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Short Communication: Gastroenterology

Intestinal Microbial and Metabolic Alterations Following Successful Fecal Microbiota Transplant for d-Lactic Acidosis

Bulik-Sullivan, Emily C.; Roy, Sayanty; Elliott, Ryan J.; Kassam, Zain; Lichtman, Steven N.; Carroll, Ian M.∗,§; Gulati, Ajay S.†,§,||

Author Information
Journal of Pediatric Gastroenterology and Nutrition: October 2018 - Volume 67 - Issue 4 - p 483-487
doi: 10.1097/MPG.0000000000002043

Abstract

What Is Known

  • d-lactic acidosis is a rare complication of short bowel syndrome, characterized by elevated plasma d-lactate and encephalopathy.
  • Present therapies focus on reducing gut bacteria (antibiotics) and eliminating the substrate for d-lactate production (dietary carbohydrate restriction).
  • Despite these treatments, a subset of patients has recurrent d-lactic acidosis episodes.

What Is New

  • Fecal microbiota transplantation was safe and effective for recurrent d-lactic acidosis in the patient described in this study.
  • Fecal microbiota transplantation resulted in changes to the composition and metabolism of the intestinal microbiota.
  • Increased fecal d-lactate (despite reduced plasma d-lactate) after fecal microbiota transplantation suggests intestinal permeability could play a role in d-lactic acidosis pathogenesis.

d-lactic acidosis (d-LA) is an uncommon but severe complication of short bowel syndrome (SBS), first described in humans in 1979 (1,2). Two enantiomers (d and l) of lactic acid exist, although humans metabolize d-lactate less efficiently than l-lactate (3). In SBS, colonic bacteria are exposed to excessive amounts of undigested carbohydrates. If high numbers of lactic acid–producing bacteria (LAB) are present in the colon, these fermentable carbohydrates may be converted to d-lactate, which passes into systemic circulation and can cause neurological symptoms. The mechanism by which d-lactate causes encephalopathy is unclear. Several theories have been proposed, including a direct effect of d-lactate on the central nervous system, associated acidosis, high colonic production of other organic acids (such as aldehydes, amines, and alcohols) that may act as false neurotransmitters, and nutritional deficiencies (4).

Patients with d-LA present with elevated plasma d-lactate levels (normal 0–0.25 mmol/L; d-LA >3 mmol/L) (5), metabolic acidosis, and encephalopathy. The latter may include altered mental status, slurred speech, ataxia, aggressive behavior, memory impairment, ptosis, somnolence, and nystagmus (3). Conventional treatments for d-LA involve rehydration, oral antibiotics, restriction of carbohydrate intake, thiamine supplementation, and in severe cases, hemodialysis (3,6). Despite these interventions, however, a subset of patients experiences recurrent episodes of d-LA, highlighting the need for novel therapies for this disorder.

Because d-LA is driven by the colonic microbiota, several microbial modulation strategies have been attempted to prevent recurrence. For example, treatment with antibiotics and l-lactate-producing probiotics (kanamycin plus Bifidobacterium breve and Lactobacillus casei) has been reported (7). Synbiotics have also been used to treat antibiotic-resistant d-LA, specifically utilizing a combination of a prebiotic (galacto-oligosaccharide) and the probiotics B. breve and L. casei(8). Finally, fecal microbiota transplantation (FMT) has also been used to treat d-LA (9). Despite these reports of successfully utilizing microbial modulation to treat d-LA, longitudinal monitoring of plasma and fecal d-lactate is sparse, and broad profiling of the intestinal microbiota before and after treatment has not been described. In the current study, we present a comprehensive characterization of the intestinal microbiota, as well as fecal and plasma d-lactate concentrations, before and after successful treatment of recurrent d-LA with FMT. These data provide insight into how microbial modulation may serve as a foundation for the future development of safe and efficacious treatment strategies for d-LA.

CASE REPORT

A 7-year-old female with SBS secondary to gastroschisis presented to the emergency department with ataxia and altered mental status. Estimated small intestinal length was 40 to 50 cm with no ileocecal valve, although the majority of the colon was intact. At presentation, parenteral nutrition provided approximately 50% of her caloric needs. Upon admission, her serum bicarbonate level was 9 mmol/L (normal 22–30 mmol/L), with a venous pH of 7.28. Her symptoms responded rapidly to intravenous hydration, and she was discharged home within 48 hours. After this initial presentation, however, the patient developed 4 additional bouts of altered mental status requiring urgent hospitalization. Her serum bicarbonate level was as low as 8 mmol/L, with a pH of 7.19 during these episodes. An extensive work up was performed, including toxicology testing (urine drug screen and ethyl alcohol levels) and computed tomography of the brain, which were normal. Notably, her plasma d-lactate levels ranged from 4.06 to 8.60 mmol/L during these episodes, leading to a diagnosis of recurrent d-LA.

In total, this patient was hospitalized 5 times for d-LA over approximately 4 months. In all cases, she responded acutely to intravenous hydration. Her mean length of stay was 2 days, and her symptom-free intervals ranged from 12 days to 2 months in between hospitalizations. During this time, numerous interventions were attempted to prevent further episodes of d-LA. These included multiple oral antibiotics (metronidazole, gentamicin, sulfamethoxazole-trimethoprim, rifaximin, neomycin, and fluconazole) and carbohydrate restriction. Despite these interventions, the patient continued to have recurrent bouts of d-LA as described.

After her fifth episode of d-LA, the decision was made with the family to proceed with FMT. Investigational new drug and institutional review board approvals were obtained. Two days before FMT, all antibiotics were stopped, and a proton pump inhibitor was initiated. One day before the procedure, the patient was admitted for bowel preparation, which included polyethylene glycol electrolyte solution administered by nasogastric tube. For the FMT, screened donor material containing 12.5 g of stool suspended in 30 mL of buffer was obtained from a stool bank (FMT Upper Delivery Microbiota Preparation, FMP30–OpenBiome, Somerville, MA) (10). This was administered via nasogastric tube over 1 hour. Following the procedure, the patient reported no adverse effects, including no abdominal pain, fever, or increased stooling. She was discharged home after 24 hours of observation. The family subsequently described an improvement in stool frequency, and the patient stopped having stool accidents during the night. Importantly, she experienced no further episodes of d-lactate encephalopathy and no adverse events in the year after FMT.

METHODS

Ethics Statement

A compassionate use investigational new drug application was approved by the Food and Drug Administration (16901), and institutional review board approval was obtained from the University of North Carolina at Chapel Hill. Informed consent for FMT and specimen collection was obtained, as well as assent from the patient.

Fecal Sample Collection

Fecal samples were collected at home by the family, stored immediately at 4°C, and delivered to University of North Carolina at Chapel Hill with frozen ice packs within 24 hours. The samples were then stored at −80°C.

Fecal Microbiota Profiling

Total stool DNA was isolated, and the V4 region of the microbial 16S rRNA gene was amplified and sequenced as described previously (11,12). Sample preparation, sequencing, and analysis are detailed in Supplemental Digital Content 1 and 2 (https://links.lww.com/MPG/B416, https://links.lww.com/MPG/B417).

Lactic Acid Concentration Determination

Plasma d-lactate levels were measured pre- and post-FMT as part of routine clinical care (Mayo Clinic Laboratories, Rochester, MN). Stool d- and l-lactate were quantified in triplicate with the d-/l-Lactic Acid Rapid Assay Kit following manufacturer's instructions (Megazyme, Bray, Ireland). Stool pH was measured with Accutest pH nitrazine indicator paper (Jant Pharmacal Corporation, Encino, CA).

RESULTS

Enteric Microbial Changes After Fecal Microbiota Transplantation

As described above, FMT resulted in lasting resolution of d-LA symptoms with no apparent side effects. This was associated with changes in the composition of the fecal microbiota post-FMT (Fig. 1). Before FMT, the predominant stool bacterial taxa were the genera Veillonella, Bifidobacterium, and Lactobacillus. Three distinct variants within the Lactobacillus genus were identified in the 30 most abundant taxa in the patient's stool, although only one could be resolved to species level (L. kitasatonis; Fig. 1A). In contrast, the FMT donor microbiota contained minimal LAB (Lactobacillus spp. and Streptococcus spp.), and was more diverse than the patient's stool as measured by the Shannon diversity index (Fig. 1B). Following FMT, there was a shift in the patient's stool microbiota. The Lactobacillus genus remained dominant, but notably more bacteria in the Escherichia/Shigella genus were present, and the relative abundance of Veillonella spp. decreased. Dorea longicatena—which was present in the donor material but not in the recipient's stool before FMT—also bloomed transiently in the week after FMT.

F1
FIGURE 1:
Fecal microbial composition of donor material and recipient stools before and after fecal microbiota transplantation (FMT) as determined from 16S rRNA gene sequence data. In both panels, data are presented chronologically from left to right and dashed red lines indicate when the patient received FMT. A, Relative abundance of the 30 most abundant taxa in each sample. Sequence variants that could not be resolved to finer taxonomic levels are demarcated with (f) for family and (g) for genus. B, Alpha diversity, as measured by the Shannon diversity index.

Alterations in Host Lactate Metabolism Following Fecal Microbiota Transplantation

To determine the impact of FMT on host lactate metabolism, we measured lactate levels in the plasma and stool pre- and post-FMT. As indicated in Figure 2A, there was a reduction in plasma d-lactate levels by 2 weeks post-FMT (up to 8-fold reduction compared to pre-FMT levels during d-LA episodes). As expected, fecal d-lactic acid concentrations were elevated pre-FMT when compared to donor stool (Fig. 2B). Intriguingly, however, FMT resulted in increased stool d-lactate levels, despite the reduction in plasma d-lactate described. Fecal l-lactic acid concentrations were consistently low at all time points. Stool pH measurements paralleled d-lactic acid levels. Specifically, the patient's stool was more acidic than healthy stool before FMT, and fecal pH continued to decrease post-FMT despite the absence of further d-LA symptoms (Fig. 2C).

F2
FIGURE 2:
Metabolite and fecal pH data before and after fecal microbiota transplantation (FMT). In all panels, data are presented chronologically from left to right and dashed red lines indicate when the patient received FMT. A, Plasma d-lactate values before and after FMT (normal: 0–0.25 mmol/L). B, Fecal d- and l-lactic acid concentration (g/L) of the patient's stool samples and the donor FMT material. C, Fecal pH of the same stool samples depicted in Figures 1 and 2B, and for donor FMT material (normal stool pH: 7.0–7.5).

DISCUSSION

In this study, we report the successful treatment of a pediatric SBS patient with recurrent d-LA using FMT. Before FMT, this patient was hospitalized for 5 episodes of d-LA, including elevated plasma d-lactate and encephalopathy. In the year after FMT, d-LA symptoms did not recur. To our knowledge, this is the second such report in the literature (9), and the first to provide a comprehensive characterization of the recipient's fecal microbiota and d-lactate levels pre- and post-FMT.

Before FMT, this patient's fecal microbiota displayed an overabundance of Lactobacillus bacteria. This is consistent with previous descriptions of the intestinal microbiota in patients with SBS and d-LA (2,13,14). In light of these findings, we hypothesized that the abundance of Lactobacillus spp. and other LAB would be diminished after FMT. Unexpectedly, however, the Lactobacillus variants remained the dominant taxa in the patient's stool even after FMT. This was despite the use of donor material that was comparatively more diverse and lacking in Lactobacillus spp. Our sequence-based analysis enabled us to identify L. kitasatonis as a predominant Lactobacillus species in the patient's stool both before and after FMT. Of note, this species is capable of producing both d- and l-lactic acid (15).

Although the Lactobacillus taxa did not change dramatically after FMT, there was a clear reduction of the Veillonella genus in the patient's stool. Interestingly, Veillonella is a known lactate-fermenting bacterial group (16,17). Given the decreased abundance of Veillonella spp. after FMT, we posit that there may have been a relationship between the lactate-producing bacteria (i.e., Lactobacillus spp.) and lactate-fermenting bacteria (Veillonella spp.) in the patient's stool that FMT interrupted. In previous research, Veillonella species in the ileum have been associated with penetrating complications in pediatric Crohn disease (18), and the combination of Lactobacillus and Veillonella bacteria has been associated with increased fecal organic acids (including lactic acid) in irritable bowel syndrome (19). Future studies will be needed to determine the biological role of Veillonella species in the pathogenesis of d-LA.

The lactic acid metabolite data collected from this patient are also intriguing. In the classical theory of d-LA pathogenesis, increased microbial production of d-lactate in the colon translates to increased plasma d-lactate levels, which in turn induce metabolic acidosis and neurological symptoms. Targeting these microbes with antibiotics is believed to reduce stool and plasma d-lactate levels, thereby ameliorating d-LA symptoms. In the present case, FMT did resolve the patient's d-LA symptoms and decrease her plasma d-lactate concentrations; however, stool d-lactate levels remarkably increased after FMT. One explanation for these findings is that intestinal permeability may play a role in the pathogenesis of d-LA. Specifically, it is possible that shifts in the microbiota (or bacterial metabolites) induced by FMT may improve (i.e., decrease) intestinal permeability, thereby “trapping” d-LA in the stool for excretion. Indeed, others have speculated that intestinal permeability may play a role in d-LA (20). Although this permeability theory must be tested further using in vivo and in vitro model systems, it represents a putative target for future therapies.

There are limitations to the present study. First, as a single-patient study, we cannot draw conclusions about the global efficacy of FMT for the treatment of d-LA, nor the precise mechanism by which it may resolve d-LA symptoms. Second, plasma and stool d-lactate levels were not measured simultaneously and therefore do not provide a direct assessment of this patient's lactic acid metabolic state at a given time. Finally, 16S rRNA gene sequencing only describes the composition of a microbiota, and does not offer insight into the function of gut bacterial communities. Further studies utilizing metagenomic and metabolomic approaches could provide additional insight into the mechanism of d-LA.

In summary, we describe the successful use of FMT to treat a case of recurrent, pediatric d-LA and provide a comprehensive characterization of donor and recipient fecal microbiota before and after FMT. These findings set the stage for further understanding the role of the microbiota in d-LA, which may lead to the development of novel therapies to treat this rare disorder.

Acknowledgments

The authors would like to acknowledge the UNC School of Medicine's High-Throughput Sequencing Facility for their sequencing assistance. OpenBiome generously provided FMT substrate through a gift from the Kellen Family Foundation.

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Keywords:

gastroschisis; gut bacteria; 16S rRNA profiling; short bowel syndrome; stool d-lactate

Supplemental Digital Content

Copyright © 2018 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition