ε all increased in the nucleus of ES-derived cardiac myocytes, compared with nuclei from undifferentiated cells. In both groups of cells, PKC-δ and -ε were mainly expressed at the nuclear level. The nuclear increase of PKC-α, -ß1, and -ß2 was due to a translocation from the cytosolic compartment. In contrast, the increase of both PKC-δ and PKC-ε in the nucleus of ES-derived cardiomyocytes occurred independently of enzyme translocation, suggesting changes in isozyme turnover and/or gene expression during cardiogenesis. No change in PKC-ζ expression was observed during cardiac differentiation. Opioid receptor antagonists prevented the nuclear increase of PKC-α, PKC-ß1, and PKC-ß2 and reduced cardiomyocyte yield but failed to affect the nuclear increase in PKC-δ and -ε. PKC inhibitors prevented the expression of cardiogenic genes and dynorphin B in ES cells and abolished their development into beating cardiomyocytes

In vitro activity of tigecycline against 313 Gram-positive and Gram-negative clinical isolates

Objectives. In this study the in vitro activity of tigecycline, member of a new class of antimicrobial agents, the glycylcyclines, was evaluated against clinical isolates collected in Italy. Study Design. A total of 313 clinical pathogens were collected and identified in our Institution during 2007-2008. Minimum inhibitory concentrations (MICs) of the antimicrobial agents were determined by the CLSI (2007) recommended broth microdilution method. Results. Globally 205 Gram-negative and 108 Gram-positive pathogens were evaluated.Tigecycline demonstrated excellent inhibitory activity against Acinetobacter spp., H. influenzae, E. coli, Enterococcus spp., S. aureus, S. agalactiae and S. pneumoniae with MIC90 ≤1mg/l. Conclusion. Tigecycline exhibited potent in vitro antibacterial activity (comparable to or greater than most commonly employed antimicrobials) against both Gram-positive and negative clinical pathogens.These data suggest that tigecycline, with an expanded broad-spectrum antimicrobial activity, may be an effective empiric therapeutic option for the treatment of serious infections caused by clinically relevant pathogens

Utilizzo del sistema Uro-Quick per l’identificazione rapida di batteri produttori di ß-lattamasi a spettro esteso (ESBL)

Objectives: To evaluate the Uro-Quick system for detection of extended-spectrum beta-lactamase (ESBL) among nosocomial strains isolated from urine during a 2 months period. Methods:A total of 221 strains collected from nosocomial patients were tested for antibiotic susceptibility by the Uro-Quick system. About 106 cell/ml were used to seed 2.5 ml of broth in vials containing ceftazidime (10 μg/mL) and ceftazidime (10 μg/mL) plus clavulanic acid (10 μg/mL). After incubation the results were plotted as growth curves. All results were confirmed by Kirby-Bauer method. Control strains were included. Results: Using an inoculum of 106 cell/ml the instrument was capable to print out the result after 5 hours of incubation.Among the pathogens studied, 46 strains resulted resistant to ceftazidime (presence of growth).These results were then compared with those obtained with the Kirby-Bauer method, and it was found that among these 46 ceftazidime-resistant clones all were ESBL-producers, even if 12 showed an intermediate, and 12 a susceptible antibiotic phenotype by Kirby-Bauer method. Conclusion: Present findings suggest that Uro-Quick represents a useful technology to detect ESBL-producing strains especially those that required further confirmatory tests for their identification.The period of time needed to achieve the results, in some cases less than 5 hours, might be an advantage over the usual methodologies

Risk of Exclusion From Stroke Rehabilitation in the Oldest Old

Objective: To investigate whether oldest-old age (\ue2\u89\ua585y) is an independent predictor of exclusion from stroke rehabilitation. Design: Retrospective cohort study. Setting: Stroke unit (SU) of a tertiary hospital. Participants: Elderly patients (N=1055; aged 65-74y, n=230; aged 75-84y, n=432; aged \ue2\u89\ua585y, n=393) who, between 2009 and 2012, were admitted to the SU with acute stroke and evaluated by a multiprofessional team for access to rehabilitation. The study excluded patients for whom rehabilitation was unnecessary or inappropriate. Interventions: Not applicable. Main Outcome Measures: Access to an early mobilization (EM) protocol during SU stay and subsequent access to postacute rehabilitation after SU discharge. Analyses were adjusted for prestroke and stroke-related characteristics. Results: 32.2% of patients were excluded from EM. Multivariable-adjusted odds ratios (ORs) of EM exclusion were 1.30 (95% confidence interval [CI], .76-2.21) for ages 75 to 84 years and 2.07 (95% CI, 1.19-3.59) for ages \ue2\u89\ua585 years compared with ages 65 to 74 years. Of 656 patients admitted to EM and who, at SU discharge, had not yet fully recovered their prestroke functional status, 18.4% were excluded from postacute rehabilitation. For patients able to walk unassisted at SU discharge, the probability of exclusion did not change across age groups. For patients unable to walk unassisted at SU discharge, ORs of exclusion from postacute rehabilitation were 3.74 (95% CI, 1.26-11.13) for ages 75 to 84 years and 9.15 (95% CI, 3.05-27.46) for ages \ue2\u89\ua585 years compared with ages 65 to 74 years. Conclusions: Oldest-old age is an independent predictor of exclusion from stroke rehabilitation