Molecular epidemiology of extended-spectrum-beta-lactamase-producing Klebsiella pneumoniae over a 10 year period in Calgary, Canada

A study was designed to investigate the molecular epidemiology of extended-spectrum -lactamase (ESBL)-producing Klebsiella pneumoniae isolated in a centralized region over a 10 year period (200009). Molecular characterization was done using isoelectric focusing, PCR and sequencing for bla(CTX-M), bla(TEM) and bla(SHV) genes and plasmid-mediated quinolone resistance determinants. Genetic relatedness was determined with PFGE using XbaI and multilocus sequencing typing. A total of 89 patients with incident infections were identified; the majority presented with hospital-onset urinary tract infections. The absolute number of ESBL-producing isolates remained very low until 2003, increased slightly in 2004, remained stable until 2008 and then in 2009 there was an abrupt increase in the numbers of ESBL producers identified. The majority of K. pneumoniae produced CTX-M-14 and -15, and have replaced SHV-12-producing isolates since 2005. We identified four different major sequence types (STs) among 32 of isolates (i.e. ST17, ST20, and the new ST573 and ST575) and provided insight into their clinical and molecular characteristics. The ST isolates were more likely to produce community-onset infections, were associated with bla(CTX-M) and emerged during the latter part of the study period. ST17 produced CTX-M-15 and SHV-12, and was more likely to be positive for qnrB; ST20 produced CTX-M-14 and was positive for qnrS. The multiresistant ST575 that produced CTX-M-15 appeared in 2009. Our study highlights the importance of molecular epidemiology in providing insight into the emergence, characteristics and distribution of STs among ESBL-producing K. pneumoniae.

Recoupled separated-local-field experiments and applications to study intermediate-regime molecular motions

A specific separated-local-field NMR experiment, dubbed Dipolar-Chemical-Shift Correlation (DIPSHIFT) is frequently used to study molecular motions by probing reorientations through the changes in XH dipolar coupling and T-2. In systems where the coupling is weak or the reorientation angle is small, a recoupled variant of the DIPSHIFT experiment is applied, where the effective dipolar coupling is amplified by a REDOR-like pi-pulse train. However, a previously described constant-time variant of this experiment is not sensitive to the motion-induced T-2 effect, which precludes the observation of motions over a large range of rates ranging from hundreds of Hz to around a MHz. We present a DIPSHIFT implementation which amplifies the dipolar couplings and is still sensitive to T-2 effects. Spin dynamics simulations, analytical calculations and experiments demonstrate the sensitivity of the technique to molecular motions, and suggest the best experimental conditions to avoid imperfections. Furthermore, an in-depth theoretical analysis of the interplay of REDOR-like recoupling and proton decoupling based on Average-Hamiltonian Theory was performed, which allowed explaining the origin of many artifacts found in literature data. (C) 2012 Elsevier Inc. All rights reserved.

Determination of chlorine in food samples via the AlCl molecule using high-resolution continuum source molecular absorption spectrometry in a graphite furnace

Determination of chlorine using the molecular absorption of aluminum mono-chloride (AlCl) at the 261.418 nm wavelength was accomplished by high-resolution continuum source molecular absorption spectrometry using a transversely heated graphite tube furnace with an integrated platform. For the analysis. 10 mu L of the sample followed by 10 mu L of a solution containing Al-Ag-Sr modifier, (1 g L-1 each), were directly injected onto the platform. A spectral interference due to the use of Al-Ag-Sr as mixed modifier was easily corrected by the least-squares algorithm present in the spectrometer software. The pyrolysis and vaporization temperatures were 500 degrees C and 2200 degrees C, respectively. To evaluate the feasibility of a simple procedure for the determination of chlorine in food samples present in our daily lives, two different digestion methods were applied, namely (A) an acid digestion method using HNO3 only at room temperature, and (B) a digestion method with Ag, HNO3 and H2O2, where chlorine is precipitated as a low-solubility salt (AgCl), which is then dissolved with ammonia solution. The experimental results obtained with method B were in good agreement with the certified values and demonstrated that the proposed method is more accurate than method A. This is because the formation of silver chloride prevented analyte losses by volatilization. The limit of detection (LOD, 3 sigma/s) for Cl in methods A and B was 18 mu g g(-1) and 9 mu g g(-1), respectively, 1.7 and 3.3 times lower compared to published work using inductively coupled plasma optical emission spectrometry, and absolute LODs were 2.4 and 1.2 ng, respectively. (C) 2012 Elsevier B.V. All rights reserved.

Biochemical, physicochemical and molecular characterization of a genuine 2-Cys-peroxiredoxin purified from cowpea [Vigna unguiculata (L.) Walpers] leaves

Background: Peroxiredoxins have diverse functions in cellular defense-signaling pathways. 2-Cys-peroxiredoxins (2-Cys-Prx) reduce H2O2 and alkyl-hydroperoxide. This study describes the purification and characterization of a genuine 2-Cys-Prx from Vigna unguiculata (Vu-2-Cys-Prx). Methods: Vu-2-Cys-Prx was purified from leaves by ammonium sulfate fractionation, chitin affinity and ion exchange chromatography. Results: Vu-2-Cys-Prx reduces H2O2 using NADPH and DTT. Vu-2-Cys-Prx is a 44 kDa (SDS-PAGE)/46 kDa (exclusion chromatography) protein that appears as a 22 kDa molecule under reducing conditions, indicating that it is a homodimer linked intermolecularly by disulfide bonds and has a pI range of 4.56-4.72; its NH2-terminal sequence was similar to 2-Cys-Prx from Phaseolus vulgaris (96%) and Populus tricocarpa (96%). Analysis by ESI-Q-TOF MS/MS showed a molecular mass/pI of 28.622 kDa/5.18. Vu-2-Cys-Prx has 8% alpha-helix, 39% beta-sheet, 22% of turns and 31% of unordered forms. Vu-2-Cys-Prx was heat stable, has optimal activity at pH 7.0, and prevented plasmid DNA degradation. Atomic force microscopy shows that Vu-2-Cys-Prx oligomerized in decamers which might be associated with its molecular chaperone activity that prevented denaturation of insulin and citrate synthase. Its cDNA analysis showed that the redox-active Cys(52) residue and the amino acids Pro(45), Thr(49) and Arg(128) are conserved as in other 2-Cys-Prx. General significance: The biochemical and molecular features of Vu-2-Cys-Prx are similar to other members of 2-Cys-Prx family. To date, only one publication reported on the purification of native 2-Cys-Prx from leaves and the subsequent analysis by N-terminal Edman sequencing, which is crucial for construction of stromal recombinant 2-Cys-Prx proteins. (C) 2012 Elsevier B.V. All rights reserved.

Genetic features and molecular epidemiology of Enterococcus faecium isolated in two university hospitals in Brazil

The global emergence of vancomycin-resistant Enterococcus faecium (VREfm) has been characterized by a clonal spread of strains belonging to clonal complex 17 (CC17). Genetic features and clonal relationships of 53 VREfm isolated from patients in 2 hospitals in Ribeirao Preto, Sao Paulo, Brazil, during 2005-2010 were determined as a contribution to the Brazilian evolutionary history of these nosocomial pathogens. All isolates were daptomycin susceptible, vancomycin-resistant, and had the vanA gene. The predominant virulence genes were acm and esp. Only 5 VREfm isolated in 2005-2006 had intact Tn1546, while 81% showed Tn1546 with deleted left extremity and insertion of IS1251 between the vanS and vanH genes. Multilocus sequence typing analysis permitted the identification of 9 different sequence types (STs), with 5 being new ones (656, 657, 658, 659, and 660). Predominant STs were ST412 and ST478, all belonging to CC17, except ST658. This is the first report of the ST78 in Brazil. (c) 2012 Elsevier Inc. All rights reserved.

Molecular measurement of BCR-ABL transcript variations in chronic myeloid leukemia patients in cytogenetic remission

Abstract Background The monitoring of BCR-ABL transcript levels by real-time quantitative polymerase chain reaction (RT-qPCR) has become important to assess minimal residual disease (MRD) and standard of care in the treatment of chronic myeloid leukemia (CML). In this study, we performed a prospective, sequential analysis using RT-qPCR monitoring of BCR-ABL gene rearrangements in blood samples from 91 CML patients in chronic phase (CP) who achieved complete cytogenetic remission (CCyR) and major molecular remission (MMR) throughout imatinib treatment. Methods The absolute level of BCR-ABL transcript from peripheral blood was serially measured every 4 to 12 weeks by RT-qPCR. Only level variations > 0.5%, according to the international scale, was considered positive. Sequential cytogenetic analysis was also performed in bone marrow samples from all patients using standard protocols. Results Based on sequential analysis of BCR-ABL transcripts, the 91 patients were divided into three categories: (A) 57 (62.6%) had no variation on sequential analysis; (B) 30 (32.9%) had a single positive variation result obtained in a single sample; and (C) 4 (4.39%) had variations of BCR-ABL transcripts in at least two consecutive samples. Of the 34 patients who had elevated levels of transcripts (group B and C), 19 (55.8%) had a < 1% of BCR-ABL/BCR ratio, 13 (38.2%) patients had a 1% to 10% increase and 2 patients had a >10% increase of RT-qPCR. The last two patients had lost a CCyR, and none of them showed mutations in the ABL gene. Transient cytogenetic alterations in Ph-negative cells were observed in five (5.5%) patients, and none of whom lost CCyR. Conclusions Despite an increase levels of BCR-ABL/BCR ratio variations by RT-qPCR, the majority of CML patients with MMR remained in CCyR. Thus, such single variations should neither be considered predictive of subsequent failure and nor an indication for altering imatinib dose or switching to second generation therapy. Changing of imatinib on the basis of BCR-ABL/BCR% sustained increase and mutational studies is a prudent approach for preserving other therapeutic options in imatinib-resistant patients.

Hepatitis B virus infection in Haemodialysis Centres from Santa Catarina State, Southern Brazil. Predictive risk factors for infection and molecular epidemiology

Abstract Background Patients under haemodialysis are considered at high risk to acquire hepatitis B virus (HBV) infection. Since few data are reported from Brazil, our aim was to assess the frequency and risk factors for HBV infection in haemodialysis patients from 22 Dialysis Centres from Santa Catarina State, south of Brazil. Methods This study includes 813 patients, 149 haemodialysis workers and 772 healthy controls matched by sex and age. Serum samples were assayed for HBV markers and viraemia was detected by nested PCR. HBV was genotyped by partial S gene sequencing. Univariate and multivariate statistical analyses with stepwise logistic regression analysis were carried out to analyse the relationship between HBV infection and the characteristics of patients and their Dialysis Units. Results Frequency of HBV infection was 10.0%, 2.7% and 2.7% among patients, haemodialysis workers and controls, respectively. Amidst patients, the most frequent HBV genotypes were A (30.6%), D (57.1%) and F (12.2%). Univariate analysis showed association between HBV infection and total time in haemodialysis, type of dialysis equipment, hygiene and sterilization of equipment, number of times reusing the dialysis lines and filters, number of patients per care-worker and current HCV infection. The logistic regression model showed that total time in haemodialysis, number of times of reusing the dialysis lines and filters, and number of patients per worker were significantly related to HBV infection. Conclusions Frequency of HBV infection among haemodialysis patients at Santa Catarina state is very high. The most frequent HBV genotypes were A, D and F. The risk for a patient to become HBV positive increase 1.47 times each month of haemodialysis; 1.96 times if the dialysis unit reuses the lines and filters ≥ 10 times compared with haemodialysis units which reuse < 10 times; 3.42 times if the number of patients per worker is more than five. Sequence similarity among the HBV S gene from isolates of different patients pointed out to nosocomial transmission.

Genetic diversity of Leishmania amazonensis strains isolated in northeastern Brazil as revealed by DNA sequencing, PCR-based analyses and molecular karyotyping

Abstract Background Leishmania (Leishmania) amazonensis infection in man results in a clinical spectrum of disease manifestations ranging from cutaneous to mucosal or visceral involvement. In the present study, we have investigated the genetic variability of 18 L. amazonensis strains isolated in northeastern Brazil from patients with different clinical manifestations of leishmaniasis. Parasite DNA was analyzed by sequencing of the ITS flanking the 5.8 S subunit of the ribosomal RNA genes, by RAPD and SSR-PCR and by PFGE followed by hybridization with gene-specific probes. Results ITS sequencing and PCR-based methods revealed genetic heterogeneity among the L. amazonensis isolates examined and molecular karyotyping also showed variation in the chromosome size of different isolates. Unrooted genetic trees separated strains into different groups. Conclusion These results indicate that L. amazonensis strains isolated from leishmaniasis patients from northeastern Brazil are genetically diverse, however, no correlation between genetic polymorphism and phenotype were found.

Cell therapy prevents structural, functional and molecular remodeling of remote non-infarcted myocardium

Background/objectives: Therapy using bone marrow (BM) cells has been tested experimentally and clinically due to the potential ability to restore cardiac function by regenerating lost myocytes or increasing the survival of tissues at risk after myocardial infarction (MI). In this study we aimed to evaluate whether BM-derived mononuclear cell (MNC) implantation can positively influence the post-MI structural remodeling, contractility and Ca(2 +)-handling proteins of the remote non-infarcted tissue in rats. Methods and results: After 48 h of MI induction, saline or BM-MNC were injected. Six weeks later, MI scars were slightly smaller and thicker, and cardiac dilatation was just partially prevented by cell therapy. However, the cardiac performance under hemodynamic stress was totally preserved in the BM-MNC treated group if compared to the untreated group, associated with normal contractility of remote myocardium as analyzed in vitro. The impaired post-rest potentiation of contractile force, associated with decreased protein expression of the sarcoplasmic reticulum Ca2 +-ATPase and phosphorylated-phospholamban and overexpression of Na(+)/Ca(2 +) exchanger, were prevented by BM-MNC, indicating preservation of the Ca(2 +) handling. Finally, pathological changes on remodeled remote tissue such as myocyte hypertrophy, interstitial fibrosis and capillary rarefaction were also mitigated by cell therapy. Conclusions: BM-MNC therapy was able to prevent cardiac structural and molecular remodeling after MI, avoiding pathological changes on Ca(2 +)-handling proteins and preserving contractile behavior of the viable myocardium, which could be the major contributor to the improvements of global cardiac performance after cell transplantation despite that scar tissue still exists.

Molecular origin of charge traps in polyfluorene-based semiconductors

The comprehensive control of morphology and structure is of extreme importance in semiconducting polymers when used as active layers in optoelectronic devices. In the work reported here, a systematic investigation of the structural and dynamical properties of poly(9,9-di-n-octyl-fluorene-alt-benzothiadiazole), known as F8BT, and their correlation with electrical properties is presented when the material is used as an active layer in optoelectronic devices. By means of X-ray diffraction, one observes that in thick layer films (thickness of about 4 μm) grown by drop-cast deposition, a solvent induced crystalline phase exists which evolves to a stable phase as the temperature is raised. This was not observed in thin films (thickness of about 250 nm) prepared by spin-coating within the investigated temperature range. By modeling the current-voltages characteristics of both thick and thin film devices, important information on the influence of crystallization on the trapping states could be drawn. Furthermore, the temperature dependence of the charge carrier mobility was found to be closely related to that of the molecular relaxation processes. The understanding of the nature of such molecular relaxations, measured by solid-state nuclear magnetic resonance methods, allows one to understand the importance of molecular relaxations and microstructure changes on the trap states of the system.