Genus
Clostridiodes, which is equivalent to
C. difficile since it only contains that species, presented the same relative abundance in infected and colonized subjects compared to non-presence, as expected in healthy controls. Therefore, the load of
C. difficile did not differentiate the state of colonization from that of infection in this study. Furthermore, the results show that the positivity of
tcdB gene also does not distinguish between colonization and infection, since 53% of the group of colonized individuals presented toxigenic strains (
Table 21). This is in line with other works in this regard
[28][29]. In addition, it has also been seen that the toxin load of
tcdB gene (colonized and infected subjects with high load
tcdB gene
versus low high
tcdB gene) did not distinguish between colonization and infection
[27][28]. Data of this study point to importance of evaluating virulence factors of
C. difficile in the future, together with studies of
16S rDNA gene and metagenomic studies. That is why factors concerning
C. difficile should be considered in the differentiation between infection and colonization. For example,
tcdA and
tcdB genes are located at
PaLoc or locus of pathogenicity. Interestingly, changes can occur in coding region of
PaLoc as insertions, deletions, and point mutations that make up genetic heterogeneity, giving rise to several different toxinotypes. This means that there could be strains with different activity and specificity of their toxins with respect to the reference strain of
C. difficile VPI 10463
[29][30]. Therefore, toxinotypes are important because they show functional properties of the
C. difficile toxin variants, that is, greater or lesser activity and greater or less production. However, the correlation between the different toxinotypes that allows us to discriminate infection from colonization, as well as the severity of CDI, has not been clarified.
Table 2. Clinical and demographic characteristics of the study groups.
Clinical and Demographic Characteristics |
Group CDI |
Group P |
Group CTRL |
Sex, number (%) |
|
Men |
4 (26%) |
10 (66%) |
7 (46%) |
Women |
11 (74%) |
5 (34%) |
8 (54%) |
Age (mean ± SD) |
69 ± 19 |
51 ± 26 |
44 ± 12 |
Antibiotics last three months, number (%) |
14 (93%) |
8 (53%) |
- |
Cephalosporins |
5 (33%) |
2 (13%) |
- |
Fluorquinolones |
4 (27%) |
2 (13%) |
- |
Β-Lactamics |
5 (33%) |
3 (20%) |
- |
Others |
5 (33%) |
4 (27%) |
- |
Without antibiotics |
1 (7%) |
2 (13%) |
- |
Unknown |
0 (0%) |
4 (27%) |
- |
Strain Type, number (%) |
|
Toxigenic |
15 (100%) |
8 (53%) |
- |
Non-toxigenic |
0 (0%) |
7 (47%) |
- |
Comorbidities |
|
Hepatic disease |
1 (7%) |
0 (0%) |
- |
Crohn’s disease |
1 (7%) |
0 (0%) |
- |
Malignant blood disease |
2 (13%) |
0 (0%) |
- |
Other intestinal disease |
3 (20%) |
0 (0%) |
- |
Other comorbidity |
13 (87%) |
6 (40%) |
- |
Previous CD, number (%) |
2 (13%) |
0 (0%) |
- |
Origin, number (%) |
|
Hospital |
7 (47%) |
2 (13%) |
- |
Community |
8 (53%) |
13 (87%) |
- |
Resolution, number (%) |
|
Complete |
11 (73%) |
- |
- |
Exitus letalis |
3 (20%) |
- |
- |
Recurrence |
1 (7%) |
- |
- |
Clinical and demographic characteristics of the study groups. Group CDI: Subjects infected by C. difficile, Group P: Subjects colonized by C. difficile, Group CTRL: Healthy controls. SD: Standard deviation.
The increase of
Bacteroides (and of phylum Bacteroidetes) in infected and colonized subjects could be surprising from the point of view that it is not a finding typically presented in previous studies, wherein decreases of this genus are normally observed
[9][18][9,19]. However, some important studies have shown an increase in
Bacteroides in patients with CDI compared to healthy controls
[13]. Through murine models, an ability to mitigate CDI has been evidenced in some species
[30][31] and it has an immunomodulatory activity in intestinal inflammatory processes that could limit the exacerbated immune response observed in patients with CDI
[31][32]. On the other hand,
Bacteroides has an unusual ability to recognize and metabolize a large quantity of polysaccharides from the diet and from the host itself. However, since competition for nutrients between members of gut microbiota is greater when groups of bacteria are more phylogenetically related
[32][33], it would imply that the depletion of genus
Bacteroides evidenced in patients with CDI and colonized subjects by many authors
[9][18][9,19], although not observed in this study, could have less a priori influence on the loss of resistance to colonization of intestinal pathogens by nutrient competition mechanisms, since this would be more accused with loss of members of the phylum Firmicutes, especially of Clostridia class, which is phylogenetically closer to
C. difficile.
An increase in Enterobacteriaceae at the expense of
Escherichia-Shigella is another common finding in patients with CDI
[14]. In this study, we also observed an increase in colonized subjects. This result has also been evidenced in recent studies
[18][27][19,28]. We assume that the increase in
Escherichia-Shigella occurs at the expense of
Escherichia coli species in the majority, as has been evidenced by metagenomic studies
[33][34]. There are different
E. coli-producing diarrhea species such as enteropathogenic
E. coli, enterotoxigenic
E. coli, enteroinvasive
E. coli, enteroaggregative
E. coli, Shiga toxin-producing
E. coli, verocytotoxigenic
E. coli, and diffusely adherent
E. coli [34][35]. Therefore, since similar pronounced increases of
Escherichia-Shigella were found in patients with CDI and colonized subjects in this study, where
E. coli would probably be a majority component of this cluster and knowing that there are several strains of enteropathogenic
E. coli, it would be interesting to know the presence of these strains in both study groups. That is why we formulate the new hypothesis that there is a colonization of pathogenic strains in the CDI group that could enhance the effects of
C. difficile toxins. On the other hand, this colonization would not occur in colonized subjects. This fact could partly explain the different clinical expressions in infection with respect to colonization of
C. difficile.
The practical eradication of
Bifidobacterium in infected subjects and the greater conservation in colonized subjects that we found in this study is an important point that differentiates the state of colonization and infection. Few microbiota studies have valued the importance of
Bifidobacterium and its potential protective role in CDI. This genus has been seen to decrease in patients with CDI
[14], and inconclusive results have been found in colonized subjects
[18][27][19,28].
Bifidobacterium is a beneficial genus of antimicrobial and anti-inflammatory properties
[35][36] whose decrease has been correlated with intestinal pathogen overgrowth
[36][37], in vivo and in vitro inhibition of growth of
C. difficile and reduced production
[37][38] and neutralization of its toxins
[38][39], and decreased tissue damage and mortality in infected mice
[39][40]. We consider that a genus of beneficial properties of
Bifidobacterium is eradicated in CDI patients and preserved in colonized subjects, an important result. This implies better control of
C. difficile in colonized subjects. To our knowledge, it is the first time that this conclusion has been reached in microbiota studies.
The increase of
Akkermansia in infected subjects and decrease in colonized subjects with respect to healthy controls is of special importance. The genus
Akkermansia contains the species
Akkermansia muciniphila (the only species isolated in humans) that has a highly effective capacity to ferment the mucin of the intestinal mucosa layer
[40][41]. Regarding CDI, only two relatively recent studies have emphasized the importance that it could present in its pathogenesis. Sangster et al. observed an increase in
A. muciniphila in 12 patients with CDI compared to 12 healthy controls. The authors highlighted that due to the ability of
A. muciniphila to degrade mucin, and since
C. difficile by itself is also capable of degrading mucin, it would provide it with a selective advantage of expanding, since it is able to adhere to a layer altered mucosa with better efficacy than other members of gut microbiota
[26][27]. Another work that was published on the same date also evidenced an increase in
A. muciniphila of 3.6% in patients with CDI, compared to 0.6% that was observed in subjects who did not receive antibiotic treatment. These authors pointed out that, although
A. muciniphila has beneficial properties, its expansion in patients with CDI could be related to the modification of the intestinal microenvironment and could reflect the inflammation of the mucosa layer
[14]. For first time, our results show an increase of
A. muciniphila in patients with CDI and a decrease in colonized subjects. Since one of functions of the intestinal mucosa layer is protection against intestinal pathogens, an alteration in the integrity means that it is more permeable and allows greater access to the epithelium and this fact could generate inflammation. On the other hand, intestinal mucosa layer is also a potential source of nutrients for intestinal pathogens. This fact is evident in antibiotic treatment, since it disturbs gut microbiota and the availability of fucose and sialic acids in mucin, which facilitates expansion of
C. difficile [41][42]. Therefore, the increase in patients with CDI reflects a greater degradation of intestinal mucosa and the decrease in colonized subjects would show a greater integrity. This finding differentiates the state of colonization from that of infection and would imply a greater control of
C. difficile in colonized subjects.
We consider that the present study has two main limitations. The first limitation is the sample size. Each study group is made up of 15 subjects. This sample size has allowed us to find statistically significant differences in terms of diversity, richness, and composition in infected and colonized subjects with respect to healthy controls. However, the differences in the composition of colonized subjects with respect to those infected are in many cases not statistically significant. We think that with a larger sample size the differences discussed would have greater statistical significance due to the low statistical power of this study. The main obstacle to increasing the sample size has been the inclusion of colonized subjects by
C. difficile, since they have been difficult to include and locate. The second limitation that we consider for interpretation of the results is diet. Diet is a factor that modulates the composition of gut microbiota, and in this study, no variables have been collected in this regard. We also think that this could have a greater influence on the gut microbiota of healthy controls.
3. Conclusions
On the basis of the main objective of the study, which was the comparison of gut microbiota of patients with CDI and colonized subjects by C. difficile with respect to a group of healthy controls, we can conclude that infected and colonized subjects present a gut microbiota with a diversity, richness, structure, and composition completely different from that of healthy controls. However, gut microbiota of infected and colonized subjects showed great similarities in terms of diversity, richness, and structure. It is in composition where we find that colonized subjects, especially in minority genera, present differences with respect to those infected. This fact explains, at least in part, the state of colonization by C. difficile.