Target enrichment improves culture-independent detection of Neisseria gonorrhoeae and antimicrobial resistance determinants direct from clinical samples with Nanopore sequencing.

Multi-drug-resistant Neisseria gonorrhoeae infection is a significant public health risk. Rapidly detecting N. gonorrhoeae and antimicrobial-resistant (AMR) determinants by metagenomic sequencing of urine is possible, although high levels of host DNA and overgrowth of contaminating species hamper sequencing and limit N. gonorrhoeae genome coverage. We performed Nanopore sequencing of nucleic acid amplification test-positive urine samples and culture-positive urethral swabs with and without probe-based target enrichment, using a custom SureSelect panel, to investigate whether selective enrichment of N. gonorrhoeae DNA improves detection of both species and AMR determinants. Probes were designed to cover the entire N. gonorrhoeae genome, with tenfold enrichment of probes covering selected AMR determinants. Multiplexing was tested in a subset of samples. The proportion of sequence bases classified as N. gonorrhoeae increased in all samples after enrichment, from a median (IQR) of 0.05 % (0.01-0.1 %) to 76 % (42-82 %), giving a corresponding median improvement in fold genome coverage of 365 times (112-720). Over 20-fold coverage, required for robust AMR determinant detection, was achieved in 13/15(87 %) samples, compared to 2/15(13 %) without enrichment. The four samples multiplexed together also achieved >20-fold genome coverage. Coverage of AMR determinants was sufficient to predict resistance conferred by changes in chromosomal genes, where present, and genome coverage also enabled phylogenetic relationships to be reconstructed. Probe-based target enrichment can improve N. gonorrhoeae genome coverage when sequencing DNA extracts directly from urine or urethral swabs, allowing for detection of AMR determinants. Additionally, multiplexing prior to enrichment provided enough genome coverage for AMR detection and reduces the costs associated with this method.

The History of Flow Chemistry at Eli Lilly and Company.

Flow chemistry was initially used for speed to early phase material delivery in the development laboratories, scaling up chemical transformations that we would not or could not scale up batch for safety reasons. Some early examples included a Newman Kwart Rearrangement, Claisen rearrangement, hydroformylation, and thermal imidazole cyclization. Next, flow chemistry was used to enable safe scale up of hazardous chemistries to manufacturing plants. Examples included high pressure hydrogenation, aerobic oxidation, and Grignard formation reactions. More recently, flow chemistry was used in Small Volume Continuous (SVC) processes, where highly potent oncolytic molecules were produced by fully continuous processes at about 10 kg/day including reaction, extraction, distillation, and crystallization, using disposable equipment contained in fume hoods.

Process Development for a 1H-Indazole Synthesis Using an Intramolecular Ullmann-Type Reaction.

Within the scope of developing a new route to an active pharmaceutical ingredient intermediate, we had need of a fluorinated indazole. Although an established route was in place, it was undesirable due to safety and selectivity concerns. A concise and improved route was developed to form the desired indazole, which takes advantage of an electronically directed metalation/formylation sequence followed by condensation with methyl hydrazine to form a hydrazone and culminates in a copper-catalyzed intramolecular Ullmann cyclization. The Ullmann reaction was plagued with difficulties ranging from poor reactivity to thermal hazard concerns, but use of high-throughput screening, statistical modeling, and an unusual isolation method for fine chemicals, safe and optimal conditions were found that produce high-purity isolated material in excellent yields at a laboratory scale.

Hospital outbreak of carbapenem-resistant Enterobacterales associated with a blaOXA-48 plasmid carried mostly by Escherichia coli ST399.

A hospital outbreak of carbapenem-resistant Enterobacterales was detected by routine surveillance. Whole genome sequencing and subsequent analysis revealed a conserved promiscuous blaOXA-48 carrying plasmid as the defining factor within this outbreak. Four different species of Enterobacterales were involved in the outbreak. Escherichia coli ST399 accounted for 35 of all the 55 isolates. Comparative genomics analysis using publicly available E. coli ST399 genomes showed that the outbreak E. coli ST399 isolates formed a unique clade. We developed a mathematical model of pOXA-48-like plasmid transmission between host lineages and used it to estimate its conjugation rate, giving a lower bound of 0.23 conjugation events per lineage per year. Our analysis suggests that co-evolution between the pOXA-48-like plasmid and E. coli ST399 could have played a role in the outbreak. This is the first study to report carbapenem-resistant E. coli ST399 carrying blaOXA-48 as the main cause of a plasmid-borne outbreak within a hospital setting. Our findings suggest complementary roles for both plasmid conjugation and clonal expansion in the emergence of this outbreak.

Stabilizing Hydrous ß-NiOOH for Efficient Electrocatalytic Water Oxidation by Integrating Y and Co into Amorphous Ni-Based Nanoparticles.

A two-stage ball milling process was used to synthesize amorphous Ni79.2Nb12.5Y8.3 and Ni74.2Co5Nb12.5Y8.3 nanoparticles from elemental powders. The two-stage ball milling process provides a scalable and industrially applicable method for producing non-metalloid amorphous nanoparticles. The amorphous nanoparticles displayed excellent catalytic performance toward the oxygen evolution reaction (OER) in 1 M KOH, displaying lower overpotentials than IrO2 at 10 mA cm-2. The addition of Co in the amorphous alloy reduced the overpotential to 288 mV at 10 mA cm-2. The pairing of X-ray photoelectron spectroscopy and in situ X-ray absorption spectroscopy revealed that the improved OER activity of amorphous Ni74.2Co5Nb12.5Y8.3 was attributed to the catalytic synergy between Y and Co. The integration of Y supported proton-coupled electron-transfer processes that assisted with the electrostatic adsorption of OH- and formation of oxyhydroxide species, while Co sites enabled metal-oxo bonding to prevent Ni overcharging and the stabilization of ß-NiOOH. The catalytic synergy between Y and Co reduces the amount of Co needed to enhance the OER activity of Ni-based alloys and lessens the dependence on Co, which is in high demand in many renewable energy and storage applications.

Antimicrobial resistance determinants are associated with Staphylococcus aureus bacteraemia and adaptation to the healthcare environment: a bacterial genome-wide association study.

Staphylococcus aureus is a major bacterial pathogen in humans, and a dominant cause of severe bloodstream infections. Globally, antimicrobial resistance (AMR) in S. aureus remains challenging. While human risk factors for infection have been defined, contradictory evidence exists for the role of bacterial genomic variation in S. aureus disease. To investigate the contribution of bacterial lineage and genomic variation to the development of bloodstream infection, we undertook a genome-wide association study comparing bacteria from 1017 individuals with bacteraemia to 984 adults with asymptomatic S. aureus nasal carriage. Within 984 carriage isolates, we also compared healthcare-associated (HA) carriage with community-associated (CA) carriage. All major global lineages were represented in both bacteraemia and carriage, with no evidence for different infection rates. However, kmers tagging trimethoprim resistance-conferring mutation F99Y in dfrB were significantly associated with bacteraemia-vs-carriage (P=10-8.9-10-9.3). Pooling variation within genes, bacteraemia-vs-carriage was associated with the presence of mecA (HMP=10-5.3) as well as the presence of SCCmec (HMP=10-4.4). Among S. aureus carriers, no lineages were associated with HA-vs-CA carriage. However, we found a novel signal of HA-vs-CA carriage in the foldase protein prsA, where kmers representing conserved sequence allele were associated with CA carriage (P=10-7.1-10-19.4), while in gyrA, a ciprofloxacin resistance-conferring mutation, L84S, was associated with HA carriage (P=10-7.2). In an extensive study of S. aureus bacteraemia and nasal carriage in the UK, we found strong evidence that all S. aureus lineages are equally capable of causing bloodstream infection, and of being carried in the healthcare environment. Genomic variation in the foldase protein prsA is a novel genomic marker of healthcare origin in S. aureus but was not associated with bacteraemia. AMR determinants were associated with both bacteraemia and healthcare-associated carriage, suggesting that AMR increases the propensity not only to survive in healthcare environments, but also to cause invasive disease.

In Situ Formation of Nano Ni-Co Oxyhydroxide Enables Water Oxidation Electrocatalysts Durable at High Current Densities.

The oxygen evolution reaction (OER) limits the energy efficiency of electrocatalytic systems due to the high overpotential symptomatic of poor reaction kinetics; this problem worsens over time if the performance of the OER electrocatalyst diminishes during operation. Here, a novel synthesis of nanocrystalline Ni-Co-Se using ball milling at cryogenic temperature is reported. It is discovered that, by anodizing the Ni-Co-Se structure during OER, Se ions leach out of the original structure, allowing water molecules to hydrate Ni and Co defective sites, and the nanoparticles to evolve into an active Ni-Co oxyhydroxide. This transformation is observed using operando X-ray absorption spectroscopy, with the findings confirmed using density functional theory calculations. The resulting electrocatalyst exhibits an overpotential of 279 mV at 0.5 A cm-2 and 329 mV at 1 A cm-2 and sustained performance for 500 h. This is achieved using low mass loadings (0.36 mg cm-2 ) of cobalt. Incorporating the electrocatalyst in an anion exchange membrane water electrolyzer yields a current density of 1 A cm-2 at 1.75 V for 95 h without decay in performance. When the electrocatalyst is integrated into a CO2 -to-ethylene electrolyzer, a record-setting full cell voltage of 3 V at current density 1 A cm-2 is achieved.

Undetected carriage explains apparent Staphylococcus aureus acquisition in a non-outbreak healthcare setting.

OBJECTIVES: Previous studies have been unable to identify patient or staff reservoirs for the majority of the nosocomial S. aureus acquisitions which occur in the presence of good infection control practice. We set out to establish the extent to which undetected pre-existing carriage explains apparent nosocomial S. aureus acquisition. METHODS: Over two years elective cardiothoracic admissions were screened for S. aureus carriage before and during hospital admission. Routine screening (nose/groin/wound sampling), was supplemented by sampling additional body sites (axilla/throat/rectum) and culture-based methods optimised to detect fastidious phenotypes (small colony variants, cell wall deficient variants) and molecular identification by PCR. RESULTS: 35% of participants (53/151) were S. aureus carriers according to routine pre-healthcare screening; increasing to 42% (63/151) when additional body sites and enhanced cultures were employed. 71% (5/7) of apparent acquisitions were explained by pre-existing carriage using augmented measures. Enhanced culture identified a minority of colonised individuals (3/151 including 1 MRSA carrier) who were undetected by routine and additional screening cultures. 4/14 (29%) participants who became culture-negative during admission had S. aureus genomic material detected at discharge. CONCLUSIONS: Conventional sampling under-estimates carriage of S. aureus and this explains the majority of apparent S. aureus acquisitions among elective cardiothoracic patients.

Genomic investigation of clinically significant coagulase-negative staphylococci.

Introduction. Coagulase-negative staphylococci have been recognized both as emerging pathogens and contaminants of clinical samples. High-resolution genomic investigation may provide insights into their clinical significance.Aims. To review the literature regarding coagulase-negative staphylococcal infection and the utility of genomic methods to aid diagnosis and management, and to identify promising areas for future research.Methodology. We searched Google Scholar with the terms (Staphylococcus) AND (sequencing OR (infection)). We prioritized papers that addressed coagulase-negative staphylococci, genomic analysis, or infection.Results. A number of studies have investigated specimen-related, phenotypic and genetic factors associated with colonization, infection and virulence, but diagnosis remains problematic.Conclusion. Genomic investigation provides insights into the genetic diversity and natural history of colonization and infection. Such information allows the development of new methodologies to identify and compare relatedness and predict antimicrobial resistance. Future clinical studies that employ suitable sampling frames coupled with the application of high-resolution whole-genome sequencing may aid the development of more discriminatory diagnostic approaches to coagulase-staphylococcal infection.

RNA polymerase mutations cause cephalosporin resistance in clinical Neisseria gonorrhoeae isolates.

Increasing Neisseria gonorrhoeae resistance to ceftriaxone, the last antibiotic recommended for empiric gonorrhea treatment, poses an urgent public health threat. However, the genetic basis of reduced susceptibility to ceftriaxone is not completely understood: while most ceftriaxone resistance in clinical isolates is caused by target site mutations in penA, some isolates lack these mutations. We show that penA-independent ceftriaxone resistance has evolved multiple times through distinct mutations in rpoB and rpoD. We identify five mutations in these genes that each increase resistance to ceftriaxone, including one mutation that arose independently in two lineages, and show that clinical isolates from multiple lineages are a single nucleotide change from ceftriaxone resistance. These RNA polymerase mutations cause large-scale transcriptional changes without altering susceptibility to other antibiotics, reducing growth rate, or deranging cell morphology. These results underscore the unexpected diversity of pathways to resistance and the importance of continued surveillance for novel resistance mutations.

Optimizing DNA Extraction Methods for Nanopore Sequencing of Neisseria gonorrhoeae Directly from Urine Samples.

Empirical gonorrhea treatment at initial diagnosis reduces onward transmission. However, increasing resistance to multiple antibiotics may necessitate waiting for culture-based diagnostics to select an effective treatment. There is a need for same-day culture-free diagnostics that identify infection and detect antimicrobial resistance. We investigated if Nanopore sequencing can detect sufficient Neisseria gonorrhoeae DNA to reconstruct whole genomes directly from urine samples. We used N. gonorrhoeae-spiked urine samples and samples from gonorrhea infections to determine optimal DNA extraction methods that maximize the amount of N. gonorrhoeae DNA sequenced while minimizing contaminating host DNA. In simulated infections, the Qiagen UCP pathogen mini kit provided the highest ratio of N. gonorrhoeae to human DNA and the most consistent results. Depletion of human DNA with saponin increased N. gonorrhoeae yields in simulated infections but decreased yields in clinical samples. In 10 urine samples from men with symptomatic urethral gonorrhea, ≥92.8% coverage of an N. gonorrhoeae reference genome was achieved in all samples, with ≥93.8% coverage breath at ≥10-fold depth in 7 (70%) samples. In simulated infections, if ≥104 CFU/ml of N. gonorrhoeae was present, sequencing of the large majority of the genome was frequently achieved. N. gonorrhoeae could also be detected from urine in cobas PCR medium tubes and from urethral swabs and in the presence of simulated Chlamydia coinfection. Using Nanopore sequencing of urine samples from men with urethral gonorrhea, sufficient data can be obtained to reconstruct whole genomes in the majority of samples without the need for culture.

Ethnically diverse urban transmission networks of Neisseria gonorrhoeae without evidence of HIV serosorting.

OBJECTIVE: We aimed to characterise gonorrhoea transmission patterns in a diverse urban population by linking genomic, epidemiological and antimicrobial susceptibility data. METHODS: Neisseria gonorrhoeae isolates from patients attending sexual health clinics at Barts Health NHS Trust, London, UK, during an 11-month period underwent whole-genome sequencing and antimicrobial susceptibility testing. We combined laboratory and patient data to investigate the transmission network structure. RESULTS: One hundred and fifty-eight isolates from 158 patients were available with associated descriptive data. One hundred and twenty-nine (82%) patients identified as male and 25 (16%) as female; four (3%) records lacked gender information. Self-described ethnicities were: 51 (32%) English/Welsh/Scottish; 33 (21%) white, other; 23 (15%) black British/black African/black, other; 12 (8%) Caribbean; 9 (6%) South Asian; 6 (4%) mixed ethnicity; and 10 (6%) other; data were missing for 14 (9%). Self-reported sexual orientations were 82 (52%) men who have sex with men (MSM); 49 (31%) heterosexual; 2 (1%) bisexual; data were missing for 25 individuals. Twenty-two (14%) patients were HIV positive. Whole-genome sequence data were generated for 151 isolates, which linked 75 (50%) patients to at least one other case. Using sequencing data, we found no evidence of transmission networks related to specific ethnic groups (p=0.64) or of HIV serosorting (p=0.35). Of 82 MSM/bisexual patients with sequencing data, 45 (55%) belonged to clusters of ≥2 cases, compared with 16/44 (36%) heterosexuals with sequencing data (p=0.06). CONCLUSION: We demonstrate links between 50% of patients in transmission networks using a relatively small sample in a large cosmopolitan city. We found no evidence of HIV serosorting. Our results do not support assortative selectivity as an explanation for differences in gonorrhoea incidence between ethnic groups.

Nitrate on a Slow Decline: Watershed Water Quality Response during Two Decades of Tallgrass Prairie Ecosystem Reconstruction in Iowa.

The Neal Smith National Wildlife Refuge was established as a tallgrass prairie ecosystem reconstruction in the Walnut Creek watershed (5238 ha), Jasper County, Iowa, with >1200 ha of prairie plantings initiated between 1993 and 2006. This study updates the documented decreases in watershed NO-N losses that accompanied this change in land cover to a 20-yr record. Annual flow-weighted NO-N concentrations declined by 0.15 mg NO-N L yr, which was not significantly different from the rate of 0.07 mg NO-N L yr reported after the first decade of monitoring. There was also evidence ( < 0.1) that prairie reconstruction led to a declining trend in annual watershed water yield, which would have contributed to the trend of decreasing NO-N loads. However, variability in climate, including 2 yr with significant flooding events followed by a major drought during the second decade of monitoring, challenged any notion that a watershed water quality record will stabilize even >10 yr after a substantial change in land cover, in this naturally drained watershed underlain by fine grained glacial deposits that exhibit multidecadal groundwater transport times. Efforts to document progress toward water quality goals will need to consider dominant flow paths and associated travel times, uncertainty in the effectiveness of management changes, and a changeable climate.

Draft Genome Sequences of 64 Type Strains of 50 Species and 25 Subspecies of the Genus Staphylococcus Rosenbach 1884.

Members of the genus Staphylococcus have been isolated from humans, animals, and the environment. Accurate identification with whole-genome sequencing requires access to data derived from type strains. We provide sequence data for type strains of 64 taxa in the genus that at the time of this writing have standing in the nomenclature.

Changes in lateral floodplain connectivity accompanying stream channel evolution: Implications for sediment and nutrient budgets.

Floodplain storage commonly represents one of the largest sediment fluxes within sediment budgets. In watersheds responding to large scale disturbance, floodplain-channel lateral connectivity may change over time with progression of channel evolution and associated changes in channel geometry. In this study we investigated the effects of channel geometry change on floodplain inundation frequency and flux of suspended sediment (SS) and total phosphorus (TP) to floodplain storage within the 52.2 km2 Walnut Creek watershed (Iowa, USA) through a combination of 25 in-field channel cross section transects, hydraulic modeling (HEC-RAS), and stream gauging station-derived water quality and quantity data. Cross sectional area of the 25 in-field channel cross sections increased by a mean of 17% over the 16 year study period (1998-2014), and field data indicate a general trend of degradation and widening to be present along Walnut Creek's main stem. Estimated stream discharge required to generate lateral overbank flow increased 15%, and floodplain inundation volume decreased by 37% over study duration. Estimated annual fluxes of SS and TP to floodplain storage decreased by 61 and 62% over study duration, respectively. The estimated reductions in flux to floodplain storage have potential to increase watershed export of SS and TP by 9 and 18%, respectively. Increased contributions to SS and TP export may continue as channel evolution progresses and floodplain storage opportunities continue to decline. In addition to loss of storage, higher discharges confined to the channel may have greater stream power, resulting in further enhancement of SS and TP export through accelerated bed and bank erosion. These increased contributions to watershed loads may mask SS and TP reductions achieved through edge of field practices, thus making it critical that stage and progression of channel evolution be taken into consideration when addressing sediment and phosphorus loading at the watershed scale.

Visible Light Mediated Aryl Migration by Homolytic C-N Cleavage of Aryl Amines.

The photocatalytic preparation of aminoalkylated heteroarenes from haloalkylamides via a 1,4-aryl migration from nitrogen to carbon, conceptually analogous to a radical Smiles rearrangement, is reported. This method enables the substitution of amino groups in heteroaromatic compounds with aminoalkyl motifs under mild, iridium(III)-mediated photoredox conditions. It provides rapid access to thienoazepinone, a pharmacophore present in multiple drug candidates for potential treatment of different conditions, including inflammation and psychotic disorders.

Distribution and mass of groundwater orthophosphorus in an agricultural watershed.

Orthophosphorus (OP) is the form of dissolved inorganic P that is commonly measured in groundwater studies, but the spatial distribution of groundwater OP across a watershed has rarely been assessed. In this study, we characterized spatial patterns of groundwater OP concentrations and loading rates within the 5218ha Walnut Creek watershed (Iowa) over a two-year period. Using a network of 24 shallow (<6m) monitoring wells established across watershed, OP concentrations ranged from <0.01 to 0.58mg/l in all samples (n=147) and averaged 0.084±0.107mg/l. Groundwater OP concentrations were higher in floodplains and OP mass loading rates were approximately three times higher than in uplands. We estimated that approximately 1231kg of OP is present in floodplain groundwater and 2869kg is present in upland groundwater within the shallow groundwater zone (0-5m depth). Assuming no new inputs of OP to shallow groundwater, we estimated it would take approximately eight years to flush out existing OP mass present in the system. Results suggest that conservation practices focused on reducing OP loading rates in floodplain areas may have a disproportionately large water quality benefit compared to upland areas.

Soil nutrient variability and groundwater nitrate-N in agricultural fields.

Landscape may result in uneven nutrient loads within a field. The objective of this study was to determine the influence of landscape on soil carbon and nutrient levels, and on levels of nitrate-N in groundwater. Soil samples were collected in three fields, two transects each, 30 sites in each field. The soil morphology was characterized for the profile, and soil organic carbon and nutrient levels were determined for 0-0.15 and 0.15-0.3 m depths. Each field had wells installed at three of the sites. One field showed a wide range of landscape variability, and significant effects of curvature on soil carbon and nutrient levels. Another field showed no significant effect of slope or curvature on soil carbon and nutrient levels because the nutrient levels were quite variable, including high spikes. The third field had less variable landscape trends but still showed a few significant effects on soil carbon and nutrient levels. Nitrate-N levels remained high in two of the nine wells (20 to 50 mg L-1), suggesting that additions of nitrate-N at the concave or converging sites replaced any losses. Median nitrate-N levels at the other seven well sites were lower, ranging from 8 to 17 mg L-1. Influence of landscape on soil carbon and nutrients was more detectable when the landscape factors were highly variable without excessive variability in soil nutrient properties.

Robust Prediction of Resistance to Trimethoprim in Staphylococcus aureus.

The rise of antibiotic resistance threatens modern medicine; to combat it new diagnostic methods are required. Sequencing the whole genome of a pathogen offers the potential to accurately determine which antibiotics will be effective to treat a patient. A key limitation of this approach is that it cannot classify rare or previously unseen mutations. Here we demonstrate that alchemical free energy methods, a well-established class of methods from computational chemistry, can successfully predict whether mutations in Staphylococcus aureus dihydrofolate reductase confer resistance to trimethoprim. We also show that the method is quantitatively accurate by calculating how much the most common resistance-conferring mutation, F99Y, reduces the binding free energy of trimethoprim and comparing predicted and experimentally measured minimum inhibitory concentrations for seven different mutations. Finally, by considering up to 32 free energy calculations for each mutation, we estimate its specificity and sensitivity.