Incidence of WHO stage 3 and 4 conditions following initiation of Anti-Retroviral Therapy in resource limited settings

To determine the incidence of WHO clinical stage 3 and 4 conditions during early anti-retroviral therapy (ART) in resource limited settings (RLS)

Agr polymorphisms and exotoxin production in Staphylococcus aureus

Staphylococcus aureus is a highly successful human pathogen capable of colonising and spreading easily between humans while causing a wide range of infections, including life threatening bacteraemias, endocarditis and pneumonia. Virulence gene expression is regulated primarily by the staphylococcal accessory gene regulator (agr) in a cell density-dependent manner termed quorum sensing. Strains of S. aureus, particularly community-associated methicillin resistant S. aureus (CA-MRSA), carrying the genes for a pore forming exotoxin, the Panton Valentine Leukocidin (PVL), have been spreading worldwide over the last 10 to 15 years. As high level production of exotoxins has been implicated in the success of the CA-MRSA clone USA300, a collection of PVL producing clinical isolates from Nottingham were studied with the aim of understanding why PVL production might vary between strains. Sequence typing of the Nottingham strain collection revealed that high PVL producing ST22 agr group 1 and low PVL producing ST30 agr group 3 strains were the most common types. PVL production was stimulated by addition of the type specific exogenous autoinducing peptide (AIP), which activates the agr sensor AgrC, and inhibited by the AgrC antagonist (ala5)AIP-1 if added before a critical cell population density was reached. Analysis of the pvl promoter identified predicted binding sites for RNA polymerase and the SarA protein family of regulators. Promoter pull down experiments confirmed the binding of several staphylococcal regulators to both the agr and pvl promoters including SarA, SarS, Rot and MgrA indicating that these are likely to play a role in the regulation of PVL production. Analysis of the agr locus of high and low PVL producing strains found that a low PVL producing ST22 agr group 1 strain TS13 had a single nucleotide polymorphism (SNP) conferring an N267I substitution in the cytoplasmic domain region of agrC not present in a high PVL producing ST22 strain TS14. Whole genome sequencing of these strains revealed them to be closely related to each other, differing by less than 200 SNPs across their core genomes. While not possessing an SCCmec element, they carried phages encoding pvl and the immune evasion complex (IEC) comprising staphylokinase, the chemotaxis inhibitory protein (CHIPS) and the staphylococcal complement inhibitor (SCIN). AgrC N267I reduced sensitivity to AIP in a bioluminescent reporter for agr which responds to but does not produce AIP. Introducing AgrC N267I into an agr reporter which both produces and senses AIP resulted in delayed autoinduction. This appeared to explain the delay in AIP autoinduction observed in TS13 as compared with TS14 with resulting low PVL production. Other naturally occurring agrC cytoplasmic mutations including T247I and I311T/A343T reduced AIP sensitivity and were associated with delayed autoinduction and reduced exotoxin production. PVL production is closely regulated by agr with expression mediated by several staphylococcal regulators in a cell population density dependent manner. Mutations in agrC occur naturally, delay autoinduction, can have a marked impact on exotoxin production and may be a form of adaptation to niches where high level agr expression is not required

Agr polymorphisms and exotoxin production in Staphylococcus aureus

Staphylococcus aureus is a highly successful human pathogen capable of colonising and spreading easily between humans while causing a wide range of infections, including life threatening bacteraemias, endocarditis and pneumonia. Virulence gene expression is regulated primarily by the staphylococcal accessory gene regulator (agr) in a cell density-dependent manner termed quorum sensing. Strains of S. aureus, particularly community-associated methicillin resistant S. aureus (CA-MRSA), carrying the genes for a pore forming exotoxin, the Panton Valentine Leukocidin (PVL), have been spreading worldwide over the last 10 to 15 years. As high level production of exotoxins has been implicated in the success of the CA-MRSA clone USA300, a collection of PVL producing clinical isolates from Nottingham were studied with the aim of understanding why PVL production might vary between strains. Sequence typing of the Nottingham strain collection revealed that high PVL producing ST22 agr group 1 and low PVL producing ST30 agr group 3 strains were the most common types. PVL production was stimulated by addition of the type specific exogenous autoinducing peptide (AIP), which activates the agr sensor AgrC, and inhibited by the AgrC antagonist (ala5)AIP-1 if added before a critical cell population density was reached. Analysis of the pvl promoter identified predicted binding sites for RNA polymerase and the SarA protein family of regulators. Promoter pull down experiments confirmed the binding of several staphylococcal regulators to both the agr and pvl promoters including SarA, SarS, Rot and MgrA indicating that these are likely to play a role in the regulation of PVL production. Analysis of the agr locus of high and low PVL producing strains found that a low PVL producing ST22 agr group 1 strain TS13 had a single nucleotide polymorphism (SNP) conferring an N267I substitution in the cytoplasmic domain region of agrC not present in a high PVL producing ST22 strain TS14. Whole genome sequencing of these strains revealed them to be closely related to each other, differing by less than 200 SNPs across their core genomes. While not possessing an SCCmec element, they carried phages encoding pvl and the immune evasion complex (IEC) comprising staphylokinase, the chemotaxis inhibitory protein (CHIPS) and the staphylococcal complement inhibitor (SCIN). AgrC N267I reduced sensitivity to AIP in a bioluminescent reporter for agr which responds to but does not produce AIP. Introducing AgrC N267I into an agr reporter which both produces and senses AIP resulted in delayed autoinduction. This appeared to explain the delay in AIP autoinduction observed in TS13 as compared with TS14 with resulting low PVL production. Other naturally occurring agrC cytoplasmic mutations including T247I and I311T/A343T reduced AIP sensitivity and were associated with delayed autoinduction and reduced exotoxin production. PVL production is closely regulated by agr with expression mediated by several staphylococcal regulators in a cell population density dependent manner. Mutations in agrC occur naturally, delay autoinduction, can have a marked impact on exotoxin production and may be a form of adaptation to niches where high level agr expression is not required

Timing Is Everything:Impact of Naturally Occurring Staphylococcus aureus AgrC Cytoplasmic Domain Adaptive Mutations on Autoinduction

Mutations in the polymorphic Staphylococcus aureus agr locus responsible for quorum sensing (QS) dependent virulence gene regulation occur frequently during host adaptation. In two genomically closely related S. aureus clinical isolates exhibiting marked differences in Pantone-Valentine leukocidin production, a mutation conferring an N267I substitution was identified in the cytoplasmic domain of the QS sensor kinase, AgrC. This natural mutation delayed the onset and accumulation of auto-inducing peptide (AIP) and showed reduced responsiveness to exogenous AIPs. Other S. aureus strains harbouring naturally occurring AgrC cytoplasmic domain mutations were identified including T247I, I311T, A343T, L245S and F264C. These mutations were associated with reduced cytotoxicity, delayed/reduced AIP production and impaired sensitivity to exogenous AIP. Molecular dynamics simulations were used to model the AgrC cytoplasmic domain conformational changes arising. While mutations were localised in different parts of the C-terminal domain, their impact on molecular structure was manifested by twisting of the leading helical hairpin α1-α2, accompanied by repositioning of the H-box and G-box along with closure of the flexible loop connecting the two and occlusion of the ATP-binding site. Such conformational rearrangements of key functional subdomains in these mutants highlight the cooperative response of molecular structure involving dimerization, ATP binding and phosphorylation, as well as the binding site for the downstream response element AgrA. These appear to increase the threshold for agr activation via AIP-dependent autoinduction so reducing virulence and maintaining S. aureus in an agr-down-regulated ‘colonization’ mode

Metagenomics reveals impact of geography and acute diarrheal disease on the Central Indian human gut microbiome

© 2020, © 2020 The Author(s). Published with license by Taylor & Francis Group, LLC. Background: The Central Indian gut microbiome remains grossly understudied. Herein, we sought to investigate the burden of antimicrobial resistance and diarrheal diseases, particularly Clostridioides difficile, in rural-agricultural and urban populations in Central India, where there is widespread unregulated antibiotic use. We utilized shotgun metagenomics to comprehensively characterize the bacterial and viral fractions of the gut microbiome and their encoded functions in 105 participants. Results: We observed distinct rural-urban differences in bacterial and viral populations, with geography exhibiting a greater influence than diarrheal status. Clostridioides difficile disease was more commonly observed in urban subjects, and their microbiomes were enriched in metabolic pathways relating to the metabolism of industrial compounds and genes encoding resistance to 3rd generation cephalosporins and carbapenems. By linking phages present in the microbiome to their bacterial hosts through CRISPR spacers, phage variation could be directly related to shifts in bacterial populations, with the auxiliary metabolic potential of rural-associated phages enriched for carbon and amino acid energy metabolism. Conclusions: We report distinct differences in antimicrobial resistance gene profiles, enrichment of metabolic pathways and phage composition between rural and urban populations, as well as a higher burden of Clostridioides difficile disease in the urban population. Our results reveal that geography is the key driver of variation in urban and rural Indian microbiomes, with acute diarrheal disease, including C. difficile disease exerting a lesser impact. Future studies will be required to understand the potential role of dietary, cultural, and genetic factors in contributing to microbiome differences between rural and urban populations

Oncology Fellows' Career Plans, Expectations, and Well-Being: Do Fellows Know What They Are Getting Into?

To evaluate the career plans, professional expectations, and well-being of oncology fellows compared with actual experiences of practicing oncologists

A diverse Late Cretaceous vertebrate tracksite from the Winton Formation of Queensland, Australia

The Upper Cretaceous ‘upper’ Winton Formation of Queensland, Australia is world famous for hosting Dinosaur Stampede National Monument at Lark Quarry Conservation Park, a somewhat controversial tracksite that preserves thousands of tridactyl dinosaur tracks attributed to ornithopods and theropods. Herein, we describe the Snake Creek Tracksite, a new vertebrate ichnoassemblage from the ‘upper’ Winton Formation, originally situated on Karoola Station but now relocated to the Australian Age of Dinosaurs Museum of Natural History. This site preserves the first sauropod tracks reported from eastern Australia, a small number of theropod and ornithopod tracks, the first fossilised crocodyliform and ?turtle tracks reported from Australia, and possible lungfish and actinopterygian feeding traces. The sauropod trackways are wide-gauge, with manus tracks bearing an ungual impression on digit I, and anteriorly tapered pes tracks with straight or concave forward posterior margins. These tracks support the hypothesis that at least one sauropod taxon from the ‘upper’ Winton Formation retained a pollex claw (previously hypothesised for Diamantinasaurus matildae based on body fossils). Many of the crocodyliform trackways indicate underwater walking. The Snake Creek Tracksite reconciles the sauropod-, crocodyliform-, turtle-, and lungfish-dominated body fossil record of the ‘upper’ Winton Formation with its heretofore ornithopod- and theropod-dominated ichnofossil record

Examining diabetic heel ulcers through an ecological lens: microbial community dynamics associated with healing and infection

Purpose: While some micro-organisms, such as Staphylococcus aureus, are clearly implicated in causing tissue damage in diabetic foot ulcers (DFUs), our knowledge of the contribution of the entire microbiome to clinical outcomes is limited. We profiled the microbiome of a longitudinal sample series of 28 people with diabetes and DFUs of the heel in an attempt to better characterize the relationship between healing, infection and the microbiome.Methodology: In total, 237 samples were analysed from 28 DFUs, collected at fortnightly intervals for 6 months or until healing. Microbiome profiles were generated by 16S rRNA gene sequence analysis, supplemented by targeted nanopore sequencing.Result/Key findings: DFUs which failed to heal during the study period (20/28, 71.4 %) were more likely to be persistently colonized with a heterogeneous community of micro-organisms including anaerobes and Enterobacteriaceae (log-likelihood ratio 9.56, P=0.008). During clinically apparent infection, a reduction in the diversity of micro-organisms in a DFU was often observed due to expansion of one or two taxa, with recovery in diversity at resolution. Modelling of the predicted species interactions in a single DFU with high diversity indicated that networks of metabolic interactions may exist that contribute to the formation of stable communities.Conclusion: Longitudinal profiling is an essential tool for improving our understanding of the microbiology of chronic wounds, as community dynamics associated with clinical events can only be identified by examining changes over multiple time points. The development of complex communities, particularly involving Enterobacteriaceae and strict anaerobes, may be contributing to poor outcomes in DFUs and requires further investigation

Metagenomics Reveals Impact of Geography and Acute Diarrhoeal Disease on the Central Indian Human Gut Microbiome

Background: The Central Indian gut microbiome remains grossly understudied. Herein, we sought to investigate the burden of antimicrobial resistance and diarrhoeal diseases, particularly Clostridioides difficile, in rural-agricultural and urban populations in Central India, where there is widespread unregulated antibiotic use. We utilised shotgun metagenomics to comprehensively characterise the bacterial and viral fractions of the gut microbiome and their encoded functions in 105 participants. Results: We observed distinct rural-urban differences in bacterial and viral populations, with geography exhibiting a greater influence than diarrhoeal status. Clostridioides difficile disease was more commonly observed in urban subjects, and their microbiomes were enriched in metabolic pathways relating to the metabolism of industrial compounds and genes encoding resistance to 3rd generation cephalosporins and carbapenems. By linking phages present in the microbiome to their bacterial hosts through CRISPR spacers, phage variation could be directly related to shifts in bacterial populations, with the auxiliary metabolic potential of rural-associated phages enriched for carbon and amino acid energy metabolism.Conclusions: We report distinct differences in antimicrobial resistance gene profiles, enrichment of metabolic pathways and phage composition between rural and urban populations, as well as a higher burden of Clostridioides difficile disease in the urban population. Our results reveal that geography is the key driver of variation in urban and rural Indian microbiomes, with acute diarrhoeal disease, including C. difficile disease exerting a lesser impact. Future studies will be required to understand the potential role of dietary, cultural and genetic factors in contributing to microbiome differences between rural and urban populations