Upper airway microbiome changes in children with sickle cell disease during vaso‐occlusive and acute chest syndrome episodes
Authors: Brett R. Loman, Chandra L. Shrestha, Kavitha Kotha, Abena Minta, Shuzhong Zhang, Asuncion Mejias, Michael T. Bailey, Susan Creary, Benjamin T. Kopp
Journal: American Journal of Hematology (2020)

Sickle cell disease (SCD) results in numerous complications, including acute chest syndrome (ACS) and vaso-occlusive crises (VOC). Beginning in infancy, patients with SCD are exposed to prophylactic antibiotics to prevent invasive pneumococcal disease from functional asplenia. Furthermore, these children commonly receive empiric broad-spectrum antibiotics when they present with febrile illnesses. Note, ACS is a leading cause of morbidity and premature death in people with SCD. Treatment with broad-spectrum systemic antibiotics is recommended for all occurrences, despite limited studies on the true occurrence of bacterial infections in ACS. VOC episodes are the most common complication in children with SCD. While antibiotics are not routinely recommended for uncomplicated VOC, they may be given if patients are febrile. The link between microbiome alterations and disease pathogenesis is established in other chronic diseases (eg, cystic fibrosis), but it is unknown if microbiome alterations are linked with SCD complications. Further, frequent antibiotic exposures can cause antibiotic-resistant pathogens. Therefore, is important to investigate if microbiome alterations occur at baseline or during acute illnesses such as ACS or VOC, given the antibiotic exposure these children frequently receive. To address this deficit, we conducted a prospective, single center one-year study of children with SCD to describe their baseline upper airway microbial composition and explore changes in microbial composition that occur early in hospitalizations for ACS or VOC. We hypothesized that the upper airway microbiome would significantly change during ACS and VOC compared to baseline. Full methods are available in the online supplement. In brief, these data were collected as part of a prospective study to explore gene expression profiles among children with SCD hospitalized for VOC and ACS.1 Deep posterior oropharyngeal swabs for microbiome analysis were obtained at baseline and placed in RNA later (ThermoFisher, AM7020). Samples were recollected within 24 hours of a hospitalization for VOC or ACS during their year of study participation. After DNA isolation and quality checks, samples were submitted to the University of Illinois for high throughput sequencing for 16S rRNA gene using the 515F (GTGYCAGCMGCCGCGGTAA) and 926R (CCGTCAATTCMTTTRAGT) primer pair and Illumina MiSeq platform. Differential abundance was analyzed via Wilcoxon test with false discovery rate P value correction, with statistical significance set at P < .05. We enrolled 83 children, and 25 had at least one hospitalization for either ACS or VOC. Fourteen participants were excluded from matched baseline to hospitalization microbiome analysis, due to low total sequences (n = 2) or >30% contaminant sequences in the baseline or hospitalization sample (n = 12). The demographics of the hospitalized cohort with acceptable sequences are in Table S1. The full cohort baseline demographics have been previously reported.1 Average counts for the overall cohort's baseline microbial composition are in Table S2. Streptococcus was the most abundant taxa present on average in all baseline samples, followed by Veillonella, Acinetobacter, Delftia, and Neisseria. Streptococcus, Veillonella and Neisseria predominance were similar to prior oropharynx microbiome studies in healthy children,2 although limited data on racially-matched children is available. We then compared the oropharyngeal microbiomes of children with SCD at baseline to early during their hospitalization for either a VOC (n = 6) or ACS (n = 5) episode. There were no baseline differences in α-diversity or β-diversity between children who were hospitalized for ACS or VOC and children not hospitalized. Compared to their baseline, children admitted for VOC experienced no changes in bacterial α-diversity (total ASVs, Pielou's evenness, Faith's, and Shannon), but demonstrated altered bacterial β-diversity of total bacterial populations as measured by Bray-Curtis Dissimilarity (Figure 1A, P = .016), but not by other metrics (weighted and unweighted UniFrac distances). Participants hospitalized with ACS had a near significant increase in α-diversity as calculated by Pielou's Evenness, a measure of evenness across represented taxa (Figure 1B, P = .057), but not by other metrics compared to their baseline. Samples from children during ACS displayed altered beta-diversity as expressed by weighted UniFrac distances (Figure 1C, P = .031), but these were not different by other metrics (Bray-Curtis Dissimilarity and unweighted UniFrac distances). Among the most abundant genera, several genera significantly changed during VOC (n = 2) and ACS (n = 3) episodes compared to matched-baseline samples (Figure 1D). Specifically, patients hospitalized for VOC had increased relative abundances of Prevotella 6 and Massilia. (Figure 1E,F). Note, ACS was associated with reduced relative abundance of Streptococcus and Gemella, but also increased Massilia (Figure 1F–H). We did not detect Mycoplasma or Chlamydia in any ACS sample. Relative abundances of Streptococcus, Gemella, Massilia, and Prevotella 6 in VOC and ACS groups at baseline were similar to baseline abundances in non-hospitalized children (Figure 1E–H). Nine children who were not hospitalized during the study had one-year follow-up samples analyzed. In contrast to the ACS and VOC groups, there was no difference in α-diversity or β-diversity, or the top genera over time for children not hospitalized (Figure 1B,E–H, control follow-up). The 50 most abundant taxa detected in all patients are presented in Table S2. Last, we performed a correlation analysis to determine if clinical factors were associated with the observed taxa changes. There were no correlations at baseline or during ACS or VOC with age, gender, genotype, hydroxyurea, prophylactic penicillin, inhaled corticosteroids, smoke exposure, hemoglobin, or LDH. Five patients had a respiratory virus isolated during their ACS, but there were no significant differences in bacteriome composition by viral positivity in this small subset of children. Although our understanding of VOC and ACS pathogenesis has evolved, treatments remain primarily supportive. However, empiric antibiotic treatment during febrile VOC or ACS episodes may be unnecessary or promote antibiotic resistance. This exploratory study identified that there may be unique alterations in the upper airway microbiome within individuals during ACS and VOC episodes. We identified alterations from the baseline microbiome of children with SCD that may be reflective of recurrent antibiotic exposure or pathogenic changes related to illness. For example, we observed an increased relative abundance of Massilia during both VOC and ACS. Massilia have been poorly characterized in humans, with rare case reports of infection3 or in nosocomial aerosols collected in a hospital. It is unclear if the increased Massilla abundance we observed may reflect hospital exposure, compensation for reduction in other bacterial taxa, or an increase in pathogenic species. Overall, the changes in Gemella and Massilia relative abundance we observed were smaller compared to Streptococcus. However, these changes could be consistent with the keystone-pathogen hypothesis, whereby low-abundance pathogens orchestrate inflammatory disease by remodeling the normal microbiome into dysbiotic communities. Our findings of reduced abundance of Gemella and Streptococcus have been reported and implicated in other diseases.4 Subsequent studies should aim to further evaluate and confirm if these taxa may have a pathogenic role in acute ACS. In contrast to prior studies indicating ACS is associated with Mycoplasma and Chlamydia infection, we identified that these pathogens were not present during hospitalization. While it is possible that these organisms were not detected by sequencing, this finding is consistent with recent studies. In a study of adults with febrile ACS who underwent nucleic acid amplification testing (NAAT), only 16% of ACS cases detected a bacterial pathogen.5 Systematic studies on the prevalence of invasive bacterial infections during febrile VOC episodes are also lacking. Combined, our results and past studies suggest a need to investigate the role of acute bacterial infection in ACS and febrile VOC episodes and whether testing-guided treatment may be appropriate. The role of the observed changes in the oropharyngeal microbiome during VOC episodes is unclear, but it is possible that these changes have implications beyond antibiotic resistance. A recent study in SCD mice demonstrated that 7 weeks of continuous antibiotic treatment enhanced osteoblast function.6 These findings support the hypothesis that altered intestinal bacteria may augment inflammatory pathways and impair osteoblast function and bone health. Whether a similar pathway of airway dysbiosis-induced inflammatory signaling promotes acute VOC episodes is unknown. Our study was limited by a single-center cohort, and lacked a second validation cohort since few participants were hospitalized. This limited our ability to identify clinical factors that correlate with microbiome changes. However, our study was strengthened by matched pairs of baseline and hospitalization samples whereby individuals could serve as their own internal controls. Also, some (n = 14) samples were inadequate for analysis. These were likely due to technical limitations that occurred because the oropharynx has a relatively low biomass compared to other body sites. Future studies evaluating the SCD upper airway microbiome should include having an experienced staff member obtain samples to prevent inadequate sampling or contamination. We also did not have invasive lower airway or sputum samples that may be more reflective of pathogenic microbiome changes during ACS. However, collecting these samples is challenging and could add additional risk to acutely ill children with SCD. In summary, these data provide the first evidence that there may be changes in the upper respiratory microbiome in patients with SCD at baseline and during ACS and VOC episodes. Future studies are needed to confirm these findings and evaluate their potential implications on ACS and VOC pathogenesis. Such studies could eventually inform prophylactic antibiotic treatment, VOC and ACS prevention, and treatment for people with SCD. We thank Anthony Villella for verification of ACS cases. The authors thank all the patients and their families for their contributions. We thank the University of Illinois Functional Genomic Lab for assistance with sequencing. Brett R. Loman, Susan Creary, Kavitha Kotha, Michael T. Bailey, and Asuncion Mejias designed the study. Brett R. Loman, Susan Creary, and Benjamin T. Kopp wrote the manuscript. Brett R. Loman performed statistical analysis and sequence identification. Chandra L. Shrestha isolated microbial samples. Shuzhong Zhang assisted with sample isolation and specimen processing. Benjamin T. Kopp and Susan Creary collected the clinical samples. Abena Minta maintained clinical data. All authors analyzed and interpreted data. All authors contributed to editing and approved the final manuscript. The authors declare no conflicts of interest. Appendix S1. Supporting Information Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.