key: cord-301649-iipyg7ab authors: Lee, John R.; Huang, Jennifer; Magruder, Matthew; Zhang, Lisa T.; Gong, Catherine; Sholi, Adam N.; Albakry, Shady; Edusei, Emmanuel; Muthukumar, Thangamani; Lubetzky, Michelle; Dadhania, Darshana M.; Taur, Ying; Pamer, Eric G.; Suthanthiran, Manikkam title: Butyrate‐producing gut bacteria and viral infections in kidney transplant recipients: A pilot study date: 2019-10-08 journal: Transpl Infect Dis DOI: 10.1111/tid.13180 sha: doc_id: 301649 cord_uid: iipyg7ab BACKGROUND: The gut microbiome is being associated increasingly with development of infections besides Clostridium difficile infection. A recent study found an association between butyrate‐producing gut (BPG) bacteria and less frequent development of lower respiratory viral infections in allogeneic hematopoietic stem cell transplant recipients (Haak et al, Blood 131(26): 2978, 2018). In this investigation, we examine the relationship between the abundance of BPG bacteria and the development of viral infections in a cohort of kidney transplant recipients. METHODS: We recruited 168 kidney transplant recipients who provided 510 fecal specimens in the first 3 months after transplantation and profiled the gut microbiota using 16S rRNA gene sequencing of the V4‐V5 hypervariable region. We classified the kidney transplant recipients into higher BPG Bacteria Group and lower BPG Bacteria Group using the same criteria of 1% relative gut abundance of BPG bacteria as the Haak et al study. RESULTS: Administration of antibiotics against anaerobes was associated with a significant decrease in the relative gut abundance of BPG bacteria. The higher BPG Bacteria Group was associated with less development of respiratory viral infections (Hazard Ratio [HR]: 0.28, P = .01) but not with less development of CMV viremia (HR: 0.38, P = .13) or BK viremia (HR: 1.02, P = .98) at 2 years post transplantation. CONCLUSION: Our pilot investigation supports future validation of the relationship between high relative gut abundance of BPG bacteria and decreased risk for development of respiratory viral infections. strains of Escherichia coli and Klebsiella pneumoniae that caused septicemia likely originated from the gut. 2 The relationship between the gut microbiota and development of viral infections, however, is not well described. Studies in mice have shown a relationship between the gut microbiota and impaired viral clearance. Abt et al investigated antibiotic administration in a mouse model of lymphocytic choriomeningitis virus and found that antibiotic administration led to decreased innate viral immunity response as well as delayed clearance. 3 Further studies have revealed that butyrate, a product of certain gut anaerobic bacteria, can have an immunomodulatory role and contributes to overall health in distant sites such as the lung. 4 Haak et al investigated the role of butyrate-producing gut (BPG) bacteria on future development of viral infections. In a cohort of 360 allogeneic HSCT recipients, they reported that having a >1% relative gut abundance of BPG bacteria is associated with 5-fold less future development of lower respiratory viral infections. 5 Based upon this study, we profiled the gut microbiota using 16S rRNA gene sequencing of the V4-V5 region in 510 fecal specimens from 168 kidney transplant recipients. We report that having a >1% relative abundance of BPG bacteria is associated with less risk for development of respiratory viral infections in kidney transplant recipients, which provides further support for the findings from the Haak et al study. 5 Kidney transplant recipients provided fecal specimens using the Fisherbrand ™ commode specimen collection kit (Thermo Fisher Science). Fecal specimens were aliquoted into approximately 200 mg aliquots and subsequently stored at −80°C. The recipients were asked to provide the specimens at post-transplant week 1, 2, 4, and 12. DNA extraction and 16S rRNA gene amplification of the 16S rRNA gene V4-V5 region (563F and 926R) were performed as described in Lee et al. 6 Sequencing of the PCR amplicons was performed on an Illumina MiSeq platform (250 base pair × 250 base pair). Bioinformatics and taxonomic classification were performed as described in Lee et al. 6 Briefly, taxonomy was determined using nucleotide BLAST 7 with the reference training set, NCBI RefSeq 8 and a minimum E-value threshold of 1 × 10 −10 . The distribution of continuous variables was analyzed using the twotailed Wilcoxon rank-sum test for unpaired values and the two-tailed Wilcoxon signed-rank test for paired values; the distribution of categorical variables was analyzed using two-tailed Fisher's exact test. A Cox Regression Hazard Model was used to estimate whether a relative gut abundance of BPG bacteria was associated with a decreased risk for development of respiratory virus infections, CMV viremia, or BK viremia. Survival curves were constructed and compared using the log-rank test. All analyses were performed in R 3.3.3 in RStudio 1.1.463. Among the 510 fecal specimens from the 168 kidney transplant recipients, the mean ± standard deviation amount of high quality 16S rRNA bacterial gene sequences was 18 179 ± 11 033. The overall demographics and transplant characteristics of these subjects are presented in Table 1 . Out of the 61 BPG species, 40 species were detected (Table S1 ). The sum relative gut abundance of BPG bacteria was calculated for each fecal specimen (defined hereafter as relative gut abundance of BPG bacteria). The mean relative gut abundance of BPG bacteria among all of the 510 fecal specimens was 14.2% with a standard deviation of 13.1%. The top BPG species were: Faecalibacterium prausnitzii (3.7%, mean), Holdemanella biformis (2.7%), Eubacterium dolichum (2.6%), Coprococcus comes (1.2%), Subdoligranulum variabile (0.8%), and Eubacterium rectale (0.7%). A heatmap of the relative gut abundance of these top species among all the fecal specimens is presented in Figure 1 . F I G U R E 1 Heatmap of the top abundant butyrate-producing gut bacteria in the kidney transplant cohort. The 510 fecal specimens from the 168 kidney transplant recipients are represented on the x-axis and the top 6 butyrateproducing gut species are on the y-axis. The relative abundance of each species is represented in blue, log-scaled. There is marked variability in the top butyrateproducing species among the specimens signed-rank test; Figure 2D ). We next evaluated the subgroup of subjects in the Paired Abx Group who received anaerobic antibiotic coverage. Anaerobic antibiotic coverage included the following antibiotics: metronidazole, carbapenems, clindamycin, combination of penicillin and beta-lactamase inhibitor, and oral vancomycin. 9, 10 The rationale for evaluating antibiotics with anaerobic coverage was based on the strict anaerobic nature of many of the BPG bacteria (eg, Faecalibacterium prausnitzii and Eubacterium rectale). 11 Among the Paired Abx Group, 6 subjects had anaerobic antibiotic coverage and all 6 had a significant decrease in the relative gut abundance of BPG bacteria from post-transplant week 1 to post-transplant week 4 (median 9.9% vs 1.9%, respectively, P = .03, Wilcoxon signed-rank test; Figure 2E ). We also evaluated whether the relative gut abundance of BPG bacteria was associated with gender, age greater than 65 years old, African American race, induction therapy, steroid maintenance therapy, and kidney function at 1 month post transplantation. As antibiotics are associated with decreased abundance of BPG bacteria, we evaluated the paired specimens from the 54 subjects who did not receive antibiotics from post-transplant week 1 and post-transplant The abundance of butyrate-producing gut bacteria over time in the kidney transplant cohort. Panel A. Each point represents a single fecal specimen with the day the specimen was produced on the x-axis and the relative gut abundance of butyrate-producing gut (BPG) bacteria on the y-axis. The line represents a locally estimated scatterplot smoothing (LOESS) curve with 95% confidence intervals indicated by the shaded band. Panel B. Each point represents a single fecal specimen with the day the specimen was produced on the x-axis and the relative gut abundance of butyrate-producing gut (BPG) bacteria on the y-axis. A point in blue represents an individual fecal specimen from the 231 specimens from the 78 patients in the No Abx Group and a point in magenta represents an individual fecal specimen from the 279 specimens from the 90 patients in the Abx Group. The line represents a LOESS curve with 95% confidence intervals indicated by the shaded bands. Panel C. Box and whisker plot with the relative gut abundance of BPG bacteria on the y-axis and the fecal specimen posttransplant week on the x-axis for the 54 subjects in the Paired No Abx Group. In each box and whisker plot, the line represents the median, the edges of the box plot 25% and 75%, the whiskers 1.5 times the median, and points represent outliers. The P value was calculated using the Wilcoxon signed-rank test. Panel D. Box and whisker plot with the relative gut abundance of BPG bacteria on the y-axis and the fecal specimen post-transplant week on the x-axis for the 22 subjects in the Paired Abx Group. In each box and whisker plot, the line represents the median, the edges of the box plot 25% and 75%, the whiskers 1.5 times the median, and points represent outliers. The P value was calculated using the Wilcoxon signed-rank test. Panel E. Box and whisker plot with the relative gut abundance of BPG bacteria on the yaxis and the fecal specimen post-transplant week on the x-axis for the 6 subjects in the Paired Abx Group who received antibiotics with anaerobic coverage. In each box and whisker plot, the line represents the median, the edges of the box plot 25% and 75%, the whiskers 1.5 times the median, and points represent outliers. The P value was calculated using the Wilcoxon signed-rank test week 4 (Paired No Abx Group). The relative gut abundance of BPG bacteria was significantly lower in the female group than in the male group (median 11.4% vs 17.7%, P = .03, Wilcoxon rank-sum test; Figure S1A ) and significantly lower in the steroid maintenance group than in the in steroid free group (median 9.4% vs 17.6%, P = .01, Wilcoxon rank-sum test; Figure S1E ). There were no significance associations between the relative gut abundance of BPG bacteria and age greater than 65 years old, African American race, induction therapy, and kidney function at 1 month post transplantation ( Figure S1B -D,F). We evaluated whether the relative gut abundance of BPG bacte- In this study, we report one of the first description of butyrate-producing gut bacteria over time in the kidney transplant population. Our data reveal that antibiotics with anaerobic coverage is associated with a decrease in relative gut abundance of BPG bacteria. Our data also support a relationship between high relative gut abundance of BPG bacteria and less risk for development of respiratory It is important to note the allogeneic HSCT subjects had a much higher proportion of patients with a less than 1% relative gut abundance of BPG bacteria than the kidney transplant recipients in our study (81% vs. 8%, respectively). We speculate that this difference is likely because of the higher number of antibiotics given to the allogeneic HSCT population. In our study, we found that an- analyses. In addition, we used the cutoff of 1% relative gut abundance of BPG bacteria, which was utilized in the allogeneic HSCT recipient population. 5 Notably, there was a significance difference in the proportion of HSCT recipients and in the proportion of kidney transplant recipients with less than 1% relative gut abundance of BPG bacteria and whether this cutoff is also relevant for the kidney transplant population is unknown. In order to address this concern, we evaluated both a bottom quartile cutoff as well as a median cutoff and we did find an association between high relative gut abundance of BPG bacteria and decreased risk for respiratory Despite these limitations, our preliminary study provides one of the first descriptions of BPG bacteria in kidney transplant recipients early after kidney transplantation. We find that antibiotics with anaerobic coverage are associated with a decrease in the relative gut abundance of BPG bacteria and we also report an association between the relative gut abundance of BPG bacteria and the risk for development of respiratory viral infections in kidney transplant recipients. Our results are preliminary in nature and require replication in a larger cohort of kidney transplant recipients. In particular, we did report a trend toward significance of the association between the relative gut abundance of BPG bacteria and CMV viremia, which would need further validation for the CMV viremia finding as well as for CMV disease. Our study examined the relative gut abundance of BPG bacteria, but future studies examining the absolute gut abundance of BPG bacteria and/or fecal butyrate concentrations may provide more insight into the relationship between BPG bacteria and development of viral complications. Nevertheless, if our findings are confirmed, our data support therapeutic options in the form of modulating the gut microbiota for decreasing viral infection rates. We thank the National Institutes of Allergy and Infectious Diseases for their research support via NIH grant K23 AI 124464 (JRL) and R37 AI 051652 (MS). The authors of this manuscript have the following competing interests to disclose. EGP has received speaker honoraria from Bristol Myers Squibb, Celgene, Seres Therapeutics, MedImmune, Novartis, and Ferring Pharmaceuticals and is an inventor on patent application # WPO2015179437A1, entitled "Methods and compositions for reducing Clostridium difficile infection" and #WO2017091753A1, enti- Intestinal domination and the risk of bacteremia in patients undergoing allogeneic hematopoietic stem cell transplantation Precision identification of diverse bloodstream pathogens in the gut microbiome Commensal bacteria calibrate the activation threshold of innate antiviral immunity Butyrate: a double-edged sword for health? 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