MMMicrobiologia MedicaMicrobiol. Med.2280-6423PAGEPress Publications, Pavia, Italy10.4081/mm.2017.6510ArticleCulturomics: Bacterial Species Isolated in 3 Healthy Donors for Faecal Microbiota Transplantation in Clostridium Difficile InfectionMasucciLuca1QuarantaGianluca1NagelDomenico1PrimusSandra2RomanoLucio1GraffeoRosalia1IaniroGianluca3GasbarriniAntonio3CammarotaGiovanni3SanguinettiMaurizio1Institute of Microbiology, Catholic University of the Sacred Heart, A. Gemelli Foundation, School of Medicine, Rome, ItalyCollege for Healthcare Professions, Catholic University of the Sacred Heart, Claudiana, Bolzano, ItalyInternal Medicine, Gastroenterology and Liver Unit, Catholic University of the Sacred Heart, School of Medicine, A. Gemelli Foundation, Rome, ItalyInstitute of Microbiology, Catholic University of the Sacred Heart, A. Gemelli Foundation, School of Medicine, L.go A. Gemelli 8, 00168 Rome, Italy. +39.06.30154218 - +39.06.3051152luca.masucci@policlinicogemelli.it
Contribution: LM, GC, study concept and design; LM, GQ, DN, SP, LR, RG, laboratory work ; GC, GI, AG, clinical activity; LM, LR, MS, drafting of the manuscript.
Clostridium difficile infections are the main cause of nosocomial acquired diarrhea, because of prolonged antibiotic regimens. In the last years, mortality has increased due to recurrent infections caused by metronidazole and vancomicin resistant hypervirulent C. difficile strain 027. Faecal Microbiota Transplantation (FMT) is an infusion of faecal material obtained from healthy donors. This procedure reduces mortality in recurrent C. difficile infections (CDI). In this study we identified bacterial species obtained from donors’ stool samples using culturomics.
Materials and methods
Three donors were selected for FMT in three recipients affected by CDI. Faces obtained for FMT underwent culturomics, applying 17 different culture methods.
Results and conclusions
Several different bacteria were isolated from donors. In two donors 4 potentially pathogenic bacteria were isolated; this suggests the use of culturomics for donors’ screening or for selection of bacteria to infuse.
Conflict of interest: the authors declare no potential conflict of interest.
Introduction
Human gut microbiota consists of approximately 1013-1014 bacteria (9). Its alteration seems to be involved in several diseases such as obesity, malnutrition, metabolic diseases and chronic inflammatory diseases. For this reason gut microbiota is considered like an organ, which in healthy humans confers metabolic role, deconjugation of bile acid, immunological effects and pathogens protection (15).
Clostridium difficile infection (CDI) is due to an alteration of microbiota homeostasis, following a long period of antibiotic therapy (6). Several intestinal complications could occur, such as recurrent CDI. Recurrent CDI is a recurrence of symptoms within days to several weeks after the first episode that occurs after discontinuation of antibiotic therapy. The risk of multiple episodes is high in patients who have already had one recurrence (5).
Faecal microbiota transplantation (FMT) is an infusion of faecal material obtained from healthy donors, we demonstrated its effectiveness in treating recurrent CDIs in a recently published clinical trial (3). Usually, after a screening to exclude microbial pathogens, feces of a healthy donor are used for FMT, without characterizing the composition of their microbiota.
In this study, we used a culturomic approach to isolate and identify the bacterial population in healthy donors’ feces to be used in FMT in recurrent CDI patients.
Culturomics is a set of culture conditions originally applied to isolate bacterial populations considered unculturable. Culture conditions include: several enrichment broth media, blood culture bottles, culture on selective and non-selective agar media, different temperatures and times of observation from few days to months (8).
Materials and methods
All aspects of this study were approved by the Ethical Committee of “Fondazione Policlinico Universitario A. Gemelli”. Written consent was obtained for all participants.
Three faecal samples obtained from three healthy donors for FMT were analysed by culturomics.
Donors screening
The donors’ health conditions were assessed as previously described (3). As regards faecal samples, they were tested for C. difficile Toxin A/B [Liaison, DiaSorin SpA, Saluggia (VC), Italy], enteric bacteria, protozoa and helminths, VRE (vancomycin-resistant Enterococci), MRSA (methicillin-resistant Staphylococcus aureus), and Gram-negative MDR (multi-drug-resistant) bacteria, according to international guideline procedures.
Donor 1 was a 45 years old daughter, donor 2 was a 42 years old daughter and donor 3 was a 43 years old brother-in law of each recipient.
Culturomics protocol
Thirty grams of each faecal sample were dissolved and re-suspended in 30 mL of saline solution. After the homogenization, there was a filtration step using patented filters, included in the Parasep® Faecal Parasite Concentrators kit (Apacor Limited, Sapphire Center, Wokingham, England). In these filters, large particles are rejected without occluding the 425μm pores, according to manufacturer’s instructions.
The filtered 30 mL were divided into three aliquots of nearly 10 mL each and then centrifuged at 3500 g for 10 minutes. Supernatants were discarded and each one of the three pellets was resuspended: one in 5 mL of Brain Hearth Infusion (BHI, Oxoid Deutschland GmbH, Wesel, Germany), one in 5 mL of supplemented Brucella Broth (BB, Remel INC., Lenexa, USA) and the last in 5 mL of rumen fluid.
Rumen fluid was obtained from a cow fed with alfalfa-hay, before slaughtering. We modified the protocol described by Santschi et al. (16); briefly rumen fluid was filtered through two layers of gauzes and it was stored refrigerated overnight at 4°C to settle down particulate material. Supernatant was submitted to filter sterilization through 0.22 μm Minisart® filters (Sartorius AG, Weender Landstr. Goettingen, Germany)
BHI suspension was divided into two aliquots of 2.5 mL each, which were inoculated into two bottles of paediatric blood cultures (Becton, Dickinson and Company, Benex Limited, Shannon, Ireland), four aliquots of 2.5 mL each were obtained from the other two suspensions inoculated into four bottles of anaerobic blood culture (Becton, Dickinson and Company, Ireland), which were incubated at 37°C, vortexed and then 100 μL plated on agar media at day 7 and day 14. Culture conditions are shown in Table 1.
Identification of isolated bacteria
Bacterial identification was performed by MALDI-TOF, Mass Spectrometry, on a Bruker MicroFlex LT (Bruker Daltonics, Billerica, MA, USA).
For plate extractions, 1 to 4 colonies from subcultures were spotted on the target plate. Each spot was overlaid with 1 μL of alpha-cyano-4-hydroxycinnamic acid (α-CHCA).
Spectra were acquired from 1000 laser shots at a minimum intensity threshold of 30 arbitrary units (A.U.). Plate calibration was performed on each run before spectral acquisition using a mixture of two proteins contained in Bacterial Test Standard (BTS) (Bruker Daltonics).
The BTS has been developed for the Quality Control (QC) process of the MALDI Biotyper system. Its specific composition covers the entire mass range of proteins used by MALDI Biotyper for precise identification of microorganisms, making BTS ideal for use with this mass spectrometry system. When BTS is used, MALDI Biotyper performs an automatic QC before each identification run. The QC process includes calibration of the mass spectrometer, a laser setting check and evaluation of spectrum quality. System performance is finally confirmed by achieving Escherichia coli identification, which must pass minimum performance criteria. The result is documented on each identification run report.
Plate calibration met acceptable specifications when at least seven of the eight proteins were detected within 150 ppm of the in last fit parameter, analysed with Flex Analysis software (Version 3.4; Bruker Daltonics).
Results
Following bacterial species were isolared from donor 1 Actinomyces neuii, Actinomyces turicensis, Alistipes shahii, Bacteroides uniformis, Bacteroides intestinalis, Bacteroides salyersiae, Bifidobacterium adolescentis, Blautia coccoides, Clostridium aldenense, Clostridium scindens, Enterococcus faecium, Escherichia coli, Eggerthella lenta, Nocardia arthritidis, Parabacteroides distasonis, Parabacteroides johnsonii and Streptococcus anginosus.
Finally, in donor 3 we found Bacteroides uniformis, Bacteroides vulgatus, Clostridium bifermentans, Clostridium clostridioforme, Clostridium perfringens, Clostridium tertium, Enterococcus faecium, Escherichia coli, Klebsiella oxytoca, Parabacteroides distasonis and Proteus mirabilis (Table 2).
Discussion
C. difficile is a Gram-positive, spore-forming, anaerobic pathogen, which is acquired via faecal-oral route, through ingestion of the bacterial spores. After reaching the anaerobic gut niche of susceptible patients, the spores germinate into metabolically active vegetative cells that produce two major exotoxins, Toxin A (TcdA) and Toxin B (TcdB) (19). Some strains of C. difficile produce a third toxin, CDT (C. difficile transferase), which may also be involved in the pathogenic process (1). These strains have been associated with severe forms of disease in humans, even if the roles of these toxins in pathogenesis aren’t clear yet (2).
Recurrent CDI is associated with prolonged antibiotic therapy that could destroy the normal intestinal flora, enhancing a decrease in colonization resistance (18). In fact the normal microbiota of adults is dominated by species of the phyla Bacteroidetes and Firmicutes (9), whereas the predominant species in patients with recurrent CDI were Proteobacteria and Verrucomicrobia, reducing dominance of Bacteroidetes (4).
The main knowledge about the composition of gut microbiota was acquired using molecular methods such as DGGE (11) and TGGE (12) analysis and recently metagenomics. For many years, several bacteria were considered non-culturable and so cultures were not used to study this microbiota, but in the last years, the cultural approach is having a rebirth with culturomics (8).
Culturomics is a laborious and time-consuming approach, which is considered very useful to study living bacteria.
Using culture methods, 90 new strains were isolated and several data were obtained about faecal bacteria in people with and without diarrhea. Langier et al. developed 18 culture methods (8) whereas Samb-Ba et al. 9(17).
In this study we applied 17 cultural conditions at 7 and 14 days on faecal samples obtained from healthy donors for FMT. We found qualitative and quantitative diversity among the bacterial species isolated in donors; according to literature this is due to different socio-economic conditions, diet, geographic areas (5) and culture methods used (8). In fact, in absence of a standardized method, various culture conditions have been used: 212 or 18(8) and 9(17), with different numbers of bacterial species isolated for each sample. We were able to cultivate several strains of Enterococci different strains of Bacteroides and Clostridia. In donor 1 particular bacteria were detected: Actinomyces neuii implicated in human infections such as abscesses, ulcer infections, cellulitis, urinary tract infections, prostatitis, chorioamnionitis or invasive infections such as bacteraemia, prostheses infections, chronic osteomyelitis, peritoneal dialysis-associated peritonitis, chronic pericarditis, lymphadenitis and endophthalmitis (20); Actinomyces turicensis reported as a pathogen in endocarditis (7); Eggerthella lenta isolated in blood samples (10). C. perfringens was isolated in donor 3 and its pathogenic effects are well known: gas gangrene and food poisoning (14).
Since 2013, in our Institution FMT has been used to treat recurrent CDI. FMT includes: donor screening by clinical history, blood and faecal tests to be carried out 7 days before FMT, to reveal possible infections; laboratory activities to prepare feces in solution for the infusion in patients; endoscopic procedure (3).
On FMT day, the donor carries faces for infusion, which is to be made within 6 hours from the emission. Culturomics could be applied as an additional screening step to avoid to infuse even potential pathogens, such as those above described in donor 1 and 3.
In 2013, a pilot study showed that a synthetic stool solution with cultured bacteria could be an alternative to the use of donor faecal sample in the treatment of recurrent CDI (13).
Conclusions
In conclusion, culturomics could be a useful technique to set up an optimal synthetic stool sample which could have several advantages in terms of safety: i) known composition; ii) absence of potential infections acquired by donor during the period between screening and donation; iii) to have readyto-use bacterial solutions for emergency infusions.
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Media and culture conditions.
1.
Pre-incubation in paediatric blood culture bottle with rumen fluid and then 5% sheep blood, Chocolate and McConkey agar under aerobic, aerobe with 2.5% CO2 and microaerophilic conditions at 37°C
2.
Pre-incubation in anaerobic blood culture bottle with rumen fluid and then Columbia and Schaedler agar under anaerobic conditions at 37°C
3.
Pre-incubation in paediatric blood culture bottle with Brucella broth and then 5% sheep blood, Chocolate and McConkey agar under aerobic, aerobe with 2.5% CO2 and microaerophilic conditions at 37°C agar under aerobic conditions at 37°C
4.
Pre-incubation in anaerobic blood culture bottle with Brucella broth and then Columbia and Schaedler agar under anaerobic conditions at 37°C
5.
Pre-incubation under aerobic conditions in BHI and then 5% sheep blood, Chocolate and McConkey agar under aerobic, aerobe with 2.5% CO2 and microaerophilic conditions at 37°C agar under aerobic conditions at 37°C
6.
Pre-incubation in anaerobic blood culture bottle with stool filtered at 5 m and then Columbia and Schaedler agar under anaerobic conditions at 37°C
7.
Pre-incubation in paediatric culture bottle with stool filtered at 5 m and then 5% sheep blood under aerobic conditions at 37°C
8.
Pre-incubation in paediatric blood culture bottle with 5 mL BHI and then 5% sheep blood, Chocolate and McConkey agar under aerobic, aerobe with 2.5% CO2 and microaerophilic conditions at 37°C agar under aerobic conditions at 37°C
9.
Pre-incubation in anaerobic blood culture bottle with 5 mL BHI and then Columbia and Schaedler agar under anaerobic conditions at 37°C
Bacteria isolated from faecal samples of the three donors.