Intestinal microbiota composition in children with short bowel syndrome (SBS) is an important factor influencing the clinical outcome. An increase of D-lactate–producing bacteria can lead to D-lactic acidosis, also referred to as D-lactate encephalopathy, with severe neurologic impairment. Antibiotic treatments for D-lactic acidosis in children with SBS offer often only short-term relief. Here, we present the case of a boy with SBS who developed recurrent episodes of D-lactic acidosis even under continuous cycling antibiotic treatment. Microbiological analyses were used to detect the presence of D-lactate–producing Lactobacillus species in the stool samples. A probiotic cocktail was introduced to alter the intestinal microbiota. During follow-up under treatment with probiotics, the patient remained stable, and there was no additional need for antibiotic therapy for more than a year. Stool composition of the patient was sequenced regularly over that period. His microbiota profile changed completely in species richness, and a clustering of species according to probiotic usage was seen. Importantly, D-lactate–producing Lactobacillus strains disappeared within a few weeks after probiotic introduction and were no longer detected in the subsequent follow-up specimens.

D-lactic acidosis is a known complication in patients with short bowel syndrome (SBS). The short transit of nutrients allows undigested carbohydrates, particularly simple sugars, to reach the lower gastrointestinal tract and become fermented by bacteria. The resulting acidic environment exemplifies the optimal growing medium for Lactobacillus species including D-lactate–producing strains.1 Primary management of D-lactic acidosis includes the restriction of oral carbohydrates and the administration of antibiotics. However, the use of antibiotics leads mostly to short-term suppression of D-lactate–producing bacteria and harbors the risk of developing antibiotic resistance with enhanced dysbiosis as an outcome. In single-case studies, probiotics were proposed as an alternative therapeutic option,2 although the use of probiotics may also be diametric. Depending on the supplemented strains, D-lactic acidosis can actually be induced by probiotics.3 Therefore, we monitored the stool bacterial composition in a pediatric patient with SBS and recurrent D-lactic acidosis during cycling antibiotics and probiotic treatment over time via 16S gene sequencing. Informed consent for sample collection and subsequent analyses was obtained from the parents.

An 18-month-old boy presented for the first time with an acute onset of ataxia, fatigue, and muscular weakness to the emergency department. He was known to have an SBS secondary to malrotation and midgut volvulus at the age of 5 days. The length of the remaining small bowel was ∼20 cm. The ileocecal valve was preserved, and he still had his entire colon. So far, he had shown excellent bowel adaptation. His nutrition consisted mainly of a combination of extensively hydrolyzed formula (Pregomin Pepti) and acid-based formula (Neocate Advance) with some additional solid foods. D-lactic acidosis was confirmed by determination of lactate in the blood (blood L-lactate: 1.70 mmol/L) and in the urine (urine total lactate: 13.5 mol/mol creatinine; normal: <0.13 mol/mol creatinine), because there was no option to measure D-lactate directly at our institution. The boy recovered quickly after fluid resuscitation, stopping his enteral nutrition for 12 hours and starting antibiotic therapy with metronidazole. Over the next 3 years, he occasionally developed further episodes of D-lactic acidosis, which were managed similarly. At the age of 4 years, the episodes became more frequent, and he was started on cyclic antibiotic treatment with gentamicin and metronidazole. During all that time, he had maintained his growth (weight and height along the 50th percentile) by increasing his energy intake to 2500 kcal per day with high amounts of amino acid–based formula (Neocate Advance) and a diet low in simple sugars. His stool output remained high, indicating a limited digestion in the small bowel and hence a higher content of undigested nutrients in the large bowel. Six months later, although under antibiotic treatment, he developed D-lactic acidosis again. In his stool, the D-lactate–producing strains Lactobacillus (L) johnsonii and L plantarum were detected. Therefore, a probiotic cocktail with non-D-lactate–producing bacteria (Pro4-50 D-Lactate Free Multistrain Probiotic capsules [Spectrumceuticals, Sydney, Australia] containing 25 billion colony-forming units [CFUs] of L rhamnosus GG, 15 billion CFUs of Bifidobacterium (B) lactis BS01, 5 billion CFUs of B breve BR03, and 5 billion CFUs of B longum BL03) was introduced into his daily treatment. Since then, the patient remained stable, and there was no additional need for antibiotic therapy for more than a year.

After initiation of a cyclic antibiotic treatment and during treatment with probiotics without any antibiotic treatment, stool samples were regularly collected and deeply profiled for stool microbiota composition. We employed 16S-based sequencing, which is used to detect highly conserved and hypervariable regions among bacterial ribosomal RNA and allows identification of bacteria, using Ion Personal Genome Machine System (Thermo Fisher Scientific, Waltham, MA).4 The raw sequences were processed in Quantitative Insights Into Microbial Ecology 1.9.1 pipeline as described,5 and microbiota composition including sample diversity and taxonomy profile (relative abundance: >0.1%) were plotted in R (https://www.r-project.org) by using the phyloseq library.6 

To identify any changes in the stool microbiota, we collected 20 stool samples over 160 days before treating the patient with probiotics and 34 stool samples over 348 days during probiotic treatment. We determined α diversity, which is used to identify the number and the proportion of each species represented in a single stool sample. Both indexes of α diversity, the Shannon and the Simpson diversity index, were calculated. The first reveals both richness and abundance, and the second gives more weight to evenness and common species. We observed a higher α diversity after introducing the probiotic to the patient expressed as increased Shannon (mean ± SD; 1.67 ± 0.6 vs 2.27 ± 0.8; P < .01) and Simpson index values (0.62 ± 0.14 vs 0.79 ± 0.23; P < .01; Fig 1A). In contrast, β diversity is used to calculate the diversity differences between samples by measuring similarity or dissimilarity of samples. A β diversity analysis using Bray–Curtis distance revealed a clustering of samples according to probiotic usage (Adonis, P < .001; Fig 1B), suggesting that microbial profiles of these groups are completely different.

FIGURE 1

The microbial characterization of the patient with SBS after probiotic supplementation. A, Shannon and Simpson indices plotted to describe the bacterial species richness in stool samples before (−) and during (+) probiotic treatment (Kruskal–Wallis test with Dunn’s multiple comparison test). B, β diversity revealing the microbial differences between stool samples before (−) and during (+) probiotic treatment plotted by using PCoA analysis of Bray–Curtis distance dissimilarities. C, The relative abundance (percentage) of microbial profile at genus rank in stool samples of the patient recorded before and during probiotic treatment. P < .05 is considered significant. PC1, principal coordinate 1; PC2, principal coordinate 2; PCoA, principal coordinates analysis; −, before treatment; +, during treatment.

FIGURE 1

The microbial characterization of the patient with SBS after probiotic supplementation. A, Shannon and Simpson indices plotted to describe the bacterial species richness in stool samples before (−) and during (+) probiotic treatment (Kruskal–Wallis test with Dunn’s multiple comparison test). B, β diversity revealing the microbial differences between stool samples before (−) and during (+) probiotic treatment plotted by using PCoA analysis of Bray–Curtis distance dissimilarities. C, The relative abundance (percentage) of microbial profile at genus rank in stool samples of the patient recorded before and during probiotic treatment. P < .05 is considered significant. PC1, principal coordinate 1; PC2, principal coordinate 2; PCoA, principal coordinates analysis; −, before treatment; +, during treatment.

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In a healthy individual, bacteria from Firmicutes and Bacteroidetes phyla dominate the intestinal microbiota, whereas Proteobacteria, Actinobacteria, and Verrucomicrobia phyla are usually found in a lesser extent.7 Hence, we analyzed the microbial composition of stool samples and the microbial features before and during the probiotic treatment (Fig 1C). Interestingly, Firmicutes, Actinobacteria, and Proteobacteria dominated the stool composition of the patient. There was no trace of Bacteroidetes in those samples. When analyzed at genus rank, we detected Lactobacillus and Bifidobacterium as the dominate genera, whereas Veillonella, Streptococcus, and Klebsiella were detected only in some of the samples over time. In a healthy gut, more than 50 different bacterial genera (>0.1% in relative abundance) can easily be detected. However, we only observed 5 genera and taxonomically unassigned genus from the Enterobacteriacae family in stool samples of this patient. During the administration of the probiotics, the microbial profile dramatically changed. The initially dominating genus, Lactobacillus, was significantly reduced in relative abundance, whereas Bifidobacterium became the dominant taxon for several days. Unassigned genus from the Enterobacteriacae family revealed no significant changes over time and probiotic usage (P > .05). To our surprise, the microbial profile went back to a Lactobacillus genus–dominant microbial profile 20 to 25 days after the start of the probiotics while the same therapy continued. The technique we used to identify the microbial composition does not allow one to determine to which extent this may be due to L rhamnosus GG provided with the probiotics.

Remarkably, we detected a change in relative abundance of L plantarum, 1 of the main causative agents of D-lactic acidosis.8 The relative abundance of L plantarum before probiotics administration did not change dramatically even under the antibiotic treatment. However, after probiotic treatment without any antibiotics, the relative abundance of this strain started to decrease drastically in a week, and within 3 weeks this strain completely disappeared from the patient’s gut (Fig 2). After 323 days of probiotics, there was still no detectable trace of L plantarum in the stool composition (Fig 2).

FIGURE 2

The relative abundance of L plantarum in the patient with SBS over 500 days. Each circle corresponds to the relative abundance of L plantarum detected in a stool sample collected in a single time point. Blue-colored circles are for stool samples collected before probiotic treatment while the patient was under antibiotic treatment, and red circles are for stool samples collected during the phase of probiotic treatment without any antibiotic treatment.

FIGURE 2

The relative abundance of L plantarum in the patient with SBS over 500 days. Each circle corresponds to the relative abundance of L plantarum detected in a stool sample collected in a single time point. Blue-colored circles are for stool samples collected before probiotic treatment while the patient was under antibiotic treatment, and red circles are for stool samples collected during the phase of probiotic treatment without any antibiotic treatment.

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Over the last years, the intestinal microbiota has become a main research focus in human health because of evolving detection techniques and its importance in better understanding health and diseases. However, until now only a few researchers have examined the microbiota in pediatric patients with SBS.

Piper et al9 investigated the gut microbiota signature in children with SBS depending on their growth trajectories. In 8 children with SBS, they found reduced phyla of Firmicutes, Bacteroidetes, and Actinobacteria in comparison with healthy children in the control group; however, the microbial composition of these patients did not follow the same pattern of dysbiosis. Patients with poor growth and cycling antibodies showed a depletion mainly of bacterial species from the phylum Firmicutes including Lactobacillus species.9 Our patient had Firmicutes as the dominant phylum, both during cycling antibiotics and probiotic therapy. This highlights the importance of personalized medicine by surveilling the intestinal microbiota of individuals and strengthens the concept of monitoring therapeutic interventions in these rare cases.

Engstrand Lilja et al10 compared the microbiota signature of children with SBS who were weaned off parenteral nutrition (PN) with those still on PN. Patients on PN received more antibiotics and showed a decrease in the diversity of the microbiota composition. The Enterobacteriacae family of Proteobacteria phylum was the most abundant family in this study. In our patient, there was also a reduced richness detected during antibiotic treatment in comparison with the probiotic phase as well as unassigned genus from the Enterobacteriacae family and Klebsiella. However, these taxa did not significantly change in relative abundance after ceasing the antibiotic usage or introducing probiotic administration, indicating that Proteobacteria is not the main phyla favoring the D-lactic acidosis in our patient.

Probiotic supplementations have been shown in many studies as an alternative treatment option to target the intestinal microbiota in a clinically beneficial way for the prevention of necrotizing enterocolitis,11 acute gastroenteritis,12 and antibiotic-associated diarrhea.13 We also showed that this is the case for a patient with SBS and several crises of D-lactic acidosis. To our knowledge, this is the first report of a patient with SDS and D-lactic acidosis, in whom the beneficial effect of probiotics on the intestinal microbiome were intensively investigated. Our patient highly benefited from the probiotic supplementation by avoiding a D-lactic acidosis outbreak during follow-up because of the disappearance of D-lactate producing L plantarum in his gut. Probiotics altered the intestinal composition of our patient in a way that L plantarum lost its beneficial environment to survive. However, the exact mechanistic necessities of L plantarum needs to be further investigated.

Microbial composition in stool samples before and during probiotic administration clustered into 2 distinct groups characterized by altered microbial composition after probiotic supplementation. Microbial richness and diversity changed favorably to stabilize the patient’s health without a need of further antibiotic treatment. Indicated in our results is that probiotic treatment in patients with SBS can be extremely beneficial to stop and prevent D-lactic acidosis crisis by directly affecting the existence of D-lactate–producing bacteria. Monitoring the microbiota profile during treatment interventions will increase our knowledge of the regulation of the gut microbiota and eventually allow us to further improve our treatment strategies.

     
  • CFU

    colony-forming unit

  •  
  • PN

    parenteral nutrition

  •  
  • SBS

    short bowel syndrome

Dr Yilmaz conducted the experiments and analyses, drafted the initial manuscript, and reviewed and revised the manuscript; Dr Schibli treated the patient and critically reviewed the manuscript for important intellectual content; Dr Macpherson critically reviewed and revised the manuscript; Dr Sokollik treated the patient, conducted the analyses, drafted the initial manuscript, and reviewed and revised the manuscript; and all authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work.

FUNDING: No external funding.

We thank Jessica Marie Rieder (Vetsuisse Faculty, University of Bern) for her linguistic and critical review of the article.

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Competing Interests

POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose.

FINANCIAL DISCLOSURE: The authors have indicated they have no financial relationships relevant to this article to disclose.