Molecular epidemiology of clinical isolates of ampc producing Klebsiella pneumoniae

Purpose: AmpC producing K. pneumoniae have been increasingly reported from India but epidemiological studies are lacking. In the present study, molecular epidemiology of extended-spectrum AmpC beta-lactamases (ESACs) producing clinical isolates of K. pneumoniae prevalent in our hospital was studied. Methods: Fifty-one non-repeat, consecutive, clinical isolates of K. pneumoniae producing AmpC enzymes, were subjected to whole cell protein profile analysis (SDS-PAGE) and ribotyping. The antimicrobial susceptibility was determined using standard disk diffusion technique. The isolates showing decreased susceptibility to cefoxitin (<18 mm) or cefotetan (<16 mm) were subjected to modified three-dimensional test for detection of AmpC enzyme. Results: Six different types of protein profiles were observed. Ribotyping could further discriminate between the strains that were clustered by protein fingerprinting. Twelve different ribo-patterns were identified. Ribotyping was found to have a better Discriminatory Index (0.98) than that of SDS-PAGE (0.78). Of the 26 isolates that showed decreased susceptibility to cefoxitin and/or cefotetan 13 isolates were found to harbour AmpC enzyme. Conclusions: The study demonstrated the usefulness of SDS-PAGE whole cell protein profile analysis and ribotyping to identify the clonality of the ESACs isolates, the latter having a higher discriminatory power. The presence of ESACs isolates in the community as well as in hospital settings emphasizes the need for regular monitoring of antimicrobial resistance.     

Plasma metabolic fingerprinting of childhood obesity by GC/MS in conjunction with multivariate statistical analysis

Metabolic fingerprinting is a powerful tool for exploring systemic metabolic perturbations and potential biomarkers, thus may shed light on the pathophysiological mechanism of diseases. In this work, a new strategy of metabolic fingerprinting was proposed to exploit the disturbances of metabolic patterns and biomarker candidates of childhood obesity. Plasma samples from children with normal weight, overweight and obesity were first profiled by GC/MS. ULDA (uncorrelated linear discriminant analysis) then revealed that the metabolic patterns of the three groups were different. Furthermore, several metabolites, say isoleucine, glyceric acid, serine, 2,3,4-trihydroxybutyric acid and phenylalanine were screened as potential biomarkers of childhood obesity by both ULDA and CCA (canonical correlation analysis). CCA also shows satisfactory correlation between the metabolic patterns and clinical parameters, and the results further suggest that WHR (waist–hip ratio) together with TG (total triglycerides), TC (total cholesterol), HDL (high density lipoprotein) and LDL (low density lipoprotein) were the most important parameters which are associated closely with the metabolic perturbations of childhood obesity, so as to be paid more attention for dealing with metabolic disturbances of childhood obesity in clinical practice rather than regularly monitored BMI (body-mass index). The results have demonstrated that the proposed metabolic fingerprinting approach may be a useful tool for discovering metabolic abnormalities and possible biomarkers for childhood obesity.     

Brain tumor cell line authentication,an efficient alternative to capillary electrophoresis by using a microfluidics-based system

Background

The current method for cell line authentication is genotyping based on short tandem repeat (STR)–PCR involving coamplification of a panel of STR loci by multiplex PCR and downstream fragment length analysis (FLA), usually performed by capillary electrophoresis. FLA by capillary electrophoresis is time-consuming and can be expensive, as the facilities are generally not accessible for many research laboratories.

Methods

In the present study, a microfluidic electrophoresis system, the Agilent 2100 Bioanalyzer, was used to analyze the STR-PCR fragments from 10 human genomic loci of a number of human cell lines, including 6 gliomas, 1 astrocyte, 1 primary lung cancer, 1 lung brain metastatic cancer, and 1 rhabdomyosarcoma; and this was compared with the standard method, that is, capillary electrophoresis, using the Applied Biosystems 3130xl Genetic Analyzer.

Results

The microfluidic electrophoresis method produced highly reproducible results with good sensitivity in sizing of multiple PCR fragments, and each cell line demonstrated a unique DNA profile. Furthermore, DNA fingerprinting of samples from 5 different passage numbers of the same cell line showed excellent reproducibility when FLA was performed with the Bioanalyzer, indicating that no cross-contamination had occurred during the culture period.

Conclusion

This novel application provides a straightforward and cost-effective alternative to STR-based cell line authentication. In addition, this application would be of great value for cell bank repositories to maintain and distribute precious cell lines.     

结核分枝杆菌广泛耐药株与耐多药株菌体蛋白质组学比较研究

目的比较和分析结核分枝杆菌广泛耐药（XDR）与耐多药（MDR）菌株在菌体蛋白质表达水平上的差异,并寻找与XDR相关的蛋白质点。方法利用双向凝胶电泳分离XDR、MDR菌株以及H37Rv标准菌株菌体蛋白质,通过计算机图像分析软件分析各菌株之间在菌体蛋白质表达水平上的差异性,并进行质谱分析。结果 XDR与MDR以及H37Rv菌株之间在菌体蛋白质表达水平上存在明显差异;与XDR相关的28个差异（新增、缺失、上调、下调）表达的蛋白质点中,质谱分析鉴定出5个相关的蛋白质点。结论结核分枝杆菌XDR与MDR菌株在菌体蛋白质表达水平上存在明显差异,为进一步研究XDR菌株耐药机理奠定了基础。     

Nontuberculous mycobacteria from household plumbing of patients with nontuberculous mycobacteria disease

To determine whether plumbing could be a source of nontuberculous mycobacteria (NTM) infection, during 2007-2009 I isolated NTM from samples from household water systems of NTM patients. Samples from 22/37 (59%) households and 109/394 (28%) total samples yielded NTM. Seventeen (46%) of the 37 households yielded ≥1 Mycobacterium spp. isolate of the same species as that found in the patient; in 7 of those households, the patient isolate and 1 plumbing isolate exhibited the same repetitive sequence-based PCR DNA fingerprint. Households with water heater temperatures ≤125 degrees C (≤50 degrees C) were significantly more likely to harbor NTM compared with households with hot water temperatures ≥130 degrees F (≥55 degrees C) (p = 0.0107). Although households with water from public or private water systems serving multiple households were more likely to have NTM (19/27, 70%) compared with households with a well providing water to only 1 household (5/12, 42%), that difference was not significant (p = 0.1532).     

MR fingerprinting ASL: Sequence characterization and comparison with dynamic susceptibility contrast (DSC) MRI

MR Fingerprinting (MRF)‐based Arterial‐Spin‐Labeling (ASL) has the potential to measure multiple parameters such as cerebral blood flow (CBF), bolus arrival time (BAT), and tissue T₁ in a single scan. However, the previous reports have only demonstrated a proof‐of‐principle of the technique but have not examined the performance of the sequence in the context of key imaging parameters. Furthermore, there has not been a study to directly compare the technique to clinically used perfusion method of dynamic‐susceptibility‐contrast (DSC) MRI. The present report consists of two studies. In the first study (N = 8), we examined the dependence of MRF‐ASL sequence on TR time pattern. Ten different TR patterns with a range of temporal characteristics were examined by both simulations and experiments. The results revealed that there was a significance dependence of the sequence performance on TR pattern (p < 0.001), although there was not a single pattern that provided dramatically improvements. Among the TR patterns tested, a sinusoidal pattern with a period of 125 TRs provided an overall best estimation in terms of spatial consistency. These experimental observations were consistent with those of numerical simulations. In the second study (N = 8), we compared MRF‐ASL results with those of DSC MRI. It was found that MRF‐ASL and DSC MRI provided highly comparable maps of cerebral blood flow (CBF) and bolus‐arrival‐time (BAT), with spatial correlation coefficients of 0.79 and 0.91, respectively. However, in terms of quantitative values, BAT obtained with MRF‐ASL was considerably lower than that from DSC (p < 0.001), presumably because of the differences in tracer characteristics in terms of diffusible versus intravascular tracers. Test–retest assessment of MRF‐ASL MRI revealed that the spatial correlations of parametric maps were 0.997, 0.962, 0.746 and 0.863 for B₁⁺, T₁, CBF, and BAT, respectively. MRF‐ASL is a promising technique for assessing multiple perfusion parameters simultaneously without contrast agent.     

Cardiac cine magnetic resonance fingerprinting for combined ejection fraction,T1 and T2 quantification

This study introduces a technique called cine magnetic resonance fingerprinting (cine‐MRF) for simultaneous T₁, T₂ and ejection fraction (EF) quantification. Data acquired with a free‐running MRF sequence are retrospectively sorted into different cardiac phases using an external electrocardiogram (ECG) signal. A low‐rank reconstruction with a finite difference sparsity constraint along the cardiac motion dimension yields images resolved by cardiac phase. To improve SNR and precision in the parameter maps, these images are nonrigidly registered to the same phase and matched to a dictionary to generate T₁ and T₂ maps. Cine images for computing left ventricular volumes and EF are also derived from the same data. Cine‐MRF was tested in simulations using a numerical relaxation phantom. Phantom and in vivo scans of 19 subjects were performed at 3 T during a 10.9 seconds breath‐hold with an in‐plane resolution of 1.6 x 1.6 mm² and 24 cardiac phases. Left ventricular EF values obtained with cine‐MRF agreed with the conventional cine images (mean bias ?1.0%). Average myocardial T₁ times in diastole/systole were 1398/1391 ms with cine‐MRF, 1394/1378 ms with ECG‐triggered cardiac MRF (cMRF) and 1234/1212 ms with MOLLI; and T₂ values were 30.7/30.3 ms with cine‐MRF, 32.6/32.9 ms with ECG‐triggered cMRF and 37.6/41.0 ms with T₂‐prepared FLASH. Cine‐MRF and ECG‐triggered cMRF relaxation times were in good agreement. Cine‐MRF T₁ values were significantly longer than MOLLI, and cine‐MRF T₂ values were significantly shorter than T₂‐prepared FLASH. In summary, cine‐MRF can potentially streamline cardiac MRI exams by combining left ventricle functional assessment and T₁‐T₂ mapping into one time‐efficient acquisition.     

Simultaneous multislice cardiac magnetic resonance fingerprinting using low rank reconstruction

This study introduces a technique for simultaneous multislice (SMS) cardiac magnetic resonance fingerprinting (cMRF), which improves the slice coverage when quantifying myocardial T_1, T₂, and M₀. The single‐slice cMRF pulse sequence was modified to use multiband (MB) RF pulses for SMS imaging. Different RF phase schedules were used to excite each slice, similar to POMP or CAIPIRINHA, which imparts tissues with a distinguishable and slice‐specific magnetization evolution over time. Because of the high net acceleration factor (R = 48 in plane combined with the slice acceleration), images were first reconstructed with a low rank technique before matching data to a dictionary of signal timecourses generated by a Bloch equation simulation. The proposed method was tested in simulations with a numerical relaxation phantom. Phantom and in vivo cardiac scans of 10 healthy volunteers were also performed at 3 T. With single‐slice acquisitions, the mean relaxation times obtained using the low rank cMRF reconstruction agree with reference values. The low rank method improves the precision in T₁ and T₂ for both single‐slice and SMS cMRF, and it enables the acquisition of maps with fewer artifacts when using SMS cMRF at higher MB factors. With this technique, in vivo cardiac maps were acquired from three slices simultaneously during a breathhold lasting 16 heartbeats. SMS cMRF improves the efficiency and slice coverage of myocardial T₁ and T₂ mapping compared with both single‐slice cMRF and conventional cardiac mapping sequences. Thus, this technique is a first step toward whole‐heart simultaneous T₁ and T₂ quantification with cMRF.