Mycobacterium tuberculosis and whole genome sequencing: a practical guide and online tools available for the clinical microbiologist

Whole genome sequencing (WGS) has the potential to revolutionize the diagnosis of Mycobacterium tuberculosis (MTB) but the lack of bioinformatic expertise among clinical microbiologists is a barrier for adoption. Software products for analysis should be simple, free of charge, able to accept data directly from the sequencer (FASTQ files) and to provide the basic functionalities all-in-one. The main aim of this narrative review is to provide a practical guide for the clinical microbiologist, with little or no practical experience of WGS analysis, with a specific focus on software products tailored made for MTB analysis. With sequencing performed by an external provider, it is now feasible to implement WGS analysis in the routine clinical practice of any microbiology laboratory, with the potential to detect resistance weeks before traditional phenotypic culture methods, but the clinical microbiologist should be aware of the limitations of this approach

The Aeromonas hydrophyla cphA gene: molecular heterogeneity among class B metallo-β-lactamases

An Aeromonas hydrophila gene, named cphA, coding for a carbapenem-hydrolyzing metallo-beta-lactamase, was cloned in Escherichia coli by screening an Aeromonas genomic library for clones able to grow on imipenem-containing medium. From sequencing data, the cloned cphA gene appeared able to code for a polypeptide of 254 amino acids whose sequence includes a potential N-terminal leader sequence for targeting the protein to the periplasmic space. These data were in agreement with the molecular mass of the original Aeromonas enzyme and of the recombinant enzyme produced in E. coli, evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude beta-lactamase preparations followed by renaturation treatment for proteins separated in the gel and localization of protein bands showing carbapenem-hydrolyzing beta-lactamase activity by a modified iodometric technique. The deduced amino acid sequence of the CphA enzyme showed regions of partial homology with both the beta-lactamase II of Bacillus cereus and the CfiA beta-lactamase of Bacteroides fragilis. Sequence homologies were more pronounced in the regions encompassing the amino acid residues known in the enzyme of B. cereus to function as ligand-binding residues for the metal cofactor. The CphA enzyme, however, appeared to share a lower degree of similarity with the two other enzymes, which, in turn, seemed more closely related to each other. These results, therefore, suggest the existence of at least two molecular subclasses within molecular class B metallo-beta-lactamases

Coarsened Lattice Spatial Disorder in the Thermodynamic Limit

In this Rapid Research Note the application of recently introduced [Physica A 277 (2000) 157] entropic measure S_Delta of spatial disorder for systems of finite-sized objects is presented. In the thermodynamic limit the critical behaviour of coarsened lattice model of random two-phase systems is illustrated for certain grain size distributions (GSDs) and chosen parameters. Also the changes of spatial disorder, quantified by S_Delta, between the limit GSDs clearly show that the topological equivalence of the two phases is broken.Comment: 3 pages, 1 figur

High specificity of cphA-encoded metallo-β-lactamase from Aeromonas hydrophila AEO36 for carbapenems and its contribution to β-lactam resistance

The Aeromonas hydrophila AE036 chromosome contains a cphA gene encoding a metallo-beta-lactamase highly active against carbapenem antibiotics. This enzyme was induced in strain AE036 to the same extent by both benzylpenicillin and imipenem. When the cphA gene was inserted into plasmid pACYC184, used to transform Escherichia coli DH5 alpha, the MICs of imipenem, meropenem, and penem HRE664 for recombinant clone DH5 alpha(pAA20R), expressing the Aeromonas metallo-beta-lactamase, were significantly increased, but those of penicillins and cephalosporins were not. When the metallo-beta-lactamase purified from E. coli DH5 alpha(pAA20R) was assayed with several beta-lactam substrates, it hydrolyzed carbapenems but not penicillins or cephalosporins efficiently. These results demonstrate that this metallo-beta-lactamase possesses an unusual spectrum of activity compared with all the other class B enzymes identified so far, being active on penems and carbapenems only. This enzyme may thus contribute to the development of resistance to penems and carbapenems but not other beta-lactams

Whole genome sequencing and prediction of antimicrobial susceptibilities in non-tuberculous mycobacteria

Non-tuberculous mycobacteria (NTM) are opportunistic pathogens commonly causing chronic, pulmonary disease which is notoriously hard to treat. Current treatment for NTM infections involves at least three active drugs (including one macrolide: clarithromycin or azithromycin) over 12 months or longer. At present there are limited phenotypic in vitro drug susceptibility testing options for NTM which are standardised globally. As seen with tuberculosis, whole genome sequencing has the potential to transform drug susceptibility testing in NTM, by utilising a genotypic approach. The Comprehensive Resistance Prediction for Tuberculosis is a database used to predict Mycobacterium tuberculosis resistance: at present there are no similar databases available to accurately predict NTM resistance. Recent studies have shown concordance between phenotypic and genotypic NTM resistance results. To benefit from the advantages of whole genome sequencing, further advances in resistance prediction need to take place, as well as there being better information on novel drug mutations and an understanding of the impact of whole genome sequencing on NTM treatment outcomes

Patient Specific Diagnostics for cardiovascular diseases based on diagnostic imaging: an application to the aneurism of the ascending aorta

In the framework of a collaboration between clinicians and engineers (namely, the Department of Radiology of the Brotzu Hospital in Cagliari and the group of experimental hydraulics at DICAAR - University of Cagliari), methodologies for the application of the in vitro study of the cardiovascular fluid mechanics to the support of the physical interpretation of the diagnostic imaging data are being tested. To this aim, we set up a mock-loop able to reproduce the physiologic pulsatile flow and designed to host a replica of aortic root made of transparent silicon rubber. Then, we developed a procedure to obtain a transparent and compliant replica of a patient specific ascending aorta from diagnostic images. The patient specific aorta model can be inserted in the mock-loop to study the fluid dynamics by means of particle image velocimetry techniques. We compared the flow in three cases, corresponding to physiological conditions, mild and severe aortic root dilation, observing significant differences in the redirection of the transvalvular jet and vortex evolution in the aortic flow. The observed fluid dynamics differences may have relevant implications on the thromboembolism and vascular tissue damage potential

Symbol–Relation Grammars: A Formalism for Graphical Languages

AbstractA common approach to the formal description of pictorial and visual languages makes use of formal grammars and rewriting mechanisms. The present paper is concerned with the formalism of Symbol–Relation Grammars (SR grammars, for short). Each sentence in an SR language is composed of a set of symbol occurrences representing visual elementary objects, which are related through a set of binary relational items. The main feature of SR grammars is the uniform way they use context-free productions to rewrite symbol occurrences as well as relation items. The clearness and uniformity of the derivation process for SR grammars allow the extension of well-established techniques of syntactic and semantic analysis to the case of SR grammars. The paper provides an accurate analysis of the derivation mechanism and the expressive power of the SR formalism. This is necessary to fully exploit the capabilities of the model. The most meaningful features of SR grammars as well as their generative power are compared with those of well-known graph grammar families. In spite of their structural simplicity, variations of SR grammars have a generative power comparable with that of expressive classes of graph grammars, such as the edNCE and the N-edNCE classes

Irritable bowel syndrome and gluten-related disorders

Background: Irritable bowel syndrome (IBS) is frequently associated with celiac disease (CD) and nonceliac gluten/wheat sensitivity (NCGS/NCWS), but epidemiological and pathophysiological aspects are still unclear. Furthermore, a gluten-free diet (GFD) can positively influence IBS symptoms. Methods: A comprehensive online search for IBS related to CD, NCGS and GFD was made using the Pubmed, Medline and Cochrane databases. Results: Although a systematic screening for CD in IBS is not recommended, CD prevalence can be increased in diarrhea-predominant IBS patients. On the other hand, IBS symptoms can be persistent in treated CD patients, and their prevalence tends to decrease on a GFD. IBS symptoms may overlap and be similar to those associated to nonceliac gluten and/or wheat sensitivity. Increased gut permeability could explain the gluten/wheat effects in IBS patients. Finally, a GFD could improve symptoms in a subgroup of IBS patients. Conclusions: The possible interplay between IBS and gluten-related disorders represents a scientifically and clinically challenging issue. Further studies are needed to confirm these data and better clarify the involved pathophysiological mechanisms