Inhibiting the Evolution of Antibiotic Resistance.

Efforts to battle antimicrobial resistance (AMR) are generally focused on developing novel antibiotics. However, history shows that resistance arises regardless of the nature or potency of new drugs. Here, we propose and provide evidence for an alternate strategy to resolve this problem: inhibiting evolution. We determined that the DNA translocase Mfd is an "evolvability factor" that promotes mutagenesis and is required for rapid resistance development to all antibiotics tested across highly divergent bacterial species. Importantly, hypermutator alleles that accelerate AMR development did not arise without Mfd, at least during evolution of trimethoprim resistance. We also show that Mfd's role in AMR development depends on its interactions with the RNA polymerase subunit RpoB and the nucleotide excision repair protein UvrA. Our findings suggest that AMR development can be inhibited through inactivation of evolvability factors (potentially with "anti-evolution" drugs)-in particular, Mfd-providing an unexplored route toward battling the AMR crisis.

Clostridium tanneri sp. nov., isolated from the faecal material of an alpaca.

A strictly anaerobic, Gram-stain-negative rod-shaped bacterium, designated A1-XYC3T, was isolated from the faeces of an alpaca (Lama pacos). On the basis of the results of a comparative 16S rRNA gene sequence analysis, the isolate was assigned to the genus Clostridium with the highest sequence similarities to Clostridium magnum DSM 2767T (96.8 %), Clostridium carboxidivorans P7T (96.3 %) and Clostridium aciditolerans JW/YJL-B3T (96.1 %). The average nucleotide identity between A1-XYC3T, C. magnum, C. carboxidivorans and C. aciditolerans was 77.4, 76.1 and 76.6  %, respectively. The predominant components of the cellular fatty acids of A1-XYC3T were C14 : 0, C16 : 0 and summed feature 10, containing C18:0/C17:0 cyclo. The DNA G+C content was 32.4 mol%. On the basis of biochemical, phylogenetic, genotypic and chemotaxonomic criteria, this isolate represents a novel species within Clostridium sensu stricto for which the name Clostridium tanneri sp. nov. is proposed. The type strain of this species is strain A1-XYC3T (=CCM 9376T=NRRL B-65691T).

Therapeutic drug monitoring of osimertinib in EGFR mutant non-small cell lung cancer by dried blood spot and plasma collection: A pilot study.

AIMS: Therapeutic drug monitoring (TDM) has led to significant improvements in individualized medical care, although its implementation in oncology has been limited to date. Tyrosine kinase inhibitors (TKIs) are a group of therapies for which TDM has been suggested. Osimertinib is one such therapy used in the treatment of epidermal growth factor receptor (EGFR) mutation-driven lung cancer. Herein, we describe a prospective pilot study involving 21 patients on osimertinib primarily as a preliminary evaluation of drug levels in a real-world setting. METHODS: Concentrations of the drug and its primary metabolites were measured with a validated liquid chromatography-mass spectrometry (LC-MS) assay across serial timepoints. As part of this study, inter-individual variability by dose and ethnicity as well as intra-individual variability across timepoints are explored. Furthermore, we attempted to validate dried blood spot (DBS)-based quantitation as an accurate alternative to plasma quantitation. RESULTS: Successful quantitation of osimertinib and primary metabolites was achieved for our subjects. Compound plasma levels were highly correlated to DBS levels. There was no significant difference in concentrations with ethnicity or dosing or intra-individual variability across timepoints. CONCLUSIONS: As such, we demonstrate that TDM for osimertinib is practical for future trials. We also validated the use of DBS as an alternative to conventional quantitation for exploration of TDM for osimertinib in larger trials and for other targeted therapies.

Comparing deposition characteristics of various embolic particles using an in vitro prostate microvasculature model.

PURPOSE: To compare spatial distributions of radiopaque glass (RG) microspheres, trisacryl gelatin (TAG) microspheres, and polyvinyl alcohol (PVA) foam particles within a planar in vitro microvascular model of the hyperplastic hemiprostate. MATERIALS AND METHODS: A microvascular model simulating hyperplastic hemiprostate was perfused with a water-glycerin mixture. A microcatheter was positioned distal to the model's prostatic artery origin and embolic particles (RG: 50 µm, 100 µm, and 150 µm; TAG: 100-300 µm and 300-500 µm; and PVA: 90-180 µm and 180-300 µm) were administered using a syringe pump. Microscopic imaging and subsequent semantic segmentation were performed to quantify particle distributions within the models. Distal penetrations were quantified statistically via modal analysis of the particle distributions. RESULTS: Maximum distal penetration was observed for RG 50, followed by RG 100 and then TAG 100-300 and RG 150. TAG 300-500, PVA 90-180, and PVA 180-300 particles exhibited the lowest distal penetrations. The distal penetration metrics between groups were significantly different (p < 0.05) except between TAG 100-300 and RG 150 and between PVA 90-180 and PVA 180-300. CONCLUSIONS: Comparing the spatial distributions of embolic particles in an in vitro microvascular model simulating the hyperplastic hemiprostate revealed that noncompressible particles and those with narrower size calibrations and smaller relative diameters exhibited higher degrees of distal packing. The embolization front was less distinct for particles with wider size calibrations, which resulted in smaller, more distal emboli along with larger, more proximal emboli. PVA and TAG 300-500 particles both exhibited relatively low overall distal penetration.

Analysis of queuosine and 2-thio tRNA modifications by high throughput sequencing.

Queuosine (Q) is a conserved tRNA modification at the wobble anticodon position of tRNAs that read the codons of amino acids Tyr, His, Asn, and Asp. Q-modification in tRNA plays important roles in the regulation of translation efficiency and fidelity. Queuosine tRNA modification is synthesized de novo in bacteria, whereas in mammals the substrate for Q-modification in tRNA is queuine, the catabolic product of the Q-base of gut bacteria. This gut microbiome dependent tRNA modification may play pivotal roles in translational regulation in different cellular contexts, but extensive studies of Q-modification biology are hindered by the lack of high throughput sequencing methods for its detection and quantitation. Here, we describe a periodate-treatment method that enables single base resolution profiling of Q-modification in tRNAs by Nextgen sequencing from biological RNA samples. Periodate oxidizes the Q-base, which results in specific deletion signatures in the RNA-seq data. Unexpectedly, we found that periodate-treatment also enables the detection of several 2-thio-modifications including τm5s2U, mcm5s2U, cmnm5s2U, and s2C by sequencing in human and E. coli tRNA. We term this method periodate-dependent analysis of queuosine and sulfur modification sequencing (PAQS-seq). We assess Q- and 2-thio-modifications at the tRNA isodecoder level, and 2-thio modification changes in stress response. PAQS-seq should be widely applicable in the biological studies of Q- and 2-thio-modifications in mammalian and microbial tRNAs.

Bacteroides vicugnae sp. nov. isolated from the fecal material of an alpaca.

Two strictly anaerobic, Gram-stain-negative rod-shaped bacterial isolates, A2-P53T and A1-P5, were isolated from an enrichment of fecal material from two alpacas (Vicugna pacos). Based on a comparative 16S rRNA gene sequence analysis, the isolates were assigned to the genus Bacteroides with the highest sequence similarities to Bacteroides koreensis YS-aM39T (A2- P53T 97.7 % and A1-P5 97.9 %). Additionally, the average nucleotide identity and digital DNA-DNA hybridization values between these isolates and their closest relatives within Bacteroides were less than 92.1 % and 49.1 %, respectively. The average nucleotide identity between isolates A2-P53T and A1-P5 was 99.9 %. The predominant cellular fatty acid for isolates A2-P53T and A1-P5 was C15:0 antesio. The G+C % content of the isolates was 41.7 %. Based on biochemical, phylogenetic, genotypic, and chemotaxonomic criteria, these isolates A2-P53T and A1-P5 represent two individual strains of a novel species within the genus Bacteroides for which the name Bacteroides vicugnae sp. nov. is proposed. The type strain of this species is strain A2-P53T (CCUG 77273T = CCM 9377T = NRRL B-65693T).

Top/Middle-Down Characterization of α-Synuclein Glycoforms.

α-Synuclein is an intrinsically disordered protein that plays a critical role in the pathogenesis of neurodegenerative disorders, such as Parkinson's disease. Proteomics studies of human brain samples have associated the modification of the O-linked N-acetyl-glucosamine (O-GlcNAc) to several synucleinopathies; in particular, the position of the O-GlcNAc can regulate protein aggregation and subsequent cell toxicity. There is a need for site specific O-GlcNAc α-synuclein screening tools to direct better therapeutic strategies. In the present work, for the first time, the potential of fast, high-resolution trapped ion mobility spectrometry (TIMS) preseparation in tandem with mass spectrometry assisted by an electromagnetostatic (EMS) cell, capable of electron capture dissociation (ECD), and ultraviolet photodissociation (213 nm UVPD) is illustrated for the characterization of α-synuclein positional glycoforms: T72, T75, T81, and S87 modified with a single O-GlcNAc. Top-down 213 nm UVPD and ECD MS/MS experiments of the intact proteoforms showed specific product ions for each α-synuclein glycoforms associated with the O-GlcNAc position with a sequence coverage of ∼68 and ∼82%, respectively. TIMS-MS profiles of α-synuclein and the four glycoforms exhibited large structural heterogeneity and signature patterns across the 8+-15+ charge state distribution; however, while the α-synuclein positional glycoforms showed signature mobility profiles, they were only partially separated in the mobility domain. Moreover, a middle-down approach based on the Val40-Phe94 (55 residues) chymotrypsin proteolytic product using tandem TIMS-q-ECD-TOF MS/MS permitted the separation of the parent positional isomeric glycoforms. The ECD fragmentation of the ion mobility and m/z separated isomeric Val40-Phe94 proteolytic peptides with single O-GlcNAc in the T72, T75, T81, and S87 positions provided the O-GlcNAc confirmation and positional assignment with a sequence coverage of ∼80%. This method enables the high-throughput screening of positional glycoforms and further enhances the structural mass spectrometry toolbox with fast, high-resolution mobility separations and 213 nm UVPD and ECD fragmentation capabilities.

An Evaluation of Sex-Based Differences in Surrogate Consent for Older Adults Undergoing Surgical Intervention.

INTRODUCTION: Differences between female and male patients have been identified in many facets of medicine. We sought to understand whether differences in frequency of surrogate consent for operation exist between older female and male patients. MATERIALS AND METHODS: A descriptive study was designed using data from the hospitals participating in the American College of Surgeons National Surgical Quality Improvement Program. Patients age 65 y and older who underwent operation between 2014 and 2018 were included. RESULTS: Of 51,618 patients identified, 3405 (6.6%) had surrogate consent for surgery. Overall, 7.7% of females had surrogate consent compared to 5.3% of males (P < 0.001). Stratified analysis based on age categories showed no difference in surrogate consent between female and male patients aged 65-74 yy (2.3% versus 2.6%, P = 0.16), but higher rates of surrogate consent in females than males among patients aged 75-84 y old (7.3% versus 5.6%, P < 0.001) and age ≥85 y (29.7% versus 20.8%, P < 0.001). A similar relationship was seen between sex and preoperative cognitive status. There was no difference in preoperative cognitive impairment in female and male patients age 65-74 y (4.4% versus 4.6%, P = 0.58), but higher rates of preoperative cognitive impairment were seen in females than males for those age 75-84 (9.5% versus 7.4%, P < 0.001) and aged ≥85 y (29.4% versus 21.3%, P < 0.001). Matching for age and cognitive impairment, there was no significant difference between rate of surrogate consent in males and females. CONCLUSIONS: Female patients are more likely than males to undergo surgery with surrogate consent. This difference is not based on patient sex alone - females undergoing operation are older than their male counterparts and more likely to be cognitively impaired.

Comparison of Bolus Versus Dual-Syringe Administration Systems on Glass Yttrium-90 Microsphere Deposition in an In Vitro Microvascular Hepatic Tumor Model.

PURPOSE: To utilize an in vitro microvascular hepatic tumor model to compare the deposition characteristics of glass yttrium-90 microspheres using the dual-syringe (DS) and traditional bolus administration methods. MATERIALS AND METHODS: The microvascular tumor model represented a 3.5-cm tumor in a 1,400-cm3 liver with a total hepatic flow of 160 mL/min and was dynamically perfused. A microcatheter was placed in a 2-mm artery feeding the tumor model and 2 additional nontarget arteries. Glass microspheres with a diameter of 20-30 µm were administered using 2 methods: (a) DS delivery at a concentration of 50 mg/mL in either a single, continuous 2-mL infusion or two 1-mL infusions and (b) bolus delivery (BD) of 100 mg of microspheres in a single 3-mL infusion. RESULTS: Overall, the degree of on-target deposition of the microspheres was 85% ± 11%, with no significant differences between the administration methods. Although the distal penetration into the tumor arterioles was approximately 15 mm (from the second microvascular bifurcation of the tumor model) for all the cases, the distal peak particle counts were significantly higher for the DS delivery case (approximately 5 × 105 microspheres achieving distal deposition vs 2 × 105 for the BD case). This resulted in significantly higher deposition uniformity within the tumor model (90% for the DS delivery case vs 80% for the BD case, α = 0.05). CONCLUSIONS: The use of this new in vitro microvascular hepatic tumor model demonstrated that the administration method can affect the deposition of yttrium-90 microspheres within a tumor, with greater distal deposition and more uniform tumor coverage when the microspheres are delivered at consistent concentrations using a DS delivery device. The BD administration method was associated with less favorable deposition characteristics of the microspheres.

Disparities in Rates of Multitarget Stool DNA Test Completion for Colorectal Cancer Screening.

GOALS: The aim was to assess patient adherence to multitarget stool DNA testing as well as factors associated with adherence. BACKGROUND: In the United States, disparities in colorectal cancer screening exist along racial and socioeconomic lines. While some studies suggest that stool-based screening tests may help reduce the screening gap, the data for multitarget stool DNA testing is unclear. STUDY: We conducted a single-center retrospective cohort study on multitarget stool DNA testing ordered between April 2020 and July 2021. We calculated the proportion of patients who completed testing and used multivariate logistic regression to identify covariates associated with test adherence. RESULTS: Among 797 patients ordered for multitarget stool DNA testing, 481 patients (60.4%) completed testing. Adherence rates by patient subgroups ranged from 35.8% to 78.1%. Higher test adherence was found in Asian patients (odds ratio 2.65, 95% CI 1.36-5.18) and those who previously completed colorectal cancer screening (OR 1.45, 95% CI 1.01-2.09), while Black patients (OR 0.58, 95% CI 0.39-0.87), patients with resident primary care physicians (OR 0.34, 95% CI 0.21-0.56), and patients contacted through an outreach program (OR 0.47, 95% CI 0.25-0.87) had lower adherence. CONCLUSIONS: A significant proportion of patients ordered for multitarget stool DNA testing did not complete testing. Differences in adherence rates among patient subgroups may be reflective of underlying disparities in health care access.

Using high-frequency monitoring data to quantify city-wide suspended-sediment load and evaluate TMDL goals.

Excess sediment is a common reason water bodies in the USA become listed as impaired resulting in total maximum daily loads (TMDL) that require municipalities to invest millions of dollars annually on management practices aimed at reducing suspended-sediment loads (SSLs), yet monitoring data are rarely used to quantify SSLs and track TMDL progress. A monitoring network was created to quantify the SSL from the City of Roanoke, Virginia, USA (CoR), to the Roanoke River and Tinker Creek and help guide TMDL assessment and implementation. Suspended-sediment concentrations were estimated between 2020 and 2022 from high-frequency turbidity data using surrogate linear-regression models. Sixty-one percent of the total three-year SSL resulted from five large storm events. The average suspended-sediment yield from the CoR (58.1 metric tons/km2/year) was similar to other urban watersheds in the Eastern United States; however, the yield was nearly five times larger than the TMDL allocation (12.2 metric tons/km2/year). The TMDL allocated load was modeled based on a predominantly forested reference watershed and may not be a practical target for highly impervious watersheds within the CoR. The TMDL model used daily input data which likely does not capture the full range of SSLs during storm events, particularly from flashy urban streams. The average SSL following the five large storm events doubled that of the CoR's annual allocated load from the TMDL. The results of this study highlight the importance of using high-frequency monitoring data to accurately estimate SSLs and evaluate TMDLs in urban areas.

Top-"Double-Down" Mass Spectrometry of Histone H4 Proteoforms: Tandem Ultraviolet-Photon and Mobility/Mass-Selected Electron Capture Dissociations.

Post-translational modifications (PTMs) on intact histones play a major role in regulating chromatin dynamics and influence biological processes such as DNA transcription, replication, and repair. The nature and position of each histone PTM is crucial to decipher how this information is translated into biological response. In the present work, the potential of a novel tandem top-"double-down" approach─ultraviolet photodissociation followed by mobility and mass-selected electron capture dissociation and mass spectrometry (UVPD-TIMS-q-ECD-ToF MS/MS)─is illustrated for the characterization of HeLa derived intact histone H4 proteoforms. The comparison between q-ECD-ToF MS/MS spectra and traditional Fourier-transform-ion cyclotron resonance-ECD MS/MS spectra of a H4 standard showed a similar sequence coverage (∼75%) with significant faster data acquisition in the ToF MS/MS platform (∼3 vs ∼15 min). Multiple mass shifts (e.g., 14 and 42 Da) were observed for the HeLa derived H4 proteoforms for which the top-down UVPD and ECD fragmentation analysis were consistent in detecting the presence of acetylated PTMs at the N-terminus and Lys5, Lys8, Lys12, and Lys16 residues, as well as methylated, dimethylated, and trimethylated PTMs at the Lys20 residue with a high sequence coverage (∼90%). The presented top-down results are in good agreement with bottom-up TIMS ToF MS/MS experiments and allowed for additional description of PTMs at the N-terminus. The integration of a 213 nm UV laser in the present platform allowed for UVPD events prior to the ion mobility-mass precursor separation for collision-induced dissociation (CID)/ECD-ToF MS. Selected c305+ UVPD fragments, from different H4 proteoforms (e.g., Ac + Me2, 2Ac + Me2 and 3Ac + Me2), exhibited multiple IMS bands for which similar CID/ECD fragmentation patterns per IMS band pointed toward the presence of conformers, adopting the same PTM distribution, with a clear assignment of the PTM localization for each of the c305+ UVPD fragment H4 proteoforms. These results were consistent with the biological "zip" model, where acetylation proceeds in the Lys16 to Lys5 direction. This novel platform further enhances the structural toolbox with alternative fragmentation mechanisms (UVPD, CID, and ECD) in tandem with fast, high-resolution mobility separations and shows great promise for global proteoform analysis.

CFTR dysregulation drives active selection of the gut microbiome.

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.

Structure and Antimicrobial Activity of Rare Lactone Lipids from the Sooty Mold (Scorias spongiosa).

Two new lactone lipids, scoriosin (1) and its methyl ester (2), with a rare furylidene ring joined to a tetrahydrofurandione ring, were isolated from Scorias spongiosa, commonly referred to as sooty mold. The planar structure of these compounds was assigned by 1D and 2D NMR. The conformational analysis of these molecules was undertaken to evaluate the relative and absolute configuration through GIAO NMR chemical shift analysis and ECD calculation. In addition to the potent antimicrobial activities, compound 2 strongly potentiated the activity of amphotericin B against Cryptococcus neoformans, suggesting the potential utility of this compound in combination therapies for treating cryptococcal infections.

Cyclic-di-GMP regulation promotes survival of a slow-replicating subpopulation of intracellular Salmonella Typhimurium.

Salmonella Typhimurium can invade and survive within macrophages where the bacterium encounters a range of host environmental conditions. Like many bacteria, S. Typhimurium rapidly responds to changing environments by the use of second messengers such as cyclic di-GMP (c-di-GMP). Here, we generate a fluorescent biosensor to measure c-di-GMP concentrations in thousands of individual bacteria during macrophage infection and to define the sensor enzymes important to c-di-GMP regulation. Three sensor phosphodiesterases were identified as critical to maintaining low c-di-GMP concentrations generated after initial phagocytosis by macrophages. Maintenance of low c-di-GMP concentrations by these phosphodiesterases was required to promote survival within macrophages and virulence for mice. Attenuation of S Typhimurium virulence was due to overproduction of c-di-GMP-regulated cellulose, as deletion of the cellulose synthase machinery restored virulence to a strain lacking enzymatic activity of the three phosphodiesterases. We further identified that the cellulose-mediated reduction in survival was constrained to a slow-replicating persister population of S. Typhimurium induced within the macrophage intracellular environment. As utilization of glucose has been shown to be required for S. Typhimurium macrophage survival, one possible hypothesis is that this persister population requires the glucose redirected to the synthesis of cellulose to maintain a slow-replicating, metabolically active state.

Infants with cystic fibrosis have altered fecal functional capacities with potential clinical and metabolic consequences.

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.

The gram-negative bacterial periplasm: Size matters.

Gram-negative bacteria are surrounded by two membrane bilayers separated by a space termed the periplasm. The periplasm is a multipurpose compartment separate from the cytoplasm whose distinct reducing environment allows more efficient and diverse mechanisms of protein oxidation, folding, and quality control. The periplasm also contains structural elements and important environmental sensing modules, and it allows complex nanomachines to span the cell envelope. Recent work indicates that the size or intermembrane distance of the periplasm is controlled by periplasmic lipoproteins that anchor the outer membrane to the periplasmic peptidoglycan polymer. This periplasm intermembrane distance is critical for sensing outer membrane damage and dictates length of the flagellar periplasmic rotor, which controls motility. These exciting results resolve longstanding debates about whether the periplasmic distance has a biological function and raise the possibility that the mechanisms for maintenance of periplasmic size could be exploited for antibiotic development.

Gastrointestinal Factors Associated With Hospitalization in Infants With Cystic Fibrosis: Results From the Baby Observational and Nutrition Study.

OBJECTIVES: To identify factors that increase the risk of gastrointestinal-related (GI-related) hospitalization of infants with cystic fibrosis (CF) during the first year of life. METHODS: The Baby Observational and Nutrition Study was a longitudinal, observational cohort of 231 infants diagnosed with CF by newborn screening. We performed a post-hoc assessment of the frequency and indications for GI-related admissions during the first year of life. RESULTS: Sixty-five participants had at least one admission in the first 12 months of life. High pancreatic enzyme replacement therapy (PERT) dosing (>2000 lipase units/kg per meal; hazard ratio [HR] = 14.75, P = 0.0005) and use of acid suppressive medications (HR = 4.94, P = 0.01) during the study period were positively associated with subsequent GI-related admissions. High levels of fecal calprotectin (fCP) (>200 µg/g) and higher relative abundance of fecal Klebsiella pneumoniae were also positively associated with subsequent GI-related admissions (HR = 2.64, P = 0.033 and HR = 4.49, P = 0.002, respectively). During the first 12 months of life, participants with any admission had lower weight-for-length z scores (WLZ) (P = 0.01). The impact of admission on WLZ was particularly evident in participants with a GI-related admission (P < 0.0001). CONCLUSIONS: Factors associated with a higher risk for GI-related admission during the first 12 months include high PERT dosing, exposure to acid suppressive medications, higher fCP levels, and/or relative abundance of fecal K pneumoniae early in life. Infants with CF requiring GI-related hospitalization had lower WLZ at 12 months of age than those not admitted as well as those admitted for non-GI-related indications.

Escherichia coli cultures maintain stable subpopulation structure during long-term evolution.

How genetic variation is generated and maintained remains a central question in evolutionary biology. When presented with a complex environment, microbes can take advantage of genetic variation to exploit new niches. Here we present a massively parallel experiment where WT and repair-deficient (∆mutL) Escherichia coli populations have evolved over 3 y in a spatially heterogeneous and nutritionally complex environment. Metagenomic sequencing revealed that these initially isogenic populations evolved and maintained stable subpopulation structure in just 10 mL of medium for up to 10,000 generations, consisting of up to five major haplotypes with many minor haplotypes. We characterized the genomic, transcriptomic, exometabolomic, and phenotypic differences between clonal isolates, revealing subpopulation structure driven primarily by spatial segregation followed by differential utilization of nutrients. In addition to genes regulating the import and catabolism of nutrients, major polymorphisms of note included insertion elements transposing into fimE (regulator of the type I fimbriae) and upstream of hns (global regulator of environmental-change and stress-response genes), both known to regulate biofilm formation. Interestingly, these genes have also been identified as critical to colonization in uropathogenic E. coli infections. Our findings illustrate the complexity that can arise and persist even in small cultures, raising the possibility that infections may often be promoted by an evolving and complex pathogen population.

Adaptation of commensal proliferating Escherichia coli to the intestinal tract of young children with cystic fibrosis.

The mature human gut microbiota is established during the first years of life, and altered intestinal microbiomes have been associated with several human health disorders. Escherichia coli usually represents less than 1% of the human intestinal microbiome, whereas in cystic fibrosis (CF), greater than 50% relative abundance is common and correlates with intestinal inflammation and fecal fat malabsorption. Despite the proliferation of E. coli and other Proteobacteria in conditions involving chronic gastrointestinal tract inflammation, little is known about adaptation of specific characteristics associated with microbiota clonal expansion. We show that E. coli isolated from fecal samples of young children with CF has adapted to growth on glycerol, a major component of fecal fat. E. coli isolates from different CF patients demonstrate an increased growth rate in the presence of glycerol compared with E. coli from healthy controls, and unrelated CF E. coli strains have independently acquired this growth trait. Furthermore, CF and control E. coli isolates have differential gene expression when grown in minimal media with glycerol as the sole carbon source. While CF isolates display a growth-promoting transcriptional profile, control isolates engage stress and stationary-phase programs, which likely results in slower growth rates. Our results indicate that there is selection of unique characteristics within the microbiome of individuals with CF, which could contribute to individual disease outcomes.