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Background The quantity of genomic data is expanding at an increasing rate. Tools for phylogenetic analysis which scale to the quantity of available data are required. To address this need, we present cognac, a user-friendly software package to rapidly generate concatenated gene alignments for phylogenetic analysis. Results We illustrate that cognac is able to rapidly identify phylogenetic marker genes using a data driven approach and efficiently generate concatenated gene alignments for very large genomic datasets. To benchmark our tool, we generated core gene alignments for eight unique genera of bacteria, including a dataset of over 11,000 genomes from the genus Escherichia producing an alignment with 1353 genes, which was constructed in less than 17 h. Conclusions We demonstrate that cognac presents an efficient method for generating concatenated gene alignments for phylogenetic analysis. We have released cognac as an R package ( https://github.com/rdcrawford/cognac ) with customizable parameters for adaptation to diverse applications.

Uropathogenic Escherichia coli (UPEC) is the major causative agent of uncomplicated urinary tract infections (UTIs). A common virulence genotype of UPEC strains responsible for UTIs is yet to be defined, due to the large variation of virulence factors observed in UPEC strains. We hypothesized that studying UPEC functional responses in patients might reveal universal UPEC features that enable pathogenesis. Here we identify a transcriptional program shared by genetically diverse UPEC strains isolated from 14 patients during uncomplicated UTIs. Strikingly, this in vivo gene expression program is marked by upregulation of translational machinery, providing a mechanism for the rapid growth within the host. Our analysis indicates that switching to a more specialized catabolism and scavenging lifestyle in the host allows for the increased translational output. Our study identifies a common transcriptional program underlying UTIs and illuminates the molecular underpinnings that likely facilitate the fast growth rate of UPEC in infected patients.

UPEC, the primary causative agent of uncomplicated UTI, is responsible for five billion dollars in health care costs in the United States each year. Rates of antibiotic resistance are on the rise; therefore, it is vital to understand the mechanisms of UPEC pathogenesis to uncover potential targets for novel therapeutics. More than half of women will experience a urinary tract infection (UTI), with uropathogenic Escherichia coli (UPEC) causing ~80% of uncomplicated cases. Iron acquisition systems are essential for uropathogenesis, and UPEC strains encode highly diverse iron acquisition systems, underlining their importance. However, a recent UPEC clinical isolate, HM7, lacks this diversity and instead encodes the synthesis pathway for a sole siderophore, enterobactin. To determine if HM7 possesses unidentified iron acquisition systems, we performed RNA sequencing under iron-limiting conditions and demonstrated that the ferric citrate uptake system ( fecABCDE and fecIR ) was highly upregulated. Importantly, there are high levels of citrate within urine, some of which is bound to iron, and the fec system is enriched in UPEC isolates compared to fecal strains. Therefore, we hypothesized that HM7 and other similar strains use the fec system to acquire iron in the host. Deletion of both enterobactin biosynthesis and ferric citrate uptake (Δ fecA /Δ entB ) abrogates use of ferric citrate as an iron source, and fecA provides an advantage in human urine in the absence of enterobactin. However, in a UTI mouse model, fecA is a fitness factor independent of enterobactin production, likely due to the action of host lipocalin-2 chelating ferrienterobactin. These findings indicate that ferric citrate uptake is used as an iron source when siderophore efficacy is limited, such as in the host during UTI. Defining these novel compensatory mechanisms and understanding the nutritional hierarchy of preferred iron sources within the urinary tract are important in the search for new approaches to combat UTI. IMPORTANCE UPEC, the primary causative agent of uncomplicated UTI, is responsible for five billion dollars in health care costs in the United States each year. Rates of antibiotic resistance are on the rise; therefore, it is vital to understand the mechanisms of UPEC pathogenesis to uncover potential targets for novel therapeutics. Iron acquisition systems used to obtain iron from sequestered host sources are essential for UPEC survival during UTI and have been used as vaccine targets to prevent infection. This study reveals the ferric citrate uptake system is another important iron acquisition system that is highly enriched in UPEC strains. Ferric citrate uptake has not previously been associated with UPEC isolates, underlining the importance of the continued study of these strains to fully understand their mechanisms of pathogenesis.

Members of the Klebsiella pneumoniae species complex frequently colonize the gut and colonization is associated with subsequent infection. To identify genes associated with progression from colonization to infection, we undertook a case-control comparative genomics study. Concordant cases ( N  = 85), where colonizing and invasive isolates were identical strain types, were matched to asymptomatically colonizing controls ( N  = 160). Thirty-seven genes are associated with infection, 27 of which remain significant following adjustment for patient variables and bacterial phylogeny. Infection-associated genes are not previously characterized virulence factors, but instead a diverse group of stress resistance, regulatory and antibiotic resistance genes, despite careful adjustment for antibiotic exposure. Many genes are plasmid borne, and for some, the relationship with infection is mediated by gut dominance. Five genes were validated in a geographically-independent cohort of colonized patients. This study identifies several genes reproducibly associated with progression to infection in patients colonized by diverse Klebsiella . Patient variables, such as comorbidities, partially explain which patients will progress to Klebsiella infection, with colonization of the gut acting as a reservoir. Little is known, however, regarding Klebsiella genes that may increase risk of disease in colonized individuals. Here, authors conduct a comparative genomics study to identify genes associated with progression from colonisation to infection.

Jails have been hypothesized to be hotspots for the spread of methicillin-resistant Staphylococcus aureus (MRSA). We integrate genomic and epidemiologic data to investigate USA300 MRSA transmission in Cook County Jail (Illinois, USA) and its connected communities. A genome-wide association study of 308 jail isolates from 2015–2018 revealed a plasmid encoding the ermC clindamycin/erythromycin resistance gene was associated with a 6-fold increased odds of MRSA genetic linkages among detainees. Additionally, 52% of jail-onset MRSA infections carried this plasmid compared to 14% of intake colonization isolates, supporting its role in MRSA spread in the jail. Extending our analysis to 774 isolates from a local healthcare system from 2011–2014, the ermC- carrying plasmid was also associated with MRSA transmission in the larger community and was enriched among former jail detainees and those with related isolates to recently incarcerated cases. Lastly, topical clindamycin exposure before MRSA infection was associated with ermC plasmid presence in both settings, but exposure prevalence was higher in jail versus community cases (7.5% vs. 0.9%), suggesting antibiotic use in the jail may have created a favorable environment for the spread of ermC -carrying strains. These findings highlight the impact of antibiotic use in jails on antibiotic resistance in both jails and their surrounding communities. Jails are high-risk settings for transmission of pathogens. Here, the authors use genomic and epidemiological data to investigate the bacterial and clinical factors affecting transmission of MRSA in jails and impacts on wider community transmission dynamics.

Uropathogenic Escherichia coli (UPEC) strains cause most uncomplicated urinary tract infections (UTIs). These strains are a subgroup of extraintestinal pathogenic E. coli (ExPEC) strains that infect extraintestinal sites, including urinary tract, meninges, bloodstream, lungs, and surgical sites. Here, we hypothesize that UPEC isolates adapt to and grow more rapidly within the urinary tract than other E. coli isolates and survive in that niche. To date, there has not been a reliable method available to measure their growth rate in vivo . Here we used two methods: segregation of nonreplicating plasmid pGTR902, and peak-to-trough ratio (PTR), a sequencing-based method that enumerates bacterial chromosomal replication forks present during cell division. In the murine model of UTI, UPEC strain growth was robust in vivo , matching or exceeding in vitro growth rates and only slowing after reaching high CFU counts at 24 and 30 h postinoculation (hpi). In contrast, asymptomatic bacteriuria (ABU) strains tended to maintain high growth rates in vivo at 6, 24, and 30 hpi, and population densities did not increase, suggesting that host responses or elimination limited population growth. Fecal strains displayed moderate growth rates at 6 hpi but did not survive to later times. By PTR, E. coli in urine of human patients with UTIs displayed extraordinarily rapid growth during active infection, with a mean doubling time of 22.4 min. Thus, in addition to traditional virulence determinants, including adhesins, toxins, iron acquisition, and motility, very high growth rates in vivo and resistance to the innate immune response appear to be critical phenotypes of UPEC strains. IMPORTANCE Uropathogenic Escherichia coli (UPEC) strains cause most urinary tract infections in otherwise healthy women. While we understand numerous virulence factors are utilized by E. coli to colonize and persist within the urinary tract, these properties are inconsequential unless bacteria can divide rapidly and survive the host immune response. To determine the contribution of growth rate to successful colonization and persistence, we employed two methods: one involving the segregation of a nonreplicating plasmid in bacteria as they divide and the peak-to-trough ratio, a sequencing-based method that enumerates chromosomal replication forks present during cell division. We found that UPEC strains divide extraordinarily rapidly during human UTIs. These techniques will be broadly applicable to measure in vivo growth rates of other bacterial pathogens during host colonization. Uropathogenic Escherichia coli (UPEC) strains cause most urinary tract infections in otherwise healthy women. While we understand numerous virulence factors are utilized by E. coli to colonize and persist within the urinary tract, these properties are inconsequential unless bacteria can divide rapidly and survive the host immune response. To determine the contribution of growth rate to successful colonization and persistence, we employed two methods: one involving the segregation of a nonreplicating plasmid in bacteria as they divide and the peak-to-trough ratio, a sequencing-based method that enumerates chromosomal replication forks present during cell division. We found that UPEC strains divide extraordinarily rapidly during human UTIs. These techniques will be broadly applicable to measure in vivo growth rates of other bacterial pathogens during host colonization.

A variety of cell surface structures dictate interactions between bacteria and their environment, including their viruses (bacteriophages). Members of the human gut Bacteroidetes characteristically produce several phase-variable capsular polysaccharides (CPSs), but their contributions to bacteriophage interactions are unknown. To begin to understand how CPSs have an impact on Bacteroides –phage interactions, we isolated 71 Bacteroides thetaiotaomicron -infecting bacteriophages from two locations in the United States. Using B. thetaiotaomicron strains that express defined subsets of CPSs, we show that CPSs dictate host tropism for these phages and that expression of non-permissive CPS variants is selected under phage predation, enabling survival. In the absence of CPSs, B. thetaiotaomicron escapes bacteriophage predation by altering expression of eight distinct phase-variable lipoproteins. When constitutively expressed, one of these lipoproteins promotes resistance to multiple bacteriophages. Our results reveal important roles for Bacteroides CPSs and other cell surface structures that allow these bacteria to persist under bacteriophage predation, and hold important implications for using bacteriophages therapeutically to target gut symbionts. Isolation of phages associated with the gut commensal Bacteroides thetaiotaomicron reveals a link between cell surface structures, including phase-variable capsular polysaccharides, lipoproteins and S-layer proteins, and susceptibility to phage infection.

In hospitalized patients, gut colonization by the bacterial pathogen Klebsiella pneumoniae ( Kp ) is a major risk factor for the development of infections. The genome of Kp varies across isolates, and the presence of certain virulence genes is associated with the ability to progress from colonization to infection. Here, we identified that virulence genes encoding capsule and lipopolysaccharide, which normally protect bacteria from the immune system, are disrupted by mutations during murine gut colonization. These mutations occurred frequently in some isolates of Kp but not others, and these virulence gene mutants from the gut were defective in causing infections. An analysis of 245 human gut isolates demonstrated that this capsule loss also occurred in patients. This work highlights that mutations that decrease virulence occur during gut colonization, the propensity for these mutations differs by isolate, and that stability of virulence genes is important to consider when assessing infection risk in patients.

Candida auris is a fungal pathogen notorious for persistent skin colonization and transmission in healthcare settings. Here, we show that a C. auris conserved adhesin, Als4112, is required for skin colonization via keratinocyte attachment and direct interactions with host extracellular matrix proteins, especially basement membrane proteins such as laminin. Deletion of ALS4112 reduces skin colonization in mouse models of epicutaneous and systemic infection. In addition, coating plastic and catheter surfaces with collagen I or III inhibits C. auris attachment and biofilm formation. Our study highlights the critical role of Als4112 in C. auris colonization and virulence, and explores potential strategies to reduce the pathogen’s adherence to abiotic surfaces and thus its spread in healthcare settings. Candida auris is a fungal pathogen notorious for persistent skin colonization and transmission in healthcare settings. Here, Zhao et al. explore the mechanisms driving pathogen’s adherence to skin, involving a conserved adhesin, as well as the potential of collagen coatings as a strategy to reduce C. auris adherence to abiotic surfaces.

Background. We examined whether disparities existed in hospital-onset (HO) Staphylococcus aureus bloodstream infections (BSIs) and used whole-genome sequencing (WGS) to identify factors associated with USA300 transmission networks. Methods. We evaluated HO methicillin-susceptible S. aureus (MSSA) and HO methicillin-resistant S. aureus (MRSA) BSIs for 2009–2013 at 2 hospitals and used an adjusted incidence for modeling. WGS and phylogenetic analyses were performed on a sample of USA300 BSI isolates. Epidemiologic data were analyzed in the context of phylogenetic reconstructions. Results. On multivariate analysis, male sex, African-American race, and non-Hispanic white race/ethnicity were significantly associated with HO-MRSA BSIs whereas Hispanic ethnicity was negatively associated (rate ratio, 0.41; P = .002). Intermixing of community-onset and HO-USA300 strains on the phylogenetic tree indicates that these strains derive from a common pool. African-American race was the only factor associated with genomic clustering of isolates. Conclusions. In a multicenter assessment of HO-S. aureus BSIs, African-American race was significantly associated with HO-MRSA but not MSSA BSIs. There appears to be a nexus of USA300 community and hospital transmission networks, with a community factor being the primary driver. Our data suggest that HO-USA300 BSIs likely are due to colonizing strains acquired in the community before hospitalization. Therefore, prevention efforts may need to extend to the community for maximal benefit.