Construction and characterization of DNA libraries from cultured phages and environmental viromes.

Despite many efforts to understand and leverage the functional potential of environmental viromes, most bacteriophage genes are largely uncharacterized. To explore novel biology from uncultivated microbes like phages, metagenomics has emerged as a powerful tool to directly mine new genes without the need to culture the diverse microbiota and the viruses within. When a pure computational approach cannot infer gene function, it may be necessary to create a DNA library from environmental genomic DNA, followed by the screening of that library for a particular function. However, these screens are often initiated without a metagenomic analysis of the completed DNA library being reported. Here, we describe the construction and characterization of DNA libraries from a single cultured phage (ΦT4), five cultured Escherichia coli phages, and three metagenomic viral sets built from freshwater, seawater, and wastewater samples. Through next-generation sequencing of five independent samplings of the libraries, we found a consistent number of recovered genes per replicate for each library, with many genes classifiable via the KEGG and Pharokka databases. By characterizing the size of the genes and inserts, we found that our libraries contain a median of one to two genes per contig with a median gene length of 303-381 bp for all libraries, reflective of the small genomes of viruses. The environmental libraries were genetically diverse compared to the single phage and multi-phage libraries. Additionally, we found reduced coverage of individual genomes when five phages were used as opposed to one. Taken together, this work provides a comprehensive analysis of the DNA libraries from phage genomes that can be used for metagenomic exploration and functional screens to infer and identify new biology.IMPORTANCEFunctional metagenomics is an approach that aims to characterize the putative biological function of genes in the microbial world. This includes an examination of the sequencing data collected from a pooled source of diverse microbes and inference of gene function by comparison to annotated and studied genes from public databases. At times, DNA libraries are made from these genes, and the library is screened for a specific function. Hits are validated using a combination of biological, computational, and structural analysis. Left unresolved is a detailed characterization of the library, both its diversity and content, for the purposes of imputing function entirely by computational means, a process that may yield findings that aid in designing useful screens to identify novel gene functions. In this study, we constructed libraries from cultured phages and uncultured viromes from the environment and characterized some important parameters, such as gene number, genes per contig, ratio of hypothetical to known proteins, total genomic coverage and recovery, and the effect of pooling genetic information from multiple sources, to provide a better understanding of the nature of these libraries. This work will aid the design and implementation of future screens of pooled DNA libraries to discover and isolate viral genes with novel biology across various biomes.

Gene discovery from microbial gene libraries I: protection against reactive oxygen species-driven DNA damage.

Reactive oxygen species (ROS) pose a lethal risk for all life forms by causing damage to cell processes, genome-wide DNA damage-driving mutation, replicative instability, and death. Thus, the development of mechanisms to resist or repair ROS-induced DNA damage is critical for the reliable replication of nucleic acids. DNA repair and protection mechanisms have been discovered in all forms of life. However, the vast array of microbes that may harbor novel repair or protection mechanisms, especially bacterial viruses, have not been adequately assessed. Here, we screened a microbial gene library composed primarily of phage open reading frames (ORFs) to uncover elements that overcome a DNA damage blockade. We report the discovery of one such protein, termed F21, which promotes bacterial survival by possibly repairing or protecting DNA in the face of ROS-induced DNA damage.IMPORTANCEDiscovery of proteins that promote DNA damage repair and protection in the face of reactive oxygen species (ROS) is of vital importance. Our group is in possession of a unique microbial DNA library with which we can screen for undiscovered genes that encode novel proteins with DNA damage repair and protective functions. This library is composed of diverse DNA from a variety of sources, namely bacteriophages, which must be assessed for their novel functions. This work focuses on the discovery of DNA damage repair and protection, but the possibilities for discovery are endless, thus highlighting the significance of this work.

RBD amplicon sequencing of wastewater reveals patterns of variant emergence and evolution.

Rapid evolution of SARS-CoV-2 has resulted in the emergence of numerous variants, posing significant challenges to public health surveillance. Clinical genome sequencing, while valuable, has limitations in capturing the full epidemiological dynamics of circulating variants in the general population. This study utilized receptor-binding domain (RBD) amplicon sequencing of wastewater samples to monitor the SARS-CoV-2 community dynamics and evolution in El Paso, TX. Over 17 months, we identified 91 variants and observed waves of dominant variants transitioning from BA.2 to BA.2.12.1, BA.4&5, BQ.1, and XBB.1.5. Our findings demonstrated early detection of variants and identification of unreported outbreaks, while showing strong consistency with clinical genome sequencing data at the local, state, and national levels. Alpha diversity analyses revealed significant periodical variations, with the highest diversity observed in winter and the outbreak lag phases, likely due to lower competition among variants before the outbreak growth phase. The data underscores the importance of low transmission periods for rapid mutation and variant evolution. This study highlights the effectiveness of integrating RBD amplicon sequencing with wastewater surveillance in tracking viral evolution, understanding variant emergence, and enhancing public health preparedness.

Enterococcus faecalis-derived adenine enhances enterohaemorrhagic Escherichia coli Type 3 Secretion System-dependent virulence.

Interactions between microbiota and enteric pathogens can promote colonization resistance or enhance pathogenesis. The pathobiont Enterococcus faecalis increases enterohaemorrhagic E. coli (EHEC) virulence by upregulating Type 3 Secretion System (T3SS) expression, effector translocation, and attaching and effacing (AE) lesion formation on enterocytes, but the mechanisms underlying this remain unknown. Using co-infection of organoids, metabolomics, supplementation experiments and bacterial genetics, here we show that co-culture of EHEC with E. faecalis increases the xanthine-hypoxanthine pathway activity and adenine biosynthesis. Adenine or E. faecalis promoted T3SS gene expression, while transcriptomics showed upregulation of adeP expression, which encodes an adenine importer. Mechanistically, adenine relieved High hemolysin activity (Hha)-dependent repression of T3SS gene expression in EHEC and promoted AE lesion formation in an AdeP-dependent manner. Microbiota-derived purines, such as adenine, support multiple beneficial host responses; however, our data show that this metabolite also increases EHEC virulence, highlighting the complexity of pathogen-microbiota-host interactions in the gut.

Vaccines against extraintestinal pathogenic Escherichia coli (ExPEC): progress and challenges.

The emergence of antimicrobial resistance (AMR) is a principal global health crisis projected to cause 10 million deaths annually worldwide by 2050. While the Gram-negative bacteria Escherichia coli is commonly found as a commensal microbe in the human gut, some strains are dangerously pathogenic, contributing to the highest AMR-associated mortality. Strains of E. coli that can translocate from the gastrointestinal tract to distal sites, called extraintestinal E. coli (ExPEC), are particularly problematic and predominantly afflict women, the elderly, and immunocompromised populations. Despite nearly 40 years of clinical trials, there is still no vaccine against ExPEC. One reason for this is the remarkable diversity in the ExPEC pangenome across pathotypes, clades, and strains, with hundreds of genes associated with pathogenesis including toxins, adhesins, and nutrient acquisition systems. Further, ExPEC is intimately associated with human mucosal surfaces and has evolved creative strategies to avoid the immune system. This review summarizes previous and ongoing preclinical and clinical ExPEC vaccine research efforts to help identify key gaps in knowledge and remaining challenges.

Progress toward a vaccine for extraintestinal pathogenic E. coli (ExPEC) II: efficacy of a toxin-autotransporter dual antigen approach.

Extraintestinal pathogenic Escherichia coli (ExPEC) is a leading cause of worldwide morbidity and mortality, the top cause of antimicrobial-resistant (AMR) infections, and the most frequent cause of life-threatening sepsis and urinary tract infections (UTI) in adults. The development of an effective and universal vaccine is complicated by this pathogen's pan-genome, its ability to mix and match virulence factors and AMR genes via horizontal gene transfer, an inability to decipher commensal from pathogens, and its intimate association and co-evolution with mammals. Using a pan virulome analysis of >20,000 sequenced E. coli strains, we identified the secreted cytolysin α-hemolysin (HlyA) as a high priority target for vaccine exploration studies. We demonstrate that a catalytically inactive pure form of HlyA, expressed in an autologous host using its own secretion system, is highly immunogenic in a murine host, protects against several forms of ExPEC infection (including lethal bacteremia), and significantly lowers bacterial burdens in multiple organ systems. Interestingly, the combination of a previously reported autotransporter (SinH) with HlyA was notably effective, inducing near complete protection against lethal challenge, including commonly used infection strains ST73 (CFT073) and ST95 (UTI89), as well as a mixture of 10 of the most highly virulent sequence types and strains from our clinical collection. Both HlyA and HlyA-SinH combinations also afforded some protection against UTI89 colonization in a murine UTI model. These findings suggest recombinant, inactive hemolysin and/or its combination with SinH warrant investigation in the development of an E. coli vaccine against invasive disease.

Wastewater analysis of Mpox virus in a city with low prevalence of Mpox disease: an environmental surveillance study.

Background: Tracking infectious diseases at the community level is challenging due to asymptomatic infections and the logistical complexities of mass surveillance. Wastewater surveillance has emerged as a valuable tool for monitoring infectious disease agents including SARS-CoV-2 and Mpox virus. However, detecting the Mpox virus in wastewater is particularly challenging due to its relatively low prevalence in the community. In this study, we aim to characterize three molecular assays for detecting and tracking the Mpox virus in wastewater from El Paso, Texas, during February and March 2023. Methods: In this study, a combined approach utilizing three real-time PCR assays targeting the C22L, F3L, and F8L genes and sequencing was employed to detect and track the Mpox virus in wastewater samples. The samples were collected from four sewersheds in the City of El Paso, Texas, during February and March 2023. Wastewater data was compared with reported clinical case data in the city. Findings: Mpox virus DNA was detected in wastewater from all the four sewersheds, whereas only one Mpox case was reported during the sampling period. Positive signals were still observed in multiple sewersheds after the Mpox case was identified. Higher viral concentrations were found in the pellet than in the supernatant of wastewater. Notably, an increasing trend in viral concentration was observed approximately 1-2 weeks before the reporting of the Mpox case. Further sequencing and epidemiological analysis provided supporting evidence for unreported Mpox infections in the city. Interpretation: Our analysis suggests that the Mpox cases in the community is underestimated. The findings emphasize the value of wastewater surveillance as a public health tool for monitoring infectious diseases even in low-prevalence areas, and the need for heightened vigilance to mitigate the spread of Mpox disease for safeguarding global health. Funding: Center of Infectious Diseases at UTHealth, the University of Texas System, and the Texas Epidemic Public Health Institute. The content of this paper is solely the responsibility of the authors and does not necessarily represent the official views of these funding organizations.

Wastewater sequencing reveals community and variant dynamics of the collective human virome.

Wastewater is a discarded human by-product, but its analysis may help us understand the health of populations. Epidemiologists first analyzed wastewater to track outbreaks of poliovirus decades ago, but so-called wastewater-based epidemiology was reinvigorated to monitor SARS-CoV-2 levels while bypassing the difficulties and pit falls of individual testing. Current approaches overlook the activity of most human viruses and preclude a deeper understanding of human virome community dynamics. Here, we conduct a comprehensive sequencing-based analysis of 363 longitudinal wastewater samples from ten distinct sites in two major cities. Critical to detection is the use of a viral probe capture set targeting thousands of viral species or variants. Over 450 distinct pathogenic viruses from 28 viral families are observed, most of which have never been detected in such samples. Sequencing reads of established pathogens and emerging viruses correlate to clinical data sets of SARS-CoV-2, influenza virus, and monkeypox viruses, outlining the public health utility of this approach. Viral communities are tightly organized by space and time. Finally, the most abundant human viruses yield sequence variant information consistent with regional spread and evolution. We reveal the viral landscape of human wastewater and its potential to improve our understanding of outbreaks, transmission, and its effects on overall population health.

Class-Driven Synergy and Antagonism between a Pseudomonas Phage and Antibiotics.

The ubiquitous bacterial pathogen Pseudomonas aeruginosa is responsible for severe infections in patients with burns, cystic fibrosis, and neutropenia. Biofilm formation gives physical refuge and a protected microenvironment for sessile cells, rendering cure by antibiotics a challenge. Bacteriophages have evolved to prey on these biofilms over millions of years, using hydrolases and depolymerases to penetrate biofilms and reach cellular targets. Here, we assessed how a newly discovered KMV-like phage (ΦJB10) interacts with antibiotics to treat P. aeruginosa more effectively in both planktonic and biofilm forms. By testing representatives of four classes of antibiotics (cephalosporins, aminoglycosides, fluoroquinolones, and carbapenems), we demonstrated class-dependent interactions between ΦJB10 and antibiotics in both biofilm clearance and P. aeruginosa killing. Despite identifying antagonism between some antibiotic classes and ΦJB10 at early time points, all classes showed neutral to favorable interactions with the phage at later time points. In one notable example where the antibiotic alone had poor activity against both biofilm and high-density planktonic cells, we found that addition of ΦJB10 demonstrated synergy and resulted in effective treatment of both. Further, ΦJB10 seemed to act as an adjuvant to several antibiotics, reducing the concentration of antibiotics required to ablate the biofilm. This report shows that phages such as ΦJB10 may be valuable additions to the armamentarium against difficult-to-treat biofilm-based infections.

Effect of various types of extracellular DNA on V. hyugaensis biofilm formation.

Marine bacteria face a constant influx of new extracellular DNA (exDNA) due to the massive viral lysis that occurs in the ocean on a daily basis. Generally, biofilms have shown to be induced by self-secreted exDNA. However, the effect of various types of exDNA with varying lengths, self vs non-self, as well as guanine-cytosine content (GC) content on biofilm formation has not been explored, despite being a critical component of the extracellular polymeric substance. To test the effect of such exDNA on biofilms, a marine bioluminescent bacterium (Vibrio hyugaensis) was isolated from the Sippewissett Salt Marsh, USA, and treated with various types of exDNA. We observed rapid pellicle formation with distinct morphologies only in cultures treated with herring sperm gDNA, another Vibrio spp. gDNA, and an oligomer of 61-80% GC content. With pH measurements before and after the treatment, we observed a positive correlation between biofilm formation and the change to a more neutral pH. Our study highlights the importance of studying DNA-biofilm interaction by carefully examining the physical properties of the DNA and by varying its content, length, and source. Our observation may serve as the basis for future studies that seek to interrogate the molecular explanation for the various types of exDNA and their effects on biofilm formation. IMPORTANCE Bacteria mostly exist as biofilm, a protective niche that promotes protection from the environment and nutrient uptake. By forming these structures, bacteria have caused recalcitrant antibiotic-resistant infections, contamination of dairy and seafood, and fouling equipment in the industry. A critical component that makes up the extracellular polymeric substances, the structural component of a biofilm, is the extracellular DNA secreted by the bacteria found in the biofilm. However, previous studies on DNA and biofilm formation have neglected the unique properties of nucleic acid and its high diversity. Our study aims at disentangling these DNA properties by monitoring their effect at inducing biofilm formation. By varying length, self vs non-self, and GC percentage, we used various microscopy techniques to visualize the structural composition of a Vibrio hyugaensis biofilm. We observed DNA-dependent biofilm stimulation in this organism, a novel function of DNA in biofilm biology.

Wastewater pandemic preparedness: Toward an end-to-end pathogen monitoring program.

Molecular analysis of public wastewater has great potential as a harbinger for community health and health threats. Long-used to monitor the presence of enteric viruses, in particular polio, recent successes of wastewater as a reliable lead indicator for trends in SARS-CoV-2 levels and hospital admissions has generated optimism and emerging evidence that similar science can be applied to other pathogens of pandemic potential (PPPs), especially respiratory viruses and their variants of concern (VOC). However, there are substantial challenges associated with implementation of this ideal, namely that multiple and distinct fields of inquiry must be bridged and coordinated. These include engineering, molecular sciences, temporal-geospatial analytics, epidemiology and medical, and governmental and public health messaging, all of which present their own caveats. Here, we outline a framework for an integrated, state-wide, end-to-end human pathogen monitoring program using wastewater to track viral PPPs.

Broad protective vaccination against systemic Escherichia coli with autotransporter antigens.

Extraintestinal pathogenic Escherichia coli (ExPEC) is the leading cause of adult life-threatening sepsis and urinary tract infections (UTI). The emergence and spread of multidrug-resistant (MDR) ExPEC strains result in a considerable amount of treatment failure and hospitalization costs, and contribute to the spread of drug resistance amongst the human microbiome. Thus, an effective vaccine against ExPEC would reduce morbidity and mortality and possibly decrease carriage in healthy or diseased populations. A comparative genomic analysis demonstrated a gene encoding an invasin-like protein, termed sinH, annotated as an autotransporter protein, shows high prevalence in various invasive ExPEC phylogroups, especially those associated with systemic bacteremia and UTI. Here, we evaluated the protective efficacy and immunogenicity of a recombinant SinH-based vaccine consisting of either domain-3 or domains-1,2, and 3 of the putative extracellular region of surface-localized SinH. Immunization of a murine host with SinH-based antigens elicited significant protection against various strains of the pandemic ExPEC sequence type 131 (ST131) as well as multiple sequence types in two distinct models of infection (colonization and bacteremia). SinH immunization also provided significant protection against ExPEC colonization in the bladder in an acute UTI model. Immunized cohorts produced significantly higher levels of vaccine-specific serum IgG and urinary IgG and IgA, findings consistent with mucosal protection. Collectively, these results demonstrate that autotransporter antigens such as SinH may constitute promising ExPEC phylogroup-specific and sequence-type effective vaccine targets that reduce E. coli colonization and virulence.

Corrigendum: Development of phage cocktails to treat E. coli catheter-associated urinary tract infection and associated biofilms.

[This corrects the article DOI: 10.3389/fmicb.2022.796132.].

Phage Resistance Accompanies Reduced Fitness of Uropathogenic Escherichia coli in the Urinary Environment.

Urinary tract infection (UTI) is among the most common infections treated worldwide each year and is caused primarily by uropathogenic Escherichia coli (UPEC). Rising rates of antibiotic resistance among uropathogens have spurred a consideration of alternative treatment strategies, such as bacteriophage (phage) therapy; however, phage-bacterial interactions within the urinary environment are poorly defined. Here, we assess the activity of two phages, namely, HP3 and ES17, against clinical UPEC isolates using in vitro and in vivo models of UTI. In both bacteriologic medium and pooled human urine, we identified phage resistance arising within the first 6 to 8 h of coincubation. Whole-genome sequencing revealed that UPEC strains resistant to HP3 and ES17 harbored mutations in genes involved in lipopolysaccharide (LPS) biosynthesis. Phage-resistant strains displayed several in vitro phenotypes, including alterations to adherence to and invasion of human bladder epithelial HTB-9 cells and increased biofilm formation in some isolates. Interestingly, these phage-resistant UPEC isolates demonstrated reduced growth in pooled human urine, which could be partially rescued by nutrient supplementation and were more sensitive to several outer membrane-targeting antibiotics than parental strains. Additionally, phage-resistant UPEC isolates were attenuated in bladder colonization in a murine UTI model. In total, our findings suggest that while resistance to phages, such as HP3 and ES17, may arise readily in the urinary environment, phage resistance is accompanied by fitness costs which may render UPEC more susceptible to host immunity or antibiotics. IMPORTANCE UTI is one of the most common causes of outpatient antibiotic use, and rising antibiotic resistance threatens the ability to control UTI unless alternative treatments are developed. Bacteriophage (phage) therapy is gaining renewed interest; however, much like with antibiotics, bacteria can readily become resistant to phages. For successful UTI treatment, we must predict how bacteria will evade killing by phage and identify the downstream consequences of phage resistance during bacterial infection. In our current study, we found that while phage-resistant bacteria quickly emerged in vitro, these bacteria were less capable of growing in human urine and colonizing the murine bladder. These results suggest that phage therapy poses a viable UTI treatment if phage resistance confers fitness costs for the uropathogen. These results have implications for developing cocktails of phage with multiple different bacterial targets, of which each is evaded only at the cost of bacterial fitness.

Development of Phage Cocktails to Treat E. coli Catheter-Associated Urinary Tract Infection and Associated Biofilms.

High rates of antimicrobial resistance and formation of biofilms makes treatment of Escherichia coli catheter-associated urinary tract infections (CAUTI) particularly challenging. CAUTI affect 1 million patients per year in the United States and are associated with morbidity and mortality, particularly as an etiology for sepsis. Phage have been proposed as a potential therapeutic option. Here, we report the development of phage cocktails that lyse contemporary E. coli strains isolated from the urine of patients with spinal cord injury (SCI) and display strong biofilm-forming properties. We characterized E. coli phage against biofilms in two in vitro CAUTI models. Biofilm viability was measured by an MTT assay that determines cell metabolic activity and by quantification of colony forming units. Nine phage decreased cell viability by >80% when added individually to biofilms of two E. coli strains in human urine. A phage cocktail comprising six phage lyses 82% of the strains in our E. coli library and is highly effective against young and old biofilms and against biofilms on silicon catheter materials. Using antibiotics together with our phage cocktail prevented or decreased emergence of E. coli resistant to phage in human urine. We created an anti-biofilm phage cocktail with broad host range against E. coli strains isolated from urine. These phage cocktails may have therapeutic potential against CAUTI.

Interactions between Enterohemorrhagic Escherichia coli (EHEC) and Gut Commensals at the Interface of Human Colonoids.

The interactions between the gut microbiota and pathogens are complex and can determine the outcome of an infection. Enterohemorrhagic Escherichia coli (EHEC) is a major human enteric pathogen that colonizes the colon through attaching and effacing (AE) lesions and uses microbiota-derived molecules as cues to control its virulence. Different gut commensals can modulate EHEC virulence. However, the lack of an animal model that recapitulates the human pathophysiology of EHEC infection makes it challenging to investigate how variations in microbiota composition could affect host susceptibility to this pathogen. Here, we addressed these interactions building from simple to more complex in vitro systems, culminating with the use of the physiological relevant human colonoids as a model to study the interactions between EHEC and different gut commensals. We demonstrated that Bacteroides thetaiotaomicron and Enterococcus faecalis enhance virulence expression and AE lesion formation in cultured epithelial cells, as well as on the colonic epithelium, while commensal E. coli did not affect these phenotypes. Importantly, in the presence of these three commensals together, virulence and AE lesion are enhanced. Moreover, we identified specific changes in the metabolic landscape promoted by different members of the gut microbiota and showed that soluble factors released by E. faecalis can increase EHEC virulence gene expression. Our study highlights the importance of interspecies bacterial interactions and chemical exchange in the modulation of EHEC virulence. IMPORTANCE Enterohemorrhagic E. coli (EHEC) is a natural human pathogen that poorly colonizes mice. Hence, the use of murine models to understand features of EHEC infection is a challenge. In this study, we use human colonoids as a physiologically relevant model to study interactions between EHEC and gut commensals. We demonstrate that the ability of EHEC to form AE lesions on the intestinal epithelium is enhanced by the presence of certain gut commensals, such as B. thetaiotaomicron and E. faecalis, while it is not affected by commensal E. coli. Furthermore, we show that commensal bacteria differently impact the metabolic landscape. These data suggest that microbiota compositions can differentially modulate EHEC-mediated disease.

Metabolic profiling reveals nutrient preferences during carbon utilization in Bacillus species.

The genus Bacillus includes species with diverse natural histories, including free-living nonpathogenic heterotrophs such as B. subtilis and host-dependent pathogens such as B. anthracis (the etiological agent of the disease anthrax) and B. cereus, a cause of food poisoning. Although highly similar genotypically, the ecological niches of these three species are mutually exclusive, which raises the untested hypothesis that their metabolism has speciated along a nutritional tract. Here, we developed a pipeline for quantitative total assessment of the use of diverse sources of carbon for general metabolism to better appreciate the "culinary preferences" of three distinct Bacillus species, as well as related Staphylococcus aureus. We show that each species has widely varying metabolic ability to utilize diverse sources of carbon that correlated to their ecological niches. This approach was applied to the growth and survival of B. anthracis in a blood-like environment and find metabolism shifts from sugar to amino acids as the preferred source of energy. Finally, various nutrients in broth and host-like environments are identified that may promote or interfere with bacterial metabolism during infection.

Drivers of transcriptional variance in human intestinal epithelial organoids.

Human intestinal epithelial organoids (enteroids and colonoids) are tissue cultures used for understanding the physiology of the human intestinal epithelium. Here, we explored the effect on the transcriptome of common variations in culture methods, including extracellular matrix substrate, format, tissue segment, differentiation status, and patient heterogeneity. RNA-sequencing datasets from 276 experiments performed on 37 human enteroid and colonoid lines from 29 patients were aggregated from several groups in the Texas Medical Center. DESeq2 and gene set enrichment analysis (GSEA) were used to identify differentially expressed genes and enriched pathways. PERMANOVA, Pearson's correlation, and dendrogram analysis of the data originally indicated three tiers of influence of culture methods on transcriptomic variation: substrate (collagen vs. Matrigel) and format (3-D, transwell, and monolayer) had the largest effect; segment of origin (duodenum, jejunum, ileum, colon) and differentiation status had a moderate effect; and patient heterogeneity and specific experimental manipulations (e.g., pathogen infection) had the smallest effect. GSEA identified hundreds of pathways that varied between culture methods, such as IL1 cytokine signaling enriched in transwell versus monolayer cultures and E2F target genes enriched in collagen versus Matrigel cultures. The transcriptional influence of the format was furthermore validated in a synchronized experiment performed with various format-substrate combinations. Surprisingly, large differences in organoid transcriptome were driven by variations in culture methods such as format, whereas experimental manipulations such as infection had modest effects. These results show that common variations in culture conditions can have large effects on intestinal organoids and should be accounted for when designing experiments and comparing results between laboratories. Our data constitute the largest RNA-seq dataset interrogating human intestinal epithelial organoids.

Development of Antimicrobial Nitric Oxide-Releasing Fibers.

Nitric oxide (NO) is a highly reactive gas molecule, exhibiting antimicrobial properties. Because of its reactive nature, it is challenging to store and deliver NO efficiently as a therapeutic agent. The objective of this study was to develop NO-releasing polymeric fibers (NO-fibers), as an effective delivery platform for NO. NO-fibers were fabricated with biopolymer solutions of polyvinyl pyrrolidone (PVP) and ethylcellulose (EC), and derivatives of N-diazeniumdiolate (NONOate) as NO donor molecules, using an electrospinning system. We evaluated in vitro NO release kinetics, along with antimicrobial effects and cytotoxicity in microorganisms and human cell culture models. We also studied the long-term stability of NONOates in NO-fibers over 12 months. We demonstrated that the NO-fibers could release NO over 24 h, and showed inhibition of the growth of Pseudomonas aeruginosa (P. aeruginosa) and methicillin-resistant Staphylococcus aureus (MRSA), without causing cytotoxicity in human cells. NO-fibers were able to store NONOates for over 12 months at room temperature. This study presents the development of NO-fibers, and the feasibility of NO-fibers to efficiently store and deliver NO, which can be further developed as a bandage.