Open-label, Randomized Study to Assess the Efficacy of a Probiotic or Fecal Microbiota Transplantation (FMT) on the Eradication of Rectal Multidrug-resistant Gram-negative Bacilli (MDR-GNB) Carriage (PROFTMDECOL)
Phase: NA
Conditions: CARRIER STATE
Sponsor: Hospital Clinic of Barcelona

Protocol 
 
PROFTMDECOL Study 
 
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CLINICAL RESEARCH PROTOCOL 
 
 
 
 
 
 
 
 
 
 
 
Open-label, randomized study to assess the efficacy of a probiotic or fecal microbiota transplantation 
(FMT) on the eradication of rectal multidrug-resistant Gram-negative bacilli (MDR-GNB) carriage 
 
 
 
 
 
 
 
Protocol: PROFTMDECOL 
Version: 1.0 (02/12/2019) 
 
Sponsor: Fundació Clínic per a la Recerca Biomèdica 
 
Principal Investigator: Dra. Ana Del Río. 
Co-principal Investigator: Dr. José Antonio Martínez. 
Infectious Diseases  
Hospital Clínic 
Villarroel, 170 
08036 Barcelona 
 

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1. Brief description 
 
1.0. Study 
 
This is an investigator-initiated, single center, parallel-group, randomized, open-label, clinical 
research with non pharmacological products. 
 
1.1. Sponsor 
 
Fundació Clínic per a la Recerca Biomédica. 
Rosselló, 149 
08036 Barcelona 
 
1.2. Study identification 
 
Title: Open-label, randomized study to assess the efficacy of a probiotic or fecal microbiota 
transplantation (FMT) on the eradication of rectal multidrug-resistant Gram-negative bacilli 
(MDR-GNB) carriage. 
 
Protocol code: PROFTMDECOL 
 
1.4. Investigators 
 
Principal investigator: Dra. Ana Del Río, Infectious Diseases 
Co-principal investigator: Dr. José Antonio Martínez, Infectious Diseases 
 
Collaborators: 
 
Dr. C. Pitart, Microbiología Clínica 
Dr. J. Mercadal, Anestesiología  
Dra. E. López, Farmacia Hospitalaria 
 
1.5. Clinical sites 
 
Single centre study: Hospital Clínic, Barcelona 
 
1.6. Ethics approval 
 
The protocol, informed consent form, and other required documents will be approved by 
the Ethics Committee (Comité de Ética de la Investigación, CEI) of the Hospital Clínic de 
Barcelona before enrolment of subjects in the study. 
 
1.7. Monitoring  
 
Dr. JA Arnaiz, Unidad de Ensayos Clínicos (CTU Clinic) 
Farmacología Clínica, Hospital Clínic, Barcelona. 
 
1.8. Treatments 
 
Eligible subjects will be randomly assigned to one of the two intervention arms (probiotic or fecal 
microbiota transplantation product) or to a control non-intervention (observation) arm in a 2:2:1 
proportion. 

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a) Probiotic (Vivomixx® 2 sachets/12h for 2 weeks)  
b) Fecal microbiota transplantation: administration of two doses (administered a week apart) 
of a fecal microbiota transplantation preparation (14-17 oral capsules per dose equivalent 
to 50 g of healthy-donor feces) 
c) No treatment (Observation) 
 
 
1.9. Primary objective 
 
To compare the decolonization efficacy at the end of the study (60 ± 7 days after 
randomization) 
 
1.10. Study population 
 
Patients with rectal colonization with MDR-GNB (ESBL-producing Klebsiella pneumoniae, CPE and 
MDR/XDR Pseudomonas aeruginosa).  
 
1.11. Subject selection  
 
 Inclusion criteria 
 
Male and female patients who meet all the following criteria will be eligible for this study: 
 
1. Adult patients (≥18 years-old). 
2. Admitted to the Hospital Clinic of Barcelona with documented rectal colonization whitin 
the previous 7 days by rectal swabbing with MDR-GNB (ESBL-producing Klebsiella 
pneumoniae, 
carbapenemase-producing 
Enterobacterial 
(CPE) 
and 
MDR/XDR 
Pseudomonas aeruginosa). 
3. Eligible for routine digestive decolonization (7 days oral administration of 
nonabsorbable antibiotics (NAA). 
4. Capable to provide informed consent (by themselves or through their legal 
representatives). 
 
 
 Exclusion criteria 
 
Patients meeting any of the following criteria will not be eligible for the study: 
 
1. Pregnant women or breastfeeding. 
2. Neutropenic patients (total neutrophil count <500 cell/mm3)*. 
3. HIV-infected patients with CD4 count <200 cell/mm3. 
4. Patients with active C. difficile infection.  
5. Patients with ileus or bowel obstruction. 
6. Patients with documented or suspected bowel perforation. 
7. Patients with a colistin-resistant MDR-GNB.  
.  
 
1.12. Assessment 
 
Decolonization rate at the end of the study (60 ± 7 days after randomization). Decolonization 
defined as a negative rectal swab sample for the target MDR-GNB at 60 ± 7 days after 
randomization. 
 

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1.13. Allocation participant 
 
 
A total of 437 eligible subjects will be randomly assigned to one of the two intervention arms (175 
patients to probiotic, 175 to fecal microbiota transplantation product) or to a control non-
intervention arm  (87 patients). Randomization will be stratified by the type of microorganism 
(ESBL-K. pneumoniae, CPE and MDR-P. aeruginosa) to ensure a balanced number of each organism 
in the study arms. 
 
 
1.14.   Follow-up  
 
Patients follow-up will be carried out 60 days (± 7 days) after randomization. 
 
1.15. End of the clinical research 
 
The end of the clinical research is defined as the date of the last visit of last subject undergoing 
the study with a recruiment period of 30 months.  
 
 
 

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2. TABLE OF CONTENTS 
 
1. RESUMEN ........................................................................................................................................................... 2 
1.0. Tipo de solicitud .............................................................................................................................................. 2 
1.1. Identificación del promotor ............................................................................................................................ 2 
1.2. Título del estudio ............................................................................................................................................ 2 
1.3. Código del protocolo ........................................................................................ ¡Error! Marcador no definido. 
1.4. Investigador coordinador e investigadores principales .................................................................................. 2 
1.5. Centros en los que se prevé realizar el ensayo ............................................................................................... 2 
1.6. Comités Éticos de Investigación que han aprobado el ensayo ....................................................................... 2 
1.7. Responsable de la monitorización .................................................................................................................. 2 
1.8. Productos en investigación ............................................................................................................................. 2 
1.9. Objetivo principal ........................................................................................................................................... 3 
1.10. Enfermedad en estudio ................................................................................................................................ 3 
1.11. Criterios de inclusión y exclusión .................................................................................................................. 3 
1.12. Variable principal de valoración ................................................................................................................... 3 
1.13. Población en estudio y número total de pacientes ...................................................................................... 4 
1.14.   Duración del tratamiento ........................................................................................................................... 4 
1.15. Calendario y fecha prevista de finalización .................................................................................................. 4 
2. TABLE OF CONTENTS ......................................................................................................................................... 5 
3. GENERAL INFORMATION ................................................................................................................................... 7 
3.1. Study identification ......................................................................................................................................... 7 
3.2. Investigators ................................................................................................................................................... 7 
3.3. Clinical sites .................................................................................................................................................... 7 
3.4. Sponsor ........................................................................................................................................................... 7 
3.5. Monitoring ...................................................................................................................................................... 7 
3.6. Study duration ................................................................................................................................................ 7 
4. BACKGROUND, RATIONALE AND OBJECTIVES ................................................................................................... 8 
4.1. Background and rationale............................................................................................................................... 8 
4.2. Objectives of the study ................................................................................................................................. 11 
4.2.1. Primary objective ................................................................................................................................... 11 
4.2.2. Secondary objectives ............................................................................................................................. 11 
5. TYPE OF STUDY AND DESIGN ........................................................................................................................... 12 
5.1. Study design.................................................................................................................................................. 12 
5.2. Criteria for termination and /or discontinuation .......................................................................................... 12 
5.3. Drug accountability ....................................................................................................................................... 12 
5.4. Allocation participant identification code .................................................................................................... 12 
5.5. Source data verification ................................................................................................................................ 13 
5.6. End of the clinical research ........................................................................................................................... 13 
6. SUBJECT SELECTION ......................................................................................................................................... 13 
6.1 Inclusion criteria ............................................................................................................................................ 13 
6.2 Exclusion criteria ............................................................................................................................................ 13 
6.3. Enrolment period .......................................................................................................................................... 14 
6.4. Patient withdrawal and study discontinuation ............................................................................................. 14 
7. DESCRIPTION OF STUDY TREATMENTS ............................................................................................................ 14 
7.1 Study arms ..................................................................................................................................................... 14 
7.2 Dose selection ................................................................................................................................................ 15 
7.3. Product information ..................................................................................................................................... 15 
7.4. Storage conditions ........................................................................................................................................ 16 
7.5. Concomitant therapies ................................................................................................................................. 16 
8. STUDY DEVELOPMENT AND ASSESSMENTS .................................................................................................... 16 
8.1. Study endpoints ............................................................................................................................................ 16 
8.2 Study development........................................................................................................................................ 17 
8.3 Procedures and follow-up ............................................................................................................................. 18 
8.3.1. Clinical variables .................................................................................................................................... 18 

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8.3.2. Microbiological studies .......................................................................................................................... 19 
8.3.3. Control rectal swabs (RS) ....................................................................................................................... 19 
8.3.4. Analysis of the microbiota ..................................................................................................................... 19 
8.4. Samples for analysis at the end of the study ................................................................................................ 19 
9. SAFETY ............................................................................................................................................................. 20 
9.1 Definitions...................................................................................................................................................... 20 
9.2 Assessment of intensity ................................................................................................................................. 20 
9.3 Assessment of causality ................................................................................................................................. 20 
9.4 Collection and follow up of adverse events ................................................................................................... 21 
9.5. Procedure for reporting serious adverse events .......................................................................................... 21 
10. DATA COLLECTION AND PROCESSING ........................................................................................................... 22 
10.1. Recording of data ....................................................................................................................................... 22 
10.2. Direct access to source data/documents ................................................................................................... 22 
10.3. Data management ...................................................................................................................................... 22 
10.4. Archiving and storage of data ..................................................................................................................... 22 
11. QUALITY CONTROL ........................................................................................................................................ 22 
12. ETHICAL AND REGULATORY CONSIDERATIONS ............................................................................................. 23 
12.1. Ethics approval ........................................................................................................................................... 23 
12.2. Reponsabilities of the investigators............................................................................................................ 23 
12.3. Patient confidentiality ................................................................................................................................ 23 
12.4. Informed consent ....................................................................................................................................... 24 
12.5. Personal data protection ............................................................................................................................ 24 
13. FINANCING, INSURANCE AND PUBLICATION ................................................................................................. 25 
13.1. Financing ..................................................................................................................................................... 25 
13.2. Insurance .................................................................................................................................................... 25 
13.3. Publication .................................................................................................................................................. 25 
14. STATISTICS ..................................................................................................................................................... 25 
14.1 Number of participants and sample size calculation ................................................................................... 25 
14.2 Statistical analysis ........................................................................................................................................ 26 
15. REFERENCES ................................................................................................................................................... 27 
 
 
Protocol appendices: 
 
Appendix 1 
WHO Toxicity grading scale 
Appendix 2 
SAE reporting form 
 
 
 

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3. GENERAL INFORMATION 
 
3.1. Study identification 
 
Title: Open-label, randomized study to assess the efficacy of a probiotic or fecal microbiota 
transplantation (FMT) on the eradication of rectal multidrug-resistant Gram-negative bacilli 
(MDR-GNB) carriage. 
 
Protocol code: PROFTMDECOL 
 
3.2. Investigators 
 
Principal investigator: Dra. Ana Del Río, Infectious Diseases 
Co-principal investigator: Dr. José Antonio Martínez, Infectious Diseases 
 
Collaborators: 
 
Dr. C. Pitart, Clinical Microbiology 
Dr. J. Mercadal, Anaesthesiology  
Dra. E. López, Hospital Pharmacy 
 
3.3. Clinical sites 
 
Single centre study: Hospital Clínic, Barcelona 
 
3.4. Sponsor 
 
Fundació Clínic per a la Recerca Biomédica. 
Rosselló, 149 
08036 Barcelona 
 
3.5. Monitoring 
 
Clinical Trials Unit (CTU Clinic) 
Clínical Pharmacology 
Hospital Clínic, Barcelona. 
 
3.6. Study duration 
 
Once approval is obtained, by January 2020, recruitment period will last 30 months.  
Patient follow-up will be 60+/- 7 days  for each patient. 
End of study corresponds to last patient-last visit and is expected by September 2022.  
The analysis of the results and the study final report will be finalised by December 2022. 
 
 
 

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4. BACKGROUND, RATIONALE AND OBJECTIVES 
 
4.1. Background and rationale 
The extension of multidrug-resistant gram-negative bacilli (MDR-GNB) in health-care 
centers is a growing problem worldwide, with economic implications that have a negative 
impact on the efficiency of health systems. The WHO considers antibiotic resistance as "a 
growing threat to global public health that requires coordinated action by governments and 
society." In our hospital, the consumption of active antibiotics against MDR-GNB increased in 
2017 with respect to the previous year by 20% in the case of meropenem and ceftazidime-
avibactam and in 140% for ceftolozane-tazobactam. The most frequent and clinically relevant 
multidrug-resistant bacteria isolated in patients admitted to our center are extended-spectrum 
beta-lactamase- and/or carbapenemase-producing (ESBL) Klebsiella pneumoniae, other 
carbapenemase-producing Enterobacterial (CPE) and MDR/XDR Pseudomonas aeruginosa. We 
will refer to all of them as multidrug-resistant gram-negative bacilli (MDR-GNB).  
 
The rectal carrier status of MDR-GNB is a risk factor for developing an infection (bacteremia, 
urinary infection, peritonitis, pneumonia) by these microorganisms. The percentage of 
colonized patients who end up developing an infection ranges between 4% and 20%, although 
this percentage can reach 50% in certain populations such as patients admitted to the ICU, 
those who undergo a transplant of hematopoietic precursors, hepatic graft recipients or those 
submitted to an intra-abdominal surgical procedure (1-4). The crude mortality of these 
patients is around 40%, which is due, at least in part, to the fact that in most cases the 
therapeutic alternatives are less effective and more toxic (colistin, amikacin, tigecycline). These 
data highlight the need of establishing strategies to prevent the transmission of MDR-GNB on 
one hand and of eradicating or reducing carriage in order to improve the prognosis of 
colonized patients on the other.  
The most effective measures to prevent horizontal patient-to-patient transmission of MDR-GNB 
include: 1) hand hygiene, as the most effective isolated measure 2) cleaning and disinfection of 
surfaces, 3) application of contact precautions (isolation) to colonized patients and 4) 
decolonization.  
 
Decolonization is the only measure designed to improve the prognosis of the patient who is 
already colonized, but there is no consensus on its widespread use mainly due to the fear of 
selecting panresistant strains (resistant to all available antibiotics) (5). Without any 
therapeutic intervention, the rate of spontaneous decolonization is low, particularly among the 
most fragile patients (tracheal intubation, decubitus ulcers, multiple vascular catheters, 
nasogastric tube) and those requiring prolonged antibiotic treatment (6,7). In this population, a 
retrospective study conducted in a hospital in Spain showed that the decolonization of patients 
with MDR-GNB with oral gentamicin significantly reduced mortality, but in up to 15% of 
patients gentamicin resistance emerged (8). These results support the need of evaluating new 
decolonization strategies that minimize the resistance selection risk. For digestive 
decolonization, oral administration of nonabsorbable antibiotics (NAA) such as colisitin or 
aminoglycosides has been the most common approach. Administered in this way they are non-
toxic and reach high concentrations in feces. A recent systematic review described an 
eradication rate at the end of treatment (7-14 days) of 65%, but at one month of follow-up the 
rate was reduced to 40% (9). In addition, among patients with CPE, resistance to colistin or 
gentimycin has been detected in 8% and 45% of those who received monotherapy with these 
antibiotics, respectively (10-12). In contrast, a prior study showed that resistance was not 
selected in any of the 5 patients who received the combination of both antibiotics (11). In this 
regards, a regimen consisting of 100 mg of colistin together with 80 mg of tobramycin and 500 
mg of amphotericin B 4 times a day has been shown to be beneficial in critically ventilated 
patients both in terms of reducing mortality and reducing the incidence of bacteremia (13,14). 

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In critically ill patients treated with this regimen, one study showed that the acquisition of 
colistin-resistant enterobacteria was 5 times higher in those previously colonized by strains 
resistant to tobramycin and therefore receiving monotherapy with colistin (15). In countries 
with a low incidence of resistance, intestinal decontamination with colistin-tobramycin has 
been associated with an increase in resistance to tobramycin, although its overall prevalence 
has remained below 1% and the efficacy of the procedure has not been compromised (16). 
Finally, in a recent randomized study on ventilated patients in ICUs with moderate to high 
prevalence of antibiotic resistance, although neither oral nor digestive decontamination with 
colistin-tobramycin was effective for reducing ICU-acquired bacteremia due to MDR-GNB, the 
prevalence of colistin resistance did not increase during the intervention periods (17).  
In our environment, the prevalence of resistance to gentamicin and tobramycin is high among 
the ESBL-producing enterobacteria (22-54% in E. coli and 31-67% in K. pneumoniae) and CPE 
(67%-74%). Amikacin maintains its activity against more than 90% of ESBL-producing 
enterobacteria and ≥80% of CPE, making it the ideal aminoglycoside to accompany colistin. The 
latter is still active against >95% of the strains of interest. Other antibiotic options have hardly 
been explored and, of these, rifaximin is probably one of the most interesting. Rifaximin is a 
non-absorbable rifamycin used for the treatment of traveler's diarrhea, hepatic 
encephalopathy, and other gastrointestinal disorders (18). Its fecal concentration after taking 
400 mg/12 h for 3 days is around 8000 mg/L and has a good activity against strains of E. coli 
producing ESBL and several CPE. In our experience in 54 patients of an hepatic ICU colonized 
by MDR-GNB during 2016-2017, the pattern of decolonization with a poly-antibiotic solution 
constituted by colistin (10 mg/mL), amikacin (8 mg/mL) and amphotericin B (50 mg/ml) 10 
mL every 6 h for 10 days (in patients with strains sensitive to colistin and amikacin) or 10 mL 
of poly-antibiotic solution every 6 h plus rifaximin 400 mg/8 h for 10 days (in patients with 
colistin-resistant strains or amikacin) was 50% at one month of follow-up (19). This antibiotic 
regimen is used routinely for the decolonization of rectal carriers of MDR-GNB in our center.  
 
Probiotics are a combination of different live microorganisms that administered orally 
produce qualitative changes in the intestinal microbiota that have been related to an efficient 
protection against Clostridium difficile. They are considered dietary supplements for 
administrative/legal purposes. There is some published experience with probiotics in the 
prevention of infections in hospitalized patients, but additional clinical trials are necessary to 
support its use.  Regarding current published experience, the combination of three 
Lactobacillus (L. acidophilus CL1285, L. casei LBC80R and L. rhamnosus CLR2) was 
administered to all patients admitted to a Quebec hospital and receiving an antibiotic (20). 
After 10 years of experience (44,835 patients included), the incidence of C. difficile decreased 
from 18/10,000 patient-days to 2.3/10,000 patient-days. On the other hand, it has been 
observed that patients acquiring a MDR-BGN during admission have a lower percentage of 
Lactobacillus spp. in the intestinal flora than those who do not become colonized and that 
Lactobacillus delbrueckii subspecies delbrueckii LDD01 (DSM22106) inhibits in vitro the 
growth of Klebsiella spp. probably through the production of a bacteriocin (21,22). Based on the 
above, it seems reasonable to hypothesize that certain probiotics can compete for adhesion to 
the intestinal mucosa and nutrients with other microorganisms of the intestinal flora and 
consequently favor the decolonization of MDR-GNB. In addition, as probiotics are almost free of 
safety concerns or resistance selection issues, they are a therapeutic option that requires 
evaluation.  
The probiotic that we propose to use in this study is Vivomixx® powder in sachets, composed of 
8 living bacterial species (L. paracasei DSM 24733, L. acidophilus DSM 24735, L. delbrueckii ssp 
bulgaricus DSM 24734, L. plantarum DSM 24730, B. brief DSM 24732, Bifidobacterium longum 
DSM 24736, B. infantis DSM 24737, Streptococcus thermophilus DSM 24731) with a 
concentration of 450 billion live lyophilized bacteria per sachet. There are numerous studies on 
the use of Vivomix® in different disorders, particulary to prevent hepatic encephalopaty in 
cirrhotic patients and in patients with inflammatory bowel disease where it has been shown to 
be very safe.  In our opinion, the lack of reports of bacteremia associated with Vivomix® 

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consumption in patients with liver cirrosis, a population at high risk of intestinal bacterial 
translocation, and the rarity of serious adverse effects associated with bacterial probiotics in 
general in other sensible population such as the critically-ill or liver transplant recipients is 
enough evidence to consider the product as probably safe for the main target population of the 
present study (23-28).  
Regarding the use of probiotics for the decolonization of MDR-GNB, clinical evidence is limited 
to three trials. A randomized study in 103 ICU patients published in 2015 did not found 
significant differences in acquisition or loss of MDR organisms, including Pseudomonas 
aeruginosa and ESBL- or carbapenemase-producing Enterobacterials between patients who 
received  L. rhamnosus GG (1 capsule containing 1010 CFU every 12h for 14 days) and those 
not receiving the probiotic (29).  In a more recently published randomized trial, the 
administration of a symbiotic (1010 CFU L. bulgaricus plus 1010 CFU L. rhamnosus plus fructo-
oligosaccharides twice a day for 1 week) to 101 evaluable hospitalized patients colonized by 
MDR-GNB was not associated with improved rates de decolonization or infection (30). It is of 
note that in both studies the number of patients was small and the probiotic consisted in one or 
two bacterial species. Lastly, in a small (80 patients) randomized comparative trial, although 
Vivomixx® was associated with a 2.7 higher rate of intestinal eradication of ESBL-producing 
enteric gram-negative bacilli (mostly E. coli) than placebo, the total proportion of decolonized 
patients whitin one year was small (12.5%) and not significatively different from placebo (31). 
However, our group has used Vivomixx® associated with NAA for the decolonization of patients 
in a recent outbreak of KPC-producing Klebsiella pneumoniae. The rate of decolonization at one 
month after diagnosis in patients who received NAA followed by Vivomixx® during 20 days was 
higher than that of those who received only NAA (88% vs. 68% p = 0.008). These results have 
been accepted as an oral communication at the 29th European Congress of Infections (ECCMID) 
(Amsterdam April 13-16, 2019) (19). We think that a rigurous further clinical evaluation of 
probiotics as a decolonization tool in rectal carriers of MDR-GNB is justified.  
 
Faecal microbiota transplantation (FMT), a highly effective treatment for recurrent 
Clostridium difficile infection, is a promising therapy for the intestinal decolonization of MDR-
GNB. Some individual cases or small series have been published in which FMT has been 
effective for the intestinal decolonization of ESBL- and CPE, carbapenemase-producing A. 
baumannii, vancomycin-resistant enterococci or methicillin-resistant Staphylococcus aureus 
(32-38). The longest prospective series published so far includes 20 cases of intestinal 
colonization with MDR-GNB in hematological patients (39). Complete decolonization at one 
month was achieved in 60% and at 6 months in 93%. In the case of Klebsiella pneumoniae (the 
most frequent in our center) the percentage of decolonization at one month was 53% (60% for 
NDM1-producing strains). The results of a small multicentre randomized open-label trial 
comparing 5 days of NAA followed by FMT versus no treatment has been recently published by 
Huttner BD et al (40). This study included 39 rectal carriers of ESBL- or CPE, of whom 22 
received FMT administered via frozen capsules or a nasogastric tube. There were no 
statistically significant differences in the rate of decolonization between the two groups at 35-
48 days after randomization, although a higher proportion of patients in the intervention arm 
was decolonized (41% vs. 29%).  In this study, it is of note that a rebound in the proportion of 
patients with a positive stool culture was observed within the two weeks after FMT. In a 
retrospective matched case-control study by Saidani N. et al. (41) analysing 10 carriers of CPE 
treated with a 5-day course of NAA followed by FMT administered via nasogastric tube, 80% of 
patients had a negative rectal swap 14 days after FMT but 5 of the 10 patients received 2 FMT, 
suggesting that in order to achive a timely high rate of decolonization, a single FMT may not be 
enough. Currently, the role of FMT for erradication of MDR organisms rectal carriage is unclear 
and many questions remain to be answered, among them which are the most effective raw 
material (from pooled or single donors), preparation (fresh, frozen, lyophilized or others), 
dosage and route of administration, if there are specific caracteristics of the donor flora 
associated with decolonization, or if there are important particularities in the entire FMT 
protocol (NAA quality, dose and duration; prior mechanical cleaning of the colon; detection and 

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decolonization of other potentially colonized sites). It seems clear from what has been 
published so far that the decolonizing effectiveness of FMT decreases significantly when the 
patient also receives systemic antibiotics and that the procedure works and is safe even in 
immunosuppressed patients. Currently, several clinical trials are underway to investigate the 
decolonization efficacy of FMT on MDR organisms that may help clarify some of these aspects. 
To date, no consensus has been reached on the cataloging of FMT for administrative-legal 
purposes, which is why it is not currently considered a medicine. 
Our group received financing from FIS (Stability and viability of liophylized intestinal 
microbiota for the treatment of Clostridium difficile infections. Fondo de Investigación Sanitaria 
(FIS) PI16/01023. Period: 2017-2019. Principal Investigator: Alex Soriano Viladomiu) to 
develop a method of processing feces to do fecal transplant in patients with C.difficile infection. 
It has been achieved to process the samples of healthy volunteers to obtain adsorbate capsules 
that can be stored at 4ºC. Furthermore, the project includes a pilot study in which patients 
treated for C.difficile will receive a dose of these capsules to prevent infection recurrence. The 
absorbate capsules are the FMT preparation intended to be used in the present clinical 
research. A brief description of the procedure to obtain them is provided in section 7 
"Description of study treatments".  
 
In summary, this project will explore the role of probiotics and FMT in eradicating the rectal 
carriage of MDR-GNB, aimed at addressing the growing problem of multiresistance in hospitals, 
which is one of most important challenges that the health system currently has to face.  
 
Hypothesis: 
 
The working hypothesis is that in patients who are rectal carriers of MDR-GNB, the rate and 
speed of erradication of the carrier status obtained with NAA regimens are insufficient and 
could be improved with additional interventions directed to restore a healthy fecal microbiota 
or to increase the colonization resistance by the putative beneficial activity of lactate-producing 
bacteria and bifidobacteria. A healthier colonic microbiota environment is expected not only to 
promote the erradication of the existing MDR organisms but also to hinder the subsequent 
recolonization and hopefully the risk of infection with gut dysbosis-associated pathogens 
(MDR-GNB, C. difficile and Candida).  
 
Obtaining decolonization rates over 75% in any of the study arms will be of potential clinical 
relevance.   
 
4.2. Objectives of the study 
 
4.2.1. Primary objective 
 
To compare the decolonization efficacy at the end of the study (60 ± 7 days after 
randomization) of the administration of a probiotic (Vivomixx® 2 sachets/12h for 2 weeks) 
versus the administration of two doses (administered a week apart) of a fecal microbiota 
transplantation preparation (14-17 oral capsules per dose equivalent to 50 g of healthy-
donor feces) and no treatment (control arm) in patients with rectal colonization with MDR-
GNB (ESBL-producing Klebsiella pneumoniae, CPE and MDR/XDR Pseudomonas aeruginosa).  
 
4.2.2. Secondary objectives 
 
1.- To determine the rate of digestive decolonization (negative fecal sample for the target 
MDR-GNB) at the end of the decolonization treatment (week 3 after randomization). 
2. - To determine the efficacy of the decontamination with NAA defined as the rate of 
digestive decolonization (negative rectal swab [RS]) for the target MDR-GNB at the end of 

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the decontamination period  (week 1 after randomization). 
3.- To determine the incidence of patients with positive clinical samples for either the same 
microorganism colonizing their digestive tract or any other MDR-GNB during the study 
period. 
4.- To determine the incidence of clinical infections (including those due to C. difficile) 
during the study period. 
5.- To determine the prevalence of resistance emergence to any of the antibiotic 
components of the NAA regimen (colistin, amikacin) in those patients with persistently 
positive control RS or fecal sample. 
6.- To assess the fecal microbiota at baseline and at the end of treatment (week 3 after 
randomization) in order to evaluate the degree of fecal microbiota restoration, engrafment 
success and relationship of fecal composition with MDR-GNB eradication.  
 
5. TYPE OF STUDY AND DESIGN 
 
5.1. Study design 
 
This is an investigator-initiated, single center, parallel-group, randomized, open-label, clinical 
research with non pharmacological products. 
 
Participants will receive treatment during two weeks. Each patient will be followed for a 
period of 60 days. Six study visits will be carried out during this period: screening, baseline, 
treatment intiation visit, treatment follow-up visit, end of treatment visit and end of study visit.   
 
Limitations of the study: The main limitation of the study is a moderate sample size that may 
raise power questions regarding comparisons stratified by bacterial species.  
 
5.2. Criteria for termination and /or discontinuation 
 
The participants will discontinue study participation if they are unwilling or unable to meet the 
protocol requirements in terms of the visit schedule or if the patient or the investigator considers it 
is best to end their participation in the study. All participants have the right to withdraw their 
consent at any time during the study without prejudice to them. 
All follow-up terminations of study subjects and the reasons for them must be reported 
immediately to the study monitor and be duly documented both in the medical records and the case 
report form. 
 
5.3. Drug accountability 
 
Drug accountability will be carried out at each study visit for those patients assigned to the 
Probiotic arm.  
FMT administration will always be performed under direct observation of one of the 
investigators and if ambulatory, the patient will remain under observation in the outpatient 
clinic during at least 2 hours after the procedure. 
 
5.4. Allocation participant identification code 
 
Eligible subjects will be randomly assigned to one of the two intervention arms (probiotic or fecal 
microbiota transplantation product) or to a control non-intervention (observation) arm in a 2:2:1 
proportion. Randomization will be stratified by the type of microorganism (ESBL-K. pneumoniae, 
CPE and MDR-P. aeruginosa) to ensure a balanced number of each organism in the study arms. 
 

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Randomization list will be computer generated. The investigators will not be aware of the 
patient assignment until the inclusion.  
 
5.5. Source data verification 
 
Source documents are defined as all observations or notes recorded on the clinical interventions, 
and all reports and notes required for assessment and reconstruction of the research study. 
Whenever possible the original document should be kept as the source document; however, 
provision of a photocopy which is clear, legible and an exact duplicate of the original document and 
signed by the principal investigator is acceptable. 
 
5.6. End of the clinical research 
 
The end of the clinical research is defined as the date of the last visit of last subject undergoing the 
study. 
 
6. SUBJECT SELECTION 
 
6.1 Inclusion criteria 
 
Male and female patients who meet all the following criteria will be eligible for this study: 
 
5. Adult patients (≥18 years-old). 
6. Admitted to the Hospital Clinic of Barcelona with documented rectal colonization whitin 
the previous 7 days by rectal swabbing with MDR-GNB (ESBL-producing Klebsiella 
pneumoniae, 
carbapenemase-producing 
Enterobacterial 
(CPE) 
and 
MDR/XDR 
Pseudomonas aeruginosa). 
7. Eligible for routine digestive decolonization (7 days oral administration of 
nonabsorbable antibiotics (NAA). 
8. Capable to provide informed consent (by themselves or through their legal 
representatives). 
 
 
6.2 Exclusion criteria 
 
Patients meeting any of the following criteria will not be eligible for the study: 
 
8. Pregnant women or breastfeeding. 
9. Neutropenic patients (total neutrophil count <500 cell/mm3)*. 
10. HIV-infected patients with CD4 count <200 cell/mm3. 
11. Patients with active C. difficile infection.  
12. Patients with ileus or bowel obstruction. 
13. Patients with documented or suspected bowel perforation. 
14. Patients with a colistin-resistant MDR-GNB.  
 
* Solid organ or haematopoietic organ transplant recipients without neutropenia will qualify 
for inclusion in the study. This is based on preliminary data on the safety of probiotics without 
Sacharomyces boulardii/cerevisae in transplant recipients of several organs (hematopoietic 
precursors, liver, kidney, small intestine) and on the preliminary safety data of FMT in non-
neutropenic 
patients 
with 
hematological 
disorders 
including 
hemotopoietic 
cell 
transplantation (25,42,43). However, the condition of transplant recipients will be assessed in 
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will be judged to be at a very low risk of complications derived from the use of the probiotic o 
fecal microbiota will be considered for inclusion in the study. 
 
 
6.3. Enrolment period 
 
In accordance with prior local data, we estimate that recruitment of 437 subjects will require 30 
months. 
 
6.4. Patient withdrawal and study discontinuation 
 
A patient will prematurely discontinue the study in case of withdrawal of informed consent. 
All patients have the right to withdraw their consent at any time during the study without prejudice 
to them.  
In addition, the investigator may decide, for reasons of medical prudence, to remove the patient 
from the study. 
The date and reasons for stopping the study will be clearly stated on the subject’s CRF and source 
document. 
 
7. DESCRIPTION OF STUDY TREATMENTS 
 
7.1 Study arms 
 
The eligible subjects will be randomly assigned to receive, after a 7-day decolonization course of 
oral non-absorbable antibiotics (NAA), as standard of care, to one of the following regimens: 
 
a) Probiotic regimen 
 
Participants assigned to the probiotic arm will receive 2 sachets of Vivomixx® dissolved in 50 L 
of warm water (according to the manufacturer indications) every 12 h orally or through a 
nasogastric tube (in this case the tube will be flushed with 50 mL of water after the 
administration). The probiotic will be started 24 h after the last dose of NAA and will be 
administered for 14 consecutive days. Each sachet of Vivomixx® contains a combination of 4 
Lactobacillus (L. paracasei DSM 24733, L. acidophilus DSM 24735, L. delbrueckii ssp bulgaricus 
DSM 24734, L. plantarum DSM 24730), 3 Bifidobacteria (B. brief DSM 24732, B. longum DSM 
24736, B. infantis DSM 24737), and Streptococcus thermophilus DSM 24731 at a concentration 
of 450 billion (45x1010) live lyophilized bacteria per sachet. 
 
b) FMT regimen 
 
The FMT preparation will be administered as 2 doses, once a week, of 14-17 capsules per dose. 
Each dose (14-17 capsules) will contain the fecal microbiota equivalent to 50 gr of stools from a 
healthy donor. If the patient is carrying a nasogastric tube, the content of capsules will be 
decapsulized (~27 gr of powder), diluted in 50 mL of water and passed thorugh it, followed by 
the administration of 50 mL of water to flush the tube. The first dose will be administered 24 h 
after the last dose of NAA and the second dose one week later. Whenever possible, the two 
doses of FMT will be originated from the same donor. According to our data done by flow 
cytometry and bacterial culture in three replicates, each absorbate capsule contains 
approximately 1.12x1010 live bacteria. 
 
 
 

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c) Control arm  
 
Patients assigned to the control arm (observation arm) will not receive other treatments 
beyond the standard 7-day course of NAA. 
 
7.2 Dose selection 
 
The most effective dose of the probiotic is unknown, hence the dosage of Vivomixx® has been 
selected to ensure an exposure to probiotic bacterial cells not inferior to that of the FTM produt 
while still being safe as proven in a prior clinical trial (31).   
 
The most efective dosage of FMT is also unclear. The one selected in the present study correspond 
to the most effective dosing strategy for the treatment of recurrent C. difficile colitis (44).  
 
 
 7.3. Product information 
 
Vivomixx® 
 
Vivomixx® is a dietary supplement. A 4.4g sackett contains 450 billions of live lactobacilli and 
bifidobacteria (Streptococcus thermophilus DSM24731®, Bifidobacterium breve DSM24732®, 
Bifidobacterium longum DSM24736®, Bifidobacterium infantis DSM24737®, Lactobacillus 
acidophilus DSM24735®, Lactobacillus plantarum DSM24730®, Lactobacillus paracasei DSM24733®, 
Lactobacillus delbrueckii ssp. bulgaricus DSM24734®), maltose, anti-caking agent: silicon dioxide. 
 
Vivomix® will be provided by the sponsor of the study. It will be administered using the common 
procedures of the clinical practice in the participating site. Patients will receive the marketed 
product. 
 
Source of FMT:  
 
Obtaining adsorbate capsules from feces 
 
The fecal mibrobiota preparation will be provided by the sponsor of the study. 
The obtention, processing feces and capsules preparation to do the fecal transplant has been 
already developed in our center. 
Healthy donors are selected after screening composed by a form, an interview and blood and fecal 
analysis. The stool sample is collected in a plastic recipient (Fecotainer®, AT Medical B.V., 
Netherlands) with BD Gaspak™ EZ anaerobe system (Becton Dickinson and Company, USA) inside 
to reach anaerobiosis, and it is received in the laboratory within 4-6 hours of collection.  
 
Each sample is separately labelled, weighed and transferred to a stomacher classic bag. Sterile saline 
is added in 10:1 proportion and the mix is homogenated in Stomacher 400 circulator (Seward Ltd., 
Sussex, United Kingdom) for 1 minute at 230rpm. Then, the slurry is transferred to a falcon tubes 
and 10% of Glycerol at 99% is added to the solution with a motorized pipette. 
 
The samples are frozen at -80ºC until its use. When required, samples are thawed overnight at 4ºC 
and 20% of Glycerol at 99% is added. They are centrifuged in Heraeus Megafuge 16R Centrifuge 
(Thermo Fisher Scientific Inc., MA, USA) for 20 minutes at 400g, 4ºC with slow deceleration to 
remove sample debris. Then, the supernatant is transferred into high resistant tubes previously 
filtered with conventional sieve to eliminate possible detritus and the volume is centrifuged for 30 
minutes at 10000g and 4ºC (Sorvall Evolution RC Centrifuge, Thermo Fisher Scientific Inc., MA, 
USA). We eliminate the supernatant leaving pellet, where we found the bacteria. The volume of 
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Magnesium stearate until achieve a homogenous powder obtaining an adsorbate product. The mix 
is encapsulated with semi-automated encapsulator FagronLAB™ FG (Fagron Iberica, Barcelona, 
Spain) into acid-resistant capsule size nº00. 
 
Around 14-17 capsules are obtained from 50g of faeces that are kept at 4ºC with Silica Gel labelled 
for its traceability until its use.  We have tested the viability and quantity of bacteria in these 
capsules by flow cytometry and bacterial cultures from 3 replicates, obtaining 1.12x1010 live 
bacteria per capsule immediately after preparation and 1.44x1010 after 3 months kept at 4ºC. 
 
The full documentation on the processing, preparation and viability control of the FMT product is 
provided elsewhere. The product is in patent proceedings. 
 
7.4. Storage conditions 
 
Vivomixx® must be stored between 2ºC and 8ºC protected from light. 
FMT capsules are stored at 4ºC. 
 
7.5. Concomitant therapies 
 
Any concomitant therapy must be recorded in the CRF detailing product, dates, dosage, route of 
administration, indication.  
 
Nonpermitted medication 
Use of antibiotics other than the 7-day decolonization course of oral non-absorbable antibiotics 
(NAA) is forbidden during the study duration. 
 
8. STUDY DEVELOPMENT AND ASSESSMENTS 
 
8.1. Study endpoints 
 
Primary endpoint 
  
Proportion of patients with digestive decolonization rate defined as negative rectal swab (RS) 
for the target MDR-GNB (ESBL-producing Klebsiella pneumoniae, CPE and MDR/XDR 
Pseudomonas aeruginosa) at the end of study (60 ± 7 days after the randomization). 
 
Secondary endpoints 
 
1.- Proportion of patients with digestive decolonization rate defined as a negative fecal sample  
for the target MDR-GNB at the end of study treatment (3 weeks after randomization). 
2.- Proportion of patients with digestive decolonization rate defined as a negative rectal swab 
for the target MDR-GNB after the decontamination period with non-absorbable antibiotics 
(week 1 after randomization). 
3.- Proportion of patients with target MDR-GNB  or any other MDR-GNB in any control RS or 
fecal sample during the study period. 
4.- Proportion of patients with clinical infections during the study period. 
5.- Proportion of patients with development of resistance to colistin or amikacin in any strain 
isolated in control RS or fecal sample during the study period. 
6.- Changes from baseline in fecal microbiota composition at the end of study treatment. 
 
 
 

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8.2 Study development 
 
Table 1 shows the summary of visits and procedures that will take place during the study. 
 
1. Inclusion visit (from day -7 to day -1). Patients admitted to the Hospital Clínic with a positive 
rectal colonization within the previous 7 days by rectal swabbing will be assessed for eligibility. 
Eligible patients will be invited to participate into the study. If they accept, informed consent 
will be obtained. After providing informed consent, the following data will be collected: 
demographic data (age and sex), medical history, concomitant medications and comorbidities 
and a pregnancy test in urine will be carried out in childbearing women. 
 
2. Baseline visit (day 0). Eligible patients will be randomized to one of the three study arms.  
During the visit, a fecal sample will be obtained for MDR-GNB detection and the analysis of 
microbiota.  Patients will be instructed to initiate the treatment with NAA during 7 days.  
 
3. Treatment initiation visit. After the decontamination period, a treatment initiation visit will 
be conducted. The visit will be carried out 1-3 days after the last dose of NAA. During the visit, 
patients assigned to the probiotic arm will start the probiotic treatment and those patients 
assigned to the FMT arm will start the first dose of the FMT preparation.  
Patients assigned to the Probiotic arm will receive 2 sachets of Vivomixx® every 12 hours 
(orally or through a nasogastric tube) during 14 consecutive days. 
Patients assigned to the FMT arm will receive the first dose of the FMT preparation (orally or 
through a nasogastric tube) consisting of 14-17 capsules.  
Patients assigned to the observation arm will neither receive probiotic nor FMT. 
Prior to the initiation of the treatment study, a rectal swab will be carried out. 
During this visit adverse events will be assessed. 
 
4. Treatment follow-up visit (from day 15 to day 16). Those patients assigned to FMT arm will 
start the second dose of the FMT preparation (orally or through a nasogastric tube) consisting 
of 14-17 capsules. A second rectal swab will be carried out prior to the initiation of the second 
dose of the FMT preparation. 
 
5. End of treatment visit. A follow-up visit will be scheduled once the 2-week treatment period 
is completed (22 to 23 days with a window of 7 days after randomization). During the visit a 
fecal sample will be obtained for MDR-GNB detection and analysis of microbiota and adverse 
events will be assessed.   
  
6. End of study visit (60 days after randomization). The visit will be carried out 60 days (± 7 
days) after randomization. A rectal swab will be carried out and adverse events will be 
assessed.   

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Table 1. Visit schedule 
 
 
VISIT 
Inclusion 
 
Baseline 
Treatment 
initiation visit 
Treatment 
follow-up visit* 
End of 
treatment visit 
End of study 
visit 
(Visit 0) 
(Visit 1) 
(Visit 2) 
(Visit 3) 
(Visit 4) 
(Visit 5) 
Day 
-7 to -1 
0 
8 
(window visit +3 
days) 
15 to 16 
 
22 to 23 
(window visit +7 
days) 
60 
(window visit ±7 
days) 
Inclusion criteria 
√ 
 
 
 
 
 
Informed consent 
√ 
 
 
 
 
 
Physical 
examination 
√ 
 
√ 
 
√ 
√ 
Medical history 
√ 
 
√ 
 
√ 
√ 
Randomization 
√ 
√ 
 
 
 
 
Rectal swab 
√ 
 
√ 
√ 
 
√ 
Fecal sample 
 
√ 
 
√ 
Treatment with 
NAA 
 
√ 
 
 
 
Probiotic/FMT 
administration 
 
 
√ 
 
√ 
 
 
Adverse events 
 
√ 
√ 
 
√ 
√ 
* only in patients assigned to TMF arm 
 
8.3 Procedures and follow-up 
 
Patients will be followed on a daily basis during admission, once per week after discharge until 
the end of treatment and once per month until the end of the study. After patient discharge, 
scheduled visits will be done at a specialized day-care clinic.  
 
Administration of FMT will always be performed under direct observation of one of the 
investigators and if ambulatory, the patient will remain under observation in the clinic during 
at least 2 h after the procedure. Accordingly to the standard FMT procedure in our Centre, on 
the evening before the the administration of the study FMT product and on the morning of the 
day of FMT product ingestion, the participant will take 20 mg of omeprazole by the oral or iv 
route. On the morning of the day of FMT, the patients will also receive a commercial laxative 
enema of 250 mL.  
 
Ambulatory patients will be instructed on the preparation of the probiotic at home to be taken 
without direct observation. The empty sachets will be spared and counted at the corresponding 
scheduled visit. 
 
In all patients included in the study and throughout the study period, a prospective registry of 
adverse events will be performed, including the presence of diarrhea and C. difficile infection 
 
8.3.1. Clinical variables 
 
From all included patients, the following variables will be collected: demographics (age and 
sex), comorbidities, date of admission to the hospital and ICU, reason for admission to the 
hospital and ICU, previous admissions in the last 6 months, antibiotics received in the previous 
month, any invasive procedure performed during the present admission (intubation and 

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mechanical ventilation, surgery, central venous catheterization, bladder catheterization, other), 
infections acquired during the study period and antibiotic treatments administered during the 
entire study period. 
 
8.3.2. Microbiological studies 
 
Rectal swabs will be seeded on selective chromogenic culture and non-selective plates to detect 
ESBL-producing, carbapenemase-producing, cefamicinase or chromosomal beta-lactamase-
producing Enterobacterials and Pseudomonas aeruginosa. The identification of microorganisms 
at gender and species level will be carried out using the MALDI-ToF system (Bruker Daltonics). 
Confirmation of antibiotic resistance will be done by phenotypic methods including the disc-
plate, E-test and/or marketed panels of the Phoenix system (Becton Dickinson) or Sensititre 
(Thermo-Fisher). Molecular epidemiology studies by using pulsed field electrophoresis and 
MLST analysis will be performed to establish the clonality of clinical and rectal isolates. The 
detection of the mecanism of resistance to betalactams will be investigated by PCR genotypic 
methods. After the detection of beta-lactamases, specific PCRs will be carried out to be able to 
sequence the detected enzyme. In addition, a group of multiresistant strains will be studied 
through massive sequencing to know the various genes involved. 
 
8.3.3. Control rectal swabs (RS) 
 
In all study arms, a RS will be obtained at the end of the NAA regimen (treatment initiation 
visitday 8 after randomization) prior to FMT or starting the probiotic and at the end of the 
study (60 ± 7 days after randomization). In patients assigned to the FMT arm, a RS prior to the 
second FMT (pre-2FMT) will also be performed to have a better dynamic profile of carriage 
after the first FMT and to confirm if there is a rebound of colonization at that point. We will 
consider the patient as decolonized when the RS is negative at the end of the study visit (60±7 
days after randomization). In all positive control rectal swabs or rectal sample, the presence of 
target MDR-GNB will be determined, as well as their susceptibility to colistin and amikacin. 
 
8.3.4. Analysis of the microbiota 
 
A microbiota analysis will be carried out in all the patients from the intervention groups to 
evaluate the degree of fecal microbiota restoration, engrafment success and relationship of fecal 
composition with MDR-GNB eradication from baseline at the end of treatment (at day 22-23 
with a window of 7 days after randomization).  
Fecal samples at baseline and at the end of study treatment will be obtained and stored to 
conduct the analysis of the microbiota.  
 
 
8.4. Samples for analysis at the end of the study 
Fecal samples for analysis of microbiota will be obtained at baseline and the end of treatment 
visit. 
Faecal samples will be frozen within the first 6h of the collection at -80 ° C. The PureLink ™ 
microbiome DNA purification kit (Invitrogen) will be used for the DNA extraction according to 
the manufacturer's instructions. The DNA concentration will be normalized for all samples after 
quantification with a Quantus™ Fluorometer (Promega) and the QuantiFluor® ONE DNA System 
(Promega). KAPA HiFi HotStart Ready Mix (Kapa Biosystems) will be used to amplify the V3-V4 
region of 16S rRNA. The PCR products will be sequenced using the 300 bp paired-end technique 
with the Nextera XT kit (Illumina) following the standard protocol provided by Illumina and 
sequenced on a MiSeq platform (Illumina) using a Miseq v3 reagent kit (Illumina). The quality 
of the sequencing data will be evaluated with the fastqc and prinseq-lite programs. R1 and R2 
will be joined using flash. The taxonomic affiliations will be assigned using the rdp_classifier 

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and the RDP database. At the same time, we will calculate the relative abundances of the 
operational taxonomic units (OTU) with the QIIME pipeline. The phylogenetic classification will 
be used to describe the intestinal composition of each subject in the different groups. The 
microbial diversity will be calculated by calculating the Shannon diversity index (SDI). All 
calculations and statistics will be carried out within the R environment. 
After analysis, the fecal samples will be destroyed in accordance with Law 14/2007 of 3 July on 
Biomedical Research. 
 
9. SAFETY 
 
9.1 Definitions 
 
Despite the study is not assessing the effect of medicinal products, the unwanted effects will be 
recorded in accordance with the existing definitions for medicines. 
 
Adverse event (AE): an AE is any untoward medical occurrence in a clinical study subject 
administered the investigational product. An AE does not necessarily have a causal relationship 
with the treatment. An AE can therefore be any unfavorable and unintended sign (including an 
abnormal finding), symptom, or disease temporally associated with the use of the 
investigational product, whether or not related to that product  
This includes any occurrence that is new in onset or aggravated in severity or frequency from 
the baseline condition, or abnormal results of diagnostic procedures, including laboratory test 
abnormalities. 
 
Serious adverse event (SAE): a SAE is any untoward medical occurrence that at any dose: 
Results in death, is life-threatening, requires inpatient hospitalization or prolongation of 
existing hospitalization, results in persistent or significant disability/incapacity, is a congenital 
anomaly/birth defect, is a suspected transmission of any infectious agent via a medicinal 
product, is medically important (according to the treating physician), other important medical 
events that may not be immediately life-threatening or result in death or hospitalization but 
may jeopardize the subject or may require intervention to prevent one of the other outcomes 
listed in the definition above. 
If a serious and unexpected AE occurs for which there is evidence suggesting a causal 
relationship between the study drug and the event (eg, death from anaphylaxis), the event must 
be reported as a serious and unexpected suspected adverse reaction even if it is a component of 
the study endpoint (eg, all-cause mortality). 
Any event requiring hospitalization (or prolongation of hospitalization) that occurs during the 
course of a subject's participation in a study must be reported as an SAE, except hospitalizations 
for the following: hospitalizations not intended to treat an acute illness or AE (eg, social reasons 
such as pending placement in long-term care facility), surgery or procedure planned before 
entry into the study. 
 
9.2 Assessment of intensity 
 
An assessment of intensity grade will be made using the general categorical descriptors 
outlined in the WHO Toxicity Grading Scale (see Appendix 1). The investigator should use 
clinical judgment in assessing the severity of events not directly experienced by the subject (eg, 
laboratory abnormalities). 
 
9.3 Assessment of causality 
 
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causal relationship and it cannot be explained by factors such as the patient's clinical condition 
or therapeutic interventions. 
 
- AE unrelated: The relationship in time of the AE with the study drug indicates an unlikely 
causal relationship, or other factors (concomitant medication or conditions) or other 
therapeutic interventions provide a satisfactory explanation for the AE. 
 
9.4 Collection and follow up of adverse events 
 
AEs may be recorded at each visit based on careful clinical observation of the patient, 
laboratory tests or spontaneously reports by the participant discovered as a result of general 
questioning by the study staff. All AEs will be recorded on the medical history and in the CRF. 
The investigator will also decide whether the adverse event is, based on his/her judgment, 
related or not to the study drug—this decision should also be noted in the medical history and 
CRF. 
The following will be recorded for each event: description, severity (grade 1, 2, 3, 4 and 5), 
duration (start and end dates), intensity, causal relationship with the drug (according to the 
previously attributability criteria) and study drug(s) for which this causal relationship is 
suspected, actions taken and outcome, using choices given on the patient’s medical history. The 
investigator should report the underlying condition when a surgical or medical procedure is 
required as the event term, and the procedure as an action taken. For a preexisting AE that has 
worsened in terms of severity or frequency, the meaning of the change should be specified. 
For all AEs, the Investigator must pursue and obtain information adequate both to determine 
the outcome of the AE and to assess whether it meets criteria for classification as a serious 
adverse event (SAE). Follow-up of the AE is required if the AE persists until the event resolves 
or stabilizes at a level acceptable to the Investigator.  
The degree of severity of an adverse event provides a qualitative assessment of the extent or 
intensity of an adverse event elicited by the investigator or reported by the patient. Severity 
does not reflect the clinical seriousness of the event, only the grade or extent of the complaint 
or incidence. 
 
9.5. Procedure for reporting serious adverse events  
 
The principal investigator or delegates will report immediately any SAE occurring from signing 
of informed consent and up to 30 days after the end of the study to the CTU Clinic.  
The initial report of SAE should be written and as complete as possible including details of the 
current disease and SAE and assessment of the causal relationship between the AE and the 
study procedure. Reporting will be made using the Serious Adverse Event Report Form 
included in Appendix 2 of this protocol within 48 hours from first knowledge by the 
investigator, completing all information on the form in the following two days.  
The information missing at the time of the initial report must be reported in the SAE follow-up 
form.  
SAEs forms should be sent immediately by fax or email to:  
 
CTU CLINIC 
Hospital Clinic de Barcelona  
Fax number: +34 93 227 98 77  
Email address: jpich@clinic.cat 
 
 
 
 

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10. DATA COLLECTION AND PROCESSING 
 
10.1. Recording of data 
 
All data required for the study will be recorded in the participating center using a case report 
form (CRF).  Completeness and plausibility checks will ensure the collection of high quality data. 
A CRF for each patient will be completed by authorized personnel who must be identified and 
authorized in writing by the Principal Investigator before they conduct any study related tasks.  
A delegation of responsibility log identifying who can enter data and/or sign off a CRF will be 
maintained by the Principal Investigator.   
The subject’s number and date of entry into the study, along with a study identifier, should be 
recorded in the subject’s study records. The following should also be recorded in the study 
records: confirmation of written consent, the subject’s clinical status, date of every study visit, 
copies of all relevant reports, comments on results and reference to serious adverse events and 
related adverse events. 
 
10.2. Direct access to source data/documents 
 
Investigators will ensure access to the source documents of the staff responsible for 
guaranteeing data quality and data analysis. In addition, access to documentation will be 
provided, if necessary, to the staff duly authorized by the sponsor (study monitors). 
 
10.3. Data management 
 
The Investigator must ensure the accuracy, completeness, legibility and timelines of data 
reported in the CRF and all required reports.  
 
10.4. Archiving and storage of data 
 
The investigator is responsible for maintaining all records which enable the conduct of the 
study at the site to be fully documented, in accordance applicable national regulatory 
requirements. Timeliness and completeness of the documentation will be regularly checked by 
the clinical monitor. All completed study related documents (e.g. eCRF, Informed consent forms, 
Subject identification log, etc) must be archived at site. 
 
11. QUALITY CONTROL 
 
The purpose of monitoring is to verify that the rights and wellbeing of human subjects are 
protected; that the study is accurate, complete and verifiable with source data and that the 
study is conducted in compliance with the protocol, and the applicable regulatory 
requirements. A monitoring plan will be designed. The monitoring plan will establish the 
guideline for conducting all the monitoring activities. 
Source data will be verified during on-site monitoring visits. During the visits, the monitor will 
compare the data entered into the CRF with the source documents. The nature and location of 
all source documents will be identified to ensure that all sources of original data required to 
complete the eCRF are known to the monitor and study-site personnel and are accessible for 
verification. During monitoring visits, the relevant study-site personnel should be available, the 
source documentation accessible, and a suitable environment provided for review of study-
related documents. 
 

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12. ETHICAL AND REGULATORY CONSIDERATIONS 
 
12.1. Ethics approval  
 
This study will be conducted in accordance with the protocol and ethical principles stated in the 
Declaration of Helsinki and all applicable local laws, rules, and regulations. 
The protocol, informed consent form, and other required documents will be approved by the 
Ethics Committee (Comité de Ética de la Investigación, CEI) of the Hospital Clínic de Barcelona 
before enrolment of subjects in the study. 
Protocol amendments must not be implemented without prior Ethics Committee approval. 
 
12.2. Reponsabilities of the investigators 
 
The investigator is responsible for ensuring that the study is performed in accordance with the 
protocol, and applicable regulatory Spanish requirements.  
In addition, the investigator is responsible for giving information about the study to all staff 
members involved in the study or in any element of subject management, before and during the 
study.  
 
12.3. Patient confidentiality 
 
The collection and processing of personal data from subjects enrolled in this study will be 
limited to those data that are necessary to fulfill the objectives of the study. Patient data will be 
anonymized to ensure confidentiality and compliance with applicable data privacy protection 
laws and regulations. 
 
Subjects will be codified with a study code that prevents their identity from being deduced. 
 
The PI and duly authorized collaborators will compromise to maintain personal data strictly 
confidential, according to the corresponding country-specific requirements. The link between 
the numeric code and real personal data from subjects will be rigorously kept by the PI. The 
informed consent obtained from the subject includes explicit consent for the processing of 
personal data and for the investigator/institution to allow direct access to his or her original 
medical records (source data/documents) for study-related monitoring, audit, Ethics 
Committee review, and regulatory inspection. 
 
In the case report form, the patient will only be identified by the assigned study code. The name 
of patients will not appear in any publication or report of the study results. 
 
The participation of the patient in the clinical research will be noted in their medical records. 
 
The investigator will complete a list which will include the names of the patients participating 
in the trial, the number of inclusion in the study, and their medical history. Only investigators 
and the staff responsible for guaranteeing data quality and data analysis will have access to the 
clinical documentation of the participants. 
 
Duly authorized persons by the sponsor and the health authorities and the Ethics Committee 
may audit or inspect the clinical research. Personal information will not be publicly available, in 
compliance with General Data Protection Regulation (GPDR) (EU) 2016/679 of 27th April 
2016). 
 
 
 

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12.4. Informed consent  
 
Informed consent will be obtained from all participating individuals or their representatives. 
Each subject must give written consent according to local requirements after the nature of the 
study has been fully explained. The ICF(s) must be signed before performance of any study 
related activity. The ICF(s) that is/are used must be approved by both the sponsor and by the 
reviewing Ethics Committee and be in a language that the patient can read and understand. The 
informed consent should be in accordance with principles that originated in the Declaration of 
Helsinki (updated according to its last version, Fortaleza, Brazil, 2013), , applicable regulatory 
and country-specific requirements, and sponsor policy.  
 
Before enrollment in the study, the investigator or an authorized member of the study-site 
personnel must explain to potential subjects the aims, methods, reasonably anticipated benefits, 
and potential hazards of the study, and any discomfort that the participation in the study may 
entail. 
 
Subjects will be informed that their participation is voluntary and that they may withdraw 
consent to participate at any time. They will be informed that choosing not to participate will 
not affect the care the subject will receive for the treatment of his or her disease. Subjects will 
be told that alternative treatments are available if they refuse to take part and that such refusal 
will not prejudice future treatment. Finally, they will be told that the investigator will maintain 
a subject identification register and that their records may be accessed by health authorities 
and authorized sponsor personnel without violating the confidentiality of the subject, to the 
extent permitted by the applicable law(s) or regulations. The subject will be given sufficient 
time to read the ICF and the opportunity to ask questions. After this explanation and before 
entry into the study, consent should be appropriately recorded by means of the subject’s 
personally dated signature. After having obtained the consent, a copy of the ICF must be given 
to the subject.   
 
12.5. Personal data protection 
 
The processing, communication and transfer of personal data of all participants will be adjusted 
to compliance with Regulation EU 2016/679 of the European Parliament and the Council of 27 
April 2016 on the protection of natural persons as to the processing of personal data and the 
free circulation of data, being mandatory as of May 25, 2018. The legal basis that justifies the 
processing of the patient data is the consent given in this act, in accordance with the provisions 
of the Article 9 of the EU Regulation 2016/679. 
The data collected for the study will be collected only identified by a code, so that no 
information will be included to identify the participants. Data will be processed with the only 
purpose of carry out all activities related to the clinical research. The legal basis for the 
processing of the data is the participant consent and Article 9.2 of the Regulation. Only the 
study doctor and his collaborators have the right to access the source data (clinical history) and 
will be able to relate the data collected in the study with the patient's medical history. 
The identity of the participants will not be available to any other person except for a medical 
emergency or legal requirement. Health authorities, the Ethics Committee and personnel 
authorized by the Sponsor of the study, may have access to the personal data identified when 
necessary to verify data and study procedures, but always maintaining confidentiality in 
accordance with current legislation. 
Only encrypted data will be transferred to third parties and to other countries, which in no case 
will contain information that can identify the participant directly (such as name and surnames, 
initials, address, social security number, etc.). In the event that this assignment occurred, it 
would be for the same purpose of the study described and guaranteeing confidentiality. 
No transfer data will be carried out outside the EU.  

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The sponsor of the trial commits to carry out the data processing according to EU Regulation 
2016/679 and, therefore, to keep a record of the processing activities to carry out and to make 
a risk assessment of the data processing, to establish what measures will be applied and how it 
will be done. 
In addition to the rights already covered by the previous legislation (access, modification, 
opposition and cancellation of data, deletion in the new Regulation) participants can now limit 
the processing of data collected for the project that has to be rectified, request a copy or move 
to a third party (portability). To exercise these rights, the participant should be directed to the 
principal investigator of the study or the Data Protection Delegate of Hospital Clínic de 
Barcelona. The participant also has the right to contact the Data Protection Agency if not 
satisfied. 
The data cannot be deleted even if a patient discontinues the study, to guarantee the validity of 
the investigation and comply with the legal duties and the medication authorization 
requirements. 
The Investigator and the Sponsor are obliged to keep the data collected for the study at least up 
to 25 years after its completion. Subsequently, personal information will only be kept by the site 
for the care of their health and by the sponsor for other scientific research purposes if the 
patient has given their consent to do so, and if the law and applicable ethical requirements so 
permit. 
In accordance with the provisions of recital 33 of the regulations and the corresponding 
provisions of each country involved in the study regulations, the data may be preserved in such 
a way that the clinical data are kept separate from the identifiers, to be used in future 
investigations, applying all technical precautions necessary to avoid their re-identification, and 
in accordance with all ethical and legal requirements. 
 
13. FINANCING, INSURANCE AND PUBLICATION 
 
13.1. Financing 
 
This study is partially funded by a public grant (ISCIII Acción Estratégica de Salud 
PI19/00842). 
 
 
13.2. Insurance 
 
No specific insurance provision is required because an increased risk for patients beyond that 
associated with standard clinical practice is not expected. 
 
13.3. Publication  
 
The main results will be published by the sponsor in accordance with the Spanish 
requirements.  
 
 
14. STATISTICS 
 
 
14.1 Number of participants and sample size calculation 
 
According to our clinical and rectal surveillance data and assuming a 75% rectal colonization 
rate in patients with positive clinical samples and a loss of 20% of putative participants, we 

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estimate to include 437 patients in total, 175 patients per treatment arm and 87 as a control 
group.  
 
In the intervention arms, the expected distribution will be 25 CPE carriers per arm, 75 patients 
carrying ESBL-Klebsiella pneumoniae per arm and 75 patients carrying MDR/XDR Pseudomonas 
aeruginosa per arm. In the control arm, half these figures are expected. With this sample size 
and taking into account a decolonizing effectiveness of 30%-50% obtained in previous studies, 
the study would have a power of 80% to detect a difference between treatment arms of 15 
percentage points and a difference between treatment and control arms of 20 percentage points 
at a significance level of 5%. 
 
Diference 50% vs 30%: 69/129 per control/treatment arms 
Diference 40% vs 20%: 59/118 per control/treatment arms 
Diference 30% vs 10%: 44/88 per control/treatment arms 
 
14.2 Statistical analysis 
 
For the primary outcome the main analysis will be performed according to the intention to 
treat principle, considering as intention-to-treat population all randomized patients. Missing RS 
will be considered as positive. In addition, we intend to do a per protocol analysis, evaluating 
only the patients who end the treatment and end the study. Categorical variables will be 
compared by the Fisher exact test and continuous ones the the student t-test or Mann-Whitney 
test. Univariate logistic regression with group assignment as predictor variable and clinical 
characteristics and decolonization as outcome will be performed to calculate OR and 95% 
confidence intervals. In the event of imbalance in the distribution of potential confounder 
despite randomization, a multivariate logistic regression analysis with decolonization as de 
dependent variable and group assignment as one of the explanatory variables will be carried 
out. 
 
All randomised patients will be included in the “intention-to-treat population” for analysis. 
Patients withdrawn for any reason or lost to follow-up will be considered as treatment failure 
in this analysis. 
 
 
Subgroup analyses 
 
An analysis of efficay for each bacterial species/resistance determinant (ESBL-producing K. 
pneumoniae, carbapenemase-producing Enterobateriaceae, MDR-P. aeruginosa) are planned   
 
Interim  analysis 
  
A safety interim analysis is planned after inclusion of the first 100 individuals in the study. 
 
 
Analysis Centre 
 
Data will be analyzed in the Institut Clínic de Medicina Interna, Dermatologia I Infeccions del 
Hospital Clínic de Barcelona.  
 
 
 

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