Biology subjects

Earl, A. M.

Publications and source records attributed to Earl, A. M..

18 recordsLinked to original sources

bioRxiv2026

Staphylococcus aureus uses a eukaryotic-like uridyltransferase to make UDP-GlcNAc for cell wall synthesis

UDP-N-acetylglucosamine (UDP-GlcNAc) is an essential metabolite used to build the peptidoglycan cell wall that protects bacteria against lysis caused by high turgor pressure. More than thirty years ago, GlmU, which contains both acetyltransferase and uridyltransferase activities, was discovered to make UDP-GlcNAc in E. coli, and it is now widely considered essential for UDP-GlcNAc synthesis across bacteria. Here we report that the clinically important pathogen Staphylococcus aureus relies on a previously uncharacterized uridyltransferase called NagU to synthesize most of its UDP-GlcNAc for cell wall synthesis. We also report that NagU binds to and negatively regulates DacA, the diadenylate cyclase that makes the essential second messenger cyclic di-AMP, which regulates turgor pressure. We propose a model in which increasing NagU expression simultaneously increases flux into peptidoglycan and increases turgor pressure to enable cell wall expansion. Finally, an evolutionary analysis shows that many bacteria beyond Staphylococcaceae encode NagU homologues but lack GlmU either entirely or in part, suggesting that alternative UDP-GlcNAc biosynthesis pathways are not rare.

microbiology↗

bioRxiv2026

Mobilome of Enterococcus faecalis from healthy nursery pigs exposed to antibiotic pressure

In response to the comparatively sudden application of industrial scale levels of antibiotics to an ecosystem where naturally produced antibiotics are scarce, namely the ecologies within and around agricultural settings, animal-associated microbes have had to rapidly adapt, mostly relying on mobile genetic elements (MGEs) taken up due to loss of CRISPR functionality. Due to selection for resistance and other adaptive traits carried by dynamic and rapidly recombining MGEs, plasmids and transposons have rapidly accumulated in this human-proximal environment. Because of the occurrence of Enterococcus faecalis in a wide range of hosts up and down the food chain, and the fact that this species represents the greatest generalist of the genus, we comprehensively examined the mobilome of multidrug-resistant E. faecalis (ST330, ST591, ST710, and ST711) from healthy piglets raised on dispersed Brazilian farms, using long-read sequencing, analysis of plasmid pangenomes, and conjugation assays with these strains serving as donors. Genomes ranged from [~]2.8-3.1 Mb, with diverse MGEs constituting [~]7-15% of those genomes. Large modular antimicrobial resistance-encoding gene blocks ([~]40 Kb) were observed to be integrated into a [~]67 Kb chromosomal segment of the pathogenicity island AF454824. Prophages contributed up to 70% of the chromosomal mobile element content, integrating into both CRISPR-deficient genomes and those with intact type II-A CRISPR1 arrays, which were enriched with Caudoviricetes phage-targeting spacers across all strains. Plasmid content showed pronounced mosaicism driven by diverse insertion sequences, transposons, and related mobile elements, many directly implicated in AMR gene cluster acquisitions. RepA_N, Inc18, and Rep3 plasmids, mostly conjugative, also carried various persistence-related traits, suggesting they may actively enhance agricultural fitness rather than passively accumulate due to loss of CRISPR protection.

microbiology↗

bioRxiv2025

Efagins evolved independently to target the enterococcal cell wall

Enterococci are major causes of multidrug-resistant infections, and antimicrobials with fundamentally new mechanisms of action are urgently needed. We identify a new class of antibacterial agents, termed efagins, which are chromosomally encoded, phage-related nanomachines that recognize cell wall carbohydrate receptors and inhibit subsets of E. faecalis, E. faecium and other enterococci selectively--a key reason they evaded prior detection. Five natural variants with distinct targeting profiles were identified - four related by sequence divergence, while one appears to have arisen through recombination of the targeting domain, likely from a phage donor. Operons encoding the corresponding carbohydrate receptors are highly variable, accounting for targeting specificity. The efagin targeting domain can be engineered to reprogram them toward alternative receptors, providing a pathway for filling critical coverage gaps. These findings advance efagins as new selective antibacterials with promise for addressing infection and spread of multidrug-resistant enterococcal infection.

microbiology↗

bioRxiv2025

Circling in on plasmids: benchmarking plasmid detection and reconstruction tools for short-read data from diverse species

The ability to detect and reconstruct plasmids from genome assemblies is crucial for studying the evolution and spread of antimicrobial resistance and virulence in bacteria. Though long-read sequencing technologies have made reconstructing plasmids easier, most (97%) of the bacterial genome assemblies in the public domain are generated from short-read data. Work to compare plasmid reconstruction tools has focused primarily on E. coli, leaving gaps in our understanding of how well these tools perform on other, less well-characterized, taxa. Using high quality assemblies as ground truth, we benchmarked 12 plasmid detection tools (which identify plasmid contigs in assemblies) and four plasmid reconstruction tools (which group contigs from the same plasmid together). We tested their ability to characterize diverse plasmids from short-read assemblies representing a wide range of Enterobacterales and Enterococcus species, including newly discovered and poorly characterized species collected from non-human hosts. Plasmer, PlasmidEC, PlaScope, and gplas2 were the highest-scoring plasmid detection tools, performing well for both Enterobacterales and enterococci. The two major determinants of accurate plasmid detection were representation in plasmid databases - with Enterobacterales plasmids being more easily detected than those from enterococci - and assembly contiguity, which was also key for successful plasmid reconstruction. Gplas2 performed best for plasmid reconstruction; however, less than half of plasmids were perfectly reconstructed, suggesting that substantial room for improvement remains in this class of tools. Key MessagesO_LIThe ability to detect and reconstruct plasmids from short-read assemblies is crucial to study the spread of antimicrobial resistance and virulence genes in bacteria. C_LIO_LIMost past comparisons of tools for plasmid detection and reconstruction have focused on plasmids from well-characterized E. coli; therefore, we broadened our benchmarking set to include plasmids from diverse Enterobacterales and Enterococcus species, collected from a wide range of hosts. C_LIO_LIWe compared the predictions of 12 recent plasmid detection and four plasmid reconstruction tools on short read assemblies, against a truth set generated from high-quality hybrid assemblies. C_LIO_LIPlasmer, PlasmidEC, PlaScope, and gplas2 were the highest-scoring plasmid detection tools. C_LIO_LIGplas2 was the highest-scoring tool for plasmid reconstruction; the quality of plasmid reconstruction was mainly determined by assembly fragmentation, with plasmids in more contiguous assemblies being easier to reconstruct. C_LI

bioinformatics↗

bioRxiv2025

An Anaerobic Pathogen Rewires Host Metabolism to Fuel Oxidative Growth in the Inflamed Gut

To colonize their host and cause disease, enteric pathogens must deploy their virulence factors to establish distinct nutrient niches. How obligate anaerobic pathogens construct nutrient niches in the densely populated large intestine remains poorly understood. Enterotoxigenic Bacteroides fragilis (ETBF) is considered an obligate anaerobic bacterium and has been implicated in inflammation-associated diseases, including colitis and colorectal cancer. Here we show that ETBF uses its virulence factor, Bacteroides fragilis toxin, to reprogram colonic epithelial cell metabolism to colonize the inflamed gut. Bacteroides fragilis toxin activates colonic epithelial signaling and hijacks the host bile acid recycling pathway, inducing a metabolic shift in the epithelium from oxidative phosphorylation to glycolysis. This shift increases local concentrations of lactate and oxygen, nutrients that support an oxidative metabolism in ETBF. These findings reveal an unexpected strategy by which a pathogenic organism, previously considered to be an obligate anaerobic bacterium, generates and exploits an oxidative niche in the inflamed gut.

microbiology↗

bioRxiv2025

Insights into global antimicrobial resistance dynamics through the sequencing of enteric bacteria from U.S. international travelers

Antimicrobial resistance (AMR) is an urgent threat to public health, but gaps in surveillance limit the detection of emergent novel threats and knowledge about the global distribution of AMR genes. International travelers frequently acquire AMR organisms, and thus may provide a window into AMR dynamics in otherwise poorly monitored regions and environments. To assess the utility of travelers as global AMR sentinels, we collected pre- and post-travel stool samples from 608 travelers, which were screened for the presence of extended-spectrum beta-lactamase producing Enterobacterales, carbapenem-resistant Enterobacterales, and mcr-mediated colistin-resistant Enterobacterales. A total of 307 distinct AMR organisms were sequenced in order to determine genotypic patterns and their association with travel region and behavior. Travel-associated AMR organisms were overwhelmingly E. coli, which exhibited considerable phylogenetic diversity regardless of travel region. However, the prevalence of resistance genes varied by region, with blaCTX-M-55 and blaCTX-M-27 significantly more common in travelers returning from South America and South-Eastern Asia, respectively. Hybrid assembly and plasmid reconstruction revealed the genomic neighborhood of blaCTX-M-55 frequently matched a motif previously linked to animal populations. Contact with animals was also associated with virulence factors in acquired AMR organisms, including carriage of the ColV plasmid, a driver of avian pathogenic E. coli. We identified novel variants of the mcr-1 gene in strains acquired from Western Africa, highlighting the potential for traveler surveillance to detect emerging clinical threats. Ongoing efforts to track travel-acquired organisms could complement existing global AMR surveillance frameworks. ImportanceWe collected pre- and post-travel stool samples from 608 U.S. international travelers, which were screened for the presence of three clinically relevant antimicrobial-resistant organisms, with the goal of understanding the utility of travelers as sentinels for emerging AMR and identifying regional resistance patterns. We sequenced 307 organisms and uncovered significant diversity and geographic heterogeneity in AMR organisms. We found significant differences in certain resistance gene alleles of travelers returning from South America and South-Eastern Asia, traveler contact with animals associated with virulence factors, including a plasmid that is a known driver of avian pathogenic E. coli, and novel variants of the colistin resistance mcr-1 gene in strains acquired from Western Africa. This work emphasizes the value of monitoring international travelers as a proxy for understanding AMR patterns globally and could be an important addition to existing global AMR surveillance frameworks.

genomics↗

bioRxiv2024

Rapid Emergence of Resistance to Broad-Spectrum Direct Antimicrobial Activity of Avibactam

Avibactam (AVI) is a diazabicyclooctane (DBO) {beta}-lactamase inhibitor used clinically in combination with ceftazidime. At concentrations higher than those typically achieved in vivo, it also has broad-spectrum direct antibacterial activity against Enterobacterales strains, including metallo-{beta}-lactamase-producing isolates, mediated by inhibition of penicillin-binding protein 2 (PBP2). This activity is mechanistically similar to that of more potent novel DBOs (zidebactam, nacubactam) in late clinical development. We found that resistance to AVI emerged readily, with a mutation frequency of 2x10-6 to 8x10-5. Whole genome sequencing of resistant isolates revealed a heterogeneous mutational target that permitted bacterial survival and replication despite PBP2 inhibition, in line with prior studies of PBP2-targeting drugs. While such mutations are believed to act by upregulating the bacterial stringent response, we found a similarly high mutation frequency in bacteria deficient in components of the stringent response, although we observed a different set of mutations in these strains. Although avibactam-resistant strains had increased lag time, suggesting a fitness cost that might render them less problematic in clinical infections, there was no statistically significant difference in growth rates between susceptible and resistant strains. The finding of rapid emergence of resistance to avibactam as the result of a large mutational target has important implications for novel DBOs with potent direct antibacterial activity, which are being developed with the goal of expanding cell wall-active treatment options for multidrug-resistant gram-negative infections but may be vulnerable to treatment-emergent resistance.

microbiology↗

bioRxiv2024

Fast and exact gap-affine partial order alignment with POASTA

MotivationPartial order alignment is a widely used method for computing multiple sequence alignments, with applications in genome assembly and pangenomics, among many others. Current algorithms to compute the optimal, gap-affine partial order alignment do not scale well to larger graphs and sequences. While heuristic approaches exist, they do not guarantee optimal alignment and sacrifice alignment accuracy. ResultsWe present POASTA, a new optimal algorithm for partial order alignment that exploits long stretches of matching sequence between the graph and a query. We benchmarked POASTA against the state-of-the-art on several diverse bacterial gene datasets and demonstrated an average speed-up of 4.1x and up to 9.8x, using less memory. POASTAs memory scaling characteristics enabled the construction of much larger POA graphs than previously possible, as demonstrated by megabase-length alignments of 342 Mycobacterium tuberculosis sequences. Availability and implementationPOASTA is available on Github at https://github.com/broadinstitute/poasta.

bioinformatics↗

bioRxiv2024

Distinct Escherichia coli transcriptional profiles in the guts of recurrent UTI sufferers revealed by pan-genome hybrid selection

Low-abundance members of microbial communities are difficult to study in their native habitats. This includes Escherichia coli, a minor, but common inhabitant of the gastrointestinal tract and opportunistic pathogen, including of the urinary tract, where it is the primary pathogen. While multi-omic analyses have detailed critical interactions between uropathogenic Escherichia coli (UPEC) and the bladder that mediate UTI outcome, comparatively little is known about UPEC in its pre-infection reservoir, partly due to its low abundance there (<1% relative abundance). To accurately and sensitively explore the genomes and transcriptomes of diverse E. coli in gastrointestinal communities, we developed E. coli PanSelect which uses a set of probes designed to specifically recognize and capture E. colis broad pangenome from sequencing libraries. We demonstrated the ability of E. coli PanSelect to enrich, by orders of magnitude, sequencing data from diverse E. coli using a mock community and a set of human stool samples collected as part of a cohort study investigating drivers of recurrent urinary tract infections (rUTI). Comparisons of genomes and transcriptomes between E. coli residing in the gastrointestinal tracts of women with and without a history of rUTI suggest that rUTI gut E. coli are responding to increased levels of oxygen and nitrate, suggestive of mucosal inflammation, which may have implications for recurrent disease. E. coli PanSelect is well suited for investigations of native in vivo biology of E. coli in other environments where it is at low relative abundance, and the framework described here has broad applicability to other highly diverse, low abundance organisms.

microbiology↗

bioRxiv2023

zol & fai: large-scale targeted detection and evolutionary investigation of gene clusters

Many universally and conditionally important genes are genomically aggregated within clusters. Here, we introduce fai and zol, which together enable large-scale comparative analysis of different types of gene clusters and mobile-genetic elements (MGEs), such as biosynthetic gene clusters (BGCs) or viruses. Fundamentally, they overcome a current bottleneck to reliably perform comprehensive orthology inference at large scale across broad taxonomic contexts and thousands of genomes. First, fai allows the identification of orthologous instances of a query gene cluster of interest amongst a database of target genomes. Subsequently, zol enables reliable, context-specific inference of ortholog groups for individual protein-encoding genes across gene cluster instances. In addition, zol performs functional annotation and computes a variety of evolutionary statistics for each inferred ortholog group. Importantly, in comparison to tools for visual exploration of homologous relationships between gene clusters, zol can scale to thousands of gene cluster instances and produce detailed reports that are easy to digest. To showcase fai and zol, we apply them for: (i) longitudinal tracking of a virus in metagenomes, (ii) discovering novel population-level genetic insights of two common BGCs in the fungal species Aspergillus flavus, and (iii) uncovering large-scale evolutionary trends of a virulence-associated gene cluster across thousands of genomes from a diverse bacterial genus.

bioinformatics↗

bioRxiv2023

The carbapenem inoculum effect provides insight into the molecular mechanisms underlying carbapenem resistance in Enterobacterales

Carbapenem-resistant Enterobacterales (CRE) are important pathogens that can develop resistance via multiple molecular mechanisms, including hydrolysis or reduced antibiotic influx. Identifying these mechanisms can improve pathogen surveillance, infection control, and patient care. We investigated, both phenomenologically and mechanistically, how resistance mechanisms influence the carbapenem inoculum effect (IE), a phenomenon where inoculum size affects antimicrobial susceptibility testing (AST). We demonstrated that any of seven different carbapenemases were sufficient to impart a meropenem IE when transformed into a laboratory strain of Escherichia coli. Across 106 clinical CRE isolates spanning 6 genera and 12 species, the carbapenem IE strictly depended on resistance mechanism: all 36 carbapenemase-producing CRE (CP-CRE) exhibited a clear IE, whereas 43 porin-deficient CRE displayed none. 27 isolates with both carbapenemase production and porin deficiency exhibited high-level resistance at all inocula, and still displayed an IE, albeit smaller in magnitude than CP-CRE with intact porins. Mechanistically, we found that clinical CP-CRE release carbapenemase activity into culture supernatant to protect other cells. Further, this released activity markedly increased upon exposure to lethal antibiotic doses, functionally consistent with altruism. Concerningly, 50% and 24% of CP-CRE isolates changed susceptibility classification to meropenem and ertapenem, respectively, across the allowable inoculum range in clinical testing guidelines. The meropenem IE, and the ratio of ertapenem to meropenem minimal inhibitory concentration (MIC) at standard inoculum, reliably identified CP-CRE. Understanding how resistance mechanisms affect AST could improve diagnosis and guide therapies for CRE infections. ImportanceInfections caused by carbapenem-resistant Enterobacterales (CRE) pose significant threats to patients and public health worldwide. Carbapenem resistance can occur through several molecular mechanisms, including enzymatic hydrolysis by carbapenemases and reduced influx via porin mutations. Identifying carbapenemase-producing isolates could enable tailored antibiotic selection to improve patient outcomes, and infection control measures to prevent further carbapenemase transmission. In a large collection of CRE isolates, we found that only carbapenemase-producing CRE exhibit an inoculum effect, in which their measured resistance varies markedly with cell density, which risks misdiagnosis. Further, this inoculum effect occurs under conditions where bacteria release carbapenemases to the community upon exposure to antibiotic that results in cell death, functionally consistent with altruism. Measuring this inoculum effect, or integrating other data from routine antimicrobial susceptibility testing, enhances carbapenem resistance detection, paving the way for more effective strategies to combat this growing public health crisis.

microbiology↗

bioRxiv2023

Global diversity of enterococci and description of 18 novel species

Enterococci are commensal gut microbes of most land animals. They diversified over hundreds of millions of years adapting to evolving hosts and host diets. Of over 60 known enterococcal species, Enterococcus faecalis and E. faecium uniquely emerged in the antibiotic era among leading causes of multidrug resistant hospital-associated infection. The basis for the association of particular enterococcal species with a host is largely unknown. To begin deciphering enterococcal species traits that drive host association, and to assess the pool of Enterococcus-adapted genes from which known facile gene exchangers such as E. faecalis and E. faecium may draw, we collected 886 enterococcal strains from nearly 1,000 specimens representing widely diverse hosts, ecologies and geographies. This provided data on the global occurrence and host associations of known species, identifying 18 new species in the process expanding genus diversity by >25%. The novel species harbor diverse genes associated with toxins, detoxification, and resource acquisition. E. faecalis and E. faecium were isolated from a wide diversity of hosts highlighting their generalist properties, whereas most other species exhibited more restricted distributions indicative of specialized host associations. The expanded species diversity permitted the Enterococcus genus phylogeny to be viewed with unprecedented resolution, allowing features to be identified that distinguish its four deeply rooted clades as well as genes associated with range expansion, such as B-vitamin biosynthesis and flagellar motility. Collectively, this work provides an unprecedentedly broad and deep view of the genus Enterococcus, potential threats to human health, and new insights into its evolution. SIGNIFICANCEEnterococci, host-associated microbes that are now leading drug-resistant hospital pathogens, arose as animals colonized land over 400 million years ago. To globally assess the diversity of enterococci now associated with land animals, we collected 886 enterococcal specimens from a wide range of geographies and ecologies, ranging from urban environments to remote areas generally inaccessible to humans. Species determination and genome analysis revealed host associations from generalists to specialists, and identified 18 new species, increasing the genus by over 25%. This added diversity provided greater resolution of the genus clade structure, identifying new features associated with species radiations. Moreover, the high rate of new species discovery shows that tremendous genetic diversity in Enterococcus remains to be discovered.

microbiology↗

bioRxiv2023

Strain Tracking with Uncertainty Quantification

The ability to detect and quantify microbiota over time has a plethora of clinical, basic science, and public health applications. One of the primary means of tracking microbiota is through sequencing technologies. When the microorganism of interest is well characterized or known a priori, targeted sequencing is often used. In many applications, however, untargeted bulk (shotgun) sequencing is more appropriate; for instance, the tracking of infection transmission events and nucleotide variants across multiple genomic loci, or studying the role of multiple genes in a particular phenotype. Given these applications, and the observation that pathogens (e.g. Clostridioides difficile, Escherichia coli, Salmonella enterica) and other taxa of interest can reside at low relative abundance in the gastrointestinal tract, there is a critical need for algorithms that accurately track low-abundance taxa with strain level resolution. Here we present a sequence quality- and time-aware model, ChronoStrain, that introduces uncertainty quantification to gauge low-abundance species and significantly outperforms the current state-of-the-art on both real and synthetic data. ChronoStrain leverages sequences quality scores and the samples temporal information to produce a probability distribution over abundance trajectories for each strain tracked in the model. We demonstrate Chronostrains improved performance in capturing post-antibiotic Escherichia coli strain blooms among women with recurrent urinary tract infections (UTIs) from the UTI Microbiome (UMB) Project. Other strain tracking models on the same data either show inconsistent temporal colonization or can only track consistently using very coarse groupings. In contrast, our probabilistic outputs can reveal the relationship between low-confidence strains present in the sample that cannot be reliably assigned a single reference label (either due to poor coverage or novelty) while simultaneously calling high-confidence strains that can be unambiguously assigned a label. We also analyze samples from the Early Life Microbiota Colonisation (ELMC) Study demonstrating the algorithms ability to correctly identify Enterococcus faecalis strains using paired sample isolates as validation.

bioinformatics↗

bioRxiv2022

Persistent Salmonella infections in humans are associated with mutations in the BarA/SirA regulatory pathway

The bacterial pathogen Salmonella enterica is able to establish persistent infections, evading antibiotics and the host immune system and providing a reservoir for recrudescence and transmission to new hosts. Non-typhoidal serovars (NTS) of S. enterica can establish and maintain symptomatic and asymptomatic long term human infections that may predispose carriers to inflammatory bowel diseases and cancer. Defining the adaptations and host-pathogen interactions enabling these persistent infections is key to devising more effective strategies to combat and prevent persistent bacterial infections. Using comparative genomics of 639 Salmonella NTS isolates belonging to 49 serovars that were longitudinally obtained from 256 salmonelosis patients during different stages of infection, we identified numerous genetic variations accruing over time in strains isolated from the same patient. Many of these changes were found in the same gene across multiple patients and serovars. Among these variant loci, genes encoding global transcriptional regulators were found to be the most commonly mutated between early and late same-patient isolates. Genetic changes in the SirA/BarA two-component regulatory system were particularly frequent, with mutations identified in 24 independent patients. Comparative RNA-Seq analysis revealed that distinct mutations in sirA/barA that arose independently in late isolates of multiple patients lead to significantly diminished expression of virulence-associated genes encoded in the Salmonella Pathogenicity Islands (SPIs) 1 and 4, many of which are known to be critical for host cell invasion and the production of enteritis. Using the salmonellosis mouse model we showed that these mutations in sirA/barA genes confer attenuated virulence in-vivo. Taken together, these data suggest that selection of mutations in the SirA/BarA pathway facilitates persistent Salmonella infection in humans, possibly by attenuating Salmonella virulence and its ability to cause inflammation.

genomics↗

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

Transcriptomics reveals how minocycline-colistin synergy overcomes antibiotic resistance in multidrug-resistant Klebsiella pneumoniae

Multidrug resistant gram-negative bacteria are a rapidly growing public health threat, and the development of novel antimicrobials has failed to keep pace with their emergence. Synergistic combinations of individually ineffective drugs present a potential solution, yet little is understood about the mechanisms of most such combinations. Here, we show that the combination of colistin (polymyxin E) and minocycline has a high rate of synergy against colistin-resistant and minocycline-intermediate or -resistant strains of Klebsiella pneumoniae. Furthermore, using RNA-Seq, we characterized the transcriptional profiles of these strains when treated with the drugs individually and in combination. We found a striking similarity between the transcriptional profiles of bacteria treated with the combination of colistin and minocycline at individually subinhibitory concentrations and those of the same isolates treated with minocycline alone. We observed a similar pattern with the combination of polymyxin B nonapeptide (a polymyxin B analogue that lacks intrinsic antimicrobial activity) and minocycline. We also found that genes involved in polymyxin resistance and peptidoglycan biosynthesis showed significant differential gene expression in the different treatment conditions, suggesting possible mechanisms for the antibacterial activity observed in the combination. These findings suggest that the synergistic activity of this combination against bacteria resistant to each drug alone involves sublethal outer membrane disruption by colistin, which permits increased intracellular accumulation of minocycline. IMPORTANCEFew treatment options exist for multidrug resistant gram-negative bacteria. Synergistic antimicrobial combinations (in which two individually inactive drugs regain activity when used in combination) can overcome resistance. In susceptible bacteria, minocycline acts inside the cell by preventing protein synthesis, while colistin disrupts the outer membrane. Even in bacteria resistant to both drugs, the combination of the two is active. To understand this activity, we studied Klebsiella pneumoniae isolates resistant to both drugs. We extracted messenger RNA during treatment with each drug alone and with the combination, then performed RNA sequencing to analyze gene expression levels. Bacteria treated with the combination had an expression profile similar to bacteria treated with minocycline alone. Our findings suggest that synergy results from low-level, subinhibitory membrane disruption by colistin, which permits sufficient accumulation of minocycline in the cell to overcome resistance. Understanding this pathway will facilitate further development of synergistic antimicrobial combination regimens.

microbiology↗

bioRxiv2021

StrainGE: A toolkit to track and characterize low-abundance strains in complex microbial communities

Human-associated microbial communities comprise not only complex mixtures of bacterial species, but also mixtures of conspecific strains, the implications of which are mostly unknown since strain level dynamics are underexplored due to the difficulties of studying them. We introduce the Strain Genome Explorer (StrainGE) toolkit, which deconvolves strain mixtures and characterizes component strains at the nucleotide level from short-read metagenomic sequencing with higher sensitivity and resolution than other tools. StrainGE is able to identify nearest known references and find variants for multiple conspecific strains within a sample at relative abundances below 0.1% in typical metagenomic datasets.

bioinformatics↗

bioRxiv2019

QuantTB - A method to classify mixed Mycobacterium tuberculosis infections within whole genome sequencing data

BackgroundMixed infections of Mycobacterium tuberculosis, and antibiotic heteroresistance, continue to complicate tuberculosis (TB) diagnosis and treatment. Detection of mixed infections has been limited to molecular genotyping techniques, which lack the sensitivity and resolution to accurately estimate the multiplicity of TB infections. In contrast, whole genome sequencing offers sensitive views of the genetic differences between strains of M. tuberculosis within a sample. Although metagenomic tools exist to classify strains in a metagenomic sample, most tools have been developed for more divergent species, and therefore cannot provide the sensitivity required to disentangle strains within closely related bacterial species such as M. tuberculosis.\n\nHere we present QuantTB, a method to identify and quantify individual M. tuberculosis strains in whole genome sequencing data. QuantTB uses SNP markers to determine the combination of strains that best explain the allelic variation observed in a sample. QuantTB outputs a list of identified strains, their corresponding relative abundances, as well as a list of drugs for which resistance-conferring mutations (or heteroresistance) has been predicted within the sample.\n\nResultsWe show that QuantTB has a high degree of resolution, and is capable of differentiating communities differing by less than 25 SNPs and identifying strains down to 1x coverage. Using simulated data, we found QuantTB outperformed other metagenomic strain identification tools at detecting strains and quantifying strain multiplicity. In a real-world scenario, using a dataset of paired clinical isolates from a study of patients with either reinfections or relapses, we found that QuantTB could detect mixed infections and reinfections at rates concordant with a manually curated approach.\n\nConclusionQuantTB can determine infection multiplicity, identify hetero-resistance patterns, enable differentiation between relapse and re-infection, and clarify transmission events across seemingly unrelated patients - even in low-coverage (1x) samples. QuantTB outperforms existing tools and promises to serve as a valuable resource for both clinicians and researchers working with clinical TB samples.

genomics↗