Generation and transmission of interlineage recombinants in the SARS-CoV-2 pandemic.

We present evidence for multiple independent origins of recombinant SARS-CoV-2 viruses sampled from late 2020 and early 2021 in the United Kingdom. Their genomes carry single-nucleotide polymorphisms and deletions that are characteristic of the B.1.1.7 variant of concern but lack the full complement of lineage-defining mutations. Instead, the remainder of their genomes share contiguous genetic variation with non-B.1.1.7 viruses circulating in the same geographic area at the same time as the recombinants. In four instances, there was evidence for onward transmission of a recombinant-origin virus, including one transmission cluster of 45 sequenced cases over the course of 2 months. The inferred genomic locations of recombination breakpoints suggest that every community-transmitted recombinant virus inherited its spike region from a B.1.1.7 parental virus, consistent with a transmission advantage for B.1.1.7's set of mutations.

Evaluating the Effects of SARS-CoV-2 Spike Mutation D614G on Transmissibility and Pathogenicity.

Global dispersal and increasing frequency of the SARS-CoV-2 spike protein variant D614G are suggestive of a selective advantage but may also be due to a random founder effect. We investigate the hypothesis for positive selection of spike D614G in the United Kingdom using more than 25,000 whole genome SARS-CoV-2 sequences. Despite the availability of a large dataset, well represented by both spike 614 variants, not all approaches showed a conclusive signal of positive selection. Population genetic analysis indicates that 614G increases in frequency relative to 614D in a manner consistent with a selective advantage. We do not find any indication that patients infected with the spike 614G variant have higher COVID-19 mortality or clinical severity, but 614G is associated with higher viral load and younger age of patients. Significant differences in growth and size of 614G phylogenetic clusters indicate a need for continued study of this variant.

Active foam: the adaptive mechanics of 2D air-liquid foam under cyclic inflation.

Foam is a canonical example of disordered soft matter where local force balance leads to the competition of many metastable configurations. We present an experimental and theoretical framework for "active foam" where an individual voxel inflates and deflates periodically. Local periodic activity leads to irreversible and reversible T1 transitions throughout the foam, eventually reaching a reversible limit cycle. Individual vertices displace outwards and subsequently return back to their approximate original radial position; this radial displacement follows an inverse law. Surprisingly, each return trajectory does not retrace its outbound path but encloses a finite area, with a clockwise (CW) or counterclockwise (CCW) direction, which we define as a local swirl. These swirls form coherent patterns spanning the scale of the material. Using a dynamical model, we demonstrate that swirl arises from disorder in the local micro-structure. We demonstrate that disorder and strain-rate control a crossover between cooperation and competition between swirls in adjacent vertices. Over 5-10 cycles, the region around the active voxel structurally adapts from a higher-energy metastable state to a lower-energy state, locally ordering and stiffening the structure. The coherent domains of CW/CCW swirl become smaller as the system stabilizes, indicative of a process similar to the Hall-Petch effect. Finally, we introduce a statistical model that evolves edge lengths with a set of rules to explore how this class of materials adapts as a function of initial structure. Adding activity to foam couples structural disorder and adaptive dynamics to encourage the development of a new class of abiotic, cellularized active matter.

Construction and evaluation of a bioluminescent Pseudomonas aeruginosa reporter for use in preservative efficacy testing.

Preservative efficacy testing (PET) is a fundamental practice in industrial microbiology used to ensure product shelf-life and quality. To improve on current growth-based PET, bioluminescence was evaluated as a real-time bacterial viability indicator using Pseudomonas aeruginosa. Random mutagenesis of an industrial P. aeruginosa strain with a promoter-less luxCDABE mini-Tn5 was used to select a stable reporter (LUX12H5) with an un-altered growth and preservative susceptibility phenotype. Bioluminescence and viability were measured with and without preservatives (isothiazolinones, phenoxyethanol, and dimethyl dimethylol hydantoin) and an antibiotic comparator (ciprofloxacin). In the absence of antimicrobials, a good correlation between bioluminescence and viability (r2=0.92) was established. However, metabolic inhibition by isothiazolinone preservatives caused a rapid decline in light output that did not correlate to a reduced viability. Conversely, after ciprofloxacin exposure, the decline in viability was greater than that of bioluminescence. A positive attribute of the bioluminescence was the early detection of metabolic recovery and re-growth of preservative injured bacteria. Overall, while initial bioluminescence read-outs were less suited to current PET requirements, it shows promise as an early, direct indicator of bacterial regrowth in the context of long-term evaluation of preservative efficacy.

Influenza classification from short reads with VAPOR facilitates robust mapping pipelines and zoonotic strain detection for routine surveillance applications.

MOTIVATION: Influenza viruses represent a global public health burden due to annual epidemics and pandemic potential. Due to a rapidly evolving RNA genome, inter-species transmission, intra-host variation, and noise in short-read data, reads can be lost during mapping, and de novo assembly can be time consuming and result in misassembly. We assessed read loss during mapping and designed a graph-based classifier, VAPOR, for selecting mapping references, assembly validation and detection of strains of non-human origin. RESULTS: Standard human reference viruses were insufficient for mapping diverse influenza samples in simulation. VAPOR retrieved references for 257 real whole-genome sequencing samples with a mean of >99.8% identity to assemblies, and increased the proportion of mapped reads by up to 13.3% compared to standard references. VAPOR has the potential to improve the robustness of bioinformatics pipelines for surveillance and could be adapted to other RNA viruses. AVAILABILITY AND IMPLEMENTATION: VAPOR is available at https://github.com/connor-lab/vapor. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Ultrafast epithelial contractions provide insights into contraction speed limits and tissue integrity.

By definition of multicellularity, all animals need to keep their cells attached and intact, despite internal and external forces. Cohesion between epithelial cells provides this key feature. To better understand fundamental limits of this cohesion, we study the epithelium mechanics of an ultrathin (∼25 µm) primitive marine animal Trichoplax adhaerens, composed essentially of two flat epithelial layers. With no known extracellular matrix and no nerves or muscles, T. adhaerens has been claimed to be the "simplest known living animal," yet is still capable of coordinated locomotion and behavior. Here we report the discovery of the fastest epithelial cellular contractions known in any metazoan, to be found in T. adhaerens dorsal epithelium (50% shrinkage of apical cell area within one second, at least an order of magnitude faster than other known examples). Live imaging reveals emergent contractile patterns that are mostly sporadic single-cell events, but also include propagating contraction waves across the tissue. We show that cell contraction speed can be explained by current models of nonmuscle actin-myosin bundles without load, while the tissue architecture and unique mechanical properties are softening the tissue, minimizing the load on a contracting cell. We propose a hypothesis, in which the physiological role of the contraction dynamics is to resist external stresses while avoiding tissue rupture ("active cohesion"), a concept that can be further applied to engineering of active materials.

Reclassification of the Specialized Metabolite Producer Pseudomonas mesoacidophila ATCC 31433 as a Member of the Burkholderia cepacia Complex.

Pseudomonas mesoacidophila ATCC 31433 is a Gram-negative bacterium, first isolated from Japanese soil samples, that produces the monobactam isosulfazecin and the ß-lactam-potentiating bulgecins. To characterize the biosynthetic potential of P. mesoacidophila ATCC 31433, its complete genome was determined using single-molecule real-time DNA sequence analysis. The 7.8-Mb genome comprised four replicons, three chromosomal (each encoding rRNA) and one plasmid. Phylogenetic analysis demonstrated that P. mesoacidophila ATCC 31433 was misclassified at the time of its deposition and is a member of the Burkholderia cepacia complex, most closely related to Burkholderia ubonensis The sequenced genome shows considerable additional biosynthetic potential; known gene clusters for malleilactone, ornibactin, isosulfazecin, alkylhydroxyquinoline, and pyrrolnitrin biosynthesis and several uncharacterized biosynthetic gene clusters for polyketides, nonribosomal peptides, and other metabolites were identified. Furthermore, P. mesoacidophila ATCC 31433 harbors many genes associated with environmental resilience and antibiotic resistance and was resistant to a range of antibiotics and metal ions. In summary, this bioactive strain should be designated B. cepacia complex strain ATCC 31433, pending further detailed taxonomic characterization.IMPORTANCE This work reports the complete genome sequence of Pseudomonas mesoacidophila ATCC 31433, a known producer of bioactive compounds. Large numbers of both known and novel biosynthetic gene clusters were identified, indicating that P. mesoacidophila ATCC 31433 is an untapped resource for discovery of novel bioactive compounds. Phylogenetic analysis demonstrated that P. mesoacidophila ATCC 31433 is in fact a member of the Burkholderia cepacia complex, most closely related to the species Burkholderia ubonensis Further investigation of the classification and biosynthetic potential of P. mesoacidophila ATCC 31433 is warranted.

Discovery and Biosynthesis of Gladiolin: A Burkholderia gladioli Antibiotic with Promising Activity against Mycobacterium tuberculosis.

An antimicrobial activity screen of Burkholderia gladioli BCC0238, a clinical isolate from a cystic fibrosis patient, led to the discovery of gladiolin, a novel macrolide antibiotic with potent activity against Mycobacterium tuberculosis H37Rv. Gladiolin is structurally related to etnangien, a highly unstable antibiotic from Sorangium cellulosum that is also active against Mycobacteria. Like etnangien, gladiolin was found to inhibit RNA polymerase, a validated drug target in M. tuberculosis. However, gladiolin lacks the highly labile hexaene moiety of etnangien and was thus found to possess significantly increased chemical stability. Moreover, gladiolin displayed low mammalian cytotoxicity and good activity against several M. tuberculosis clinical isolates, including four that are resistant to isoniazid and one that is resistant to both isoniazid and rifampicin. Overall, these data suggest that gladiolin may represent a useful starting point for the development of novel drugs to tackle multidrug-resistant tuberculosis. The B. gladioli BCC0238 genome was sequenced using Single Molecule Real Time (SMRT) technology. This resulted in four contiguous sequences: two large circular chromosomes and two smaller putative plasmids. Analysis of the chromosome sequences identified 49 putative specialized metabolite biosynthetic gene clusters. One such gene cluster, located on the smaller of the two chromosomes, encodes a trans-acyltransferase (trans-AT) polyketide synthase (PKS) multienzyme that was hypothesized to assemble gladiolin. Insertional inactivation of a gene in this cluster encoding one of the PKS subunits abrogated gladiolin production, confirming that the gene cluster is responsible for biosynthesis of the antibiotic. Comparison of the PKSs responsible for the assembly of gladiolin and etnangien showed that they possess a remarkably similar architecture, obfuscating the biosynthetic mechanisms responsible for most of the structural differences between the two metabolites.

Molecular epidemiology of Pseudomonas aeruginosa in an unsegregated bronchiectasis cohort sharing hospital facilities with a cystic fibrosis cohort.

While Pseudomonas aeruginosa (PA) cross-infection is well documented among patients with cystic fibrosis (CF), the equivalent risk among patients with non-CF bronchiectasis (NCFB) is unclear, particularly those managed alongside patients with CF. We performed analysis of PA within a single centre that manages an unsegregated NCFB cohort alongside a segregated CF cohort. We found no evidence of cross-infection between the two cohorts or within the segregated CF cohort. However, within the unsegregated NCFB cohort, evidence of cross-infection was found between three (of 46) patients. While we do not presently advocate any change in the management of our NCFB cohort, longitudinal surveillance is clearly warranted.

Optimizing 1-µs-Resolution Single-Molecule Force Spectroscopy on a Commercial Atomic Force Microscope.

Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is widely used to mechanically measure the folding and unfolding of proteins. However, the temporal resolution of a standard commercial cantilever is 50-1000 µs, masking rapid transitions and short-lived intermediates. Recently, SMFS with 0.7-µs temporal resolution was achieved using an ultrashort (L = 9 µm) cantilever on a custom-built, high-speed AFM. By micromachining such cantilevers with a focused ion beam, we optimized them for SMFS rather than tapping-mode imaging. To enhance usability and throughput, we detected the modified cantilevers on a commercial AFM retrofitted with a detection laser system featuring a 3-µm circular spot size. Moreover, individual cantilevers were reused over multiple days. The improved capabilities of the modified cantilevers for SMFS were showcased by unfolding a polyprotein, a popular biophysical assay. Specifically, these cantilevers maintained a 1-µs response time while eliminating cantilever ringing (Q ≅ 0.5). We therefore expect such cantilevers, along with the instrumentational improvements to detect them on a commercial AFM, to accelerate high-precision AFM-based SMFS studies.

Rapid Detection of Emerging Pathogens and Loss of Microbial Diversity Associated with Severe Lung Disease in Cystic Fibrosis.

Respiratory infection in cystic fibrosis (CF) is polymicrobial, but standard sputum microbiology does not account for the lung microbiome or detect changes in microbial diversity associated with disease. As a clinically applicable CF microbiome surveillance scheme, total sputum nucleic acids isolated by a standard high-throughput robotic method for accredited viral diagnosis were profiled for bacterial diversity using ribosomal intergenic spacer analysis (RISA) PCR. Conventional culture and RISA were performed on 200 paired sputum samples from 93 CF adults; pyrosequencing of the 16S rRNA gene was applied to 59 patients to systematically determine bacterial diversity. Compared to the microbiology data, RISA profiles clustered into two groups: the emerging nonfermenting Gram-negative organisms (eNFGN) and Pseudomonas groups. Patients who were culture positive for Burkholderia, Achromobacter, Stenotrophomonas, and Ralstonia clustered within the eNFGN group. Pseudomonas group RISA profiles were associated with Pseudomonas aeruginosa culture-positive patients. Sequence analysis confirmed the abundance of eNFGN genera and Pseudomonas within these respective groups. Low bacterial diversity was associated with severe lung disease (P < 0.001) and the presence of Burkholderia (P < 0.001). An absence of Streptococcus (P < 0.05) occurred in individuals with lung function in the lowest quartile. In summary, nucleic acids isolated from CF sputum can serve as a single template for both molecular virology and bacteriology, with a RISA PCR rapidly detecting the presence of dominant eNFGN pathogens or P. aeruginosa missed by culture (11% of cases). We provide guidance for how this straightforward CF microbiota profiling scheme may be adopted by clinical laboratories.

Ultrastable measurement platform: sub-nm drift over hours in 3D at room temperature.

Advanced optical traps can probe single molecules with Ångstrom-scale precision, but drift limits the utility of these instruments. To achieve Å-scale stability, a differential measurement scheme between a pair of laser foci was introduced that substantially exceeds the inherent mechanical stability of various types of microscopes at room temperature. By using lock-in detection to measure both lasers with a single quadrant photodiode, we enhanced the differential stability of this optical reference frame and thereby stabilized an optical-trapping microscope to 0.2 Å laterally over 100 s based on the Allan deviation. In three dimensions, we achieved stabilities of 1 Å over 1,000 s and 1 nm over 15 h. This stability was complemented by high measurement bandwidth (100 kHz). Overall, our compact back-scattered detection enables an ultrastable measurement platform compatible with optical traps, atomic force microscopy, and optical microscopy, including super-resolution techniques.

Spatial methods for infectious disease outbreak investigations: systematic literature review.

Investigations of infectious disease outbreaks are conventionally framed in terms of person, time and place. Although geographic information systems have increased the range of tools available, spatial analyses are used relatively infrequently. We conducted a systematic review of published reports of outbreak investigations worldwide to estimate the prevalence of spatial methods, describe the techniques applied and explore their utility. We identified 80 reports using spatial methods published between 1979 and 2013, ca 0.4% of the total number of published outbreaks. Environmental or waterborne infections were the most commonly investigated, and most reports were from the United Kingdom. A range of techniques were used, including simple dot maps, cluster analyses and modelling approaches. Spatial tools were usefully applied throughout investigations, from initial confirmation of the outbreak to describing and analysing cases and communicating findings. They provided valuable insights that led to public health actions, but there is scope for much wider implementation and development of new methods.

Atomic force microscopy with sub-picoNewton force stability for biological applications.

Atomic force microscopy (AFM) is widely used in the biological sciences. Despite 25 years of technical developments, two popular modes of bioAFM, imaging and single molecule force spectroscopy, remain hindered by relatively poor force precision and stability. Recently, we achieved both sub-pN force precision and stability under biologically useful conditions (in liquid at room temperature). Importantly, this sub-pN level of performance is routinely accessible using a commercial cantilever on a commercial instrument. The two critical results are that (i) force precision and stability were limited by the gold coating on the cantilevers, and (ii) smaller yet stiffer cantilevers did not lead to better force precision on time scales longer than 25 ms. These new findings complement our previous work that addressed tip-sample stability. In this review, we detail the methods needed to achieve this sub-pN force stability and demonstrate improvements in force spectroscopy and imaging when using uncoated cantilevers. With this improved cantilever performance, the widespread use of nonspecific biomolecular attachments becomes a limiting factor in high-precision studies. Thus, we conclude by briefly reviewing site-specific covalent-immobilization protocols for linking a biomolecule to the substrate and to the AFM tip.

Genomic epidemiology reveals geographical clustering of multidrug-resistant Escherichia coli ST131 associated with bacteraemia in Wales.

Antibiotic resistance is a significant global public health concern. Uropathogenic Escherichia coli sequence type (ST)131, a widely prevalent multidrug-resistant clone, is frequently associated with bacteraemia. This study investigates third-generation cephalosporin resistance in bloodstream infections caused by E. coli ST131. From 2013-2014 blood culture surveillance in Wales, 142 E. coli ST131 genomes were studied alongside global data. All three major ST131 clades were represented across Wales, with clade C/H30 predominant (n = 102/142, 71.8%). Consistent with global findings, Welsh strains of clade C/H30 contain ß-lactamase genes from the blaCTX-M-1 group (n = 65/102, 63.7%), which confer resistance to third-generation cephalosporins. Most Welsh clade C/H30 genomes belonged to sub-clade C2/H30Rx (58.3%). A Wales-specific sub-lineage, named GB-WLS.C2, diverged around 1996-2000. An introduction to North Wales around 2002 led to a localised cluster by 2009, depicting limited genomic diversity within North Wales. This investigation emphasises the value of genomic epidemiology, allowing the detection of genetically similar strains in local areas, enabling targeted and timely public health interventions.

Intravenous antibiotics reduce the presence of Aspergillus in adult cystic fibrosis sputum.

BACKGROUND: Pseudomonas aeruginosa and Aspergillus fumigatus frequently co-colonise the airways of patients with cystic fibrosis (CF). This study aimed to assess the impact of short-term administration of intravenous antipseudomonal antibiotics during CF exacerbations on the presence of Aspergillus. METHODS: Pre- and post-antibiotic sputum samples from 26 adult patients with CF and chronic Pseudomonas colonisation were analysed for the presence of Aspergillus by fungal culture, real-time PCR and galactomannan antigen (GM). Lung function (forced expiratory volume in 1 s and forced vital capacity % predicted) and blood levels of total IgE, specific A fumigatus IgE and specific A fumigatus IgG were measured at the start and end of antibiotics. Respiratory viral real-time PCR and bacterial community profiling using ribosomal intergenic spacer analysis (RISA) were performed to estimate concurrent changes in the lung microbiome. RESULTS: Aspergillus PCR and GM were more sensitive than culture in detecting Aspergillus species (culture 8%, GM 31%, PCR 77%). There was a significant decline in the presence of Aspergillus, measured both by PCR and GM index, following antibacterial therapy (PCR: median increase in crossing threshold 1.7 (IQR 0.5-3.8), p<0.001; GM: median fall in GM index 0.7 (IQR 0.4-1.6), p=0.016). All patients improved clinically with a significant increase in lung function (p<0.0001). RISA community analysis showed large changes in bacterial community similarity in 67% of patients following antibiotics. Viral RT-PCR demonstrated the presence of a concurrent respiratory virus in 27% of patients. CONCLUSIONS: Intravenous antibiotics targeting Pseudomonas during CF pulmonary exacerbations have a negative impact on the presence of Aspergillus in sputum samples.

Optimizing bead size reduces errors in force measurements in optical traps.

Optical traps are used to measure force (F) over a wide range (0.01 to 1,000 pN). Variations in bead radius (r) hinder force precision since trap stiffness (k(trap)) varies as r3 when r is small. Prior work has shown k(trap) is maximized when r is approximately equal to the beam waist (w0), which on our instrument was ~400 nm when trapping with a 1064-nm laser. In this work, we show that by choosing r ≈w0, we improved the force precision by 2.8-fold as compared to a smaller bead (250 nm). This improvement in force precision was verified by pulling on a canonical DNA hairpin. Thus, by using an optimum bead size, one can simultaneously maximize k(trap) while minimizing errors in F.

Routine and timely sub-picoNewton force stability and precision for biological applications of atomic force microscopy.

Force drift is a significant, yet unresolved, problem in atomic force microscopy (AFM). We show that the primary source of force drift for a popular class of cantilevers is their gold coating, even though they are coated on both sides to minimize drift. Drift of the zero-force position of the cantilever was reduced from 900 nm for gold-coated cantilevers to 70 nm (N = 10; rms) for uncoated cantilevers over the first 2 h after wetting the tip; a majority of these uncoated cantilevers (60%) showed significantly less drift (12 nm, rms). Removing the gold also led to ∼10-fold reduction in reflected light, yet short-term (0.1-10 s) force precision improved. Moreover, improved force precision did not require extended settling; most of the cantilevers tested (9 out of 15) achieved sub-pN force precision (0.54 ± 0.02 pN) over a broad bandwidth (0.01-10 Hz) just 30 min after loading. Finally, this precision was maintained while stretching DNA. Hence, removing gold enables both routine and timely access to sub-pN force precision in liquid over extended periods (100 s). We expect that many current and future applications of AFM can immediately benefit from these improvements in force stability and precision.

Design and Biological Evaluation of Piperazine-Bearing Nitrobenzamide Hypoxia/GDEPT Prodrugs: The Discovery of CP-506.

Off-target aerobic activation of PR-104A by human aldo-keto reductase 1C3 (AKR1C3) has confounded the development of this dual hypoxia/gene therapy prodrug. Previous attempts to design prodrugs resistant to AKR1C3 activation have resulted in candidates that require further optimization. Herein we report the evaluation of a lipophilic series of PR-104A analogues in which a piperazine moiety has been introduced to improve drug-like properties. Octanol-water partition coefficients (LogD7.4) spanned >2 orders of magnitude. 2D antiproliferative and 3D multicellular clonogenic assays using isogenic HCT116 and H1299 cells confirmed that all examples were resistant to AKR1C3 metabolism while producing an E. coli NfsA nitroreductase-mediated bystander effect. Prodrugs 16, 17, and 20 demonstrated efficacy in H1299 xenografts where only a minority of tumor cells express NfsA. These prodrugs and their bromo/mesylate counterparts (25-27) were also evaluated for hypoxia-selective cell killing in vitro. These results in conjunction with stability assays recommended prodrug 26 (CP-506) for Phase I/II clinical trial.