Hematologic side effects are associated with prolonged antibiotic exposure in up to 34% of patients. Neutropenia, reported in 10-15% of patients, increases the risk of sepsis and death. Murine studies have established a link between the intestinal microbiota and normal hematopoiesis. We sought to identify predisposing factors, presence of microbiota-derived metabolites, and changes in intestinal microbiota composition in otherwise healthy pediatric patients who developed neutropenia after prolonged courses of antibiotics. In this multi-center study, patients with infections requiring anticipated antibiotic treatment of two or more weeks were enrolled. Stool samples were obtained at the start and completion of antibiotics and at the time of neutropenia. We identified 10 patients who developed neutropenia on antibiotics and 29 controls matched for age, sex, race, and ethnicity. Clinical data demonstrated no association between neutropenia and type of infection or type of antibiotic used; however intensive care unit admission and length of therapy were associated with neutropenia. Reduced intestinal microbiome richness and decreased abundance of Lachnospiraceae family members correlated with neutropenia. Untargeted stool metabolomic profiling revealed several metabolites that were depleted exclusively in patients with neutropenia, including members of the urea cycle pathway, pyrimidine metabolism and fatty acid metabolism that are known to be produced by Lachnospiraceae . Our study confirms a relationship between intestinal microbiota disruption and abnormal hematopoiesis and identifies taxa and metabolites likely to contribute to microbiota-sustained hematopoiesis. As the microbiome is a key determinant of stem cell transplant and immunotherapy outcomes, these findings are likely to be of broad significance. Key Points:Neutropenia occurred in 17% of patients receiving prolonged antibiotic therapy.We found no association between neutropenia and type of infection or class of antibiotic used. Development of neutropenia after prolonged antibiotic treatment was associated with decreased prevalence of Lachnospiraceae and Lachnospiraceae metabolites such as citrulline.

Prolonged antibiotic exposure causes dangerous hematologic side effects, including neutropenia, in up to 34% of patients. Murine studies established a link between the intestinal microbiota and hematopoiesis. To identify factors that predispose to neutropenia in pediatric patients, we evaluated changes in microbiota-derived metabolites and intestinal microbiota composition after prolonged courses of antibiotics. In this multi-center study, patients with infections requiring anticipated antibiotic treatment of two or more weeks were enrolled. Stool samples were obtained at the start and completion of antibiotics or at neutropenia onset (prospective arm). Some patients were enrolled in a retrospective arm in which a stool sample was collected at the time of neutropenia during antibiotic therapy and 2-4 weeks after completion of antibiotics with recovery of blood counts. We identified 10 patients who developed neutropenia on antibiotics and 29 controls matched for age, sex, race, and ethnicity. Clinical data demonstrated no association between neutropenia and the type of infection or antibiotic used; however, patients with neutropenia were admitted to the intensive care unit more often and received longer courses of antibiotics. Reduced intestinal microbiome richness and, specifically, decreased abundance of Lachnospiraceae family members correlated with neutropenia. Untargeted stool metabolomic profiling revealed several metabolites that were depleted exclusively in patients with neutropenia, including members of the urea cycle pathway, pyrimidine metabolism, and fatty acid metabolism that are known to be produced by Lachnospiraceae. Our study shows a relationship between intestinal microbiota disruption and abnormal hematopoiesis and identifies taxa and metabolites likely to contribute to microbiota-sustained hematopoiesis.

Prolonged antibiotic administration has been associated with peripheral cytopenias in up to 10-15% of patients. Of these, neutropenia is particularly notable because of the associated risk of severe bacterial infections. Yet, the mechanisms underlying antibiotic-associated neutropenia remain poorly understood. Recent studies in murine models demonstrate that antibiotics suppress hematopoiesis by depletion of the intestinal microbiota, with concomitant suppression of basal Type I interferon (IFN) and STAT1 signaling. Oral administration of the microbial metabolites that induce Type I IFN restores normal hematopoiesis in antibiotic-treated mice. These findings support a paradigm in which products of the intestinal microbiota enter the bloodstream and travel to the bone marrow where they promote production of growth cytokines that support normal hematopoiesis. To identify microbiota and microbiota-derived metabolites altered in patients with antibiotic-associated neutropenia, we enrolled pediatric patients receiving antibiotic therapy for a planned duration of fourteen days or more in a multicenter-IRB approved study. We analyzed pre- and post-treatment stool samples from 51 subjects by 16S rRNA sequencing and untargeted metabolomics profiling. Neutropenia occurred in 13% of prospectively enrolled subjects. In total, 10 neutropenic patients were included in the study, with 29 control patients selected by frequency matching for age, race, and ethnicity. Length of intravenous antibiotic treatment, total length of antibiotic treatment, length of stay and admission to the intensive care unit were correlated with the development of neutropenia, whereas type of antibiotic or type of infection were not. Based on 16S rRNA sequencing, alpha diversity was significantly decreased in the stool of patients with neutropenia but not in controls (p= 0.027 vs p= 0.059). Analysis of taxa abundance showed significant depletion of Lachnospiraceae in patients with neutropenia (p=0.043), while no significant difference was identified in controls. Lachnospiraceae are a main producer of short chain fatty acids and have been linked to hematopoietic stem cell transplant outcomes. Partial least square-discriminant analysis (sPLS-DA) showed that the metabolomes of neutropenic and control stools were similar at baseline. However, 17 metabolites were differentially abundant after antibiotic treatment in the neutropenic group but not in the control group including citrulline, tyrosine, and N-acetylglucosamine/N-acetylgalactosamine. Future work will assess whether these compounds can rescue antibiotic-associated neutropenia in the animal model. This is the first clinical study linking changes in the intestinal microbiota with antibiotic associated neutropenia. An improved understanding of the molecular mechanisms by which the microbiome sustains normal hematopoiesis will lead to ways to prevent and treat antibiotic-associated bone marrow suppression.

Prolonged antibiotic administration has been associated with bone marrow suppression in approximately 10-15% of patients. The mechanisms that underlie the association between prolonged antibiotic therapy and bone marrow suppression, including neutropenia, remain poorly understood. Our recent studies in murine models demonstrated that prolonged broad-spectrum antibiotics suppress hematopoiesis by depletion of the intestinal microbiota. Furthermore, oral administration of the microbial metabolites nucleotide-binding oligomerization domain-containing protein 1 ligand (NOD1L) and desaminotyrosine (DAT) was sufficient to restore normal hematopoiesis in antibiotic-treated mice. Additionally, Type I IFN signaling in hematopoietic cells was noted to be critical for the intestinal microbiota to promote hematopoiesis, as administration of IFNα during antibiotic therapy rescued normal hematopoiesis. These findings support a paradigm in which products of the intestinal microbiota enter the bloodstream and travel to the bone marrow where they promote the production of growth cytokines that support normal hematopoiesis. To elucidate whether changes in the microbiome similar to those observed in mice also occur in humans treated with prolonged courses of antibiotics, we carried out a multicenter non-treatment, non-intervention study. We enrolled patients aged 0-17 years receiving long-term (>2 weeks) intravenous (IV) or oral (PO) antibiotic courses across two tertiary children's hospitals. We excluded patients with history of abnormal blood counts, bone marrow failure, gastrointestinal infection / syndromes / abnormalities, immunodeficiency, malignancy, severe malnutrition, use of prophylactic antibiotics or use of antibiotics immediately before admission. Patients noted to develop neutropenia while receiving antibiotics were enrolled in the retrospective arm. A stool sample was collected at 2 timepoints: at the start and at the completion of antibiotics for the prospective arm of the study; at the onset of neutropenia while on antibiotics and at resolution of neutropenia after completion of antibiotics for the retrospective arm. Here, we report an interim analysis performed for an initial cohort of 5 patients with antibiotic-associated neutropenia and 10 controls matched for age and race/ethnicity. For clinical data analysis, we assessed continuous variables using parametric and non-parametric tests when appropriate while using χ2 or Fisher's exact tests for categorical variables. Stool samples were sent for 16s rDNA sequencing to determine whether there are significant changes in the composition (individual microbial taxa) and diversity (both alpha and beta) of the gut microbiome between groups; as well as for untargeted metabolomic profiling to assess whether changes in the relative abundance of specific microbiome-related metabolites, including but not limited to NOD1L and DAT, are associated with the development of neutropenia with prolonged antibiotic treatment. Analyses of these data are currently ongoing. Neutropenia occurred in 9.4% of our prospective patient samples (3 out of 32 patients). Median age was 13y (range 6 mo. - 17y) for the neutropenic group and 11y (range 1-15y) for the control group. Forty percent of patients were female in both groups (Table 1). Total length of IV antibiotic and ICU admission were positively correlated with neutropenia in this cohort (Table 2). To our knowledge, this is the first clinical study linking changes in the intestinal microbiota with antibiotic associated neutropenia. The incidence of antibiotic-associated neutropenia was comparable to what has been previously reported. The analysis of stool samples from this cohort through 16sDNA sequencing and untargeted metabolomic profiling will outline specific changes in microbiome load, diversity, as well as specific taxa and metabolites potentially related to the development of neutropenia. Altogether, we hope to utilize an improved understanding of the molecular pathways, mediators and receptors by which the microbiome sustains normal hematopoiesis to prevent and treat antibiotic-associated bone marrow suppression. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract Background To characterize how antibiotic exposure impacts development and durability of intestinal dysbiosis and the acquisition of antibiotic resistance genes (ARGs) in the intestinal flora of premature infants in the Neonatal Intensive Care Unit (NICU), we established an infant gut microbiome biorepository (IGMB). Methods We performed prospective weekly stool collection in NICU patients meeting the following criteria: birthweight < 2000 g, postnatal age < 2 months and no diagnoses of congenital gut malformation or cyanotic heart disease. Cases were infants with bloodstream infections (BSI), defined as bacterial growth from blood culture; controls were infants with < 5 days of antibiotic exposure, no BSI nor necrotizing enterocolitis. We performed metagenomic analysis on 5–6 serial stool samples from each of the 10 cases and 10 controls (n= 100 stools). We used Wilcoxon rank sum tests for pairwise comparisons. Results From July 2021 to May 2022, 265 infants contributed 1,300 stool samples to the IGMB. In 7 of 8 BSI cases the causative pathogen was identified in the pre-BSI stool sample. Two more BSI cases did not have a pre-BSI stool sample. Microbiome species α-diversity increased with advancing postnatal age in controls but not in cases (Fig. 1). Among 6 cases with high beta lactam exposure ( >14 cumulative days by last stool collection), 3 had notable increases in the relative abundance of Enterococcus spp. (Fig. 2). Controls did not have similar trends in Enterococcus spp., but did have higher relative abundance of facultative anaerobes including Bifidobacterium, Lactobacillus, and Veillonella spp. (Fig. 3). Antibiotic resistance gene (ARG) abundance did not differ significantly between cases and controls. Conclusion Compared to controls, neonates with BSI have increased antibiotic intensity throughout their NICU admissions, reduction of microbial species diversity and overabundance of specific organisms in their gut microbiomes. We observed increased Enterococcus spp. prevalence with higher beta lactam exposure. ARG abundance did not differ between cases and controls. The clinical implications of this microbiome dysbiosis warrant further study. Disclosures Jörn-Hendrik Weitkamp, MD, Roche Diagnostics: Advisor/Consultant.

Patients with sickle cell disease are already at high risk for respiratory complications, which SARS-CoV-2 can rapidly worsen. The case emphasizes the importance of efficiently maximizing standard therapies in sickle cell patients with COVID-19.