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Diphtheria is a respiratory disease caused by Corynebacterium diphtheriae. While the toxin-based vaccine has helped control outbreaks of the disease since the mid-20th century there has been an increase in cases in recent years, including systemic infections caused by non-toxigenic C. diphtheriae strains. Here we describe the first study of gene essentiality in C. diphtheriae, providing the most-dense Transposon Directed Insertion Sequencing (TraDIS) library in the phylum Actinobacteriota. This high-density library has allowed the identification of conserved genes across the genus and phylum with essential function and enabled the elucidation of essential domains within the resulting proteins including those involved in cell envelope biogenesis. Validation of these data through protein mass spectrometry identified hypothetical and uncharacterized proteins in the proteome which are also represented in the vaccine. These data are an important benchmark and useful resource for the Corynebacterium, Mycobacterium, Nocardia and Rhodococcus research community. It enables the identification of novel antimicrobial and vaccine targets and provides a basis for future studies of Actinobacterial biology. Author summary: Corynebacterium diphtheriae causes both toxin-mediated diphtheria and non-toxigenic invasive infections. Despite a vaccine to protect against diphtheria, case numbers for both invasive and diphtherial disease have increased over the last decade. Furthermore, an increase in antibiotic resistant strains are being isolated from patients. It's clear that additional treatment strategies for this organism will be needed in the future. Using high-throughput mutagenesis, this work presents the densest library of mutants for any Corynebacterium sp.. This work identifies the essential genome of C. diphtheriae; an important classification as these genes are often the target of therapeutic intervention. We identify highly conserved genes and species-specific genes unique to pathogens. This data presents an important benchmark and focus for the future development of therapeutic options. Of particular significance is the identification of uncharacterized, conserved proteins within the Diphtheria vaccine. [ABSTRACT FROM AUTHOR]

The gram-positive filamentous bacterium Streptomyces coelicolor has a complex developmental cycle with three distinct phases: growth of the substrate mycelium, development of reproductive structures called aerial hyphae, and differentiation of these aerial filaments into long chains of exospores. During a transposon mutagenesis screen, we identified a novel gene (devA) required for proper development. The devA mutant produced only rare aerial hyphae, and those that were produced developed aberrant spore chains that were much shorter than wild-type chains and had misplaced septa. devA encodes a member of the GntR superfamily, a class of transcriptional regulators that typically respond to metabolite effector molecules. devA forms an operon with the downstream gene devB, which encodes a putative hydrolase that is also required for aerial mycelium formation on R5 medium. S1 nuclease protection analysis showed that transcription from the single devA promoter was temporally associated with vegetative growth, and enhanced green fluorescent protein transcriptional fusions showed that transcription was spatially confined to the substrate hyphae in the wild type. In contrast, devAB transcript levels were dramatically upregulated in a devA mutant and the devA promoter was also active in aerial hyphae and spores in this background, suggesting that DevA might negatively regulate its own production. This suggestion was confirmed by gel mobility shift assays that showed that DevA binds its own promoter region in vitro. [ABSTRACT FROM AUTHOR]

Plant growth-promoting endophytic (PGPE) actinomycetes have been known to enhance plant growth and mitigate plant from abiotic stresses via their PGP-traits. In this study, PGPE Streptomyces sp. GKU 895 promoted growth and alleviated salt tolerance of salt-susceptible rice cultivar IR29 by augmentation of plant weight and declined ROS after irrigation with 150 mM NaCl in a pot experiment. Transcriptome analysis of IR29 exposed to the combination of strain GKU 895 and salinity demonstrated up and downregulated differentially expressed genes (DEGs) classified by gene ontology and plant reactome. Streptomyces sp. GKU 895 induced changes in expression of rice genes including transcription factors under salt treatment which involved in growth and development, photosynthesis, plant hormones, ROS scavenging, ion transport and homeostasis, and plant–microbe interactions regarding pathogenesis- and symbiosis-related proteins. Taken together, these data demonstrate that PGPE Streptomyces sp. GKU 895 colonized and enhanced growth of rice IR29 and triggered salt tolerance phenotype. Our findings suggest that utilisation of beneficial endophytes in the saline fields could allow for the use of such marginal soils for growing rice and possibly other crops. [ABSTRACT FROM AUTHOR]

The global increase in antimicrobial-resistant infections means that there is a need to develop new antimicrobial molecules and strategies to combat the issue. Aurodox is a linear polyketide natural product that is produced by Streptomyces goldiniensis, yet little is known about aurodox biosynthesis or the nature of the biosynthetic gene cluster (BGC) that encodes its production. To gain a deeper understanding of aurodox biosynthesis by S. goldiniensis, the whole genome of the organism was sequenced, revealing the presence of an 87 kb hybrid polyketide synthase/non-ribosomal peptide synthetase (PKS/NRPS) BGC. The aurodox BGC shares significant homology with the kirromycin BGC from S. collinus T= 365. However, the genetic organization of the BGC differs significantly. The candidate aurodox gene cluster was cloned and expressed in a heterologous host to demonstrate that it was responsible for aurodox biosynthesis and disruption of the primary PKS gene (aurAI) abolished aurodox production. These data supported a model whereby the initial core biosynthetic reactions involved in aurodox biosynthesis followed that of kirromycin. Cloning aurM* from S. goldiniensis and expressing this in the kirromycin producer S. collinus T= 365 enabled methylation of the pyridone group, suggesting this is the last step in biosynthesis. This methylation step is also sufficient to confer the unique type III secretion system inhibitory properties to aurodox. [ABSTRACT FROM AUTHOR]

The results of infrared spectroscopic investigations into the band assignments, vibrational relaxation, and solvation dynamics of the common anti-tuberculosis treatment Isoniazid (INH) are reported. INH is known to inhibit InhA, a 2-trans-enoyl-acyl carrier protein reductase enzyme responsible for the maintenance of cell walls in Mycobacterium tuberculosis but as new drug-resistant strains of the bacterium appear, next-generation therapeutics will be essential to combat the rise of the disease. Small molecules such as INH offer the potential for use as a biomolecular marker through which ultrafast multidimensional spectroscopies can probe drug binding and so inform design strategies but a complete characterization of the spectroscopy and dynamics of INH in solution is required to inform such activity. Infrared absorption spectroscopy, in combination with density functional theory calculations, is used to assign the vibrational modes of INH in the 1400-1700 cm-1 region of the infrared spectrum while ultrafast multidimensional spectroscopy measurements determine the vibrational relaxation dynamics and the effects of solvation via spectral diffusion of the carbonyl stretching vibrational mode. These results are discussed in the context of previous linear spectroscopy studies on solid-phase INH and its usefulness as a biomolecular probe. [ABSTRACT FROM AUTHOR]

Accurate and rapid diagnostic tests are critical to reducing the impact of SARS-CoV-2. This study presents early, but promising measurements of SARS-CoV-2 using the ACE2 enzyme as the recognition element to achieve clinically relevant detection. The test provides a scalable route to sensitive, specific, rapid and low cost mass testing. [ABSTRACT FROM AUTHOR]

The transport of charged molecules across biological membranes faces the dual problem of accommodating charges in a highly hydrophobic environment while maintaining selective substrate translocation. This has been the subject of a particular controversy for the exchange of ammonium across cellular membranes, an essential process in all domains of life. Ammonium transport is mediated by the ubiquitous Amt/Mep/Rh transporters that includes the human Rhesus factors. Here, using a combination of electrophysiology, yeast functional complementation and extended molecular dynamics simulations, we reveal a unique two-lane pathway for electrogenic NH4 + transport in two archetypal members of the family, the transporters AmtB from Escherichia coli and Rh50 from Nitrosomonas europaea. The pathway underpins a mechanism by which charged H+ and neutral NH3 are carried separately across the membrane after NH4 + deprotonation. This mechanism defines a new principle of achieving transport selectivity against competing ions in a biological transport process. [ABSTRACT FROM AUTHOR]

Covering: 2008 up to 2019 The forces of biochemical adaptive evolution operate at the level of genes, manifesting in complex phenotypes and the global biodiversity of proteins and metabolites. While evolutionary histories have been deciphered for some other complex traits, the origins of natural product biosynthesis largely remain a mystery. This fundamental knowledge gap is surprising given the many decades of research probing the genetic, chemical, and biophysical mechanisms of bacterial natural product biosynthesis. Recently, evolutionary thinking has begun to permeate this otherwise mechanistically dominated field. Natural products are now sometimes referred to as 'specialized' rather than 'secondary' metabolites, reinforcing the importance of their biological and ecological functions. Here, we review known evolutionary mechanisms underlying the overwhelming chemical diversity of bacterial secondary metabolism, focusing on enzyme promiscuity and the evolution of enzymatic domains that enable metabolic traits. We discuss the mechanisms that drive the assembly of natural product biosynthetic gene clusters and propose formal definitions for 'specialized' and 'secondary' metabolism. We further explore how biosynthetic gene clusters evolve to synthesize related molecular species, and in turn how the biological and ecological roles that emerge from metabolic diversity are acted on by selection. Finally, we reconcile chemical, functional, and genetic data into an evolutionary model, the dynamic chemical matrix evolutionary hypothesis, in which the relationships between chemical distance, biomolecular activity, and relative fitness shape adaptive landscapes. [ABSTRACT FROM AUTHOR]

Improvements in hygiene and health management have driven significant increases in human lifespan over the last 50 years. Frustratingly however, this extension of lifespan has not been matched by equivalent improvements in late-life health, not least due to the global pandemic in type-2 diabetes, obesity and cardiovascular disease, all ageing-associated conditions exacerbated and accelerated by widespread adoption of the high calorie Western diet (HCD). Recently, evidence has begun to emerge that parasitic worm infection might protect against such ageing-associated co-morbidities, as a serendipitous side-effect of their evolution of pro-survival, anti-inflammatory mechanisms. As a novel therapeutic strategy, we have therefore investigated the potential of ES-62, an anti-inflammatory secreted product of the filarial nematode Acanthocheilonema viteae, to improve healthspan (the period of life before diseases of ageing appear) by targeting the chronic inflammation that drives metabolic dysregulation underpinning ageing-induced ill-health. We administered ES-62 subcutaneously (at a dose of 1 μg/week) to C57BL/6 mice undergoing HCD-accelerated ageing throughout their lifespan, while subjecting the animals to analysis of 120 immunometabolic responses at various time-points. ES-62 improved a number of inflammatory markers, but markedly, a range of pathophysiological, metabolic and microbiome parameters of ageing were also successfully targeted. Notably, ES-62-mediated promotion of healthspan in male and female HCD-mice was associated with different mechanisms and reflecting this, machine learning modelling identified sex-specific signatures predictive of ES-62 action against HCD-accelerated ageing. Remarkably, ES-62 substantially increased the median survival of male HCD-mice. This was not the case with female animals and unexpectedly, this difference between the two sexes could not be explained in terms of suppression of the chronic inflammation driving ageing, as ES-62 tended to be more effective in reducing this in female mice. Rather, the difference appeared to be associated with ES-62's additional ability to preferentially promote a healthier gut-metabolic tissue axis in male animals. Author summary: Infection with parasitic worms is increasingly considered to protect against chronic inflammatory disorders, including obesity, type-2 diabetes and cardiovascular disease. Although previously associated with old age, these ailments are ever more prevalent even in young people, due to the global adoption of the "Western" lifestyle and high calorie diet (HCD; high sugar and high fat). Rather than developing live worm therapies, our approach focuses on the potential of ES-62, a molecule secreted by the parasitic worm Acanthocheilonema viteae that we have previously shown to protect against inflammatory conditions like allergic asthma and arthritis in mice. We found that a small weekly dose of ES-62 promoted late-life wellbeing in HCD-fed mice of both sexes and substantially extended the life of obese male mice (by over 70 days). Machine learning modelling of its impact on the complex immunometabolic networks underpinning obesity showed that health-promoting effects of ES-62 could not simply be explained by its anti-inflammatory actions but encompassed protection against adipose, liver and gut pathology. The target signatures identified may thus represent a starting point for developing novel ES-62-based approaches to improving health during ageing and increasing lifespan in humans. [ABSTRACT FROM AUTHOR]

A tandem enzymatic strategy to enhance the scope of C‐alkylation of small molecules via the in situ formation of S‐adenosyl methionine (SAM) cofactor analogues is described. A solvent‐exposed channel present in the SAM‐forming enzyme SalL tolerates 5′‐chloro‐5′‐deoxyadenosine (ClDA) analogues modified at the 2‐position of the adenine nucleobase. Coupling SalL‐catalyzed cofactor production with C‐(m)ethyl transfer to coumarin substrates catalyzed by the methyltransferase (MTase) NovO forms C‐(m)ethylated coumarins in superior yield and greater substrate scope relative to that obtained using cofactors lacking nucleobase modifications. Establishing the molecular determinants that influence C‐alkylation provides the basis to develop a late‐stage enzymatic platform for the preparation of high value small molecules. [ABSTRACT FROM AUTHOR]

Abstract This paper outlines a simple label-free sensor system for the sensitive, real time measurement of an important protein biomarker of sepsis, using a novel microelectrode integrated onto a needle shaped substrate. Sepsis is a life threatening condition with a high mortality rate, which is characterised by dysregulation of the immune response following infection, leading to organ failure and cardiovascular collapse if untreated. Currently, sepsis testing is typically carried out by taking blood samples which are sent to a central laboratory for processing. Analysis times can be between 12 and 72 h making it notoriously difficult to diagnose and treat patients in a timely manner. The pathobiology of sepsis is becoming increasingly well understood and clinically relevant biomarkers are emerging, which could be used in conjunction with a biosensor to support real time diagnosis of sepsis. In this context, microelectrodes have the analytical advantages of reduced iR drop, enhanced signal to noise ratio, simplified quantification and the ability to measure in hydrodynamic situations, such as the bloodstream. In this study, arrays of eight (r = 25 µm) microelectrodes were fabricated onto needle shaped silicon substrates and electrochemically characterised in order to confirm successful sensor production and to verify whether the observed behaviour agreed with established theory. After this, the electrodes were functionalised with an antibody for interleukin-6 (IL-6) which is a protein involved in the immune response to infection and whose levels in the blood increase during progression of sepsis. The results show that IL-6 is detectable at physiologically relevant levels (pg/mL) with incubation times as short as 2.5 min. Electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) measurements were taken and DPV was concluded to be the more suitable form of measurement. In contrast to the accepted view for macro electrodes that the impedance increases upon antigen bind, we report herein a decrease in the micro electrode impedance upon binding. The small size of the fabricated devices and their needle shape make them ideal for either point of care testing or insertion into blood vessels for continuous sepsis monitoring. Highlights • An array of 8 × 50 µm gold electrodes were fabricated onto a needle shaped substrate. • The device was electrochemically characterised to confirm successful fabrication and demonstrate microelectrode behaviour. • Interleukin 6 (IL-6) antibody was immobilised onto the electrode surface. • EIS & DPV measurements were demonstrated as effective methods for detection of antibody- antigen binding. • A dose response curve was established showing IL-6 could be measured at normal and elevated levels using DPV. [ABSTRACT FROM AUTHOR]

The flagellar systems of Escherichia coli and Salmonella enterica exhibit a significant level of genetic and functional synteny. Both systems are controlled by the flagellar specific master regulator FlhD4C2. Since the early days of genetic analyses of flagellar systems it has been known that E. coli flhDC can complement a ∆flhDC mutant in S. enterica. The genomic revolution has identified how genetic changes to transcription factors and/or DNA binding sites can impact the phenotypic outcome across related species. We were therefore interested in asking: using modern tools to interrogate flagellar gene expression and assembly, what would the impact be of replacing the flhDC coding sequences in S. enterica for the E. coli genes at the flhDC S. entercia chromosomal locus? We show that even though all strains created are motile, flagellar gene expression is measurably lower when flhDCEC are present. These changes can be attributed to the impact of FlhD4C2 DNA recognition and the protein-protein interactions required to generate a stable FlhD4C2 complex. Furthermore, our data suggests that in E. coli the internal flagellar FliT regulatory feedback loop has a marked difference with respect to output of the flagellar systems. We argue due diligence is required in making assumptions based on heterologous expression of regulators and that even systems showing significant synteny may not behave in exactly the same manner. [ABSTRACT FROM AUTHOR]

Background: Diphtheria remains a major public health concern with multiple recent outbreaks around the world. Moreover, invasive non-toxigenic strains have emerged globally causing severe infections. A diphtheria epidemic in the former Soviet Union in the 1990s resulted in ~5000 deaths. In this study, we analysed the genome sequences of a collection of 93 C. diphtheriae strains collected during and after this outbreak (1996 - 2014) in a former Soviet State, Belarus to understand the evolutionary dynamics and virulence capacities of these strains. Results: C. diphtheriae strains from Belarus belong to ten sequence types (STs). Two major clones, non-toxigenic ST5 and toxigenic ST8, encompassed 76% of the isolates that are associated with sore throat and diphtheria in patients, respectively. Core genomic diversity is limited within outbreak-associated ST8 with relatively higher mutation rates (8.9 × 10-7 substitutions per strain per year) than ST5 (5.6 × 10-7 substitutions per strain per year) where most of the diversity was introduced by recombination. A variation in the virulence gene repertoire including the presence of tox gene is likely responsible for pathogenic differences between different strains. However, strains with similar virulence potential can cause disease in some individuals and remain asymptomatic in others. Eight synonymous single nucleotide polymorphisms were observed between the tox genes of the vaccine strain PW8 and other toxigenic strains of ST8, ST25, ST28, ST41 and non-toxigenic tox gene-bearing (NTTB) ST40 strains. A single nucleotide deletion at position 52 in the tox gene resulted in the frameshift in ST40 isolates, converting them into NTTB strains. Conclusions: Non-toxigenic C. diphtheriae ST5 and toxigenic ST8 strains have been endemic in Belarus both during and after the epidemic in 1990s. A high vaccine coverage has effectively controlled diphtheria in Belarus; however, non-toxigenic strains continue to circulate in the population. Recombination is an important evolutionary force in shaping the genomic diversity in C. diphtheriae. However, the relative role of recombination and mutations in diversification varies between different clones. [ABSTRACT FROM AUTHOR]

Rhodococcus defluvii strain Ca11 was isolated from a bioreactor involved in extensive phosphorus removal. We have sequenced the whole genome of this strain, and our comparative genomic and phylogenetic analyses confirm its close relatedness with Rhodococcus equi ( Rhodococcus hoagii) strains, which share >80 % of the gene content. The R. equi virulence plasmid is absent though most of the chromosomal R. equi virulence-associated genes are present in R. defluvii Ca11. These data suggest that although R. defluvii is an environmental organism, it has the potential to colonize animal hosts. [ABSTRACT FROM AUTHOR]

Rhodococcus equi (' Prescottella equi') is a pathogenic actinomycete primarily infecting horses but has emerged as an opportunistic human pathogen. We have sequenced the genome of the type strain of this species, R. equi strain C7T, and compared the genome with that of another foal isolate 103S and of a human isolate ATCC 33707. The R. equi strains are closely related to each other and yet distantly related to other rhodococci and Nocardia brasiliensis. The comparison of gene contents among R. equi strains revealed minor differences that could be associated with host adaptation from foals to humans, including the presence of a paa operon in the human isolate, which is potentially involved in pathogenesis. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen particularly associated with the inherited disease cystic fibrosis ( CF). Pseudomonas aeruginosa is well known to have a large and adaptable genome that enables it to colonise a wide range of ecological niches. Here, we have used a comparative genomics approach to identify changes that occur during infection of the CF lung. We used the mucoid phenotype as an obvious marker of host adaptation and compared these genomes to analyse SNPs, indels and islands within near-isogenic pairs. To commence the correction of the natural bias towards clinical isolates in genomics studies and to widen our understanding of the genomic diversity of P. aeruginosa, we included four environmental isolates in our analysis. Our data suggest that genome plasticity plays an important role in chronic infection and that the strains sequenced in this study are representative of the two major phylogenetic groups as determined by core genome SNP analysis. [ABSTRACT FROM AUTHOR]

Antibiotic resistance is one of the most pressing scientific and societal issues of our age. There is an urgent need to develop new diagnostic technologies which can quickly determine whether an infection is susceptible or resistant to different treatments so that rational antibiotic prescribing can take place. The main objective of the study was therefore to develop a rapid, simple, cost effective and comprehensive antibiotic susceptibility/resistance test based on rapid nucleic acid profiling. To do so, we integrated a microelectrode sensor within a microfluidic chip that combined bacterial incubation, lysis, and electrochemical detection chambers in a single simple set-up. As a case study, Mycobacterium smegmatis was investigated as a surrogate organism for Mycobacterium tuberculosis. The novelty of the work lies in developed capability of performing incubation, lysis, fragmentation, and detection process in a comprehensive yet simple lab-on-a-chip device called 'MycoCHIP'. A gold microelectrode in combination with a specifically developed nucleic acid probe sequence for the 16SrRNA region of the mycobacterial genome were employed to monitor M. smegmatis nucleic acid sequences using Differential Pulse Voltammetry (DPV) and Square-Wave Voltammetry (SWV). The results demonstrated that it was possible to detect bacterial nucleic acid sequences and distinguish antibiotic incubated (Ab-i) cells from nonincubated (Ab-n) cells on MycoCHIP with a label-free molecular detection. The antibiotic susceptibility test showed that through measuring 16SrRNA levels from M. smegmatis ,sensitivity to antibiotic was apparent after 24 h incubation, with a developed protocol representing a potential approach to determining antibiotic susceptibility more quickly, reliable and economically than current methods. [Display omitted] • Development of an on-chip label-free molecular-based antibiotic susceptibility test for the compariatively slow-growing M. smegmatis. • MycoCHIP consists of an incubation incubation , lysis and a microelectrode functionalised with nucleic acid sequences specific for the bacteria. • Electrochemistry measurements proved that the rapid, qualitative analysis of such slow-growing bacteria was possible. • MycoCHIP proves to be capable of performing antibiotic susceptibility testing of M. smegmatis on a single chip within 24 h. • The valves developed on the chip made the opening and closing operations of the microchannels. [ABSTRACT FROM AUTHOR]

Branching networks are ubiquitous in nature and their growth often responds to environmental cues dynamically. Using the antibiotic-producing soil bacterium Streptomyces as a model we have developed a flexible mathematical model platform for the study of branched biological networks. Streptomyces form large aggregates in liquid culture that can impair industrial antibiotic fermentations. Understanding the features of these could aid improvement of such processes. The model requires relatively few experimental values for parameterisation, yet delivers realistic simulations of Streptomyces pellet and is able to predict features, such as the density of hyphae, the number of growing tips and the location of antibiotic production within a pellet in response to pellet size and external nutrient supply. The model is scalable and will find utility in a range of branched biological networks such as angiogenesis, plant root growth and fungal hyphal networks. [ABSTRACT FROM AUTHOR]

Abstract: In the post-genomic area, the prediction of transcription factor regulons by position weight matrix-based programmes is a powerful approach to decipher biological pathways and to modelize regulatory networks in bacteria. The main difficulty once a regulon prediction is available is to estimate its reliability prior to start expensive experimental validations and therefore trying to find a way how to identify true positive hits from an endless list of potential target genes of a regulatory protein. Here we introduce PREDetector (Prokaryotic Regulatory Elements Detector), a tool developed for predicting regulons of DNA-binding proteins in bacterial genomes that, beside the automatic prediction, scoring and positioning of potential binding sites and their respective target genes in annotated bacterial genomes, it also provides an easy way to estimate the thresholds where to find reliable possible new target genes. PREDetector can be downloaded freely at http://www.montefiore.ulg.ac.be/~hiard/PreDetector/PreDetector.php. [Copyright &y& Elsevier]

• Adsorption of PCR amplicons electrochemically detected on low-cost PCB electrodes with methlyene blue (MB). • Amplicon length and MB concentration are both critical to achieving linear sensor performance. • Detection of SARS-CoV-2 nucleocapsid gene amplicons from 10 pg/μl (1.7 fM). • N1 fragment successfully amplified and detected in wastewater spiked with SARS-CoV-2 control RNA. We present a low-cost electrochemical DNA biosensor based on printed circuit board (PCB) electrodes for wastewater monitoring using portable PCR instruments, such as miniPCR®, without the requirement for qPCR reagents. PCB electrodes are attractive candidates for low-cost and sensitive DNA biosensors of relevance in a pandemic such as COVID-19, and facilitate the opportunity to map disease spread in Low-Middle Income Countries (LMICs) through monitoring of environmental samples such as wastewater. The biosensor reported in this work is capable of detecting PCR amplicons through the intercalation of methylene blue (MB) with DNA, which increases the voltammogram peak current at the redox potential of MB. We describe how these changes are likely to result from the adsorption of MB-DNA complex on the electrode surface. The electrodes are reusable, easy to clean, do not undergo any surface modification and represent a cost-effective solution with long shelf-life. We also explore the impact that MB concentration and DNA length have upon our biosensor performance and provide insights useful to other investigators in the field. The sensor reported here is capable of detecting SARS-CoV-2 nucleocapsid gene amplicons at concentrations as low as 10 pg/μl (approximately 1.7 fM) and can detect nucleotides amplified after 10 PCR cycles. Furthermore, using the PCB electrode and approaches described here, SARS-CoV-2 amplicons were detected in simulated wastewater sample, by spiking wastewater collected from a sewage treatment plant in Mumbai, India with SARS-CoV-2 RNA. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance (AMR) is an issue of upmost global importance, with an annually increasing mortality rate and growing economic burden. Poor antimicrobial stewardship has resulted in an abundance and diverse range of antimicrobial resistance mechanisms. To tackle AMR effectively, better diagnostic tests must be developed in order to improve antibiotic stewardship and reduce the emergence of antibiotic resistant organisms. This study employs a low-cost, commercially available screen printed electrode modified with an agarose-based hydrogel deposit to monitor bacterial growth using the techniques of electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) giving rise to a new approach to measuring susceptibility. Susceptible and drug resistant Staphylococcus aureus strains were deposited onto agarose gel modified electrodes which contained clinically important antibiotics to establish growth profiles for each bacterial strain and monitor the influence of the antibiotic on bacterial growth. The results show that S. aureus is able to grow on electrodes modified with gel containing no antibiotic, but is inhibited when the gel modified electrode is seeded with antibiotic. Conversely, methicillin-resistant S. aureus (MRSA; the drug resistant strain) is able to grow on gel modified electrodes containing clinically relevant concentrations of antibiotic. Results show rapid growth profiles, with possible time to results for antibiotic susceptibility <45 min, a significant improvement on the current gold standards of at least 1–2 days. Image 1 • A gold screen printed electrode modified with a hydrogel deposit was developed. • The sensor was electrochemically characterised to monitor bacterial growth. • Growth profiles were developed for susceptible and drug resistant S. aureus. • Rapid growth profiles established; antibiotic susceptibility results <45 min. [ABSTRACT FROM AUTHOR]

Prokaryotic systematics provides the fundamental framework for microbiological research but remains a discipline that relies on a labour- and time-intensive polyphasic taxonomic approach, including DNA-DNA hybridization, variation in 16S rRNA gene sequence and phenotypic characteristics. These techniques suffer from poor resolution in distinguishing between closely related species and often result in misclassification and misidentification of strains. Moreover, guidelines are unclear for the delineation of bacterial genera. Here, we have applied an innovative phylogenetic and taxogenomic approach to a heterogeneous actinobacterial taxon, Rhodococcus, to identify boundaries for intrageneric and supraspecific classification. Seven species-groups were identified within the genus Rhodococcus that are as distantly related to one another as they are to representatives of other mycolic acid containing actinobacteria and can thus be equated with the rank of genus. It was also evident that strains assigned to rhodococcal species-groups are underspeciated with many misclassified using conventional taxonomic criteria. The phylogenetic and taxogenomic methods used in this study provide data of theoretical value for the circumscription of generic and species boundaries and are also of practical significance as they provide a robust basis for the classification and identification of rhodococci of agricultural, industrial and medical/veterinary significance. [ABSTRACT FROM AUTHOR]

We report an annotated draft genome of the human pathogen Corynebacterium diphtheriae bv. intermedius NCTC 5011. This strain is the first C. diphtheriae bv. intermedius strain to be sequenced, and our results provide a useful comparison to the other primary disease-causing biovars, C. diphtheriae by. gravis and C. diphtheriae bv. mitis. The sequence has been deposited at DDBJ/EMBL/GenBank with the accession number AJVH01000000. [ABSTRACT FROM AUTHOR]

Recent mesoscopic characterization of nutrient-transporting channels in Escherichia coli has allowed the identification and measurement of individual channels in whole mature colony biofilms. However, their complexity under different physiological and environmental conditions remains unknown. Analysis of confocal micrographs of colony biofilms formed by cell shape mutants of E. coli shows that channels have high fractal complexity, regardless of cell phenotype or growth medium. In particular, colony biofilms formed by the mutant strain Δ ompR , which has a wide-cell phenotype, have a higher fractal dimension when grown on rich medium than when grown on minimal medium, with channel complexity affected by glucose and agar concentrations in the medium. Osmotic stress leads to a dramatic reduction in the Δ ompR cell size but has a limited effect on channel morphology. This work shows that fractal image analysis is a powerful tool to quantify the effect of phenotypic mutations and growth environment on the morphological complexity of internal E. coli biofilm structures. If applied to a wider range of mutant strains, this approach could help elucidate the genetic determinants of channel formation in E. coli colony biofilms.

We provide a brief review of the development and application of the Mesolens and its impact on microbiology. Microbial specimens such as infected tissue samples, colonies surfaces, and biofilms are routinely collected at the mesoscale. This means that they are relatively large multimillimetre-sized samples which contain microscopic detail that must be observed to answer important questions across various sectors. The Mesolens presents the ideal imaging method to study these specimens as no other optical microscope can thanks to its unique combination of low magnification and high numerical aperture providing large field-of-view, high-resolution imaging. We demonstrate the current applications of the Mesolens to microbial imaging and go on to outline the huge potential of the Mesolens to impact other key areas of microbiology., (© 2023 The Author(s). Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.)

Streptomyces is among the most extensively studied genera of bacteria but its complex taxonomy remains contested and is suspected to contain significant species-level misclassification. Resolving the classification of Streptomyces would benefit many areas of applied microbiology that rely on an accurate ground truth for grouping of related organisms, including comparative genomics-based searches for novel antimicrobials. We survey taxonomic conflicts between 16S rRNA and whole genome-based Streptomyces classifications using 2276 publicly available Streptomyces genome assemblies and 48 981 publicly available full-length 16S rRNA Streptomyces sequences from silva, Greengenes, Ribosomal Database Project (RDP), and NCBI (National Centre for Biotechnology Information) databases. We construct a full-length 16S gene tree for 14 239 distinct Streptomyces sequences that resolves three major lineages of Streptomyces , but whose topology is not consistent with existing taxonomic assignments. We use these sequence data to delineate 16S and whole genome landscapes for Streptomyces , demonstrating that 16S and whole-genome classifications are frequently in disagreement, and that 16S zero-radius Operational Taxonomic Units (zOTUs) are often inconsistent with Average Nucleotide Identity (ANI)-based taxonomy. Our results strongly imply that 16S rRNA sequence data does not map to taxonomy sufficiently well to delineate Streptomyces species routinely. We propose that alternative marker sequences should be adopted by the community for classification and metabarcoding. Insofar as Streptomyces taxonomy has been determined or supported by 16S sequence data and may in parts be in error, we also propose that reclassification of the genus by alternative approaches may benefit the Streptomyces community.

Nucleosides are pervasive building blocks that are found throughout nature and used extensively in medicinal chemistry and biotechnology. However, the preparation of base-modified analogues using conventional synthetic methodology poses challenges in scale-up and purification. In this work, an integrated approach involving structural analysis, screening and reaction optimization, is established to prepare 2'-deoxyribonucleoside analogues catalysed by the type II nucleoside 2'-deoxyribosyltransferase from Lactobacillus leichmannii ( Ll NDT-2). Structural analysis in combination with substrate profiling, identified the constraints on pyrimidine and purine acceptor bases by Ll NDT2. A solvent screen identifies pure water as a suitable solvent for the preparation of high value purine and pyrimidine 2'-deoxyribonucleoside analogues on a gram scale under optimized reaction conditions. This approach provides the basis to establish a convergent, step-efficient chemoenzymatic platform for the preparation of high value 2'-deoxyribonucleosides., Competing Interests: The authors declare no competing financial conflict of interest., (This journal is © The Royal Society of Chemistry.)

Chemical gradients and the emergence of distinct microenvironments in biofilms are vital to the stratification, maturation and overall function of microbial communities. These gradients have been well characterised throughout the biofilm mass but the microenvironment of recently discovered nutrient transporting channels in Escherichia coli biofilms remains unexplored. This study employs three different oxygen sensing approaches to provide a robust quantitative overview of the oxygen gradients and microenvironments throughout the biofilm transport channel networks formed by E. coli macrocolony biofilms. Oxygen nanosensing combined with confocal laser scanning microscopy established that the oxygen concentration changes along the length of biofilm transport channels. Electrochemical sensing provided precise quantification of the oxygen profile in the transport channels, showing similar anoxic profiles compared with the adjacent cells. Anoxic biosensing corroborated these approaches, providing an overview of the oxygen utilisation throughout the biomass. The discovery that transport channels maintain oxygen gradients contradicts the previous literature that channels are completely open to the environment along the apical surface of the biofilm. We provide a potential mechanism for the sustenance of channel microenvironments via orthogonal visualisations of biofilm thin sections showing thin layers of actively growing cells. This complete overview of the oxygen environment in biofilm transport channels primes future studies aiming to exploit these emergent structures for new bioremediation approaches., Competing Interests: Competing Interests We declare no competing interests

Conjugation of plasmids from Escherichia coli is essential for the genetic manipulation of Streptomyces spp. To facilitate intergeneric conjugation from E. coli to Streptomyces the conjugative machinery required for genetic transfer is usually provided by the non-transferable helper plasmid, pUZ8002. Here we present the complete nucleotide sequence of pUZ8002, describe the previously undocumented creation process, and provide details of the sequence relative to the parental pUZ8 plasmid and another previously published pUZ8002 sequence., Competing Interests: The authors declare that there are no conflicts of interest., (Copyright © 2024 The Authors.)

Streptomyces are a large genus of multicellular bacteria best known for their prolific production of bioactive natural products. In addition, they play key roles in the mineralisation of insoluble resources, such as chitin and cellulose. Because of their multicellular mode of growth, colonies of interconnected hyphae extend over a large area that may experience different conditions in different parts of the colony. Here, we argue that within-colony phenotypic heterogeneity can allow colonies to simultaneously respond to divergent inputs from resources or competitors that are spatially and temporally dynamic. We discuss causal drivers of heterogeneity, including competitors, precursor availability, metabolic diversity and division of labour, that facilitate divergent phenotypes within Streptomyces colonies. We discuss the adaptive causes and consequences of within-colony heterogeneity, highlight current knowledge (gaps) and outline key questions for future studies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

tGrowth of microorganisms and interpretation of growth data are core skills required by microbiologists. While science moves forward, it is of paramount importance that essential skills are not lost. The bacterial growth curve and the information that can gleaned from it is of great value to all of microbiology, whether this be a simple growth experiment, comparison of mutant strains or the establishment of conditions for a large-scale multi-omics experiment. Increasingly, the basics of plotting and interpreting growth curves and growth data are being overlooked. This primer article serves as a refresher for microbiologists on the fundamentals of microbial growth kinetics.

The parasitic worm-derived immunomodulator, ES-62 rescues defective levels of IL-10-producing regulatory B cells (Bregs) and suppresses chronic Th1/Th17-driven inflammation to protect against joint destruction in the mouse collagen-induced arthritis (CIA) model of rheumatoid arthritis. Such autoimmune arthritis is also associated with dysbiosis of the gut microbiota and disruption of intestinal barrier integrity. We recently further exploited the CIA model to show that ES-62's prevention of joint destruction is associated with protection of intestinal barrier integrity and normalization of the gut microbiota, thereby suppressing the gut pathology that precedes the onset of autoimmunity and joint damage in CIA-mice. As the status of the gut microbiota impacts on immune responses by influencing haematopoiesis, we have therefore investigated whether ES-62 harnesses the homeostatic mechanisms regulating this gut-bone marrow (BM) axis to resolve the chronic inflammation promoting autoimmunity and joint destruction in CIA. Reflecting this, ES-62 was found to counteract the BM myeloid/lymphoid bias typically associated with chronic inflammation and infection. This was achieved primarily by ES-62 acting to maintain the levels of lymphoid lineages (B220 + and CD3 + cells) observed in naïve, healthy mice but lost from the BM of CIA-mice. Moreover, ES-62's ability to prevent bone-destroying osteoclastogenesis was found to be associated with its suppression of CIA-induced upregulation of osteoclast progenitors (OCPs) in the BM. Critically, and supporting ES-62's targeting of the gut-BM axis, this rewiring of inflammatory haematopoiesis was lost in mice with a depleted microbiome. Underlining the importance of ES-62's actions in restoring steady-state haematopoiesis, the BM levels of B and T lymphoid cells were shown to be inversely correlated, whilst the levels of OCPs positively correlated, with the severity of joint damage in CIA-mice., Competing Interests: Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The exchange of ammonium across cellular membranes is a fundamental process in all domains of life and is facilitated by the ubiquitous Amt/Mep/Rh transporter superfamily. Remarkably, despite a high structural conservation in all domains of life, these proteins have gained various biological functions during evolution. It is tempting to hypothesise that the physiological functions gained by these proteins may be explained at least in part by differences in the energetics of their translocation mechanisms. Therefore, in this review, we will explore our current knowledge of energetics of the Amt/Mep/Rh family, discuss variations in observations between different organisms, and highlight some technical drawbacks which have hampered effects at mechanistic characterisation. Through the review, we aim to provide a comprehensive overview of current understanding of the mechanism of transport of this unique and extraordinary Amt/Mep/Rh superfamily of ammonium transporters., (© 2024 The Author(s).)

Polymicrobial infection with Candida albicans and Staphylococcus aureus may result in a concomitant increase in virulence and resistance to antimicrobial drugs. This enhanced pathogenicity phenotype is mediated by numerous factors, including metabolic processes and direct interaction of S. aureus with C. albicans hyphae. The overall structure of biofilms is known to contribute to their recalcitrance to treatment, although the dynamics of direct interaction between species and how it contributes to pathogenicity is poorly understood. To address this, a novel time-lapse mesoscopic optical imaging method was developed to enable the formation of C. albicans / S. aureus whole dual-species biofilms to be followed. It was found that yeast-form or hyphal-form C. albicans in the biofilm founder population profoundly affects the structure of the biofilm as it matures. Different sub-populations of C. albicans and S. aureus arise within each biofilm as a result of the different C. albicans morphotypes, resulting in distinct sub-regions. These data reveal that C. albicans cell morphology is pivotal in the development of global biofilm architecture and the emergence of colony macrostructures and may temporally influence synergy in infection.

We have developed a tuneable workflow for the study of soil microbes in an imitative 3D soil environment that is compatible with routine and advanced optical imaging, is chemically customisable, and is reliably refractive index matched based on the carbon catabolism of the study organism. We demonstrate our transparent soil pipeline with two representative soil organisms, Bacillus subtilis and Streptomyces coelicolor , and visualise their colonisation behaviours using fluorescence microscopy and mesoscopy. This spatially structured, 3D approach to microbial culture has the potential to further study the behaviour of bacteria in conditions matching their native environment and could be expanded to study microbial interactions, such as competition and warfare.

Extra-intestinal pathogenic Escherichia coli (ExPEC) can cause a variety of infections outside of the intestine and are a major causative agent of urinary tract infections. Treatment of these infections is increasingly frustrated by antimicrobial resistance (AMR) diminishing the number of effective therapies available to clinicians. Incidence of multidrug resistance (MDR) is not uniform across the phylogenetic spectrum of E. coli. Instead, AMR is concentrated in select lineages, such as ST131, which are MDR pandemic clones that have spread AMR globally. Using a gnotobiotic mouse model, we demonstrate that an MDR E. coli ST131 is capable of out-competing and displacing non-MDR E. coli from the gut in vivo. This is achieved in the absence of antibiotic treatment mediating a selective advantage. In mice colonised with non-MDR E. coli strains, challenge with MDR E. coli either by oral gavage or co-housing with MDR E. coli colonised mice results in displacement and dominant intestinal colonisation by MDR E. coli ST131. To investigate the genetic basis of this superior gut colonisation ability by MDR E. coli, we assayed the metabolic capabilities of our strains using a Biolog phenotypic microarray revealing altered carbon metabolism. Functional pangenomic analysis of 19,571 E. coli genomes revealed that carriage of AMR genes is associated with increased diversity in carbohydrate metabolism genes. The data presented here demonstrate that independent of antibiotic selective pressures, MDR E. coli display a competitive advantage to colonise the mammalian gut and points to a vital role of metabolism in the evolution and success of MDR lineages of E. coli via carriage and spread., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Connor et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The exchange of ammonium across cellular membranes is a fundamental process in all domains of life. In plants, bacteria and fungi, ammonium represents a vital source of nitrogen, which is scavenged from the external environment. In contrast, in animal cells ammonium is a cytotoxic metabolic waste product and must be excreted to prevent cell death. Transport of ammonium is facilitated by the ubiquitous Amt/Mep/Rh transporter superfamily. In addition to their function as transporters, Amt/Mep/Rh proteins play roles in a diverse array of biological processes and human physiopathology. Despite this clear physiological importance and medical relevance, the molecular mechanism of Amt/Mep/Rh proteins has remained elusive. Crystal structures of bacterial Amt/Rh proteins suggest electroneutral transport, whilst functional evidence supports an electrogenic mechanism. Here, focusing on bacterial members of the family, we summarize the structure of Amt/Rh proteins and what three decades of research tells us concerning the general mechanisms of ammonium translocation, in particular the possibility that the transport mechanism might differ in various members of the Amt/Mep/Rh superfamily.

Natural products are the raw material for drug discovery programmes. Bioactive natural products are used extensively in medicine and agriculture and have found utility as antibiotics, immunosuppressives, anti-cancer drugs and anthelminthics. Remarkably, the natural role and what mechanisms drive evolution of these molecules is relatively poorly understood. The exponential increase in genome and chemical data in recent years, coupled with technical advances in bioinformatics and genetics have enabled progress to be made in understanding the evolution of biosynthetic gene clusters and the products of their enzymatic machinery. Here we discuss the diversity of natural products, incorporating the mechanisms that govern evolution of metabolic pathways and how this can be applied to biosynthetic gene clusters. We build on the nomenclature of natural products in terms of primary, integrated, secondary and specialised metabolism and place this within an ecology-evolutionary-developmental biology framework. This eco-evo-devo framework we believe will help to clarify the nature and use of the term specialised metabolites in the future., (Copyright © 2023. Published by Elsevier Inc.)

Nutrient-transporting channels have been recently discovered in mature Escherichia coli biofilms, however the relationship between intra-colony channel structure and the surrounding environmental conditions is poorly understood. Using a combination of fluorescence mesoscopy and a purpose-designed open-source quantitative image analysis pipeline, we show that growth substrate composition and nutrient availability have a profound effect on the morphology of intra-colony channels in mature E. coli biofilms. Under all nutrient conditions, intra-colony channel width was observed to increase non-linearly with radial distance from the centre of the biofilm. Notably, the channels were around 25% wider at the centre of carbon-limited biofilms compared to nitrogen-limited biofilms. Channel density also differed in colonies grown on rich and minimal media, with the former creating a network of tightly packed channels and the latter leading to well-separated, wider channels with defined edges. Our approach paves the way for measurement of internal patterns in a wide range of biofilms, offering the potential for new insights into infection and pathogenicity., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

We report the genome sequence of Streptomyces goldiniensis ATCC 21386, a strain which produces the anti-bacterial and anti-virulence polyketide, aurodox. The genome of S. goldiniensis ATCC 21386 was sequenced using a multiplatform hybrid approach, revealing a linear genome of ~10 Mbp with a G+C content of 71%. The genome sequence revealed 36 putative biosynthetic gene clusters (BGCs), including a large region of 271 Kbp that was rich in biosynthetic capability. The genome sequence is deposited in DDBJ/EMBL/GenBank with the accession number PRJNA602141., Competing Interests: The authors declare that there are no conflicts of interest., (© 2022 The Authors.)

Actinobacteria is an ancient phylum of Gram-positive bacteria with a characteristic high GC content to their DNA. The ActinoBase Wiki is focused on the filamentous actinobacteria, such as Streptomyces species, and the techniques and growth conditions used to study them. These organisms are studied because of their complex developmental life cycles and diverse specialised metabolism which produces many of the antibiotics currently used in the clinic. ActinoBase is a community effort that provides valuable and freely accessible resources, including protocols and practical information about filamentous actinobacteria. It is aimed at enabling knowledge exchange between members of the international research community working with these fascinating bacteria. ActinoBase is an anchor platform that underpins worldwide efforts to understand the ecology, biology and metabolic potential of these organisms. There are two key differences that set ActinoBase apart from other Wiki-based platforms: [1] ActinoBase is specifically aimed at researchers working on filamentous actinobacteria and is tailored to help users overcome challenges working with these bacteria and [2] it provides a freely accessible resource with global networking opportunities for researchers with a broad range of experience in this field.

Ammonium translocation through biological membranes, by the ubiquitous Amt-Mep-Rh family of transporters, plays a key role in all domains of life. Two highly conserved histidine residues protrude into the lumen of the pore of these transporters, forming the family's characteristic Twin-His motif. It has been hypothesized that the motif is essential to confer the selectivity of the transport mechanism. Here, using a combination of in vitro electrophysiology on Escherichia coli AmtB, in silico molecular dynamics simulations, and in vivo yeast functional complementation assays, we demonstrate that variations in the Twin-His motif trigger a mechanistic switch between a specific transporter, depending on ammonium deprotonation, to an unspecific ion channel activity. We therefore propose that there is no selective filter that governs specificity in Amt-Mep-Rh transporters, but the inherent mechanism of translocation, dependent on the fragmentation of the substrate, ensures the high specificity of the translocation. We show that coexistence of both mechanisms in single Twin-His variants of yeast Mep2 transceptors disrupts the signaling function and so impairs fungal filamentation. These data support a signaling process driven by the transport mechanism of the fungal Mep2 transceptors. IMPORTANCE Fungal infections represent a significant threat to human health and cause huge damage to crop yields worldwide. The dimorphic switch between yeast and filamentous growth is associated with the virulence of pathogenic fungi. Of note, fungal Mep2 proteins of the conserved Amt-Mep-Rh family play a transceptor role in the induction of filamentation; however, the signaling mechanism remains largely unknown. Amt-Mep-Rh proteins ensure the specific scavenging of NH 4 through a mechanism relying on substrate deprotonation, thereby preventing competition and translocation of similar-sized K + . Our multidisciplinary approaches using E. coli AmtB, Saccharomyces cerevisiae, and Candida albicans Mep2 show that double variation of the family-defining Twin-His motif triggers a mechanistic switch from a specific transporter to an unspecific ion channel with both mechanisms still coexisting in single variants. Moreover, we show that this mechanistic alteration is associated with loss of signaling ability of Mep2, supporting a transport mechanism-driven process in filamentation induction.+ . Our multidisciplinary approaches using E. coli AmtB, Saccharomyces cerevisiae, and Candida albicans Mep2 show that double variation of the family-defining Twin-His motif triggers a mechanistic switch from a specific transporter to an unspecific ion channel with both mechanisms still coexisting in single variants. Moreover, we show that this mechanistic alteration is associated with loss of signaling ability of Mep2, supporting a transport mechanism-driven process in filamentation induction.

SARS-CoV-2 diagnostic practices broadly involve either quantitative polymerase chain reaction (qPCR)-based nucleic amplification of viral sequences or antigen-based tests such as lateral flow assays (LFAs). Reverse transcriptase-qPCR can detect viral RNA and is the gold standard for sensitivity. However, the technique is time-consuming and requires expensive laboratory infrastructure and trained staff. LFAs are lower in cost and near real time, and because they are antigen-based, they have the potential to provide a more accurate indication of a disease state. However, LFAs are reported to have low real-world sensitivity and in most cases are only qualitative. Here, an antigen-based electrochemical aptamer sensor is presented, which has the potential to address some of these shortfalls. An aptamer, raised to the SARS-CoV-2 spike protein, was immobilized on a low-cost gold-coated polyester substrate adapted from the blood glucose testing industry. Clinically relevant detection levels for SARS-CoV-2 are achieved in a simple, label-free measurement format using sample incubation times as short as 15 min on nasopharyngeal swab samples. This assay can readily be optimized for mass manufacture and is compatible with a low-cost meter.