Subjects and study design
This was a prospective, single-centre cohort study performed between May 2018 and April 2019 at Severance Hospital in Seoul, South Korea. Patients older than 6 months colonized with CPE, VRE, or CPE and VRE in the GI tract were enrolled in this study. CPE or VRE colonisation was determined by at least one positive result of rectal swab culture a week prior to FMT. Patients carrying MDROs in a location other than the GI tract or who had immunosuppression, food allergy, or high risk associated with study participation were excluded. Patients who used antibiotics were allowed to continue to do so at the time of FMT, and there were no restrictions on the use of antibiotics after FMT. The sample size of this study was determined using the GPower 3.1 Software. Power analysis indicated that a total of 25 participants (number of measurements = 5) were needed for effect size (0.25) when α = 0.05 for a power of 0.8. Therefore, a total of 27 patients were enrolled taking dropout rate into consideration.
The primary endpoint of the study was the complete elimination of antibiotic resistance genes 1 month after FMT. The secondary endpoint included decreased expression of resistance genes. All faecal samples, regardless of MDRO subtype, were tested using real-time reverse-transcription polymerase chain reaction (PCR) for the quantification of antibiotic resistance genes such as VanA, blaKPC, blaOXA, and blaNDM. Genetic response was defined as a decrease in the number of genes.
Faecal microbiota transplantation
Faecal material was obtained from healthy, unrelated donors. All volunteers were screened based on their history and clinical examination for antibiotic use within 3 months, GI symptoms, and any risk of infectious disease. Donors were excluded if they had taken any antibiotics in the past 3 months. Donors were tested for hepatitis (A, B, and C), human immunodeficiency virus, syphilis, bacteria (stool culture), rotavirus/norovirus/adenovirus (stool PCR), C. difficile toxin, parasites and their eggs (rectal exam), and VRE and CPE (stool culture). Stool samples were donated, and 100 g samples were mixed with 200 mL of sterile normal saline and stored as concentrated glycerol stocks at − 70 °C.
FMT was performed using a preparation of the frozen faecal solution via colonoscopy, duodenoscopy, a percutaneous jejunostomy tube, or an gastric capsule. The FMT delivery modality was selected based on the clinicians’ assessment as per the patient’s clinical condition. Patients under colonoscopy took 4 L of bowel preparations 1 day prior to FMT. Only patients who were less likely to aspirate were treated with capsules, which were taken after fasting for 2 h following breakfast for 2 consecutive days. After taking capsules with cranberry juice, patients were advised not to ingest any food and to sit in Fowler’s position for at least 2 h.
Real-time reverse-transcription PCR for detection of antibiotic resistance genes
Faecal samples of subjects were obtained before FMT and 1, 7, 14, and 28 days after FMT and stored at − 80 °C until used for DNA extraction. The efficacy of FMT was assessed by real-time PCR to detect expression of antibiotic resistance genes encoding VanA and carbapenemase. The DNA was extracted from faecal samples using the FastDNA® SPIN Kit for Soli (MP Biomedicals, Solon, OH, USA), which is well-suited for use with faecal samples, as per the manufacturer’ s instructions. Based on the available literature data, PCR was performed using the primers VanAF 5ʹ-ATCAACCATGTTGATGTAGC-3ʹ for VanA, KPC-rtF 5ʹ-CAGCTCATTCAAGGGCTTTC-3ʹ for blaKPC, Oxa-rtF 5ʹ-AGGCACGTATGAGCAAGATG-3ʹ for blaOXA, and Ndm-rtF 5ʹ-GATTGCGACTTATGCCAATG-3ʹ for blaNDM. PCR was performed using 2 × SYBR Green Master Mix (Applied Biosystems) on a 7300 Real-Time PCR system (Applied Biosystems). Amplifications were carried out under the following conditions: 95 °C for 30 s; 40 cycles of 95 °C for 30 s, 60 °C for 34 s, and 95 °C for 15 s, 60 °C for 60 s, and 95 °C for 15 s. Amplification was verified by running the products on a 1% agarose gel. Standard curves were generated using the reference quantities of the cloned resistance genes.
The analysis was performed in two ways. One was culture-dependent PCR, which detects resistance genes based on the resistance the strains demonstrate in cultures. The other is culture-independent PCR, which quantifies resistance genes in polymicrobial samples regardless of their phenotypes. In the first case, for example, VanA gene quantified using PCR confirmed the presence of VRE. In the latter case, all resistance genes such as VanA, blaKPC, blaOXA, and blaNDM were quantified regardless of the MDRO type.
Data collection
Clinical and laboratory data at each follow-up point were collected as follows: age, sex, body mass index, type of MDRO carriage, duration of carriage before FMT, whether antibiotic treatment was used either before or after FMT, laboratory findings such as white blood cells, haemoglobin, platelets, blood urine nitrogen, creatinine, aspartate transaminase, alanine transaminase, total cholesterol, low-density lipoprotein, albumin, fasting glucose, and C-reactive protein. All DNA values were log10 transformed before analysis.
Statistical analysis
All variables are presented as mean ± standard deviation, unless otherwise indicated. Comparisons were performed using Mann–Whitney U test, χ2 analysis, or Fisher’s exact test, as appropriate. A linear mixed model was used to confirm significant decrease in gene expression after FMT. The number of genes at each point was compared using the Wilcoxon signed-rank test. Statistical significance was set at p < 0.05. All statistical analyses were conducted using the Statistical Package for the Social Sciences version 25.0 (IBM Corporation, Armonk, NY, USA).