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A comparison of small bowel and fecal microbiota in children with short bowel syndrome
YJPSU-59576; No of Pages 5 Journal of Pediatric Surgery xxx (xxxx) xxx
Contents lists available at ScienceDirect
Journal of Pediatric Surgery journal homepage: www.elsevier.com/locate/jpedsurg
A comparison of small bowel and fecal microbiota in children with short bowel syndrome☆ Hannah G. Piper a,⁎, Laura A. Coughlin b, Van Nguyen b, Nandini Channabasappa b, Andrew Y. Koh b,c,d a
Department of Surgery, University of British Columbia, Vancouver, BC, Canada Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, USA Harold C. Simmons Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX, USA d Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX, USA b c
a r t i c l e
i n f o
Article history: Received 18 January 2020 Accepted 25 January 2020 Available online xxxx Key words: Short bowel syndrome Intestinal microbiota Small intestinal bacterial overgrowth
a b s t r a c t Background: Babies with short bowel syndrome (SBS) have small intestinal microbial disturbances that impact gut function. Characterizing the small bowel microbiota is challenging, and the utility of sampling stool is unclear. This study compares the microbiota from fecal samples and the small bowel. Methods: Stool samples were collected (2016–2017) from infants with SBS and colon in continuity (COLON) or SBS with small bowel ostomy (sbSTOMA). The abundance and quantity of major bacterial genera was compared between groups and to healthy controls using 16S rRNA sequencing and qPCR. Kruskall-Wallis test was used for analysis with P values b0.05 considered significant. Results: Samples (n = 41) were collected from 15 SBS infants (b2 years) (9 sbSTOMA, 6 COLON) and 3 healthy infants. Demographics and small intestinal length did not differ between sbSTOMA and COLON infants. The microbiota of SBS groups differed significantly from healthy controls. Fecal samples contained higher quantities of bacteria, but there were no significant differences between sbSTOMA and COLON groups in the abundance of facultative or obligate anaerobes, anti-inflammatory Clostridia, Enterobacteriaceae, or Bifidobacterium. Conclusion: Infants with SBS have disturbances to their intestinal microbiota. Sampling small intestinal effluent is challenging. Stool samples may provide a window into the more proximal microbial community. Type of Study: Diagnostic. Level of Evidence: Level II. © 2020 Elsevier Inc. All rights reserved.
The impact of changes to the intestinal microbiota on overall health in children is becoming increasingly recognized. This is particularly true early in life when the gut microbiota is in transition. In infancy, colonization with commensal bacteria is easily disrupted by external factors including diet and antibiotic exposure which can result in long-term alterations [1,2]. Infants who undergo significant intestinal resection with resultant short bowel syndrome (SBS) are at particularly high risk for disturbances to microbial homeostasis within the gut [3,4]. After intestinal resection babies are frequently exposed to prolonged antibiotic therapy, are restricted in their enteral intake, and have alterations to their intestinal anatomy resulting in some degree of intestinal dilation over time - all of which can predispose to microbial dysbiosis. One of the long-term concerns for infants and children with SBS is the development of small intestinal bacterial overgrowth (SIBO). Traditionally SIBO ☆ Declarations of interest: none. ⁎ Corresponding author at: University of British Columbia, Division of Pediatric Surgery, 4480 Oak Street, Vancouver, BC, V6H 3V4, Canada. Tel.: +1 604 875 3744; fax: +1 604 875 2721. E-mail address: [email protected] (H.G. Piper).
is defined as 105 CFU/mL of bacteria in the small bowel, or N 103 CFU/mL of bacterial species that normally populate the colon [6]. SIBO can occur at any time, although intestinal dilation and poor motility are identified risk factors. When present, SIBO places children at risk for bacterial bloodstream infection, poor absorption and growth, and D-lactic acidosis [6,7]. However, making a quantitative diagnosis of SIBO is challenging and frequently impractical relying on upper endoscopy or breath testing both of which have inherent inaccuracies [8]. More commonly, the diagnosis is based primarily on clinical symptoms including bloating, diarrhea and abdominal pain, and is treated empirically. This is problematic in that many children are exposed to long-term antibiotics without a quantitative means of assessing efficacy. The idea of using stool samples, which are much easier to obtain, to characterize the microbiota of the more proximal small bowel, has been questioned because it is unclear if the stool provides an accurate representation of the bacterial community in the small bowel. However, many of these patients have rapid intestinal transit and a loss of the normal physiologic breaks between the small and large bowel. In addition, there is some evidence that SBS patients with symptoms of overgrowth do have distinct changes to their distal gut microbiota as characterized by
https://doi.org/10.1016/j.jpedsurg.2020.01.032 0022-3468/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: H.G. Piper, L.A. Coughlin, V. Nguyen, et al., A comparison of small bowel and fecal microbiota in children with short bowel syndrome, Journal of Pediatric Surgery, https://doi.org/10.1016/j.jpedsurg.2020.01.032
2
H.G. Piper et al. / Journal of Pediatric Surgery xxx (xxxx) xxx
16S rRNA analysis of bacterial populations from stool samples [5]. The purpose of this study was to determine if the microbiota from stool samples differs significantly from samples obtained from more proximal small bowel stomas in infants with short bowel syndrome. If small bowel stoma samples show a similar microbiota composition, stool samples could potentially be used more reliably to detect changes to the microbiota more proximally, and to guide therapeutic interventions.
Sequences were quality filtered and then analyzed using the open source software package Quantitative Insights Into Microbial Ecology (QIIME). 16S rRNA gene sequences were assigned to operational taxonomic units (OTUs) using UCLUST (www.drive5.com/usearch), with a threshold of 97% pair-wise identify, then classified taxonomically using Greengenes (greengenes.lbl.gov). 1.3. Statistical analyses
1. Methods 1.1. Patients and Study Design This was a prospective cohort study using 16S rRNA sequencing to compare the intestinal microbiota in infants with SBS with either a small bowel stoma or intact colon. All subjects were enrolled under the IRB approved human study protocol (122015–032). Infants (less than 24 months of age) were eligible for the study if they required parenteral nutrition (PN) for at least 3 months secondary to intestinal loss and continued to require PN support for the duration of the study. Patients were recruited from the Center for Intestinal Rehabilitation. Enrolled infants with SBS were further categorized by intestinal anatomy. Patients with a small bowel ostomy (including jejunostomy or ileostomy) were included in the stoma group and patients who did not have a small bowel stoma and had not previously undergone a colonic resection, were included in the intact colon group. Clinical characteristics between groups were compared. Stool samples were collected monthly over three months when possible. At each time point patient anthropometrics including weight, length and head circumference were recorded. Expected and median values were obtained from the World Health Organization growth charts. Nutritional intake from both parenteral and enteral routes and serum markers of hepatic cholestasis were documented at all time points. Additionally, stool samples were collected from 3 healthy control infants with intact intestinal tracts to which samples from both groups of infants with SBS were compared. 1.2. Isolation of fecal gDNA and 16S rRNA analysis Fresh samples were collected and stored at −80 °C for further analysis. Bacterial gDNA was isolated from fecal or stomal specimens as previously described [9]. 16S rRNA genes (variable region 4, V4) were amplified from each sample using a composite forward primer and a reverse primer containing a unique 10-base barcode that was used to tag PCR products from respective samples [9]. Pooled PCR products were sequenced using the Illumina HiSeq 2000.
A comparison of bacterial taxonomic abundance, alpha-diversity metrics, and bacterial 16S rRNA abundance were analyzed by Mann– Whitney or Kruskall-Wallis tests, and when multiple comparisons were analyzed, Bonferroni's correction with the significance level α = 0.05 was invoked. Clinical correlate analysis was performed using Chi-square test and Fischer's exact test. Statistical analyses were carried out using the GraphPad Prism Software (San Diego, CA). 1.4. qPCR for Microbiota Analysis The quantity of bacteria present in representative samples (first collected sample) from both groups of infants with SBS were compared using qPCR analysis (SsoAdvanced SYBR Green Supermix, BioRad, Hercules, CA) using group-specific 16S rRNA gene primers, as previously described [9,10]: Eubacteria (EUBAC), all bacteria; Enterobacteriaceae (ENTERO); Clostridium leptum group (CLEPT), Clostridial Cluster IV; and Clostridium coccoides-Eubacterium rectale group (EREC). Bacterial abundance was determined using standard curves constructed with reference to cloned DNA corresponding to a short segment of the 16S rRNA gene that was amplified using conserved specific primers. Measures are in gene copies/g and not colony-forming units. 2. Results A total of 15 infants, with SBS, younger than 2 years of age, were enrolled between 2016 and 2017. This included 9 infants with small bowel stomas (sbSTOMA) and 6 infants with intact colons (COLON). Within the two groups there were no significant differences in gestational age at birth, age at enrollment, or small bowel length. All of the infants with intact colon had the ileocecal valve in continuity, compared to none of the infants with small bowel stomas. Both groups had similar total and direct bilirubin levels at the start of the study, however the sbSTOMA group had a higher mean GGT level (Table 1). All of the Infants were receiving a portion of their calories from PN, however those with sbSTOMA were receiving fewer enteral calories compared to the
Table 1 Clinical Characteristics of SBS patients with small bowel stoma and intact colon.
Total Patients Male (%) Median GA in weeks at birth (range) Median age in months at enrollment (range) Diagnosis leading to SBS (n) NEC Atresia Volvulus Anatomy Median small bowel length (range) Median % of expected length based on GA (range) Ileocecal valve in continuity (%) Markers of cholestasis at study start Mean total/direct bilirubin (mg/dL) Mean AST (U/L) Mean ALT (U/L) Mean GGT (U/L) Mean AP (U/L)
Sb stoma
Intact colon
9 4 (44) 32 (23–38) 4 (1–15)
6 0 (0) 32 (24–38) 10 (3–22)
p value
(5) 56% (3) 33% (1) 11%
(3) 50% (3) 50% (0) 0%
53 cm (40–140 cm) 44 (5–75) 0 (0)
47 cm (25–84 cm) 28 (10–57) 6 (100%)
0.44 0.58 0.00
6.0/5.0 135 112 119 381
3.3/2.5 95 117 46 327
0.43 0.42 0.93 0.01 0.52
0.06 0.81 0.07 0.72
Statistical analyses by t-test, GA = gestational age, ICV = ileocecal valve, AST = aspartate aminotransferase, ALT = alanine aminotransferase, GGT = gamma-glutamyl transferase, AP = alkaline phosphatase.
Please cite this article as: H.G. Piper, L.A. Coughlin, V. Nguyen, et al., A comparison of small bowel and fecal microbiota in children with short bowel syndrome, Journal of Pediatric Surgery, https://doi.org/10.1016/j.jpedsurg.2020.01.032
H.G. Piper et al. / Journal of Pediatric Surgery xxx (xxxx) xxx
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Table 2 Nutritional and Anthropometric Parameters.
Nutrition at study start Mean calories given as PN (kcal/kg/d) (range) Mean % PN of total caloric intake PN composition (mean) Lipid (g/kg/d) Glucose infusion rate (mg/kg/min) Protein (g/kg) Mean Z-score weight Mean Z-score length Mean Z-score weight for length
Sb stoma
Intact colon
P value
N=9 77 (29–104) 79
N=6 49 (27–97) 52
0.04 0.05
1.3 11.2 3.4 −0.94 −2.21 −0.26
1.0 9.2 2.1 −0.68 −0.90 0.17
0.26 0.31 0.03 0.18 0.06 0.93
Statistical analysis by t-test, PN = parenteral nutrition.
COLON group. There were no significant differences in anthropometrics between groups (Table 2). One patient in each group received antibiotics during the study. Twenty-five sbSTOMA samples and 17 COLON samples were collected over three months. Following 16S rRNA sequencing, both groups were found to have similar abundance of the major represented bacteria including anti-inflammatory Clostrida (defined as Clostridiaceae, Erysipelotrichaceae, Eubacteriaceae, Lachnospiraceae and Ruminococcaceae) Enterobacteriaceae, Bifidobacterium, Eubacterium and Lactobacillaceae (Figs. 1, Table 3). Additionally, when comparing the relative abundance of obligate and facultative anaerobes, no significant differences were seen between the two groups (Fig. 1). Infants in the sbSTOMA group were found to have an increased abundance of Bacteroides compared to those in the COLON group (28% vs. 15%, p = 0.01, Table 3), however both groups had a significantly reduced abundance compared to healthy children
(44%, p = 0.036, p = 0.001). Healthy controls also had significantly more obligate anaerobes, Bifidobacterium, Eubacterium and Clostridial Firmicutes and significantly fewer Enterobacteriaceae than all SBS patients. When quantitative levels of bacteria in the specimens were compared between the sbSTOMA and COLON groups using qPCR, fecal samples contained significantly higher levels of ENTERO (pro-inflammatory bacteria), CLEPT and EREC (commensal, anti-inflammatory bacteria) and Eubacteria (all bacteria, median of 13.5 X 10 8 vs. 29.5 X 10 10 copies/g, p = 0.036) than those from small bowel stomas (Fig. 2). 3. Discussion This study demonstrates that in infants with short bowel syndrome, both fecal samples and small bowel stoma samples have a similar overall distribution of bacterial phyla. Although the bacterial load was greater in
Fig. 1. Comparison of gut microbial populations in infants with SBS from either small bowel stoma or fecal samples, and healthy controls. Relative abundance of bacterial phyla and families as determined by 16S sequencing collected from infants with SBS and small bowel stoma (sb stoma) or an intact colon in continuity, compared to healthy controls. NS = non significant, ⁎ p b 0.05, ⁎⁎ p b 0.01, ⁎⁎⁎ p b 0.001, Mann–Whitney test.
Please cite this article as: H.G. Piper, L.A. Coughlin, V. Nguyen, et al., A comparison of small bowel and fecal microbiota in children with short bowel syndrome, Journal of Pediatric Surgery, https://doi.org/10.1016/j.jpedsurg.2020.01.032
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H.G. Piper et al. / Journal of Pediatric Surgery xxx (xxxx) xxx
Table 3 Relative Abundance of common intestinal bacterial. Bacterial genus
Sb stoma
Intact colon
P value
Bifidobacterium (%) Enterobacteriaceae (%) Lactobacillaceae (%) Clostridial Firmicutes (%) Eubacterium (%) Bacteroides (%)
20.6 12.7 22.7 8.3 24.7 28.1
18.2 16.7 27.9 7.5 21.5 14.8
0.54 0.23 0.73 1.00 0.81 0.01
Statistical analysis by Kruskal-Wallis test.
fecal samples compared to stoma samples, the relative abundance of genera and families commonly associated with short bowel syndrome was not significantly different between the two groups. Initial studies looking at the intestinal microbiota in children with SBS identified the characteristic findings of increased Proteobacteria (specifically Enterobacteriaceae) and Lactobacillus in this patient population [3,4]. Similarly, a more recent study by Engelstad et al. also found that children with SBS had an overabundance of Proteobacteria, particularly in those with small bowel length b 35 cm [11]. Both Enterobacteriaceae and Lactobacillus had a similar abundance in stoma vs. fecal samples in this study. Interestingly, several of the published series to date use a combination of both stool and stoma samples and do not distinguish between the two when characterizing the microbiota [3,11,12]. Certainly, children with short bowel
syndrome frequently have rapid transit through the small intestine in addition to a foreshortened colon. Additionally, these patients may have lost the ileocecal valve, potentially resulting in a more similar microbial distribution between the distal small bowel and the colon. However, a direct comparison of the microbiota from these two sources has not been extensively studied in children with SBS and has several important clinical implications. In humans it is well known that in the presence of normal intestinal anatomy and motility, the intestinal microbiota changes considerably from the mouth to the anus. In general, there is a gradual transition from mostly gram-positive aerobic and facultative anaerobes in the proximal GI tract, to a more diverse and populated bacterial environment dominated largely by gram-negative anaerobes in the colon [6,13,14]. However, after intestinal resection the transition may be less abrupt or predictable. In a study comparing fecal samples from adults with SBS (average of 90-100 cm of small bowel) who either had a jejunal/colonic anastomosis or a jejunal/ileal anastomosis with intact colon, there was a significantly higher abundance of Enterobacteriaceae in patients with a jejunal/colonic anastomosis (and therefore no ileocecal valve and shorter colon) compared to both those with jejunal/ileal anastomoses and healthy controls [15]. This finding is consistent with the previously published work in children, suggesting that in some subsets of patients with SBS, more proximal intestinal microbiota may not differ significantly from distal samples. Furthermore, in a piglet model of short
Fig. 2. Analysis of absolute levels of bacteria in children with SBS with either a small bowel stoma or intact colon. The absolute levels of all bacteria (Eubacteria), Enterobacteriaceae (ENTERO), Clostridium leptum group (CLEPT), Clostridial Cluster IV; and Clostridium coccoides-Eubacterium rectale group (EREC) were compared as determined by group-specific qPCR (Mann–Whitney test).
Please cite this article as: H.G. Piper, L.A. Coughlin, V. Nguyen, et al., A comparison of small bowel and fecal microbiota in children with short bowel syndrome, Journal of Pediatric Surgery, https://doi.org/10.1016/j.jpedsurg.2020.01.032
H.G. Piper et al. / Journal of Pediatric Surgery xxx (xxxx) xxx
bowel syndrome, the animals were also found to have a high relative abundance of Enterobacteriaceae (up to 50% in some animals) when ileal mucosal scrapings were sequenced [16]. This again suggests that some of the pathologic shifts seen in this population may occur in both the proximal and distal intestine. This is further supported by two recent studies, one that found both jejunal aspirates and stool samples from children with short bowel syndrome had increased abundance of Klebsiella, Enterococcus and Enterobacteriacieae compared to samples from healthy children [17], and the other that demonstrated that Proteobacteria dominated both small bowel stoma effluent and colonic samples in a small group of children with SBS [18]. If fecal samples are representative of the bacterial composition of more proximal intestine, fecal samples could then be used to help with diagnosis and treatment of children who fail to make steady progress towards enteral independence. A significant challenge in caring for children with short bowel syndrome is that much of the intestinal pathology occurs in the small bowel, both initially and later during the intestinal adaptation phase which can continue for several years. However, the small intestine is notoriously difficult to access both for diagnostic and therapeutic purposes. For example, a portion of patients with SBS develop a variety of gastrointestinal symptoms including abdominal bloating, pain, feed intolerance and/or frequent loose stools that are ascribed to overpopulation of the small intestine with primarily colonic bacteria, also known as small intestinal bacterial overgrowth (SIBO). SIBO can increase the risk of infection, result in metabolic abnormalities and make it more challenging to advance enteral nutrition in these patients, However, accurately making this diagnosis by analyzing the small intestinal microbiota is difficult. Proposed diagnostic methods include duodenal aspirates or hydrogen breath testing [8,19,20]. Both strategies have limitations in this patient population, and in a recent study there was poor correlation between breath test results and bacterial numbers both using culture based methods and bacterial DNA sequencing [21]. Most commonly, a clinical diagnosis is made, followed by empiric antibiotic treatment. As we gain more insight into the risks of prolonged antibiotic therapy for children, it would be helpful to have a more practical means of assessing the microbiota in symptomatic patients before and after treatment. Perhaps establishing new diagnostic criteria for SIBO based on both clinical symptoms and the relative abundance of certain bacterial groups within fecal samples would be useful when managing children with short bowel syndrome. This study had several limitations that must be considered when interpreting the results. There was some variability in both diet and environmental exposures during the study, both of which can impact the microbiota, and the sample size was small. However, the included patients were homogenous in that they were all under 2 years of age, had approximately half of their expected small bowel length, and required at least a third of their daily calories from PN throughout the study. In this specific population, there may be value in using more distal samples as a reflection of the more proximal microbiota. Young children with SBS are frequently a group that have great potential for intestinal adaptation, and many will be able to successfully wean from PN within the first 2 years. However, there is considerable clinical variability seen in enteral tolerance. For those who are not able to wean from PN, symptoms are often felt to be due to bacterial overgrowth, but serial assessment of the proximal gut can be invasive and challenging. By defining clinically relevant microbial
5
signatures from stool samples, treatment response could potentially become easier to quantify. Although real time bacterial sequencing from stool samples if not currently widely available in the clinical realm, further studies correlating changes to the bacterial communities to clinical symptoms could help guide treatment strategies. Although results from this study cannot necessarily be generalized to a more heterogeneous population of SBS patients, it does help to emphasize the value in obtaining stool samples as a means of analyzing overall gut health in this population. References [1] Spor A, Koren O, Ley R. Unravelling the effects of the environment and host genotype on the gut microbiome. Nat Rev Microbiol 2011;9(4):279–90. [2] Guarner F, Malagelada JR. Gut flora in health and disease. Lancet 2003;361(9356): 512–9. [3] Korpela K, Mutanen A, Salonen A, et al. Intestinal microbiota signatures associated with histological liver Steatosis in pediatric-onset intestinal failure. JPEN J Parenter Enteral Nutr 2015;41:238–48. [4] Engstrand Lilja H, Wefer H, Nystrom N, et al. Intestinal dysbiosis in children with short bowel syndrome is associated with impaired outcome. Microbiome 2015;3:18. [5] Piper HG, Fan D, Coughlin LA, et al. Severe gut microbiota Dysbiosis is associated with poor growth in patients with short bowel syndrome. JPEN J Parenter Enteral Nutr 2016;41:1202–12. [6] Dibaise JK, Young RJ, Vanderhoof JA. Enteric microbial flora, bacterial overgrowth, and short-bowel syndrome. Clin Gastroenterol Hepatol 2006;4(1):11–20. [7] Cole CR, Frem JC, Schmotzer B, Gewirtz AT, Meddings JB, Gold BD, Ziegler TR. The rate of bloodstream infection is high in infants with short bowel syndrome: relationship with small bowel bacterial overgrowth, enteral feeding, and inflammatory and immune responses. J Pediatr 2010;156(6):941–7 947 e941. [8] Gutierrez IM, Kang KH, Calvert CE, et al. Risk factors for small bowel bacterial overgrowth and diagnostic yield of duodenal aspirates in children with intestinal failure: a retrospective review. J Pediatr Surg 2012;47(6):1150–4. [9] Fan D, Coughlin LA, Neubauer MM, et al. Activation of HIF-1alpha and LL-37 by commensal bacteria inhibits Candida albicans colonization. Nat Med 2015;21(7): 808–14. [10] Simms-Waldrip TR, Sunkersett G, Coughlin LA, et al. Antibiotic-induced depletion of anti-inflammatory clostridia is associated with the development of graft-versus-host disease in pediatric stem cell transplantation patients. Biol Blood Marrow Transplant :J Am Soc Blood Marrow Transplant 2017;23(5):820–9. [11] Engelstad HJ, Barron L, Moen J, et al. Remnant small bowel length in pediatric short bowel syndrome and the correlation with intestinal Dysbiosis and linear growth. J Am Coll Surg 2018;227(4):439–49. [12] Wang P, Wang Y, Lu L, et al. Alterations in intestinal microbiota relate to intestinal failure-associated liver disease and central line infections. J Pediatr Surg 2017;52 (8):1318–26. [13] Sieczkowska A, Landowski P, Kaminska B, et al. Small bowel bacterial overgrowth in children. J Pediatr Gastroenterol Nutr 2016;62(2):196–207. [14] Palmer C, Bik EM, DiGiulio DB, et al. Development of the human infant intestinal microbiota. PLoS Biol 2007;5(7):e177. [15] Huang Y, Guo F, Li Y, et al. Fecal microbiota signatures of adult patients with different types of short bowel syndrome. J Gastroenterol Hepatol 2017;32 (12):1949–57. [16] Lavallee CM, MacPherson JAR, Zhou M, et al. Lipid emulsion formulation of parenteral nutrition affects intestinal microbiota and host responses in neonatal piglets. JPEN J Parenter Enteral Nutr 2017;41(8):1301–9. [17] Zeichner SL, Mongodin EF, Hittle L, et al. The bacterial communities of the small intestine and stool in children with short bowel syndrome. PLoS One 2019;14(5): e0215351. [18] Zhang T, Wang Y, Yan W, et al. Microbial alteration of small bowel stoma effluents and colonic feces in infants with short bowel syndrome. J Pediatr Surg 2019;19 [Epub ahead of print]. [19] Adike A, DiBaise JK. Small intestinal bacterial overgrowth: nutritional implications, diagnosis and Management. Gastroenterol Clin North Am 2018;47(1):193–208. [20] Bohm M, Siwiec RM, Wo JM. Diagnosis and management of small intestinal bacterial overgrowth. Nutr Clin Pract 2013;28(3):289–99. [21] Sundin OH, Mendoza-Ladd A, Morales E, et al. Does a glucose-based hydrogen and methane breath test detect bacterial overgrowth in the jejunum? Neurogastroenterol Motil :Off J Eur Gastrointest Motil Soc 2018;30(11): e13350.
Please cite this article as: H.G. Piper, L.A. Coughlin, V. Nguyen, et al., A comparison of small bowel and fecal microbiota in children with short bowel syndrome, Journal of Pediatric Surgery, https://doi.org/10.1016/j.jpedsurg.2020.01.032
Contents lists available at ScienceDirect
Journal of Pediatric Surgery journal homepage: www.elsevier.com/locate/jpedsurg
A comparison of small bowel and fecal microbiota in children with short bowel syndrome☆ Hannah G. Piper a,⁎, Laura A. Coughlin b, Van Nguyen b, Nandini Channabasappa b, Andrew Y. Koh b,c,d a
Department of Surgery, University of British Columbia, Vancouver, BC, Canada Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, USA Harold C. Simmons Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX, USA d Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX, USA b c
a r t i c l e
i n f o
Article history: Received 18 January 2020 Accepted 25 January 2020 Available online xxxx Key words: Short bowel syndrome Intestinal microbiota Small intestinal bacterial overgrowth
a b s t r a c t Background: Babies with short bowel syndrome (SBS) have small intestinal microbial disturbances that impact gut function. Characterizing the small bowel microbiota is challenging, and the utility of sampling stool is unclear. This study compares the microbiota from fecal samples and the small bowel. Methods: Stool samples were collected (2016–2017) from infants with SBS and colon in continuity (COLON) or SBS with small bowel ostomy (sbSTOMA). The abundance and quantity of major bacterial genera was compared between groups and to healthy controls using 16S rRNA sequencing and qPCR. Kruskall-Wallis test was used for analysis with P values b0.05 considered significant. Results: Samples (n = 41) were collected from 15 SBS infants (b2 years) (9 sbSTOMA, 6 COLON) and 3 healthy infants. Demographics and small intestinal length did not differ between sbSTOMA and COLON infants. The microbiota of SBS groups differed significantly from healthy controls. Fecal samples contained higher quantities of bacteria, but there were no significant differences between sbSTOMA and COLON groups in the abundance of facultative or obligate anaerobes, anti-inflammatory Clostridia, Enterobacteriaceae, or Bifidobacterium. Conclusion: Infants with SBS have disturbances to their intestinal microbiota. Sampling small intestinal effluent is challenging. Stool samples may provide a window into the more proximal microbial community. Type of Study: Diagnostic. Level of Evidence: Level II. © 2020 Elsevier Inc. All rights reserved.
The impact of changes to the intestinal microbiota on overall health in children is becoming increasingly recognized. This is particularly true early in life when the gut microbiota is in transition. In infancy, colonization with commensal bacteria is easily disrupted by external factors including diet and antibiotic exposure which can result in long-term alterations [1,2]. Infants who undergo significant intestinal resection with resultant short bowel syndrome (SBS) are at particularly high risk for disturbances to microbial homeostasis within the gut [3,4]. After intestinal resection babies are frequently exposed to prolonged antibiotic therapy, are restricted in their enteral intake, and have alterations to their intestinal anatomy resulting in some degree of intestinal dilation over time - all of which can predispose to microbial dysbiosis. One of the long-term concerns for infants and children with SBS is the development of small intestinal bacterial overgrowth (SIBO). Traditionally SIBO ☆ Declarations of interest: none. ⁎ Corresponding author at: University of British Columbia, Division of Pediatric Surgery, 4480 Oak Street, Vancouver, BC, V6H 3V4, Canada. Tel.: +1 604 875 3744; fax: +1 604 875 2721. E-mail address: [email protected] (H.G. Piper).
is defined as 105 CFU/mL of bacteria in the small bowel, or N 103 CFU/mL of bacterial species that normally populate the colon [6]. SIBO can occur at any time, although intestinal dilation and poor motility are identified risk factors. When present, SIBO places children at risk for bacterial bloodstream infection, poor absorption and growth, and D-lactic acidosis [6,7]. However, making a quantitative diagnosis of SIBO is challenging and frequently impractical relying on upper endoscopy or breath testing both of which have inherent inaccuracies [8]. More commonly, the diagnosis is based primarily on clinical symptoms including bloating, diarrhea and abdominal pain, and is treated empirically. This is problematic in that many children are exposed to long-term antibiotics without a quantitative means of assessing efficacy. The idea of using stool samples, which are much easier to obtain, to characterize the microbiota of the more proximal small bowel, has been questioned because it is unclear if the stool provides an accurate representation of the bacterial community in the small bowel. However, many of these patients have rapid intestinal transit and a loss of the normal physiologic breaks between the small and large bowel. In addition, there is some evidence that SBS patients with symptoms of overgrowth do have distinct changes to their distal gut microbiota as characterized by
https://doi.org/10.1016/j.jpedsurg.2020.01.032 0022-3468/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: H.G. Piper, L.A. Coughlin, V. Nguyen, et al., A comparison of small bowel and fecal microbiota in children with short bowel syndrome, Journal of Pediatric Surgery, https://doi.org/10.1016/j.jpedsurg.2020.01.032
2
H.G. Piper et al. / Journal of Pediatric Surgery xxx (xxxx) xxx
16S rRNA analysis of bacterial populations from stool samples [5]. The purpose of this study was to determine if the microbiota from stool samples differs significantly from samples obtained from more proximal small bowel stomas in infants with short bowel syndrome. If small bowel stoma samples show a similar microbiota composition, stool samples could potentially be used more reliably to detect changes to the microbiota more proximally, and to guide therapeutic interventions.
Sequences were quality filtered and then analyzed using the open source software package Quantitative Insights Into Microbial Ecology (QIIME). 16S rRNA gene sequences were assigned to operational taxonomic units (OTUs) using UCLUST (www.drive5.com/usearch), with a threshold of 97% pair-wise identify, then classified taxonomically using Greengenes (greengenes.lbl.gov). 1.3. Statistical analyses
1. Methods 1.1. Patients and Study Design This was a prospective cohort study using 16S rRNA sequencing to compare the intestinal microbiota in infants with SBS with either a small bowel stoma or intact colon. All subjects were enrolled under the IRB approved human study protocol (122015–032). Infants (less than 24 months of age) were eligible for the study if they required parenteral nutrition (PN) for at least 3 months secondary to intestinal loss and continued to require PN support for the duration of the study. Patients were recruited from the Center for Intestinal Rehabilitation. Enrolled infants with SBS were further categorized by intestinal anatomy. Patients with a small bowel ostomy (including jejunostomy or ileostomy) were included in the stoma group and patients who did not have a small bowel stoma and had not previously undergone a colonic resection, were included in the intact colon group. Clinical characteristics between groups were compared. Stool samples were collected monthly over three months when possible. At each time point patient anthropometrics including weight, length and head circumference were recorded. Expected and median values were obtained from the World Health Organization growth charts. Nutritional intake from both parenteral and enteral routes and serum markers of hepatic cholestasis were documented at all time points. Additionally, stool samples were collected from 3 healthy control infants with intact intestinal tracts to which samples from both groups of infants with SBS were compared. 1.2. Isolation of fecal gDNA and 16S rRNA analysis Fresh samples were collected and stored at −80 °C for further analysis. Bacterial gDNA was isolated from fecal or stomal specimens as previously described [9]. 16S rRNA genes (variable region 4, V4) were amplified from each sample using a composite forward primer and a reverse primer containing a unique 10-base barcode that was used to tag PCR products from respective samples [9]. Pooled PCR products were sequenced using the Illumina HiSeq 2000.
A comparison of bacterial taxonomic abundance, alpha-diversity metrics, and bacterial 16S rRNA abundance were analyzed by Mann– Whitney or Kruskall-Wallis tests, and when multiple comparisons were analyzed, Bonferroni's correction with the significance level α = 0.05 was invoked. Clinical correlate analysis was performed using Chi-square test and Fischer's exact test. Statistical analyses were carried out using the GraphPad Prism Software (San Diego, CA). 1.4. qPCR for Microbiota Analysis The quantity of bacteria present in representative samples (first collected sample) from both groups of infants with SBS were compared using qPCR analysis (SsoAdvanced SYBR Green Supermix, BioRad, Hercules, CA) using group-specific 16S rRNA gene primers, as previously described [9,10]: Eubacteria (EUBAC), all bacteria; Enterobacteriaceae (ENTERO); Clostridium leptum group (CLEPT), Clostridial Cluster IV; and Clostridium coccoides-Eubacterium rectale group (EREC). Bacterial abundance was determined using standard curves constructed with reference to cloned DNA corresponding to a short segment of the 16S rRNA gene that was amplified using conserved specific primers. Measures are in gene copies/g and not colony-forming units. 2. Results A total of 15 infants, with SBS, younger than 2 years of age, were enrolled between 2016 and 2017. This included 9 infants with small bowel stomas (sbSTOMA) and 6 infants with intact colons (COLON). Within the two groups there were no significant differences in gestational age at birth, age at enrollment, or small bowel length. All of the infants with intact colon had the ileocecal valve in continuity, compared to none of the infants with small bowel stomas. Both groups had similar total and direct bilirubin levels at the start of the study, however the sbSTOMA group had a higher mean GGT level (Table 1). All of the Infants were receiving a portion of their calories from PN, however those with sbSTOMA were receiving fewer enteral calories compared to the
Table 1 Clinical Characteristics of SBS patients with small bowel stoma and intact colon.
Total Patients Male (%) Median GA in weeks at birth (range) Median age in months at enrollment (range) Diagnosis leading to SBS (n) NEC Atresia Volvulus Anatomy Median small bowel length (range) Median % of expected length based on GA (range) Ileocecal valve in continuity (%) Markers of cholestasis at study start Mean total/direct bilirubin (mg/dL) Mean AST (U/L) Mean ALT (U/L) Mean GGT (U/L) Mean AP (U/L)
Sb stoma
Intact colon
9 4 (44) 32 (23–38) 4 (1–15)
6 0 (0) 32 (24–38) 10 (3–22)
p value
(5) 56% (3) 33% (1) 11%
(3) 50% (3) 50% (0) 0%
53 cm (40–140 cm) 44 (5–75) 0 (0)
47 cm (25–84 cm) 28 (10–57) 6 (100%)
0.44 0.58 0.00
6.0/5.0 135 112 119 381
3.3/2.5 95 117 46 327
0.43 0.42 0.93 0.01 0.52
0.06 0.81 0.07 0.72
Statistical analyses by t-test, GA = gestational age, ICV = ileocecal valve, AST = aspartate aminotransferase, ALT = alanine aminotransferase, GGT = gamma-glutamyl transferase, AP = alkaline phosphatase.
Please cite this article as: H.G. Piper, L.A. Coughlin, V. Nguyen, et al., A comparison of small bowel and fecal microbiota in children with short bowel syndrome, Journal of Pediatric Surgery, https://doi.org/10.1016/j.jpedsurg.2020.01.032
H.G. Piper et al. / Journal of Pediatric Surgery xxx (xxxx) xxx
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Table 2 Nutritional and Anthropometric Parameters.
Nutrition at study start Mean calories given as PN (kcal/kg/d) (range) Mean % PN of total caloric intake PN composition (mean) Lipid (g/kg/d) Glucose infusion rate (mg/kg/min) Protein (g/kg) Mean Z-score weight Mean Z-score length Mean Z-score weight for length
Sb stoma
Intact colon
P value
N=9 77 (29–104) 79
N=6 49 (27–97) 52
0.04 0.05
1.3 11.2 3.4 −0.94 −2.21 −0.26
1.0 9.2 2.1 −0.68 −0.90 0.17
0.26 0.31 0.03 0.18 0.06 0.93
Statistical analysis by t-test, PN = parenteral nutrition.
COLON group. There were no significant differences in anthropometrics between groups (Table 2). One patient in each group received antibiotics during the study. Twenty-five sbSTOMA samples and 17 COLON samples were collected over three months. Following 16S rRNA sequencing, both groups were found to have similar abundance of the major represented bacteria including anti-inflammatory Clostrida (defined as Clostridiaceae, Erysipelotrichaceae, Eubacteriaceae, Lachnospiraceae and Ruminococcaceae) Enterobacteriaceae, Bifidobacterium, Eubacterium and Lactobacillaceae (Figs. 1, Table 3). Additionally, when comparing the relative abundance of obligate and facultative anaerobes, no significant differences were seen between the two groups (Fig. 1). Infants in the sbSTOMA group were found to have an increased abundance of Bacteroides compared to those in the COLON group (28% vs. 15%, p = 0.01, Table 3), however both groups had a significantly reduced abundance compared to healthy children
(44%, p = 0.036, p = 0.001). Healthy controls also had significantly more obligate anaerobes, Bifidobacterium, Eubacterium and Clostridial Firmicutes and significantly fewer Enterobacteriaceae than all SBS patients. When quantitative levels of bacteria in the specimens were compared between the sbSTOMA and COLON groups using qPCR, fecal samples contained significantly higher levels of ENTERO (pro-inflammatory bacteria), CLEPT and EREC (commensal, anti-inflammatory bacteria) and Eubacteria (all bacteria, median of 13.5 X 10 8 vs. 29.5 X 10 10 copies/g, p = 0.036) than those from small bowel stomas (Fig. 2). 3. Discussion This study demonstrates that in infants with short bowel syndrome, both fecal samples and small bowel stoma samples have a similar overall distribution of bacterial phyla. Although the bacterial load was greater in
Fig. 1. Comparison of gut microbial populations in infants with SBS from either small bowel stoma or fecal samples, and healthy controls. Relative abundance of bacterial phyla and families as determined by 16S sequencing collected from infants with SBS and small bowel stoma (sb stoma) or an intact colon in continuity, compared to healthy controls. NS = non significant, ⁎ p b 0.05, ⁎⁎ p b 0.01, ⁎⁎⁎ p b 0.001, Mann–Whitney test.
Please cite this article as: H.G. Piper, L.A. Coughlin, V. Nguyen, et al., A comparison of small bowel and fecal microbiota in children with short bowel syndrome, Journal of Pediatric Surgery, https://doi.org/10.1016/j.jpedsurg.2020.01.032
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H.G. Piper et al. / Journal of Pediatric Surgery xxx (xxxx) xxx
Table 3 Relative Abundance of common intestinal bacterial. Bacterial genus
Sb stoma
Intact colon
P value
Bifidobacterium (%) Enterobacteriaceae (%) Lactobacillaceae (%) Clostridial Firmicutes (%) Eubacterium (%) Bacteroides (%)
20.6 12.7 22.7 8.3 24.7 28.1
18.2 16.7 27.9 7.5 21.5 14.8
0.54 0.23 0.73 1.00 0.81 0.01
Statistical analysis by Kruskal-Wallis test.
fecal samples compared to stoma samples, the relative abundance of genera and families commonly associated with short bowel syndrome was not significantly different between the two groups. Initial studies looking at the intestinal microbiota in children with SBS identified the characteristic findings of increased Proteobacteria (specifically Enterobacteriaceae) and Lactobacillus in this patient population [3,4]. Similarly, a more recent study by Engelstad et al. also found that children with SBS had an overabundance of Proteobacteria, particularly in those with small bowel length b 35 cm [11]. Both Enterobacteriaceae and Lactobacillus had a similar abundance in stoma vs. fecal samples in this study. Interestingly, several of the published series to date use a combination of both stool and stoma samples and do not distinguish between the two when characterizing the microbiota [3,11,12]. Certainly, children with short bowel
syndrome frequently have rapid transit through the small intestine in addition to a foreshortened colon. Additionally, these patients may have lost the ileocecal valve, potentially resulting in a more similar microbial distribution between the distal small bowel and the colon. However, a direct comparison of the microbiota from these two sources has not been extensively studied in children with SBS and has several important clinical implications. In humans it is well known that in the presence of normal intestinal anatomy and motility, the intestinal microbiota changes considerably from the mouth to the anus. In general, there is a gradual transition from mostly gram-positive aerobic and facultative anaerobes in the proximal GI tract, to a more diverse and populated bacterial environment dominated largely by gram-negative anaerobes in the colon [6,13,14]. However, after intestinal resection the transition may be less abrupt or predictable. In a study comparing fecal samples from adults with SBS (average of 90-100 cm of small bowel) who either had a jejunal/colonic anastomosis or a jejunal/ileal anastomosis with intact colon, there was a significantly higher abundance of Enterobacteriaceae in patients with a jejunal/colonic anastomosis (and therefore no ileocecal valve and shorter colon) compared to both those with jejunal/ileal anastomoses and healthy controls [15]. This finding is consistent with the previously published work in children, suggesting that in some subsets of patients with SBS, more proximal intestinal microbiota may not differ significantly from distal samples. Furthermore, in a piglet model of short
Fig. 2. Analysis of absolute levels of bacteria in children with SBS with either a small bowel stoma or intact colon. The absolute levels of all bacteria (Eubacteria), Enterobacteriaceae (ENTERO), Clostridium leptum group (CLEPT), Clostridial Cluster IV; and Clostridium coccoides-Eubacterium rectale group (EREC) were compared as determined by group-specific qPCR (Mann–Whitney test).
Please cite this article as: H.G. Piper, L.A. Coughlin, V. Nguyen, et al., A comparison of small bowel and fecal microbiota in children with short bowel syndrome, Journal of Pediatric Surgery, https://doi.org/10.1016/j.jpedsurg.2020.01.032
H.G. Piper et al. / Journal of Pediatric Surgery xxx (xxxx) xxx
bowel syndrome, the animals were also found to have a high relative abundance of Enterobacteriaceae (up to 50% in some animals) when ileal mucosal scrapings were sequenced [16]. This again suggests that some of the pathologic shifts seen in this population may occur in both the proximal and distal intestine. This is further supported by two recent studies, one that found both jejunal aspirates and stool samples from children with short bowel syndrome had increased abundance of Klebsiella, Enterococcus and Enterobacteriacieae compared to samples from healthy children [17], and the other that demonstrated that Proteobacteria dominated both small bowel stoma effluent and colonic samples in a small group of children with SBS [18]. If fecal samples are representative of the bacterial composition of more proximal intestine, fecal samples could then be used to help with diagnosis and treatment of children who fail to make steady progress towards enteral independence. A significant challenge in caring for children with short bowel syndrome is that much of the intestinal pathology occurs in the small bowel, both initially and later during the intestinal adaptation phase which can continue for several years. However, the small intestine is notoriously difficult to access both for diagnostic and therapeutic purposes. For example, a portion of patients with SBS develop a variety of gastrointestinal symptoms including abdominal bloating, pain, feed intolerance and/or frequent loose stools that are ascribed to overpopulation of the small intestine with primarily colonic bacteria, also known as small intestinal bacterial overgrowth (SIBO). SIBO can increase the risk of infection, result in metabolic abnormalities and make it more challenging to advance enteral nutrition in these patients, However, accurately making this diagnosis by analyzing the small intestinal microbiota is difficult. Proposed diagnostic methods include duodenal aspirates or hydrogen breath testing [8,19,20]. Both strategies have limitations in this patient population, and in a recent study there was poor correlation between breath test results and bacterial numbers both using culture based methods and bacterial DNA sequencing [21]. Most commonly, a clinical diagnosis is made, followed by empiric antibiotic treatment. As we gain more insight into the risks of prolonged antibiotic therapy for children, it would be helpful to have a more practical means of assessing the microbiota in symptomatic patients before and after treatment. Perhaps establishing new diagnostic criteria for SIBO based on both clinical symptoms and the relative abundance of certain bacterial groups within fecal samples would be useful when managing children with short bowel syndrome. This study had several limitations that must be considered when interpreting the results. There was some variability in both diet and environmental exposures during the study, both of which can impact the microbiota, and the sample size was small. However, the included patients were homogenous in that they were all under 2 years of age, had approximately half of their expected small bowel length, and required at least a third of their daily calories from PN throughout the study. In this specific population, there may be value in using more distal samples as a reflection of the more proximal microbiota. Young children with SBS are frequently a group that have great potential for intestinal adaptation, and many will be able to successfully wean from PN within the first 2 years. However, there is considerable clinical variability seen in enteral tolerance. For those who are not able to wean from PN, symptoms are often felt to be due to bacterial overgrowth, but serial assessment of the proximal gut can be invasive and challenging. By defining clinically relevant microbial
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signatures from stool samples, treatment response could potentially become easier to quantify. Although real time bacterial sequencing from stool samples if not currently widely available in the clinical realm, further studies correlating changes to the bacterial communities to clinical symptoms could help guide treatment strategies. Although results from this study cannot necessarily be generalized to a more heterogeneous population of SBS patients, it does help to emphasize the value in obtaining stool samples as a means of analyzing overall gut health in this population. References [1] Spor A, Koren O, Ley R. Unravelling the effects of the environment and host genotype on the gut microbiome. Nat Rev Microbiol 2011;9(4):279–90. [2] Guarner F, Malagelada JR. Gut flora in health and disease. Lancet 2003;361(9356): 512–9. [3] Korpela K, Mutanen A, Salonen A, et al. Intestinal microbiota signatures associated with histological liver Steatosis in pediatric-onset intestinal failure. JPEN J Parenter Enteral Nutr 2015;41:238–48. [4] Engstrand Lilja H, Wefer H, Nystrom N, et al. Intestinal dysbiosis in children with short bowel syndrome is associated with impaired outcome. Microbiome 2015;3:18. [5] Piper HG, Fan D, Coughlin LA, et al. Severe gut microbiota Dysbiosis is associated with poor growth in patients with short bowel syndrome. JPEN J Parenter Enteral Nutr 2016;41:1202–12. [6] Dibaise JK, Young RJ, Vanderhoof JA. Enteric microbial flora, bacterial overgrowth, and short-bowel syndrome. Clin Gastroenterol Hepatol 2006;4(1):11–20. [7] Cole CR, Frem JC, Schmotzer B, Gewirtz AT, Meddings JB, Gold BD, Ziegler TR. The rate of bloodstream infection is high in infants with short bowel syndrome: relationship with small bowel bacterial overgrowth, enteral feeding, and inflammatory and immune responses. J Pediatr 2010;156(6):941–7 947 e941. [8] Gutierrez IM, Kang KH, Calvert CE, et al. Risk factors for small bowel bacterial overgrowth and diagnostic yield of duodenal aspirates in children with intestinal failure: a retrospective review. J Pediatr Surg 2012;47(6):1150–4. [9] Fan D, Coughlin LA, Neubauer MM, et al. Activation of HIF-1alpha and LL-37 by commensal bacteria inhibits Candida albicans colonization. Nat Med 2015;21(7): 808–14. [10] Simms-Waldrip TR, Sunkersett G, Coughlin LA, et al. Antibiotic-induced depletion of anti-inflammatory clostridia is associated with the development of graft-versus-host disease in pediatric stem cell transplantation patients. Biol Blood Marrow Transplant :J Am Soc Blood Marrow Transplant 2017;23(5):820–9. [11] Engelstad HJ, Barron L, Moen J, et al. Remnant small bowel length in pediatric short bowel syndrome and the correlation with intestinal Dysbiosis and linear growth. J Am Coll Surg 2018;227(4):439–49. [12] Wang P, Wang Y, Lu L, et al. Alterations in intestinal microbiota relate to intestinal failure-associated liver disease and central line infections. J Pediatr Surg 2017;52 (8):1318–26. [13] Sieczkowska A, Landowski P, Kaminska B, et al. Small bowel bacterial overgrowth in children. J Pediatr Gastroenterol Nutr 2016;62(2):196–207. [14] Palmer C, Bik EM, DiGiulio DB, et al. Development of the human infant intestinal microbiota. PLoS Biol 2007;5(7):e177. [15] Huang Y, Guo F, Li Y, et al. Fecal microbiota signatures of adult patients with different types of short bowel syndrome. J Gastroenterol Hepatol 2017;32 (12):1949–57. [16] Lavallee CM, MacPherson JAR, Zhou M, et al. Lipid emulsion formulation of parenteral nutrition affects intestinal microbiota and host responses in neonatal piglets. JPEN J Parenter Enteral Nutr 2017;41(8):1301–9. [17] Zeichner SL, Mongodin EF, Hittle L, et al. The bacterial communities of the small intestine and stool in children with short bowel syndrome. PLoS One 2019;14(5): e0215351. [18] Zhang T, Wang Y, Yan W, et al. Microbial alteration of small bowel stoma effluents and colonic feces in infants with short bowel syndrome. J Pediatr Surg 2019;19 [Epub ahead of print]. [19] Adike A, DiBaise JK. Small intestinal bacterial overgrowth: nutritional implications, diagnosis and Management. Gastroenterol Clin North Am 2018;47(1):193–208. [20] Bohm M, Siwiec RM, Wo JM. Diagnosis and management of small intestinal bacterial overgrowth. Nutr Clin Pract 2013;28(3):289–99. [21] Sundin OH, Mendoza-Ladd A, Morales E, et al. Does a glucose-based hydrogen and methane breath test detect bacterial overgrowth in the jejunum? Neurogastroenterol Motil :Off J Eur Gastrointest Motil Soc 2018;30(11): e13350.
Please cite this article as: H.G. Piper, L.A. Coughlin, V. Nguyen, et al., A comparison of small bowel and fecal microbiota in children with short bowel syndrome, Journal of Pediatric Surgery, https://doi.org/10.1016/j.jpedsurg.2020.01.032