Oxygen-insensitive nitroreductase E. coli NfsA, but not NfsB, is inhibited by fumarate.

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.

Structure and Dynamics of Three Escherichia coli NfsB Nitro-Reductase Mutants Selected for Enhanced Activity with the Cancer Prodrug CB1954.

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.

The structures of E. coli NfsA bound to the antibiotic nitrofurantoin; to 1,4-benzoquinone and to FMN.

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.

ESBL-producing strains isolated from imported cases of enteric fever in England and Wales reveal multiple chromosomal integrations of blaCTX-M-15 in XDR Salmonella Typhi.

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.

Azithromycin susceptibility testing for Salmonella enterica isolates: discordances in results using MIC gradient strips.

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.

Comparison of phenotypic and WGS-derived antimicrobial resistance profiles of Campylobacter jejuni and Campylobacter coli isolated from cases of diarrhoeal disease in England and Wales, 2015-16.

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.

Epidemiology and Outcomes of Nontyphoidal Salmonella Bacteremias from England, 2004 to 2015.

Nontyphoidal Salmonella (NTS) bacteremia causes hospitalization and high morbidity and mortality. We linked Gastrointestinal Bacteria Reference Unit (GBRU) data to the Hospital Episode Statistics (HES) data set to study the trends and outcomes of NTS bacteremias in England between 2004 and 2015. All confirmed NTS isolates from blood from England submitted to GBRU between 1 January 2004 and 31 December 2015 were deterministically linked to HES records. Adjusted odds ratios (AOR), proportions, and confidence intervals (CI) were calculated to describe differences in age, sex, antibiotic resistance patterns, and serotypes over time. Males, neonates, and adults above 65 years were more likely to have NTS bacteremia (AOR, 1.54 [95% CI, 1.46 to 1.67]; 2.57 [95% CI, 1.43 to 4.60]; and 3.56 [95% CI, 3.25 to 3.90], respectively). Proportions of bacteremia increased from 1.41% in 2004 to 2.67% in 2015. Thirty-four percent of all blood isolates were resistant to a first-line antibiotic, and 1,397 (56%) blood isolates were linked to an HES record. Of the patients with NTS bacteremia, 969 (69%) had a cardiovascular condition and 155 (12%) patients died, out of which 120 (77%) patients were age 65 years and above. NTS bacteremia mainly affects older people with comorbidities placing them at increased risk of prolonged hospital stay and death. Resistance of invasive NTS to first-line antimicrobial agents appeared to be stable in England, but the emergence of resistance to last-resort antibiotics, such as colistin, requires careful monitoring.

Changes in Metal Ion Concentrations in a Chardonnay Wine Related to Oxygen Exposure during Vinification.

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.

Comparison of phenotypic and WGS-derived antimicrobial resistance profiles of Salmonella enterica serovars Typhi and Paratyphi.

Objectives: Surveillance of antimicrobial resistance (AMR) in Salmonella enterica serovars Typhi and Paratyphi is essential to provide an evidence base for empirical treatment protocols and to monitor emerging AMR. We sought to compare phenotypic and WGS-based genotypic methods for the detection of AMR in Salmonella Typhi and Salmonella Paratyphi. Methods: WGS data from 603 isolates of Salmonella Typhi (n = 332) and Salmonella Paratyphi (n = 271) were mapped to genes or chromosomal mutations known to be associated with phenotypic AMR and compared with phenotypic susceptibility data interpreted using breakpoints recommended by EUCAST. Results: There were two (0.03%) discordant interpretations out of a possible 6030 isolate/antimicrobial class combinations. MDR (resistant to three or more classes of antimicrobial) was detected in 83/332 (25.0%) Salmonella Typhi isolates, but was not detected in Salmonella Paratyphi. Thirty-six (10.8%) isolates of Salmonella Typhi were resistant to ciprofloxacin (MIC >0.5 mg/L), with 33 (9.9%) of 332 exhibiting mutations in gyrA and parC, and 244 (73.5%) isolates had reduced susceptibility to ciprofloxacin (MIC 0.06-0.25 mg/L). In comparison, 209/227 (92.1%) isolates of Salmonella Paratyphi A exhibited resistance to ciprofloxacin (MIC >0.5 mg/L). No resistance to azithromycin or the third-generation cephalosporins was detected. Conclusions: WGS data provided a robust and informative approach for monitoring MDR and emerging resistance to ciprofloxacin in Salmonella Typhi and Salmonella Paratyphi. Phenotypic antimicrobial susceptibility testing continues to be performed to guide targeted individual patient treatment, but inferred AMR profiles from WGS data may be used for surveillance and to guide empirical therapy.

Comparison of phenotypic and WGS-derived antimicrobial resistance profiles of enteroaggregative Escherichia coli isolated from cases of diarrhoeal disease in England, 2015-16.

OBJECTIVES: Phenotypic and genotypic methods for the detection of antimicrobial resistance (AMR) in enteroaggregative Escherichia coli (EAEC) were compared and evaluated. METHODS: WGS data from 155 isolates of EAEC isolated between June 2015 and December 2016 were mapped to genes known to be associated with phenotypic AMR. RESULTS: Phenotypic and genotypic testing of 155 isolates against 10 antimicrobial classes resulted in a total of 25 (1.6%) discordant results of a possible 1550 isolate/antimicrobial combinations. Twenty-three of the mismatches were observed in streptomycin or sulphonamide resistance profiles. These discrepancies were associated with either insertions or truncations in the genes predicted to confer resistance, or in their promotors, rendering them non-functional, or with the presence of aadA variants associated with reduced expression. The most common resistances detected were to ampicillin (56.1%), the sulphonamides (49.7%) and trimethoprim (48.4%). The presence of CTX-M ESBL variants and/or acquired AmpC was detected in 87 of 155 (56.1%) isolates and 18 of 155 (11.6%) isolates were resistant to ciprofloxacin. Eighty-eight (56.8%) isolates were MDR. CONCLUSIONS: Phenotypic and genome-derived AMR comparisons showed good correlation for EAEC. A better understanding of the role of allelic variants, specific gene combinations and promoter/attenuator mechanisms in the phenotypic manifestation will improve our ability to provide a robust interpretation of the data for surveillance purposes and, ultimately, in the clinical setting.

Antimicrobial resistance in Shiga toxin-producing Escherichia coli serogroups O157 and O26 isolated from human cases of diarrhoeal disease in England, 2015.

OBJECTIVES: Shiga toxin-producing Escherichia coli (STEC) are zoonotic and transmission to humans occurs via contaminated food or contact with infected animals. In this study, WGS data were used to predict antimicrobial resistance (AMR) in STEC from symptomatic human cases to assess the extent of transmission of antibiotic-resistant E. coli from animals to humans. METHODS: WGS data from 430 isolates of STEC were mapped to genes known to be associated with phenotypic AMR. Susceptibility testing was performed by a breakpoint method on all viable isolates exhibiting resistance to at least one antimicrobial. RESULTS: 327/396 (82.6%) of STEC O157 and 22/34 (64.7%) of STEC O26 lacked identifiable resistance genes and were predicted to be fully susceptible to 11 diverse classes of antimicrobials. For the remaining 81 isolates, 74 were phenotypically tested and there was concordance between WGS-predicted resistance and expression of phenotypic resistance. The most common resistance profile was ampicillin, streptomycin, trimethoprim/sulphonamide and tetracycline occurring in 25 (5.8%) isolates. Resistance to other antimicrobials, including resistance to chloramphenicol (2.1%), resistance to azithromycin (0.2%) and reduced susceptibility to ciprofloxacin (2.6%), was less frequent. Three isolates were identified as ESBL producers. CONCLUSIONS: ß-Lactams, trimethoprim/sulphonamides and tetracyclines account for the majority of therapeutic antimicrobials sold for veterinary use and this may be a risk factor for the presence of AMR in domestically acquired human clinical isolates of STEC. Isolates that were resistant to ampicillin, streptomycin, sulphonamide, tetracycline and azithromycin and had reduced susceptibility to ciprofloxacin were associated with cases who reported recent travel abroad.

Comparison of phenotypic and WGS-derived antimicrobial resistance profiles of Shigella sonnei isolated from cases of diarrhoeal disease in England and Wales, 2015.

Objectives: Phenotypic and genotypic methods for the detection of antimicrobial resistance (AMR) in Shigella sonnei in England and Wales were compared and evaluated. Methods: WGS data from 341 isolates of S. sonnei isolated between June 2015 and January 2016 were mapped to genes known to be associated with phenotypic AMR. Antimicrobial susceptibility testing was performed on all viable isolates (n = 335). Results: Fifteen of 335 isolates had a discrepancy between phenotypic and genotypic testing for 1 of the 10 antimicrobial classes tested, equating to 15 (0.45%) discordant results out of a possible 3350 isolate/antimicrobial combinations. All 15 mismatched results were genotypically resistant but phenotypically susceptible. Eleven of the 15 discrepancies were observed in streptomycin resistance profiles. The most common resistance profile was trimethoprim, sulphonamides, tetracyclines and streptomycin, occurring in 97 (28.4%) isolates. Resistances to ciprofloxacin and the third-generation cephalosporins, not detected in England and Wales prior to 2002, were identified in 18.2% and 12% of isolates, respectively. Three hundred and four (89.1%) isolates were MDR. There was no significant association between any of the AMR determinants tested and recent foreign travel in male or female cases. The number of isolates of S. sonnei harbouring blaTEM-1 and ermB/mphA was significantly higher in men who reported no recent travel outside the UK. Conclusions: The use of WGS for routine public health surveillance is a reliable method for rapid detection of emerging AMR in isolates of S. sonnei.

ESBL-Producing and Macrolide-Resistant Shigella sonnei Infections among Men Who Have Sex with Men, England, 2015.

In England in 2015, Shigella sonnei isolates from men who have sex with men produced extended-spectrum ß-lactamases and exhibited macrolide resistance. Whole-genome sequencing showed a close relationship among the isolates, which harbored a plasmid that was previously identified in a shigellosis outbreak among this population but has acquired a mobile element.

WGS for surveillance of antimicrobial resistance: a pilot study to detect the prevalence and mechanism of resistance to azithromycin in a UK population of non-typhoidal Salmonella.

OBJECTIVES: WGS and phenotypic methods were used to determine the prevalence of azithromycin resistance in Salmonella enterica isolates from the UK and to identify the underlying mechanisms of resistance. METHODS: WGS by Illumina HiSeq was carried out on 683 Salmonella spp. isolates. Known genes associated with azithromycin resistance were detected by WGS using a mapping-based approach. Macrolide resistance determinants were identified and the genomic context of these elements was assessed by various bioinformatics tools. Susceptibility testing was in accordance with EUCAST methodology (MIC ≤16 mg/L). RESULTS: Fifteen isolates of non-typhoidal Salmonella enterica belonging to serovars Salmonella Blockley, Salmonella Typhimurium, Salmonella Thompson, Salmonella Ridge and Salmonella Kentucky showed resistance or decreased susceptibility to azithromycin (from 6 to >16 mg/L) due to the presence of macrolide resistance genes mphA, mphB or mefB. These genes were either plasmid or chromosomally mediated. Azithromycin-resistant Salmonella Blockley isolates harboured a macrolide inactivation gene cluster, mphA-mrx-mphr(A), within a novel Salmonella azithromycin resistance genomic island (SARGI) determined by MinION sequencing. This is the first known chromosomally mediated mphA gene cluster described in salmonellae. Phylogenetic analysis and epidemiological information showed that mphA Salmonella Blockley isolates were not derived from a single epidemiologically related event. The azithromycin MICs of the 15 Salmonella spp. isolates showed that the presence of the mphA gene was associated with MIC ≥16 mg/L, while the presence of mefB or mphB was not. CONCLUSIONS: Azithromycin resistance due to acquisition of known macrolide resistance genes was seen in four different Salmonella serovars and can be either plasmid-encoded or chromosomally encoded.

Detection of the plasmid-mediated mcr-1 gene conferring colistin resistance in human and food isolates of Salmonella enterica and Escherichia coli in England and Wales.

OBJECTIVES: In response to the first report of transmissible colistin resistance mediated by the mcr-1 gene in Escherichia coli and Klebsiella spp. from animals and humans in China, we sought to determine its presence in Enterobacteriaceae isolated in the UK. METHODS: The PHE archive of whole-genome sequences of isolates from surveillance collections, submissions to reference services and research projects was retrospectively analysed for the presence of mcr-1 using Genefinder. The genetic environment of the gene was also analysed. RESULTS: Rapid screening of the genomes of ∼24 000 Salmonella enterica, E. coli, Klebsiella spp., Enterobacter spp., Campylobacter spp. and Shigella spp. isolated from food or humans identified 15 mcr-1-positive isolates. These comprised: 10 human S. enterica isolates submitted between 2012 and 2015 (8 Salmonella Typhimurium, 1 Salmonella Paratyphi B var Java and 1 Salmonella Virchow) from 10 patients; 3 isolates of E. coli from 2 patients; and 2 isolates of Salmonella Paratyphi B var Java from poultry meat imported from the EU. The mcr-1 gene was located on diverse plasmids belonging to the IncHI2, IncI2 and IncX4 replicon types and its association with ISApl1 varied. Six mcr-1-positive S. enterica isolates were from patients who had recently travelled to Asia. CONCLUSIONS: Analysis of WGS data allowed rapid confirmation of the presence of the plasmid-mediated colistin resistance gene mcr-1 in diverse genetic environments and plasmids. It has been present in E. coli and Salmonella spp. harboured by humans in England and Wales since at least 2012.

The extant World War 1 dysentery bacillus NCTC1: a genomic analysis.

BACKGROUND: Shigellosis (previously bacillary dysentery) was the primary diarrhoeal disease of World War 1, but outbreaks still occur in military operations, and shigellosis causes hundreds of thousands of deaths per year in developing nations. We aimed to generate a high-quality reference genome of the historical Shigella flexneri isolate NCTC1 and to examine the isolate for resistance to antimicrobials. METHODS: In this genomic analysis, we sequenced the oldest extant Shigella flexneri serotype 2a isolate using single-molecule real-time (SMRT) sequencing technology. Isolated from a soldier with dysentery from the British forces fighting on the Western Front in World War 1, this bacterium, NCTC1, was the first isolate accessioned into the National Collection of Type Cultures. We created a reference sequence for NCTC1, investigated the isolate for antimicrobial resistance, and undertook comparative genetics with S flexneri reference strains isolated during the 100 years since World War 1. FINDINGS: We discovered that NCTC1 belonged to a 2a lineage of S flexneri, with which it shares common characteristics and a large core genome. NCTC1 was resistant to penicillin and erythromycin, and contained a complement of chromosomal antimicrobial resistance genes similar to that of more recent isolates. Genomic islands gained in the S flexneri 2a lineage over time were predominately associated with additional antimicrobial resistances, virulence, and serotype conversion. INTERPRETATION: This S flexneri 2a lineage is a well adapted pathogen that has continued to respond to selective pressures. We have created a valuable historical benchmark for shigellae in the form of a high-quality reference sequence for a publicly available isolate. FUNDING: The Wellcome Trust.

Clinical isolates of Salmonella enterica serovar Agona producing NDM-1 metallo-ß-lactamase: first report from Pakistan.

We report two cases of infantile diarrhea due to multidrug-resistant, NDM-1 metallo-ß-lactamase-producing Salmonella enterica serovar Agona from Pakistan. This study alerts toward possible risk of NDM-1 transmission to enteric fever pathogens and encourages microbiologists to consider active screening of carbapenem resistance in nontyphoidal Salmonella isolates.

Designing and creating Saccharomyces interspecific hybrids for improved, industry relevant, phenotypes.

To remain competitive in increasingly overcrowded markets, yeast strain development programmes are crucial for fermentation-based food and beverage industries. In a winemaking context, there are many yeast phenotypes that stand to be improved. For example, winemakers endeavouring to produce sweet dessert wines wrestle with fermentation challenges particular to fermenting high-sugar juices, which can lead to elevated volatile acidity levels and extended fermentation times. In the current study, we used natural yeast breeding techniques to generate Saccharomyces spp. interspecific hybrids as a non-genetically modified (GM) strategy to introduce targeted improvements in important, wine-relevant traits. The hybrids were generated by mating a robust wine strain of Saccharomyces cerevisiae with a wine isolate of Saccharomyces bayanus, a species previously reported to produce wines with low concentrations of acetic acid. Two hybrids generated from the cross showed robust fermentation properties in high-sugar grape juice and produced botrytised Riesling wines with much lower concentrations of acetic acid relative to the industrial wine yeast parent. The hybrids also displayed suitability for icewine production when bench-marked against an industry standard icewine yeast, by delivering icewines with lower levels of acetic acid. Additionally, the hybrid yeast produced wines with novel aroma and flavour profiles and established that choice of yeast strain impacts on wine colour. These new hybrid yeasts display the desired targeted fermentation phenotypes from both parents, robust fermentation in high-sugar juice and the production of wines with low volatile acidity, thus establishing their suitability for wine styles that are traditionally troubled by excessive volatile acidity levels.

Predicting confidence in flashbulb memories.

Years after a shocking news event many people confidently report details of their flashbulb memories (e.g., what they were doing). People's confidence is a defining feature of their flashbulb memories, but it is not well understood. We tested a model that predicted confidence in flashbulb memories. In particular we examined whether people's social bond with the target of a news event predicts confidence. At a first session shortly after the death of Michael Jackson participants reported their sense of attachment to Michael Jackson, as well as their flashbulb memories and emotional and other reactions to Jackson's death. At a second session approximately 18 months later they reported their flashbulb memories and confidence in those memories. Results supported our proposed model. A stronger sense of attachment to Jackson was related to reports of more initial surprise, emotion, and rehearsal during the first session. Participants' bond with Michael Jackson predicted their confidence but not the consistency of their flashbulb memories 18 months later. We also examined whether participants' initial forecasts regarding the persistence of their flashbulb memories predicted the durability of their memories. Participants' initial forecasts were more strongly related to participants' subsequent confidence than to the actual consistency of their memories.