Characterization of Bacterial and Fungal Microbiome in Children with Hirschsprung Disease with and without a History of Enterocolitis: A Multicenter Study

Development of potentially life-threatening enterocolitis is the most frequent complication in children with Hirschsprung disease (HSCR), even after definitive corrective surgery. Intestinal microbiota likely contribute to the etiology of enterocolitis, so the aim of this study was to compare the fecal bacterial and fungal communities of children who developed Hirschsprung-associated enterocolitis (HAEC) with HSCR patients who had never had enterocolitis. Eighteen Hirschsprung patients who had completed definitive surgery were enrolled: 9 had a history of HAEC and 9 did not. Fecal DNA was isolated and 16S and ITS-1 regions sequenced using Next Generation Sequencing and data analysis for species identification. The HAEC group bacterial composition showed a modest reduction in Firmicutes and Verrucomicrobia with increased Bacteroidetes and Proteobacteria compared with the HSCR group. In contrast, the fecal fungi composition of the HAEC group showed marked reduction in diversity with increased Candida sp., and reduced Malassezia and Saccharomyces sp. compared with the HSCR group. The most striking finding within the HAEC group is that the Candida genus segregated into “high burden” patients with 97.8% C. albicans and 2.2% C. tropicalis compared with “low burden” patients 26.8% C. albicans and 73% C. tropicalis. Interestingly even the low burden HAEC group had altered Candida community structure with just two species compared to more diverse Candida populations in the HSCR patients. This is the first study to identify Candida sp. as potentially playing a role in HAEC either as expanded commensal species as a consequence of enterocolitis (or treatment), or possibly as pathobioants contributing to the pathogenesis of HAEC. These findings suggest a dysbiosis in the gut microbial ecosystem of HAEC patients, such that there may be dominance of fungi and bacteria predisposing patients to development of HAEC.     

Cytometric fingerprints of gut microbiota predict Crohn’s disease state

Variations in the gut microbiome have been associated with changes in health state such as Crohn’s disease (CD). Most surveys characterize the microbiome through analysis of the 16S rRNA gene. An alternative technology that can be used is flow cytometry. In this report, we reanalyzed a disease cohort that has been characterized by both technologies. Changes in microbial community structure are reflected in both types of data. We demonstrate that cytometric fingerprints can be used as a diagnostic tool in order to classify samples according to CD state. These results highlight the potential of flow cytometry to perform rapid diagnostics of microbiome-associated diseases.Subject terms: Cellular microbiology, Crohn''s disease, Infectious-disease diagnostics     

Vertical vs. horizontal transmission of the microbiome in a key disease vector,Ixodes pacificus

Vector‐borne pathogens are increasingly found to interact with the vector's microbiome, influencing disease transmission dynamics. However, the processes that regulate the formation and development of the microbiome are largely unexplored for most tick species, an emerging group of disease vectors. It is not known how much of the tick microbiome is acquired through vertical transmission vs. horizontally from the environment or interactions with bloodmeal sources. Using 16S rRNA sequencing, we examined the microbiome of Ixodes pacificus, the vector of Lyme disease in the western USA, across life stages and infection status. We also characterized microbiome diversity in field and laboratory‐collected nymphal ticks to determine how the surrounding environment affects microbiome diversity. We found a decrease in both species richness and evenness as the tick matures from larva to adult. When the dominant Rickettsial endosymbiont was computationally removed from the tick microbial community, we found that infected nymphs had lower species evenness than uninfected ticks, suggesting that lower microbiome diversity is associated with pathogen transmission in wild‐type ticks. Furthermore, laboratory‐reared nymph microbiome diversity was found to be compositionally distinct and significantly depauperate relative to field‐collected nymphs. These results highlight unique patterns in the microbial community of I. pacificus that is distinct from other tick species. We provide strong evidence that ticks acquire a significant portion of their microbiome through exposure to their environment despite a loss of overall diversity through life stages. We provide evidence that loss of microbial diversity is at least in part due to elimination of microbial diversity with bloodmeal feeding but other factors may also play a role.     

Gut Microbiome Alterations in COVID-19

Since the outset of the coronavirus disease 2019 (COVID-19) pandemic, the gut microbiome in COVID-19 has garnered substantial interest, given its significant roles in human health and pathophysiology. Accumulating evidence is unveiling that the gut microbiome is broadly altered in COVID-19, including the bacterial microbiome, mycobiome, and virome. Overall, the gut microbial ecological network is significantly weakened and becomes sparse in patients with COVID-19, together with a decrease in gut microbiome diversity. Beyond the existence of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), the gut microbiome of patients with COVID-19 is also characterized by enrichment of opportunistic bacteria, fungi, and eukaryotic viruses, which are also associated with disease severity and presentation. Meanwhile, a multitude of symbiotic bacteria and bacteriophages are decreased in abundance in patients with COVID-19. Such gut microbiome features persist in a significant subset of patients with COVID-19 even after disease resolution, coinciding with ‘long COVID’ (also known as post-acute sequelae of COVID-19). The broadly-altered gut microbiome is largely a consequence of SARS-CoV-2 infection and its downstream detrimental effects on the systemic host immunity and the gut milieu. The impaired host immunity and distorted gut microbial ecology, particularly loss of low-abundance beneficial bacteria and blooms of opportunistic fungi including Candida, may hinder the reassembly of the gut microbiome post COVID-19. Future investigation is necessary to fully understand the role of the gut microbiome in host immunity against SARS-CoV-2 infection, as well as the long-term effect of COVID-19 on the gut microbiome in relation to the host health after the pandemic.     

A Compositional Look at the Human Gastrointestinal Microbiome and Immune Activation Parameters in HIV Infected Subjects

HIV progression is characterized by immune activation and microbial translocation. One factor that may be contributing to HIV progression could be a dysbiotic microbiome. We therefore hypothesized that the GI mucosal microbiome is altered in HIV patients and this alteration correlates with immune activation in HIV. 121 specimens were collected from 21 HIV positive and 22 control human subjects during colonoscopy. The composition of the lower gastrointestinal tract mucosal and luminal bacterial microbiome was characterized using 16S rDNA pyrosequencing and was correlated to clinical parameters as well as immune activation and circulating bacterial products in HIV patients on ART. The composition of the HIV microbiome was significantly different than that of controls; it was less diverse in the right colon and terminal ileum, and was characterized by loss of bacterial taxa that are typically considered commensals. In HIV samples, there was a gain of some pathogenic bacterial taxa. This is the first report characterizing the terminal ileal and colonic mucosal microbiome in HIV patients with next generation sequencing. Limitations include use of HIV-infected subjects on HAART therapy.     

Community Structure and Function of Amphibian Skin Microbes: An Experiment with Bullfrogs Exposed to a Chytrid Fungus

The vertebrate microbiome contributes to disease resistance, but few experiments have examined the link between microbiome community structure and disease resistance functions. Chytridiomycosis, a major cause of amphibian population declines, is a skin disease caused by the fungus, Batrachochytrium dendrobatidis (Bd). In a factorial experiment, bullfrog skin microbiota was reduced with antibiotics, augmented with an anti-Bd bacterial isolate (Janthinobacterium lividum), or unmanipulated, and individuals were then either exposed or not exposed to Bd. We found that the microbial community structure of individual frogs prior to Bd exposure influenced Bd infection intensity one week following exposure, which, in turn, was negatively correlated with proportional growth during the experiment. Microbial community structure and function differed among unmanipulated, antibiotic-treated, and augmented frogs only when frogs were exposed to Bd. Bd is a selective force on microbial community structure and function, and beneficial states of microbial community structure may serve to limit the impacts of infection.     

Analysis of the Airway Microbiota of Healthy Individuals and Patients with Chronic Obstructive Pulmonary Disease by T-RFLP and Clone Sequencing

Chronic obstructive pulmonary disease (COPD) is a progressive, inflammatory lung disease that affects a large number of patients and has significant impact. One hallmark of the disease is the presence of bacteria in the lower airways. Objective: The aim of this study was to analyze the detailed structure of microbial communities found in the lungs of healthy individuals and patients with COPD. Nine COPD patients as compared and 9 healthy individuals underwent flexible bronchoscopy and BAL was performed. Bacterial nucleic acids were subjected to terminal restriction fragment (TRF) length polymorphism and clone library analysis. Overall, we identified 326 T-RFLP band, 159 in patients and 167 in healthy controls. The results of the TRF analysis correlated partly with the data obtained from clone sequencing. Although the results of the sequencing showed high diversity, the genera Prevotella, Sphingomonas, Pseudomonas, Acinetobacter, Fusobacterium, Megasphaera, Veillonella, Staphylococcus, and Streptococcus constituted the major part of the core microbiome found in both groups. A TRF band possibly representing Pseudomonas sp. monoinfection was associated with a reduction of the microbial diversity. Non-cultural methods reveal the complexity of the pulmonary microbiome in healthy individuals and in patients with COPD. Alterations of the microbiome in pulmonary diseases are correlated with disease.     

Comparison of Microbiomes from Different Niches of Upper and Lower Airways in Children and Adolescents with Cystic Fibrosis

Changes in the airway microbiome may be important in the pathophysiology of chronic lung disease in patients with cystic fibrosis. However, little is known about the microbiome in early cystic fibrosis lung disease and the relationship between the microbiomes from different niches in the upper and lower airways. Therefore, in this cross-sectional study, we examined the relationship between the microbiome in the upper (nose and throat) and lower (sputum) airways from children with cystic fibrosis using next generation sequencing. Our results demonstrate a significant difference in both α and β-diversity between the nose and the two other sampling sites. The nasal microbiome was characterized by a polymicrobial community while the throat and sputum communities were less diverse and dominated by a few operational taxonomic units. Moreover, sputum and throat microbiomes were closely related especially in patients with clinically stable lung disease. There was a high inter-individual variability in sputum samples primarily due to a decrease in evenness linked to increased abundance of potential respiratory pathogens such as Pseudomonas aeruginosa. Patients with chronic Pseudomonas aeruginosa infection exhibited a less diverse sputum microbiome. A high concordance was found between pediatric and adult sputum microbiomes except that Burkholderia was only observed in the adult cohort. These results indicate that an adult-like lower airways microbiome is established early in life and that throat swabs may be a good surrogate in clinically stable children with cystic fibrosis without chronic Pseudomonas aeruginosa infection in whom sputum sampling is often not feasible.     

Successional dynamics of the cultivated kelp microbiome

Kelp are important primary producers that are colonized by diverse microbes that can have both positive and negative effects on their hosts. The kelp microbiome could support the burgeoning kelp cultivation sector by improving host growth, stress tolerance, and resistance to disease. Fundamental questions about the cultivated kelp microbiome still need to be addressed before microbiome-based approaches can be developed. A critical knowledge gap is how cultivated kelp microbiomes change as hosts grow, particularly following outplanting to sites that vary in abiotic conditions and microbial source pools. In this study we assessed if microbes that colonize kelp in the nursery stage persist after outplanting. We characterized microbiome succession over time on two species of kelp, Alaria marginata and Saccharina latissima, outplanted to open ocean cultivation sites in multiple geographic locations. We tested for host-species specificity of the microbiome and the effect of different abiotic conditions and microbial source pools on kelp microbiome stability during the cultivation process. We found the microbiome of kelp in the nursery is distinct from that of outplanted kelp. Few bacteria persisted on kelp following outplanting. Instead, we identified significant microbiome differences correlated with host species and microbial source pools at each cultivation site. Microbiome variation related to sampling month also indicates that seasonality in host and/or abiotic factors may influence temporal succession and microbiome turnover in cultivated kelps. This study provides a baseline understanding of microbiome dynamics during kelp cultivation and highlights research needs for applying microbiome manipulation to kelp cultivation.     

Are you what you eat? A highly transient and prey‐influenced gut microbiome in the grey house spider Badumna longinqua

Stable core microbial communities have been described in numerous animal species and are commonly associated with fitness benefits for their hosts. Recent research, however, highlights examples of species whose microbiota are transient and environmentally derived. Here, we test the effect of diet on gut microbial community assembly in the spider Badumna longinqua. Using 16S rRNA gene amplicon sequencing combined with quantitative PCR, we analyzed diversity and abundance of the spider's gut microbes, and simultaneously characterized its prey communities using nuclear rRNA markers. We found a clear correlation between community similarity of the spider's insect prey and gut microbial DNA, suggesting that microbiome assembly is primarily diet‐driven. This assumption is supported by a feeding experiment, in which two types of prey—crickets and fruit flies—both substantially altered microbial diversity and community similarity between spiders, but did so in different ways. After cricket consumption, numerous cricket‐derived microbes appeared in the spider's gut, resulting in a rapid homogenization of microbial communities among spiders. In contrast, few prey‐associated bacteria were detected after consumption of fruit flies; instead, the microbial community was remodelled by environmentally sourced microbes, or abundance shifts of rare taxa in the spider's gut. The reshaping of the microbiota by both prey taxa mimicked a stable core microbiome in the spiders for several weeks post feeding. Our results suggest that the spider's gut microbiome undergoes pronounced temporal fluctuations, that its assembly is dictated by the consumed prey, and that different prey taxa may remodel the microbiota in drastically different ways.     

Impacts of abiotic factors on the fungal communities of ‘Honeycrisp’ apples in Canada

The maintenance of the beneficial plant microbiome to control plant pathogens is an emerging concept of disease management, and necessitates a clear understanding of these microbial communities and the environmental factors that affect their diversity and compositional structure. As such, studies investigating the microbiome of economically significant cultivars within each growing region are necessary to develop adequate disease management strategies. Here, we assessed the relative impacts of growing season, management strategy, and geographical location on the fungal microbiome of ‘Honeycrisp’ apples from seven different orchard locations in the Atlantic Maritime Ecozone for two consecutive growing years. Though apple fruit tissue was dominated by relatively few fungal genera, significant changes in their fungal communities were observed as a result of environmental factors, including shifts in genera with plant-associated lifestyles (symbionts and pathogens), such as Aureobasidium, Alternaria, Penicillium, Diplodia, and Mycosphaerella. Variation in fungal composition between different tissues of fruit was also observed. We demonstrate that growing season is the most significant factor affecting fungal community structure and diversity of apple fruit, suggesting that future microbiome studies should take place for multiple growing seasons to better represent the host–microbiome of perennial crops under different environmental conditions.     

Conditional Regression Based on a Multivariate Zero-Inflated Logistic-Normal Model for Microbiome Relative Abundance Data

The human microbiome plays critical roles in human health and has been linked to many diseases. While advanced sequencing technologies can characterize the composition of the microbiome in unprecedented detail, it remains challenging to disentangle the complex interplay between human microbiome and disease risk factors due to the complicated nature of microbiome data. Excessive numbers of zero values, high dimensionality, the hierarchical phylogenetic tree and compositional structure are compounded and consequently make existing methods inadequate to appropriately address these issues. We propose a multivariate two-part zero-inflated logistic-normal model to analyze the association of disease risk factors with individual microbial taxa and overall microbial community composition. This approach can naturally handle excessive numbers of zeros and the compositional data structure with the discrete part and the logistic-normal part of the model. For parameter estimation, an estimating equations approach is employed that enables us to address the complex inter-taxa correlation structure induced by the hierarchical phylogenetic tree structure and the compositional data structure. This model is able to incorporate standard regularization approaches to deal with high dimensionality. Simulation shows that our model outperforms existing methods. Our approach is also compared to others using the analysis of real microbiome data.     

Molecular tools for probing the microbiome

The microbiome plays essential roles in health and disease. Our understanding of the imbalances that can arise in the microbiome and their consequences is held back by a lack of technologies that selectively knock out members of these microbial communities. Antibiotics and fecal transplants, the existing methods for manipulating the microbiota of the gastrointestinal tract, are not sufficiently pinpointed to reveal how particular microbial genes, strains, or species affect human health. A toolset for the precise manipulation of the microbiome could significantly advance disease diagnosis and treatment. Here, we provide an overview of current and future strategies for the development of molecular tools that can be used to probe the microbiome without producing off-target effects.     

The gut microbiome reflects ancestry despite dietary shifts across a hybrid zone

The microbiome is critical to an organism's phenotype, and its composition is shaped by, and a driver of, eco-evolutionary interactions. We investigated how host ancestry, habitat and diet shape gut microbial composition in a mammalian hybrid zone between Neotoma lepida and N. bryanti that occurs across an ecotone between distinct vegetation communities. We found that habitat is the primary determinant of diet, while host genotype is the primary determinant of the gut microbiome—a finding further supported by intermediate microbiome composition in first-generation hybrids. Despite these distinct primary drivers, microbial richness was correlated with diet richness, and individuals that maintained higher dietary richness had greater gut microbial community stability. Both relationships were stronger in the relative dietary generalist of the two parental species. Our findings show that host ancestry interacts with dietary habits to shape the microbiome, ultimately resulting in the phenotypic plasticity that host–microbial interactions allow.     

The Dental Plaque Microbiome in Health and Disease

Dental decay is one of the most prevalent chronic diseases worldwide. A variety of factors, including microbial, genetic, immunological, behavioral and environmental, interact to contribute to dental caries onset and development. Previous studies focused on the microbial basis for dental caries have identified species associated with both dental health and disease. The purpose of the current study was to improve our knowledge of the microbial species involved in dental caries and health by performing a comprehensive 16S rDNA profiling of the dental plaque microbiome of both caries-free and caries-active subjects. Analysis of over 50,000 nearly full-length 16S rDNA clones allowed the identification of 1,372 operational taxonomic units (OTUs) in the dental plaque microbiome. Approximately half of the OTUs were common to both caries-free and caries-active microbiomes and present at similar abundance. The majority of differences in OTU’s reflected very low abundance phylotypes. This survey allowed us to define the population structure of the dental plaque microbiome and to identify the microbial signatures associated with dental health and disease. The deep profiling of dental plaque allowed the identification of 87 phylotypes that are over-represented in either caries-free or caries-active subjects. Among these signatures, those associated with dental health outnumbered those associated with dental caries by nearly two-fold. A comparison of this data to other published studies indicate significant heterogeneity in study outcomes and suggest that novel approaches may be required to further define the signatures of dental caries onset and progression.     

Longitudinal Analysis of the Premature Infant Intestinal Microbiome Prior to Necrotizing Enterocolitis: A Case-Control Study

Necrotizing enterocolitis (NEC) is an inflammatory disease of the newborn bowel, primarily affecting premature infants. Early intestinal colonization has been implicated in the pathogenesis of NEC. The objective of this prospective case-control study was to evaluate differences in the intestinal microbiota between infants who developed NEC and unaffected controls prior to disease onset. We conducted longitudinal analysis of the 16S rRNA genes of 312 samples obtained from 12 NEC cases and 26 age-matched controls with a median frequency of 7 samples per subject and median sampling interval of 3 days. We found that the microbiome undergoes dynamic development during the first two months of life with day of life being the major factor contributing to the colonization process. Depending on when the infant was diagnosed with NEC (i.e. early vs. late onset), the pattern of microbial progression was different for cases and controls. The difference in the microbiota was most overt in early onset NEC cases and controls. In proximity to NEC onset, the abundances of Clostridium sensu stricto from Clostridia class were significantly higher in early onset NEC subjects comparing to controls. In late onset NEC, Escherichia/Shigella among Gammaproteobacteria, showed an increasing pattern prior to disease onset, and was significantly higher in cases than controls six days before NEC onset. Cronobacter from Gammaproteobacteria was also significantly higher in late onset NEC cases than controls 1-3 days prior to NEC onset. Thus, the specific infectious agent associated with NEC may vary by the age of infant at disease onset. We found that intravenously administered antibiotics may have an impact on the microbial diversity present in fecal material. Longitudinal analysis at multiple time points was an important strategy utilized in this study, allowing us to appreciate the dynamics of the premature infant intestinal microbiome while approaching NEC at various points.