Development of advanced control material for reverse transcription-mediated bacterial nucleic acid amplification tests.

Detection of bacterial RNA by nucleic acid amplification tests (NAATs), such as reverse transcription PCR (RT-PCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP), offers distinct advantages over DNA-based methods. However, such assays also present challenges in ascertaining positive and internal control material that can reliably monitor success over all phases of testing (bacterial lysis, nucleic acid recovery, reverse transcription, amplification, and signal detection): since they are unable to distinguish between amplification of bacterial RNA transcripts and the DNA templates that encode them, using intact organisms as controls can inform cell lysis but not successful detection of RNA. We developed a control strategy for RNA-based bacterial NAATs that allows ready discrimination of RNA from DNA templates using self-splicing bacterial introns, such that those nucleic acids ultimately encode different sequences. We engineered two vectors encoding synthetic transgenes based on this principle, one that is active in the Gram-negative bacterium Escherichia coli and one that functions in both E. coli and the Gram-positive organism Staphylococcus aureus. We subsequently designed RT-LAMP assays that either target RNA and DNA from transgenic organisms or target RNA exclusively and demonstrated the specificity of amplification using purified nucleic acids. Using multiplex fluorescent RT-LAMP of heat-lysed specimens, we showed the practicality of deploying such transgenic organisms as an internal control to ascertain sample integrity and assay performance during clinical diagnostic testing. Our approach has broad utility for RNA-based bacterial NAATs, especially point-of-care assays and other applications where nucleic acids are nonspecifically liberated for testing.

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.

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.

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.

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.