heuristic-mining-hierarchical-genotypes-accessory-genome-loci

Heuristic Mining of Hierarchical Genotypes and Accessory Genome Loci in Bacterial Populations

Routine and systematic use of bacterial whole-genome sequencing (WGS) is enhancing the accuracy and resolution of epidemiological investigations carried ...

University of Nebraska-Lincoln

 is known to colonize and cause disease in growing-finishing pigs. In this study, two clinical isolates of  were compared by inoculating cesarean-derived colostrum-deprived and specific-pathogen-free growing pigs. After intranasal or intravenous inoculation, the proportion and distribution pattern of clinical cases was compared in addition to the severity of lameness. Tonsils were found to be the primary site of colonization, while bacteremia was rarely detected prior to the observation of clinical signs. Regardless of the clinical isolate, route of inoculation, or volume of inocula, histopathological alterations and tissue invasion were detected in multiple joints, indicating an apparent lack of specific joint tropism. Acute disease was primarily observed 7 to 10 days post-inoculation. The variability in the severity of synovial microscopic lesions and pathogen detection in joint cavities suggests that the duration of joint infection may influence the diagnostic accuracy. In summary, these findings demonstrate that diagnosis of -associated arthritis can be influenced by the clinical isolate, and provides a study platform to investigate the colonization and virulence potential of field isolates. This approach can be particularly relevant to auxiliate in surveillance and testing of therapeutic and/or vaccine candidates.

Two clinical isolates of  showed differing pattern of lameness and pathogen detection in experimentally challenged pigs.

Small bowel transplantation (SBT) can be a life-saving medical procedure. However, these recipients experience high risk of bloodstream infections caused by Candida. This research aims to characterise the SBT recipient gut microbiota over time following transplantation and investigate the epidemiology of candidaemia in seven paediatric patients. Candida species from the recipients' ileum and bloodstream were identified by internal transcribed spacer sequence and distinguished to strain by multilocus sequence typing and randomly amplified polymorphic DNA. Antifungal susceptibility of bloodstream isolates was determined against nine antifungals. Twenty-two ileostomy samples harboured at least one Candida species. Fungaemia were caused by Candida parapsilosis, Candida albicans, Candida glabrata, Candida orthopsilosis and Candida pelliculosa. All but three bloodstream isolates showed susceptibility to all the antifungals tested. One C. glabrata isolate showed multidrug resistance to itraconazole, amphotericin B and posaconazole and intermediate resistance to caspofungin. Results are congruent with both endogenous (C. albicans, C. glabrata) and exogenous (C. parapsilosis) infections; results also suggest two patients were infected by the same strain of C. parapsilosis. Continuing to work towards a better understanding of sources of infection-particularly the exogenous sources-would lead to targeted prevention strategies.

Epidemiological investigation of Candida species causing bloodstream infection in paediatric small bowel transplant recipients.

Obesity-related diseases, including type 2 diabetes and cardiovascular disease, have reached epidemic proportions in industrialized nations, and dietary interventions for their prevention are therefore important. Resistant starches (RS) improve insulin sensitivity in clinical trials, but the mechanisms underlying this health benefit remain poorly understood. Because RS fermentation by the gut microbiota results in the formation of physiologically active metabolites, we chose to specifically determine the role of the gut microbiota in mediating the metabolic benefits of RS. To achieve this goal, we determined the effects of RS when added to a Western diet on host metabolism in mice with and without a microbiota.

Resistant starch can improve insulin sensitivity independently of the gut microbiota.

 is an abundant inhabitant of the intestinal mucus layer of rodents and other animals and has been suggested to be a pathobiont, a commensal that plays a role in disease. In order to gain insights into its lifestyle, we analyzed the genome and transcriptome of  ASF 457 and performed physiological experiments to test traits predicted by its genome. Although described as a mucus inhabitant,  has limited capacity for degrading host-derived mucosal glycans and other complex polysaccharides. Additionally,  reduces nitrate and expresses systems for scavenging oxygen and reactive oxygen species , which may account for its localization close to the mucosal tissue and expansion during inflammation. Also of note,  harbors a type VI secretion system and putative effector proteins and can modify gene expression in mucosal tissue, suggesting intimate interactions with its host and a possible role in inflammation. The  genome has been shaped by extensive horizontal gene transfer, primarily from intestinal - and , indicating that horizontal gene transfer has played a key role in defining its niche in the gut ecosystem.  Shifts in gut microbiota composition have been associated with intestinal inflammation, but it remains unclear whether inflammation-associated bacteria are commensal or detrimental to their host. Here, we studied the lifestyle of the gut bacterium , which is associated with inflammation in widely used mouse models. We found that  has specialized systems to handle oxidative stress during inflammation. Additionally, it expresses secretion systems and effector proteins and can modify the mucosal gene expression of its host. This suggests that  undergoes intimate interactions with its host and may play a role in inflammation. The insights presented here aid our understanding of how commensal gut bacteria may be involved in altering susceptibility to disease.

Lifestyle and Horizontal Gene Transfer-Mediated Evolution of , a Core Member of the Murine Gut Microbiota.

Changes in the gastrointestinal microbial community are frequently associated with chronic diseases such as Inflammatory Bowel Diseases. However, understanding the relationship of any individual taxon within the community to host physiology is made complex due to the diversity and individuality of the gut microbiota. Defined microbial communities such as the Altered Schaedler Flora (ASF) help alleviate the challenges of a diverse microbiota by allowing one to interrogate the relationship between individual bacterial species and host responses. An important aspect of studying these relationships with defined microbial communities is the ability to measure the population abundance and dynamics of each member. Herein, we describe the development of an improved ASF species-specific and sensitive real-time quantitative polymerase chain reaction (qPCR) for use with SYBR Green chemistry to accurately assess individual ASF member abundance. This approach targets hypervariable regions V1 through V3 of the 16S rRNA gene of each ASF taxon to enhance assay specificity. We demonstrate the reproducibility, sensitivity and application of this new method by quantifying each ASF bacterium in two inbred mouse lines. We also used it to assess changes in ASF member abundance before and after acute antibiotic perturbation of the community as well as in mice fed two different diets. Additionally, we describe a nested PCR assay for the detection of lowly abundant ASF members. Altogether, this improved qPCR method will facilitate gnotobiotic research involving the ASF community by allowing for reproducible quantification of its members under various physiological conditions.

A real-time PCR assay for accurate quantification of the individual members of the Altered Schaedler Flora microbiota in gnotobiotic mice.

The community of microorganisms in the mammalian gastrointestinal tract, referred to as the gut microbiota, influences host physiology and immunity. The last decade of microbiome research has provided significant advancements for the field and highlighted the importance of gut microbes to states of both health and disease. Novel molecular techniques have unraveled the tremendous diversity of intestinal symbionts that potentially influence the host, many proof-of-concept studies have demonstrated causative roles of gut microbial communities in various pathologies, and microbiome-based approaches are beginning to be implemented in the clinic for diagnostic purposes or for personalized treatments. However, several challenges for the field remain: purely descriptive reports outnumbering mechanistic studies and slow translation of experimental results obtained in animal models into the clinics. Moreover, there is a dearth of knowledge regarding how gut microbes, including novel species that have yet to be identified, impact host immune responses. The sheer complexity of the gut microbial ecosystem makes it difficult, in part, to fully understand the microbiota-host networks that regulate immunity. In the present manuscript, we review key findings on the interactions between gut microbiota members and the immune system. Because culturing microbes allows performing functional studies, we have emphasized the impact of specific taxa or communities thereof. We also highlight underlying molecular mechanisms and discuss opportunities to implement minimal microbiome-based strategies.

Deciphering interactions between the gut microbiota and the immune system via microbial cultivation and minimal microbiomes.

Inflammatory bowel diseases (IBD) are likely driven by aberrant immune responses directed against the resident microbiota. Although IBD is commonly associated with a dysbiotic microbiota enriched in putative pathobionts, the etiological agents of IBD remain unknown. Using a pathobiont-induced intestinal inflammation model and a defined bacterial community, we provide new insights into the immune-microbiota interactions during disease. In this model system, the pathobiont Helicobacter bilis instigates disease following sub-pathological dextran sulfate sodium treatment. We show that H. bilis causes mild inflammation in mono-associated mice, but severe disease in the presence of a microbiota, demonstrating synergy between the pathobiont and microbiota in exacerbating pathology. Remarkably, inflammation depends on the presence of H. bilis, but is marked by a predominant Th17 response against specific members of the microbiota and not the pathobiont, even upon the removal of the most immune-dominant taxa. Neither increases in pathobiont burden nor unique changes in immune-targeted microbiota member abundances are observed during disease. Collectively, our findings demonstrate that a pathobiont instigates inflammation without being the primary target of a Th17 response or by altering the microbiota community structure. Moreover, our findings point toward monitoring pathobiont-induced changes in microbiota immune targeting as a new concept in IBD diagnotics.

A gut pathobiont synergizes with the microbiota to instigate inflammatory disease marked by immunoreactivity against other symbionts but not itself.

Many enteric pathogens, including Salmonella and enteropathogenic and enterohemorrhagic Escherichia coli, express adhesins that recognize and bind to carbohydrate moieties expressed on epithelial cells. An attractive strategy for inhibiting bacterial adherence employs molecules that mimic these epithelial binding sites. Prebiotic oligosaccharides are non-digestible, fermentable fibres capable of modulating the gut microbiota. Moreover, they may act as molecular decoys that competitively inhibit adherence of pathogens to host cells. In particular, galactooligosaccharides (GOS) and other prebiotic fibres have been shown to inhibit pathogen adherence to epithelial cells in vitro. In the present study, we determined the ability of prophylactic GOS administration to reduce enteric pathogen adherence both in vitro and in vivo as well as protect against intestinal inflammation. GOS supplementation significantly reduced the adherence of the epithelial-adherent murine bacterial pathogen Citrobacter rodentium in a dose-dependent manner to the surface of epithelial cells in vitro. A 1- to 2-log reduction in bacterial adherence was observed at the lowest and highest doses tested, respectively. However, mouse studies revealed that treatment with GOS neither reduced the adherence of C. rodentium to the distal colon nor decreased its dissemination to systemic organs. Despite the absence of adherence inhibition, colonic disease scores for GOS-treated, C. rodentium-infected mice were significantly lower than those of untreated C. rodentium-infected animals (P=0.028). Together, these data suggest that GOS has a direct protective effect in ameliorating disease severity following C. rodentium infection through an anti-adherence-independent mechanism.

Galactooligosaccharide supplementation provides protection against Citrobacter rodentium-induced colitis without limiting pathogen burden.

The factors that govern assembly of the gut microbiota are insufficiently understood. Here, we test the hypothesis that inter-individual microbiota variation can arise solely from differences in the order and timing by which the gut is colonized early in life. Experiments in which mice were inoculated in sequence either with two complex seed communities or a cocktail of four bacterial strains and a seed community revealed that colonization order influenced both the outcome of community assembly and the ecological success of individual colonizers. Historical contingency and priority effects also occurred in  mice, suggesting that the adaptive immune system is not a major contributor to these processes. In conclusion, this study established a measurable effect of colonization history on gut microbiota assembly in a model in which host and environmental factors were strictly controlled, illuminating a potential cause for the high levels of unexplained individuality in host-associated microbial communities.

Experimental evaluation of the importance of colonization history in early-life gut microbiota assembly.

 is a facultative anaerobic symbiont found widely among mammalian gastrointestinal tracts. Several human studies have reported increased commensal  abundance in the intestine during inflammation; however, host immunological responses toward commensal  during inflammation are not well-defined. Here, we show that colonization of gnotobiotic mice with different genotypes of commensal  isolated from healthy conventional microbiota mice and representing distinct populations of  elicited strain-specific disease phenotypes and immunopathological changes following treatment with the inflammatory stimulus, dextran sulfate sodium (DSS). Production of the inflammatory cytokines GM-CSF, IL-6, and IFN-γ was a hallmark of the severe inflammation induced by  strains of Sequence Type 129 (ST129) and ST375 following DSS administration. In contrast, colonization with  strains ST150 and ST468 caused mild intestinal inflammation and triggered only low levels of pro-inflammatory cytokines, a response indistinguishable from that of -free control mice treated with DSS. The disease development observed with ST129 and ST375 colonization was not directly associated with their abundance in the GI tract as their levels did not change throughout DSS treatment, and no major differences in bacterial burden in the gut were observed among the strains tested. Data mining and  neutralization identified IL-6 as a key cytokine responsible for the observed differential disease severity. Collectively, our results show that the capacity to exacerbate acute intestinal inflammation is a strain-specific trait that can potentially be overcome by blocking the pro-inflammatory immune responses that mediate intestinal tissue damage.

Commensal  Strains Can Promote Intestinal Inflammation via Differential Interleukin-6 Production.

Whole Genome Sequence (WGS) data from bacterial species is used for a variety of applications ranging from basic microbiological research, diagnostics, and epidemiological surveillance. The availability of WGS data from hundreds of thousands of individual isolates of individual microbial species poses a tremendous opportunity for discovery and hypothesis-generating research into ecology and evolution of these microorganisms. Flexibility, scalability, and user-friendliness of existing pipelines for population-scale inquiry, however, limit applications of systematic, population-scale approaches. Here, we present ProkEvo, an automated, scalable, reproducible, and open-source framework for bacterial population genomics analyses using WGS data. ProkEvo was specifically developed to achieve the following goals: (1) Automation and scaling of complex combinations of computational analyses for many thousands of bacterial genomes from inputs of raw Illumina paired-end sequence reads; (2) Use of workflow management systems (WMS) such as Pegasus WMS to ensure reproducibility, scalability, modularity, fault-tolerance, and robust file management throughout the process; (3) Use of high-performance and high-throughput computational platforms; (4) Generation of hierarchical-based population structure analysis based on combinations of multi-locus and Bayesian statistical approaches for classification for ecological and epidemiological inquiries; (5) Association of antimicrobial resistance (AMR) genes, putative virulence factors, and plasmids from curated databases with the hierarchically-related genotypic classifications; and (6) Production of pan-genome annotations and data compilation that can be utilized for downstream analysis such as identification of population-specific genomic signatures. The scalability of ProkEvo was measured with two datasets comprising significantly different numbers of input genomes (one with ~2,400 genomes, and the second with ~23,000 genomes). Depending on the dataset and the computational platform used, the running time of ProkEvo varied from ~3-26 days. ProkEvo can be used with virtually any bacterial species, and the Pegasus WMS uniquely facilitates addition or removal of programs from the workflow or modification of options within them. To demonstrate versatility of the ProkEvo platform, we performed a hierarchical-based population structure analyses from available genomes of three distinct pathogenic bacterial species as individual case studies. The specific case studies illustrate how hierarchical analyses of population structures, genotype frequencies, and distribution of specific gene functions can be integrated into an analysis. Collectively, our study shows that ProkEvo presents a practical viable option for scalable, automated analyses of bacterial populations with direct applications for basic microbiology research, clinical microbiological diagnostics, and epidemiological surveillance.

ProkEvo: an automated, reproducible, and scalable framework for high-throughput bacterial population genomics analyses.

The common marmoset (Callithrix jacchus) is an omnivorous New World primate whose diet in the wild includes large amounts of fruit, seeds, flowers, and a variety of lizards and invertebrates. Marmosets also feed heavily on tree gums and exudates, and they have evolved unique morphological and anatomical characteristics to facilitate gum feeding (gummivory). In this study, we characterized the fecal microbiomes of adult and infant animals from a captive population of common marmosets at the Callitrichid Research Center at the University of Nebraska at Omaha under their normal dietary and environmental conditions. The microbiomes of adult animals were dominated by species of , , , , , and . Culturing and genomic analysis of the  populations from adult animals identified four known marmoset-associated species (, , , and ) and three unclassified taxa of  that are phylogenetically distinct. Species-specific quantitative PCR (qPCR) confirmed that these same species of  are abundant members of the microbiome throughout the lives of the animals. Genomic loci in each  species encode enzymes to support growth and major marmoset milk oligosaccharides during breastfeeding; however, metabolic islands that can support growth on complex polysaccharide substrates in the diets of captive adults (pectin, xyloglucan, and xylan), including loci in  that can support its unique ability to grow on arabinogalactan-rich tree gums, were species-specific.   species are recognized as important, beneficial microbes in the human gut microbiome, and their ability colonize individuals at different stages of life is influenced by host, dietary, environmental, and ecological factors, which is poorly understood. The common marmoset is an emerging nonhuman primate model with a short maturation period, making this model amenable to study the microbiome throughout a life history. Features of the microbiome in captive marmosets are also shared with human gut microbiomes, including abundant populations of  species. Our studies show that several species of  are dominant members of the captive marmoset microbiome throughout their life history. Metabolic capacities in genomes of the marmoset  species suggest species-specific adaptations to different components of the captive marmoset diet, including the unique capacity in  for degradation of gum arabic, suggesting that regular dietary exposure in captivity may be important for preserving gum-degrading species in the microbiome.

Captive Common Marmosets (Callithrix jacchus) Are Colonized throughout Their Lives by a Community of  Species with Species-Specific Genomic Content That Can Support Adaptation to Distinct Metabolic Niches.

Article	Year
Two clinical isolates of showed differing pattern of lameness and pathogen detection in experimentally challenged pigs. Journal of veterinary science\| PubMed ID: 27297416	2016
Epidemiological investigation of Candida species causing bloodstream infection in paediatric small bowel transplant recipients. Mycoses\| PubMed ID: 28139856	2017
Resistant starch can improve insulin sensitivity independently of the gut microbiota. Microbiome\| PubMed ID: 28166818	2017
Lifestyle and Horizontal Gene Transfer-Mediated Evolution of , a Core Member of the Murine Gut Microbiota. mSystems\| PubMed ID: 28168224	2017
A real-time PCR assay for accurate quantification of the individual members of the Altered Schaedler Flora microbiota in gnotobiotic mice. Journal of microbiological methods\| PubMed ID: 28189782	2017
Deciphering interactions between the gut microbiota and the immune system via microbial cultivation and minimal microbiomes. Immunological reviews\| PubMed ID: 28856739	2017
A gut pathobiont synergizes with the microbiota to instigate inflammatory disease marked by immunoreactivity against other symbionts but not itself. Scientific reports\| PubMed ID: 29255158	2017
Galactooligosaccharide supplementation provides protection against Citrobacter rodentium-induced colitis without limiting pathogen burden. Microbiology (Reading, England)\| PubMed ID: 29256851	2018
Experimental evaluation of the importance of colonization history in early-life gut microbiota assembly. eLife\| PubMed ID: 30226190	2018
Commensal Strains Can Promote Intestinal Inflammation via Differential Interleukin-6 Production. Frontiers in immunology\| PubMed ID: 30356663	2018
ProkEvo: an automated, reproducible, and scalable framework for high-throughput bacterial population genomics analyses. PeerJ\| PubMed ID: 34055480	2021
Captive Common Marmosets (Callithrix jacchus) Are Colonized throughout Their Lives by a Community of Species with Species-Specific Genomic Content That Can Support Adaptation to Distinct Me... mBio\| PubMed ID: 34340536	2021

Joao Carlos Gomes-Neto

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