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.

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.

Improved Species-Level Clinical Identification of Enterobacteriaceae through Broad-Range dnaJ PCR and Sequencing.

Enterobacteriaceae represent a diverse and medically important family of bacteria that are difficult to identify to the species level using the standard molecular method of 16S rRNA gene sequencing. Prior work has demonstrated the value of dnaJ gene sequence analysis in resolving different members of the family. However, existing protocols are not optimized for clinical use and exhibit several limitations in practice. Here, we describe an improved assay for dnaJ-based identification of Enterobacteriaceae which boasts increased broad-range specificity across genera, shorter amplicon sizes that are suitable for use with formalin-fixed or direct patient specimens, and enhanced amplification efficiency and assay sensitivity through the incorporation of locked nucleic acid chemistries. Sequence analysis of public databases indicates that the partial dnaJ sequence interrogated by this design retains high discriminatory power among Enterobacteriaceae genera and species, with only particular lineages of Shigella sp. and Escherichia coli proving unresolvable. Limits of detection studies using 8 disparate species indicated that amplification was consistently achievable across organisms and allowed robust dideoxynucleotide chain terminator sequencing from as little as 10 genome equivalents of template, depending on the species interrogated. Retrospective application of the dnaJ assay to patient specimens enabled unambiguous classification of Enterobacteriaceae to the species level in 22 of 27 (81.5%) positive specimens examined, with most remaining cases representing unresolvable calls between closely related Escherichia coli and Shigella species. We expect that this assay will facilitate the accurate molecular identification of species from the Enterobacteriaceae family in a variety of clinical specimens and diagnostic contexts.

Diagnosis of Ovarian Carcinoma Homologous Recombination DNA Repair Deficiency From Targeted Gene Capture Oncology Assays.

PURPOSE: Homologous recombination DNA repair deficiency (HRD) is a therapeutic biomarker for sensitivity to platinum and poly(ADP-ribose) polymerase inhibitor therapies in breast and ovarian cancers. Several molecular phenotypes and diagnostic strategies have been developed to assess HRD; however, their clinical implementation remains both technically challenging and methodologically unstandardized. METHODS: We developed and validated an efficient and cost-effective strategy for HRD determination on the basis of calculation of a genome-wide loss of heterozygosity (LOH) score through targeted, hybridization capture and next-generation DNA sequencing augmented with 3,000 common, polymorphic single-nucleotide polymorphism (SNP) sites distributed genome-wide. This approach requires minimal sequence reads and can be readily integrated into targeted gene capture workflows already in use for molecular oncology. We interrogated 99 ovarian neoplasm-normal pairs using this method and compared results with patient mutational genotypes and orthologous predictors of HRD derived from whole-genome mutational signatures. RESULTS: LOH scores of ≥11% had >86% sensitivity for identifying tumors with HRD-causing mutations in an independent validation set (90.6% sensitivity for all specimens). We found strong agreement of our analytic approach with genome-wide mutational signature assays for determining HRD, yielding an estimated 96.7% sensitivity and 50% specificity. We observed poor concordance with mutational signatures inferred using only mutations detected by the targeted gene capture panel, suggesting inadequacy of the latter approach. LOH score did not significantly correlate with treatment outcomes. CONCLUSION: Targeted sequencing of genome-wide polymorphic SNP sites can be used to infer LOH events and subsequently diagnose HRD in ovarian tumors. The methods presented here are readily generalizable to other targeted gene oncology assays and could be adapted for HRD diagnosis in other tumor types.

Deep-Structure Adaptations and Culturally Grounded Prevention Interventions for Native Hawaiians: a Systematic Review of the Literature.

Research has shown that Native Hawaiians disproportionately suffer from behavioral disorders and chronic physical diseases, yet they have historically lacked effective and culturally relevant prevention interventions to address their pervasive health disparities. This article systematically reviewed the recent culturally relevant prevention intervention literature focused on Native Hawaiians. In this review, we assessed 14 peer-reviewed articles published between 2015 and 2020 that met inclusion and exclusion criteria pertaining to the development and/or evaluation of prevention interventions for Native Hawaiians. The reviewed studies evaluated ten different interventions that were developed using deep-structure adaptation or culturally grounded procedures, and primarily focused on prevention of substance use, obesity/diabetes, and pregnancy/sexually transmitted infections (STIs). Compared with the prior related literature reviews, the present review suggests an overall advancement in prevention science for Native Hawaiians, evidenced by an increase in federal funding and randomized controlled clinical trials of prevention interventions for the population. This review provides an update to the state of the science for Native Hawaiian prevention interventions and points to areas of future research and development.

Placental IL-10 dysregulation and association with bronchopulmonary dysplasia risk.

Cytokine profiles in amniotic fluid, cord serum, and tracheal aspirate of premature infants suggest a shift toward a proinflammatory state. Cytokines also contribute to the pathogenesis of bronchopulmonary dysplasia (BPD). We hypothesize that the initiating events for BPD are reflected in the placenta and propose that placental expression of cytokines provide a blueprint of events leading to BPD. This is a retrospective, case-controlled study of placental cytokines of premature infants with (n = 49) and without (n = 49) BPD, matched for gender, birth weight, and year of birth at Women and Infants Hospital between 2003 and 2005. Cytokine expression, including IL-6 and IL-10, was determined by immunohistochemistry in membrane rolls, umbilical cords, and placentas. IL-6 was similarly expressed in all tissues of infants with and without BPD. In contrast, anti-inflammatory cytokine IL-10 was less prominent in the placenta of patients with BPD compared with those without BPD. IL-10 expression in the villous trophoblast layer was associated with a reduced odds ratio of developing BPD (adjusted OR 0.08, 95% CI 0.01-0.70, p = 0.02). These results suggest that a placental balance between inflammatory and anti-inflammatory cytokines is crucial to normal lung organogenesis. Importantly, IL-10 seems to be protective against the development of BPD.

Gallium disrupts bacterial iron metabolism and has therapeutic effects in mice and humans with lung infections.

The lack of new antibiotics is among the most critical challenges facing medicine. The problem is particularly acute for Gram-negative bacteria. An unconventional antibiotic strategy is to target bacterial nutrition and metabolism. The metal gallium can disrupt bacterial iron metabolism because it substitutes for iron when taken up by bacteria. We investigated the antibiotic activity of gallium ex vivo, in a mouse model of airway infection, and in a phase 1 clinical trial in individuals with cystic fibrosis (CF) and chronic Pseudomonas aeruginosa airway infections. Our results show that micromolar concentrations of gallium inhibited P. aeruginosa growth in sputum samples from patients with CF. Ex vivo experiments indicated that gallium inhibited key iron-dependent bacterial enzymes and increased bacterial sensitivity to oxidants. Furthermore, gallium resistance developed slowly, its activity was synergistic with certain antibiotics, and gallium did not diminish the antibacterial activity of host macrophages. Systemic gallium treatment showed antibiotic activity in murine lung infections. In addition, systemic gallium treatment improved lung function in people with CF and chronic P. aeruginosa lung infection in a preliminary phase 1 clinical trial. These findings raise the possibility that human infections could be treated by targeting iron metabolism or other nutritional vulnerabilities of bacterial pathogens.

Evolved Aztreonam Resistance Is Multifactorial and Can Produce Hypervirulence in Pseudomonas aeruginosa.

While much attention has been focused on acquired antibiotic resistance genes, chromosomal mutations may be most important in chronic infections where isolated, persistently infecting lineages experience repeated antibiotic exposure. Here, we used experimental evolution and whole-genome sequencing to investigate chromosomally encoded mutations causing aztreonam resistance in Pseudomonas aeruginosa and characterized the secondary consequences of resistance development. We identified 19 recurrently mutated genes associated with aztreonam resistance. The most frequently observed mutations affected negative transcriptional regulators of the mexAB-oprM efflux system and the target of aztreonam, ftsI While individual mutations conferred modest resistance gains, high-level resistance (1,024 µg/ml) was achieved through the accumulation of multiple variants. Despite being largely stable when strains were passaged in the absence of antibiotics, aztreonam resistance was associated with decreased in vitro growth rates, indicating an associated fitness cost. In some instances, evolved aztreonam-resistant strains exhibited increased resistance to structurally unrelated antipseudomonal antibiotics. Surprisingly, strains carrying evolved mutations which affected negative regulators of mexAB-oprM (mexR and nalD) demonstrated enhanced virulence in a murine pneumonia infection model. Mutations in these genes, and other genes that we associated with aztreonam resistance, were common in P. aeruginosa isolates from chronically infected patients with cystic fibrosis. These findings illuminate mechanisms of P. aeruginosa aztreonam resistance and raise the possibility that antibiotic treatment could inadvertently select for hypervirulence phenotypes.IMPORTANCE Inhaled aztreonam is a relatively new antibiotic which is being increasingly used to treat cystic fibrosis patients with Pseudomonas aeruginosa airway infections. As for all antimicrobial agents, bacteria can evolve resistance that decreases the effectiveness of the drug; however, the mechanisms and consequences of aztreonam resistance are incompletely understood. Here, using experimental evolution, we have cataloged spontaneous mutations conferring aztreonam resistance and have explored their effects. We found that a diverse collection of genes contributes to aztreonam resistance, each with a small but cumulative effect. Surprisingly, we found that selection for aztreonam resistance mutations could confer increased resistance to other antibiotics and promote hypervirulence in a mouse infection model. Our study reveals inherent mechanisms of aztreonam resistance and indicates that aztreonam exposure can have unintended secondary effects.

Severe umbilical cord inflammation-a predictor of periventricular leukomalacia in very low birth weight infants.

Chorioamnionitis has been associated with periventricular leukomalacia (PVL) in very low birth weight (VLBW) infants. We examined the association between the pathological severity of chorioamnionitis and PVL in VLBW infants. Thirty-four VLBW infants with PVL and 34 control infants matched for gestational age without a diagnosis of PVL or intraventricular hemorrhage were obtained from the Women and Infants' Hospital of Rhode Island's Neonatal Follow-up Clinic database. Placental samples, including the amnion/chorion, chorionic plate, and umbilical cord, were examined microscopically. Statistical analysis included Mantel-Haenszel chi-square, and Student's t-test. Severe inflammation in the umbilical cord was observed in 53% of infants with PVL and 32% without PVL (p<0.05). Severe umbilical cord inflammation is one of the risk factors associated with the development of PVL in VLBW infants.

Osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a rare congenital disorder of collagen production that results in brittle bones and affects other body systems containing collagen. This article reviews the current body of knowledge about OI and the management of infants with the disorder. Relieving pain, reducing the incidence of new fractures, establishing adequate follow-up, and connecting parents with community resources are the goals of management during the neonatal period. A case study illustrates management and the discharge process.