The impact of the gut microbiota on drug metabolism and clinical outcome

The significance of the gut microbiota as a determinant of drug pharmacokinetics and accordingly therapeutic response is of increasing importance with the advent of modern medicines characterised by low solubility and/or permeability, or modified-release. These physicochemical properties and release kinetics prolong drug residence times within the gastrointestinal tract, wherein biotransformation by commensal microbes can occur. As the evidence base in support of this supplementary metabolic “organ” expands, novel opportunities to engineer the microbiota for clinical benefit have emerged. This review provides an overview of microbe-mediated alteration of drug pharmacokinetics, with particular emphasis on studies demonstrating proof of concept in vivo. Additionally, recent advances in modulating the microbiota to improve clinical response to therapeutics are explored.

Growing up in a bubble: using germ-free animals to assess the influence of the gut microbiota on brain and behavior

There is a growing recognition of the importance of the commensal intestinal microbiota in the development and later function of the central nervous system. Research using germ-free mice (mice raised without any exposure to microorganisms) has provided some of the most persuasive evidence for a role of these bacteria in gut-brain signalling. Key findings show that the microbiota is necessary for normal stress responsivity, anxiety-like behaviors, sociability, and cognition. Furthermore, the microbiota maintains central nervous system homeostasis by regulating immune function and blood brain barrier integrity. Studies have also found that the gut microbiota influences neurotransmitter, synaptic, and neurotrophic signalling systems and neurogenesis. The principle advantage of the germ-free mouse model is in proof-of-principle studies and that a complete microbiota or defined consortiums of bacteria can be introduced at various developmental time points. However, a germ-free upbringing can induce permanent neurodevelopmental deficits that may deem the model unsuitable for specific scientific queries that do not involve early-life microbial deficiency. As such, alternatives and complementary strategies to the germ-free model are warranted and include antibiotic treatment to create microbiota-deficient animals at distinct time points across the lifespan. Increasing our understanding of the impact of the gut microbiota on brain and behavior has the potential to inform novel management strategies for stress-related gastrointestinal and neuropsychiatric disorders.

Behavioural and neurochemical consequences of chronic gut microbiota depletion during adulthood in the rat

Gut microbiota colonization is a key event for host physiology that occurs early in life. Disruption of this process leads to altered brain development which ultimately manifests as changes in brain function and behaviour in adulthood. Studies using germ-free mice highlight the extreme impact on brain health that results from life without commensal microbes, however the impact of microbiota disturbances occurring in adulthood is less studied. To this end, we depleted the gut microbiota of 10-week-old male Sprague Dawley rats via chronic antibiotic treatment. Following this marked, sustained depletion of the gut bacteria, we investigated behavioural and molecular hallmarks of gut-brain communication. Our results reveal that depletion of the gut microbiota during adulthood results in deficits in spatial memory as tested by Morris water maze, increased visceral sensitivity and a greater display of depressive-like behaviours in the forced swim test. In tandem with these clear behavioural alterations we found change in altered CNS serotonin concentration along with changes in the mRNA levels of corticotrophin releasing hormone receptor 1 and glucocorticoid receptor. Additionally, we found changes in the expression of BDNF, a hallmark of altered microbiota-gut-brain axis signaling. In summary, this model of antibiotic-induced depletion of the gut microbiota can be used for future studies interested in the impact of the gut microbiota on host health without the confounding developmental influence of early-life microbial alterations.

Establishment and development of infant gut microbiota

The nascent gut microbiota at birth is established in concert with numerous developmental parameters. Here, in the INFAMTET study, we chronicled the impact of some factors which are key determinants of the infant gut microbiota, namely; mode of birth, gestational age, and type of feeding. We determined that the aggregated microbiota profile of naturally delivered, initially breastfed infants are relatively stable from one week to six months of age and are not significantly altered by increased duration of breastfeeding. Contrastingly, there is significant development of the microbiota profile of C-section delivered infants, and this development is significantly influenced by breastfeeding duration. Preterm infants, born by either mode of birth, initially have a high proportion of Proteobacteria, and demonstrate significant development of the gut microbiota from week 1 to later time-points. The microbiota is still slightly, but significantly, affected by birth mode at one year of age although no specific genera were found to be significantly altered in relative abundance. By two years of age, there is no effect of either birth mode or gestational age. However this does not preclude the possibility that symptoms developed later in life, which are associated with preterm or C-section birth, are as a result of the early perturbation of the neonatal gut microbiota. It is likely that the combination of relatively low exposure (breast fed), high exposure (formula fed) or delayed exposure (C-section and preterm) to specific antigens and the resulting inflammatory responses, in this crucial window of host-microbiota interaction, influence systemic health of the individual throughout life.

Can gut microbiota analysis be beneficial for ex-situ and in-situ conservation of threatened animal species?

Ex-situ conservation and the in-situ conservation of natural habitats are the tools to conserve biodiversity. Habitats and ecosystems have been becoming altered by human activities and a growing number of species requires form of management to ensure their survival. Conservation queries become more complex and urgent. Developing scientifically based and innovative approaches to ex-situ conservation is necessary. Recent studies underline importance of gut microbiome in animal health with implications for animal conservation and management. Animal and human studies have demonstrated that environmental factors can impact gut microbiome composition. Within this scenario, the present work focused on species belonging to different taxa, reptiles and mammals: Aldabrachelys gigantea, the giant tortoise of the Seychelles islands and Indri indri, the greatest leaving lemur of Madagascar. The Seychelles giant tortoise is vulnerable species with declining population, whereas the indri is a critically endangered species that could reach the extinction within 25 years. Both need research to help them to survive. Tortoises live for very long time and to observe how they can afford the environmental changes is very difficult. Indris, instead, are able to survive only in a small area of the Madagascar forest, with a very strong link between the species’ survival and the environment. The obtained results underline importance of environmental factors, both in-situ and ex-situ, for species conservation. Microbiome could help the organisms to respond on a short timescale and cope with, environmental changes. However, species with long generation time might not be able to adapt to fast changes but bacteria with a short generation time can adapt on a shorter timescale allowing the host to cope with fluctuating environment. Gut microbiome plays an important role in an animal’s health and has the potential to improve the management of individuals under human care for conservation purposes.

Gut microbiota perturbation induced by emerging dietary habits: an in vitro colon model to predict their effects by a microbiomic and metabolomic approach

Over the past 30 years, unhealthy diets and lifestyles have increased the incidence of noncommunicable diseases and are culprits of diffusion on world’s population of syndromes as obesity or other metabolic disorders, reaching pandemic proportions. In order to comply with such scenario, the food industry has tackled these challenges with different approaches, as the reformulation of foods, fortification of foods, substitution of ingredients and supplements with healthier ingredients, reduced animal protein, reduced fats and improved fibres applications. Although the technological quality of these emerging food products is known, the impact they have on the gut microbiota of consumers remains unclear. In the present PhD thesis, the recipient work was conducted to study different foods with the substitution of the industrial and market components to that of novel green oriented and sustainable ingredients. So far, this thesis included eight representative case studies of the most common substitutions/additions/fortifications in dairy, meat, and vegetable products. The products studied were: (i) a set of breads fortified with polyphenol-rich olive fiber, to replace synthetic antioxidant and preservatives, (ii) a set of Gluten-free breads fortified with algae powder, to fortify the protein content of standard GF products, (iii) different formulations of salami where nitrates were replaced by ascorbic acid and vegetal extract antioxidants and nitrate-reducers starter cultures, (iv) chocolate fiber plus D-Limonene food supplement, as a novel prebiotic formula, (v) hemp seed bran and its alkalase hydrolysate, to introduce as a supplement, (vi) milk with and without lactose, to evaluate the different impact on human colonic microbiota of healthy or lactose-intolerants, (vii) lactose-free whey fermented and/or with probiotics added, to be introduced as an alternative beverage, exploring its impact on human colonic microbiota from healthy or lactose-intolerants, and (viii) antibiotics, to assess whether maternal amoxicillin affects the colon microbiota of piglets.

The predictive role of the human gut microbiota in covid-19: implications for disease severity and post-covid syndrome

The COVID-19 pandemic, sparked by the SARS-CoV-2 virus, stirred global comparisons to historical pandemics. Initially presenting a high mortality rate, it later stabilized globally at around 0.5-3%. Patients manifest a spectrum of symptoms, necessitating efficient triaging for appropriate treatment strategies, ranging from symptomatic relief to antivirals or monoclonal antibodies. Beyond traditional approaches, emerging research suggests a potential link between COVID-19 severity and alterations in gut microbiota composition, impacting inflammatory responses. However, most studies focus on severe hospitalized cases without standardized criteria for severity. Addressing this gap, the first study in this thesis spans diverse COVID-19 severity levels, utilizing 16S rRNA amplicon sequencing on fecal samples from 315 subjects. The findings highlight significant microbiota differences correlated with severity. Machine learning classifiers, including a multi-layer convoluted neural network, demonstrated the potential of microbiota compositional data to predict patient severity, achieving an 84.2% mean balanced accuracy starting one week post-symptom onset. These preliminary results underscore the gut microbiota's potential as a biomarker in clinical decision-making for COVID-19. The second study delves into mild COVID-19 cases, exploring their implications for ‘long COVID’ or Post-Acute COVID-19 Syndrome (PACS). Employing longitudinal analysis, the study unveils dynamic shifts in microbial composition during the acute phase, akin to severe cases. Innovative techniques, including network approaches and spline-based longitudinal analysis, were deployed to assess microbiota dynamics and potential associations with PACS. The research suggests that even in mild cases, similar mechanisms to hospitalized patients are established regarding changes in intestinal microbiota during the acute phase of the infection. These findings lay the foundation for potential microbiota-targeted therapies to mitigate inflammation, potentially preventing long COVID symptoms in the broader population. In essence, these studies offer valuable insights into the intricate relationships between COVID-19 severity, gut microbiota, and the potential for innovative clinical applications.

Effects of resistant starch type III polymorphs on human colon microbiota and short chain fatty acids in human gut models

This study probed the possible effects of type III resistant starch (RS) crystalline polymorphism on RS fermentability by human gut microbiota and the short chain fatty acids production in vitro. Human fecal pH-controlled batch cultures showed RS induces an ecological shift in the colonic microbiota with polymorph B inducing Bifidobacterium spp. and polymorph A inducing Atopobium spp. Interestingly, polymorph B also induced higher butyrate production to levels of 0.79 mM. In addition, human gut simulation demonstrated that polymorph B promotes the growth of bifidobacteria in the proximal part of the colon and double their relative proportion in the microbiota in the distal colon. These findings suggest that RS polymorph B may promote large bowel health. While the findings are limited by study constraints, they do raise the possibility of using different thermal processing to delineate differences in the prebiotic capabilities of RS, especially its butryrogenicity in the human colon.

Depletion of murine intestinal microbiota: effects on gut mucosa and epithelial gene expression

Background Inappropriate cross talk between mammals and their gut microbiota may trigger intestinal inflammation and drive extra-intestinal immune-mediated diseases. Epithelial cells constitute the interface between gut microbiota and host tissue, and may regulate host responses to commensal enteric bacteria. Gnotobiotic animals represent a powerful approach to study bacterial-host interaction but are not readily accessible to the wide scientific community. We aimed at refining a protocol that in a robust manner would deplete the cultivable intestinal microbiota of conventionally raised mice and that would prove to have significant biologic validity. Methodology/Principal Findings Previously published protocols for depleting mice of their intestinal microbiota by administering broad-spectrum antibiotics in drinking water were difficult to reproduce. We show that twice daily delivery of antibiotics by gavage depleted mice of their cultivable fecal microbiota and reduced the fecal bacterial DNA load by 400 fold while ensuring the animals' health. Mice subjected to the protocol for 17 days displayed enlarged ceca, reduced Peyer's patches and small spleens. Antibiotic treatment significantly reduced the expression of antimicrobial factors to a level similar to that of germ-free mice and altered the expression of 517 genes in total in the colonic epithelium. Genes involved in cell cycle were significantly altered concomitant with reduced epithelial proliferative activity in situ assessed by Ki-67 expression, suggesting that commensal microbiota drives cellular proliferation in colonic epithelium. Conclusion We present a robust protocol for depleting conventionally raised mice of their cultivatable intestinal microbiota with antibiotics by gavage and show that the biological effect of this depletion phenocopies physiological characteristics of germ-free mice.

Pathogenetic mechanisms of gastrointestinal symptoms in a mouse model of Fabry disease: insights on the microbiota-gut-brain axis

Fabry disease (FD), X-linked metabolic disorder caused by a deficiency in α-galactosidase A activity, leads to the accumulation of glycosphingolipids, mainly Gb3 and lyso-Gb3, in several organs. Gastrointestinal (GI) symptoms are among the earliest and most common, strongly impacting patients’ quality of life. However, the origin of these symptoms and the exact mechanisms of pathogenesis are still poorly understood, thus the pressing need to improve their knowledge. Here we aimed to evaluate whether a FD murine model (α-galactosidase A Knock-Out) captures the functional GI issues experienced by patients. In particular, the potential mechanisms involved in the development and maintenance of GI symptoms were explored by looking at the microbiota-gut-brain axis involvement. Moreover, we sought to examine the effects of lyso-Gb3 on colonic contractility and the intestinal epithelium and the enteric nervous system, which together play important roles in regulating intestinal ion transport and fluid and electrolyte homeostasis. Fabry mice revealed visceral hypersensitivity and a diarrhea-like phenotype accompanied by anxious-like behavior and reduced locomotor activity. They reported also an imbalance of SCFAs and an early compositional and functional dysbiosis of the gut microbiota, which partly persisted with advancing age. Moreover, overexpression of TRPV1 was found in affected mice, and partial alteration of TRPV4 and TRPA1 as well, identifying them as possible therapeutic targets. The Ussing chamber results after treatment with lyso-Gb3 showed an increase in Isc (likely mediated by HCO3- ions movement) which affects neuron-mediated secretion, especially capsaicin- and partly veratridine-mediated. This first characterization of gut-brain axis dysfunction in FD mouse provides functional validation of the model, suggesting new targets and possible therapeutic approaches. Furthermore, lyso-Gb3 is confirmed to be not only a marker for the diagnosis and follow-up of FD but also a possible player in the alteration of the FD colonic ion transport process.

Influence of a probiotic soy product on fecal microbiota and its association with cardiovascular risk factors in an animal model

Background: Previous work showed that daily ingestion of an aqueous soy extract fermented with Enterococcus faecium CRL 183 and Lactobacillus helveticus 416, supplemented or not with isoflavones, reduced the total cholesterol and non-HDL-cholesterol levels, increased the high-density lipoprotein (HDL) concentration and inhibited the raising of autoantibody against oxidized low-density lipoprotein (ox-LDL Ab) and the development of atherosclerotic lesions. Objective: The aim of this study was to characterize the fecal microbiota in order to investigate the possible correlation between fecal microbiota, serum lipid parameters and atherosclerotic lesion development in rabbits with induced hypercholesterolemia, that ingested the aqueous soy extract fermented with Enterococcus faecium CRL 183 and Lactobacillus helveticus 416. Methods: The rabbits were randomly allocated to five experimental groups (n = 6): control (C), hypercholesterolemic (H), hypercholesterolemic plus unfermented soy product (HUF), hypercholesterolemic plus fermented soy product (HF) and hypercholesterolemic plus isoflavone-supplemented fermented soy product (HIF). Lipid parameters and microbiota composition were analyzed on days 0 and 60 of the treatment and the atherosclerotic lesions were quantified at the end of the experiment. The fecal microbiota was characterized by enumerating the Lactobacillus spp., Bifidobacterium spp., Enterococcus spp., Enterobacteria and Clostridium spp. populations. Results: After 60 days of the experiment, intake of the probiotic soy product was correlated with significant increases (P < 0.05) on Lactobacillus spp., Bifidobacterium spp. and Enterococcus spp. and a decrease in the Enterobacteria population. A strong correlation was observed between microbiota composition and lipid profile. Populations of Enterococcus spp., Lactobacillus spp. and Bifidobacterium spp. were negatively correlated with total cholesterol, non-HDL-cholesterol, autoantibodies against oxidized LDL (ox-LDL Ab) and lesion size. HDL-C levels were positively correlated with Lactobacillus spp., Bifidobacterium spp., and Enterococcus spp. populations. Conclusion: In conclusion, daily ingestion of the probiotic soy product, supplemented or not with isoflavones, may contribute to a beneficial balance of the fecal microbiota and this modulation is associated with an improved cholesterol profile and inhibition of atherosclerotic lesion development.

Neural Mechanisms of Exercise: Effects on Gut Miccrobiota and Depression

Microbiota is a set of microorganisms resident in gut ecosystem that reacts to psychological stressful stimuli, and is involved in depressed or anxious status in both animals and human being. Interestingly, a series of studies have shown the effects of physical exercise on gut microbiota dynamics, suggesting that gut microbiota regulation might act as one mediator for the effects of exercise on the brain. Recent studies found that gut microbiota dynamics are also regulated by metabolism changes, such as through physical exercise or diet change. Interestingly, physical exercise modulates different population of gut bacteria in compared to food restriction or rich diet, and alleviates gut syndromes to toxin intake. Gut microbiota could as well contribute to the beneficial effects of exercise on cognition and emotion, either directly through serotonin signaling or indirectly by modulating metabolism and exercise performance.

Early antibiotic administration affects the gut barrier function and the immune response to oral antigen in suckling rats

Abstract: The increasingly high hygienic standards characterizing westernized societies correlate with an increasingly high prevalence of allergic disease. Initially based on these observations, the hygiene hypothesis postulates that reduced microbial stimulation during infancy impairs the immune system development and increases the risk of allergy. Moreover, there is increasing evidence that the crosstalk existing between the intestine and the resident microbiota is crucial for gut homeostasis. In particular, bacterial colonization of the gut affects the integrity of the gut barrier and stimulates the development of the gut associated immune tissue, both phenomena being essential for the immune system to mount a controlled response to food antigens. Therefore, alterations in the microbial colonization process, by compromising the barrier homeostasis, may increase the risk of food allergy. In this context, antibiotic treatment, frequently prescribed during infancy, affects gut colonization by bacteria. However, little is known about the impact of alterations in the colonization process on the maturation of the gut barrier and on the immunological response to oral antigens. The objective of this work was to determine the impact of a commercial antibiotic preparation employed in pediatric settings on the gut barrier status at the critical period of the suckling/weaning transition and to evaluate the physiological consequences of this treatment in terms of immune response to food antigens. We established an antibiotic-treated suckling rat model relevant to the pediatric population in terms of type, dose and route of administration of the antibiotic and of changes in the patterns of microbial colonization. Oral tolerance to a novel luminal antigen (ovalbumin) was impaired when the antigen was introduced during antibiotic treatment. These results paralleled to alterations in the intestinal permeability to macromolecules and reduced intestinal expression of genes coding for the major histocomptatibility complex II molecules, which suggest a reduced capacity of antigen handling and presentation in the intestine of the antibiotic-treated animals. In addition, low luminal IgA levels and reduced intestinal expression of genes coding for antimicrobial proteins suggest that protection against pathogens was reduced under antibiotic treatment. In conclusion, we observed in suckling rats that treatment with abroad-spectrum antibiotic commonly used in pediatric practices reduced the capacity of the immune system to develop tolerance. The impact of the antibiotic treatment on the immune response to the antigen-was likely mediated by the alterations of the gut microbiota, through impairment in the mechanisms of antigen handling and presentation. This work reinforces the body of data supporting a key role of the intestinal microbiota modulating the risk of allergy development and leads us to propose that the introduction of new food antigens should be avoided during antibiotic treatment in infants. Résumé: L'augmentation du niveau d'hygiène caractérisant les sociétés occidentales semble être fortement corrélée avec l'augmentation des cas d'allergie dans ces pays. De cette observation est née l'hypothèse qu'une diminution des stimuli microbiens pendant l'enfance modifie le développement du système immunitaire augmentant ainsi le risque d'allergie. En ce sens, un nombre croissant de données indiquent que les interactions existant entre l'intestin et les bactéries résidantes sont cruciales pour l'équilibre du système. En effet, la présence de bactéries dans l'intestin affecte l'intégrité de sa fonction de barrière et stimule le développement du système immunitaire intestinal. Ces deux paramètres étant essentiels à la mise en place d'une réponse contrôlée vis à vis d'un antigène reçu oralement, toute modification du processus naturel de colonisation compromettant l'équilibre intestinal pourrait augmenter le risque d'allergie. Les traitements aux antibiotiques, fréquemment prescrits en pédiatrie, modifient de façon conséquente le processus de colonisation bactérienne. Cependant peu de données existent concernant l'impact d'une altération du processus de colonisation sur la maturation de la barrière intestinale et de la réponse immunitaire dirigée contre un antigène. L'objectif de ce travail était de déterminer l'impact d'un antibiotique commercial et employé en pédiatrie sur l'état de la barrière intestinale au moment critique du sevrage et d'évaluer les conséquences physiologiques d'un tel traitement sur la réponse immune à un antigène alimentaire. Nous avons mis en place un modèle de rats allaités, traités à l'antibiotique, le plus proche possible des pratiques pédiatriques, en terme de nature, dose et voie d'administration de l'antibiotique. Nous avons constaté que l'établissement de la tolérance orale à un nouvel antigène (l'ovalbumine) est altéré quand celui-ci est donné pour la première fois au cours du traitement antibiotique. Ces résultats coïncident avec une diminution de la perméabilité intestinale aux macromolécules, ainsi qu'avec une diminution de l'expression des gènes codant pour les molécules du complexe majeur d'histocomptatibilité de classe II, suggérant une modification de l'apprêtement et de la présentation de l'antigène au niveau intestinal chez les rats traités à l'antibiotique. De plus, un faible taux d'IgA et une diminution de l'expression des gènes codant pour des protéines antimicrobiennes, observés après l'administration d'antibiotique, laissent à penser que la protection contre un pathogène est diminuée lors d'un traitement antibiotique. En conclusion, nous avons observé qu'un traitement antibiotique à large spectre d'activité, couramment utilisé en pédiatrie, réduit la capacité d'induction de la tolérance orale chez le rat allaité. L'impact du traitement antibiotique sur la réponse immune semble induite par l'altération de la flore intestinale via son effet sur les mécanismes d'apprêtement et de présentation de l'antigène. Ce travail renforce l'ensemble des données existantes qui accorde à la flore intestinale un rôle clef dans la modulation du risque de développement d'allergie et nous amène à recommander d'éviter l'introduction d'un nouvel aliment lorsqu'un enfant est traité aux antibiotiques.

Metabolomic insights into the intricate gut microbial-host interaction in the development of obesity and type 2 diabetes.

Gut microbiota has recently been proposed as a crucial environmental factor in the development of metabolic diseases such as obesity and type 2 diabetes, mainly due to its contribution in the modulation of several processes including host energy metabolism, gut epithelial permeability, gut peptide hormone secretion, and host inflammatory state. Since the symbiotic interaction between the gut microbiota and the host is essentially reflected in specific metabolic signatures, much expectation is placed on the application of metabolomic approaches to unveil the key mechanisms linking the gut microbiota composition and activity with disease development. The present review aims to summarize the gut microbial-host co-metabolites identified so far by targeted and untargeted metabolomic studies in humans, in association with impaired glucose homeostasis and/or obesity. An alteration of the co-metabolism of bile acids, branched fatty acids, choline, vitamins (i.e., niacin), purines, and phenolic compounds has been associated so far with the obese or diabese phenotype, in respect to healthy controls. Furthermore, anti-diabetic treatments such as metformin and sulfonylurea have been observed to modulate the gut microbiota or at least their metabolic profiles, thereby potentially affecting insulin resistance through indirect mechanisms still unknown. Despite the scarcity of the metabolomic studies currently available on the microbial-host crosstalk, the data-driven results largely confirmed findings independently obtained from in vitro and animal model studies, putting forward the mechanisms underlying the implication of a dysfunctional gut microbiota in the development of metabolic disorders.

Hidden diversity in honey bee gut symbionts detected by single-cell genomics.

Microbial communities in animal guts are composed of diverse, specialized bacterial species, but little is known about how gut bacteria diversify to produce genetically and ecologically distinct entities. The gut microbiota of the honey bee, Apis mellifera, presents a useful model, because it consists of a small number of characteristic bacterial species, each showing signs of diversification. Here, we used single-cell genomics to study the variation within two species of the bee gut microbiota: Gilliamella apicola and Snodgrassella alvi. For both species, our analyses revealed extensive variation in intraspecific divergence of protein-coding genes but uniformly high levels of 16S rRNA similarity. In both species, the divergence of 16S rRNA loci appears to have been curtailed by frequent recombination within populations, while other genomic regions have continuously diverged. Furthermore, gene repertoires differ markedly among strains in both species, implying distinct metabolic capabilities. Our results show that, despite minimal divergence at 16S rRNA genes, in situ diversification occurs within gut communities and generates bacterial lineages with distinct ecological niches. Therefore, important dimensions of microbial diversity are not evident from analyses of 16S rRNA, and single cell genomics has potential to elucidate processes of bacterial diversification.