Clinical and in vitro models identify distinct adaptations enhancing Staphylococcus aureus pathogenesis in human macrophages.

Staphylococcus aureus is a facultative intracellular pathogen of human macrophages, which facilitates chronic infection. The genotypes, pathways, and mutations influencing that phenotype remain incompletely explored. Here, we used two distinct strategies to ascertain S. aureus gene mutations affecting pathogenesis in macrophages. First, we analyzed isolates collected serially from chronic cystic fibrosis (CF) respiratory infections. We found that S. aureus strains evolved greater macrophage invasion capacity during chronic human infection. Bacterial genome-wide association studies (GWAS) identified 127 candidate genes for which mutation was significantly associated with macrophage pathogenesis in vivo. In parallel, we passaged laboratory S. aureus strains in vitro to select for increased infection of human THP-1 derived macrophages, which identified 15 candidate genes by whole-genome sequencing. Functional validation of candidate genes using isogenic transposon mutant knockouts and CRISPR interference (CRISPRi) knockdowns confirmed virulence contributions from 37 of 39 tested genes (95%) implicated by in vivo studies and 7 of 10 genes (70%) ascertained from in vitro selection, with one gene in common to the two strategies. Validated genes included 17 known virulence factors (39%) and 27 newly identified by our study (61%), some encoding functions not previously associated with macrophage pathogenesis. Most genes (80%) positively impacted macrophage invasion when disrupted, consistent with the phenotype readily arising from loss-of-function mutations in vivo. This work reveals genes and mechanisms that contribute to S. aureus infection of macrophages, highlights differences in mutations underlying convergent phenotypes arising from in vivo and in vitro systems, and supports the relevance of S. aureus macrophage pathogenesis during chronic respiratory infection in CF. Additional studies will be needed to illuminate the exact mechanisms by which implicated mutations affect their phenotypes.

Transposon sequencing identifies genes impacting Staphylococcus aureus invasion in a human macrophage model.

Staphylococcus aureus is a facultative intracellular pathogen in many host cell types, facilitating its persistence in chronic infections. The genes contributing to intracellular pathogenesis have not yet been fully enumerated. Here, we cataloged genes influencing S. aureus invasion and survival within human THP-1 derived macrophages using two laboratory strains (ATCC2913 and JE2). We developed an in vitro transposition method to produce highly saturated transposon mutant libraries in S. aureus and performed transposon insertion sequencing (Tn-Seq) to identify candidate genes with significantly altered abundance following macrophage invasion. While some significant genes were strain-specific, 108 were identified as common across both S. aureus strains, with most (n = 106) being required for optimal macrophage infection. We used CRISPR interference (CRISPRi) to functionally validate phenotypic contributions for a subset of genes. Of the 20 genes passing validation, seven had previously identified roles in S. aureus virulence, and 13 were newly implicated. Validated genes frequently evidenced strain-specific effects, yielding opposing phenotypes when knocked down in the alternative strain. Genomic analysis of de novo mutations occurring in groups (n = 237) of clonally related S. aureus isolates from the airways of chronically infected individuals with cystic fibrosis (CF) revealed significantly greater in vivo purifying selection in conditionally essential candidate genes than those not associated with macrophage invasion. This study implicates a core set of genes necessary to support macrophage invasion by S. aureus, highlights strain-specific differences in phenotypic effects of effector genes, and provides evidence for selection of candidate genes identified by Tn-Seq analyses during chronic airway infection in CF patients in vivo.

In Vitro Selection Identifies Staphylococcus aureus Genes Influencing Biofilm Formation.

Staphylococcus aureus generates biofilms during many chronic human infections, which contributes to its growth and persistence in the host. Multiple genes and pathways necessary for S. aureus biofilm production have been identified, but knowledge is incomplete, and little is known about spontaneous mutations that increase biofilm formation as infection progresses. Here, we performed in vitro selection of four S. aureus laboratory strains (ATCC 29213, JE2, N315, and Newman) to identify mutations associated with enhanced biofilm production. Biofilm formation increased in passaged isolates from all strains, exhibiting from 1.2- to 5-fold the capacity of parental lines. Whole-genome sequencing identified nonsynonymous mutations affecting 23 candidate genes and a genomic duplication encompassing sigB. Six candidate genes significantly impacted biofilm formation as isogenic transposon knockouts: three were previously reported to impact S. aureus biofilm formation (icaR, spdC, and codY), while the remaining three (manA, narH, and fruB) were newly implicated by this study. Plasmid-mediated genetic complementation of manA, narH, and fruB transposon mutants corrected biofilm deficiencies, with high-level expression of manA and fruB further enhancing biofilm formation over basal levels. This work recognizes genes not previously identified as contributing to biofilm formation in S. aureus and reveals genetic changes able to augment biofilm production by that organism.

Emergence of a Homo sapiens-specific gene family and chromosome 16p11.2 CNV susceptibility.

Genetic differences that specify unique aspects of human evolution have typically been identified by comparative analyses between the genomes of humans and closely related primates, including more recently the genomes of archaic hominins. Not all regions of the genome, however, are equally amenable to such study. Recurrent copy number variation (CNV) at chromosome 16p11.2 accounts for approximately 1% of cases of autism and is mediated by a complex set of segmental duplications, many of which arose recently during human evolution. Here we reconstruct the evolutionary history of the locus and identify bolA family member 2 (BOLA2) as a gene duplicated exclusively in Homo sapiens. We estimate that a 95-kilobase-pair segment containing BOLA2 duplicated across the critical region approximately 282 thousand years ago (ka), one of the latest among a series of genomic changes that dramatically restructured the locus during hominid evolution. All humans examined carried one or more copies of the duplication, which nearly fixed early in the human lineage--a pattern unlikely to have arisen so rapidly in the absence of selection (P < 0.0097). We show that the duplication of BOLA2 led to a novel, human-specific in-frame fusion transcript and that BOLA2 copy number correlates with both RNA expression (r = 0.36) and protein level (r = 0.65), with the greatest expression difference between human and chimpanzee in experimentally derived stem cells. Analyses of 152 patients carrying a chromosome 16p11. rearrangement show that more than 96% of breakpoints occur within the H. sapiens-specific duplication. In summary, the duplicative transposition of BOLA2 at the root of the H. sapiens lineage about 282 ka simultaneously increased copy number of a gene associated with iron homeostasis and predisposed our species to recurrent rearrangements associated with disease.

Polyclonality, Shared Strains, and Convergent Evolution in Chronic Cystic Fibrosis Staphylococcus aureus Airway Infection.

Rationale:Staphylococcus aureus is the most common respiratory pathogen isolated from patients with cystic fibrosis (CF) in the United States. Although modes of acquisition and genetic adaptation have been described for Pseudomonas aeruginosa, resulting in improved diagnosis and treatment, these features remain more poorly defined for S. aureus.Objectives: To characterize the molecular epidemiology and genetic adaptation of S. aureus during chronic CF airway infection and in response to antibiotic therapy.Methods: We performed whole-genome sequencing of 1,382 S. aureus isolates collected longitudinally over a mean 2.2 years from 246 children with CF at five U.S. centers between 2008 and 2017. Results were integrated with clinical and demographic data to characterize bacterial population dynamics and identify common genetic targets of in vivo adaptation.Measurements and Main Results: Results showed that 45.5% of patients carried multiple, coexisting S. aureus lineages, often having different antibiotic susceptibility profiles. Adaptation during the course of infection commonly occurred in a set of genes related to persistence and antimicrobial resistance. Individual sequence types demonstrated wide geographic distribution, and we identified limited strain-sharing among children linked by common household or clinical exposures. Unlike P. aeruginosa, S. aureus genetic diversity was unconstrained, with an ongoing flow of new genetic elements into the population of isolates from children with CF.Conclusions: CF airways are frequently coinfected by multiple, genetically distinct S. aureus lineages, indicating that current clinical procedures for sampling isolates and selecting antibiotics are likely inadequate. Strains can be shared by patients in close domestic or clinical contact and can undergo convergent evolution in key persistence and antimicrobial-resistance genes, suggesting novel diagnostic and therapeutic approaches for future study.

Identification of a novel tedizolid resistance mutation in rpoB of MRSA after in vitro serial passage.

OBJECTIVES: Tedizolid is an oxazolidinone antimicrobial with activity against Gram-positive bacteria, including MRSA. Tedizolid resistance is uncommon and tedizolid's capacity to select for cross-resistance to other antimicrobials is incompletely understood. The objective of this study was to further explore the phenotypic and genetic basis of tedizolid resistance in MRSA. METHODS: We selected for tedizolid resistance in an MRSA laboratory strain, N315, by serial passage until an isolate with an MIC ≥1 log2 dilution above the breakpoint for resistance (≥2 mg/L) was recovered. This isolate was subjected to WGS and susceptibility to a panel of related and unrelated antimicrobials was tested in order to determine cross-resistance. Homology modelling was performed to evaluate the potential impact of the mutation on target protein function. RESULTS: After 10 days of serial passage we recovered a phenotypically stable mutant with a tedizolid MIC of 4 mg/L. WGS revealed only one single nucleotide variant (A1345G) in rpoB, corresponding to amino acid substitution D449N. MICs of linezolid, chloramphenicol, retapamulin and quinupristin/dalfopristin increased by ≥2 log2 dilutions, suggesting the emergence of the so-called 'PhLOPSa' resistance phenotype. Susceptibility to other drugs, including rifampicin, was largely unchanged. Homology models revealed that the mutated residue of RNA polymerase would be unlikely to directly affect oxazolidinone action. CONCLUSIONS: To the best of our knowledge, this is the first time that an rpoB mutation has been implicated in resistance to PhLOPSa antimicrobials. The mechanism of resistance remains unclear, but is likely indirect, involving σ-factor binding or other alterations in transcriptional regulation.

Recurrent structural variation, clustered sites of selection, and disease risk for the complement factor H (CFH) gene family.

Structural variation and single-nucleotide variation of the complement factor H (CFH) gene family underlie several complex genetic diseases, including age-related macular degeneration (AMD) and atypical hemolytic uremic syndrome (AHUS). To understand its diversity and evolution, we performed high-quality sequencing of this ∼360-kbp locus in six primate lineages, including multiple human haplotypes. Comparative sequence analyses reveal two distinct periods of gene duplication leading to the emergence of four CFH-related (CFHR) gene paralogs (CFHR2 and CFHR4 ∼25-35 Mya and CFHR1 and CFHR3 ∼7-13 Mya). Remarkably, all evolutionary breakpoints share a common ∼4.8-kbp segment corresponding to an ancestral CFHR gene promoter that has expanded independently throughout primate evolution. This segment is recurrently reused and juxtaposed with a donor duplication containing exons 8 and 9 from ancestral CFH, creating four CFHR fusion genes that include lineage-specific members of the gene family. Combined analysis of >5,000 AMD cases and controls identifies a significant burden of a rare missense mutation that clusters at the N terminus of CFH [P = 5.81 × 10-8, odds ratio (OR) = 9.8 (3.67-Infinity)]. A bipolar clustering pattern of rare nonsynonymous mutations in patients with AMD (P < 10-3) and AHUS (P = 0.0079) maps to functional domains that show evidence of positive selection during primate evolution. Our structural variation analysis in >2,400 individuals reveals five recurrent rearrangement breakpoints that show variable frequency among AMD cases and controls. These data suggest a dynamic and recurrent pattern of mutation critical to the emergence of new CFHR genes but also in the predisposition to complex human genetic disease phenotypes.

Occurrence of cross-resistance and ß-lactam seesaw effect in glycopeptide-, lipopeptide- and lipoglycopeptide-resistant MRSA correlates with membrane phosphatidylglycerol levels.

BACKGROUND: Glycopeptides (GPs), lipopeptides (LPs) and lipoglycopeptides (LGPs) are related antimicrobials important for the management of invasive MRSA infections. Cross-resistance among these antibiotics in MRSA is well documented, as is the observation that susceptibility of MRSA to ß-lactams increases as susceptibility to GPs and LPs decreases (i.e. the seesaw effect). Efforts to understand the relationship between GP/LP/LGP cross-resistance and the seesaw effect have focused on the PBPs, but the role of lipid metabolism has not been investigated. OBJECTIVES: Since the cell membrane is structurally and metabolically integrated with the cell wall and anchors associated proteins, including PBPs, we examined the relationship between membrane lipid composition and the phenomena of cross-resistance among GPs/LPs/LGPs and the ß-lactam seesaw effect. METHODS: We selected for daptomycin, vancomycin and dalbavancin resistance using the USA300 strain JE2 and evaluated the resulting mutants by WGS, MS-based lipidomics and antimicrobial susceptibility testing to assess the relationship between membrane composition, cross-resistance, and the seesaw effect. RESULTS: We observed cross-resistance to GPs/LPs/LGPs among the selected strains and the seesaw effect against various ß-lactams, depending on the PBP targets of the particular ß-lactam. We found that modification of membrane composition occurs not only in daptomycin-selected strains, but also vancomycin- and dalbavancin-selected strains. Significantly, we observed that the abundance of most phosphatidylglycerols positively correlates with MICs of GPs/LPs/LGPs and negatively correlates with the MICs of ß-lactams. CONCLUSIONS: These studies demonstrate a major association between membrane remodelling, cross-resistance and the seesaw effect.

Artificial Selection for Pathogenicity Mutations in Staphylococcus aureus Identifies Novel Factors Relevant to Chronic Infection.

Adaptation of Staphylococcus aureus to host microenvironments during chronic infection involves spontaneous mutations, yet changes underlying adaptive phenotypes remain incompletely explored. Here, we employed artificial selection and whole-genome sequencing to better characterize spontaneous chromosomal mutations that alter two pathogenicity phenotypes relevant to chronic infection in S. aureus: intracellular invasiveness and intracellular cytotoxicity. We identified 23 genes whose alteration coincided with enhanced virulence, 11 that were previously known and 12 (52%) that had no previously described role in S. aureus pathogenicity. Using precision genome editing, transposon mutants, and gene complementation, we empirically assessed the contributions of individual genes to the two virulence phenotypes. We functionally validated 14 of 21 genes tested as measurably influencing invasion and/or cytotoxicity, including 8 newly implicated by this study. We identified inactivating mutations (murA, ndhC, and a hypothetical membrane protein) and gain-of-function mutations (aroE Thr182Ile, yhcF Thr74Ile, and Asp486Glu in a hypothetical peptidase) in previously unrecognized S. aureus virulence genes that enhance pathogenesis when introduced into a clean genetic background, as well as a novel activating mutation in the known virulence regulator gene saeS (Ala106Thr). Investigation of potentially epistatic interactions identified a tufA mutation (Ala271Val) that enhances virulence only in the context of purine operon repressor gene (purR) inactivation. This project reveals a functionally diverse range of genes affected by gain- or loss-of-function mutations that contribute to S. aureus adaptive virulence phenotypes. More generally, the work establishes artificial selection as a means to determine the genetic mechanisms underlying complex bacterial phenotypes relevant to adaptation during infection.

Genomic Analysis Identifies Novel Pseudomonas aeruginosa Resistance Genes under Selection during Inhaled Aztreonam Therapy In Vivo.

Inhaled aztreonam is increasingly used for chronic Pseudomonas aeruginosa suppression in patients with cystic fibrosis (CF), but the potential for that organism to evolve aztreonam resistance remains incompletely explored. Here, we performed genomic analysis of clonally related pre- and posttreatment CF clinical isolate pairs to identify genes that are under positive selection during aztreonam therapy in vivo We identified 16 frequently mutated genes associated with aztreonam resistance, the most prevalent being ftsI and ampC, and 13 of which increased aztreonam resistance when introduced as single gene transposon mutants. Several previously implicated aztreonam resistance genes were found to be under positive selection in clinical isolates even in the absence of inhaled aztreonam exposure, indicating that other selective pressures in the cystic fibrosis airway can promote aztreonam resistance. Given its potential to confer plasmid-mediated resistance, we further characterized mutant ampC alleles and performed artificial evolution of ampC for maximal activity against aztreonam. We found that naturally occurring ampC mutants conferred variably increased resistance to aztreonam (2- to 64-fold) and other ß-lactam agents but that its maximal evolutionary capacity for hydrolyzing aztreonam was considerably higher (512- to 1,024-fold increases) and was achieved while maintaining or increasing resistance to other drugs. These studies implicate novel chromosomal aztreonam resistance determinants while highlighting that different mutations are favored during selection in vivo and in vitro, show that ampC has a high maximal potential to hydrolyze aztreonam, and provide an approach to disambiguate mutations promoting specific resistance phenotypes from those more generally increasing bacterial fitness in vivo.

The Vaginal Microbiome of Transgender Men.

BACKGROUND: Hormonal changes influence the composition of vaginal flora, which is directly related to the health of an individual. Transgender men prescribed testosterone experience a vaginal hormone composition that differs from cisgender women. To the author's knowledge, there are no clinical studies evaluating the influence that testosterone administration has on the vaginal microbiome. METHODS: Vaginal swabs were self-collected by a cohort of self-identified healthy transgender men prescribed testosterone for at least 1 year (n = 28) and from cisgender women who were used as the comparator (n = 8). Participants completed a questionnaire to indicate the mode and dose of testosterone administration, sexual history, and vaginal health. Serum was collected for hormone analysis. Bacterial community profiles were assessed with broad-range PCR primers targeting the V3-V4 hypervariable region of the 16S bacterial rRNA, next-generation sequencing, and analysis by phylogenetic placement. RESULTS: Compared to cisgender women, the vaginal floras of transgender men were less likely to have Lactobacillus as their primary genus. Intravaginal estrogen administration was positively associated with the presence of Lactobacillus in transgender men (P = 0.045). Transgender men had a significantly increased relative abundance of >30 species and a significantly higher α diversity (P = 0.0003). The presence of Lactobacillus was significantly associated with a lower α diversity index (P = 0.017). CONCLUSIONS: The vaginal microbiome of transgender men who were assigned a female sex at birth and use testosterone may differ from that of cisgender women. Intravaginal estrogen administration may reduce these differences by promoting colonization with Lactobacillus species and decreasing α diversity.

Ultrasensitive Detection of Chimerism by Single-Molecule Molecular Inversion Probe Capture and High-Throughput Sequencing of Copy Number Deletion Polymorphisms.

BACKGROUND: Genomic chimerism, the co-occurrence of cells from different genetic origins, provides important diagnostic information in diverse clinical contexts, including graft injury detection and longitudinal surveillance of hematopoietic stem cell transplantation patients, but existing assays are limiting. Here we applied single-molecule molecular inversion probes (smMIPs), a high-throughput sequencing technology combining multiplexed target capture with read quantification mediated by unique molecular identifiers, to detect chimerism based on the presence or absence of polymorphic genomic loci. METHODS: We designed a 159-smMIP panel targeting 40 autosomal regions of frequent homozygous deletion across human populations and 2 sex-linked loci. We developed methods for detecting and quantitating loci absent from 1 cell population but present in another, which could be used to sensitively identify chimeric cell populations. RESULTS: Unrelated individuals and first-degree relatives were highly polymorphic across the loci examined. Using synthetic DNA mixtures, limits of detection of at least 1 in 10000 chimeric cells were demonstrated without prior knowledge of genotypes, and mixtures of up to 4 separate donors could be deconvoluted. Quantitative linearity over 4 orders of magnitude and false-positive rates <1 in 85000 events were achieved. Eleven of 11 posttransplant clinical specimens from patients with hematological malignancies testing positive for residual cancer by conventional methods had detectable chimeric populations by smMIP, whereas 11 of 11 specimens testing negative by conventional methods were low-positive for chimerism by smMIP. CONCLUSIONS: smMIPs are scalable to high sensitivity and large numbers of informative markers, enabling ultrasensitive chimerism detection for many clinical purposes.

Accurate Pan-Cancer Molecular Diagnosis of Microsatellite Instability by Single-Molecule Molecular Inversion Probe Capture and High-Throughput Sequencing.

BACKGROUND: Microsatellite instability (MSI) is an emerging actionable phenotype in oncology that informs tumor response to immune checkpoint pathway immunotherapy. However, there remains a need for MSI diagnostics that are low cost, highly accurate, and generalizable across cancer types. We developed a method for targeted high-throughput sequencing of numerous microsatellite loci with pan-cancer informativity for MSI using single-molecule molecular inversion probes (smMIPs). METHODS: We designed a smMIP panel targeting 111 loci highly informative for MSI across cancers. We developed an analytical framework taking advantage of smMIP-mediated error correction to specifically and sensitively detect instability events without the need for typing matched normal material. RESULTS: Using synthetic DNA mixtures, smMIPs were sensitive to at least 1% MSI-positive cells and were highly consistent across replicates. The fraction of identified unstable microsatellites discriminated tumors exhibiting MSI from those lacking MSI with high accuracy across colorectal (100% diagnostic sensitivity and specificity), prostate (100% diagnostic sensitivity and specificity), and endometrial cancers (95.8% diagnostic sensitivity and 100% specificity). MSI-PCR, the current standard-of-care molecular diagnostic for MSI, proved equally robust for colorectal tumors but evidenced multiple false-negative results in prostate (81.8% diagnostic sensitivity and 100% specificity) and endometrial (75.0% diagnostic sensitivity and 100% specificity) tumors. CONCLUSIONS: smMIP capture provides an accurate, diagnostically sensitive, and economical means to diagnose MSI across cancer types without reliance on patient-matched normal material. The assay is readily scalable to large numbers of clinical samples, enables automated and quantitative analysis of microsatellite instability, and is readily standardized across clinical laboratories.

Ultrasensitive detection of acute myeloid leukemia minimal residual disease using single molecule molecular inversion probes.

The identification of minimal residual disease is the primary diagnostic finding which predicts relapse in patients treated for acute myeloid leukemia. Ultrasensitive detection of minimal residual disease would enable better patient risk stratification and could open opportunities for early therapeutic intervention. Herein we apply single molecule molecular inversion probe capture, a technology combining multiplexed targeted sequencing with error correction schemes based on molecular barcoding, in order to detect mutations identifying minimal residual disease with ultrasensitive and quantitative precision. We designed a single molecule molecular inversion probe capture panel spanning >50 kb and targeting 32 factors relevant to acute myeloid leukemia pathogenesis. We demonstrate linearity and quantitative precision over 100-fold relative abundance of mutant cells (1 in 100 to 1 in 1,500), with estimated error rates approaching 1 in 1,200 base pairs sequenced and maximum theoretical limits of detection exceeding 1 in 60,000 mutant alleles. In 3 of 4 longitudinally collected specimens from patients with acute myeloid leukemia, we find that single molecule molecular inversion probe capture detects somatic mutations identifying minimal residual disease at substantially earlier time points and with greater sensitivity than clinical diagnostic approaches used as current standard of care (flow cytometry and conventional molecular diagnosis), and identifies persisting neoplastic cells during clinical remission. In 2 patients, single molecule molecular inversion probe capture detected heterogeneous, subclonal acute myeloid leukemia populations carrying distinct mutational signatures. Single molecule molecular inversion probe technology uniquely couples scalable target enrichment with sequence read error correction, providing an integrated, ultrasensitive approach for detecting minimal residual disease identifying mutations.

Longitudinal profiling of the intestinal microbiome in children with cystic fibrosis treated with elexacaftor-tezacaftor-ivacaftor.

The intestinal microbiome influences growth and disease progression in children with cystic fibrosis (CF). Elexacaftor-tezacaftor-ivacaftor (ELX/TEZ/IVA), the newest pharmaceutical modulator for CF, restores the function of the pathogenic mutated CF transmembrane conductance regulator (CFTR) channel. We performed a single-center longitudinal analysis of the effect of ELX/TEZ/IVA on the intestinal microbiome, intestinal inflammation, and clinical parameters in children with CF. Following ELX/TEZ/IVA, children with CF had significant improvements in body mass index and percent predicted forced expiratory volume in one second, and required fewer antibiotics for respiratory infections. Intestinal microbiome diversity increased following ELX/TEZ/IVA coupled with a decrease in the intestinal carriage of Staphylococcus aureus, the predominant respiratory pathogen in children with CF. There was a reduced abundance of microbiome-encoded antibiotic resistance genes. Microbial pathways for aerobic respiration were reduced after ELX/TEZ/IVA. The abundance of microbial acid tolerance genes was reduced, indicating microbial adaptation to increased CFTR function. In all, this study represents the first comprehensive analysis of the intestinal microbiome in children with CF receiving ELX/TEZ/IVA.IMPORTANCECystic fibrosis (CF) is an autosomal recessive disease with significant gastrointestinal symptoms in addition to pulmonary complications. Recently approved treatments for CF, CF transmembrane conductance regulator (CFTR) modulators, are anticipated to substantially improve the care of people with CF and extend their lifespans. Prior work has shown that the intestinal microbiome correlates with health outcomes in CF, particularly in children. Here, we study the intestinal microbiome of children with CF before and after the CFTR modulator, ELX/TEZ/IVA. We identify promising improvements in microbiome diversity, reduced measures of intestinal inflammation, and reduced antibiotic resistance genes. We present specific bacterial taxa and protein groups which change following ELX/TEZ/IVA. These results will inform future mechanistic studies to understand the microbial improvements associated with CFTR modulator treatment. This study demonstrates how the microbiome can change in response to a targeted medication that corrects a genetic disease.

Contribution of the patient microbiome to surgical site infection and antibiotic prophylaxis failure in spine surgery.

Despite modern antiseptic techniques, surgical site infection (SSI) remains a leading complication of surgery. However, the origins of SSI and the high rates of antimicrobial resistance observed in these infections are poorly understood. Using instrumented spine surgery as a model of clean (class I) skin incision, we prospectively sampled preoperative microbiomes and postoperative SSI isolates in a cohort of 204 patients. Combining multiple forms of genomic analysis, we correlated the identity, anatomic distribution, and antimicrobial resistance profiles of SSI pathogens with those of preoperative strains obtained from the patient skin microbiome. We found that 86% of SSIs, comprising a broad range of bacterial species, originated endogenously from preoperative strains, with no evidence of common source infection among a superset of 1610 patients. Most SSI isolates (59%) were resistant to the prophylactic antibiotic administered during surgery, and their resistance phenotypes correlated with the patient's preoperative resistome (P = 0.0002). These findings indicate the need for SSI prevention strategies tailored to the preoperative microbiome and resistome present in individual patients.

Genomic reconstruction and directed interventions in a multidrug-resistant Shigellosis outbreak in Seattle, WA, USA: a genomic surveillance study.

BACKGROUND: Shigella spp have been associated with community-wide outbreaks in urban settings. We analysed a sustained shigellosis outbreak in Seattle, WA, USA, to understand its origins and mechanisms of antimicrobial resistance, define ongoing transmission patterns, and optimise strategies for treatment and infection control. METHODS: We did a retrospective study of all Shigella isolates identified from stool samples at the clinical laboratories at Harborview Medical Center and University of Washington Medical Center (Seattle, WA, USA) from May 1, 2017, to Feb 28, 2022. We characterised isolates by species identification, phenotypic susceptibility testing, and whole-genome sequencing. Demographic characteristics and clinical outcomes of the patients were retrospectively examined. FINDINGS: 171 cases of shigellosis were included. 78 (46%) patients were men who have sex with men (MSM), and 88 (52%) were people experiencing homelessness (PEH). Although 84 (51%) isolates were multidrug resistant, 100 (70%) of 143 patients with data on antimicrobial therapy received appropriate empirical therapy. Phylogenomic analysis identified sequential outbreaks of multiple distinct lineages of Shigella flexneri and Shigella sonnei. Discrete clonal lineages (ten in S flexneri and nine in S sonnei) and resistance traits were responsible for infection in different at-risk populations (ie, MSM, PEH), enabling development of effective guidelines for empirical treatment. The most prevalent lineage in Seattle was probably introduced to Washington State via international travel, with subsequent domestic transmission between at-risk groups. INTERPRETATION: An outbreak in Seattle was driven by parallel emergence of multidrug-resistant strains involving international transmission networks and domestic transmission between at-risk populations. Genomic analysis elucidated not only outbreak origin, but directed optimal approaches to testing, treatment, and public health response. Rapid diagnostics combined with detailed knowledge of local epidemiology can enable high rates of appropriate empirical therapy even in multidrug-resistant infection. FUNDING: None.

Longitudinal Profiling of the Intestinal Microbiome in Children with Cystic Fibrosis Treated with Elexacaftor-Tezacaftor-Ivacaftor.

The intestinal microbiome influences growth and disease progression in children with cystic fibrosis (CF). Elexacaftor-tezacaftor-ivacaftor (ELX/TEZ/IVA), the newest pharmaceutical modulator for CF, restores function of the pathogenic mutated CFTR channel. We performed a single-center longitudinal analysis of the effect of ELX/TEZ/IVA on the intestinal microbiome, intestinal inflammation, and clinical parameters in children with CF. Following ELX/TEZ/IVA, children with CF had significant improvements in BMI, ppFEV1 and required fewer antibiotics for respiratory infections. Intestinal microbiome diversity increased following ELX/TEZ/IVA coupled with a decrease in the intestinal carriage of Staphylococcus aureus, the predominant respiratory pathogen in children with CF. There was a reduced abundance of microbiome-encoded antibiotic-resistance genes. Microbial pathways for aerobic respiration were reduced after ELX/TEZ/IVA. The abundance of microbial acid tolerance genes was reduced, indicating microbial adaptation to increased CFTR function. In all, this study represents the first comprehensive analysis of the intestinal microbiome in children with CF receiving ELX/TEZ/IVA.

Discovery of a glutathione utilization pathway in Francisella that shows functional divergence between environmental and pathogenic species.

Glutathione (GSH) is an abundant metabolite within eukaryotic cells that can act as a signal, a nutrient source, or serve in a redox capacity for intracellular bacterial pathogens. For Francisella, GSH is thought to be a critical in vivo source of cysteine; however, the cellular pathways permitting GSH utilization by Francisella differ between strains and have remained poorly understood. Using genetic screening, we discovered a unique pathway for GSH utilization in Francisella. Whereas prior work suggested GSH catabolism initiates in the periplasm, the pathway we define consists of a major facilitator superfamily (MFS) member that transports intact GSH and a previously unrecognized bacterial cytoplasmic enzyme that catalyzes the first step of GSH degradation. Interestingly, we find that the transporter gene for this pathway is pseudogenized in pathogenic Francisella, explaining phenotypic discrepancies in GSH utilization among Francisella spp. and revealing a critical role for GSH in the environmental niche of these bacteria.

Recombineering in Staphylococcus aureus.

Recombineering has proven to be an extraordinarily powerful and versatile approach for the modification of bacterial genomes, but has historically not been possible in the important opportunistic pathogen Staphylococcus aureus. After evaluating the activity of various recombinases in S. aureus, we developed methods for recombineering in that organism using synthetic, single-stranded DNA oligonucleotides. This approach can be coupled to CRISPR/Cas9-mediated lethal counterselection in order to improve the efficiency with which recombinant S. aureus are recovered, which is especially useful in instances where mutants lack a selectable phenotype. These methods provide a rapid, scalable, precise, and inexpensive means to engineer point mutations, variable-length deletions, and short insertions into the S. aureus genome.