The CF gastrointestinal microbiome: Structure ... - Wiley Online Library

Pediatric Pulmonology 51:S35–S44 (2016)

The CF Gastrointestinal Microbiome: Structure and Clinical Impact Geraint B. Rogers,1 Michael R. Narkewicz,2,3 and Lucas R. Hoffman4,5* Summary. The gastrointestinal (GI) microbiome is shaped by host diet, immunity, and other physicochemical characteristics of the GI tract, and perturbations such as antibiotic treatments can lead to persistent changes in microbial constituency and function. These GI microbes also play critical roles in host nutrition and health. A growing body of evidence suggests that the GI microbiome in people with CF is altered, and that these dysbioses contribute to disease manifestations in many organs, both within and beyond the GI tract. Therapies that people with CF receive, even those targeting the respiratory tract, may impact the CF GI microbiome in ways that can influence the outcome of treatment. These new perspectives on the microbial contents of the CF intestine offer new opportunities for preventing a variety of CF-associated disorders. Pediatr Pulmonol. 2016;51:S35–S44. ß 2016 Wiley Periodicals, Inc. Key words: cystic fibrosis (CF); antibiotic therapy; gastrointestinal; microbiome. Funding source: NIH, Numbers: 1R01DK095869-01A1, P30 DK089507.

INTRODUCTION

Mentioning the words “CF microbiome” to a group of interested clinicians, microbiologists, or patients is likely to evoke images of purulent sputum and inflamed airways. However, while the bulk of the illness and shortened life expectancy conferred by cystic fibrosis (CF) is attributable to respiratory disease, many of the earliest and most severe manifestations of CF afflict the gastrointestinal (GI) tract.1,2 In both sickness and health, the GI tract hosts a diverse assortment of microbes, comprising trillions of cells, and recent advancements in molecular microbiological methods have afforded a greatly enhanced appreciation for the roles of these GI microbiota in homeostasis and disease, both in the gut and beyond. These microbes are essential for host metabolism, nutrient harvest and synthesis, and the development and homeostasis of the GI tract and general host immunity, among other tasks. In turn, the GI physicochemical environment, which is decidedly altered in people with CF, shapes both the constituency and behavior of the GI microbiota. Recent evidence shows that CF GI microbiota are indeed different from people without CF, potentially with pathological consequences within and beyond the CF GI tract.3–8 In this article, we will review the recent literature on the CF GI microbiome and its clinical impact, as well as how we might use these recent discoveries to prevent a host of CF disease manifestations. We will focus largely on the structure, development, and behavior of the CF GI microbiota as compared with people without CF, and the drivers of the intestinal “dysbiosis” that is common in people and animals with ß 2016 Wiley Periodicals, Inc.

CF. First, however, we will outline the terminology and methods commonly used in the rapidly expanding field of microbiomics. MICROBIOME: TERMINOLOGY, TECHNIQUES, AND THEORIES

The external surfaces of our bodies, particularly the mucosae, are associated with an abundance of commensal

1

SAHMRI Infection and Immunity Theme, School of Medicine, Flinders University, Adelaide, South Australia, Australia. 2 Section of Pediatric Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, University of Colorado School of Medicine, Aurora, Colorado. 3

Digestive Health Institute, Children’s Hospital Colorado, Aurora, Colorado.

4 Departments of Pediatrics and Microbiology, University of Washington, Seattle, Washington. 5

Seattle Children’s Hospital, Seattle, Washington.

Conflicts of interest: None.

Correspondence to: Lucas R. Hoffman, Departments of Pediatrics and Microbiology, University of Washington, Seattle, WA. E-mail: [email protected] Received 2 May 2016; Revised 8 July 2016; Accepted 11 July 2016. DOI 10.1002/ppul.23544 Published online in Wiley Online Library (wileyonlinelibrary.com).

S36

Rogers et al.

microorganisms. These microbes, along with their genetic material and the products that it encodes, are collectively referred to as the human microbiome. Our ability to describe these microbial systems in detail and to explore their role in human development and physiology is relatively recent and owes much to advances in highthroughput DNA sequencing technologies. For example, sequencing of the bacterial 16S ribosomal RNA gene allows the identification and relative enumeration of the bacterial taxa within a clinical sample (the microbiota) in a way that would be simply unachievable using culturebased approaches, while analyzing all of the genes in a sample (shotgun metagenomic sequencing) defines the functional potential encoded within the collective genomes of the microbes present. Metabolomic analysis, which characterizes microbial chemical products using techniques such as nuclear magnetic resonance and mass spectrometry, further enables assessment of the collective behavior of the microbiota. Microbiome analysis generates data of far greater complexity than does traditional diagnostic microbiology. Describing these intricate communities requires concepts and terminology borrowed from the field of macroecology. Descriptions of microbiota typically focus on assessments of diversity, based on numbers and relative abundances of microbial taxa, rather than the presence of particular species or strains. These measures can provide insight into characteristics of the niche that particular microbiota inhabit, the mechanisms by which they influence the health of the host, and the manner in which they change in response to disease and therapy. THE GASTROINTESTINAL (GI) MICROBIOME: STRUCTURE, DEVELOPMENT, AND ROLE IN HEALTH

Commensal microbiota are established in early life, with their composition strongly influenced by the local environment. For example, differences in the microbial (and antibiotic) exposures of infants delivered vaginally or by caesarean section are reflected in the composition of nascent respiratory and GI microbial communities.9 However, as they mature, the composition of microbial communities at particular anatomical sites is increasingly determined by location-specific characteristics. In the GI tract, for example, differences in factors such as pH, mucin composition, and nutrient availability are reflected in the number and type of bacteria that occur at different locations.10,11 The development of the intestinal microbiota occurs in parallel to the establishment of normal metabolic and immunological regulation in the neonate. Breast milk oligosaccharides provide a substrate for the growth of bacteria that contribute both to immune development12,13 and to the biosynthesis of important nutrients, such as Pediatric Pulmonology

folate.14 Complex and site-specific mucosal microbiota provide abundant opportunity for compartmentalized host–microbe communication. The capacity for the microbiome to influence human physiology is perhaps greatest in the colon, which houses a bacterial community estimated to contain more than 50 bacterial phyla,15 at densities exceeding 1012 cells per gram,16 overlaying a vast epithelial surface rich in cell types that respond to microbial stimuli and well-adapted for the uptake of microbe-derived compounds. The benefits of the commensal GI microbiome to the host are manifold and include effective nutrient and energy harvest,17 suppression of enteric pathogens,18 and the maintenance of GI epithelial homeostasis through promotion of epithelial cell turnover19 and gut barrier function.20 By interacting with a system of host regulatory networks, the GI microbiome is also an important regulator of systemic processes, including glucose and lipid metabolism,21,22 and innate and adaptive immunity.23 The disruption of the GI microbiota, or dysbiosis, is not only associated with a reduction in these beneficial functions, but also an increase in microbiome traits that stimulate local and systemic inflammation and dysregulation of other aspects of host physiology. THE GI MICROBIOME IN GI AND RESPIRATORY DISEASE

Given the intimate physical relationship between the GI tract and its microbial contents, a close relationship between dysbiosis and GI disease is not difficult to imagine. Indeed, GI dysbiosis has been linked to diverse GI diseases,24 including inflammatory bowel disease, necrotizing enterocolitis, irritable bowel syndrome, and other intestinal disorders. GI dysbiosis has also been linked to a number of common diseases of the liver, including non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, alcoholic liver disease, and cirrhosis.25,26 For example, the dysbiosis that arises as a result of increased luminal fat, as is the case in CF,6 results in enrichment of bacteria that convert primary bile acids into hepatotoxic secondary bile acids,27 as well as in increased gut permeability28 and, therefore, greater liver exposure to gut-derived endotoxins.29 However, GI dysbiosis has also been shown to be associated with a range of diseases beyond the GI tract, often those in which chronic inflammation or metabolic dysregulation is implicated in pathogenesis.10,30–36 Dysbiosis in the neonate appears to be particularly disruptive, taking place when microbial colonization of the gut occurs in parallel with the development of host systems that detect, respond to, and regulate the microbiota. GI dysbiosis in early life is associated with lasting dysregulation of systemic immunity, deranged metabolic control, and nervous system dysfunction33,37–40 and has been linked with an increased

CF Gastrointestinal Microbiome 41

risk of airway allergen sensitization and susceptibility to lung infection.42 There are many potential causes of these alterations in the GI microbiota, and we will now consider some of the most common factors. IMPACT OF MEDICATIONS AND DIET ON THE GI MICROBIOME

The physicochemical conditions of the GI lumen (a short phrase that belies the staggering complexity of this space, comprising viscous and antimicrobial host secretions; the nutrients provided by diet and host fluids; gradients of oxygen and other molecules; and drugs like antibiotics, all encased in an anatomically heterogeneous, muscular, constantly moving tubular organ) select specific microbes from the many to which we are exposed daily. Variations in these selective characteristics, as a result of environmental, genetic, dietary, or therapeutic influences, contribute to the composition a patient’s of the GI microbiota. Perturbations by two of these factors, dietary changes and antibiotic treatment, have been wellstudied and found to deeply influence the constituency and behavior of the GI microbiota. A number of studies have demonstrated the important role of diet in shaping the GI microbiome. For example, David et al.43 demonstrated reproducibly dramatic and rapid changes in fecal microbiota in people with a variety of starting diets upon even short-term alterations in those diets, an observation recapitulated in mouse models by Carmody et al.44 In a complementary study, De Filippo et al.45 found that the gut microbiota in children from rural Africa and from Europe were well-suited genetically for metabolizing the nutrients in their respective diets only. This profound impact of diet is particularly evident in studies of the development of the infant GI microbiome; as shown by B€ackhed et al.,46 the shift from breast feeding to other foods plays a major role in microbiota maturation during the first year of life. Many different antibiotics, with divergent antimicrobial spectra and a range of penetration into the GI lumen, have been examined for their effects on the GI microbiota (reviewed in Modi et al.47). Antibiotic treatment has rapid, widespread, and often long-lasting impacts on the composition of the GI microbiome, often leading to a post-antibiotic dysbiotic state with decreases both in diversity and resistance to invading pathogens, changes in metabolic capacity, and complex effects on interactions with host immunity. Often, antibiotic-induced dysbiosis is characterized by reductions in the phyla Firmicutes and Bacteroides, with compensatory expansion in the family Enterobacteriaceae; each of these changes has been associated with GI dysfunction.48 Moreover, the detrimental effects of antibiotics can extend beyond the GI microbiome: in a recent CF mouse model study,49 the GI dysbiosis induced by oral treatment with the non-

S37

absorbable antibiotic streptomycin was accompanied by alterations in pulmonary inflammatory cell profile, as well as airway hyperresponsiveness, underscoring our growing appreciation for a mechanistic link between GI microbiology, antibiotic treatment, and disease manifestations at other mucosal surfaces, including the airways. CYSTIC FIBROSIS: RESPIRATORY AND GI MANIFESTATIONS

CF respiratory disease is characterized by progressive obstruction of the airways with purulent, sticky mucus. These viscous, airway luminal secretions are infected with a steady progression of microbes, beginning with a diverse collection of bacteria in early disease, and culminating most often in high densities of one or a few types of antibiotic-resistant pathogens at the final stages of lung disease.50–53 With disease advancement, the airways become progressively damaged and dilated as the integrity of their elastic walls is compromised by chronic inflammation and infection, a structural defect known as bronchiectasis. There is considerable controversy regarding whether these microbial changes cause, or reflect, the advancing destruction of the airways, and the role of therapeutic antibiotics given for worsening symptoms in shaping the respiratory microbiota.51–56 By comparison, the levels of inflammation in the CF GI tract are more subtle. However, several functions and structures of the complete GI tract (which includes the liver, pancreas, and other organs) are grossly altered at birth in CF,1,2 and others are severely impacted by CFTR dysfunction. Most infants with CF are born with pancreatic exocrine deficiency, as manifested by a paucity of several pancreatic digestive enzymes—lipase, proteases, and amylase—in the small intestine. This deficiency leads to inadequate digestion and nutrient absorption, particularly of fats and fat-soluble vitamins, resulting in nutritional failure without oral pancreatic enzyme supplementation (and often even despite this treatment). Many neonates with CF suffer from intestinal obstruction by inspissated feces—known as meconium ileus—that requires surgical intervention, and in the most severe cases in intestinal resection. Intestinal obstruction can and often does occur even after the neonatal period for a variety of as-yet poorly defined reasons. There is also evidence for more subtle intestinal dysfunction in CF; for example, fecal measures show that the CF GI tract has chronic, low-level inflammation.5,8,57 The small intestines of many people with CF are overgrown by bacteria, with related slow transit of intestinal contents through this organ.2,56 In addition, there is evidence of altered intestinal permeability in CF56,58 and the presence of macroscopic intestinal epithelial ulcerations and other lesions.56,59 Pediatric Pulmonology

S38

Rogers et al.

With increasing life expectancy, people with CF are more apt to experience additional complications of the organs of the GI system. Specifically, diabetes due to pancreatic endocrine failure is increasingly likely with advancing age. CF-related liver disease (CFRLD) is also increasingly recognized,60 and is thought to be associated with an increased frequency of cirrhosis and portal hypertension. We are only beginning to understand the pathogenesis of the majority of these GI manifestations of CF, and for many, the GI microbiota are thought to play an important part. ANIMAL MODELS OF CF GI DISEASE: PATHOLOGY AND MICROBIOTA

Much of our current and emerging understanding of the pathogenesis of CF GI disease, and the contribution of the GI microbiota, comes from observations of animals with engineered CFTR mutations. Perhaps the best-studied are mouse models; there have been many such models, yielding a wealth of information (reviewed in De Lisle and Borowitz2). While most murine transgenic models of CF exhibit both pancreatic sufficiency and at worst mild airway disease,61 they suffer from severe intestinal obstruction, delayed transit times, mucus accumulation, and nutrient malabsorption, offering a useful opportunity to study the effects of these characteristics on GI microbiota. In general, mice with engineered CF mutations exhibit stark differences in their GI microbiomes compared with non-CF littermates. However, the types and severity of the abovementioned GI manifestations differ depending on mouse genetic background62,63; accordingly, the alterations in GI microbiomes differ by mouse strain, as well. In general, CF mice have higher overall GI bacterial load, consistent with bacterial overgrowth.63,64 In at least one mouse model, the CF GI microbiota were of lower diversity65 than in non-CF mice. All models exhibited significant differences compared with non-CF mice in the relative abundances of specific taxa thought to be important for GI health, including Lactobacillus and Bifidobacterium species.63,65 In one study, a single course of two antibiotics was shown to substantially alter the GI microbiota, in particular suppressing the taxa associated with small intestinal bacterial overgrowth.63,65 This same treatment was shown in another study to improve mouse somatic growth.64 These observations provide a convincing link between dysbiosis, antibiotic treatment, and GI manifestations of CF disease. Similarly, ferrets with engineered CF mutations exhibit significant GI disease.66 In the intestine, these animals had gastric ulceration, intestinal bacterial overgrowth with villous atrophy, and rectal prolapse; pancreatic fibrosis was also common, while hepatic and biliary manifestations were present somewhat more variably. Pediatric Pulmonology

Aside from overgrowth, however, and while CF ferrets’ GI microbiota always differed from those of their non-CF littermates, these differences did not consistently involve the same taxa. Pigs with CF mutations also develop many of the same manifestations of GI disease as people with CF, including exocrine pancreatic insufficiency, focal biliary cirrhosis, and intestinal obstruction.67–69 Of these, meconium ileus is the most striking; 100% of newborn CF pigs have this complication (compared with 15% of newborn human babies with CF). To date, we are unaware of any studies of the GI microbiology in the CF pig. Together, these and other animal models70 provide a nuanced and complex view of the relationships between CF GI dysfunction, antibiotic treatment, and intestinal microbiota. While the location and load of bacteria in the CF GI tract is clearly altered, the imperfect association of microbial taxonomic dysbiosis and CF genotype in even these well characterized, carefully curated, and otherwise isogenic animal models highlight the likely important role of environmental and stochastic factors in shaping the CF intestinal microbiome. This concept is borne out by observations of the fecal microbiota from people with CF. THE MICROBIOTA IN THE HUMAN CF GI TRACT

Studies of the GI microbiota of people with CF have generally focused on the most convenient type of “intestinal” sample: stool. While this consistency in specimen type makes it easier to compare results from different studies, they provide at best a limited snapshot of intestinal microbiology, and they offer no reasonable insights into the microbiome of the small intestine, which is known to have significant pathology and bacterial overgrowth. By comparison, the microbiome studies of the CF GI tract published to date have used vastly diverse analytical methods as these techniques have evolved, and the ages, disease manifestations, and treatment regimens of the study subjects have also differed considerably between studies. Despite these methodological and study population differences, however, these studies made a few similar observations that are likely instructive. Two studies by the Vandamme group used a gel electrophoretic method and quantitative PCR to characterize the microbiota in children and teenagers with CF and their healthy siblings and found relative depletions in several taxa in the Bifidobacterium and Clostridium genera as well as lower overall species richness.7,71 Scanlan et al.4 made similar observations using different methodology. The Vandamme group also found, using a group of subjects similar to those in their prior studies, that isolates of Enterobacteriaceae cultured from the stool of children with CF tended to be less susceptible to betalactams,72 highlighting the markedly higher antibiotic exposure children with CF experience (and thus the

CF Gastrointestinal Microbiome

importance of considering antibiotics as important selective pressures in shaping CF GI microbiomes). Schippa et al.73 found in a group of fasting CF patients that the fecal microbiota correlated with severity of CFTR mutation, with expansions among those with diseasecausing mutations of potentially harmful Proteobacteria species such as Escherichia coli and Eubacterium biforme, and depletions in health-associated taxa such as Eubacterium limosum, Faecalibacterium prasunitzii, and Bifidobacterium spp. These dysbioses were particularly marked among people who with the most severe CFTR dysfunction, such as those homozygous for the F508del CFTR mutation. Hoffman et al.5,6 showed that these effects were already present in young children with CF, and even in the absence of recent antibiotic treatment; this dysbiosis was significantly associated with fecal measures of both fat malabsorption and inflammation, suggesting a relationship between the microbiota and GI dysfunction in CF supported by observations from additional studies of microbiota, probiotics, and GI dysfunction, to be described below. THE CF RESPIRATORY MICROBIOTA AND RELATIONSHIP WITH GI MICROBIOTA

Modulation of local immune response is influenced by host–microbe interactions in both the GI and respiratory tracts, with systemic immune pathways providing a basis for bi-directional communication between these two mucosal surfaces (Fig. 1). The airway and gastrointestinal epithelia share many structural and physiological features, and each undergo an ordered colonization in early life that shapes local immune regulation.74 Not only does the colonization of the gut and upper respiratory tract (URT) occur at the same time, but there is evidence of substantial cross-talk between the two sites.75 This effect has been demonstrated in longitudinal studies of both microbiota of children with CF, revealing synchronized fluctuations in the abundance of a number of bacterial taxa.76 This same group showed a closer relationship between respiratory disease symptoms with the GI microbiomes of children with CF than with their respiratory microbiomes,77 suggesting that the interaction between the early CF GI and respiratory tracts is more complex than simply sharing microbes. Similarly, treatments such as antibiotics given for CF respiratory disease also impact the gut microbiota, whether through the direct antimicrobial impact of antibiotics,78 or through their effect on gastrointestinal physiology (for example, the motor-stimulating activity of macrolide antibiotics).79 RELATIONSHIP BETWEEN GI MICROBIOTA AND CF GI DISEASE

Considering what is known about the functions of GI microbiota in healthy people, there are several disorders

S39

of the CF GI tract that are likely to either influence or be influenced by gut microbes. Currently, perhaps the best evidence for these interactions comes from studies of GI inflammation, GI luminal erosive lesions, and CF-related liver disease. Manor et al.6 found that fecal dysbiosis in young children with CF resulted in enrichment in bacterial genes for degrading lipids (which are abundant due to malabsorption) relative to children without CF. Conversely, the same metagenomic analysis showed that CF fecal microbiota are predicted to be relatively deficient for synthesizing specific microbial-derived small molecules, known as short-chain fatty acids (SCFAs), which are important for regulating epithelial cell health and mucosal immune responses in the GI tract. These metagenomic alterations correlated with both fecal fat content and measures of inflammation, suggesting that enteral lipid abundance may enrich for pro-inflammatory microbiota in children with CF. This model is supported by observations from clinical trials of probiotics in CF patients,8,80 as will be discussed in more detail below, as well as from a study of bacterial and host proteins (i.e., using proteomics) in CF fecal samples.81 Similarly, observations by Flass et al.56 provide strong evidence of a role for the GI microbiota in CFRLD. Contrary to current models of CFLD development, which hold that biliary obstruction by inspissated secretions ultimately leads to hepatic fibrosis and cirrhosis, these investigators presented observations from children with CF and cirrhosis and others without liver disease suggesting that subjects with cirrhosis have a pathogenic dysbiosis that, coupled with increased intestinal permeability, mucosal inflammation, and altered intestinal transit, sets the stage for the translocation of profibrotic bacterial factors into the portal circulation and consequent portal fibrosis (Fig. 1). This hypothesis is supported by observations by Blanco et al., who showed that induction of colitis in the CF mouse leads to the development of liver disease.82 Therefore, addressing CF GI dysbiosis could impact not only GI inflammation, but also risk of developing liver disease. There are also good reasons to hypothesize that the CF GI microbiota contribute to one of the most troubling manifestations of CF in children: nutritional failure. As described above, the GI microbiota are important for harvesting and metabolizing nutrients for the host, and studies of both humans83 and animals84 with malnourishment have demonstrated key roles for the microbiota in body habitus. Antibiotic treatment is also known to lead to weight gain in mice85 and people86 with CF. Furthermore, nutritional outcomes are known to be maddeningly variable and unpredictable in CF, suggesting that factors beyond inadequate supplementation or genetics play a part. Pediatric Pulmonology

S40

Rogers et al.

Fig. 1. A model of pathways thought to link the GI microbiome and CF-related pathophysiology.

EFFECTS OF CURRENT AND POTENTIAL TREATMENTS ON CF GI MICROBIOTA

Given the deleterious health impact of dybiosis, promotion of a more beneficial GI microbiome is an important therapeutic goal, particularly in patients who are already under immune and metabolic stress. In mice, restoration of the gut microbiota through fecal microbiota transplantation (FMT) following antibiotic ablation leads to normalization of respiratory immune function and protection against respiratory Streptococcus pneumoniae infection.87 While FMT may be impractical in the context of chronic respiratory disease, the introduction of preparations of live potentially beneficial microbes, or probiotics, has been shown to have immunomodulatory Pediatric Pulmonology

effects through direct interaction with host cells. For example, in patients with CF, pilot studies suggest administration of Lactobacillus GG or a multispecies probiotic results in reduced rates of pulmonary exacerbation.88,89 However, while probiotic modulation of host immunity can be effective, particularly where commensal microbiota are substantially depleted, it does not fulfill the many other beneficial functions of a diverse commensal microbiota. As mentioned above, the composition of the GI microbiota and its metabolic output can be more readily altered by changes in diet.43 Prebiotics, specific nutrients (particularly fermentable oligo- and polysaccharides) that promote the proliferation of commensal species or their production of beneficial metabolites, have been shown to be effective in reversing dysbioses.90 Such changes result


in altered metabolic and immune-modulatory properties of the microbiome, including reducing the abundance of Proteobacteria90 and increasing production of SCFAs.91 Typically, prebiotics are inexpensive, readily available, and already approved for dietary use. As such, the potential for prebiotics to provide therapeutic benefit in CF warrants further investigation. Further, pre- and pro-biotics can be combined in symbiotic preparations, potentially important where the commensal GI microbiota have been depleted, for example, through antibiotic exposure. There is also a suggestion that early antibiotic treatments may have more global effects on health in CF. In a large prospective study of children with CF who underwent screening liver ultrasounds, early (2 years of age) Pseudomonas infection, which usually results in more antibiotic exposure, was protective for the findings of an abnormal ultrasound.92 This finding provides further evidence that early alterations of the GI microbiota may have long-term impacts distant from the intestinal tract. Given the critical importance of host-microbiota interactions during early childhood, the potential benefits of supporting the parallel development of GI health and microbiota through early (i.e., during infancy) probiotic or prebiotic supplementation warrant careful consideration. The immunomodulatory qualities of bacterial taxa able to ferment the milk oligosaccharides that are so important for shaping the early microbiome differ substantially,93 suggesting that introducing more beneficial strains in the form of probiotic preparations may preclude the need for environmental acquisition by chance. Further, supplementation with prebiotics that are able to offset altered intestinal physicochemical conditions may provide additional support for microbiota establishment, for example by improving stool frequency and consistency.94–96 Early life microbiome modification through dietary supplementation might have additional advantages, such as reducing the incidence of intestinal obstruction and other gastrointestinal complications, and increased energy and nutrient harvest.

S41

Beneficial changes in the GI microbiome may also arise as a result of potentiator and corrector therapies that target CFTR dysfunction directly.97,98 Evidence of resolution of intestinal histopathology with CFTR modulator therapy99 supports the impact of these treatments on the GI lumen. The close relationship between the intestinal environment and the GI microbiota means that changes in factors such as mucin hydration, fat absorption, or luminal transit are likely to result in substantial changes in microbiota composition and function. The associated reduction in proinflammatory pathways and an increase in beneficial immune-modulatory behaviors could result in significant microbiota-mediated beneficial effects on inflammation and disease at many mucosal surfaces. To date, we could find no published reports of the effects of CFTR modulators on GI microbiota. SUMMARY, CONCLUSIONS, AND FUTURE DIRECTIONS

There is ample evidence that the microbes that inhabit the GI tracts of people with CF are not simply passive spectators, nor are they impervious to the altered GI luminal physical conditions and many treatments that they encounter. The CF GI microbiome is characterized by alterations in taxonomic makeup, in behavior, and even in microbial density compared with people without CF, and a growing body of evidence suggests a schema involving markedly complex interactions between the GI microbiota, therapy, and multiorgan disease manifestations of this genetic disorder (Fig. 1). Beyond simply providing a new perspective on CF disease pathogenesis, these models also provide promising new avenues for therapeutic interventions that aim to improve many measures of health by targeting the CF GI microbiota. However, many questions remain regarding the makeup and roles of the GI microbiota in CF; examples of some of the most important questions for future research are listed in (Table 1).

TABLE 1— Future Research Directions for the CF GI Microbiome Disease pathogenesis: What is the relationship between the GI microbiota and nutritional failure? Does inflammation drive dysbiosis, vice-versa, or both? Does dysbiosis cause development of liver disease? Does dysbiosis contribute to lung disease? Does altered intestinal peristalsis cause or reflect bacterial overgrowth? Mechanisms of dysbiosis: What are the relative roles of antibiotics and malabsorption in shaping the CF GI microbiota? How do alterations in intestinal mucus, pH, and other chemical changes contribute to dysbiosis? Therapeutic interventions: Are there antibiotic, probiotic, prebiotic, or other therapies that could improve CF outcomes through the GI microbiota? Do antibiotics improve nutritional and/or metabolic outcomes through the GI microbiota? How do laxatives impact the CF GI microbiota? How do CFTR modulators affect the GI microbiota?

Pediatric Pulmonology

S42

Rogers et al.

ACKNOWLEDGMENTS

The authors would like to thank the patients and participants of the many studies who contributed to the concepts in this manuscript. LRH was supported by a grant from the NIH (1R01DK095869-01A1 and P30 DK089507). REFERENCES 1. Borowitz D, Gelfond D. Intestinal complications of cystic fibrosis. Curr Opin Pulm Med 2013;19:676–680. 2. De Lisle RC, Borowitz D. The cystic fibrosis intestine. Cold Spring Harb Perspect Med 013;3:a009753. 3. Madan JC. Neonatal gastrointestinal and respiratory microbiome in cystic fibrosis: potential interactions and implications for systemic health. Clin Ther 2016;38:740–746. 4. Scanlan PD, Buckling A, Kong W, Wild Y, Lynch SV, Harrison F. Gut dysbiosis in cystic fibrosis. J Cyst Fibros 2012;11:454–455. 5. Hoffman LR, Pope CE, Hayden HS, Heltshe S, Levy R, McNamara S, Jacobs MA, Rohmer L, Radey M, Ramsey BW, et al. Escherichia coli dysbiosis correlates with gastrointestinal dysfunction in children with cystic fibrosis. Clin Infect Dis 2014; 58:396–399. 6. Manor O, Levy R, Pope CE, Hayden HS, Brittnacher MJ, Carr R, Radey MC, Hager KR, Heltshe SL, Ramsey BW, et al. Metagenomic evidence for taxonomic dysbiosis and functional imbalance in the gastrointestinal tracts of children with cystic fibrosis. Sci Rep 2016;6:22493. 7. Duytschaever G, Huys G, Bekaert M, Boulanger L, De Boeck K, Vandamme P. Dysbiosis of Bifidobacteria and Clostridium cluster XIVa in the cystic fibrosis fecal microbiota. J Cyst Fibros 2013;12:206–215. 8. Bruzzese E, Callegari ML, Raia V, Viscovo S, Scotto R, Ferrari S, Morelli L, Buccigrossi V, Lo Vecchio A, Ruberto E, et al. Disrupted intestinal microbiota and intestinal inflammation in children with cystic fibrosis and its restoration with Lactobacillus GG: a randomised clinical trial. PLoS ONE 2014;9:e87796. 9. Dominguez-Bello MG, Costello EK, Contreras M, Magris M, Hidalgo G, Fierer N, Knight R. Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns. Proc Natl Acad Sci USA 2010;107: 11971–11975. 10. Zhang Z, Geng J, Tang X, Fan H, Xu J, Wen X, Ma ZS, Shi P. Spatial heterogeneity and co-occurrence patterns of human mucosal-associated intestinal microbiota. ISME J 2014;8: 881–893. 11. Stearns JC, Lynch MDJ, Senadheera DB, Tenenbaum HC, Goldberg MB, Cvitkovitch DG, Croitoru K, Moreno-Hagelsieb G, Neufeld JD. Bacterial biogeography of the human digestive tract. Sci Rep 2011;1:170. 12. Marcobal A, Barboza M, Sonnenburg ED, Pudlo N, Martens EC, Desai P, Lebrilla CB, Weimer BC, Mills DA, German JB, et al. Bacteroides in the infant gut consume milk oligosaccharides via mucus-utilization pathways. Cell Host Microbe 2011;10:507–514. 13. Sela DA, Mills DA. Nursing our microbiota: molecular linkages between bifidobacteria and milk oligosaccharides. Trends Microbiol 2010;18:298–307. 14. Klaassens ES, Boesten RJ, Haarman M, Knol J, Schuren FH, Vaughan EE, de Vos WM. Mixed-species genomic microarray analysis of fecal samples reveals differential transcriptional responses of bifidobacteria in breast- and formula-fed infants. Appl Environ Microbiol 2009;75:2668–2676.


15. Schloss PD, Handelsman J. Status of the microbial census. Microbiol Mol Biol Rev 2004;68:686–691. 16. O’Hara AM, Shanahan F. The gut flora as a forgotten organ. EMBO Rep 2006;7:688–693. 17. Krajmalnik-Brown R, Ilhan Z-E, Kang D-W, DiBaise JK. Effects of gut microbes on nutrient absorption and energy regulation. Nutr Clin Pract 2012;27:201–214. 18. Kamada N, Chen GY, Inohara N, Nun˜ez G. Control of pathogens and pathobionts by the gut microbiota. Nat Immunol 2013;14: 685–690. 19. Kelly CJ, Zheng L, Campbell EL, Saeedi B, Scholz CC, Bayless AJ, Wilson KE, Glover LE, Kominsky DJ, Magnuson A, et al. Crosstalk between microbiota-derived short-chain fatty acids and intestinal epithelial HIF augments tissue barrier function. Cell Host Microbe 2015;17:662–671. 20. Louis P, Flint HJ. Diversity, metabolism and microbial ecology of butyrate-producing bacteria from the human large intestine. FEMS Microbiol Lett 2009;294:1–8. 21. Yano JM, Yu K, Donaldson GP, Shastri GG, Ann P, Ma L, Nagler CR, Ismagilov RF, Mazmanian SK, Hsiao EY. Indigenous bacteria from the gut microbiota regulate host serotonin biosynthesis. Cell 2015;161:264–276. 22. Joyce SA, MacSharry J, Casey PG, Kinsella M, Murphy EF, Shanahan F, Hill C, Gahan CGM. Regulation of host weight gain and lipid metabolism by bacterial bile acid modification in the gut. Proc Natl Acad Sci USA 2014;111:7421–7426. 23. Jarchum I, Pamer EG. Regulation of innate and adaptive immunity by the commensal microbiota. Curr Opin Immunol 2011;23: 353–360. 24. Chang C, Lin H. Dysbiosis in gastrointestinal disorders. Best Pract Res Clin Gastroenterol 2016;30:3–15. 25. Goel A, Gupta M, Aggarwal R. Gut microbiota and liver disease: gut microbiota and liver disease. J Gastroenterol Hepatol 2014;29: 1139–1148. 26. Schnabl B, Brenner DA. Interactions between the intestinal microbiome and liver diseases. Gastroenterology 2014;146: 1513–1524. 27. Yoshimoto S, Loo TM, Atarashi K, Kanda H, Sato S, Oyadomari S, Iwakura Y, Oshima K, Morita H, Hattori M, et al. Obesityinduced gut microbial metabolite promotes liver cancer through senescence secretome. Nature 2013;499:97–101. 28. Cani PD, Amar J, Iglesias MA, Poggi M, Knauf C, Bastelica D, Neyrinck AM, Fava F, Tuohy KM, Chabo C, et al. Metabolic endotoxemia initiates obesity and insulin resistance. Diabetes 2007;56:1761–1772. 29. Mouzaki M, Comelli EM, Arendt BM, Bonengel J, Fung SK, Fischer SE, McGilvray ID, Allard JP. Intestinal microbiota in patients with nonalcoholic fatty liver disease. Hepatology 2013; 58:120–127. 30. Vijay-Kumar M, Aitken JD, Carvalho FA, Cullender TC, Mwangi S, Srinivasan S, Sitaraman SV, Knight R, Ley RE, Gewirtz AT. Metabolic syndrome and altered gut microbiota in mice lacking Toll-like receptor 5. Science 2010;328: 228–231. 31. Serino M, Blasco-Baque V, Nicolas S, Burcelin R. Far from the eyes, close to the heart: dysbiosis of gut microbiota and cardiovascular consequences. Curr Cardiol Rep 2014;16:540. 32. Mira-Pascual L, Cabrera-Rubio R, Ocon S, Costales P, Parra A, Suarez A, Moris F, Rodrigo L, Mira A, Collado MC. Microbial mucosal colonic shifts associated with the development of colorectal cancer reveal the presence of different bacterial and archaeal biomarkers. J Gastroenterol 2015;50:167–179. 33. Rogers GB, Keating DJ, Young RL, Wong M-L, Licinio J, Wesselingh S. From gut dysbiosis to altered brain function and


34.

35.

36.

37.

38.

39. 40.

41.

42.

43.

44.

45.

46.

47. 48. 49.

50.

mental illness: mechanisms and pathways. Mol Psychiatry 2016;21:738–748. Boursier J, Mueller O, Barret M, Machado M, Fizanne L, Araujo-Perez F, Guy CD, Seed PC, Rawls JF, David LA, et al. The severity of nonalcoholic fatty liver disease is associated with gut dysbiosis and shift in the metabolic function of the gut microbiota. Hepatology 2016;63:764–775. Wood NJ. Microbiota: dysbiosis driven by inflammasome deficiency exacerbates hepatic steatosis and governs rate of NAFLD progression. Nat Rev Gastroenterol Hepatol 2012; 9:123. Lurie I, Yang Y-X, Haynes K, Mamtani R, Boursi B. Antibiotic exposure and the risk for depression, anxiety, or psychosis: a nested case-control study. J Clin Psychiatry 2015;76:1522–1528. Kaplan JL, Shi HN, Walker WA. The role of microbes in developmental immunologic programming. Pediatr Res 2011;69:465–472. Karmarkar D, Rock KL. Microbiota signalling through MyD88 is necessary for a systemic neutrophilic inflammatory response. Immunology 2013;140:483–492. Goulet O. Potential role of the intestinal microbiota in programming health and disease. Nutr Rev 2015;73:32–40. Zeissig S, Blumberg RS. Life at the beginning: perturbation of the microbiota by antibiotics in early life and its role in health and disease. Nat Immunol 2014;15:307–310. Russell SL, Gold MJ, Reynolds LA, Willing BP, Dimitriu P, Thorson L, Redpath SA, Perona-Wright G, Blanchet M-R, Mohn WW, et al. Perinatal antibiotic-induced shifts in gut microbiota have differential effects on inflammatory lung diseases. J Allergy Clin Immunol 2015;135:100–109. Biesbroek G, Tsivtsivadze E, Sanders EAM, Montijn R, Veenhoven RH, Keijser BJF, Bogaert D. Early respiratory microbiota composition determines bacterial succession patterns and respiratory health in children. Am J Respir Crit Care Med 2014;190:1283–1292. David LA, Maurice CF, Carmody RN, Gootenberg DB, Button JE, Wolfe BE, Ling AV, Devlin AS, Varma Y, Fischbach MA, et al. Diet rapidly and reproducibly alters the human gut microbiome. Nature 2013;505:559–563. Carmody RN, Gerber GK, Luevano JM, Gatti DM, Somes L, Svenson KL, Turnbaugh PJ. Diet dominates host genotype in shaping the murine gut microbiota. Cell Host Microbe 2015;17:72–84. De Filippo C, Cavalieri D, Di Paola M, Ramazzotti M, Poullet JB, Massart S, Collini S, Pieraccini G, Lionetti P. Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa. Proc Natl Acad Sci USA 2010;107:14691–14696. Backhed F, Ley RE, Sonnenburg JL, Peterson DA, Gordon JI. Host-bacterial mutualism in the human intestine. Science 2005;307:1915–1920. Modi SR, Collins JJ, Relman DA. Antibiotics and the gut microbiota. J Clin Invest 2014;124:4212–4218. Lange K, Buerger M, Stallmach A, Bruns T. Effects of antibiotics on gut microbiota. Digest Dis 2016;34:260–268. Bazett M, Bergeron M-E, Haston CK. Streptomycin treatment alters the intestinal microbiome, pulmonary T cell profile and airway hyperresponsiveness in a cystic fibrosis mouse model. Sci Rep 2016;6:19189. Goddard AF, Staudinger BJ, Dowd SE, Joshi-Datar A, Wolcott RD, Aitken ML, Fligner CL, Singh PK. Direct sampling of cystic fibrosis lungs indicates that DNA-based analyses of upper-airway specimens can misrepresent lung microbiota. Proc Natl Acad Sci USA 2012;109:13769–13774.

S43

51. Caverly LJ, Zhao J, LiPuma JJ. Cystic fibrosis lung microbiome: opportunities to reconsider management of airway infection. Pediatr Pulmonol 2015;50:S31–S38. 52. Chmiel JF, Aksamit TR, Chotirmall SH, Dasenbrook EC, Elborn JS, LiPuma JJ, Ranganathan SC, Waters VJ, Ratjen FA. Antibiotic management of lung infections in cystic fibrosis. I. The microbiome, methicillin-resistant Staphylococcus aureus, gramnegative bacteria, and multiple infections. Ann Am Thorac Soc 2014;11:1120–1129. 53. Huang YJ, LiPuma JJ. The microbiome in cystic fibrosis. Clin Chest Med 2016;37:59–67. 54. Zhao J, Murray S, Lipuma JJ. Modeling the impact of antibiotic exposure on human microbiota. Sci Rep 2014;4:4345. 55. Coburn B, Wang PW, Diaz Caballero J, Clark ST, Brahma V, Donaldson S, Zhang Y, Surendra A, Gong Y, Elizabeth Tullis D, et al. Lung microbiota across age and disease stage in cystic fibrosis. Sci Rep 2015;5:10241. 56. Flass T, Tong S, Frank DN, Wagner BD, Robertson CE, Kotter CV, Sokol RJ, Zemanick E, Accurso F, Hoffenberg EJ, et al. Intestinal lesions are associated with altered intestinal microbiome and are more frequent in children and young adults with cystic fibrosis and cirrhosis. PLoS ONE 2015;10:e0116967. 57. Norkina O, Kaur S, Ziemer D, De Lisle RC. Inflammation of the cystic fibrosis mouse small intestine. Am J Physiol Gastrointest Liver Physiol 2004;286:G1032–G1041. 58. van Elburg RM, Uil JJ, van Aalderen WM, Mulder CJ, Heymans HS. Intestinal permeability in exocrine pancreatic insufficiency due to cystic fibrosis or chronic pancreatitis. Pediatr Res 1996;39:985–991. 59. Werlin SL, Benuri-Silbiger I, Kerem E, Adler SN, Goldin E, Zimmerman J, Malka N, Cohen L, Armoni S, Yatzkan-Israelit Y, et al. Evidence of intestinal inflammation in patients with cystic fibrosis. J Pediatr Gastroenterol Nutr 2010;51:304–308. 60. Flass T, Narkewicz MR. Cirrhosis and other liver disease in cystic fibrosis. J Cyst Fibros 2013;12:116–124. 61. Grubb BR, Gabriel SE. Intestinal physiology and pathology in gene-targeted mouse models of cystic fibrosis. Am J Physiol 1997;273:G258–G266. 62. Norkina O, De Lisle RC. Potential genetic modifiers of the cystic fibrosis intestinal inflammatory phenotype on mouse chromosomes 1, 9, and 10. BMC Genet 2005;6:29. 63. Bazett M, Honeyman L, Stefanov AN, Pope CE, Hoffman LR, Haston CK. Cystic fibrosis mouse model-dependent intestinal structure and gut microbiome. Mamm Genome 2015;26:222–234. 64. Norkina O, Burnett TG, De Lisle RC. Bacterial overgrowth in the cystic fibrosis transmembrane conductance regulator null mouse small intestine. Infect Immun 2004;72:6040–6049. 65. Lynch SV, Goldfarb KC, Wild YK, Kong W, De Lisle RC, Brodie EL. Cystic fibrosis transmembrane conductance regulator knockout mice exhibit aberrant gastrointestinal microbiota. Gut Microbes 2013;4:41–47. 66. Sun X, Olivier AK, Yi Y, Pope CE, Hayden HS, Liang B, Sui H, Zhou W, Hager KR, Zhang Y, et al. Gastrointestinal pathology in juvenile and adult CFTR-knockout ferrets. Am J Pathol 2014;184:1309–1322. 67. Meyerholz DK, Stoltz DA, Pezzulo AA, Welsh MJ. Pathology of gastrointestinal organs in a porcine model of cystic fibrosis. Am J Pathol 2010;176:1377–1389. 68. Stoltz DA, Rokhlina T, Ernst SE, Pezzulo AA, Ostedgaard LS, Karp PH, Samuel MS, Reznikov LR, Rector MV, Gansemer ND, et al. Intestinal CFTR expression alleviates meconium ileus in cystic fibrosis pigs. J Clin Invest 2013;123:2685–2693. 69. Rogers CS, Stoltz DA, Meyerholz DK, Ostedgaard LS, Rokhlina T, Taft PJ, Rogan MP, Pezzulo AA, Karp PH, Itani OA, et al.


S44

70.

71.

72.

73.

74.

75. 76.

77.

78. 79.

80.

81.

82.

83.

Rogers et al. Disruption of the CFTR gene produces a model of cystic fibrosis in newborn pigs. Science 2008;321:1837–1841. Olivier AK, Gibson-Corley KN, Meyerholz DK. Animal models of gastrointestinal and liver diseases. Animal models of cystic fibrosis: gastrointestinal, pancreatic, and hepatobiliary disease and pathophysiology. Am J Physiol Gastrointest Liver Physiol 2015;308:G459–G471. Duytschaever G, Huys G, Bekaert M, Boulanger L, De Boeck K, Vandamme P. Cross-sectional and longitudinal comparisons of the predominant fecal microbiota compositions of a group of pediatric patients with cystic fibrosis and their healthy siblings. Appl Environ Microbiol 2011;77:8015–8024. Duytschaever G, Huys G, Boulanger L, De Boeck K, Vandamme P. Amoxicillin-clavulanic acid resistance in fecal Enterobacteriaceae from patients with cystic fibrosis and healthy siblings. J Cyst Fibros 2013;12:780–783. Schippa S, Iebba V, Santangelo F, Gagliardi A, De Biase RV, Stamato A, Bertasi S, Lucarelli M, Conte MP, Quattrucci S. Cystic fibrosis transmembrane conductance regulator (CFTR) allelic variants relate to shifts in faecal microbiota of cystic fibrosis patients. PLoS ONE 2013;8:e61176. Taylor SL, Wesselingh S, Rogers GB. Host-microbiome interactions in acute and chronic respiratory infections: host-microbe interactions in respiratory disease. Cell Microbiol 2016;18: 652–662. Keely S, Talley NJ, Hansbro PM. Pulmonary-intestinal cross-talk in mucosal inflammatory disease. Mucosal Immunol 2012;5:7–18. Madan JC, Koestler DC, Stanton BA, Davidson L, Moulton LA, Housman ML, Moore JH, Guill MF, Morrison HG, Sogin ML, et al. Serial analysis of the gut and respiratory microbiome in cystic fibrosis in infancy: interaction between intestinal and respiratory tracts and impact of nutritional exposures. mBio 2012;3:e00251-12. Hoen AG, Li J, Moulton LA, O’Toole GA, Housman ML, Koestler DC, Guill MF, Moore JH, Hibberd PL, Morrison HG, et al. Associations between gut microbial colonization in early life and respiratory outcomes in cystic fibrosis. J Pediatr 2015;167: 138–147.e1–e3. Bartlett JG. Clinical practice. Antibiotic-associated diarrhea. N Engl J Med 2002;346:334–339. Itoh Z, Suzuki T, Nakaya M, Inoue M, Mitsuhashi S. Gastrointestinal motor-stimulating activity of macrolide antibiotics and analysis of their side effects on the canine gut. Antimicrob Agents Chemother 1984;26:863–869. Del Campo R, Garriga M, Perez-Aragon A, Guallarte P, Lamas A, Maiz L, Bayon C, Roy G, Canton R, Zamora J, et al. Improvement of digestive health and reduction in proteobacterial populations in the gut microbiota of cystic fibrosis patients using a Lactobacillus reuteri probiotic preparation: a double blind prospective study. J Cyst Fibros 2014;13:716–722. Debyser G, Mesuere B, Clement L, Van de Weygaert J, Van Hecke P, Duytschaever G, Aerts M, Dawyndt P, De Boeck K, Vandamme P, et al. Faecal proteomics: a tool to investigate dysbiosis and inflammation in patients with cystic fibrosis. J Cyst Fibros 2016; 15:242–250. Blanco PG, Zaman MM, Junaidi O, Sheth S, Yantiss RK, Nasser IA, Freedman SD. Induction of colitis in cftr / mice results in bile duct injury. Am J Physiol Gastrointest Liver Physiol 2004; 287:G491–G496. Blanton LV, Charbonneau MR, Salih T, Barratt MJ, Venkatesh S, Ilkaveya O, Subramanian S, Manary MJ, Trehan I, Jorgensen JM, et al. Gut bacteria that prevent growth impairments transmitted by microbiota from malnourished children. Science 2016;351:pii: aad3311.


84. Ridaura VK, Faith JJ, Rey FE, Cheng J, Duncan AE, Kau AL, Griffin NW, Lombard V, Henrissat B, Bain JR, et al. Gut microbiota from twins discordant for obesity modulate metabolism in mice. Science 2013;341:1241214. 85. De Lisle RC. Altered transit and bacterial overgrowth in the cystic fibrosis mouse small intestine. Am J Physiol Gastrointest Liver Physiol 2007;293:G104–G111. 86. Dror T, Dickstein Y, Dubourg G, Paul M. Microbiota manipulation for weight change. Microb Pathog 2016 pii: S0882-4010(15) 30206-0. DOI: 10.1016/j.micpath.2016.01.002 [Epub ahead of print]. 87. Schuijt TJ, Lankelma JM, Scicluna BP, de Sousa Melo EF, Roelofs JJTH, de Boer JD, Hoogendijk AJ, de Beer R, de Vos A, Belzer C, et al. The gut microbiota plays a protective role in the host defence against pneumococcal pneumonia. Gut 2016;65:575–583. 88. Bruzzese E, Raia V, Spagnuolo MI, Volpicelli M, De Marco G, Maiuri L, Guarino A. Effect of Lactobacillus GG supplementation on pulmonary exacerbations in patients with cystic fibrosis: a pilot study. Clin Nutr 2007;26:322–328. 89. Weiss B, Bujanover Y, Yahav Y, Vilozni D, Fireman E, Efrati O. Probiotic supplementation affects pulmonary exacerbations in patients with cystic fibrosis: a pilot study. Pediatr Pulmonol 2010; 45:536–540. 90. Phillips J, Muir JG, Birkett A, Lu ZX, Jones GP, O’Dea K, Young GP. Effect of resistant starch on fecal bulk and fermentationdependent events in humans. Am J Clin Nutr 1995;62:121–130. 91. Karimi P, Farhangi MA, Sarmadi B, Gargari BP, Zare Javid A, Pouraghaei M, Dehghan P. The therapeutic potential of resistant starch in modulation of insulin resistance, endotoxemia, oxidative stress and antioxidant biomarkers in women with type 2 diabetes: a randomized controlled clinical trial. Ann Nutr Metab 2016;68: 85–93. 92. Leung DH, Ye W, Molleston JP, Weymann A, Ling S, Paranjape SM, Romero R, Schwarzenberg SJ, Palermo J, Alonso EM, et al. Baseline ultrasound and clinical correlates in children with cystic fibrosis. J Pediatr 2015;167:862–868.e2. 93. Vatanen T, Kostic AD, d’Hennezel E, Siljander H, Franzosa EA, Yassour M, Kolde R, Vlamakis H, Arthur TD, H€am€al€ainen A-M, et al. Variation in microbiome LPS immunogenicity contributes to autoimmunity in humans. Cell 2016;165:842–853. 94. Westerbeek EAM, Hensgens RL, Mihatsch WA, Boehm G, Lafeber HN, van Elburg RM. The effect of neutral and acidic oligosaccharides on stool viscosity, stool frequency and stool pH in preterm infants. Acta Paediatr 2011;100:1426–1431. 95. Oozeer R, van Limpt K, Ludwig T, Ben Amor K, Martin R, Wind RD, Boehm G, Knol J. Intestinal microbiology in early life: specific prebiotics can have similar functionalities as human-milk oligosaccharides. Am J Clin Nutr 2013;98:561S–571S. 96. Holscher HD, Faust KL, Czerkies LA, Litov R, Ziegler EE, Lessin H, Hatch T, Sun S, Tappenden KA. Effects of prebiotic-containing infant formula on gastrointestinal tolerance and fecal microbiota in a randomized controlled trial. JPEN J Parenter Enteral Nutr 2012;36:95S–105S. 97. Wainwright CE, Elborn JS, Ramsey BW. Lumacaftor-ivacaftor in patients with cystic fibrosis homozygous for phe508del CFTR. N Engl J Med 2015;373:1783–1784. 98. Ramsey BW, Davies J, McElvaney NG, Tullis E, Bell SC, Drevı´nek P, Griese M, McKone EF, Wainwright CE, Konstan MW, et al. A CFTR potentiator in patients with cystic fibrosis and the G551D mutation. N Engl J Med 2011;365:1663–1672. 99. Safe M, Gifford AJ, Jaffe A, Ooi CY. Resolution of intestinal histopathology changes in cystic fibrosis after treatment with ivacaftor. Ann Am Thorac Soc 2016;13:297–298.