A 37-year-old Chinese woman suffering from end-stage renal disease underwent a cadaveric kidney transplantation in November 2018. Postoperative recovery was uneventful and the serum creatinine level of the patient returned to normal three days after the transplantation. On postoperative day 8, the patient experienced acute rejection, which was treated with high-dose prednisone and plasma exchange, and after treatment, her renal function became normal. The patient developed CRKP infection one month after the kidney transplantation. The CRKP could be detected in incision secretion, urine, blood and anal swab cultures of the patient, and resulted in impaired surgical incision healing. Multi-locus sequence typing (MLST) of seven sets of DNA fragments derived from housekeeping genes was performed to examine the genetic diversity of CRKP strains isolated from the incision, urine, and fecal samples of the patient, and the results revealed that all the three strains were exclusively grouped into ST11 (Table S1 and Figure S1). The hypermucoviscous and hypervirulent nature of the Klebsiella pneumoniae were also checked using the "string test" and qRT-PCR, respectively. The results showed that the CRKP isolate exhibited a positive string test (Figure S2), and expressed virulence-associated genes (rmpA2, ybtS, iucA, rmpA and peg344; Table S2 and Figure S3), indicating its hypermucoviscous and hypervirulent phenotype. Since the CRKP isolated from the patient was resistant against most antibiotics except tigecycline, fosfomycin and polymyxin (Table S3), the combination treatment of with tigecycline (50 mg/12 hours), fosfomycin (4 g/8 hours) and meropenem (1~2 g/8 hours) and was utilized for treating the infection. The detailed treatment timeline for the CRKP infection is shown in Figure 1. After 46 days of antibiotic treatment, the incision of the patient could still not heal. The CRKP converted to be negative in the patient blood cultures, but positive in the purulent incision secretion, urine and anal swab cultures. The patient had normal body temperature and white blood cell (WBC) count (4.6x109/L), but the C-reactive protein (CRP) level was elevated (24.79 mg/L). Symptoms of urinary tract infection such as frequent and urgent urination, and pelvic pain could also be observed. Under this condition, FMT was performed, and antibiotics were stopped one day before FMT. Preoperatively, the patient took proton pump inhibitor, and polyethylene glycol electrolyte solution was given for bowel preparation. Washed microbiota suspensions from a healthy volunteer (screening protocol was shown in Table S4) obtained from fmtBank.org were used for FMT. The preparation of the washed microbiota suspensions was conducted as described previously. Briefly, 10 cm3 (about 1.0x1013 bacteria) of the final precipitated microbiota is the basic unit dose for clinical use, and the volume ratio of the final precipitation/vector solution for making suspensions is 1:2. In this study, 10.3 basic units of the washed microbiota suspensions (about 1.03x1014 bacteria) were given to the patient via nasogastric tube. The patient was not given any antimicrobial agent postoperatively. One week after FMT, the patient's urine and anal swab cultures returned negative for CRKP. The patient's incision began to heal three days after FMT, and showed complete healing 17 days after FMT (Figure 2A). The patient tolerated the FMT procedure without complications including abdominal pain, diarrhea and fever. Moreover, there was no obvious increase in serum creatinine and blood urea nitrogen (BUN) after FMT (Figure 2B), suggesting FMT did not have adverse effect on the patient's renal function. The patient had no symptoms of infection two months after FMT, indicating a positive clinical outcome.
We performed a microbiota analysis of the fecal samples from the donor and the patient. Alpha diversity analyses showed that before FMT, the patient was associated with obviously lower species richness (Chao1 index, Figure 3A) and diversity (Shannon index, Figure 3B) than the donor, which significantly increased at one week, three weeks and two months after FMT (Figure 3A and B), indicating that FMT could lead to an increase in the richness and diversity of the patient fecal microbiota. Next, beta diversity analysis was calculated using weighted and unweighted UniFrac distances, and visualized via principle coordinate analyses (PCoA) to compare microbial communities between samples. Both the weighted (Figure 3C) and unweighted (Figure 3D) UniFrac methods revealed significant divergence of the microbial communities between the donor and the patient before FMT. It should be noted that though the patient microbial community post-FMT still differed from the donor one, their distances visibly decreased, especially at one week and three weeks after FMT (Figure 3C and D).
Furthermore, we analyzed the microbial compositions in detail, and the results were visualized as cumulative histograms (Figure 4A). At genus level, Prevotella_9 (relative abundance 35.9%), Ruminococcaceae_NK4A214_group (relative abundance 12.9%) and Faecalibacterium (relative abundance 10.0%) accounted for the major bulk of the donor microbiota, while Bacteroides (relative abundance 60.6%) and Klebsiella (relative abundance 38.9%) were predominant in the recipient's sample, which accounted for nearly 90% of the total taxa. At one and three weeks after FMT, Phascolarctobacterium increased significantly in relative abundance (28.8% and 26.1%), and became the most dominant taxon. Besides Phascolarctobacterium, the proportions of [Ruminococcus]_gnavus_group, Alistipes, and Lachnoclostridium were also observed to increase after FMT. Meanwhile, at two months after FMT, Bacteroides, Lachnoclostridium and Phascolarctobacterium were the predominant populations in the fecal samples of the patient. Results of the cluster analysis showed that the fecal microbial composition of the patient following FMT was more similar to the donor microbial composition than that of the patient collected prior to FMT (Figure 4A). It should be noted that the relative abundance of Klebsiella significantly decreased at one week after FMT (61.15% vs 1.5%), and remained low at three weeks (1.4%) and two months (6.0%) after FMT (Figure 4B). Moreover, the relative abundances of Ruminococcus, Coprococcus, and Clostridium were negatively correlated with that of Klebsiella, indicating that these genera may competitively interact with Klebsiella (Figure 4C).