Biology subjects

Hung, D. T.

Publications and source records attributed to Hung, D. T..

At least 19 recordsLinked to original sources

bioRxiv2026

Type 1 immunity linked to CMV exposure improves long-term survival in sepsis without shock

Dysregulated immune responses are at the core of sepsis pathology and can drive both acute organ dysfunction and long-term morbidity and mortality. To better understand the association between specific immune histories, perturbations and sepsis outcomes, we combined immune phenotyping, functional cytokine production assays and antibody repertoire profiling. While sepsis immune profiles were highly heterogeneous, we identified a strong association between the frequency of CXCR3-expressing CD4 memory T cells at time of enrollment and six-month survival in sepsis without shock. Surprisingly, the same population was linked to improved organ function trajectories in septic shock patients but did not influence six-month survival, highlighting the importance of considering physiological states to define immune predictors. Finally, phage display profiling revealed that these Th1 cells were generally expanded in CMV-seropositive individuals, suggesting that previous and latent infections may modulate sepsis outcomes.

immunology↗

bioRxiv2026

Kainate receptors coordinate primitive hematopoiesis and hemogenic niche organization to promote hematopoietic stem cell development

The adult hematopoietic system is established during embryogenesis through the generation of hematopoietic stem and progenitor cells (HSPCs) within specialized transient niches, yet the signaling mechanisms that coordinate this process remain poorly understood. Here, we identify a developmental role for kainate receptors (KARs), glutamate-gated ion channels classically associated with excitatory neurotransmission, in coordinating primitive hematopoiesis and hemogenic niche formation in zebrafish. Loss of the KAR subunits encoded by grik1b or grik5 depletes primitive blood cells and disrupts hemogenic niche organization. These defects are accompanied by endothelial oxidative stress and impaired Notch signaling, which together reduce HSPC emergence and limit the clonal diversity of the adult hematopoietic stem cell pool. Shifting primitive hematopoietic output away from erythropoiesis and toward myelopoiesis restored macrophage niche colonization, endothelial Notch activity, and HSPC formation in KAR-deficient embryos. Moreover, hemogenic cell-specific Notch reactivation rescued HSPC production, demonstrating that KAR-dependent primitive hematopoiesis regulates this process through endothelial Notch signaling. Lastly, conserved KAR expression in endothelial and hematopoietic populations across zebrafish, mouse, and human suggests that these receptors may regulate blood development across multiple cellular components. Together, these findings reveal that glutamate receptor signaling extends beyond its well-established functions in neuronal transmission to coordinate the developmental processes that establish the blood system.

developmental biology↗

bioRxiv2026

Large-Scale Chemical-Genetic Interaction Profiling Identifies a Novel Small-Molecule Inhibitor of Mycobacterium tuberculosis Polyketide Synthase 13

PROSPECT (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets) is an antimicrobial discovery platform based on chemical-genetic interaction (CGI) profiling of compounds against a pool of Mycobacterium tuberculosis (Mtb) hypomorphs, each depleted of an essential gene. From prior screening data, we have now identified a novel N-oxolan-3-yl pyrazole carboxamide inhibitor (BRD1554) that had increased, selective activity against strains depleted of polyketide synthase 13 (Pks13), an essential enzyme in mycolic acid synthesis, and Rv2581c, an uncharacterized protein similar to glyoxylase II enzymes. Perturbagen CLass (PCL) analysis, a reference-based approach to mechanism of action (MOA) assignment from PROSPECT, predicted Pks13, a polyketide synthase with five catalytic domains responsible for the terminal condensation step in mycolic acid biosynthesis, was the likely target, potentially implicating the thioesterase domain. We synthesized a more active analogue while assigning the absolute stereochemistry of the active diastereomer, resulting in 1554-06-3R,4S with an MIC90 of 3.0 {micro}M against Mtb H37Rv. Exposure to 1554-06 led to the upregulation of the pks13 operon along with the iniBAC operon and other genes linked to mycolic acid synthesis. Isolation of mutants resistant to 1554-06 revealed single nucleotide polymorphisms in the thioesterase domain of Pks13. Finally, we biochemically confirmed that 1554-06 inhibits the activity of recombinant Pks13 thioesterase domain, with computational docking of 1554-06 steroisomers consistent with the stereospecific activity seen in whole cell assays. We found unique chemical genetic interactions between inhibitors of the different Pks13 domains and different detoxifying enzymes of Mtb, thus revealing novel gene-gene interactions. These results highlight how PROSPECT can not only immediately reveal, with domain-level resolution, the MOA of new whole-cell active chemical inhibitors of Mtb, allowing the integration of biological insight into compound triage and accelerated early development, but can also illuminate genetic interactions linked to those mechanisms that could inform predictions of synergy for antitubercular drug development. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/704361v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@9a4f38org.highwire.dtl.DTLVardef@c70cd2org.highwire.dtl.DTLVardef@1ac2e1org.highwire.dtl.DTLVardef@f06df0_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Mapping genetic and phenotypic diversity of Pseudomonas aeruginosa across clinical and environmental isolation sites

Pseudomonas aeruginosa is a clinically significant opportunistic pathogen adept at thriving in both host-associated and environmental settings. To define the extent to which P. aeruginosa isolates specialize across niches and identify genotype-phenotype correlates, we performed whole genome sequencing and comprehensive phenotypic characterization of 125 P. aeruginosa isolates from diverse clinical and environmental sites, evaluating virulence-associated traits, including motility, cytotoxicity, biofilm formation, pyocyanin production, and antimicrobial susceptibility. We identify that genomic diversity does not correlate with isolation source or most virulence phenotypes. Instead, we find that the two major P. aeruginosa clades (Groups A and B) delineate phylogeny and cytotoxicity, with Group B strains showing significantly higher cytotoxicity than Group A. Sequence analysis revealed previously uncharacterized alleles of genes encoding type III secretion effector proteins. We observed high variability amongst strains and isolation sources in all four assayed virulence phenotypes. Antimicrobial resistance (AMR) is exclusively observed in clinical isolates, not environmental, reflecting antibiotic exposure-driven selection. Bacterial GWAS revealed a statistically significant association between cytotoxicity and exoU presence, and we identified a novel exoU allelic variant with decreased cytotoxicity, demonstrating that functional diversity within well-characterized virulence factors may still influence pathogenic outcomes. In summary, our analyses of 125 diverse isolates suggest that the ability of P. aeruginosa to thrive across diverse niches is driven by broadly conserved genetic repertoire rather than niche-specific accessory genes. ImportancePseudomonas aeruginosa is a clinically significant opportunistic pathogen adept at thriving in both host-associated and environmental niches. A major gap in our understanding of this difficult-to-treat pathogen is whether niche specialization occurs in the context of human disease. Addressing this question is critical for guiding effective infection control strategies. Previous large-scale studies have focused solely on genotypic or phenotypic analyses; when paired, they have been limited to a single phenotypic assay or to a small number of isolates from one source, or relied on PCR-based methods targeting a restricted set of genes. To comprehensively uncover niche specialization and pathogenic versatility, we performed whole genome sequencing and phenotypic characterization of 5 virulence-associated traits including AST of 125 clinical and environmental P. aeruginosa isolates. Our systems-level findings challenge reductionist models of bacterial niche specialization, instead supporting an integrated view where conserved genomic systems enable opportunistic pathogenesis across diverse environments.

microbiology↗

bioRxiv2025

An Inducible CRISPRi system for phenotypic analysis of essential genes in Pseudomonas aeruginosa

Precise and tunable genetic tools are essential for high throughput functional genomics. To address this need in the important gram-negative pathogen Pseudomonas aeruginosa, we developed and characterized a tightly regulated CRISPRi system that enables precise and tunable repression of essential genes. The system utilizes a rhamnose-inducible promoter to control both the Streptococcus pasteurianus-derived dCas9 and gene-specific sgRNAs, each encoded on separate plasmids for modularity and efficiency. The combination of tight regulation and high conjugation efficiency facilitated the rapid and facile construction of strains with regulated depletion of 16 essential genes spanning diverse pathways. Comparison of phenotypes across the different genetically depleted strains, including growth rate, susceptibility to antibiotics, and changes in transcriptional programs, revealed novel aspects of gene function or small molecule mechanism of action. Finally, the rhamnose-inducible CRISPRi system supports the generation and stable maintenance of pooled mutant libraries, thereby paving the way for future genome-wide, systematic assessment of individual gene vulnerabilities, which will provide critical insights for target prioritization in antibiotic discovery efforts against this recalcitrant pathogen. IMPORTANCECRISPRi has become an invaluable tool for studying genetics. In particular, the ability to knockdown genes enables the study of essential genes and their role in cell survival. However, a tightly regulated gene knockdown system is required to gain valuable insights into these vulnerable genes by virtue of their essentiality. We report a tightly regulated CRISPRi system to study the biology of essential gene perturbations in Pseudomonas aeruginosa, an important gram-negative pathogen that causes severe infections and is increasingly resistant to current antibiotics. This system enables characterization of both chemical genetic interactions between small molecules and specific gene depletions, and the impact of genetic perturbations on transcriptional networks. Genetic perturbations using CRISPRi can thus further our understanding of basic biology with translation towards future antimicrobial development.

microbiology↗

bioRxiv2025

Kmo restricts Salmonella in a whole organism infection model by promoting macrophage lysosomal acidification through kainate receptor antagonism.

The kynurenine pathway of tryptophan degradation has been implicated in various diseases, including cancer, neurodegenerative disorders, and infectious diseases. A key branchpoint in this pathway is production of the metabolite 3-hydroxy-kynurenine (3-HK) by the enzyme kynurenine 3-monooxygenase (Kmo). We have previously reported that administration of exogenous 3-HK promotes survival of zebrafish larvae to Salmonella Typhimurium infection by restricting bacterial expansion via a systemic mechanism that targets kainate sensitive glutamate receptor (KAR) ion channels and that the endogenous production of 3-HK by Kmo is required for defense against systemic Salmonella infection. Here we show that endogenous 3-HK promotes lysosomal acidification to contribute to macrophage microbicidal activity, with its absence leading to increased host susceptibility to infection. Further, 3-HK promotes lysosomal acidification in a KAR-dependent manner. We thus reveal a novel link between KARs and macrophage lysosomal acidification, and a novel mechanism by which 3-HK promotes control of bacterial infection. Author SummaryStandard therapy for bacterial infections involves antibiotics to clear pathogens. However, the host immune system can also efficiently eliminate bacteria. We have recently shown that a metabolite of the kynurenine metabolic pathway, 3-hydroxy-kynurenine (3-HK), plays a role in the innate immune response to bacterial infection. Here, we show that the kynurenine pathway promotes macrophage clearance of intracellular bacteria by increasing lysosomal acidification of engulfed bacteria and that 3-HK does so by antagonizing kainate receptors. Together, this adds to our understanding of how multiple biological systems, including metabolic and immune pathways, interact to boost defense against bacteria.

immunology↗

bioRxiv2025

A multiplexed, target-based phenotypic screening platform using CRISPR interference in Mycobacterium abscessus

The rise of difficult-to-treat Mycobacterium abscessus infections presents a growing clinical challenge due to the immense arsenal of intrinsic, inducible and acquired antibiotic resistance mechanisms that render many existing antibiotics ineffective against this pathogen. Moreover, the limited success in discovery of novel compounds that inhibit novel pathways underscores the need for innovative drug discovery strategies. Here, we report a strategic advancement in PROSPECT (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets), which is an antimicrobial discovery strategy that measures chemical-genetic interactions between small molecules and a pool of bacterial mutants, each depleted of a different essential protein target, to identify whole-cell active compounds with high sensitivity. Applying this modified strategy to M. abscessus, in contrast to previously described versions of PROSPECT which utilized protein degradation or promoter replacement strategies for generating engineered hypomorphic strains, here we leveraged CRISPR interference (CRISPRi) to more efficiently generate mutants each depleted of a different essential gene involved in cell wall synthesis or located at the bacterial surface. We applied this platform to perform a pooled PROSPECT pilot screen of a library of 809 compounds using CRISPRi guides as mutant barcodes. We identified a range of active hits, including compounds targeting InhA, a well-known mycobacterial target but under-explored in the M. abscessus space. The unexpected susceptibility to isoniazid, traditionally considered to be ineffective in M. abscessus, suggested a complex interplay of several intrinsic resistance mechanisms. While further complementary efforts will be needed to change the landscape of therapeutic options for M. abscessus, we propose that PROSPECT with CRISPRi engineering provides an increasingly accessible, high-throughput target-based phenotypic screening platform and thus represents an important step towards accelerating early-stage drug discovery.

microbiology↗

bioRxiv2025

Reference-based chemical-genetic interaction profiling to elucidate small molecule mechanism of action in Mycobacterium tuberculosis

In an era of increasing resistance, new and effective strategies are needed for antibiotic discovery. Whole-cell active screens yield candidate compounds lacking mechanism-of-action (MOA) information and thus do not provide biological insight for prioritization. We previously reported PROSPECT (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets), an antimicrobial discovery strategy that measures chemical-genetic interactions between small molecules and a pool of Mycobacterium tuberculosis mutants, each depleted of a different essential protein target. PROSPECT facilitates efficient hit prioritization by simultaneously identifying whole-cell active compounds with high sensitivity and providing early insights into their MOA. Here, we report a reference-based approach to infer MOA from often complex PROSPECT data. For this aim, we curated a reference set of 437 compounds with published, annotated MOA and known or suspected antitubercular activity, and applied PROSPECT to it. We then developed Perturbagen CLass (PCL) analysis, a computational method that predicts MOA by comparing chemical-genetic interaction profiles of unknown compounds to those of this reference set. In leave-one-out cross-validation, PCL analysis correctly predicted MOA with 70% sensitivity and 75% precision. When applied to 75 antitubercular leads with known MOA previously reported by GlaxoSmithKline (GSK), PCL analysis similarly achieved 69% sensitivity and 87% precision. We also analyzed 98 GSK compounds lacking MOA information, predicting 60 of them to act via a reference MOA, and followed up with functional validation of 29 compounds predicted to target respiration-related MOAs. Finally, we applied PROSPECT and PCL analysis to [~]5,000 compounds from larger unbiased libraries that had not been preselected for antitubercular activity. PCL analysis identified a novel scaffold lacking wild-type activity but predicted to inhibit respiration via QcrB, and we confirmed this prediction while chemically optimizing this scaffold to achieve wild-type activity. PCL analysis of PROSPECT data thus enables rapid MOA assignment and hit prioritization, advancing the discovery of new, potent antitubercular compounds.

microbiology↗

bioRxiv2024

Structure and inhibition mechanisms of Mycobacterium tuberculosis essential transporterefflux protein A

A broad chemical genetics screen in Mycobacterium tuberculosis (Mtb) to identify inhibitors of established or previously untapped targets for therapeutic development yielded compounds (BRD-8000.3 and BRD-9327) that inhibit the essential efflux pump EfpA. To understand the mechanisms of inhibition by these compounds, we determined the structures of EfpA with inhibitors bound at 2.7 -3.4 [A] resolution. Our structures reveal different mechanisms of inhibition for the two inhibitors. BRD-8000.3 binds in a tunnel making contact with the lipid bilayer and extending toward the central cavity to displace the fatty acid chain of a lipid molecule bound in the apo structure, suggesting its blocking of an access route for a natural lipidic substrate, in contrast to its uncompetitive mechanism for the small molecule substrate ethidium bromide which likely enters through an alternative tunnel. Meanwhile, BRD-9327 binds in the outer vestibule without complete blockade of the substrate path to the outside, suggesting its possible inhibition of the dynamical motion necessary for "alternate access" to the two different sides of the membrane, as is characteristic of major facilitator superfamily (MFS) transporters. Both inhibitors may have a role in inhibiting the "alternate access" mechanism that could account for the uncompetitive nature of their efflux of some substrates. Our results explain the basis of the synergy of these inhibitors and their potential for combination in a multi drug strategy for anti-tuberculosis therapy. They also potentially point to a possible function for this essential efflux pump as a lipid transporter. The structures provide a foundation for rational modification of these inhibitors to increase potency.

biochemistry↗

bioRxiv2024

Essential and virulence-related protein interactions of pathogens revealed through deep learning

Identification of bacterial protein-protein interactions and predicting the structures of the complexes could aid in the understanding of pathogenicity mechanisms and developing treatments for infectious diseases. Here, we developed a deep learning-based pipeline that leverages residue-residue coevolution and protein structure prediction to systematically identify and structurally characterize protein-protein interactions at the proteome-wide scale. Using this pipeline, we searched through 78 million pairs of proteins across 19 human bacterial pathogens and identified 1923 confidently predicted complexes involving essential genes and 256 involving virulence factors. Many of these complexes were not previously known; we experimentally tested 12 such predictions, and half of them were validated. The predicted interactions span core metabolic and virulence pathways ranging from post-transcriptional modification to acid neutralization to outer membrane machinery and should contribute to our understanding of the biology of these important pathogens and the design of drugs to combat them.

biochemistry↗

bioRxiv2024

Massively parallel combination screen reveals small molecule sensitization of antibiotic-resistant Gram-negative ESKAPE pathogens

Antibiotic resistance, especially in multidrug-resistant ESKAPE pathogens, remains a worldwide problem. Combination antimicrobial therapies may be an important strategy to overcome resistance and broaden the spectrum of existing antibiotics. However, this strategy is limited by the ability to efficiently screen large combinatorial chemical spaces. Here, we deployed a high-throughput combinatorial screening platform, DropArray, to evaluate the interactions of over 30,000 compounds with up to 22 antibiotics and 6 strains of Gram-negative ESKAPE pathogens, totaling to over 1.3 million unique strain-antibiotic-compound combinations. In this dataset, compounds more frequently exhibited synergy with known antibiotics than single-agent activity. We identified a compound, P2-56, and developed a more potent analog, P2-56-3, which potentiated rifampin (RIF) activity against Acinetobacter baumannii and Klebsiella pneumoniae. Using phenotypic assays, we showed P2-56-3 disrupts the outer membrane of A. baumannii. To identify pathways involved in the mechanism of synergy between P2-56-3 and RIF, we performed genetic screens in A. baumannii. CRISPRi-induced partial depletion of lipooligosaccharide transport genes (lptA-D, lptFG) resulted in hypersensitivity to P2-56-3/RIF treatment, demonstrating the genetic dependency of P2-56-3 activity and RIF sensitization on lpt genes in A. baumannii. Consistent with outer membrane homeostasis being an important determinant of P2-56-3/RIF tolerance, knockout of maintenance of lipid asymmetry complex genes and overexpression of certain resistance-nodulation-division efflux pumps - a phenotype associated with multidrug-resistance - resulted in hypersensitivity to P2-56-3. These findings demonstrate the immense scale of phenotypic antibiotic combination screens using DropArray and the potential for such approaches to discover new small molecule synergies against multidrug-resistant ESKAPE strains. Significance StatementThere is an unmet need for new antibiotic therapies effective against the multidrug-resistant, Gram-negative ESKAPE pathogens. Combination therapies have the potential to overcome resistance and broaden the spectrum of existing antibiotics. In this study, we use DropArray, a massively parallel combinatorial screening tool, to assay more than 1.3 million combinations of small molecules against the Gram-negative ESKAPE pathogens, Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa. We discovered a synthetic small molecule potentiator, P2-56, of the antibiotic rifampin effective in A. baumannii and K. pneumoniae. We generated P2-56-3, a more potent derivative of P2-56, and found that it likely potentiates rifampin by compromising the outer membrane integrity. Our study demonstrates a high-throughput strategy for identifying antibiotic potentiators against multidrug-resistant bacteria.

microbiology↗

bioRxiv2024

Multiplexed screen identifies a Pseudomonas aeruginosa-specific small molecule targeting the outer membrane protein OprH and its interaction with LPS

SUMMARYThe surge of antimicrobial resistance threatens efficacy of current antibiotics, particularly against Pseudomonas aeruginosa, a highly resistant gram-negative pathogen. The asymmetric outer membrane (OM) of P. aeruginosa combined with its array of efflux pumps provide a barrier to xenobiotic accumulation, thus making antibiotic discovery challenging. We adapted PROSPECT1, a target-based, whole-cell screening strategy, to discover small molecule probes that kill P. aeruginosa mutants depleted for essential proteins localized at the OM. We identified BRD1401, a small molecule that has specific activity against a P. aeruginosa mutant depleted for the essential lipoprotein, OprL. Genetic and chemical biological studies identified that BRD1401 acts by targeting the OM {beta}-barrel protein OprH to disrupt its interaction with LPS and increase membrane fluidity. Studies with BRD1401 also revealed an interaction between OprL and OprH, directly linking the OM with peptidoglycan. Thus, a whole-cell, multiplexed screen can identify species-specific chemical probes to reveal novel pathogen biology.

microbiology↗

bioRxiv2023

A tryptophan metabolite modulates the host response to bacterial infection via kainate receptors

Bacterial infection involves a complex interaction between the pathogen and host where the outcome of infection is not solely determined by pathogen eradication. To identify small molecules that promote host survival by altering the host-pathogen dynamic, we conducted an in vivo chemical screen using zebrafish embryos and found that treatment with 3-hydroxy-kynurenine protects from lethal gram-negative bacterial infection. 3-hydroxy-kynurenine, a metabolite produced through host tryptophan metabolism, has no direct antibacterial activity but enhances host survival by restricting bacterial expansion in macrophages by targeting kainate-sensitive glutamate receptors. These findings reveal new mechanisms by which tryptophan metabolism and kainate-sensitive glutamate receptors function and interact to modulate immunity, with significant implications for the coordination between the immune and nervous systems in pathological conditions.

immunology↗

bioRxiv2022

Rapid Single-Shot Synthesis of the 217 Amino Acid-Long N-Terminal Domain of Pyocin S2

The impermeable outer membrane of Pseudomonas aeruginosa is bypassed by antibacterial proteins known as S-type pyocins. Because of their properties, pyocins are investigated as a potential new class of antimicrobials against Pseudomonas infections. Their production and modification, however, remains challenging. To address this limitation, we employed automated fast-flow peptide synthesis (AFPS) for the rapid production of a pyocin S2 import domain. The N-terminal domain sequence (PyS2NTD) was synthesized in under 10 hours and purified to yield milligrams quantities of the desired product. To our knowledge, the 217 amino acid sequence of PyS2NTD is among the longest peptides produced from a "single-shot" synthesis, i.e., made in a single stepwise route without the use of ligation techniques. Biophysical characterization of the PyS2NTD with circular dichroism was consistent with the literature reports. Fluorescently labeled PyS2NTD binds to P. aeruginosa expressing the cognate ferripyoverdine receptor (FpvA) and is taken up into the periplasm. This selective uptake was validated with confocal and super resolution microscopy, flow cytometry, and fluorescence recovery after photobleaching (FRAP). These modified, synthetic S-type pyocins domains can be used to probe import mechanisms of P. aeruginosa and leveraged to develop selective antimicrobial agents that bypass the outer membrane.

synthetic biology↗

bioRxiv2022

Bacterial droplet-based single-cell RNA-seq reveals heterogeneity in bacterial populations and in response to antibiotic perturbation

We introduce BacDrop, a highly scalable technology for bacterial single-cell RNA sequencing that has overcome many challenges hindering the development of scRNA-seq in bacteria. BacDrop can be applied to thousands or millions of cells from both gram-negative and gram-positive species. It features universal ribosomal RNA depletion and combinatorial barcodes that enable multiplexing and massively parallel sequencing. We applied BacDrop to study Klebsiella pneumoniae clinical isolates and to elucidate their heterogeneous responses to antibiotic stress. In an unperturbed population presumed to be homogenous, we find within- population heterogeneity largely driven by the expression of mobile genetic elements that promote the evolution of antibiotic resistance. Under antibiotic perturbation, BacDrop revealed transcriptionally distinct subpopulations associated with different phenotypic outcomes including antibiotic persistence. BacDrop thus can capture cellular states that cannot be detected by bulk RNA-seq, which will unlock new microbiological insights into bacterial responses to perturbations and larger bacterial communities such as the microbiome.

microbiology↗

bioRxiv2022

Antibiotic action revealed by real-time imaging of the mycobacterial membrane

The current understanding of mycobacterial cell envelope remodeling in response to antibiotics is limited. Chemical tools that report on phenotypic changes with minimal cell wall perturbation are critical to understanding such time-dependent processes. We employed a fluorogenic chemical probe to image how antibiotics perturb mycobacterial cell envelope assembly in real-time. Time-lapse microscopy revealed that differential antibiotic treatment elicited unique cellular phenotypes, providing a platform for simultaneously monitoring cell envelope construction and remodeling responses. Our data show that rifampicin, which does not directly inhibit cell wall biosynthesis, affords a readily detected mycomembrane phenotype. The fluorogenic probe revealed the production of extracellular vesicles in response to antibiotics, and analyses of these vesicles indicate that antibiotic treatment elicits the release of agents that attenuate macrophage activation.

microbiology↗

bioRxiv2021

Inter-species geographic signatures for tracing horizontal gene transfer and long-term persistence of carbapenem resistance

BackgroundCarbapenem-resistant Enterobacterales (CRE) are an urgent global health threat. Inferring the dynamics of local CRE dissemination is currently limited by our inability to confidently trace the spread of resistance determinants to unrelated bacterial hosts. Whole genome sequence comparison is useful for identifying CRE clonal transmission and outbreaks, but high-frequency horizontal gene transfer (HGT) of carbapenem resistance genes and subsequent genome rearrangement complicate tracing the local persistence and mobilization of these genes across organisms. MethodsTo overcome this limitation, we developed a new approach to identify recent HGT of large, near-identical plasmid segments across species boundaries, which also allowed us to overcome technical challenges with genome assembly. We applied this to complete and near-complete genome assemblies to examine the local spread of CRE in a systematic, prospective collection of all CRE, as well as time- and species-matched carbapenem susceptible Enterobacterales, isolated from patients from four U.S. hospitals over nearly five years. ResultsOur CRE collection comprised a diverse range of species, lineages and carbapenem resistance mechanisms, many of which were encoded on a variety of promiscuous plasmid types. We found and quantified rearrangement, persistence, and repeated transfer of plasmid segments, including those harboring carbapenemases, between organisms over multiple years. Some plasmid segments were found to be strongly associated with specific locales, thus representing geographic signatures that make it possible to trace recent and localized HGT events. Functional analysis of these signatures revealed genes commonly found in plasmids of nosocomial pathogens, such as functions required for plasmid retention and spread, as well survival against a variety of antibiotic and antiseptics common to the hospital environment. ConclusionsCollectively, the framework we developed provides a clearer, high resolution picture of the epidemiology of antibiotic resistance importation, spread, and persistence in patients and healthcare networks.

genomics↗

bioRxiv2021

Multiplexed detection of bacterial nucleic acids using Cas13 in droplet microarrays

Rapid and accurate diagnosis of infections is fundamental to individual patient care and public health management. Nucleic acid detection methods are critical to this effort, but are limited either in the breadth of pathogens targeted or by the expertise and infrastructure required. We present here a high-throughput system that enables rapid identification of bacterial pathogens, bCARMEN, which utilizes: (1) modular CRISPR-Cas13-based nucleic acid detection with enhanced sensitivity and specificity; and (2) a droplet microfluidic system that enables thousands of simultaneous, spatially multiplexed detection reactions at nanoliter volumes; and (3) a novel pre-amplification strategy that further enhances sensitivity and specificity. We demonstrate bCARMEN is capable of detecting and discriminating 52 clinically relevant bacterial species and several key antibiotic resistance genes. We further develop a proof of principle system for use with stabilized reagents and a simple workflow with optical readout using a cell phone camera, opening up the possibility of a rapid point-of-care multiplexed bacterial pathogen identification and antibiotic susceptibility testing. Significance StatementIn this paper, we use a novel primer design method combined with droplet-based CRISPR Cas13 detection to distinguish 52 clinically relevant bacterial pathogens in a single assay. We also apply the method to detect and distinguish a panel of major antibiotic resistance genes, which is of critical importance in this era of rising antibiotic resistance. Finally, we make key advances towards making our diagnostic assay deployable at the point-of-care, with a simplified emulsion-free assay process that uses mobile phone camera for detection and reduces infrastructure/skilled labor requirements.

microbiology↗