Figure 1. Figure 1. ESBL-Producing E. coli Antimicrobial Resistance Patterns and Sequence Variant Analysis Based on Whole-Genome Sequencing. Panel A shows antimicrobial resistance patterns of extended-spectrum beta-lactamase (ESBL)–producing Escherichia coli. The testing method and interpretive categories were based on the recommendations of the Clinical and Laboratory Standards Institute.17 Panel B shows sequence variant analysis based on whole-genome sequencing. Patient 1 was enrolled in an open-label trial of fecal microbiota transplantation (FMT) oral capsules for the treatment of refractory hepatic encephalopathy (HE) (ClinicalTrials.gov number, NCT03420482). Patient 2 was enrolled in a phase 2 trial to preemptively administer FMT oral capsules before and after allogeneic hematopoietic-cell transplantation (HCT) (NCT03720392). Numbers denote single-nucleotide polymorphism distances between pairs of isolates, as computed by counting the number of variable sites. The yellow-tinted boxes indicate clonal isolates.

The occurrence of these two bacteremias with similar resistance patterns (Figure 1A) prompted investigation. FMT capsules from all lots are routinely frozen for possible future analyses. We confirmed that Patients 1 and 2 both received FMT capsules from the same lot from the same donor. Each of three lots of capsules from that donor was found to contain ESBL-producing E. coli with a resistance pattern similar, but not identical, to the blood isolates from the patients, as determined by means of culture (Figure 1A). Frozen fecal samples that were obtained from Patients 1 and 2 before FMT were cultured and found to be negative for ESBL-producing organisms.

Genomes of ESBL-producing E. coli isolated in blood samples from the patients and in a stool sample from the donor and a control strain were sequenced by personnel at Day Zero Diagnostics. Genomic relatedness between samples was calculated by means of whole-genome sequencing and single-nucleotide polymorphism (SNP)-based analysis. Genomic DNA from the bacterial samples was sequenced on the iSeq 100 System (Illumina), reads were mapped to a closely related reference genome, and high-quality SNPs were identifed in each sample. SNP distances between pairs of isolates were computed by counting the number of variable sites (see Section 2 in the Supplementary Appendix). In silico multilocus sequence typing and serotyping showed that the three isolates belong to the same multilocus sequence type 131 and serotype O25:H4. SNP analysis revealed high genetic similarity among the three isolates, which either had no SNP differences or differed by 1 SNP across 4.5 million base pairs examined (Figure 1B). The isolates exhibited much lower genetic relatedness (distance of 121 to 124 SNPs) to the control E. coli isolate of multilocus sequence type 131 and serotype O25:H4 derived from a blood culture that had been obtained at our hospital in 2017 (Figure 1B). Similarly, a genomic analysis of nine previously sequenced multilocus sequence type 131 isolates (six isolates of O25:H4 serotype and three of O16:H5 serotype) derived from clinical urine samples with no known epidemiologic links (also obtained at our hospital) revealed that they all had a distance of more than 100 SNPs from the FMT-linked isolates. Genomic studies of E. coli outbreaks have shown epidemiologically linked isolates that had a distance from each other of less than 10 SNPs to be a part of a transmission cluster.18-20 Because there was a distance of at most 1 SNP, the isolates from the donor and the patients were deemed with high confidence to be clonal organisms.

Table 2. Table 2. Results of Stool Screening for ESBL-Producing Organisms.

A total of 22 patients received FMT capsules generated from this donor (6 recipients in the two trials described above and 16 recipients who received FMT for the treatment of recurrent or refractory C. difficile infection). All recipients were subsequently contacted and informed, and stool screening with an ESBL selective medium was offered. The results are shown in Table 2. For the patients in the two trials discussed herein, stool samples that had been obtained before FMT were available; the samples were cultured, and all showed no growth on screening plates. No pre-FMT samples were available for the 16 recipients who underwent FMT for the treatment of C. difficile infection. Of the 12 tested patients who received FMT capsules generated from this donor, 5 had post-FMT samples that grew organisms on an ESBL selective medium. Of the 7 tested recipients who underwent FMT for recurrent or refractory C. difficile infection, 4 had post-FMT samples that showed growth on an ESBL selective medium that was morphologically consistent with E. coli. The number of antibiotic-resistant genes contained within the fecal microbial metagenome has been shown to be much higher in patients with recurrent or refractory C. difficile infection than in healthy persons, and screened donors have even lower numbers of resistant genes.21 When FMT is successful, the recipient’s metagenomic burden of antimicrobial resistance genes mimics that of the donor.21 Although we cannot conclusively attribute positive screening results for ESBL-producing organisms in other asymptomatic recipients to FMT, the rates of positive tests are, in our opinion, unexpectedly high and probably represent transmission through FMT. Stored capsules from 32 FMT preparations from 10 other donors (2014 to 2018) were subsequently screened, and none showed any growth on ESBL screening plates.