BACKGROUND:Relatively little is known about Pseudomonas aeruginosa lineages during early infection in individuals with cystic fibrosis (CF). The Early Pseudomonas Infection Control Clinical Trial (EPIC-CT), which treated individuals with CF and newly detected P. aeruginosa infections, presents an ideal opportunity to study this phenomenon. METHODS:We performed whole-genome sequencing on 572 P. aeruginosa isolates from 190 EPIC-CT subjects. RESULTS:We identified 203 P. aeruginosa lineages causing newly detected infections near the time of enrollment of these subjects. Twelve subjects were initially infected with more than one P. aeruginosa lineage, and 20 subjects had different PA lineages detected during the follow-up period of the EPIC-CT. Of the 203 lineages causing initial infections, 144 were not detected again by culture and 59 were detected again despite antibiotic therapy. Multilocus sequence typing was as accurate as whole genome single nucleotide variant (SNV) typing in discriminating lineages, but pulsed-field gel electrophoresis inaccurately classified 17 genetically related isolates as distinct. Although subjects were infected for a relatively short time, 23 P. aeruginosa genes acquired nonsynonymous SNVs in at least 2 subjects. Of these, 14 had been previously identified as pathoadaptive, confirming that such mutations can emerge early in CF infections. CONCLUSIONS:Our study demonstrates the complexity of early P. aeruginosa infections in people with CF and the potential of these complexities to confound interpretation of antibiotic efficacy studies.

BACKGROUND:Ventricular reservoir infections and cerebrospinal fluid (CSF) shunt infections are diagnosed when bacteria are recovered from microbiological cultures of CSF samples from these devices. We applied high throughput sequencing (HTS) to understand the course of changes in ventricular reservoir and shunt infection microbiota. OBJECTIVES:Evaluate the utility of monitoring microbiota in CSF (1) from ventricular reservoirs to detect development of an infection and (2) during treatment of CSF shunt infections to assess treatment response. METHODS:Study populations included (1) neonates with temporizing ventricular reservoirs who developed reservoir infection and (2) children undergoing treatment for conventional culture-confirmed CSF shunt infection. The V4 region of the 16S ribosomal RNA gene was amplified and sequenced. Comparison of taxonomic results of HTS with standard microbiological culture results (when available) was described for each CSF sample. A robust HTS signal was defined by a microbial load of ≥1e5 microbial genome equivalents/mL. RESULTS:In none of the five ventricular reservoir infection cases was there a robust HTS signal for the responsible bacteria immediately prior to infection. In six of the seven CSF shunt infection cases, there was a robust HTS signal for the genus of the responsible bacteria in the sample at the time of positive CSF culture. The proportion of sequences from the genus associated with the responsible bacteria decreased during infection treatment. CONCLUSIONS:These pilot data suggest limited utility in using HTS for surveillance for ventricular reservoir infections, as they emerge abruptly. In CSF shunt infection, HTS demonstrates a return to heterogeneous microbiota when bacterial cultures become negative.

Current approaches for computationally analyzing viruses within human microbiomes often rely on databases largely composed of fragmented viral genomes from gastrointestinal samples, limiting identification of viruses exclusively found outside the gastrointestinal tract and analyses requiring high-quality genomes. To address these issues, we created the Unified Human Virome Database (UHVDB), comprising 575,497 high-quality, annotated viral genomes from human gastrointestinal, airway, skin, and urogenital sample metagenomes. We developed an associated toolkit that uses UHVDB to characterize viruses and their potential activity from metagenomes, then applied this toolkit to 1,983 airway sample metagenomes from people with cystic fibrosis. Over half of detected viruses lacked evidence of potential activity and were detected transiently. UHVDB is nearly three times larger than prior viral databases and its ability to identify likely active viruses enables rigorous analysis of viruses from diverse human sample types, expanding the capacity to define virus contributions to health and disease.

BACKGROUND:People with cystic fibrosis (PwCF) often have fecal dysbioses relative to those without CF, characterized by increased pro-inflammatory microbiota and gastrointestinal (GI) inflammation as measured by fecal calprotectin, suggesting that inflammation contributes to CF GI disease. The multicenter observational PROMISE study (NCT04038047) found that calprotectin decreased in PwCF treated with elexacaftor/tezacaftor/ivacaftor (ETI). To better understand the dynamics between fecal dysbiosis and GI inflammation, we characterized the microbiomes of fecal samples from PROMISE and the relationships with calprotectin before, 1-month post, and 6-months post ETI. METHODS:Fecal microbiota from participants ≥12 y/o were determined by shotgun metagenomic sequencing with random forest modeling and multivariate linear regression analysis to define relationships between microbiota, calprotectin, and deltaF508 genotype before and after ETI. RESULTS:We analyzed 345 samples from 124 participants. At baseline, we observed community-level differences in the fecal microbiota among participants with abnormal compared to normal calprotectin. With ETI, the relative abundances of 7 bacterial species - Escherichia coli, Staphylococcus aureus, Clostridium scindens, Enterocloster clostridioformis, Clostridium butyricum, Anaeroglobus geminatus, and Ruminococcus gnavus - decreased significantly, correlating with calprotectin decrease. We detected community-level differences in the fecal microbiota based on CFTR genotype and a distinct pattern of microbiota change in F508del homozygous compared to heterozygous participants after ETI. CONCLUSIONS:We identified 7 species for which fecal abundances decreased with ETI and correlated with calprotectin decrease, supporting a close relationship between fecal microbiota and inflammation in PwCF. Future work will define these relationships with metabolites and GI symptoms during long-term ETI therapy.

BackgroundCystic fibrosis associated liver disease (CFLD) carries a significant disease burden with no effective preventive therapies. According to the gut-liver axis hypothesis for CFLD pathogenesis, dysbiosis and increased intestinal inflammation and permeability permit pathogenic bacterial translocation into the portal circulation, leading to hepatic inflammation and fibrosis. Evaluating the effect of CFTR (cystic fibrosis transmembrane conductance regulator) modulation with elexacaftor/tezacaftor/ivacaftor (ETI) may help determine the role of CFTR in CFLD and increase understanding of CFLD pathogenesis, which is critical for developing therapies. We aimed to characterize the fecal microbiota in participants with CF with and without advanced CFLD (aCFLD) before and after ETI.MethodsThis is an ancillary analysis of stool samples from participants ages ≥12 y/o enrolled in PROMISE (NCT04038047). Included participants had aCFLD (cirrhosis with or without portal hypertension, or non-cirrhotic portal hypertension) or CF without liver disease (CFnoLD). Fecal microbiota were defined by shotgun metagenomic sequencing at baseline and 1 and 6 months post-ETI.ResultsWe analyzed 93 samples from 34 participants (11 aCFLD and 23 CFnoLD). Compared to CFnoLD, aCFLD had significantly higher baseline relative abundances of potential pathogens Streptococcus salivarius and Veillonella parvula. Four of 11 aCFLD participants had an initially abnormal fecal calprotectin that normalized 6 months post-ETI, correlating with a significant decrease in S. salivarius and a trend towards decreasing V. parvula.ConclusionsThese results support an association between dysbiosis and intestinal inflammation in CFLD with improvements in both post-ETI, lending further support to the gut-liver axis in aCFLD.

Background Gastrointestinal (GI) complications are a significant source of morbidity for people with cystic fibrosis (PwCF). Historically, dietary recommendations in CF have focused on calories, typically emphasizing a high fat diet. The changing landscape of CF highlights the need to update this nutritional strategy. There is little research into how the quality of calories consumed by PwCF influences nutritional outcomes, GI symptoms, or likely contributors: intestinal inflammation and GI microbiology. We assessed the feasibility of a whole foods-based diet (WFD) and avoidance of ultra-processed foods, measuring safety/tolerability, adherence, and GI symptoms, as well as fecal measures of inflammation and microbiota among children with CF (CwCF) with GI symptoms. Methods Single center, 4-week dietary intervention involving CwCF aged 5-14 years who screened positive on GI symptom questionnaire. Assessments included weight, symptom questionnaires and adverse events (AEs). Stool was analyzed for microbiota (16S rRNA) and calprotectin. Results 108 children were pre-screened, 9 enrolled and 8 initiated and completed the study. There were no significant changes in weight and no AEs. PEDS-QL GI identified overall improvement in symptoms. Certain symptom domains (constipation, diarrhea, gas/bloating, stomach pain and hurt) demonstrated significant improvement on the WFD. Of two participants with abnormal fecal calprotectin at enrollment, both exhibited decreased values on WFD. There was no significant change in microbiota diversity. Conclusion A WFD diet was feasible and safe in CwCF. There was improvement in GI symptom scores based on both parent and child assessments. Larger studies are needed to further investigate effects on intestinal inflammation and microbiota.

Shunt infections are a common complication when treating hydrocephalus by cerebrospinal fluid (CSF) shunt placement. The source of infecting pathogens is not well understood. One hypothesis, which we explored here, is that microorganisms persist chronically in the host long before a symptomatic infection occurs and may be detectable in surgical events preceding infection. A cohort of 13 patients was selected, for which CSF samples were available from an infection episode and from a previous surgery event, which was either an initial shunt placement or a revision. Microbiota were analyzed both directly from CSF and from isolates cultured from CSF on aerobic and anaerobic media. The detection and identification of bacteria was done with high throughput DNA sequencing methods and mass spectrometry. The presence of bacteria was confirmed in 4 infection samples, of which 2 were after initial placement and 2 after revision surgery. Taxonomic identification was consistent with clinical microbiology laboratory results. Bacteria were not detected in any of the CSF samples collected at the time of the previous surgical events. While our findings do not provide direct evidence for long-term persistence of pathogens, they suggest the need for consideration of additional source material, such as biofilm and environmental swabs, and/or the use of more sensitive and specific analytical methods.

BACKGROUND:In pediatric Crohn's disease (CD), commercial formulas used as exclusive enteral nutrition (EEN) are effective at inducing remission. This study aims to assess the impact of a whole-food blended smoothie as EEN on CD activity and the intestinal microbiome. METHODS:A 4-week prospective trial assessed the impact of EEN with a whole-food smoothie on newly diagnosed mild-to-moderate active pediatric CD. The smoothie with a multivitamin were developed to meet age-appropriate nutritional requirements. Assessment over 4 weeks included Pediatric Crohn's Disease Activity Index (PCDAI), serum laboratories, fecal calprotectin (FCP), and stool collection for metagenomic shotgun sequencing and microbiota composition analysis. Clinical remission was defined as PCDAI ≤ 10 at week 4. RESULTS:Ten participants were enrolled with median age 14.5 years, and 8 completed the trial. Baseline mean PCDAI was 26.3 ± 9.1 and mean FCP 1149 ± 718 µg/g. At week 4, 80% of participants achieved clinical remission. FCP decreased by over half in 60% of participants, with FCP below 250 µg/g in 60% and below 100 µg/g in 40%. Microbiome analysis showed a significant increase in species richness over 4 weeks (p = 0.01). Compared to baseline, the relative abundance at week 2 and at week 4 was significantly increased for Bifidobacterium and Streptococcus and decreased for Blautia (p < 0.05 for all). CONCLUSION:A whole-food blended smoothie was effective for inducing clinical remission and decreasing FCP in pediatric CD similar to commercial EEN formulas. Further research may give insight into data-driven whole-food dietary approaches for CD management. CLINICALTRIALS:gov NCT03508193.

Advances in both laboratory and computational components of high-throughput 16S amplicon sequencing (16S HTS) have markedly increased its sensitivity and specificity. These refinements have better delineated the limits of sensitivity, and contributions of contamination to these limits, for 16S HTS that are particularly relevant for samples with low bacterial loads such as human cerebrospinal fluid (CSF).

Background: Elexacaftor/tezacaftor/ivacaftor (ETI) improves pulmonary disease in people with cystic fi-brosis (PwCF), but its effect on gastrointestinal symptoms, which also affect quality of life, is not clear.Methods: PROMISE is a 56-center prospective, observational study of ETI in PwCF > 12 years and at least one F508del allele. Gastrointestinal symptoms, evaluated by validated questionnaires: Patient Assessment of Upper Gastrointestinal Disorders-Symptom (PAGI-SYM), Patient Assessment of Constipation-Symptom (PAC-SYM), Patient Assessment of Constipation-Quality of Life (PAC-QOL)), fecal calprotectin, steatocrit and elastase-1 were measured before and 6 months after ETI initiation. Mean difference and 95% confi-dence intervals were obtained from linear regression with adjustment for age and sex.Results: 438 participants fully completed at least 1 questionnaire. Mean (SD) for baseline PAGI-SYM, PAC-SYM, and PAC-QOL total scores were 0.56 (0.59), 0.47 (0.45), and 0.69 (0.53) out of maximum 5, 4, and 5, respectively (higher score indicates greater severity). Corresponding age-and sex-adjusted 6 months

Objective To characterize the microbiota of the cerebrospinal fluid (CSF) from children with hydrocephalus at the time of initial surgical intervention. Study design CSF was obtained at initial surgical intervention. One aliquot was stored in skim milk-tryptone-glucose-glycerol (STGG) medium and the second was unprocessed; both were then stored at –70°C. Bacterial growth for CSF samples stored in STGG were subsequently characterized using aerobic and anaerobic culture on blood agar and MALDI-TOF sequencing. All unprocessed CSF samples underwent 16S quantitative polymerase chain reaction (qPCR) sequencing, and a subset underwent standard clinical microbiological culture. CSF with culture growth (either after storage in STGG or standard clinical) were further analyzed using whole-genome amplification sequencing (WGAS). Results 11/66 (17%) samples stored in STGG and 1/36 (3%) that underwent standard clinical microbiological culture demonstrated bacterial growth. Of the organisms present, 8 were common skin flora and 4 were potential pathogens; only 1 was also qPCR positive. WGAS findings and STGG culture findings were concordant for only 1 sample, identifying Staphylococcus epidermidis. No significant difference in time to second surgical intervention was observed between the STGG culture-positive and negative groups. Conclusion(s) Using high sensitivity methods, we detected the presence of bacteria in a subset of CSF samples at the time of first surgery. Therefore, the true presence of bacteria in CSF of children with hydrocephalus cannot be ruled out, though our findings may suggest these bacteria are contaminants or false positives of the detection methods. Regardless of origin, the detection of microbiota in the CSF of these children may not have any clinical significance.

Understanding the etiology of cerebrospinal fluid (CSF) shunt infections and reinfections requires detailed characterization of associated microorganisms. Traditionally, identification of bacteria present in the CSF has relied on culture methods, but recent studies have used high throughput sequencing of 16S rRNA genes. Here we evaluated the method of shotgun DNA sequencing for its potential to provide additional genomic information. CSF samples were collected from 3 patients near the beginning and end of each of 2 infection episodes. Extracted total DNA was sequenced by: (1) whole genome amplification followed by shotgun sequencing (WGA) and (2) high-throughput sequencing of the 16S rRNA V4 region (16S). Taxonomic assignments of sequences from WGA and 16S were compared with one another and with conventional microbiological cultures. While classification of bacteria was consistent among the 3 approaches, WGA provided additional insights into sample microbiological composition, such as showing relative abundances of microbial versus human DNA, identifying samples of questionable quality, and detecting significant viral load in some samples. One sample yielded sufficient non-human reads to allow assembly of a high-quality Staphylococcus epidermidis genome, denoted CLIMB1, which we characterized in terms of its MLST profile, gene complement (including putative antimicrobial resistance genes), and similarity to other annotated S. epidermidis genomes. Our results demonstrate that WGA directly applied to CSF is a valuable tool for the identification and genomic characterization of dominant microorganisms in CSF shunt infections, which can facilitate molecular approaches for the development of better diagnostic and treatment methods.

Background Infants with cystic fibrosis (CF) suffer from gastrointestinal (GI) complications, including pancreatic insufficiency and intestinal inflammation, which have been associated with impaired nutrition and growth. Recent evidence identified altered fecal microbiota taxonomic compositions in infants with CF relative to healthy infants that were characterized by differences in the abundances of taxa associated with GI health and nutrition. Furthermore, these taxonomic differences were more pronounced in low length infants with CF, suggesting a potential link to linear growth failure. We hypothesized that these differences would entail shifts in the microbiome's functional capacities that could contribute to inflammation and nutritional failure in infants with CF. Results To test this hypothesis, we compared fecal microbial metagenomic content between healthy infants and infants with CF, supplemented with an analysis of fecal metabolomes in infants with CF. We identified notable differences in CF fecal microbial functional capacities, including metabolic and environmental response functions, compared to healthy infants that intensified during the first year of life. A machine learning-based longitudinal metagenomic age analysis of healthy and CF fecal metagenomic functional profiles further demonstrated that these differences are characterized by a CF-associated delay in the development of these functional capacities. Moreover, we found metagenomic differences in functions related to metabolism among infants with CF that were associated with diet and antibiotic exposure, and identified several taxa as potential drivers of these functional differences. An integrated metagenomic and metabolomic analysis further revealed that abundances of several fecal GI metabolites important for nutrient absorption, including three bile acids, correlated with specific microbes in infants with CF. Conclusions Our results highlight several metagenomic and metabolomic factors, including bile acids and other microbial metabolites, that may impact nutrition, growth, and GI health in infants with CF. These factors could serve as promising avenues for novel microbiome-based therapeutics to improve health outcomes in these infants.

Background Nearly 20% of patients with cerebrospinal fluid (CSF) shunt infection develop reinfection. It is unclear whether reinfections are caused by an organism previously present or are independent infection events. Objective We used bacterial culture and high throughput sequencing (HTS) of 16S ribosomal RNA (rRNA) genes to identify bacteria present in serial CSF samples obtained from children who failed CSF shunt infection treatment. We hypothesized that organisms that persist in CSF despite treatment would be detected upon reinfection. Design/methods Serial CSF samples were obtained from 6 patients, 5 with 2 infections and 1 with 3 infections; the study was limited to those for which CSF samples were available from the end of infection and beginning of reinfection. Amplicons of the 16S rRNA gene V4 region were sequenced. Taxonomic assignments of V4 sequences were compared with bacterial species identified in culture. Results Seven infection dyads averaging 13.5 samples per infection were analyzed. A median of 8 taxa [interquartile range (IQR) 5–10] were observed in the first samples from reinfection using HTS. Conventional culture correlated with high abundance of an organism by HTS in all but 1 infection. In 6 of 7 infection dyads, organisms identified by culture at reinfection were detected by HTS of culture-negative samples at the end of the previous infection. The median Chao-Jaccard abundance-based similarity index for matched infection pairs at end of infection and beginning of reinfection was 0.57 (IQR 0.07–0.87) compared to that for unmatched pairs of 0.40 (IQR 0.10–0.60) [p = 0.46]. Conclusion(s) HTS results were generally consistent with culture-based methods in CSF shunt infection and reinfection, and may detect organisms missed by culture at the end of infection treatment but detected by culture at reinfection. However, the CSF microbiota did not correlate more closely within patients at the end of infection and beginning of reinfection than between any two unrelated infections. We cannot reject the hypothesis that sequential infections were independent.

Patients with cystic fibrosis (CF) have altered fecal microbiomes compared to those of healthy controls. The magnitude of this dysbiosis correlates with measures of CF gastrointestinal (GI) disease, including GI inflammation and nutrient malabsorption. However, whether this dysbiosis is caused by mutations in the CFTR gene, the underlying defect in CF, or whether CF-associated dysbiosis augments GI disease was not clear. To test the relationships between CFTR dysfunction, microbes, and intestinal health, we established a germ-free (GF) CF mouse model and demonstrated that CFTR gene mutations are sufficient to alter the GI microbiome. Furthermore, flow cytometric analysis demonstrated that colonized CF mice have increased mesenteric lymph node and spleen TH17+ cells compared with non-CF mice, suggesting that CFTR defects alter adaptive immune responses. Our findings demonstrate that CFTR mutations modulate both the host adaptive immune response and the intestinal microbiome.

Most infants with cystic fibrosis (CF) have pancreatic exocrine insufficiency that results in nutrient malabsorption and requires oral pancreatic enzyme replacement. Newborn screening for CF has enabled earlier diagnosis, nutritional intervention and enzyme replacement for these infants, allowing most infants with CF to achieve their weight goals by 12 months of age(1). Nevertheless, most infants with CF continue to have poor linear growth during their first year of life(1). Although this early linear growth failure is associated with worse long-term respiratory function and survival(2,3), the determinants of body length in infants with CF have not been defined. Several characteristics of the CF gastrointestinal (GI) tract, including inflammation, maldigestion and malabsorption, may promote intestinal dysbiosis(4,5). As GI microbiome activities are known to affect endocrine functions(6,7), the intestinal microbiome of infants with CF may also impact growth. We identified an early, progressive fecal dysbiosis that distinguished infants with CF and low length from infants with CF and normal length. This dysbiosis included altered abundances of taxa that perform functions that are important for GI health, nutrient harvest and growth hormone signaling, including decreased abundance of Bacteroidetes and increased abundance of Proteobacteria. Thus, the GI microbiota represent a potential therapeutic target for the correction of low linear growth in infants with CF. Most infants with cystic fibrosis have poor early linear growth in their first year despite nutritional supplementation and treatment. Intestinal dysbiosis in these infants is associated with low length, suggesting a path for intervention.

Culture and sequencing have produced divergent hypotheses about cystic fibrosis (CF) lung infections. Culturing suggests that CF lungs are uninfected before colonization by a limited group of CF pathogens. Sequencing suggests diverse communities of mostly oral bacteria inhabit lungs early on and diversity decreases as disease progresses. We studied the lung microbiota of CF children using bronchoscopy and sequencing, with measures to reduce contamination. We found no evidence for oral bacterial communities in lung lavages that lacked CF pathogens. Lavage microbial diversity varied widely, but decreases in diversity appeared to be driven by increased CF pathogen abundance, which reduced the signal from contaminants. Streptococcus, Prevotella, and Veillonella DNA was detected in some lavages containing CF pathogens, but DNA from these organisms was vastly exceeded by CF pathogen DNA and was not associated with inflammation. These findings support the hypothesis that established CF pathogens are primarily responsible for CF lung infections.

Metagenomic sequencing is a promising approach for identifying and characterizing organisms and their functional characteristics in complex, polymicrobial infections, such as airway infections in people with cystic fibrosis. These analyses are often hampered, however, by overwhelming quantities of human DNA, yielding only a small proportion of microbial reads for analysis. In addition, many abundant microbes in respiratory samples can produce large quantities of extracellular bacterial DNA originating either from biofilms or dead cells. We describe a method for simultaneously depleting DNA from intact human cells and extracellular DNA (human and bacterial) in sputum, using selective lysis of eukaryotic cells and endonuclease digestion. We show that this method increases microbial sequencing depth and, consequently, both the number of taxa detected and coverage of individual genes such as those involved in antibiotic resistance. This finding underscores the substantial impact of DNA from sources other than live bacteria in microbiological analyses of complex, chronic infection specimens.