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Escherichia coli NfsB has been studied extensively for its potential for cancer gene therapy by reducing the prodrug CB1954 to a cytotoxic derivative. We have previously made several mutants with enhanced activity for the prodrug and characterised their activity in vitro and in vivo. Here, we determine the X-ray structure of our most active triple and double mutants to date, T41Q/N71S/F124T and T41L/N71S. The two mutant proteins have lower redox potentials than wild-type NfsB, and the mutations have lowered activity with NADH so that, in contrast to the wild-type enzyme, the reduction of the enzyme by NADH, rather than the reaction with CB1954, has a slower maximum rate. The structure of the triple mutant shows the interaction between Q41 and T124, explaining the synergy between these two mutations. Based on these structures, we selected mutants with even higher activity. The most active one contains T41Q/N71S/F124T/M127V, in which the additional M127V mutation enlarges a small channel to the active site. Molecular dynamics simulations show that the mutations or reduction of the FMN cofactors of the protein has little effect on its dynamics and that the largest backbone fluctuations occur at residues that flank the active site, contributing towards its broad substrate range.
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Proteínas de Escherichia coli , Neoplasias , Pró-Fármacos , Humanos , Escherichia coli/metabolismo , Pró-Fármacos/química , NAD , Neoplasias/tratamento farmacológico , Oxirredutases , Nitrorredutases/metabolismo , Proteínas de Escherichia coli/genéticaRESUMO
Escherichia coli NfsA and NfsB are founding members of two flavoprotein families that catalyze the oxygen-insensitive reduction of nitroaromatics and quinones by NAD(P)H. This reduction is required for the activity of nitrofuran antibiotics and the enzymes have also been proposed for use with nitroaromatic prodrugs in cancer gene therapy and biocatalysis, but the roles of the proteins in vivo in bacteria are not known. NfsA is NADPH-specific whereas NfsB can also use NADH. The crystal structures of E. coli NfsA and NfsB and several analogs have been determined previously. In our crystal trials, we unexpectedly observed NfsA bound to fumarate. We here present the X-ray structure of the E. coli NfsA-fumarate complex and show that fumarate acts as a weak inhibitor of NfsA but not of NfsB. The structural basis of this differential inhibition is conserved in the two protein families and occurs at fumarate concentrations found in vivo, so impacting the efficacy of these proteins.
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Proteínas de Escherichia coli , Nitrofuranos , Escherichia coli/metabolismo , Oxigênio , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Nitrorredutases/químicaRESUMO
Whether and which university to attend are among the most financially consequential choices most people make. Universities with relatively larger endowments can offer better education experiences, which can drive inequality in students' subsequent outcomes. We first explore three interrelated questions: the current educational inequality across U.S. universities, people's perceptions of this inequality, and their desired inequality. Educational inequality is large: the top 20% of universities have 80% of the total university endowment wealth while the bottom 20% have around 1%. Studies 1 to 3 demonstrated that people underestimate university endowment inequality and desire more equality. These perceptions and ideals were mostly unaffected by contextual factors (e.g., salience of endowment consequences, distribution range) and were not well explained by participants' demographics. Finally, Study 4 revealed that learning about current endowment inequality decreased tolerance of the distribution of university wealth. We discuss the implications of awareness of educational inequality for behaviors and educational policies.
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Administração Financeira , Estudantes , Humanos , Estados Unidos , Universidades , Escolaridade , AprendizagemRESUMO
Nitroreductases activate nitroaromatic antibiotics and cancer prodrugs to cytotoxic hydroxylamines and reduce quinones to quinols. Using steady-state and stopped-flow kinetics, we show that the Escherichia coli nitroreductase NfsA is 20-50 fold more active with NADPH than with NADH and that product release may be rate-limiting. The crystal structure of NfsA with NADP+ shows that a mobile loop forms a phosphate-binding pocket. The nicotinamide ring and nicotinamide ribose are mobile, as confirmed in molecular dynamics (MD) simulations. We present a model of NADPH bound to NfsA. Only one NADP+ is seen bound to the NfsA dimers, and MD simulations show that binding of a second NADP(H) cofactor is unfavourable, suggesting that NfsA and other members of this protein superfamily may have a half-of-sites mechanism.
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Proteínas de Escherichia coli , Pró-Fármacos , Antibacterianos , Escherichia coli/genética , Escherichia coli/metabolismo , Hidroquinonas , Hidroxilaminas , Cinética , NAD/metabolismo , NADP/metabolismo , Niacinamida , Nitrorredutases/química , Nitrorredutases/metabolismo , Fosfatos , Pró-Fármacos/química , Pró-Fármacos/metabolismo , QuinonasRESUMO
Background: IgG4-related disease (IgG4-RD) is an autoimmune condition affecting almost every organ system, with an early inflammatory phase and later fibrotic consequences. Vascular manifestations, particularly, large-vessel involvement in IgG4-RD, are well described. However, important IgG4-related effects on medium-sized arteries and the pericardium are less well recognized. These less frequently reported cardiovascular effects of IgG4-RD include coronary artery stenosis, pericardial disease, cardiac masses, and valvular heart disease. Case summary: This case series focuses on three patients that demonstrate the cardiovascular effects of IgG4-RD and the pitfalls and importance of early diagnosis. Cases 1 and 2 presented with cardiac manifestations prior to more typical organ systems being affected which led to a delay in diagnosis. Case 1 presented with an acute myocardial infarction secondary to IgG4-RD of the coronary arteries and Case 2 presented with pericarditis which progressed to pericardial constriction due to IgG4-RD. Case 3 already had a diagnosis of IgG4-RD from a prior renal biopsy which raised the index of suspicion that his pericardial disease and thoracic mass were also related to IgG4-RD. Discussion: Cardiac manifestations of IgG4-RD remain under-recognized and include coronary artery and pericardial disease. These manifestations often precede more typical manifestations in other organ systems. Recognizing cardiac manifestations of IgG4-RD on cardiac imaging can raise clinical suspicion and act as a catalyst to ascertain a confirmatory diagnosis. Early diagnosis and treatment are crucial to prevent potentially fatal outcomes and irreversible fibrosis.
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The emergence of antimicrobial resistance (AMR) to first- and second-line treatment regimens of enteric fever is a global public-health problem, and routine genomic surveillance to inform clinical and public-health management guidance is essential. Here, we present the prospective analysis of genomic data to monitor trends in incidence, AMR and travel, and assess hierarchical clustering (HierCC) methodology of 1742 isolates of typhoidal salmonellae. Trend analysis of Salmonella Typhi and S. Paratyphi A cases per year increased 48 and 17.3%, respectively, between 2016 and 2019 in England, mainly associated with travel to South Asia. S. Paratyphi B cases have remained stable and are mainly associated with travel to the Middle East and South America. There has been an increase in the number of S. Typhi exhibiting a multidrug-resistant (MDR) profile and the emergence of extensively drug resistant (XDR) profiles. HierCC was a robust method to categorize clonal groups into clades and clusters associated with travel and AMR profiles. The majority of cases that had XDR S. Typhi reported recent travel to Pakistan (94â%) and belonged to a subpopulation of the 4.3.1 (H58) clone (HC5_1452). The phenotypic and genotypic AMR results showed high concordance for S. Typhi and S. Paratyphi A, B and C, with 99.99â% concordance and only three (0.01â%) discordant results out of a possible 23â178 isolate/antibiotic combinations. Genomic surveillance of enteric fever has shown the recent emergence and increase of MDR and XDR S. Typhi strains, resulting in a review of clinical guidelines to improve management of imported infections.
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Antibacterianos , Filogenia , Salmonella typhi/classificação , Salmonella typhi/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/farmacologia , Criança , Pré-Escolar , Farmacorresistência Bacteriana , Inglaterra , Feminino , Genótipo , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Oriente Médio , Paquistão , Salmonella typhi/efeitos dos fármacos , Febre Tifoide/epidemiologia , Febre Tifoide/microbiologia , Adulto JovemRESUMO
NfsA is a dimeric flavoprotein that catalyses the reduction in nitroaromatics and quinones by NADPH. This reduction is required for the activity of nitrofuran antibiotics. The crystal structure of free Escherichia coli NfsA and several homologues have been determined previously, but there is no structure of the enzyme with ligands. We present here crystal structures of oxidised E. coli NfsA in the presence of several ligands, including the antibiotic nitrofurantoin. Nitrofurantoin binds with the furan ring, rather than the nitro group that is reduced, near the N5 of the FMN. Molecular dynamics simulations show that this orientation is only favourable in the oxidised enzyme, while potentiometry suggests that little semiquinone is formed in the free protein. This suggests that the reduction occurs by direct hydride transfer from FMNH- to nitrofurantoin bound in the reverse orientation to that in the crystal structure. We present a model of nitrofurantoin bound to reduced NfsA in a viable hydride transfer orientation. The substrate 1,4-benzoquinone and the product hydroquinone are positioned close to the FMN N5 in the respective crystal structures with NfsA, suitable for reaction, but are mobile within the active site. The structure with a second FMN, bound as a ligand, shows that a mobile loop in the free protein forms a phosphate-binding pocket. NfsA is specific for NADPH and a similar conformational change, forming a phosphate-binding pocket, is likely to also occur with the natural cofactor.
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Antibacterianos/metabolismo , Benzoquinonas/metabolismo , Proteínas de Escherichia coli/metabolismo , Mononucleotídeo de Flavina/metabolismo , Nitrofurantoína/metabolismo , Nitrorredutases/metabolismo , Antibacterianos/química , Benzoquinonas/química , Sítios de Ligação/genética , Biocatálise , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Mononucleotídeo de Flavina/química , Cinética , Simulação de Dinâmica Molecular , Estrutura Molecular , NADP/metabolismo , Nitrofurantoína/química , Nitrorredutases/química , Nitrorredutases/genética , Oxirredução , Ligação Proteica , Domínios Proteicos , Especificidade por SubstratoRESUMO
BACKGROUND: There are approximately 300 cases of enteric fever reported annually from England and Wales; most are imported infections. Clinical management of enteric fever remains a challenge with the emergence of ESBL-producing strains, especially XDR Salmonella Typhi from Sindh, Pakistan. METHODS: All strains of S. Typhi and Salmonella Paratyphi A isolated from cases presenting with symptoms of enteric fever in England and Wales, between 1 April 2014 and 31 March 2020, were characterized using WGS. Antibiotic susceptibility testing was performed using an agar dilution method. RESULTS: ESBL strains contributed to 69 cases of enteric fever (S. Typhi n = 68, S. Paratyphi A n = 1); 68 were imported (Pakistan n = 64, Iraq n = 2, Bangladesh n = 1 and India n = 1). Ages ranged from 1 to 56 years, 36/69 (52%) were children, 52% were female and the duration of hospital stay ranged from 1 to 23 days. The ESBL phenotype was conferred by the presence of blaCTX-M-15 (S. Typhi n = 67 and S. Paratyphi A n = 1) or blaCTX-M-55 (S. Typhi n = 1). An IncY plasmid harbouring blaCTX-M-15 and qnr was detected in 56 strains from Pakistan. The IncY plasmid was absent in the remaining strains and there was evidence of a 4 kb ISEcpl-blaCTX-M-15-tnp gene cassette insertion into the chromosome at one of three integration points. CONCLUSIONS: Chromosomal integration of blaCTX-M-15 within the XDR Sindh strains may lead to the maintenance of resistance in the absence of antibiotic selection pressure. Empirical treatment of cases of complicated enteric fever returning from Pakistan will henceforth have to include a carbapenem.
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Salmonella typhi , Febre Tifoide , Adolescente , Adulto , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Bangladesh , Criança , Pré-Escolar , Cromossomos , Inglaterra/epidemiologia , Feminino , Humanos , Índia , Lactente , Pessoa de Meia-Idade , Paquistão , Salmonella typhi/genética , Febre Tifoide/epidemiologia , País de Gales/epidemiologia , Adulto Jovem , beta-Lactamases/genéticaRESUMO
Ever feel concerned that you may not achieve your career goals or feel worried about where your life is going? Such examples may reflect the experience of status anxiety, that is, concerns that one may be stuck or not able to move up in life, or worries that one may be too low in standing compared to society's standards. Status anxiety is believed to be exacerbated by economic inequality and negatively affect well-being. While job satisfaction is an important determinant of well-being, no research has examined whether status anxiety can also help explain people's satisfaction with their jobs. We tested whether status anxiety differs from other organizational constructs and uniquely relates to job satisfaction among full-time working adults. In a pilot study, we found that status anxiety is separate from the concept of job insecurity (e.g., perceived threat of job loss). Results of our main study also indicated that higher status anxiety significantly predicted lower job satisfaction beyond several other indicators of organizational attitudes (job insecurity, occupational self-efficacy, distributive, procedural, and interactional justice), as well as the tendency to seek status and several background factors (e.g., income, education, perceived socioeconomic status). We discuss the unique role of status anxiety in job satisfaction and the implications of this research to our understanding of status concerns, as well as organizational attitudes and policies.
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Introduction. Diarrhoeagenic Escherichia coli (DEC) are difficult to distinguish from non-pathogenic commensal E. coli using traditional culture methods. The implementation of PCR targeting specific virulence genes characteristic of the five DEC pathotypes, has improved the detection of DEC in faecal specimens from patients with symptoms of gastrointestinal disease.Aim. Antimicrobial resistance (AMR) profiles of 660 strains of DEC isolated between 2015 and 2017 from UK travellers reporting symptoms of gastrointestinal disease were reviewed to look for evidence of emerging AMR associated with travellers' diarrhoea.Methodology. All isolates of DEC were sequenced, and sequence type, serotype, pathotype markers and AMR profiles were derived from the genome data.Results. A travel history was provided for 54.1â% (357/660) of cases, of which 77.0â% (275/357) reported travel outside the UK within 7 days of onset of symptoms, and 23.0â% (82/357) reported no travel in that time frame. Of the 660 strains of DEC in this study, 265 (40.2â%) samples were identified as EAEC, 48 (7.3â%) as EIEC, 61 (9.2â%) were ETEC and 286 (43.3â%) were EPEC. EPEC caused the highest percentage of infections in children (40.6â%) whilst the highest proportion of cases reporting recent travel were infected with ETEC (86.1â%). There were 390/660 (59.0â%) isolates resistant to at least one antimicrobial on the panel tested (EIEC, 81.3â%; ETEC, n=65.6â%; EAEC, n=73.2â%; EPEC, 40.9â%) and 265/660 (40.2â%) were multidrug-resistant (EIEC, 33.3â%; ETEC, 32.8â%; EAEC, 56.2â%; EPEC, 28.0â%). Genes conferring resistance to the beta-lactams and fluroquinolones were highest in the EAEC pathotype, 56.6 and 60.7%, respectively.Conclusions. Increasing MDR, along with resistance to the fluroquinolones and the third-generation cephalosporins, in DEC causing travellers' diarrhoea provides further evidence for the need to restrict the use of antimicrobial agents and continuous monitoring.
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Farmacorresistência Bacteriana/genética , Disenteria/genética , Escherichia coli/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/farmacologia , Criança , Pré-Escolar , Diarreia/patologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Disenteria/epidemiologia , Disenteria/microbiologia , Escherichia coli/isolamento & purificação , Escherichia coli/patogenicidade , Infecções por Escherichia coli/genética , Fezes/microbiologia , Feminino , Gastroenteropatias/epidemiologia , Gastroenteropatias/genética , Gastroenteropatias/microbiologia , Humanos , Lactente , Recém-Nascido , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/métodos , Viagem , Reino Unido , Virulência/efeitos dos fármacosRESUMO
BACKGROUND: Azithromycin resistance is emerging in typhoidal Salmonella. Confirmation of azithromycin MIC is the most frequent antibiotic susceptibility request made to the Gastrointestinal Bacteria Reference Unit (GBRU) laboratory in England by local diagnostic laboratories. OBJECTIVES: (i) Determine concordance between local diagnostic and reference laboratory estimations of azithromycin MIC by gradient strip in Salmonella enterica serovars Typhi and Paratyphi. (ii) Consider causes of variation. METHODS: Isolates from patients with enteric fever attending a central London hospital between May 2011 and April 2019 were tested for azithromycin susceptibility using gradient strips, according to EUCAST methodology. Matched local diagnostic and reference laboratory estimations of azithromycin and ciprofloxacin (as a comparator) MICs were included; concordance in estimations was examined. RESULTS: Local diagnostic laboratory readings overestimated azithromycin MIC values compared with the reference laboratory, resulting in poor concordance in susceptibility/resistance attribution (concordant susceptibility interpretation in 8/19, κ = 0). In contrast, ciprofloxacin MIC estimation demonstrated superior concordance (concordant susceptibility interpretation in 16/17, κ = 0.85). None of the isolates was resistant to azithromycin at the reference laboratory and no known genes associated with azithromycin resistance were detected in any isolate using WGS. CONCLUSIONS: Overestimation of azithromycin resistance is likely to be due to difficulty in interpreting the point of intersection of the 'trailing edge' with the gradient strip, used to determine MIC. We advise local diagnostic laboratories to review their experience and consider adopting a 'second reader' system to mitigate this.
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Azitromicina , Salmonella enterica , Antibacterianos/farmacologia , Azitromicina/farmacologia , Ciprofloxacina/farmacologia , Farmacorresistência Bacteriana , Inglaterra , Humanos , Londres , Testes de Sensibilidade Microbiana , Salmonella enterica/genética , Salmonella typhiRESUMO
Introduction. Despite many ongoing surveillance projects and the recent focus on the veterinary and clinical 'One Health' aspects of antimicrobial resistance (AMR), evidence of the extent of any public health risk posed by animal reservoirs with respect to the transmission of resistant strains of Escherichia coli to humans remains varied and contentious. In the UK, the main zoonotic reservoir for the foodborne pathogen Shiga toxin-producing E. coli (STEC) is cattle and sheep. In this study, we adopt an alternative approach to the risk assessment of transmission of AMR E. coli from animals to humans, involving monitoring AMR in isolates of STEC, an established zoonotic, foodborne pathogen, from human cases of gastrointestinal disease.Aim. The aim of this study was to determine the genome-derived AMR profiles for STEC from human cases to assess the risk of transmission of multidrug-resistant STEC from ruminants to humans.Methodology. STEC belonging to 10 different clonal complexes (CCs) (n=457) isolated from human faecal specimens were sequenced and genome-derived AMR profiles were determined. Phenotypic susceptibility testing was undertaken on all isolates (n=100) predicted to be resistant to at least one class of antimicrobial.Results. Of the 457 isolates, 332 (72.7â%) lacked identifiable resistance genes and were predicted to be fully susceptible to 11 classes of antimicrobials; 125/332 (27.3â%) carried 1 or more resistance genes, of which 83/125 (66.4â%) were resistant to 3 or more classes of antibiotic. The percentage of isolates harbouring AMR determinants varied between CCs, from 4% in CC25 to 100% in CC504. Forty-six different AMR genes were detected, which conferred resistance to eight different antibiotic classes. Resistance to ampicillin, streptomycin, tetracyclines and sulphonamides was most commonly detected. Four isolates were identified as extended-spectrum ß-lactamase producers. An overall concordance of 97.7â% (n=1075/1100) was demonstrated between the phenotypic and genotypic methods.Conclusion. This analysis provided an indirect assessment of the risk of transmission of AMR gastrointestinal pathogens from animals to humans, and revealed a subset of human isolates of the zoonotic pathogen STEC were resistant to the antimicrobials used in animal husbandry. However, this proportion has not increased over the last three decades, and thismay provide evidence that guidancepromoting responsible practice has been effective.
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Antibacterianos/farmacologia , Reservatórios de Doenças/microbiologia , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Escherichia coli/epidemiologia , Genoma Bacteriano/genética , Escherichia coli Shiga Toxigênica/genética , Animais , Bovinos , Inglaterra/epidemiologia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/transmissão , Fezes/microbiologia , Genótipo , Humanos , Masculino , Saúde Única , Minorias Sexuais e de Gênero , Ovinos , Escherichia coli Shiga Toxigênica/isolamento & purificação , ZoonosesRESUMO
To establish the prevalence of mobile colistin resistance (mcr) genes amongst Salmonella enterica isolates obtained through public health surveillance in England (April 2014 to September 2017), 33 205 S. enterica genome sequences obtained from human, food, animal and environmental isolates were screened for the presence of mcr variants 1 to 8. The mcr-positive genomes were assembled, annotated and characterized according to plasmid type. Nanopore sequencing was performed on six selected isolates with putative novel plasmids, and phylogenetic analysis was used to provide an evolutionary context for the most commonly isolated clones. Fifty-two mcr-positive isolates were identified, of which 32 were positive for mcr-1, 19 for mcr-3 and 1 for mcr-5. The combination of Illumina and Nanopore sequencing identified three novel mcr-3 plasmids and one novel mcr-5 plasmid, as well as the presence of chromosomally integrated mcr-1 and mcr-3. Monophasic S. enterica serovar Typhimurium accounted for 27/52 (52 %) of the mcr-positive isolates, with the majority clustering in clades associated with travel to Southeast Asia. Isolates in these clades were associated with a specific plasmid range and an additional extended-spectrum beta-lactamase genotype. Routine whole-genome sequencing for public health surveillance provides an effective screen for novel and emerging antimicrobial determinants, including mcr. Complementary long-read technologies elucidated the genomic context of resistance determinants, offering insights into plasmid dissemination and linkage to other resistance genes.
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Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Colistina/farmacologia , Genoma Bacteriano , Salmonella enterica/genética , Animais , Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana , Inglaterra/epidemiologia , Microbiologia Ambiental , Microbiologia de Alimentos , Humanos , Testes de Sensibilidade Microbiana , Filogenia , Plasmídeos/genética , Plasmídeos/metabolismo , Vigilância em Saúde Pública , Infecções por Salmonella/epidemiologia , Infecções por Salmonella/microbiologia , Salmonelose Animal/epidemiologia , Salmonelose Animal/microbiologia , Salmonella enterica/classificação , Salmonella enterica/efeitos dos fármacos , Salmonella enterica/isolamento & purificaçãoRESUMO
OBJECTIVES: To compare and evaluate phenotypic and genotypic methods for the detection of antimicrobial resistance (AMR) in Campylobacter jejuni and Campylobacter coli in England and Wales. METHODS: WGS data from 528 isolates of Campylobacter spp. (452 C. jejuni and 76 C. coli) from human (494), food (21) and environmental (2) sources, collected between January 2015 and December 2016, and from the PHE culture collection (11) were mapped to genes known to be associated with phenotypic resistance to antimicrobials in the genus. Phenotypic antibiotic susceptibility (erythromycin, ciprofloxacin, tetracycline, gentamicin and streptomycin) testing using an in-agar dilution method was performed on all isolates. RESULTS: Concordance between phenotypic resistance and the presence of corresponding AMR determinants was 97.5% (515/528 isolates). Only 13 out of 528 isolates (10 C. jejuni and 3 C. coli) had discordant interpretations for at least one of the five antibiotics tested, equating to a total of 15 (0.6%) discrepancies out of 2640 isolate/antimicrobial combinations. Seven discrepant results were genotypically resistant but phenotypically susceptible (major errors) and eight discrepant results were genotypically susceptible but phenotypically resistant (very major errors). CONCLUSIONS: The use of this bioinformatics approach for predicting AMR from WGS data for routine public health surveillance is a reliable method for real-time monitoring of changing AMR patterns in isolates of C. jejuni and C. coli.
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Infecções por Campylobacter , Campylobacter coli , Campylobacter jejuni , Campylobacter , Antibacterianos/farmacologia , Infecções por Campylobacter/epidemiologia , Campylobacter coli/genética , Campylobacter jejuni/genética , Diarreia , Farmacorresistência Bacteriana , Inglaterra/epidemiologia , Humanos , Testes de Sensibilidade Microbiana , País de Gales/epidemiologiaRESUMO
Whole genome sequencing (WGS) has been used routinely by Public Health England (PHE) for identification, surveillance and monitoring of resistance determinants in referred Salmonella isolates since 2015. We report the first identified case of extended-spectrum-ß-lactamase (ESBL) Salmonella enterica serovar Paratyphi A (S. Paratyphi A) isolated from a traveller returning to England from Bangladesh in November 2017. The isolate (440915) was resistant to ciprofloxacin and harboured both the mobile element ISEcp9 -blaCTX-M-15-hp-tnpA and blaTEM-191, associated with ESBL production. Phenotypic resistance was subsequently confirmed by Antimicrobial Susceptibility Testing (AST). S. Paratyphi A 440915 harboured an IncI1 plasmid previously reported to encode ESBL elements in Enterobacteriaceae and recently described in a S. Typhi isolate from Bangladesh. Results from this study indicate the importance of monitoring imported drug resistance for typhoidal salmonellae as ceftriaxone is the first line antibiotic treatment for complicated enteric fever in England. We conclude that WGS provides a rapid, accurate method for surveillance of drug resistance genes in Salmonella, leading to the first reported case of ESBL producing S. Paratyphi A and continues to inform the national treatment guidelines for management of enteric fever.
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Febre Paratifoide/diagnóstico , Fatores R , Salmonella paratyphi A/genética , Salmonella paratyphi A/isolamento & purificação , Viagem , Adulto , Bangladesh , DNA Bacteriano , Inglaterra , Humanos , Masculino , Febre Paratifoide/microbiologia , Salmonella paratyphi A/efeitos dos fármacos , Sequenciamento Completo do Genoma , Resistência beta-Lactâmica/genéticaRESUMO
The use of whole genome sequencing (WGS) as a method for supporting outbreak investigations, studying Salmonella microbial populations and improving understanding of pathogenicity has been well-described (1-3). However, performing WGS on a discrete dataset does not pose the same challenges as implementing WGS as a routine, reference microbiology service for public health surveillance. Challenges include translating WGS data into a useable format for laboratory reporting, clinical case management, Salmonella surveillance, and outbreak investigation as well as meeting the requirement to communicate that information in an understandable and universal language for clinical and public health action. Public Health England have been routinely sequencing all referred presumptive Salmonella isolates since 2014 which has transformed our approach to reference microbiology and surveillance. Here we describe an overview of the integrated methods for cross-disciplinary working, describe the challenges and provide a perspective on how WGS has impacted the laboratory and surveillance processes in England and Wales.
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Antibacterianos/uso terapêutico , Disenteria Bacilar/diagnóstico , Disenteria Bacilar/tratamento farmacológico , Shigella/isolamento & purificação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/farmacologia , Estudos de Coortes , Resistência Microbiana a Medicamentos , Disenteria Bacilar/epidemiologia , Feminino , Hospitalização , Humanos , Londres/epidemiologia , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Estudos Retrospectivos , Shigella/classificação , Shigella/efeitos dos fármacos , Shigella/genética , Sequenciamento Completo do Genoma , Adulto JovemRESUMO
Salmonella enterica serovar Typhi (S. Typhi) is the causative agent of typhoid fever, a systemic human infection with a burden exceeding 20 million cases each year that occurs disproportionately among children in low and middle income countries. Antimicrobial therapy is the mainstay for treatment, but resistance to multiple agents is common. Here we report genotypes and antimicrobial resistance (AMR) determinants detected from routine whole-genome sequencing (WGS) of 533 S. Typhi isolates referred to Public Health England between April 2014 and March 2017, 488 (92%) of which had accompanying patient travel information obtained via an enhanced surveillance questionnaire. The majority of cases involved S. Typhi 4.3.1 (H58) linked with travel to South Asia (59%). Travel to East and West Africa were associated with genotypes 4.3.1 and 3.3.1, respectively. Point mutations in the quinolone resistance determining region (QRDR), associated with reduced susceptibility to fluoroquinolones, were very common (85% of all cases) but the frequency varied significantly by region of travel: 95% in South Asia, 43% in East Africa, 27% in West Africa. QRDR triple mutants, resistant to ciprofloxacin, were restricted to 4.3.1 lineage II and associated with travel to India, accounting for 23% of cases reporting travel to the country. Overall 24% of isolates were MDR, however the frequency varied significantly by region and country of travel: 27% in West Africa, 52% in East Africa, 55% in Pakistan, 24% in Bangladesh, 3% in India. MDR determinants were plasmid-borne (IncHI1 PST2 plasmids) in S. Typhi 3.1.1 linked to West Africa, but in all other regions MDR was chromosomally integrated in 4.3.1 lineage I. We propose that routine WGS data from travel-associated cases in industrialised countries could serve as informal sentinel AMR genomic surveillance data for countries where WGS is not available or routinely performed.
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Farmacorresistência Bacteriana/genética , Salmonella typhi/genética , Febre Tifoide/epidemiologia , Genótipo , Humanos , Testes de Sensibilidade Microbiana , Quinolonas/farmacologia , Inquéritos e Questionários , Doença Relacionada a Viagens , Febre Tifoide/microbiologia , Sequenciamento Completo do GenomaRESUMO
Red wines injected with nitrogen or oxygen during fermentation were used to identify the effect of gas exposure on tannin structure and reactivity with poly-l-proline. Tannin was purified from wine after fermentation and after three years of bottle storage. Tannin from nitrogen-treated wine had a lower percentage of galloylation and were less pigmented than tannin from oxygen-exposed wine. Self-aggregation of tannin was measured by nanoparticle tracking analysis and a larger particle size was observed for the oxidized treatment. The interaction of tannin and poly-l-proline was measured by isothermal titration calorimetry, and involved more hydrogen bonding than hydrophobic interactions in the case of nitrogen-treated wine tannin. Conversely, oxidized tannin was more hydrophobic and the association with poly-l-proline was entropy-driven due to a change of solvation. The results show meaningful changes in the structure and reactivity of tannin as a result of oxygen exposure during fermentation, which may impact astringency perception.
Assuntos
Oxigênio/química , Peptídeos/química , Taninos/química , Vinho/análise , Reatores Biológicos , Calorimetria , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Oxirredução , Tamanho da Partícula , Taninos/análise , TermodinâmicaRESUMO
The impact of oxygen exposure during winemaking on metal ion concentrations in wine were investigated throughout the winemaking process in a Chardonnay wine. The concentrations of Al, Ca, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, Sn, and Zn were determined using inductively coupled plasma-mass spectrometry. Oxygen exposure significantly impacted 13 metal ions at different phases of winemaking. However, only the concentrations of Cr, Cu, and Fe were impacted by early oxygen exposure during pressing, with lower Cr and Cu concentrations in wines that were aerobically pressed and lower concentrations of Fe in wines that were inertly pressed. The sequestering of Al, Cu, Ni, and Zn by wine lees was significantly affected by oxygen treatment, with lees collected from wines that were treated oxidatively sequestering significantly greater amounts of Cu and Zn and removing these metals from the wine supernatant. The metal ion that was most affected by oxygen exposure during pressing and handling was Cu, with significantly lower Cu measured in wines that were produced under oxidative conditions. It is known that elevated Cu concentrations have negative implications for wine aroma and flavour. This study demonstrated that oxygen management during winemaking significantly impacts metal ion concentrations in lees and wine, which may decrease the risk of developing taints and faults.