Biology subjects

Van Tyne, D.

Publications and source records attributed to Van Tyne, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Functional genomic characterization of anti-phage defense systems in vancomycin-resistant Enterococcus faecium

Vancomycin-resistant Enterococcus faecium (VREfm) is a significant public health threat, demonstrating high rates of antibiotic resistance and frequently causing hospital-acquired infections. Bacteriophages (phages) are a promising adjunctive therapy to reduce bacterial load and manage VREfm infections. Many bacteria encode anti-phage defense systems--groups of genes that can counteract phage infection--but these systems have not been characterized in VREfm. Here we explored the diversity of anti-phage defense systems within clinical VREfm isolate genomes and examined their impact on phage susceptibility. We first used two available bioinformatic tools (DefenseFinder and PADLOC) to identify putative anti-phage defense systems in 21 hybrid-assembled VREfm clinical isolate genomes, and observed that most anti-phage defense systems were located on plasmids, defense islands, and prophages. Combining this information with functional testing of phage susceptibility did not reveal robust associations between specific defense systems and resistance to four VREfm-targeting lytic phages. Due to inherent limitations of phage susceptibility testing, we next developed an assay to assess phage survival after co-incubation with bacteria, and identified over two dozen genes associated with phage killing, including a potentially novel defense island. Lastly, we attempted to functionally validate 10 candidate anti-phage defense systems, only four of which were ultimately tested in enterococci. Expression of one of these candidates, AbiE, decreased bacterial susceptibility to two Enterococcus-targeting phages. As VREfm-targeting phage therapy becomes more widely available, identification of genes contributing to phage resistance can aid in streamlining the provision of phages to patients who could benefit from this new therapeutic modality.

microbiology↗

bioRxiv2026

Diverse transcriptomic effects of evolved imipenem-relebactam resistance in Pseudomonas aeruginosa

Imipenem-relebactam (Imi/Rel) is a {beta}-lactam/{beta}-lactamase inhibitor combination used for the treatment of multidrug-resistant Pseudomonas aeruginosa infections. We previously reported that treatment-emergent resistance to Imi/Rel is associated with mutations in the AmpC beta-lactamase and/or the MexAB-OprM and MexEF-OprN efflux operons. However, the impact of these mutations on bacterial gene expression has not been explored extensively, particularly among P. aeruginosa from patients treated with Imi/Rel. To determine the effect of treatment-emergent Imi/Rel resistance on P. aeruginosa global transcription, we performed RNA sequencing on paired P. aeruginosa clinical isolates from six patients collected before and after Imi/Rel treatment. Transcriptional responses varied substantially, with no conserved changes in gene expression identified across all six patients. Three isolate pairs showed significant upregulation of previously characterized Imi/Rel resistance-associated genes in the treatment-emergent resistant isolate, while two resistant isolates displayed significant downregulation of ampC. Comparisons of the top 10 differentially regulated genes in the resistant isolate from each patient revealed only one gene that was shared between all six patients. Pathway enrichment analysis using Clusters of Orthologous Genes (COG) categories suggested that Imi/Rel exposure impacts transcription of genes involved in translation and metabolism, but these changes are highly variable between patients and isolates. Overall, we find that the transcriptional response of clinical P. aeruginosa to Imi/Rel exposure appears to be highly diverse and likely dependent on the genetic background of the infecting P. aeruginosa strain.

microbiology↗

bioRxiv2026

Variable phage susceptibility of Pseudomonas aeruginosa from patients with and without cystic fibrosis following treatment-emergent resistance to ceftolozane-tazobactam

Background: Pseudomonas aeruginosa is a ubiquitous opportunistic bacterial pathogen associated with nosocomial infections and is a leading cause of infection in persons with cystic fibrosis (pwCF). The front-line treatment for multidrug-resistant P. aeruginosa infections is ceftolozane-tazobactam (C/T). While previous research has characterized clinical P. aeruginosa isolates that evolved resistance to C/T, the collateral effect of evolved resistance on susceptibility to bacteriophages has not been explored. Methods: We collected paired P. aeruginosa clinical isolates from 10 pwCF and 18 non-pwCF who developed treatment-emergent C/T resistance. We compared genetic relatedness, acute and chronic virulence phenotypes, and antibiotic and phage susceptibilities between each pair of susceptible baseline and treatment-emergent C/T-resistant isolates. Results: Treatment-emergent C/T-resistant isolates were genetically closely related to baseline isolates in all patients. Virulence phenotypes did not differ between pre- and post-C/T exposure isolates, but isolates from pwCF demonstrated differences in protease production, twitching motility, and amino acid auxotrophy compared to isolates from non-pwCF. Treatment-emergent C/T resistance was associated with increased resistance to ceftazidime and ceftazidime/avibactam, but no other trends in antibiotic or phage susceptibility were detected. Conclusions: Treatment-emergent resistance to C/T does not cause predictable alterations in phage susceptibility across genotypically and phenotypically diverse multidrug-resistant P. aeruginosa clinical isolates.

microbiology↗

bioRxiv2026

Evaluation of phage activity against diverse Enterococcus faecalis from infective endocarditis

Enterococcus faecalis is a leading cause of infective endocarditis, particularly in older and hospitalized patients. Treatment is challenging due to the intrinsic resistance of E. faecalis to many clinically important antibiotics and the increasing prevalence of multidrug-resistant strains. Bacteriophage (phage)-based therapies, including phage cocktails and phage-antibiotic combinations, offer a promising approach for treating resistant bacterial infections. In this study, we isolated ten novel phages from wastewater by screening on E. faecalis clinical isolates and tested phage activity both individually and in phage-phage and phage-antibiotic combinations against 18 E. faecalis clinical isolates collected from patients with infective endocarditis. We found that 15/18 (83%) isolates were susceptible to at least one phage tested, and that the same proportion of isolates were susceptible to a cocktail of three phages with complementary activities. We also observed improved bacterial growth inhibition and increased killing when phages were combined with ampicillin or daptomycin, two antibiotics commonly used to treat E. faecalis infections. Finally, we found that treatment with the three-phage cocktail improved the survival of Galleria mellonella infected with a pathogenic E. faecalis endocarditis isolate. Overall, these findings suggest that phages could be a helpful addition to the therapeutic repertoire for treating E. faecalis infective endocarditis. ImportanceNew approaches are needed for treating infective endocarditis, a life-threatening heart infection. Here we focus on Enterococcus faecalis, a resilient bacterium that is increasingly resistant to standard-of care antibiotic regimens, and explore the use of phage therapy for treating E. faecalis endocarditis. We found that combining phages together in a cocktail or pairing them with existing antibiotics can effectively kill E. faecalis clinical isolates from patients with infective endocarditis, and that treatment of infected waxworm moth larvae with a phage cocktail improved their survival. The results of this study contribute to the development of phage-based therapeutic approaches for E. faecalis endocarditis, which could ultimately lead to improved outcomes for patients facing this severe infection.

microbiology↗

bioRxiv2026

High-depth whole genome sequencing of blood culture plates reveals evolutionary dynamics in cases of persistent bacteremia due to methicillin-resistant Staphylococcus aureus

Within a single bacterial strain, DNA sequence variation is expected between individual clones. Whole genome sequencing (WGS) can be applied to clinical cultures to detect this polyclonal variation, enabling tracking of within-host evolution and transmission. Culture isolates from infected patients are often sequenced as individual colonies (c-seq). To increase the sensitivity of variant detection, cultures can also be sequenced to a high depth of coverage as a pool (p-seq), but the utility of this approach is not clear for most clinical specimens. To understand the performance of high-depth WGS in bacteremia, we applied p-seq to blood culture plates for 10 patients with persistent bacteremia due to methicillin-resistant Staphylococcus aureus. As a comparison, for six patients, we also applied c-seq to five colonies (c5-seq) from the same plates. p-seq was more sensitive than c5-seq for detecting low frequency variant alleles; however, the most important factor for new variant detection was the number of culture plates analyzed rather than the sequencing method used. We also used these data to construct Muller plots for three patients with especially diverse infecting populations, which enabled visualization of rapid evolutionary dynamics in response to antibiotic exposures. We identified 204 unique variant alleles, and our analysis provides additional evidence for parallel evolution of several different genes during S. aureus bacteremia. Overall, these data provide a detailed view of evolutionary dynamics during clinical cases of MRSA bacteremia and describe the merits and limitations of a c-seq versus p-seq strategy for analyzing blood culture plates using WGS. ImportanceAs bacterial whole genome sequencing (WGS) is increasingly used as a research tool for clinical samples, it is important to understand the pros and cons of different culture sampling methodologies. Here, we analyzed cases of persistent bacteremia due to methicillin-resistant Staphylococcus aureus by applying WGS to either each of five individual colonies isolated on blood culture plates (c5-seq) or the pooled bacterial population on each plate (p-seq). We found that c5-seq was a more practical and informative method to understand evolutionary dynamics.

microbiology↗

bioRxiv2025

Within-Patient Evolution of Pseudomonas aeruginosa Populations During Antimicrobial Treatment

Multidrug-resistant (MDR) Pseudomonas aeruginosa infections pose a major challenge to effective treatment. Understanding genomic adaptations during antimicrobial therapy in patients infected with this pathogen is crucial for preventing therapeutic failure. Here we investigated the population diversity and evolution of P. aeruginosa collected longitudinally from six patients who evolved multidrug-resistant infections. Serial P. aeruginosa clinical isolates (n=63) and culture-enriched metagenomic population samples (n=39) were collected and subjected to whole-genome sequencing. The resulting data were used to characterize and compare the species composition, multi-locus sequence types (STs), and resistance-associated mutations present within each sample type. Single-isolate sequencing showed that each patient was infected with a single P. aeruginosa strain that accumulated mutations and became increasingly more resistant over time. Mutations in genes associated with beta-lactam resistance, including ampC, ftsI, and mexR, arose over time and corresponded with changes in antimicrobial susceptibility in single isolates. Species profiling of culture-enriched metagenomic populations revealed that all samples contained P. aeruginosa, but also additional Gram-negative pathogens. Metagenomic analysis of culture-enriched populations identified resistance-associated mutations at low frequency, many of which were not identified in single isolates from the same sample. In some cases, resistance-associated mutations initially detected at low frequency rose to fixation after antimicrobial treatment. Overall, this study shows that population-based metagenomic sequencing effectively captures within-patient genomic diversity of P. aeruginosa during antimicrobial therapy, and could aid the detection and interpretation of resistance-associated mutations in this pathogen. ImportancePseudomonas aeruginosa infections are notoriously difficult to treat and are associated with high rates of morbidity and mortality. While the genetic basis of resistance in P. aeruginosa is well documented in vitro, less is known about how resistance evolves within patients during antibiotic therapy. Standard approaches based on analysis of clonal isolates may miss within-patient diversity, potentially overlooking low-frequency mutations that contribute to treatment failure. In this study, we integrated single-colony whole-genome sequencing with culture-enriched metagenomic sequencing to monitor the evolution of P. aeruginosa populations in patients receiving antibiotic therapy. This approach enabled the detection of emerging resistance mutations, such as low-frequency variants in ampC and ftsI, before these variants rose to fixation. It also revealed genetically resistant subpopulations missed by isolate sequencing alone. Together, our findings highlight the value of population-based metagenomic sequencing in capturing bacterial adaptation during infection, and underscore its potential to improve resistance surveillance and guide personalized antimicrobial therapy.

evolutionary biology↗

bioRxiv2025

Inactivation of peptidoglycan remodeling promotes antibiotic susceptibility in vancomycin-resistant Enterococcus faecium

Vancomycin-resistant Enterococcus faecium (VREfm) is a leading cause of healthcare-associated infections globally and demands new approaches for treatment. Here we show that genetic and pharmacological inactivation of a highly conserved NlpC/P60 peptidoglycan hydrolase, secreted antigen A (SagA), enhanced vancomycin susceptibility of VREfm ex vivo and in vivo. Notably, genetic deletion of sagA impaired VREfm peptidoglycan remodeling, growth and increased the activity of vancomycin. We then identified first-in-class covalent NlpC/P60 peptidoglycan hydrolase inhibitors and demonstrated that pharmacological inactivation of SagA activity also impaired peptidoglycan remodeling and increased the efficacy of vancomycin across genetically distinct VREfm clinical isolates. Our study reveals peptidoglycan hydrolases are druggable targets whose inactivation improves the efficacy of vancomycin against VREfm.

microbiology↗

bioRxiv2025

Allele-Specific Effects of Mutations in the Rifampin Resistance-Determining Region (RRDR) of RpoB on Physiology and Antibiotic Resistance in Enterococcus faecium

Enterococcus faecium is a member of the human gut microbiota that has evolved into a problematic nosocomial pathogen and leading cause of infections in hospitalized patients. Treatment of E. faecium infections is complicated by antibiotic resistance, making it important to understand resistance mechanisms and their broader consequences in this pathogen. Here we explored the collateral effects of rifampin resistance-associated mutations in the E. faecium RNA polymerase {beta}-subunit (RpoB). Of 14,384 publicly available E. faecium genomes, nearly one-third carried a mutation in the rifampin resistance-determining region (RRDR) of RpoB. In a local population of 710 E. faecium clinical isolates collected from patients at a single hospital, we found significant associations between the presence of RRDR mutations and prior exposure to rifamycin antibiotics, as well as associations between RRDR mutations and altered daptomycin susceptibility. To investigate the phenotypic impacts of RRDR mutations, we generated and studied four isogenic strains with distinct RRDR mutations (Q473K, G482D, H486Y, S491L) that overlapped with clinical isolate variants. Transcriptomic and phenotypic analyses revealed allele-specific effects on E. faecium gene expression, growth dynamics, antibiotic susceptibility, isopropanol tolerance, and cell wall physiology. One frequently observed mutation, H486Y, caused minimal transcriptional changes and enhanced bacterial fitness under antibiotic stress. In contrast, the S491L mutation induced extensive transcriptional changes and slowed bacterial growth, but also conferred increased isopropanol tolerance, potentially enhancing bacterial survival in the hospital. Overall, our findings highlight the multifaceted impacts of RRDR mutations in shaping E. faecium physiology and antibiotic resistance, two important features of this hospital-associated pathogen. ImportanceUnderstanding how antimicrobial resistance affects bacterial physiology is critical for developing effective therapeutics against bacterial infections. In this study, we found that rifampin resistance-associated mutations in RpoB are widespread in Enterococcus faecium, a leading multidrug-resistant pathogen. By studying isogenic wild-type and RpoB mutant strains, we discovered that RpoB mutations, while conferring resistance to rifampin, have distinct allele-specific effects on other bacterial phenotypes. Some of these collateral effects appear to promote E. faecium resistance to antibiotics and survival in the hospital environment, raising questions about the selective pressures driving their emergence. Overall, our study underscores the importance of examining the collateral effects of resistance-associated mutations in multidrug-resistant pathogens, which could help mitigate their persistence and spread among vulnerable patients.

microbiology↗

bioRxiv2025

Fsr quorum sensing system restricts biofilm growth and activates inflammation in enterococcal infective endocarditis

Infective endocarditis (IE) is a life-threatening biofilm-associated infection, yet the factors driving biofilm formation remain poorly understood. Here, we identified the Fsr quorum sensing (QS) system of Enterococcus faecalis as a potent negative regulator of IE pathogenesis. Using microfluidic and in vivo models, we show that Fsr is induced in late IE when bacteria become shielded from blood flow. Deleting Fsr altered biofilm metabolism and promoted robust biofilm growth and gentamicin tolerance in vivo. Furthermore, Fsr inactivation attenuated inflammation by disrupting IL-1{beta} cleavage and activation via the Fsr-regulated gelatinase (gelE), allowing biofilm to grow unchecked by the immune system. Consistent with our pre-clinical findings, analysis of two IE patient cohorts linked naturally occurring Fsr-deficient E. faecalis to prolonged bacteremia. Overall, our findings provide insights into the role of QS in biofilm growth, persistence, and immune evasion in enterococcal IE.

microbiology↗

bioRxiv2024

Mutations Leading to Ceftolozane/Tazobactam and Imipenem/Cilastatin/Relebactam Resistance During in vivo exposure to Ceftazidime/Avibactam in Pseudomonas aeruginosa

Identifying resistance mechanisms to novel antimicrobials informs treatment and antimicrobial development, but frequently identifies multiple candidate resistance mutations without resolving the driver mutation. Using whole genome sequencing of longitudinal Pseudomonas aeruginosa that developed imipenem/cilastatin/relebactam and ceftolozane/tazobactam resistance during ceftazidime/avibactam treatment, we determined mutations resulting in cross-resistance. Penicillin-binding protein ftsI, transcriptional repressor bepR, and virulence regulator pvdS were found in resistant isolates. We conclude that peptidoglycan synthesis gene mutations can alter the efficacy of multiple antimicrobials.

microbiology↗

bioRxiv2024

Lytic bacteriophages interact with respiratory epithelial cells and induce the secretion of antiviral and proinflammatory cytokines

Phage therapy is a therapeutic approach to treat multidrug resistant infections that employs lytic bacteriophages (phages) to eliminate bacteria. Despite the abundant evidence for its success as an antimicrobial in Eastern Europe, there is scarce data regarding its effects on the human host. Here, we aimed to understand how lytic phages interact with cells of the airway epithelium, the tissue site that is colonized by bacterial biofilms in numerous chronic respiratory disorders. We determined that interactions between phages and epithelial cells depend on specific phage properties as well as physiochemical features of the microenvironment. Although poor at internalizing phages, the airway epithelium responds to phage exposure by changing its transcriptional profile and secreting antiviral and proinflammatory cytokines that correlate with specific phage families. Overall, our findings indicate that mammalian responses to phages are heterogenous and could potentially alter the way that respiratory local defenses aid in bacterial clearance during phage therapy. Thus, besides phage receptor specificity in a particular bacterial isolate, the criteria to select lytic phages for therapy should be expanded to include mammalian cell responses.

microbiology↗

bioRxiv2024

Harnessing the diversity of Burkholderia spp. prophages for therapeutic potential

Burkholderia spp. are often resistant to antibiotics, and infections with these organisms are difficult to treat. A potential alternative treatment for Burkholderia spp. infections is bacteriophage (phage) therapy; however, it can be difficult to locate phages that target these bacteria. Prophages incorporated into the bacterial genome have been identified within Burkholderia spp. and may represent a source of useful phages for therapy. Here we investigate whether prophages within Burkholderia spp. clinical isolates can kill conspecific and heterospecific isolates. Thirty-two Burkholderia spp. isolates were induced for prophage release, and harvested prophages were tested for lytic activity against the same 32 isolates. Lytic phages were passaged and their host ranges were determined, resulting in four unique phages of prophage origin that showed different ranges of lytic activity. We also analyzed the prophage content of 35 Burkholderia spp. clinical isolate genomes, and identified several prophages present in the genomes of multiple isolates of the same species. Finally, we observed that B. cenocepacia isolates were more phage-susceptible than Burkholderia multivorans isolates. Overall, our findings suggest that prophages present within Burkholderia spp. genomes are a potentially useful starting point for the isolation and development of novel phages for use in phage therapy.

microbiology↗

bioRxiv2023

Evolution of extended-spectrum β-lactamase-producing ST131 Escherichia coli at a single hospital over 15 years

Escherichia coli belonging to sequence type ST131 constitute a globally distributed pandemic lineage that causes multidrug-resistant extra-intestinal infections. ST131 E. coli frequently produce extended-spectrum {beta}-lactamases (ESBLs), which confer resistance to many {beta}-lactam antibiotics and make infections difficult to treat. We sequenced the genomes of 154 ESBL-producing E. coli clinical isolates belonging to the ST131 lineage from patients at the University of Pittsburgh Medical Center (UPMC) between 2004 and 2018. Isolates belonged to the well described ST131 clades A (8%), B (3%), C1 (33%), and C2 (54%). An additional four isolates belonged to another distinct subclade within clade C and encoded genomic characteristics that have not been previously described. Time-dated phylogenetic analysis estimated that the most recent common ancestor (MRCA) for all clade C isolates from UPMC emerged around 1989, consistent with previous studies. We identified multiple genes potentially under selection in clade C, including the cell wall assembly gene ftsI, the LPS biosynthesis gene arnC, and the yersiniabactin uptake receptor fyuA. Diverse ESBL genes belonging to the blaCTX- M, blaSHV, and blaTEM families were identified; these genes were found at varying numbers of loci and in variable numbers of copies across isolates. Analysis of ESBL flanking regions revealed diverse mobile elements that varied by ESBL type. Overall, our findings show that ST131 subclades C1 and C2 dominated and were stably maintained among patients in the same hospital and uncover possible signals of ongoing adaptation within the clade C ST131 lineage.

microbiology↗

bioRxiv2023

In vivo evolution of a Klebsiella pneumoniae capsule defect promotes complement-mediated opsono-phagocytosis and persistence during recurrent infection

Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) bloodstream infections rarely overwhelm the host but are associated with high mortality. The complement system is a key host defense against bloodstream infection. However, there are varying reports of serum resistance among KPC-Kp isolates. We assessed growth of 59 KPC-Kp clinical isolates in human serum and found increased resistance in 16/59 (27%). We identified five genetically-related bloodstream isolates with varying serum resistance profiles collected from a single patient during an extended hospitalization marked by recurrent KPC-Kp bloodstream infections. We noted a loss-of-function mutation in the capsule biosynthesis gene, wcaJ, that emerged during infection was associated with decreased polysaccharide capsule content, and resistance to complement-mediated killing. Surprisingly, disruption of wcaJ increased deposition of complement proteins on the microbial surface compared to the wild-type strain and led to increased complement-mediated opsono-phagocytosis in human whole blood. Disabling opsono-phagocytosis in the airspaces of mice impaired in vivo control of the wcaJ loss-of-function mutant in an acute lung infection model. These findings describe the rise of a capsular mutation that promotes KPC-Kp persistence within the host by enabling co-existence of increased bloodstream fitness and reduced tissue virulence. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/542722v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@155736eorg.highwire.dtl.DTLVardef@10d47c0org.highwire.dtl.DTLVardef@e1ab83org.highwire.dtl.DTLVardef@1c33131_HPS_FORMAT_FIGEXP M_FIG Graphical abstract Created with BioRender.com C_FIG

microbiology↗

bioRxiv2023

The Type VI Secretion System of the Emerging Pathogen Stenotrophomonas maltophilia has Antibacterial Properties

Antagonistic behaviors between bacterial cells can have profound effects on microbial populations and disease outcomes. Polymicrobial interactions may be mediated by contact-dependent proteins with antibacterial properties. The Type VI Secretion System (T6SS) is a macromolecular weapon used by Gram-negative bacteria to translocate proteins into adjacent cells. The T6SS is used by pathogens to escape immune cells, eliminate commensal bacteria, and facilitate infection. Stenotrophomonas maltophilia is a Gram-negative opportunistic pathogen that causes a wide range of infections in immunocompromised patients and infects the lungs of patients with cystic fibrosis. Infections with the bacterium can be deadly and are challenging to treat because many isolates are multidrug-resistant. We found that globally dispersed S. maltophilia clinical and environmental strains possess T6SS genes. We demonstrate that the T6SS of an S. maltophilia patient isolate is active and can eliminate other bacteria. Furthermore, we provide evidence that the T6SS contributes to the competitive fitness of S. maltophilia against a co-infecting Pseudomonas aeruginosa isolate, and that the T6SS alters the cellular organization of S. maltophilia and P. aeruginosa co-cultures. This study expands our knowledge of the mechanisms employed by S. maltophilia to secrete antibacterial proteins and compete against other bacteria. IMPORTANCEInfections with the opportunistic pathogen Stenotrophomonas maltophilia can be fatal for immunocompromised patients. The mechanisms used by the bacterium to compete against other prokaryotes are not well understood. We found that the T6SS allows S. maltophilia to eliminate other bacteria and contributes to the competitive fitness against a co-infecting isolate. The presence of T6SS genes in isolates across the globe highlights the importance of this apparatus as a weapon in the antibacterial arsenal of S. maltophilia. The T6SS may confer survival advantages to S. maltophilia isolates in polymicrobial communities in both environmental settings and during infections.

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↗

bioRxiv2022

Targeted IS-element sequencing uncovers transposition dynamics during selective pressure in enterococci

Insertion sequences (IS) are simple transposons implicated in the genome evolution of diverse pathogenic bacterial species. Enterococci have emerged as important human intestinal pathogens with newly adapted virulence potential and antibiotic resistance. These genetic features arose in tandem with large-scale genome evolution mediated by mobile elements. Pathoadaptation in enterococci is thought to be mediated in part by the IS element IS256 through gene inactivation and recombination events. However, the regulation of IS256 and the mechanisms controlling its activation are not well understood. Here, we adapt an IS256-specfic deep sequencing method to describe how chronic lytic phage infection drives widespread diversification of IS256 in E. faecalis and how antibiotic exposure is associated with IS256 diversification in both E. faecalis and E. faecium during a clinical human infection. We show through comparative genomics that IS256 is primarily found in hospital-adapted enterococcal isolates. Analyses of IS256 transposase gene levels reveal that IS256 mobility is regulated at the transcriptional level by multiple mechanisms in E. faecalis, indicating tight control of IS256 activation in the absence of selective pressure. Our findings reveal that stressors such as phages and antibiotic exposure drives rapid genome-scale transposition in the enterococci. IS256 diversification can therefore explain how selective pressures mediate evolution of the enterococcal genome, ultimately leading to the emergence of dominant nosocomial lineages that threaten human health. Author SummaryInsertion sequence (IS) elements are simple transposons that are ubiquitous in bacteria. In the enterococci, which includes medically relevant species such as Enterococcus faecalis and Enterococcus faecium, the IS element IS256 is widespread and has been implicated in pathogenesis and antibiotic resistance. Despite the importance of IS256 to the biology of the enterococci, we know very about how this element is regulated and diversifies enterococcal genomes. Here, we show that IS256 is preferentially found in hospital-adapted and virulent strains of the enterococci. In E. faecalis V583, a vancomycin resistant blood isolate, IS256 is regulated by multiple transcriptional mechanisms. To understand how IS256 is mobilized, we adapted an Illumina-based deep sequencing method called IS-Seq to find novel IS256 insertions when applying the selective pressure of bacteriophage (phage) predation. Using this method, we found that chronic phage infection drives IS256 diversification of the E. faecalis V583 genome. Additionally, we tracked IS256 insertional activity during a human E. faecium infection and found that increased IS256 diversity was associated with specific antibiotic usage. Together, our results demonstrate that the enterococci control IS256 activity to diversify their genomes which may lead to the emergence of hospital-adapted strains that threaten human health.

microbiology↗

bioRxiv2021

Genomic diversity of hospital-acquired infections revealed through prospective whole genome sequencing-based surveillance

Healthcare-associated infections (HAIs) cause mortality, morbidity, and waste of healthcare resources. HAIs are also an important driver of antimicrobial resistance, which is increasing around the world. Beginning in November 2016, we instituted an initiative to detect outbreaks of HAI using prospective whole genome sequencing-based surveillance of bacterial pathogens collected from hospitalized patients. Here we describe the biodiversity of bacteria sampled from hospitalized patients at a single center, as revealed through systematic analysis of their genomes. We sequenced the genomes of 3,004 bacterial isolates from hospitalized patients collected over a 25-month period. We identified bacteria belonging to 97 distinct species, which were distributed among 14 species groups. Within these groups, isolates could be distinguished from one another by both average nucleotide identity (ANI) and principal component analysis of accessory genes (PCA-A). Genetic distances between isolates and rates of evolution varied between different species, which has implications for the selection of distance cut-offs for outbreak analysis. Antimicrobial resistance genes and the sharing of mobile genetic elements between different species were frequently observed. Overall, this study describes the population structure of pathogens circulating in a single healthcare setting, and shows how investigating microbial population dynamics can inform genomic epidemiology studies. ImportanceHospitalized patients are at increased risk of becoming infected with antibiotic-resistant organisms. We used whole-genome sequencing to survey and compare over 3,000 bacterial isolates collected from hospitalized patients at a large medical center over a two-year period. We identified nearly 100 different bacterial species, suggesting that patients can be infected with a wide variety of different organisms. When we examined how genetic relatedness differed between species, we found that different species are likely evolving at different rates within our hospital. This is significant because the identification of bacterial outbreaks in the hospital currently relies on genetic similarity cut-offs, which are often applied uniformly across organisms. Finally, we found that antibiotic resistance genes and mobile genetic elements were abundant among the bacterial isolates we sampled. Overall, this study provides an in-depth view of the genomic diversity and evolution of bacteria sampled from hospitalized patients, as well as genetic similarity estimates that can inform hospital outbreak detection and prevention efforts.

microbiology↗