- Email: [email protected]
Clostridium difficile infection and gut microbiota
Author's Accepted Manuscript
Clostridium difficile infection and gut microbiota Sabina Zalig, Maja Rupnik PhD
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PII: DOI: Reference:
S1043-1489(14)00028-1 http://dx.doi.org/10.1053/j.scrs.2014.05.005 YSCRS463
To appear in:
Seminars in Colon and Rectal Surgery
Cite this article as: Sabina Zalig, Maja Rupnik PhD, Clostridium difficile infection and gut microbiota, Seminars in Colon and Rectal Surgery, http://dx.doi. org/10.1053/j.scrs.2014.05.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Clostridium difficile infection and gut microbiota
Sabina Zalig1, Maja Rupnik, PhD1,2,3 1
University of Maribor, Faculty of medicine, Maribor, Slovenia; 2National laboratory for health,
environment and food, Maribor, Slovenia; 3Centre of excellence for integrated approaches in chemistry and biology of proteins, Ljubljana, Slovenia;
Acknowledgement: SZ and MR are supported by Slovenian Research Agency (grant P30387). Mailing address for corresponding author: Maja Rupnik NLZOH Prvomajska 1 2000 Maribor Slovenia phone: 00386 2 4500 183 fax: 00386 2 4500 193 Email: [email protected]
Abstract Clostridium difficile infection (CDI) is associated with disturbance of intestinal microbiota. Microbiota of CDI patients usually shows decreased diversity, increase of facultative anaerobes and decreased levels of bifidobacteria, Bacteriodetes, Lachnospiraceae and butyrate producing bacteria. Studies including symptomatic, asymptomatic, recurrent and fecal therapy treated patients could result in the recognition of microbial markers for CDI risk or could provide the combination of beneficial microbes for a C. difficile specific probiotic.
Key words: Clostridium difficile infection, gut, intestinal microbiota, dysbiosis
Clostridium difficile infection (CDI) can range from self limiting diarrhea to colitis or pseudomembranous colitis and is currently one of the major pathogens causing intestinal infections. The hallmark of CDI is its association with antibiotic therapy and C. difficile is the major pathogen causing the antibiotic associated diarrhea. Therefore it was assumed already very early that the disturbance of gut microbiota is necessary for successful colonization of C. difficile (1). Gut microbiota is a complex community of bacteria, archaea, fungi and protozoan microorganisms populating the intestines. The composition and numbers are typical for each part of the intestinal system. Within each part there are further differences in microbes colonizing the mucosal epithelium or the lumen (2). Gut microbiota has essential role in host development and in maintenance of several important functions (immune system, nutrition, gut-brain axis, colonization resistance) (2, 3). A state of imbalance in composition of gut microbiota is called dysbiosis and can result in different disorders, C. difficile infection being only one of them (2). The aim of this paper is to give current overview on studies on gut microbiota in humans colonized with C. difficile or patients with CDI (Table 1). For further general information on gut microbiota several recent reviews are suggested: the role of microbiota in health and diasease (1-3), overview of methods used in gut microbiota studies and overview of recent large international consortia (4), the effect of antibiotics on the gut microbiota (5), importance of culturing gut microorganisms (culturomics; 6), possibilities for modulation of
gut microbiota (2), probiotics, prebiotics, phages and CDI (7-9), and in vivo and in vitro gut models for CDI (10).
Studies on gut microbiota in association with C. difficile Studies on characterization of microbiota in association with C. difficile can include only infants, only elderly or population of various ages (Table 1) and can be broadly organized into several groups. Some of them compare CDI patients with healthy individuals, while others compare only C. difficile positive (colonized) and noncolonized individuals without clinical data (Table 1). Couple of studies have compared effect of different antibiotic therapies for CDI on gut microbiota (11, 12) and several studies have looked at the changes of gut microbiota after fecal transplantation (13-16). Only a single study (17) has included in the analysis also adjustment of epidemiological risk factors for CDI. They found that some risk factors (certain antibiotics and non steroid anti inflammatory treatment) may result in changes in gut microbiota. In addition to these studies looking specifically at C. difficile and microbiota, C. difficile is mentioned also in some large microbiota studies with more broad aims, for instance in large studies on of infant microbiota development and association with feeding, delivery mode or allergic disease (18, 19). Also murine models are used for analysis of the gut microbiota role in CDI (20-22).
How was gut microbiota analyzed Fecal samples are usually used in the studies and are assumed a good marker for colonic microbiota. Most of the studies are focusing on bacterial populations and their numbers and types can be determined in two principal ways: with culturing and with
molecular methods. In earlier studies this molecular approaches were based either on specific detection of the main groups of fecal microbiota or were using different ways to analyze amplified bacterial marker gene pool (16S rDNA) with gel gradients (TTGE), chromatographically (DHPLC) or by clone libraries (Table 1). Currently, most studies are using sequencing approaches.
The major changes of gut microbiota composition associated with C. difficile Although there is a huge variability between individuals, studies agree that gut microbiota of an adult healthy human has following characteristics: high microbial numbers (1012 bacteria/g feces) and high species diversity, outnumbering of anaerobic bacteria over facultative anaerobic bacteria (eg. Enterobacteriaceae, Enterococcus) and predominance of two or three main phyla (Firmicutes, Bacteroidetes, Actinobacteria) (2, 6, 23, 24). All those characteristics are changed in individuals symptomatically or asymptomatically colonized with C. difficile. Main changes observed in all but one study (17) were decrease of bacterial counts and decrease of diversity. Overall species richness (i.e., the total number of phylotypes) is highest in healthy individuals, lower in individuals with first CDI episode and the lowest in individuals with recurrent CDI (25).
The other study revealed that the diversity and
composition of fecal microbiota is almost identical in non-colonized and colonized subjects, because the bacterial families that were changed significantly between colonized (but asymptomatic) and non-colonized individuals comprised only small part (up to 0.05%) of entire identified microbiota (26). The same study also describes that patients with the history of CDI have yet another type of microbiota which is different to healthy and active CDI (26). Furthermore, some C. difficile ribotypes (eg. R027) might be associated with more disturbed microbiota than the others (22, 26, 27).
Although different studies have identified different bacterial groups associated either with presence or absence of C. difficile there are also some common features (Table 1). These common features include an important role of Bacteroides spp., bifidobacteria, Lachnospiraceae and butyrate producing bacteria in colonization resistance against C. difficile and increase in enterobacteria and enterococci in disturbed microbiota associated with C. difficile presence. Most of the disagreement between studies seems to result from different phylogenetic levels analyzed or reported in the publications (Table 2). As an example, in symptomatic patients Actinobacteria as a broad group (phylum) are reported to increase, while a specific species within Actinobacteria (Bifidobacterium longum) is decreasing. Not only changes within the individual groups of bacteria, but also specific gut microbiota patterns associated with C. difficile colonization are important (27).
C. difficile, microbiota and multidrug resistant pathogens Increase of Gram positive cocci including enterocococci in C. difficile positive individuals is in agreement with some publications on association of C. difficile and vancomycin-resistant enterococci (VRE). C. difficile patients were found to have higher risk for colonization with VRE and other multi drug resistant bacteria (MRSA, Actinobacter) (28). The changes in microbiota are probably associated to other risk factors for colonization with multidrug resistant bacteria, such as long term care facility or prior hospitalization, described by those authors. Also, Choi et al., 2011, found that VRE colonization is a risk factor for recurrence (29). It is possible that both, VRE and C. difficile colonization, are markers for disbalanced gut microbiota.
Fecal transplantation and changes in gut microbiota in CDI patients
Fecal transplantation (FT) or bacteriotherapy is a procedure where a homogenate of faecal sample from a healthy donor is introduced to recipient using a colonoscope or via nasogastric tube. In recent years it has received much attention as a treatment option for CDI patients with multiple recurrences (16, 30, 31). Studies on FT in which also gut microbiota was followed (Table 1) report on individual differences, but in general recipient microbiota changes after FT, diversity is increased, some bacterial groups are replaced by others and composition resembles more the healthy microbiota (13-16).
Conclusions. The correlation of disbalanced gut microbiota and CDI was known for a long time. But only in recent years studies are focusing on precise characterization of changes in gut microbiota associated with presence of C. difficile, either in symptomatic or asymptomatic individuals. In parallel with these studies much attention was drawn towards restoring gut microbiota with fecal transplantation, which is becoming a treatment option for recurrent CDI. Results of gut microbiota in CDI could finally result in the microbial markers for risk of CDI/recurrent CDI or could provide the combination of beneficial microbes for a C. difficile specific probiotic.
Acknowledgements. SZ and MR are supported by Slovenian Research Agency grant P30387.
References 1. Britton RA, Young VB: Interaction between the intestinal microbiota and host in Clostridium difficile colonization resistance. Trends Microbiol 20(7):313-319, 2012 2. Walker AW, Lawley TD: Therapeutic modulation of intestinal dysbiosis. Pharmacol Res 69(1):75-86, 2013
3. Sommer F, Bäckhed F: The gut microbiota--masters of host development and physiology. Nat Rev Microbiol 11(4):227-238, 2013 4. Lepage P, Leclerc MC, Joossens M, et al: A metagenomic insight into our gut's microbiome. Gut 62(1):146-158, 2013 5. Willing BP, Russell SL, Finlay BB: Shifting the balance: antibiotic effects on hostmicrobiota mutualism. Nat Rev Microbiol 9(4):233-243, 2011 6. Lagier JC, Million M, Hugon P, et al: Human gut microbiota: repertoire and variations. Front Cell Infect Microbiol 2:136, 2012 7. Steer T, Carpenter H, Tuohy K, et al: Perspectives on the role of the human gut microbiota and its modulation by pro- and prebiotics. Nutr Res Rev 13(2):229-254, 2000 8. Hell M, Bernhofer C, Stalzer P, et al: Probiotics in Clostridium difficile infection: reviewing the need for a multistrain probiotic. Benef Microbes 4(1):39-51, 2013 9. Rea MC, Alemayehu D, Ross RP, et al: Gut solutions to a gut problem: bacteriocins, probiotics and bacteriophage for control of Clostridium difficile infection. J Med Microbiol 62(Pt 9):1369-1378, 2013 10. Best EL, Freeman J, Wilcox MH: Models for the study of Clostridium difficile infection. Gut Microbes 3(2):145-167, 2012 11. Tannock GW, Munro K, Taylor C, et al: A new macrocyclic antibiotic, fidaxomicin (OPT-80), causes less alteration to the bowel microbiota of Clostridium difficileinfected patients than does vancomycin. Microbiology 156:3354-3359, 2010 12. Louie TJ, Cannon K, Byrne B, et al: Fidaxomicin preserves the intestinal microbiome during and after treatment of Clostridium difficile Infection (CDI) and reduces both toxin reexpression and recurrence of CDI. Clin Infect Dis 55(S2):S132-S142, 2012 (suppl)
13. Khoruts A, Dicksved J, Jansson JK, et al: Changes in the composition of the human fecal microbiome following bacteriotherapy for recurrent Clostridium difficileassociated diarrhea. J Clin Gastroenterol 44(5):354-360, 2010 14. Shahinas D, Silverman M, Sittler T, et al: Toward an understanding of changes in diversity associated with fecal microbiome transplantation based on 16S rRNA gene deep sequencing. mBio 3(5):e00338-12, 2012 15. Song Y, Garg S, Girotra M, et al: Microbiota dynamics in patients treated with fecal microbiota transplantation for recurrent Clostridium difficile infection. PLoS ONE 8(11): e81330, 2013 16. Van Nood E, Vrieze A, Nieuwdorp M, et al: Duodenal infusion of donor feces for recurrent Clostridium difficile. N Engl J Med 368(5):407-415, 2013 17. Manges AR, Labbe A, Loo VG, et al: Comparative metagenomic study of alterations to the intestinal microbiota and risk of nosocomial Clostridum difficile–associated disease. J Infect Dis 202(12):1877–1884, 2010 18. Kirjavainen PV, Apostolou E, Arvola T, et al: Characterizing the composition of intestinal microflora as a prospective treatment target in infant allergic disease. FEMS Immunol Med Microbiol 32(1):1–7, 2001 19. Azad MB, Konya T, Mauhan H, et al: Gut microbiota of healthy Canadian infants:profiles by mode of delivery and infant diet at 4 months. CMAJ 185(5):385394, 2013 20. Reeves AE, Theriot CM, Bergin IL, et al: The interplay between microbiome dynamics and pathogen dynamics in a murine model of Clostridium difficile infection. Gut Microbes 2(3):145-158, 2011
21. Buffie CG, Jarchum I, Equinda M, et al: Profound alterations of intestinal microbiota following a single dose of clindamycin results in sustained susceptibility to Clostridium difficile-induced colitis. Infect Immun 80(1):62-73, 2012 22. Lawley TD, Clare S, Walker AW, et al: Targeted restoration of the intestinal microbiota with a simple, defined bacteriotherapy resolves relapsing Clostridium difficile disease in mice. PLoS Pathog 8(10):e1002995, 2012 23. Andersson AF, Lindberg M, Jakobsson H, et al: Comparative analysis of human gut microbiota by barcoded pyrosequencing. PloS ONE 3(7): e2836, 2008 24. Lozupone CA, Stombaugh JI, Gordon JI, et al: Diversity, stability and resilience of the human gut microbiota. Nature 489(7415):220-230, 2012 25. Chang JY, Antonopoulos DA, Kalra A, et al: Decreased diversity of the fecal microbiome in recurrent Clostridium difficile–associated diarrhea. J Infect Dis 197(3):435-438, 2008 26. Rea MC, O'Sullivan O, Shanahan F, et al: Clostridium difficile carriage in elderly subjects and associated changes in the intestinal microbiota. J Clin Microbiol 50(3):867-875, 2012 27. Skraban J, Dzeroski S, Zenko B, et al: Gut microbiota patterns associated with colonization of different Clostridium difficile ribotypes. PLoS ONE 8(2):e58005, 2013 28. Fujitani S, George WL, Morgan MA, et al: Implications for vancomycin-resistant Enterococcus colonization associated with Clostridium difficile infections. Am J Infect Control 39(3):188-193, 2011 29. Choi HK, Kim KH, Lee SH, et al: Risk Factors for recurrence of Clostridium difficile infection: effect of vancomycin-resistant enterococci colonization. J Korean Med Sci 26(7):859-864, 2011
30. Brandt LJ, Aroniadis OC: An overview of fecal microbiota transplantation: techniques, indications, and outcomes. Gastrointest Endosc 78(2):240-249, 2013 31. Smits LP, Bouter KEC, De Vos WM, et al: Therapeutic potential of fecal microbiota transplantation. Gastroenterology 145(5):946-953, 2013 32. Fallani M, Rigottier-Gois L, Aguilera M, et al: Clostridium difficile and Clostridium perfringens species detected in infant faecal microbiota using 16S rRNA targeted probes. J Microbiol Methods 67(1):150-161, 2006 33. Rousseau C, Levenez F, Fouqueray C, et al: Clostridium difficile colonization in early infancy is accompanied by changes in intestinal microbiota composition. J Clin Microbiol 49(3):858-865, 2011 34. Ozaki E, Kato H, Kita H, et al: Clostridium difficile colonization in healthy adults: transient colonization and correlation with enterococcal colonisation. J Med Microbiol 53(2):167-172, 2004 35. De La Cochetière MF, Durand T, Lalande V, et al: Effect of antibiotic therapy on human fecal microbiota and the relation to the development of Clostridium difficile. Microb Ecol 56(3):395-402, 2008 36. Antharam VC, Li EC, Ishmael A, et al: Intestinal dysbiosis and depletion of butyrogenic bacteria in Clostridium difficile infection and nosocomial diarrhea. J Clin Microbiol 51(9):2884–2892, 2013 37. Hopkins MJ, Macfarlane GT: Changes in predominant bacterial populations in human faeces with age and with Clostridium difficile infection. J Med Microbiol 51(5):448454, 2002 38. Sepp E, Štšepetova J, Smidt I, et al: Intestinal lactoflora in Estonian and Norwegian patients with antibiotic associated diarrhea. Anaerobe 17(6):407-409, 2011
Table 1. Overview of studies on C. difficile and gut microbiota CDI status
asympto matic carriage
Number of individuals
33 (9 CD+)
1.5 to 18.5 months
53 (27 CD neg, 26 CD poz)
0 to 13 months
139 (16)*
19 y to 65 y
18 CD carriers + 252 controls
≥ 65 y
208 (105 CDI + 103 controls)
14y to 72y
11 CDI 38 controls
28y to 78 y
39 CDI 40 controls symptom atic 15 (4 CDI + 11 controls)
recurrent
Age
54.7 +/20.1 (CDI) 60.9 +/9.1 (controls) 67y to 73y (CDI) 21y to 88y (controls)
Method used fluorescent in situ hybridization and flow cytometry (FISH/FC) PCRtemporal temperature gradient gel electrophores is (TTGE) cultivation methods pyrosequenci ng (Roche 454) denaturing high pressure liquid chromatograp hy (DHPLC) temporal temperature gradient electrophores is (TTGE) of amplified 16S rDNA
Bifidobacteria
32
Ruminococcus gnavus, Klebsiella pneumoniae
Bifidobacterium longum, E. coli, S.epidermidis
33
Enterococcus
/
34
Clostridia
Bifidobacterium, Eubacterium
26
Ruminococcus bromii, Peptostreptococcaceae, Streptococcus sp./Enterococcus sp.
Bifidobacterium longum, Bacteroides, Enterobacteriaceae
27
Clostridiales (Clostridiaceae, Eubacteria, Lachnospiraceae)
/
35
pyrosequenci ng (Roche 454)
Veillonella, Enterococcus, Lactobacillus, Gammaproteobacteria
Ruminococcaceae, Lachnospiraceae, butyrogenic bacteria belonging to Clostridium clusters IV and XIVa, Deltaproteobacteria
36
cultivation methods
Enterobacteriaceae, Enterococcus, Lactobacillus, Clostridia
Bacteroides, Prevotella, Bifidobacteria
37
Proteobacteria, Actinobacteria, Firmicutes (Lactobacillaceae, Enterococcaceae)
Bacteroides
17
Parabacteroides, Enterococcus
Bifidobacterium, Facealibacterium
26
/
lactobacilli (Lactobacillus plantarum)
38
enterobacteria, lactobacilli/enterococci
clostridial clusters, Bifidobacteria
11
64y to 75y
16S rRNA microarray
2 CD positive simpt + 252 CD negative
≥ 65 y
pyrosequenci ng (Roche 454)
3y to 89 y
31 CDI (8 receiving vancomycin, 23 receiving fidaxomicin) ** + 8 healthy
nd
Reference
Bacteroides
24 CDI 48 controls
74
Microbiota modifications (compared to healthy control subjects) INCREASED DECREASED groups/species groups/species
cultivation methods and real-time PCR PCRtemporal temperature gradient electrophores is (TTGE), fluorescent in situ hybridization
and flow cytometry (FISH/FC)
3 CDI 3 controls
80 y to 84 y (CDI) 65 y to 71 y (controls)
16S rRNAclone libraries
Clostridia, Proteobacteria, Verrucomicrobia
/
25
nd
real-time qPCR
Veillonella, Enterobacteriaceae
Bacteroides/Prevotella, Clostridium coccoides group, Clostridium leptum group, Bifidobacteria
12
Lactobacillus, Streptococcus, Veillonella, Eubacterium
Bacteroides
13
Streptococcaceae, Enterococcaceae, Enterobacteriaceae
Lachnospiraceae, Ruminococcaceae
15
20 CDI (10 receiving vancomycin, 10 receiving fidaxomicin) ** + 10 controls
FT recurrent
terminalrestriction fragment lenght polymorphis m (T-RFLP) pyrosequenci ng (Roche 454)
1 CDI + 1 donor
61y CDI 44y donor
14 CDI + 14 donors
41 y to 79 y (CDI)
16 CDI + 15 donors
73 +/- 13 y (CDI) 44 +/18.1 (donors)
16S rRNA microarray
Proteobacteria
Bacteroidetes, Clostridium clusters IV and XIVa
16
6
nd
pyrosequenci ng (Roche 454)
Proteobacteria
Bacteroidetes
14
* - only in 16 individuals the microbiota was studied ** - results given only for vancomycin treated patients nd - not defined; CD – C. difficile; CDI – C. difficile infection
Table 2. Overview of bacterial phylotypes reported to change in different studies on gut microbiota and C. difficile Phylum
Family
Genus
Species
Actinobacteria Bifidobacteriaceae Bifidobacteri Bifidobacterium um longum (A) Bacteroidetes
Bacteroidaceae
Bacteroides
/
Prevotellaceae
Prevotella
/
Microbiota modifications (compared to healthy control subjects) increased when symptomatic (A; phylum level); decreased when asymptomatic, symptomatic and/or (FT) recurrent increased when asymptomatic; decreased when symptomatic and/or recurrent decreased when symptomatic
Firmicutes
increased when symptomatic; Clostridium decreased when symptomatic and/or (FT) recurrent (B; coccoides group, Clostridium leptum group level) group (B)
Clostridiaceae
Clostridium
Enterococcaceae (C)
Clostridium clusters IV and XIVa (B) Enterococcu / s
Eubacteriaceae
/
/
Lachnospiraceae
/
/
Lactobacillaceae
Lactobacillu Lactobacillus s plantarum (D)
increased when symptomatic and/or recurrent; decreased when symptomatic (D; species level)
Peptostreptococca ceae Ruminococcaceae (E)
/
increased when symptomatic
Streptococcaceae Veillonellaceae
/
Ruminococc us
Ruminococcus bromii (F) Ruminococcus gnavus (G) Streptococcu / s Veillonella /
increased when asymptomatic, symptomatic and/or (FT) recurrent; decreased when symptomatic (C; family level) increased when symptomatic increased when symptomatic; decreased when symptomatic and/or FT recurrent
increased when asymptomatic (G; species level) and symptomatic (F; species level); decreased when FT recurrent (E; family level) increased when symptomatic and/or FT recurrent increased when symptomatic and/or (FT) recurrent
Proteobacteria Enterobacteriacea e (H)
Klebsiella
Klebsiella pneumoniae
increased when asymptomatic; decreased when symptomatic (H; family level)
Verrucomicrob / ia
/
/
increased when recurrent
Clostridium difficile infection and gut microbiota Sabina Zalig, Maja Rupnik PhD
www.elsevier.com/locate/yscrs
PII: DOI: Reference:
S1043-1489(14)00028-1 http://dx.doi.org/10.1053/j.scrs.2014.05.005 YSCRS463
To appear in:
Seminars in Colon and Rectal Surgery
Cite this article as: Sabina Zalig, Maja Rupnik PhD, Clostridium difficile infection and gut microbiota, Seminars in Colon and Rectal Surgery, http://dx.doi. org/10.1053/j.scrs.2014.05.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Clostridium difficile infection and gut microbiota
Sabina Zalig1, Maja Rupnik, PhD1,2,3 1
University of Maribor, Faculty of medicine, Maribor, Slovenia; 2National laboratory for health,
environment and food, Maribor, Slovenia; 3Centre of excellence for integrated approaches in chemistry and biology of proteins, Ljubljana, Slovenia;
Acknowledgement: SZ and MR are supported by Slovenian Research Agency (grant P30387). Mailing address for corresponding author: Maja Rupnik NLZOH Prvomajska 1 2000 Maribor Slovenia phone: 00386 2 4500 183 fax: 00386 2 4500 193 Email: [email protected]
Abstract Clostridium difficile infection (CDI) is associated with disturbance of intestinal microbiota. Microbiota of CDI patients usually shows decreased diversity, increase of facultative anaerobes and decreased levels of bifidobacteria, Bacteriodetes, Lachnospiraceae and butyrate producing bacteria. Studies including symptomatic, asymptomatic, recurrent and fecal therapy treated patients could result in the recognition of microbial markers for CDI risk or could provide the combination of beneficial microbes for a C. difficile specific probiotic.
Key words: Clostridium difficile infection, gut, intestinal microbiota, dysbiosis
Clostridium difficile infection (CDI) can range from self limiting diarrhea to colitis or pseudomembranous colitis and is currently one of the major pathogens causing intestinal infections. The hallmark of CDI is its association with antibiotic therapy and C. difficile is the major pathogen causing the antibiotic associated diarrhea. Therefore it was assumed already very early that the disturbance of gut microbiota is necessary for successful colonization of C. difficile (1). Gut microbiota is a complex community of bacteria, archaea, fungi and protozoan microorganisms populating the intestines. The composition and numbers are typical for each part of the intestinal system. Within each part there are further differences in microbes colonizing the mucosal epithelium or the lumen (2). Gut microbiota has essential role in host development and in maintenance of several important functions (immune system, nutrition, gut-brain axis, colonization resistance) (2, 3). A state of imbalance in composition of gut microbiota is called dysbiosis and can result in different disorders, C. difficile infection being only one of them (2). The aim of this paper is to give current overview on studies on gut microbiota in humans colonized with C. difficile or patients with CDI (Table 1). For further general information on gut microbiota several recent reviews are suggested: the role of microbiota in health and diasease (1-3), overview of methods used in gut microbiota studies and overview of recent large international consortia (4), the effect of antibiotics on the gut microbiota (5), importance of culturing gut microorganisms (culturomics; 6), possibilities for modulation of
gut microbiota (2), probiotics, prebiotics, phages and CDI (7-9), and in vivo and in vitro gut models for CDI (10).
Studies on gut microbiota in association with C. difficile Studies on characterization of microbiota in association with C. difficile can include only infants, only elderly or population of various ages (Table 1) and can be broadly organized into several groups. Some of them compare CDI patients with healthy individuals, while others compare only C. difficile positive (colonized) and noncolonized individuals without clinical data (Table 1). Couple of studies have compared effect of different antibiotic therapies for CDI on gut microbiota (11, 12) and several studies have looked at the changes of gut microbiota after fecal transplantation (13-16). Only a single study (17) has included in the analysis also adjustment of epidemiological risk factors for CDI. They found that some risk factors (certain antibiotics and non steroid anti inflammatory treatment) may result in changes in gut microbiota. In addition to these studies looking specifically at C. difficile and microbiota, C. difficile is mentioned also in some large microbiota studies with more broad aims, for instance in large studies on of infant microbiota development and association with feeding, delivery mode or allergic disease (18, 19). Also murine models are used for analysis of the gut microbiota role in CDI (20-22).
How was gut microbiota analyzed Fecal samples are usually used in the studies and are assumed a good marker for colonic microbiota. Most of the studies are focusing on bacterial populations and their numbers and types can be determined in two principal ways: with culturing and with
molecular methods. In earlier studies this molecular approaches were based either on specific detection of the main groups of fecal microbiota or were using different ways to analyze amplified bacterial marker gene pool (16S rDNA) with gel gradients (TTGE), chromatographically (DHPLC) or by clone libraries (Table 1). Currently, most studies are using sequencing approaches.
The major changes of gut microbiota composition associated with C. difficile Although there is a huge variability between individuals, studies agree that gut microbiota of an adult healthy human has following characteristics: high microbial numbers (1012 bacteria/g feces) and high species diversity, outnumbering of anaerobic bacteria over facultative anaerobic bacteria (eg. Enterobacteriaceae, Enterococcus) and predominance of two or three main phyla (Firmicutes, Bacteroidetes, Actinobacteria) (2, 6, 23, 24). All those characteristics are changed in individuals symptomatically or asymptomatically colonized with C. difficile. Main changes observed in all but one study (17) were decrease of bacterial counts and decrease of diversity. Overall species richness (i.e., the total number of phylotypes) is highest in healthy individuals, lower in individuals with first CDI episode and the lowest in individuals with recurrent CDI (25).
The other study revealed that the diversity and
composition of fecal microbiota is almost identical in non-colonized and colonized subjects, because the bacterial families that were changed significantly between colonized (but asymptomatic) and non-colonized individuals comprised only small part (up to 0.05%) of entire identified microbiota (26). The same study also describes that patients with the history of CDI have yet another type of microbiota which is different to healthy and active CDI (26). Furthermore, some C. difficile ribotypes (eg. R027) might be associated with more disturbed microbiota than the others (22, 26, 27).
Although different studies have identified different bacterial groups associated either with presence or absence of C. difficile there are also some common features (Table 1). These common features include an important role of Bacteroides spp., bifidobacteria, Lachnospiraceae and butyrate producing bacteria in colonization resistance against C. difficile and increase in enterobacteria and enterococci in disturbed microbiota associated with C. difficile presence. Most of the disagreement between studies seems to result from different phylogenetic levels analyzed or reported in the publications (Table 2). As an example, in symptomatic patients Actinobacteria as a broad group (phylum) are reported to increase, while a specific species within Actinobacteria (Bifidobacterium longum) is decreasing. Not only changes within the individual groups of bacteria, but also specific gut microbiota patterns associated with C. difficile colonization are important (27).
C. difficile, microbiota and multidrug resistant pathogens Increase of Gram positive cocci including enterocococci in C. difficile positive individuals is in agreement with some publications on association of C. difficile and vancomycin-resistant enterococci (VRE). C. difficile patients were found to have higher risk for colonization with VRE and other multi drug resistant bacteria (MRSA, Actinobacter) (28). The changes in microbiota are probably associated to other risk factors for colonization with multidrug resistant bacteria, such as long term care facility or prior hospitalization, described by those authors. Also, Choi et al., 2011, found that VRE colonization is a risk factor for recurrence (29). It is possible that both, VRE and C. difficile colonization, are markers for disbalanced gut microbiota.
Fecal transplantation and changes in gut microbiota in CDI patients
Fecal transplantation (FT) or bacteriotherapy is a procedure where a homogenate of faecal sample from a healthy donor is introduced to recipient using a colonoscope or via nasogastric tube. In recent years it has received much attention as a treatment option for CDI patients with multiple recurrences (16, 30, 31). Studies on FT in which also gut microbiota was followed (Table 1) report on individual differences, but in general recipient microbiota changes after FT, diversity is increased, some bacterial groups are replaced by others and composition resembles more the healthy microbiota (13-16).
Conclusions. The correlation of disbalanced gut microbiota and CDI was known for a long time. But only in recent years studies are focusing on precise characterization of changes in gut microbiota associated with presence of C. difficile, either in symptomatic or asymptomatic individuals. In parallel with these studies much attention was drawn towards restoring gut microbiota with fecal transplantation, which is becoming a treatment option for recurrent CDI. Results of gut microbiota in CDI could finally result in the microbial markers for risk of CDI/recurrent CDI or could provide the combination of beneficial microbes for a C. difficile specific probiotic.
Acknowledgements. SZ and MR are supported by Slovenian Research Agency grant P30387.
References 1. Britton RA, Young VB: Interaction between the intestinal microbiota and host in Clostridium difficile colonization resistance. Trends Microbiol 20(7):313-319, 2012 2. Walker AW, Lawley TD: Therapeutic modulation of intestinal dysbiosis. Pharmacol Res 69(1):75-86, 2013
3. Sommer F, Bäckhed F: The gut microbiota--masters of host development and physiology. Nat Rev Microbiol 11(4):227-238, 2013 4. Lepage P, Leclerc MC, Joossens M, et al: A metagenomic insight into our gut's microbiome. Gut 62(1):146-158, 2013 5. Willing BP, Russell SL, Finlay BB: Shifting the balance: antibiotic effects on hostmicrobiota mutualism. Nat Rev Microbiol 9(4):233-243, 2011 6. Lagier JC, Million M, Hugon P, et al: Human gut microbiota: repertoire and variations. Front Cell Infect Microbiol 2:136, 2012 7. Steer T, Carpenter H, Tuohy K, et al: Perspectives on the role of the human gut microbiota and its modulation by pro- and prebiotics. Nutr Res Rev 13(2):229-254, 2000 8. Hell M, Bernhofer C, Stalzer P, et al: Probiotics in Clostridium difficile infection: reviewing the need for a multistrain probiotic. Benef Microbes 4(1):39-51, 2013 9. Rea MC, Alemayehu D, Ross RP, et al: Gut solutions to a gut problem: bacteriocins, probiotics and bacteriophage for control of Clostridium difficile infection. J Med Microbiol 62(Pt 9):1369-1378, 2013 10. Best EL, Freeman J, Wilcox MH: Models for the study of Clostridium difficile infection. Gut Microbes 3(2):145-167, 2012 11. Tannock GW, Munro K, Taylor C, et al: A new macrocyclic antibiotic, fidaxomicin (OPT-80), causes less alteration to the bowel microbiota of Clostridium difficileinfected patients than does vancomycin. Microbiology 156:3354-3359, 2010 12. Louie TJ, Cannon K, Byrne B, et al: Fidaxomicin preserves the intestinal microbiome during and after treatment of Clostridium difficile Infection (CDI) and reduces both toxin reexpression and recurrence of CDI. Clin Infect Dis 55(S2):S132-S142, 2012 (suppl)
13. Khoruts A, Dicksved J, Jansson JK, et al: Changes in the composition of the human fecal microbiome following bacteriotherapy for recurrent Clostridium difficileassociated diarrhea. J Clin Gastroenterol 44(5):354-360, 2010 14. Shahinas D, Silverman M, Sittler T, et al: Toward an understanding of changes in diversity associated with fecal microbiome transplantation based on 16S rRNA gene deep sequencing. mBio 3(5):e00338-12, 2012 15. Song Y, Garg S, Girotra M, et al: Microbiota dynamics in patients treated with fecal microbiota transplantation for recurrent Clostridium difficile infection. PLoS ONE 8(11): e81330, 2013 16. Van Nood E, Vrieze A, Nieuwdorp M, et al: Duodenal infusion of donor feces for recurrent Clostridium difficile. N Engl J Med 368(5):407-415, 2013 17. Manges AR, Labbe A, Loo VG, et al: Comparative metagenomic study of alterations to the intestinal microbiota and risk of nosocomial Clostridum difficile–associated disease. J Infect Dis 202(12):1877–1884, 2010 18. Kirjavainen PV, Apostolou E, Arvola T, et al: Characterizing the composition of intestinal microflora as a prospective treatment target in infant allergic disease. FEMS Immunol Med Microbiol 32(1):1–7, 2001 19. Azad MB, Konya T, Mauhan H, et al: Gut microbiota of healthy Canadian infants:profiles by mode of delivery and infant diet at 4 months. CMAJ 185(5):385394, 2013 20. Reeves AE, Theriot CM, Bergin IL, et al: The interplay between microbiome dynamics and pathogen dynamics in a murine model of Clostridium difficile infection. Gut Microbes 2(3):145-158, 2011
21. Buffie CG, Jarchum I, Equinda M, et al: Profound alterations of intestinal microbiota following a single dose of clindamycin results in sustained susceptibility to Clostridium difficile-induced colitis. Infect Immun 80(1):62-73, 2012 22. Lawley TD, Clare S, Walker AW, et al: Targeted restoration of the intestinal microbiota with a simple, defined bacteriotherapy resolves relapsing Clostridium difficile disease in mice. PLoS Pathog 8(10):e1002995, 2012 23. Andersson AF, Lindberg M, Jakobsson H, et al: Comparative analysis of human gut microbiota by barcoded pyrosequencing. PloS ONE 3(7): e2836, 2008 24. Lozupone CA, Stombaugh JI, Gordon JI, et al: Diversity, stability and resilience of the human gut microbiota. Nature 489(7415):220-230, 2012 25. Chang JY, Antonopoulos DA, Kalra A, et al: Decreased diversity of the fecal microbiome in recurrent Clostridium difficile–associated diarrhea. J Infect Dis 197(3):435-438, 2008 26. Rea MC, O'Sullivan O, Shanahan F, et al: Clostridium difficile carriage in elderly subjects and associated changes in the intestinal microbiota. J Clin Microbiol 50(3):867-875, 2012 27. Skraban J, Dzeroski S, Zenko B, et al: Gut microbiota patterns associated with colonization of different Clostridium difficile ribotypes. PLoS ONE 8(2):e58005, 2013 28. Fujitani S, George WL, Morgan MA, et al: Implications for vancomycin-resistant Enterococcus colonization associated with Clostridium difficile infections. Am J Infect Control 39(3):188-193, 2011 29. Choi HK, Kim KH, Lee SH, et al: Risk Factors for recurrence of Clostridium difficile infection: effect of vancomycin-resistant enterococci colonization. J Korean Med Sci 26(7):859-864, 2011
30. Brandt LJ, Aroniadis OC: An overview of fecal microbiota transplantation: techniques, indications, and outcomes. Gastrointest Endosc 78(2):240-249, 2013 31. Smits LP, Bouter KEC, De Vos WM, et al: Therapeutic potential of fecal microbiota transplantation. Gastroenterology 145(5):946-953, 2013 32. Fallani M, Rigottier-Gois L, Aguilera M, et al: Clostridium difficile and Clostridium perfringens species detected in infant faecal microbiota using 16S rRNA targeted probes. J Microbiol Methods 67(1):150-161, 2006 33. Rousseau C, Levenez F, Fouqueray C, et al: Clostridium difficile colonization in early infancy is accompanied by changes in intestinal microbiota composition. J Clin Microbiol 49(3):858-865, 2011 34. Ozaki E, Kato H, Kita H, et al: Clostridium difficile colonization in healthy adults: transient colonization and correlation with enterococcal colonisation. J Med Microbiol 53(2):167-172, 2004 35. De La Cochetière MF, Durand T, Lalande V, et al: Effect of antibiotic therapy on human fecal microbiota and the relation to the development of Clostridium difficile. Microb Ecol 56(3):395-402, 2008 36. Antharam VC, Li EC, Ishmael A, et al: Intestinal dysbiosis and depletion of butyrogenic bacteria in Clostridium difficile infection and nosocomial diarrhea. J Clin Microbiol 51(9):2884–2892, 2013 37. Hopkins MJ, Macfarlane GT: Changes in predominant bacterial populations in human faeces with age and with Clostridium difficile infection. J Med Microbiol 51(5):448454, 2002 38. Sepp E, Štšepetova J, Smidt I, et al: Intestinal lactoflora in Estonian and Norwegian patients with antibiotic associated diarrhea. Anaerobe 17(6):407-409, 2011
Table 1. Overview of studies on C. difficile and gut microbiota CDI status
asympto matic carriage
Number of individuals
33 (9 CD+)
1.5 to 18.5 months
53 (27 CD neg, 26 CD poz)
0 to 13 months
139 (16)*
19 y to 65 y
18 CD carriers + 252 controls
≥ 65 y
208 (105 CDI + 103 controls)
14y to 72y
11 CDI 38 controls
28y to 78 y
39 CDI 40 controls symptom atic 15 (4 CDI + 11 controls)
recurrent
Age
54.7 +/20.1 (CDI) 60.9 +/9.1 (controls) 67y to 73y (CDI) 21y to 88y (controls)
Method used fluorescent in situ hybridization and flow cytometry (FISH/FC) PCRtemporal temperature gradient gel electrophores is (TTGE) cultivation methods pyrosequenci ng (Roche 454) denaturing high pressure liquid chromatograp hy (DHPLC) temporal temperature gradient electrophores is (TTGE) of amplified 16S rDNA
Bifidobacteria
32
Ruminococcus gnavus, Klebsiella pneumoniae
Bifidobacterium longum, E. coli, S.epidermidis
33
Enterococcus
/
34
Clostridia
Bifidobacterium, Eubacterium
26
Ruminococcus bromii, Peptostreptococcaceae, Streptococcus sp./Enterococcus sp.
Bifidobacterium longum, Bacteroides, Enterobacteriaceae
27
Clostridiales (Clostridiaceae, Eubacteria, Lachnospiraceae)
/
35
pyrosequenci ng (Roche 454)
Veillonella, Enterococcus, Lactobacillus, Gammaproteobacteria
Ruminococcaceae, Lachnospiraceae, butyrogenic bacteria belonging to Clostridium clusters IV and XIVa, Deltaproteobacteria
36
cultivation methods
Enterobacteriaceae, Enterococcus, Lactobacillus, Clostridia
Bacteroides, Prevotella, Bifidobacteria
37
Proteobacteria, Actinobacteria, Firmicutes (Lactobacillaceae, Enterococcaceae)
Bacteroides
17
Parabacteroides, Enterococcus
Bifidobacterium, Facealibacterium
26
/
lactobacilli (Lactobacillus plantarum)
38
enterobacteria, lactobacilli/enterococci
clostridial clusters, Bifidobacteria
11
64y to 75y
16S rRNA microarray
2 CD positive simpt + 252 CD negative
≥ 65 y
pyrosequenci ng (Roche 454)
3y to 89 y
31 CDI (8 receiving vancomycin, 23 receiving fidaxomicin) ** + 8 healthy
nd
Reference
Bacteroides
24 CDI 48 controls
74
Microbiota modifications (compared to healthy control subjects) INCREASED DECREASED groups/species groups/species
cultivation methods and real-time PCR PCRtemporal temperature gradient electrophores is (TTGE), fluorescent in situ hybridization
and flow cytometry (FISH/FC)
3 CDI 3 controls
80 y to 84 y (CDI) 65 y to 71 y (controls)
16S rRNAclone libraries
Clostridia, Proteobacteria, Verrucomicrobia
/
25
nd
real-time qPCR
Veillonella, Enterobacteriaceae
Bacteroides/Prevotella, Clostridium coccoides group, Clostridium leptum group, Bifidobacteria
12
Lactobacillus, Streptococcus, Veillonella, Eubacterium
Bacteroides
13
Streptococcaceae, Enterococcaceae, Enterobacteriaceae
Lachnospiraceae, Ruminococcaceae
15
20 CDI (10 receiving vancomycin, 10 receiving fidaxomicin) ** + 10 controls
FT recurrent
terminalrestriction fragment lenght polymorphis m (T-RFLP) pyrosequenci ng (Roche 454)
1 CDI + 1 donor
61y CDI 44y donor
14 CDI + 14 donors
41 y to 79 y (CDI)
16 CDI + 15 donors
73 +/- 13 y (CDI) 44 +/18.1 (donors)
16S rRNA microarray
Proteobacteria
Bacteroidetes, Clostridium clusters IV and XIVa
16
6
nd
pyrosequenci ng (Roche 454)
Proteobacteria
Bacteroidetes
14
* - only in 16 individuals the microbiota was studied ** - results given only for vancomycin treated patients nd - not defined; CD – C. difficile; CDI – C. difficile infection
Table 2. Overview of bacterial phylotypes reported to change in different studies on gut microbiota and C. difficile Phylum
Family
Genus
Species
Actinobacteria Bifidobacteriaceae Bifidobacteri Bifidobacterium um longum (A) Bacteroidetes
Bacteroidaceae
Bacteroides
/
Prevotellaceae
Prevotella
/
Microbiota modifications (compared to healthy control subjects) increased when symptomatic (A; phylum level); decreased when asymptomatic, symptomatic and/or (FT) recurrent increased when asymptomatic; decreased when symptomatic and/or recurrent decreased when symptomatic
Firmicutes
increased when symptomatic; Clostridium decreased when symptomatic and/or (FT) recurrent (B; coccoides group, Clostridium leptum group level) group (B)
Clostridiaceae
Clostridium
Enterococcaceae (C)
Clostridium clusters IV and XIVa (B) Enterococcu / s
Eubacteriaceae
/
/
Lachnospiraceae
/
/
Lactobacillaceae
Lactobacillu Lactobacillus s plantarum (D)
increased when symptomatic and/or recurrent; decreased when symptomatic (D; species level)
Peptostreptococca ceae Ruminococcaceae (E)
/
increased when symptomatic
Streptococcaceae Veillonellaceae
/
Ruminococc us
Ruminococcus bromii (F) Ruminococcus gnavus (G) Streptococcu / s Veillonella /
increased when asymptomatic, symptomatic and/or (FT) recurrent; decreased when symptomatic (C; family level) increased when symptomatic increased when symptomatic; decreased when symptomatic and/or FT recurrent
increased when asymptomatic (G; species level) and symptomatic (F; species level); decreased when FT recurrent (E; family level) increased when symptomatic and/or FT recurrent increased when symptomatic and/or (FT) recurrent
Proteobacteria Enterobacteriacea e (H)
Klebsiella
Klebsiella pneumoniae
increased when asymptomatic; decreased when symptomatic (H; family level)
Verrucomicrob / ia
/
/
increased when recurrent