Real-time whole-genome sequencing for routine typing, surveillance, and outbreak detection of verotoxigenic Escherichia coli.

Fast and accurate identification and typing of pathogens are essential for effective surveillance and outbreak detection. The current routine procedure is based on a variety of techniques, making the procedure laborious, time-consuming, and expensive. With whole-genome sequencing (WGS) becoming cheaper, it has huge potential in both diagnostics and routine surveillance. The aim of this study was to perform a real-time evaluation of WGS for routine typing and surveillance of verocytotoxin-producing Escherichia coli (VTEC). In Denmark, the Statens Serum Institut (SSI) routinely receives all suspected VTEC isolates. During a 7-week period in the fall of 2012, all incoming isolates were concurrently subjected to WGS using IonTorrent PGM. Real-time bioinformatics analysis was performed using web-tools (www.genomicepidemiology.org) for species determination, multilocus sequence type (MLST) typing, and determination of phylogenetic relationship, and a specific VirulenceFinder for detection of E. coli virulence genes was developed as part of this study. In total, 46 suspected VTEC isolates were characterized in parallel during the study. VirulenceFinder proved successful in detecting virulence genes included in routine typing, explicitly verocytotoxin 1 (vtx1), verocytotoxin 2 (vtx2), and intimin (eae), and also detected additional virulence genes. VirulenceFinder is also a robust method for assigning verocytotoxin (vtx) subtypes. A real-time clustering of isolates in agreement with the epidemiology was established from WGS, enabling discrimination between sporadic and outbreak isolates. Overall, WGS typing produced results faster and at a lower cost than the current routine. Therefore, WGS typing is a superior alternative to conventional typing strategies. This approach may also be applied to typing and surveillance of other pathogens

Original Article

Objective: Glucagon is well known to regulate blood glucose but may be equally important for amino acid metabolism. Plasma levels of amino acids are regulated by glucagon-dependent mechanism(s), while amino acids stimulate glucagon secretion from alpha cells, completing the recently described liver-alpha cell axis. The mechanisms underlying the cycle and the possible impact of hepatic steatosis are unclear. Methods: We assessed amino acid clearance in vivo in mice treated with a glucagon receptor antagonist (GRA), transgenic mice with 95% reduction in alpha cells, and mice with hepatic steatosis. In addition, we evaluated urea formation in primary hepatocytes from ob/ob mice and humans, and we studied acute metabolic effects of glucagon in perfused rat livers. We also performed RNA sequencing on livers from glucagon receptor knock-out mice and mice with hepatic steatosis. Finally, we measured individual plasma amino acids and glucagon in healthy controls and in two independent cohorts of patients with biopsy-verified non-alcoholic fatty liver disease (NAFLD). Results: Amino acid clearance was reduced in mice treated with GRA and mice lacking endogenous glucagon (loss of alpha cells) concomitantly with reduced production of urea. Glucagon administration markedly changed the secretion of rat liver metabolites and within minutes increased urea formation in mice, in perfused rat liver, and in primary human hepatocytes. Transcriptomic analyses revealed that three genes responsible for amino acid catabolism (Cps1, Slc7a2, and Slc38a2) were downregulated both in mice with hepatic steatosis and in mice with deletion of the glucagon receptor. Cultured ob/ob hepatocytes produced less urea upon stimulation with mixed amino acids, and amino acid clearance was lower in mice with hepatic steatosis. Glucagon-induced ureagenesis was impaired in perfused rat livers with hepatic steatosis. Patients with NAFLD had hyperglucagonemia and increased levels of glucagonotropic amino acids, including alanine in particular. Both glucagon and alanine levels were reduced after diet-induced reduction in Homeostatic Model Assessment for Insulin Resistance (HOMA-IR, a marker of hepatic steatosis). Conclusions: Glucagon regulates amino acid metabolism both non-transcriptionally and transcriptionally. Hepatic steatosis may impair glucagon-dependent enhancement of amino acid catabolism. (C) 2020 The Author(s). Published by Elsevier GmbH

Phylogenetic and pathotype analysis of Escherichia coli swine isolates from Southern Brazil

The current study evaluated the presence of virulence factors by a multiplex PCR technique and then phylogenetically classified the studied strains into groups A, B1, B2 and D, according to Clermont et al. (2000), in 152 intestinal and extraintestinal swine isolates of Escherichia coli. Seventy seven isolates tested were positive for virulence factors. Phylogenetic characterization placed 21 samples into group A, 65 into B1, 19 into B2 and 47 into D. Fourteen urine samples were classified as uropathogenic E. coli (UPEC), nine were both UPEC and enterotoxigenic E. coli (ETEC) and four were ETEC only. The most common phylogenetic classifications were B1 and D groups. Of the analyzed fecal samples, 25 were classified as ETEC. Phylogenetically, the group of higher occurrence was B1, followed by B2, A and D. For the small intestine samples, 20 were classified as ETEC. Phylogenetic analysis found groups B1 and A to be the most commons in these samples. Six isolated tissue samples were classified as ETEC and most of them were designated as group D by phylogenetic classification. The phylogenetic analysis could be employed in veterinary laboratories in the E. coli isolates screening, including the possibility of vaccine strain selection and epidemiological searches

Sitagliptin reduces cardiac apoptosis, hypertrophy and fibrosis primarily by insulin-dependent mechanisms in experimental type-II diabetes. Potential roles of GLP-1 isoforms

Background:Myocardial fibrosis is a key process in diabetic cardiomyopathy. However, their underlying mechanisms have not been elucidated, leading to a lack of therapy. The glucagon-like peptide-1 (GLP-1) enhancer, sitagliptin, reduces hyperglycemia but may also trigger direct effects on the heart.Methods:Goto-Kakizaki (GK) rats developed type-II diabetes and received sitagliptin, an anti-hyperglycemic drug (metformin) or vehicle (n=10, each). After cardiac structure and function assessment, plasma and left ventricles were isolated for biochemical studies. Cultured cardiomyocytes and fibroblasts were used for in vitro assays.Results:Untreated GK rats exhibited hyperglycemia, hyperlipidemia, plasma GLP-1 decrease, and cardiac cell-death, hypertrophy, fibrosis and prolonged deceleration time. Moreover, cardiac pro-apoptotic/necrotic, hypertrophic and fibrotic factors were up-regulated. Importantly, both sitagliptin and metformin lessened all these parameters. In cultured cardiomyocytes and cardiac fibroblasts, high-concentration of palmitate or glucose induced cell-death, hypertrophy and fibrosis. Interestingly, GLP-1 and its insulinotropic-inactive metabolite, GLP-1(9-36), alleviated these responses. In addition, despite a specific GLP-1 receptor was only detected in cardiomyocytes, GLP-1 isoforms attenuated the pro-fibrotic expression in cardiomyocytes and fibroblasts. In addition, GLP-1 receptor signalling may be linked to PPARδ activation, and metformin may also exhibit anti-apoptotic/necrotic and anti-fibrotic direct effects in cardiac cells.Conclusions:Sitagliptin, via GLP-1 stabilization, promoted cardioprotection in type-II diabetic hearts primarily by limiting hyperglycemia e hyperlipidemia. However, GLP-1 and GLP-1(9-36) promoted survival and anti-hypertrophic/fibrotic effects on cultured cardiac cells, suggesting cell-autonomous cardioprotective actionsThis work was supported by national funding from Ministerio de Educación y Ciencia (SAF2009-08367), Comunidad de Madrid (CCG10-UAM/ BIO-5289), and a unrestricted grant from by Merck/MS

Transcriptomics and adaptive genomics of the asymptomatic bacteriuria Escherichia coli strain 83972

Escherichia coli strains are the major cause of urinary tract infections in humans. Such strains can be divided into virulent, UPEC strains causing symptomatic infections, and asymptomatic, commensal-like strains causing asymptomatic bacteriuria, ABU. The best-characterized ABU strain is strain 83972. Global gene expression profiling of strain 83972 has been carried out under seven different sets of environmental conditions ranging from laboratory minimal medium to human bladders. The data reveal highly specific gene expression responses to different conditions. A number of potential fitness factors for the human urinary tract could be identified. Also, presence/absence data of the gene expression was used as an adaptive genomics tool to model the gene pool of 83972 using primarily UPEC strain CFT073 as a scaffold. In our analysis, 96% of the transcripts filtered present in strain 83972 can be found in CFT073, and genes on six of the seven pathogenicity islands were expressed in 83972. Despite the very different patient symptom profiles, the two strains seem to be very similar. Genes expressed in CFT073 but not in 83972 were identified and can be considered as virulence factor candidates. Strain 83972 is a deconstructed pathogen rather than a commensal strain that has acquired fitness properties

Genomic Characterization of Enteroaggregative Escherichia coli From Children in Mali

Background. Enteroaggregative Escherichia coli (EAEC) is a cause of epidemic and sporadic diarrhea, yet its role as an enteric pathogen is not fully understood