Fecal microbiota transplantation restores dysbiosis in patients with methicillin resistant Staphylococcus aureus enterocolitis

Patients

This study was approved by the Institutional Ethics Committee of Jinglin hosipital.
Five cases from July, 2013 to February, 2014 were collected in Jinling hospital. Written
informed consent was obtained from all the patients and the donors. There were three
males and two females with an age range from19–45 years (mean age, 28 years) (Table 1). No patients were on proton pump inhibitors (PPI) or had a history of MRSA prior
to operation. Antibiotics given at induction and continued in the postoperative period
were showed in detail (Table 2). The patients were placed on the open ward prior to development of symptoms.

Table 1. Basic clinical data of the patients

Table 2. Drugs given at induction and continued in the postoperative period

All the patients developed unexplained high fever (over 39°), bloating, nausea, vomit,
a high stoma output or diarrhea in the color of yellow-green with copious amounts
of mucus leading to dehydration and tachycardia after short time of operation (2-4d).
Full septic screen was performed. This included bacterial swabs of stoma as well as
blood, urine, ascites and gastric juice cultures. These were repeated throughout the
diseased state. Fecal was investigated for Salmonella spp. and Clostridia difficile. Radiological studies included plain radiographs, ultrasonography, contrast computerized
tomography (CT) to exclude intraabdominal or other infections. We got the etiology
diagnosis from all the ptients’ gastric juice cultures which revealed MRSA and collected
MRSA strain for further genotype identification. One patient (Patient 2) also underwent
colonoscopy correspondingly showed mucosal edema and large quantity of pseudomembrane
(Fig. 1).

Fig. 1. Patient 2’s colonoscopy before and after treatment. a. showed mucosal edema and large quantity of pseudomembrane in the colon before FMT
and vancomycin. b. the pseudomembrane disappeared and mucosa healed well after the treatment of FMT
combined with vancomycin for three days. FMT?=?fecal microbiota transplantation

Donor

The intended stool donors had received no antibiotic therapy within the last 6 months.
To avoid a transmission of other diseases, donors had to have a negative history for
intestinal diseases or recent gastrointestinal infections, autoimmune or other immune-mediated
diseases, or any kind of malignancies. Chronic hepatitis B and C, human immunodeficiency
virus, and syphilis were excluded and the donors’ stool was tested for C. difficile, enterohemorrhagic Escherichia coli, Salmonella, Shigella, Yersinia, and Campylobacter as well as parasites. Stool samples of four donors were collected for further analysis
of the fecal microbiota, unfortunately we lost a sample stool from one donor.

Donor material preparation

Donors produced stool samples within 6 h before FMT. 60 g fresh fecal samples were
blended with 350 ml sterile saline for 10 min in a designated GI laboratory space.
This blended fecal mixture was then filtered through 3 gauze pieces to remove larger
sediments. Filtered fecal preparation was then kept at 4 °C until FMT was performed.

Transplantation procedure

All patients had been informed and agreed to undergo FMT. All but one (who accepted
vancomycin for 10 days before FMT) were maintained on full dose of vancomycin (Vancocin
cp) (500 mg, twice a day for continuous three days) until 12 h before the FMT procedure.
The prepared donor microbiota was administered via nasojejunal tube once a day for
3 consecutive days (Table 3). Since the patients all suffered from severe diarrhea, no mechanical bowel preparation
were done prior to FMT as usually recommended 4].

Table 3. Data of fecal mircobiota transplantation

Microbial diversity and type of MRSA analysis

Microbial diversity analysis

Microbial DNA was extracted from fecal samples using the E.Z.N.A. ® DNA Kit (Omega
Bio-tek, Norcross, GA, U.S.) according to manufacturer’s protocols. The V4-V5 region
of the bacteria 16S ribosomal RNA gene were amplified by PCR (95 °C for 2 min, followed
by 25 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s and a final extension
at 72 °C for 5 min) using primers 515 F 5’-barcode- GTGCCAGCMGCCGCGG)-3’ and 907R
5’-CCGTCAATTCMTTTRAGTTT-3’, where barcode is an eight-base sequence unique to each
sample. PCR reactions were performed in triplicate 20 ?L mixture containing 4 ?L of
5?×?FastPfu Buffer, 2 ?L of 2.5 mM dNTPs, 0.8 ?L of each primer (5 ?M), 0.4 ?L of
FastPfu Polymerase, and 10 ng of template DNA. Amplicons were extracted from 2 % agarose
gels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union
City, CA, U.S.) according to the manufacturer’s instructions and quantified using
QuantiFluorâ„¢ -ST (Promega, U.S.). Purified amplicons were pooled in equimolar and
paired-end sequenced (2?×?250) on an Illumina MiSeq platform according to the standard
protocols. The raw reads were deposited into the NCBI Sequence Read Archive (SRA)
database.

Type of MRSA analysis

DNA were extracted from different specimens.9 primers of SCCmecI-V were synthesized
with multiple PCR technique according to the reference 5] and for STR analysis to make out the type of MRSA.