Staphylococcus aureus nosocomial infections: The role of a rapid and low-cost characterization for the establishment of a surveillance system

Continuous surveillance on resistance patterns and characterization of Staphylococcus aureus represent simple and low-cost techniques to understand and evaluate the effectiveness of infection control and antimicrobial prescribing measures. In this study we analyzed the antibiotic susceptibility and trends for S. aureus strains collected from bacteraemia cases in a five year period. Between 2004 and 2008 we noted a progressive decrease in the number of S. aureus isolates compared to all pathogens from clinical specimens and S. aureus bloodstream infections (BSI) reflected a similar trend. In particular we analyzed 185 isolates from blood cultures: 89 isolates were MSSA and 96 isolates were MRSA. Molecular SCCmec typing of these strains showed an absolute prevalence of types I and II, whereas five spa types from 96 isolates were obtained. Resistance pattern analysis allowed us to place MRSA strains into 12 antibiotypes and the major antibiotype was resistant to penicillin, gentamicin, erythromycin, clindamycin and ciprofloxacin. The predominant antibiotype among the MSSA isolates was resistant only to penicillin. In addition, 19.1% of MSSA are susceptible to all antibiotics tested. We also found a close association between antibiotyping 1 and genotyping t002/SCCmecI of MRSA strains, suggesting a nosocomial scenario dominated by a few particular clones

SNPs and real-time quantitative PCR method for constitutional allelic copy number determination, the VPREB1 marker case

<p>Abstract</p> <p>Background</p> <p>22q11.2 microdeletion is responsible for the DiGeorge Syndrome, characterized by heart defects, psychiatric disorders, endocrine and immune alterations and a 1 in 4000 live birth prevalence. Real-time quantitative PCR (qPCR) approaches for allelic copy number determination have recently been investigated in 22q11.2 microdeletions detection. The qPCR method was performed for 22q11.2 microdeletions detection as a first-level screening approach in a genetically unknown series of patients with congenital heart defects. A technical issue related to the <it>VPREB1 </it>qPCR marker was pointed out.</p> <p>Methods</p> <p>A set of 100 unrelated Italian patients with congenital heart defects were tested for 22q11.2 microdeletions by a qPCR method using six different markers. Fluorescence In Situ Hybridization technique (FISH) was used for confirmation.</p> <p>Results</p> <p>qPCR identified six patients harbouring the 22q11.2 microdeletion, confirmed by FISH. The <it>VPREB1 </it>gene marker presented with a pattern consistent with hemideletion in one 3 Mb deleted patient, suggestive for a long distal deletion, and in additional five non-deleted patients. The long distal 22q11.2 deletion was not confirmed by Comparative Genomic Hybridization. Indeed, the <it>VPREB1 </it>gene marker generated false positive results in association with the rs1320 G/A SNP, a polymorphism localized within the <it>VPREB1 </it>marker reverse primer sequence. Patients heterozygous for rs1320 SNP, showed a qPCR profile consistent with the presence of a hemideletion.</p> <p>Conclusions</p> <p>Though the qPCR technique showed advantages as a screening approach in terms of cost and time, the <it>VPREB1 </it>marker case revealed that single nucleotide polymorphisms can interfere with qPCR data generating erroneous allelic copy number interpretations.</p

CRISPR/Cas9-Mediated Deletion of CTG Expansions Recovers Normal Phenotype in Myogenic Cells Derived from Myotonic Dystrophy 1 Patients

Myotonic dystrophy type 1 (DM1) is the most common adult-onset muscular dystrophy, characterized by progressive myopathy, myotonia, and multi-organ involvement. This dystrophy is an inherited autosomal dominant disease caused by a (CTG)n expansion within the 3′ untranslated region of the DMPK gene. Expression of the mutated gene results in production of toxic transcripts that aggregate as nuclear foci and sequester RNA-binding proteins, resulting in mis-splicing of several transcripts, defective translation, and microRNA dysregulation. No effective therapy is yet available for treatment of the disease. In this study, myogenic cell models were generated from myotonic dystrophy patient-derived fibroblasts. These cells exhibit typical disease-associated ribonuclear aggregates, containing CUG repeats and muscleblind-like 1 protein, and alternative splicing alterations. We exploited these cell models to develop new gene therapy strategies aimed at eliminating the toxic mutant repeats. Using the CRISPR/Cas9 gene-editing system, the repeat expansions were removed, therefore preventing nuclear foci formation and splicing alterations. Compared with the previously reported strategies of inhibition/degradation of CUG expanded transcripts by various techniques, the advantage of this approach is that affected cells can be permanently reverted to a normal phenotype

Overexpression of CUGBP1 in skeletal muscle from adult classic myotonic dystrophy type 1 but not from myotonic dystrophy type 2.

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are progressive multisystemic disorders caused by similar mutations at two different genetic loci. The common key feature of DM pathogenesis is nuclear accumulation of mutant RNA which causes aberrant alternative splicing of specific pre-mRNAs by altering the functions of two RNA binding proteins, MBNL1 and CUGBP1. However, DM1 and DM2 show disease-specific features that make them clearly separate diseases suggesting that other cellular and molecular pathways may be involved. In this study we have analysed the histopathological, and biomolecular features of skeletal muscle biopsies from DM1 and DM2 patients in relation to presenting phenotypes to better define the molecular pathogenesis. Particularly, the expression of CUGBP1 protein has been examined to clarify if this factor may act as modifier of disease-specific manifestations in DM. The results indicate that the splicing and muscle pathological alterations observed are related to the clinical phenotype both in DM1 and in DM2 and that CUGBP1 seems to play a role in classic DM1 but not in DM2. In conclusion, our results indicate that multisystemic disease spectrum of DM pathologies may not be explained only by spliceopathy thus confirming that the molecular pathomechanism of DM is more complex than that actually suggested

Emergence of Carbapenem-Resistant Klebsiella pneumoniae: Progressive Spread and Four-Year Period of Observation in a Cardiac Surgery Division

Frequent use of carbapenems has contributed to the increase to K. pneumoniae strains resistant to this class of antibiotics (CRKP), causing a problem in the clinical treatment of patients. This investigation reports the epidemiology, genetic diversity, and clinical implication of the resistance to drugs mediated by CRKP in our hospital. A total of 280 K. pneumoniae strains were collected; in particular 98/280 (35%) were CRKP. Sequencing analysis of CRKP isolated strains showed that 9/98 of MBL-producing strains carried the VIM-1 gene and 89/98 of the isolates were positive for KPC-2 . Antimicrobial susceptibility tests revealed a complete resistance to third-generation cephalosporins and a moderate resistance to tigecycline, gentamicin, and fluoroquinolones with percentages of resistance of 61%, 64%, and 98%, respectively. A resistance of 31% was shown towards trimethoprimsulfamethoxazole. Colistin was the most active agent against CRKP with 99% of susceptibility. Clonality was evaluated by PFGE and MLST: MLST showed the same clonal type, ST258, while PFGE analysis indicated the presence of a major clone, namely, pulsotype A. This finding indicates that the prevalent resistant isolates were genetically related, suggesting that the spread of these genes could be due to clonal dissemination as well as to genetic exchange between different clones

Long noncoding RNA dysregulation in ischemic heart failure

12nonenoneGreco, Simona; Zaccagnini, Germana; Perfetti, Alessandra; Fuschi, Paola; Valaperta, Rea; Voellenkle, Christine; Castelvecchio, Serenella; Gaetano, Carlo; Finato, Nicoletta; Beltrami, Antonio Paolo; Menicanti, Lorenzo; Martelli, Fabio*Greco, Simona; Zaccagnini, Germana; Perfetti, Alessandra; Fuschi, Paola; Valaperta, Rea; Voellenkle, Christine; Castelvecchio, Serenella; Gaetano, Carlo; Finato, Nicoletta; Beltrami, Antonio Paolo; Menicanti, Lorenzo; Martelli, Fabi

Cardiac involvement in myotonic dystrophy: The role of troponins and N-terminal pro B-type natriuretic peptide

Background and aims Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are dominant inherited muscular dystrophies with multiple systemic involvement, often producing cardiac injury. This study sought to determine the clinical significance of elevated high sensitivity cardiac troponin T and I (hs-cTnT and hs-cTnI), and N-terminal pro B-type natriuretic peptide (NT-pro-BNP) in this population. Methods Sixty DM patients (35 men and 25 women; mean age: 45.1 years, range: 12â\u80\u9373 years) underwent clinical cardiac investigations and measurements of serum hs-cTnT, hs-cTnI, creatine kinase (CK), and NT-proBNP. Left ventricular (LV) ejection fraction (EF) was assessed by echocardiography. Results Genetic analysis revealed that 46 of the 60 patients were DM1, and 14 DM2. Blood measurements showed persistent elevation of hs-cTnT and CK in 55/60 DM patients (91.73%). In contrast, hs-cTnI values were persistently normal throughout the study. Only 2 patients showed an EF <50%, being the overall range of this population between 40% and 79%. We found ECG abnormalities in 19 patients. Of these patients, 13 showed first or second-degree atrio ventricular (AV) blocks (PR interval â\u89¥ 200 ms), 4 showed a left bundle branch block (LBBB) prolonged (QRS duration â\u89¥120 ms), and 2 had an incomplete bundle branch block (QRS duration between 110 and 119 ms). After excluding patients with EF <50%, NT-pro-BNP measurement > 125 pg/mL was an independent predictor of ECG abnormalities. Conclusions NT-pro-BNP levels may be considered to be used clinically to identify DM patients at increased risk of developing myocardial conduction abnormalities

Circulating Irisin Is Reduced in Male Patients with Type 1 and Type 2 Myotonic Dystrophies

ContextMyotonic dystrophies (DM) are dominantly inherited muscle disorders characterized by myotonia, muscle weakness, and wasting. The reasons for sarcopenia in DMs are uncleared and multiple factors are involved. Irisin, a positive hormone regulator of muscle growth and bone, may play a role.ObjectivesTo investigate (1) circulating irisin in a series of DM1 and DM2 male patients compared with healthy controls and (2) the relationships between irisin and anthropometric, metabolic and hormonal parameters.Design and study participantsThis is a cross-sectional study. Fasting blood samples for glucometabolic, gonadic, bone markers, and irisin were collected from 28 ambulatory DM1, 10 DM2, and 23 age-matched healthy male subjects. Body composition and bone mineralization [bone mineral density (BMD)] were measured by DEXA. Echocardiographic assessment and visceral adiposity, namely, liver and epicardial fat, were investigated by ultrasound. Irisin released from cultured myotubes derived from 3 DM1, 3 DM2, and 3 healthy donors was assayed.ResultsPlasma irisin levels were definitely lower in both DM1 and DM2 patients than in controls with no difference between DM1 and DM2. Irisin released from DM1 and DM2 myotubes was similar to that released from myotubes of the non-DM donors, though diabetic DM2 myotubes released more irisin than DM1 myotubes. There was no correlation between irisin and muscle strength or lean mass in both DM1 and DM2 patients. In DM1 patients, plasma irisin levels correlated negatively with oxygen consumption and positively with insulin resistance, while in DM2 patients plasma irisin levels positively correlated with fat mass at arms and legs levels. No correlation with visceral fat, left ventricular mass, and gonadal hormones could be detected. In both DM1 and DM2 patients, legs BMD parameters positively correlated with plasma irisin levels.ConclusionPlasma irisin is reduced in both DM1 and DM2 male patients likely reflecting muscle mass reduction. Moreover, insulin resistance may contribute to modulation of plasma irisin in DM1 patients. The irisin-mediated cross talk muscle–adipose tissue–bone may be active also in the male myotonic dystrophies’ model

Fast myosin immunostaining of skeletal muscle transversal sections obtained from a healthy patient (A), DM1 patients (B-D) and DM2 patients (E-G).

<p>Type 2 fibers (fast positive fibers) are stained in brown. Muscle from DM1-E1 (B) and DM2-PS (E) patients show a normal histological muscle pattern similar to those observed in control muscle section (A). Muscle from DM1-E2 (C) and DM1-CDM (D) patients show a high fiber size variability with both type 1 (unstained fibers; white arrows) and type 2 (black arrows) atrophic fibers, fast positive nuclear clumps (arrowheads) and a preferential type 1 fiber central nucleation (asterisks). Muscle from DM2-PDM and DM2-PROMM patients also show high fiber size variability with very small type 2 fibers (black arrows), type 2 nuclear clumps (arrowheads) and a preferential type 2 fiber central nucleation (asterisks).</p