Biology subjects

van Opijnen, T.

Publications and source records attributed to van Opijnen, T..

At least 19 recordsLinked to original sources

bioRxiv2026

Translation Inhibiting Antibiotics Induce a Strain Specific asRNA Regulating Purine Metabolism in S. pneumoniae TIGR4

Translation inhibiting antibiotics (TIA) are an important class of antibiotics that elicit a wide variety of transcriptional responses. In this work we characterize the transcriptional responses of Streptococcus pneumoniae TIGR4, via RNA-seq, 5-, and 3-end mapping, to sub-MIC levels of three TIA, chloramphenicol, tetracycline, and kasugamycin. We find that kasugamycin (initiation inhibitor) displays a distinct transcriptomic profile compared to chloramphenicol and tetracycline (elongation inhibitors). However, genes in nucleotide metabolism were consistently downregulated. We also detected a TIA induced antisense transcript complementary to downregulated genes for purine metabolism, which we term SP_0835as. Mutating the promoter for SP_0835as eliminates both induction of SP_0835as and repression of the complementary genes in response to chloramphenicol. The mutated strain also displays slower growth rates than control strains in conditions that strongly induce SP_0835as, implying biologically relevant activity. Furthermore, the SP_0835as promoter straddles the boundary between SP_0835 and an insertion sequence (ISL3) immediately downstream. A survey of S. pneumoniae genomes shows the promoter is present in several phylogenetically distinct strain clusters. However, other common laboratory strains of S. pneumoniae lack the promoter, SP_0835as expression, and TIA repression of the complementary genes. Thus, SP_0835as represents a nascent RNA regulator that significantly remodels the metabolic response to TIA.

microbiology↗

bioRxiv2026

A soluble host signal drives rapid, brain-predominant capsular thickening in Streptococcus pneumoniae via a putative sodium-dependent transporter (SPD_0642) and capsular prepromoter sequence

The Streptococcus pneumoniae capsule is a major determinant of virulence, yet whether bacteria actively remodel it during infection remains unclear. Studying Swiss and South African clinical isolates (serotypes 1, 6B, 8, 12F, 19F, and 35B), we identified a rapid, tissue-specific response: capsule thickness increased within hours upon co-exposure to host cells and tissues. Only two 12F strains failed to thicken. Thickening was greatest in brain tissue, moderate in serum, and absent on the epithelium. This adaptation occurred independently of cod locus phase variation and nutritional factors, and was instead driven by a soluble, thermostable host signal (<3 kDa). Thickening correlated with neuroinflammation but did not require it, as it also occurred in contact with resting brain immune cells. It exacerbated meningitis in mice and enhanced bacteremia. Once induced, capsule thickening dampened inflammatory responses, coinciding with downregulation of pneumolysin, a major pro-inflammatory toxin. Genetic analysis of the non-thickening 12F isolates, together with targeted mutagenesis, identified two independent determinants of capsule-thickness modulation: a specific promoter-proximal element and SPD_0642, a conserved putative transporter encoded outside the capsule operon. Both contributed to the host-induced thickening phenotype. Pneumococci therefore rapidly remodel their surface in response to tissue-specific cues within the host, in a manner distinct from stochastic phase variation outside it. ImportanceMany bacteria are covered by a slimy outer layer, known as a capsule, that helps them evade the immune system. The amount of this layer can influence how easily harmful bacteria cause disease. Until now, scientists knew that bacteria can turn capsule production on or off through changes in their DNA. In this study, we show that Streptococcus pneumoniae, a common cause of serious infections, can also adjust its capsule in another way. It senses soluble signals from the tissues it enters, allowing it to recognize where it is in the body and to gradually change the thickness of its protective outer layer. This finding offers a new way of understanding how bacterial infections develop and may point to new treatment strategies.

microbiology↗

bioRxiv2026

The transcriptional response of Yersinia pseudotuberculosis to macrophage-released chemicals during growth within synthetic microcolonies

Yersinia pseudotuberculosis (Yptb) replicates in immune cell-encompassed microcolonies within tissues. Bacterial replication is controlled by protection against neutrophil attack and by macrophage-released antimicrobial factors, such as nitric oxide (NO). During these attacks, bacteria located on the microcolony periphery encounter extracellular signals that differ from those in the interior. To dissect individual microbial populations, {gamma} interferon-activated macrophages were used to challenge microdroplet-grown Yptb harboring an NO-responsive mCherry reporter. Subsequently, bacterial subpopulations that hyperactivated the reporter were isolated from droplets composed of a reversible polymer matrix. RNA-seq analysis indicated that induction of nitrosative stress-associated genes was the primary determinant distinguishing peripheral bacteria from the remaining population. In addition, a secondary stress response that induced prophage-associated genes was detected, which could not be traced to either DNA damage or nitrosative stress responses. Activated macrophages also induced the expression of the Yptb itaconate degradation enzyme-encoding transcript throughout the entire colony. To determine if itaconate production by the interferon-activated Irg1 protein played a role in restricting Yptb, bacteria harboring an itaconate-responsive reporter and Yptb mutants defective for itaconate degradation were analyzed during bacterial colonization of the murine spleen. Only a subset of colonies appeared to be exposed to itaconate, which may explain the very small defects exhibited by mutants unable to degrade the interferon-induced macrophage product. These results indicate that the primary response of bacteria to macrophage-elicited factors is likely associated with protection against NO-derived metabolites.

microbiology↗

bioRxiv2026

RNA Quality Control Enables Antibiotic Tolerance

The mechanisms underlying adaptation to antibiotic pressure within complex host environments remain incompletely understood. By experimentally evolving Streptococcus pneumoniae subjected to various antibiotics and immune states, we demonstrate populations adopting distinct adaptive strategies depending on specific selective context. General antibiotic stress drives convergent mutations in rny, encoding the RNA degradosome scaffold RNase Y, that exhibit broad-spectrum antibiotic tolerance and accelerated recovery. Single-cell transcriptomics revealed antibiotic-induced death is driven by transcriptional collapse, a catastrophic loss of RNA quantity and integrity. In contrast, rny mutants avert this via a bet-hedging strategy: a resilient minority maintains a baseline transcriptional profile, while a quiescent majority undergoes selective RNA degradation to preserve transcript fidelity. Upon stress removal, these populations execute a prioritized transcriptional ribosomal reboot, facilitating accelerated recovery. These findings identify RNA turnover as a tunable master regulator to survive combined pressures of antibiotics and immunity.

microbiology↗

bioRxiv2024

Dual CRISPRi-Seq for genome-wide genetic interaction studies identifies key genes involved in the pneumococcal cell cycle

A major goal in biology is to uncover the relationship between genotype and phenotype. However, identifying gene function is often hampered by genetic redundancy. For example, under standard laboratory conditions, three-quarters of the genes in the human pathogen Streptococcus pneumoniae are non-essential. A powerful approach to unravel genetic redundancy is by identifying gene-gene interactions. To uncover genetic interactions (GIs) in S. pneumoniae on a genome-wide scale, a generally applicable dual CRISPRi-Seq method and associated analysis pipeline was developed. Specifically, we created a library of 869 dual sgRNAs targeting high-confidence operons that encode essential and non-essential genes, covering over 70% of the genetic elements in the pneumococcal genome. Testing these 378,015 unique combinations, 4,026 significant GIs were identified, including 1,935 negative and 2,091 positive interactions. Besides known GIs, we found and confirmed previously unknown interactions involving genes responsible for fundamental cellular processes such as cell division, cell shape maintenance, and chromosome segregation. The presented methods and bioinformatic approaches can serve as a roadmap for genome-wide gene interaction studies in other organisms. Lastly, all interactions are available for exploration via the Pneumococcal Genetic Interaction Network (PneumoGIN) at https://veeninglab.shinyapps.io/PneumoGIN, which can serve as a starting point for new biological discoveries and translational research.

microbiology↗

bioRxiv2024

Diclofenac sensitizes multi-drug resistant Acinetobacter baumannii to colistin

Acinetobacter baumannii causes life-threatening infections that are becoming difficult to treat due to increasing rates of multi-drug resistance (MDR) among clinical isolates. This has led the World Health Organization and the CDC to categorize MDR A. baumannii as a top priority for the research and development of new antibiotics. Colistin is the last-resort antibiotic to treat carbapenem-resistant A. baumannii. Not surprisingly, reintroduction of colistin has resulted in the emergence of colistin-resistant strains. Diclofenac is a nonsteroidal anti-inflammatory drug used to treat pain and inflammation associated with arthritis. In this work, we show that diclofenac sensitizes colistin-resistant A. baumannii clinical strains to colistin, in vitro and in a murine model of pneumonia. Diclofenac also reduced the colistin MIC of Klebsiella pneumoniae and Pseudomonas aeruginosa isolates. Transcriptomic and proteomic analyses revealed an upregulation of oxidative stress-related genes and downregulation of type IV pili induced by the combination treatment. Notably, the concentrations of colistin and diclofenac effective in the murine model were substantially lower than those determined in vitro, implying a stronger synergistic effect in vivo compared to in vitro. A pilA mutant strain, lacking the primary component of the type IV pili, became sensitive to colistin in the absence of diclofenac. This suggest that the downregulation of type IV pili is key for the synergistic activity of these drugs in vivo and indicates that colistin and diclofenac exert an anti-virulence effect. Together, these results suggest that the diclofenac can be repurposed with colistin to treat MDR A. baumannii.

microbiology↗

bioRxiv2024

Directed evolution of a bacterial leucyl tRNA in mammalian cells for enhanced noncanonical amino acid mutagenesis

The E. coli leucyl-tRNA synthetase (EcLeuRS)/tRNAEcLeu pair has been engineered to genetically encode a structurally diverse group of enabling noncanonical amino acids (ncAAs) in eukaryotes, including those with bioconjugation handles, environment-sensitive fluorophores, photocaged amino acids, and native post-translational modifications. However, the scope of this toolbox in mammalian cells is limited by the poor activity of tRNAEcLeu. Here, we overcome this limitation by evolving tRNAEcLeu directly in mammalian cells using a virus-assisted selection scheme. This directed evolution platform was optimized for higher throughput such that the entire acceptor stem of tRNAEcLeu could be simultaneously engineered, which resulted in the identification of several variants with remarkably improved efficiency for incorporating a wide range of ncAAs. The advantage of the evolved leucyl tRNAs was demonstrated by expressing ncAA mutants in mammalian cells that were challenging to express before using the wild-type tRNAEcLeu, by creating viral vectors that facilitated ncAA mutagenesis at a significantly lower dose, and by creating more efficient mammalian cell lines stably expressing the ncAA-incorporation machinery.

synthetic biology↗

bioRxiv2023

Evaluation of the fitness benefit conferred by RNA cis-regulators to Streptococcus pneumoniae during infection

Bacteria have evolved complex transcriptional regulatory networks, as well as many diverse regulatory strategies at the RNA level, to enable more efficient use of metabolic resources and rapid response to changing conditions. However, most RNA-based regulatory mechanisms are not well conserved across different bacterial species despite controlling genes important for virulence or essential biosynthetic processes. Here, we characterize the activity of, and assess the fitness benefit conferred by, twelve cis-acting regulatory RNAs (including several riboswitches and a T-box), in the opportunistic pathogen Streptococcus pneumoniae TIGR4. By evaluating native locus mutants of each regulator that result in constitutively active and repressed expression, we establish that growth defects in planktonic culture are associated with constitutive repression of gene expression, while constitutive activation of gene expression is rarely deleterious. In contrast, in mouse nasal carriage and pneumonia models, strains with both constitutively active and repressed gene expression are significantly less fit than matched control strains. Furthermore, two RNA-regulated pathways, FMN synthesis/transport and pyrimidine synthesis/transport display the greatest sensitivity to mis-regulation or constitutive gene repression in both planktonic culture and in vivo environments. Our findings suggest that despite lack of an obvious phenotypes associated with gene over-expression in vitro, the fitness benefit conferred on bacteria via fine-tuned metabolic regulation through cis-acting regulatory RNAs is often substantial in vivo, and therefore easily sufficient to drive the evolution and maintenance of diverse RNA regulatory mechanisms.

microbiology↗

bioRxiv2023

Identification of Determinants that Allow Maintenance of High-Level Fluoroquinolone Resistance in Acinetobacter baumannii

Acinetobacter baumannii is associated with multidrug resistant (MDR) infections in healthcare settings, with fluoroquinolones such as ciprofloxacin being currently ineffective. Clinical isolates largely harbor mutations in the GyrA and TopoIV fluoroquinolone targets, as well as mutations that increase expression of drug resistance-nodulation-division (RND) efflux pumps. Factors critical for maintaining fitness levels of pump overproducers are uncharacterized despite their prevalence in clinical isolates. We here identify proteins that contribute to the fitness of FQR strains overexpressing three known RND systems using high-density insertion mutagenesis. Overexpression of the AdeFGH efflux pump caused hypersensitization to defects in outer membrane homeostatic regulation, including lesions that reduced LOS biosynthesis and blocked production of the major A. baumannii porin. In contrast, AdeAB pump hyperexpression, in the absence of elevated adeC expression (the outer membrane component of the pump), was relatively tolerant to loss of these functions, consistent with the outer membrane protein being the primary disruptive component. Surprisingly, overexpression of proton-transporting efflux pumps had little impact on cytosolic pH, consistent with a compensatory response to pump activity. The most striking transcriptional changes were associated with AdeFGH pump overexpression, including the activation of the phenylacetate (PAA) degradation regulon. Disruption of the PAA pathway resulted in cytosolic acidification and defective expression of genes involved in protection from oxidative stress. These results indicate that RND efflux pump overproduction is compensated by maintenance of outer membrane integrity in A. baumannii to facilitate fitness of FQR isolates. ImportanceAcinetobacter baumannii is a pathogen that often causes multidrug resistant (MDR) infections in healthcare settings, presenting a threat to the efficacy of known therapeutic interventions. Fluoroquinolones such as ciprofloxacin are currently ineffective against a majority of clinical A. baumannii isolates, many of which express pumps that remove this antibiotic class from within the bacterium. Three of these pumps can be found in most clinical isolates, with one of the three often hyperproduced at all times. In this study we identify proteins that are necessary for the fitness of pump hyperproducers. The identified proteins are necessary to stabilize the outer membrane and allow the cytoplasm to tolerate the accumulation of ions as a consequence of excess pump activity. These results point to strategies for developing therapies that combine known antibiotics with drugs that target proteins important for survival of strains hyperexpressing efflux pumps.

microbiology↗

bioRxiv2023

Dual Membrane-Sspanning Anti-Sigma Factors Regulate Vesiculation in Gut Bacteroidota

Bacteroidota are abundant members of the human gut microbiota that shape the enteric landscape by modulating host immunity and degrading dietary- and host-derived glycans. These processes are at least partially mediated by Outer Membrane Vesicles (OMVs). In this work, we developed a high-throughput screen to identify genes required for OMV biogenesis and its regulation in Bacteroides thetaiotaomicron (Bt). Our screening led us to the identification of a novel family of Dual Membrane-spanning Anti-sigma factors (Dma), which regulate OMV biogenesis in Bt. We employed molecular and multiomic analyses to demonstrate that deletion of Dma1, the founding member of the Dma family, results in hypervesiculation by modulating the expression of NigD1, which belongs to a family of uncharacterized proteins found throughout Bacteroidota. Dma1 has an unprecedented domain organization: it contains a C-terminal {beta}-barrel embedded in the OM; its N-terminal domain interacts with its cognate sigma factor in the cytoplasm, and both domains are tethered via an intrinsically disordered region that traverses the periplasm. Phylogenetic analyses reveal that the Dma family is a unique feature of Bacteroidota. This study provides the first mechanistic insights into the regulation of OMV biogenesis in human gut bacteria.

microbiology↗

bioRxiv2023

CRISPRi-TnSeq: A genome-wide high-throughput tool for bacterial essential-nonessential genetic interaction mapping

Genetic interaction networks can help identify functional connections between genes and pathways, which can be leveraged to establish (new) gene function, drug targets, and fill pathway gaps. Since there is no optimal tool that can map genetic interactions across many different bacterial strains and species, we develop CRISPRi-TnSeq, a genome-wide tool that maps genetic interactions between essential genes and nonessential genes through the knockdown of a targeted essential gene (CRISPRi) and the simultaneous knockout of individual nonessential genes (Tn-Seq). CRISPRi-TnSeq thereby identifies, on a genome-wide scale, synthetic and suppressor-type relationships between essential and nonessential genes, enabling the construction of essential-nonessential genetic interaction networks. To develop and optimize CRISPRi-TnSeq, CRISPRi strains were obtained for 13 essential genes in Streptococcus pneumoniae, involved in different biological processes including metabolism, DNA replication, transcription, cell division and cell envelope synthesis. Transposon-mutant libraries were constructed in each strain enabling screening of [~]24,000 gene-gene pairs, which led to the identification of 1,334 genetic interactions, including 754 negative and 580 positive genetic interactions. Through extensive network analyses and validation experiments we identify a set of 17 pleiotropic genes, of which a subset tentatively functions as genetic capacitors, dampening phenotypic outcomes and protecting against perturbations. Furthermore, we focus on the relationships between cell wall synthesis, integrity and cell division and highlight: 1) how essential gene knockdown can be compensated by rerouting flux through nonessential genes in a pathway; 2) the existence of a delicate balance between Z-ring formation and localization, and septal and peripheral peptidoglycan (PG) synthesis to successfully accomplish cell division; 3) the control of c-di-AMP over intracellular K+ and turgor, and thereby modulation of the cell wall synthesis machinery; 4) the dynamic nature of cell wall protein CozEb and its effect on PG synthesis, cell shape morphology and envelope integrity; 5) functional dependency between chromosome decatenation and segregation, and the critical link with cell division, and cell wall synthesis. Overall, we show that CRISPRi-TnSeq uncovers genetic interactions between closely functionally linked genes and pathways, as well as disparate genes and pathways, highlighting pathway dependencies and valuable leads for gene function. Importantly, since both CRISPRi and Tn-Seq are widely used tools, CRISPRi-TnSeq should be relatively easy to implement to construct genetic interaction networks across many different microbial strains and species.

genetics↗

bioRxiv2022

Genome-wide phage susceptibility analysis in Acinetobacter baumannii reveals capsule modulation as a central determinant of phage defense

Phage have gained renewed interest as an adjunctive treatment for life-threatening infections with the resistant nosocomial pathogen Acinetobacter baumannii. Our understanding of how A. baumannii defends against phage remains limited, although this information could lead to improved antimicrobial therapies. To address this problem, we identified genome-wide determinants of phage susceptibility in A. baumannii using Tn-seq. These studies focused on the lytic phage Loki, which targets Acinetobacter by unknown mechanisms. We identified 41 candidate loci that increase susceptibility to Loki when disrupted, and 10 that decrease susceptibility. Combined with spontaneous resistance mapping, our results support the model that Loki uses the K3 capsule as an essential receptor, and that capsule modulation provides A. baumannii with strategies to control vulnerability to phage. A key center of this control is transcriptional regulation of capsule synthesis and phage virulence by the global regulator BfmRS. Mutations hyperactivating BfmRS simultaneously increase capsule levels, Loki replication, and host killing, while BfmRS-inactivating mutations have the opposite effect, reducing capsule and blocking Loki infection. We identified novel BfmRS-activating mutations, including knockouts of a T2 RNase protein and the disulfide formation enzyme DsbA, that hypersensitize bacteria to phage challenge. We further found that mutation of a glycosyltransferase known to alter capsule structure and bacterial virulence can also cause complete phage resistance. Finally, additional factors including lipooligosaccharide and Lon protease act independently of capsule modulation to interfere with Loki infection. This work demonstrates that regulatory and structural modulation of capsule, known to alter A. baumannii virulence, is also a major determinant of susceptibility to phage. Author SummaryAntibiotic-resistant infections with Acinetobacter baumannii are a major problem in critical care units and have increased in frequency during the COVID-19 pandemic. The virulence of these infections depends on a polysaccharide capsule surrounding the bacterium. Phage, or viruses that kill bacteria, represent a promising alternative therapy against highly antibiotic-resistant A. baumannii infections, and A. baumannii-specific phage often target the capsule. Here, we use high-throughput genetics to analyze how A. baumannii defends against phage and identify ways to potentiate their killing activity. We found that stressing the bacteria in ways that cause augmented production of capsule also causes hyper-susceptibility to phage. By contrast, turning off the stress response, or mutating the capsule structure, causes complete phage resistance. Altering another surface structure, lipooligosaccharide, or an intracellular protease also enhances phage attack. Modulating the amounts or makeup of capsular polysaccharide is known to influence virulence in A. baumannii. This work thus uncovers a connection between phage pressure and the evolution of virulence in A. baumannii, and it identifies control mechanisms that may be leveraged for improving future phage-based antimicrobial therapies.

microbiology↗

bioRxiv2022

A bacterial pan-genome makes gene essentiality strain-dependent and evolvable

Many bacterial species are represented by a pan-genome, whose genetic repertoire far outstrips that of any single bacterial genome. Here we investigate how a bacterial pan-genome might influence gene essentiality, and whether essential genes that are initially critical for the survival of an organism can evolve to become non-essential. By using Tn-Seq, whole-genome sequencing, and RNA-Seq on a set of 36 clinical Streptococcus pneumoniae strains representative of >68% of the species pan-genome, we identify a species-wide essentialome that can be subdivided into universal, strain-specific and accessory essential genes. By employing forced-evolution experiments we show that specific genetic changes allow bacteria to bypass essentiality. Moreover, by untangling several genetic mechanisms we show that gene-essentiality can be highly influenced and/or dependent on: 1) the composition of the accessory genome; 2) the accumulation of toxic intermediates; 3) functional redundancy; 4) efficient recycling of critical metabolites; and 5) pathway rewiring. While this functional characterization underscores the evolvability-potential of many essential genes, we also show that genes with differential essentiality remain important antimicrobial drug target candidates, as their inactivation almost always has a severe fitness cost in vivo.

microbiology↗

bioRxiv2022

A genome-wide atlas of antibiotic susceptibility targets and pathways to tolerance.

Detailed knowledge on how bacteria evade antibiotics and eventually develop resistance could open avenues for novel therapeutics and diagnostics. It is thereby key to develop a comprehensive genome-wide understanding of how bacteria process antibiotic stress, and how modulation of the involved processes affects their ability to overcome said stress. Here we undertake a comprehensive genetic analysis of how the major human pathogen Streptococcus pneumoniae responds to 20 antibiotics. We built a genome-wide atlas of drug susceptibility determinants and generate a genetic interaction network that connects cellular processes and genes of unknown function, which we show can be used as therapeutic targets. Pathway analysis reveals a genome-wide "tolerome", defined by cellular processes that can make a bacterium less susceptible, and often tolerant, in an antibiotic specific manner. Importantly, modulation of these processes confers fitness benefits during active infections under antibiotic selection. Moreover, screening of sequenced clinical isolates demonstrates that mutations in tolerome genes readily evolve and are frequently associated with resistant strains, indicating such mutations may be an important harbinger for the emergence of antibiotic resistance.

microbiology↗

bioRxiv2022

Virus-assisted directed evolution of enhanced suppressor tRNAs in mammalian cells

Site-specific incorporation of unnatural amino acids (Uaas) in living cells relies on engineered aminoacyl-tRNA synthetase/tRNA pairs borrowed from a distant domain of life. Such heterologous suppressor tRNAs often show poor intrinsic activity, presumably due to the failure to optimally interact with a non-native translation system. This limitation can be addressed in E. coli using directed evolution. However, no suitable selection system is currently available to do the same in mammalian cells. Here we report virus-assisted directed evolution of tRNAs (VADER) in mammalian cells, which employs a double-sieve selection scheme to facilitate single-step enrichment of active-yet-orthogonal tRNA mutants from naive libraries. Using VADER, we developed improved mutants of M. mazei pyrrolysyl-tRNA, the most popular Uaa mutagenesis platform in eukaryotes. We also show that the higher activity of the most efficient mutants is specific for mammalian cells, alluding to an improved interaction with the unique mammalian translation system.

synthetic biology↗

bioRxiv2021

Yersinia pseudotuberculosis doxycycline tolerance strategies include modulating expression of genes involved in cell permeability and tRNA modifications

Antibiotic tolerance is typically associated with a phenotypic change within a bacterial population, resulting in a transient decrease in antibiotic susceptibility that can contribute to treatment failure and recurrent infections. Although tolerant cells may emerge prior to treatment, the stress of prolonged antibiotic exposure can also promote tolerance. Here, we sought to determine how Yersinia pseudotuberculosis responds to doxycycline exposure, to then verify if these gene expression changes could promote doxycycline tolerance in culture and in our mouse model of infection. Only four genes were differentially regulated in response to a physiologically-relevant dose of doxycycline: osmB and ompF were upregulated, tusB and cnfy were downregulated; differential expression also occurred during doxycycline treatment in the mouse. ompF, tusB and cnfy were also differentially regulated in response to chloramphenicol, indicating these could be general responses to ribosomal inhibition. cnfy has previously been associated with persistence and was not a major focus here. We found deletion of the OmpF porin resulted in increased antibiotic accumulation, suggesting expression may promote diffusion of doxycycline out of the cell, while OsmB lipoprotein had a minor impact on antibiotic permeability. Overexpression of tusB significantly impaired bacterial survival in culture and in the mouse, suggesting that tRNA modification by tusB, and the resulting impacts on translational machinery, may play an important role in promoting tolerance. We believe this is the first observation of bactericidal activity of doxycycline, which was revealed by reversing tusB downregulation.

microbiology↗

bioRxiv2021

Rapid, multianalyte detection of opioid metabolites in wastewater

By monitoring opioid metabolites, wastewater-based epidemiology (WBE) could be an excellent tool for real-time information on consumption of illicit drugs. A key limitation of WBE is the reliance on costly laboratory-based techniques that require substantial infrastructure and trained personnel, resulting in long turnaround times. Here, we present an aptamer-based graphene field effect transistor (AptG-FET) platform for simultaneous detection of three different opioid metabolites. This platform provides a reliable, rapid, and inexpensive method for quantitative analysis of opioid metabolites in wastewater (WW). The platform delivers a limit of detection (LOD) 2-3 orders of magnitude lower than previous reports, but in line with the concentrations range (pg/ml to ng/ml) of these opioid metabolites present in real samples. To enable multianalyte detection we developed a facile, reproducible, and high yield fabrication process producing twenty G-FETs with integrated side gate platinum (Pt) electrodes on a single chip. Our devices achieved the simultaneous and selective multianalyte detection of three different metabolites: Noroxycodone (NX), 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), and Norfentanyl (NF) in wastewater.

biophysics↗

bioRxiv2021

Immunosuppression broadens evolutionary pathways to treatment failure during Acinetobacter baumannii pneumonia

Acinetobacter baumannii is increasingly refractory to antibiotic treatment in healthcare settings. As is true of most human pathogens, the genetic path to antimicrobial resistance (AMR) and the role that the immune system plays in modulating AMR during disease are poorly understood. Here we reproduced several routes to fluoroquinolone resistance, performing evolution experiments using sequential lung infections in mice that are replete or depleted of neutrophils, providing two key insights into the evolution of drug resistance. First, neutropenic hosts acted as reservoirs for the accumulation of drug resistance during drug treatment. Selection for variants with altered drug sensitivity profiles arose readily in the absence of neutrophils, while immunocompetent animals restricted the appearance of these variants. Secondly, antibiotic treatment failure in the immunocompromised host was shown to occur without clinically defined resistance, an unexpected result that provides a model for how antibiotic failure occurs clinically in the absence of AMR. The genetic mechanism underlying both these results is initiated by mutations activating the drug egress pump regulator AdeL, which drives persistence in the presence of antibiotic. Therefore, antibiotic persistence mutations present a two-pronged risk during disease, causing drug treatment failure in the immunocompromised host while simultaneously increasing the emergence of high-level AMR.

microbiology↗