1805Use of Stool Transplant to Clear Fecal Colonization with Carbapenem-Resistant Enterobacteraciae (CRE): Proof of Concept

Abstract

Background. Clostridium difficile infection (CDI) causes 3 million cases of diarrhea and colitis in the US annually. Although standard antibiotic is effective in suppressing C. difficile, it does not prevent relapse. Indeed, 15 to 30% of patients experience recurrence within 3 months following antibiotic treatment. Recovery of the gut microbiota has been proposed as an important key factor in preventing recurrent C. difficile. However, the stability and responsiveness of gut microbiota following C. difficile treatment and their relationship to recurrence is poorly understood. We have previously showed that Lachnospiraceae, Ruminococcaceae and butyrogenic bacteria are depleted in patients with CDI. We hypothesize that persistent depletion of these organisms is associated with recurrent CDI.Methods. Culture-independent high-density Roche/454 pyrosequencing was used to characterize distal gut microbiota of 30 adults following CDI sampled longitudinally for up to 1 year, and compared to fecal microbiota from 14 healthy controls.Results. We analyzed ∼2 million partial V1-V3 16S rRNA sequences from ∼500 longitudinal fecal samples, identifying a total of 5,904 bacterial phylotypes. Phylogenetics-based analysis revealed that the gut microbiome undergoes a slow but steady recovery in microbial diversity and species richness over a period of a few months following C. difficile therapy. However, in patients who developed recurrent CDI, the recovery of gut microbial diversity and richness was slower and incomplete. There was a paucity of phylotypes within the Lachnospiraceae and Ruminococcaceae family in the Firmicutes Phylum prior to C. difficile recurrence.Conclusion. These results implicate members of the Lachnospiraceae and Ruminococcaceae family in the pathogenesis of recurrent C. difficile and suggest a potential role in colonization resistance against C. difficile. The organisms identified here may lead to probiotic-based therapy for recurrent C. difficle and the development of a novel diagnostic test for predicting C. difficile recurrence.Disclosures. G. Wang, Merck: Grant Investigator, Research grantBackground. Bladders of chronically catheterized persons are usually colonized with potentially pathogenic organisms, yet such patients are often asymptomatic. We characterized the microbiome through Next Generation sequencing in such patients who did/did not develop symptomatic urinary tract infections.Methods. Adults over age 50 and with indwelling catheters and baseline bacteriuria were enrolled in a clinical trial of urinary catheters pre-coated with potentially probiotic E. coli HU2117. Serial urine cultures were collected after insertion of a study catheter. Urine specimens were split for traditional microbiology methods and 16S rRNA gene profiling. Subjects with E. coli HU2117 in their urine were classified as colonized with E. coli HU2117, while subjects who developed fever related to the urinary tract were classified as infected with a UTI.Results. 10 subjects received a study catheter, 9 males and 1 female. We performed paired microbiological/genomic analysis on 17 urine samples from 4 subjects, including 2 colonized with E. coli HU2117 and 2 infected with UTI. All 4 subjects had other organisms in their urine detected by standard microbiology methods throughout the course of the study. 100% of organisms detected by microbiology were also detected by sequencing, but only 11 of 30 (37%) of the genera identified by sequencing in abundance of ≥0.5% were also identified by microbiology. Frequently missed organisms included Actinobaculum, Aerococcus, Lactobacillus, and Prevotella; 12 of the 19 missed organisms were anaerobes. Subjects who developed an infection had significantly lower urinary bacterial diversity than subjects who did not develop an infection (Figure 1A, p = 0.001). Bacterial diversity did not differ by colonization status with E. coli HU2117 (p = 0.43), nor did rates of UTI differ before, during and after colonization in all 10 subjects (p = 0.5).Conclusion. Bladder colonization with E. coli HU2117 did not protect against symptomatic infection, nor did colonization impact bladder bacterial diversity. The role of anaerobes in bladder health may be underappreciated. The bacterial diversity of the bladder microbiota may play a role in protection from invasive disease.Disclosures. All authors: No reported disclosures.Background. Acquisition of microbes by the neonate is influenced by mother's microbiota and modified by antibiotics. Prolonged duration of antibiotic therapy in neonates is associated with increased risk of late-onset sepsis, a disorder critically controlled by neutrophils. However, the role of gut microbiota in regulating neutrophil behavior in the neonate is unknown.Methods. We exposed pregnant dams to antibiotics in drinking water beginning 5 days before delivery and measured bone marrow (BM) and peripheral neutrophils and IL17 producing cells in the gut and determined composition of gut microbiota in their neonates at PN 3-14d by 16S rDNA sequencing. We inoculated pups (PN 3d) with Escherichia coli K1 (104 CFU/g) via i.p. route. We transferred the intestinal contents from no antibiotic-exposed (control) mice (PN 3d) to age-matched antibiotic-exposed mice by gavage, to reconstitute the microbiota in antibiotic-exposed mice. We interrogated the role of IL17 or toll like receptor (TLR) 4 by using gene targeted neonatal mice (PN 3d) (Il17ra−/− or Tlr4−/−) or by using anti-IL17A or anti-TLR4 neutralizing antibody.Results. Perinatal antibiotic exposure altered the pattern of microbial colonization, abolished the early appearance of Gammaproteobacteria in the gut and increased the susceptibility of neonatal mice to E. coli as compared to controls (median survival 8h vs. >72h). These changes were associated with decreased number of circulating and BM neutrophils, reduced number of IL17 producing cells in the gut. Transfer of normal intestinal microbiota restored the circulating and BM neutrophil counts, IL17 producing cells and restored the host resistance to E. coli (median survival 36h). The numbers of circulating and BM neutrophils were significantly lower in neonatal Il17ra−/− or Tlr4−/−mice. Pretreatment of antibiotic-exposed mice (PN 3d) with anti-IL17A or anti-TLR4 neutralizing antibody blocked the increase in circulating and BM neutrophil counts in antibiotic-exposed neonatal mice after transfer of normal microbiota.Conclusion. Microbiota are critical in regulating postnatal granulocytosis and host resistance to E. coli via TLR4 and IL17A-dependent mechanisms raising the possibility that therapies to ameliorate antibiotic-induced microbiota dysbiosis might improve neonatal mortality.Disclosures. All authors: No reported disclosures.Background. USA300 S. aureus (SA) is the major cause of skin and soft tissue infections in the USA, many of which are difficult to eradicate despite appropriate antibiotic treatment. We postulated that human keratinocytes could provide a reservoir for staphylococcal persistence.Methods. We infected human skin grafts maintained on SCID mice, as well as keratinocytes in organotypic culture and cell lines with wt USA300 MRSA, agr null mutants and clinical isolates. Using a gentamicin protection assay, we recovered intracellular SA at times intervals, and performed histological studies.Results. We found that accessory gene regulator (agr) null mutants were recovered from within keratinocytes in significantly higher numbers than WT USA300 strains (pConclusion. The dynamic selection of SA mutants that can persist within human keratinocytes provides a source for persistent and/or recurrent skin infection. (funding Ð NIH R21 AI105978, RO1 AI103854)Disclosures. All authors: No reported disclosures.Background. Staphylococcus aureus superinfection contributed to high morbidity during the 2009 H1N1 influenza epidemic. In this setting, co-infection with methicillin-resistant S. aureus (MRSA) is specifically associated with increased mortality in pediatric patients. The molecular pathogenesis of co-infection is poorly understood, precluding the development of preventive or therapeutic interventions. S. aureus α-toxin (Hla) is a key virulence factor in acute lung injury and a focus of anti-staphylococcal clinical trials. Whereas past studies suggest that humans develop Hla-specific antibody after invasive infection, data is lacking for the current MRSA epidemic in the context of influenza co-infection.Methods. Quantitative in vitro assays measuring anti-Hla IgG and Hla-neutralizing activity were developed. In collaboration with the Pediatric Acute Lung Injury and Sepsis Investigators, sera from 57 severely-ill influenza patients admitted to pediatric intensive care units were prospectively collected and analyzed. Serial specimens were available for 34 children.Results. Day 0 sera (indicating pre- or early disease exposure) displayed anti-Hla activity that spanned 1–2 orders of magnitude, with no difference in serologic activity in patients mono-infected with influenza versus those co-infected with either methicillin-sensitive (MS-) or MRSA. However, Hla-antibody significantly increased in those with MRSA co-infection (median 16-fold increase from baseline, p < 0.001 v. MSSA or influenza only). In some children, anti-Hla IgG and neutralization activity increased over 100-fold in 2 weeks.Conclusion. Hla is a potent immunogen in influenza-S. aureus co-infection, consistent with toxin production early in disease. The observation of elevated anti-toxin titers in influenza-MRSA co-infected children suggests that toxin-mediated lung injury may exacerbate disease in this population, highlighting potential benefit from Hla-neutralization therapy in early influenza infection. These data provide impetus for ongoing studies to understand pathogen synergy, the role of Hla, and targeted disease interventions.Project support is from the Depts. of Microbiology and Pediatrics (Universit