A sensitive method for the recovery of Escherichia coli serogroup O55 including Shiga toxin-producing variants for potential use in outbreaks

AIM: Shiga toxin-producing Escherichia coli (STEC) cause bloody diarrhoea, kidney failure and occasionally death. However, identifying the source of infection caused by STEC other than serogroup O157 is hampered by availability of sensitive methods for detecting these pathogens. In this study we developed novel tools for detecting E. coli O55 that is potentially associated with human outbreaks. METHOD AND RESULTS: Overall specificity of immuno-magnetic separation (IMS) beads coated with anti-O55 serum was good with exception of cross reactivity with E. coli O22 and O23, which was eliminated using an O55 specific PCR. Limit of detection for E. coli O55 using O55-IMS-beads in spiked cattle faeces was on average 50 CFU ml-1 (range 1-90), and improved to <10 CFU ml-1 using the O55 specific PCR, following IMS on samples enriched for 2h with E. coli O55. Application of these tools to test cattle faeces collected on-farm allowed the isolation of O55:H19, which through whole genome sequencing was compared to STEC O55:H7 human outbreak strains. CONCLUSION: These tools provide a sensitive method which could be used to screen samples for STEC O55, whether environmental or human clinical. SIGNIFICANCE AND IMPACT OF THE STUDY: Several human outbreaks reported in England were caused by STEC O55:H7. Tools developed here could assist in identification of the environmental source for these isolates, which has not yet been established. This article is protected by copyright. All rights reserved

Carriage of antimicrobial resistant Escherichia coli in dogs: prevalence, associated risk factors and molecular characteristics

Resistance to antimicrobials, in particular that mediated by extended spectrum β-lactamases (ESBL) and AmpC β-lactamases are frequently reported in bacteria causing canine disease as well as in commensal bacteria, which could be a potential health risk for humans they come into contact with. This cross-sectional study aimed to estimate the prevalence and investigate the molecular characteristics of ESBL and plasmid encoded AmpC (pAmpC)-producing E. coli in the mainland UK vet-visiting canine population and, using responses from detailed questionnaires identify factors associated with their carriage. Faecal samples were cultured for antimicrobial resistant (AMR), ESBL and pAmpC-producing E. coli. A subset of ESBL and pAmpC-producing isolates were subjected to multi-locus sequence typing and DNA microarray analyses. Multivariable logistic regression analysis was used to construct models to identify risk factors associated with multidrug resistant (MDR, resistance to three or more antimicrobial classes), fluoroquinolone resistant, ESBL and AmpC-producing E. coli. AMR E.coli were isolated from 44.8% (n = 260) of samples, with 1.9% and 7.1% of samples carrying ESBL and pAmpC-producing E. coli, respectively. MDR E. coli were identified in 18.3% of samples. Recent use of antimicrobials and being fed raw poultry were both identified as risk factors in the outcomes investigated. A number of virulence and resistance genes were identified, including genes associated with extra-intestinal and enteropathogenic E. coli genotypes. Considering the close contact that people have with dogs, the high levels of AMR E. coli in canine faeces may be a potential reservoir of AMR bacteria or resistance determinants

Molecular characterization of extended spectrum cephalosporin resistant Escherichia coli isolated from livestock and in-contact humans in Southeast Nigeria

The rise in antimicrobial resistance (AMR) in bacteria is reducing therapeutic options for livestock and human health, with a paucity of information globally. To fill this gap, a One-Health approach was taken by sampling livestock on farms (n = 52), abattoir (n = 8), and animal markets (n = 10), and in-contact humans in Southeast Nigeria. Extended spectrum cephalosporin (ESC)-resistant (ESC-R) Escherichia coli was selectively cultured from 975 healthy livestock faecal swabs, and hand swabs from in-contact humans. Antimicrobial susceptibility testing (AST) was performed on all ESC-R E. coli. For isolates showing a multi-drug resistance (MDR) phenotype (n = 196), quantitative real-time PCR (qPCR) was performed for confirmation of extended-spectrum β-lactamase (ESBL) and carbapenemase genes. Whole-genome sequencing (WGS) was performed on a subset (n = 157) for detailed molecular characterisation. The results showed ESC-R E. coli was present in 41.2% of samples, with AST results indicating 48.8% of isolates were phenotypically MDR. qPCR confirmed presence of ESBL genes, with bla(CTX-M) present in all but others in a subset [bla(TEM) (62.8%) and bla(SHV) (0.5%)] of isolates; none harboured transferable carbapenemase genes. Multi-locus sequence typing identified 34 Sequence Types (ST) distributed among different sampling levels; ST196 carrying bla(CTX-M-55) was predominant in chickens. Large numbers of single nucleotide polymorphisms (SNPs) in the core genome of isolates, even within the same clade by phylogenetic analysis, indicated high genetic diversity. AMR genotyping indicated the predominant bla(CTX-M) variant was bla(CTX-M-15) (87.9%), although bla(CTX-M-55), bla(CTX-M-64,) and bla(CTX-M-65) were present; it was notable that bla(CTX-M-1), common in livestock, was absent. Other predominant AMR genes included: sul2, qnrS1, strB, bla(TEM-1b), tetA-v2, and dfrA14, with prevalence varying according to host livestock species. A bla(CTX-M-15) harbouring plasmid from livestock isolates in Ebonyi showed high sequence identity to one from river/sewage water in India, indicating this ESBL plasmid to be globally disseminated, being present beyond the river environment. In conclusion, ESC-R E. coli was widespread in livestock and in-contact humans from Southeast Nigeria. WGS data indicated the isolates were genetically highly diverse, probably representing true diversity of wild type E. coli; they were likely to be MDR with several harbouring bla(CTX-M-15.) Surprisingly, human isolates had highest numbers of AMR genes and pigs the least

Reset of inflammatory priming of joint tissue and reduction of the severity of arthritis flares by bromodomain inhibition

OBJECTIVE: We have recently shown that priming of synovial fibroblasts (SFs) drives arthritis flares. Pathogenic priming of SFs is essentially mediated by epigenetic reprogramming. Bromodomain and extra-terminal motif (BET) proteins translate epigenetic changes into transcription. Here we used a BET inhibitor to target inflammatory tissue priming and reduce flare severity in experimental arthritis. METHODS: BALB/c mice were treated intraperitoneally or locally into the paw with I-BET151, which blocks interaction of BET proteins with acetylated histones. Effect of I-BET151 on acute arthritis and/or inflammatory tissue priming was assessed in a model of repeated injections of monosodium urate crystals or zymosan into the paw. I-BET151 was given either from before arthritis induction, at peak inflammation, or after healing of the first arthritis bout. Transcriptomic (RNA-Seq), epigenomic (ATAC-Seq) and functional analysis (invasion, cytokine production, migration, senescence, metabolic flux) was performed on murine and human SFs treated with I-BET151 in vitro or in vivo. RESULTS: Systemic I-BET151 administration did not affect acute inflammation but abolished inflammatory tissue priming and diminished flare severity in both preventive and therapeutic treatment settings. I-BET151 was also effective when applied locally in the joint. BET inhibition also inhibited osteoclast differentiation, while macrophage activation in the joint was not affected. Flare reduction after BET inhibition was mediated, at least in part, by rolling back the primed transcriptional, metabolic and pathogenic phenotype of SFs. CONCLUSION: Inflammatory tissue priming is dependent on transcriptional regulation by BET proteins, which makes them promising therapeutic targets for preventing arthritis flares in previously affected joints

Mutations in disordered regions can cause disease by creating dileucine motifs

Many disease-causing missense mutations affect intrinsically disordered regions (IDRs) of proteins, but the molecular mechanism of their pathogenicity is enigmatic. Here, we employ a peptide-based proteomic screen to investigate the impact of mutations in IDRs on protein-protein interactions. We find that mutations in disordered cytosolic regions of three transmembrane proteins (GLUT1, ITPR1, and CACNA1H) lead to an increased clathrin binding. All three mutations create dileucine motifs known to mediate clathrin-dependent trafficking. Follow-up experiments on GLUT1 (SLC2A1), the glucose transporter causative of GLUT1 deficiency syndrome, revealed that the mutated protein mislocalizes to intracellular compartments. Mutant GLUT1 interacts with adaptor proteins (APs) in vitro, and knocking down AP-2 reverts the cellular mislocalization and restores glucose transport. A systematic analysis of other known disease-causing variants revealed a significant and specific overrepresentation of gained dileucine motifs in structurally disordered cytosolic domains of transmembrane proteins. Thus, several mutations in disordered regions appear to cause "dileucineopathies.

Molecular characterization of extended spectrum cephalosporin resistant Escherichia coli isolated from livestock and in-contact humans in Southeast Nigeria

The rise in antimicrobial resistance (AMR) in bacteria is reducing therapeutic options for livestock and human health, with a paucity of information globally. To fill this gap, a One-Health approach was taken by sampling livestock on farms (n = 52), abattoir (n = 8), and animal markets (n = 10), and in-contact humans in Southeast Nigeria. Extended spectrum cephalosporin (ESC)-resistant (ESC-R) Escherichia coli was selectively cultured from 975 healthy livestock faecal swabs, and hand swabs from in-contact humans. Antimicrobial susceptibility testing (AST) was performed on all ESC-R E. coli. For isolates showing a multi-drug resistance (MDR) phenotype (n = 196), quantitative real-time PCR (qPCR) was performed for confirmation of extended-spectrum β-lactamase (ESBL) and carbapenemase genes. Whole-genome sequencing (WGS) was performed on a subset (n = 157) for detailed molecular characterisation. The results showed ESC-R E. coli was present in 41.2% of samples, with AST results indicating 48.8% of isolates were phenotypically MDR. qPCR confirmed presence of ESBL genes, with blaCTX-M present in all but others in a subset [blaTEM (62.8%) and blaSHV (0.5%)] of isolates; none harboured transferable carbapenemase genes. Multi-locus sequence typing identified 34 Sequence Types (ST) distributed among different sampling levels; ST196 carrying blaCTX-M-55 was predominant in chickens. Large numbers of single nucleotide polymorphisms (SNPs) in the core genome of isolates, even within the same clade by phylogenetic analysis, indicated high genetic diversity. AMR genotyping indicated the predominant blaCTX-M variant was blaCTX-M-15 (87.9%), although blaCTX-M-55, blaCTX-M-64, and blaCTX-M-65 were present; it was notable that blaCTX-M-1, common in livestock, was absent. Other predominant AMR genes included: sul2, qnrS1, strB, blaTEM-1b, tetA-v2, and dfrA14, with prevalence varying according to host livestock species. A blaCTX-M-15 harbouring plasmid from livestock isolates in Ebonyi showed high sequence identity to one from river/sewage water in India, indicating this ESBL plasmid to be globally disseminated, being present beyond the river environment. In conclusion, ESC-R E. coli was widespread in livestock and in-contact humans from Southeast Nigeria. WGS data indicated the isolates were genetically highly diverse, probably representing true diversity of wild type E. coli; they were likely to be MDR with several harbouring blaCTX-M-15. Surprisingly, human isolates had highest numbers of AMR genes and pigs the least

Introdução: de acordo com donabedian, o estudo do processo de assistência é a melhor forma de melhorar a qualidade do serviço

Telemedicin

Neutral sphingomyelinase mediates the co-morbidity trias of alcohol abuse, major depression and bone defects

Mental disorders are highly comorbid and occur together with physical diseases, which are often considered to arise from separate pathogenic pathways. We observed in alcohol-dependent patients increased serum activity of neutral sphingomyelinase. A genetic association analysis in 456,693 volunteers found associations of haplotypes of SMPD3 coding for NSM-2 (NSM) with alcohol consumption, but also with affective state, and bone mineralisation. Functional analysis in mice showed that NSM controls alcohol consumption, affective behaviour, and their interaction by regulating hippocampal volume, cortical connectivity, and monoaminergic responses. Furthermore, NSM controlled bone–brain communication by enhancing osteocalcin signalling, which can independently supress alcohol consumption and reduce depressive behaviour. Altogether, we identified a single gene source for multiple pathways originating in the brain and bone, which interlink disorders of a mental–physical co-morbidity trias of alcohol abuse—depression/anxiety—bone disorder. Targeting NSM and osteocalcin signalling may, thus, provide a new systems approach in the treatment of a mental–physical co-morbidity trias