Clostridioides (formerly Clostridium) difficile is a Gram-positive, obligate anaerobe, spore-forming bacteria, harboring a plethora of surface and secreted proteins responsible for the colonic colonization and subsequent inflammation characteristic for C. difficile infection (CDI), among which the most important are the clostridial toxins: toxin A (TcdA) and toxin B (TcdB), and in some bacterial strains, the binary toxin CDT (Smits et al., 2016). Clinical symptoms range from mild diarrhea to fulminant colitis, known as pseudomembranous colitis, with its complications – toxic megacolon and large bowel perforation (McDonald et al., 2018; Dieterle et al., 2019). C. difficile is the number one causative agent of nosocomial post-antibiotic colitis, associated with high morbidity and mortality (Ghose, 2013). In the last decade, both frequency and severity of CDI have increased, largely due to the emergence of a hypervirulent strain called NAP1 (North American pulsed-field gel electrophoresis type 1 strain) (Depestel and Aronoff, 2013). Moreover, in the last two decades, there has been a significant increase in the incidence of CDI in previously considered low-risk population categories, including community-associated-Clostridioides difficile infections (CA-CDI), with more than 30% of cases not showing typical CDI risk factors, such as antibiotic treatment or recent hospitalization (Wilcox et al., 2008; Hensgens et al., 2011). Although CDI is the preserve of the elderly population, it can also affect other age segments. In a retrospective survey performed by U.S. National Hospital Discharge Surveys from 2001 to 2010 revealed that CDI incidence was highest among elderly adults (11.6 CDI discharges/1,000 total discharges), followed by adults (3.5 CDI discharges/1,000 total discharges) and pediatrics (<12 years) (1.2 CDI discharges/1,000 total discharges). The mortality rates attributable to CDI in the elderly were significantly higher (8.8%) compared to adults (3.1%) and pediatrics (1.4%) (Pechal et al., 2016). Although there is not much information available about the epidemiology of infection in infants, however, the carriage rate of non-toxigenic C. difficile is very high in newborns, suggesting the commensal status of this bacterium in this population segment (Sammons et al., 2013; Borali and De Giacomo, 2016). For this reason, C. difficile is not considered an enteric pathogen in infants and children affected by bloody diarrhea and younger than 12 months (Cama et al., 2019). However, asymptomatic infants could be also infected by toxigenic adult infectious strains both after hospitalization and even in the community, thus, constituting a reservoir for toxigenic strains (Rousseau et al., 2011; Ferraris et al., 2020).

This review aims to present some particular aspects of gut microbiota in CDI infants and adults, taking into account that the pathophysiology of this disease suggests that the clinical manifestations occur in cases of an imbalance of the intestinal microbiota, known as dysbiosis. In this purpose, studies performing culture-dependent (C. difficile cultivation) and independent (16S rRNA and metagenomics) have been analyzed.

Some of the underlying causes of intestinal dysbiosis are antibiotic treatments (Kukla et al., 2020), advanced age (over 65 years), hospitalization (particularly in patients sharing the hospital room with an infected patient, in intensive care units, during prolonged hospitalization), nursing home stay, severe associated diseases, immunological suppression, gastric acidity suppression by proton pump inhibitors or histamine₂-receptor antagonists and prolonged use of elemental diet in the context of enteral nutrition, inflammatory bowel diseases, gastrointestinal surgery (in particular colectomy, small-bowel resection, and gastric resection were associated with the highest risk while patients undergoing cholecystectomy and appendectomy had the lowest risk), all of these circumstances being associated with characteristic changes in the configuration of the gut microbiota and with an increased CDI risk (O’Keefe, 2010; Fashner et al., 2011; Nitzan et al., 2013; Rodriguez et al., 2016; Sartelli et al., 2019; Avni et al., 2020). A strong argument regarding the impact of dysbiosis on CDI risk is offered by animal studies proving that correcting dysbiosis by administration of different substances such as phytophenolic compounds or carvacrol has been shown to decrease susceptibility to CDI. The gut dysbiosis of 6-week-old C57BL/6 mice was induced by the oral administration of an antibiotic cocktail in water simultaneously with the intra-peritoneal injection of clindamycin. The mice were infected with 10⁵ CFU/ml of hypervirulent C. difficile ATCC 1870 spores. Carvacrol supplementation significantly reduced the incidence of diarrhea and improved mice’s clinical and diarrhea scores. Microbiome analysis revealed that carvacrol increased the abundance of Bacteroidetes and Firmicutes. An increased abundance of Lactobacillaceae and Lachnospiraceae was noticed among the beneficial taxa in carvacrol treated mice. Also, carvacrol decreased the proportion of pro-inflammatory microbiota, such as Proteobacteria (i.e., Enterobacteriaceae) and Verrucomicrobia, without significantly affecting the gut microbiome diversity compared to the control (Mooyottu et al., 2017).

Five clinical features (potential risk factors) predict dysbiosis in CDI patients: antibiotic use within the previous 3 weeks, immunosuppression, multimorbidity, recent/multiple hospitalization, and prior CDI (Battaglioli et al., 2018). In individuals whose normal intestinal microbiota has been disrupted, ingested C. difficile spores germinate in the presence of bile salts in the small intestine and colonize the colon epithelial cells, releasing the inflammatory enterotoxins, which are primarily and largely responsible for the colonic inflammation in C. difficile diseases, inducing cytoskeletal changes, disruption of tight junctions, and induction of inflammatory cytokine production (Shen, 2012; Winston and Theriot, 2016). The C. difficile spores are released by the patient facilitating CDI transmission to susceptible hosts (Czepiel et al., 2019).

The intestinal environment represents a complex network of bacterial cells and metabolic products and/or other unknown substances derived from their own structures or metabolisms, which are in close and continuous interaction, both with each other, as well as with the human intestinal cells and the host’s immune system (Lazar et al., 2018, 2019).

The characterization of the baseline healthy microbiota and differences that are associated with various diseases has been possible with the contribution of large-scale projects, such as Meta-HIT and the Human Microbiome Project (HMP), using different omics technologies (Qin et al., 2010; Human Microbiome Project, 2012).

During early development, the gut microbiota undergoes subsequent changes until a stable adult state is reached. The adult microbiota has three basic characteristics: diversity (a high microbiota diversity defined by high species richness and high functional diversity being generally associated with the health condition), resilience (the property of the gut microbiota to resist to an impact and to recover and to baseline after the disturbance cessation; the capacity of a microbial community to reach a stable state in response to chemical, physical or biological perturbations of different intensities is achieved through genetically diverse resident clonal populations and population-level dynamics) and long-term stability of high taxonomic level components (Levine and d’Antonio, 1999; Lozupone et al., 2012; McBurney et al., 2019; Priya and Blekhman, 2019; Dogra et al., 2020).

Regarding the diversity, human microbiota displays a remarkable heterogeneity within and between individuals, the results of the culture-independent studies leading to the generally accepted idea that we rather share a functional core microbiome, than a core microbiota (Lozupone et al., 2012). The >1,000 estimated species-level phylotypes are belonging to few microbial phyla, which are Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, Fusobacteria, and Verrucomicrobia; among these, the two Firmicutes and Bacteroidetes phyla are representing 90% of gut microbiota (Magne et al., 2020). Firmicutes comprises more than 200 different genera, such as Lactobacillus, Bacillus, Clostridium, Enterococcus, and Ruminococcus (Kachrimanidou and Tsintarakis, 2020). Clostridium genera represent 95% of the Firmicutes, while Bacteroidetes include as predominant genera Bacteroidetes and Prevotella (Rinninella et al., 2019). Actinobacteria are proportionally less abundant and mainly represented by Bifidobacterium species (Rinninella et al., 2019). From the mentioned components, Ruminococcus and Bifidobacterium have been reported to exhibit protective roles against CDI (Seekatz and Young, 2014; Kho and Lal, 2018).

The gut microbiota community shows resilience to different perturbations, such as those induced by different diets, antibiotic administration, invasion by new species (called colonization resistance) (Folke et al., 2004). Under the impact of a certain disturbances, such as antibiotic administration, microbiota enters an unstable state that progresses to a new stable state. When the latter is highly similar to the pre-disturbance state, this indicates a complete recovery. However, sometimes the post-disturbance stable state is distinct and this unfortunately can be both abnormal and resilient, as a response to the perturbation persistence (e.g., poor diet, antibiotic treatments etc) (Dupont, 2011). An example of gut microbiota resilience is the success of bacteriotherapy or microbiota transplantation in treating recurrent CDI. In this case, the gut microbiota switches from an initial dysbiosis state, favoring the CDI (e.g., increased abundance of Veillonella and Streptococcus) to a baseline state, in which the taxa from the healthy donor (e.g., Bacteroidetes) persisted 1 month after transplantation (Gough et al., 2011). A high diversity (high species richness, α-diversity) and host immune effectors, e.g., nucleotide-binding oligomerization domain (Nod2), an intracellular innate immune sensor involved in the anti-infectious host defense, were linked to gut microbiota resilience (Tap et al., 2015; Raymond et al., 2016; Goethel et al., 2019). A low microbiota diversity was correlated with recurrent CDI, but with unknown effects on microbiota resilience (Chang et al., 2008; Khoruts et al., 2010). However, a comparable phylum-level diversity was observed in individuals with initial CDI and healthy controls, while in case of recurrent CDI the phylum-level diversity switched to different highly divergent profiles very different from the healthy state other (Chang et al., 2008).

The stability of gut microbiota is affected by different factors, including genetic factors, early-life events, travel, dietary changes, weight loss or gain, diarrheal disease, antibiotics, immunosuppressants, premeditated interventions to influence the microbiota by administration of prebiotics, probiotics, postbiotics and symbiotics, as well as fecal transplantation (Faith et al., 2013; Britton and Young, 2014).

During childhood, the intestinal microbiota is subject to many factors that shape the microbiota on short- and long-term. Apart from the maternal-fetal transmission of certain bacterial components, the microbiota is influenced by the type and the time of birth, the place of birth (hospitals or home births), the type of feeding (breastfeeding or artificial feeding), administration of probiotic and prebiotic supplements, dietary factors, antibiotics and other drugs, sex, and other genetic differences and environmental factors, such as exposure to pets, number of family members, rural or urban environment, hygiene, geographical factors (Combellick et al., 2018; Mohammadkhah et al., 2018; Akagawa et al., 2019).

Taking into account the numerous factors that could influence the intestinal microbiota of the newborns, the healthy profile of this age group is considered to be represented by the types of gut bacteria and the abundances found in vaginally delivered, exclusively breastfed and not exposed to antibiotics neonates (Arboleya et al., 2015; Combellick et al., 2018; Mohammadkhah et al., 2018; Akagawa et al., 2019). It has been hypothesized that bacterial colonization of the digestive tract begins in utero, the healthy placenta bearing a low biomass microbiome, composed of non-pathogenic species belonging to the Tenericutes, Firmicutes, Bacteroidetes, Proteobacteria, and Fusobacteria phyla, while in the amniotic fluid predominate Proteobacteria. However, the results reported by different studies are contradictory and depend on the sampling method and the culture-based or molecular-based approaches. Recent metagenomic studies conclude that the placenta does not harbor a specific, consistent and functional microbiota (Gschwind et al., 2020). The meconium harbors a cultivable microbiota, initially dominated by Bacteroides-Prevotella. The digestive contamination of the fetus occurs most probably by swallowing the amniotic fluid, starting with the 10th week after conception (Chong et al., 2018). The transfer of Enterococcus faecium from pregnant female mice into meconium was also demonstrated experimentally (Jimenez et al., 2008). All these lead to the conclusion that changes in the maternal internal environment may affect both fetal and newborn development (Zhuang et al., 2019).

The first step in C. difficile treatment is the de-escalation of antibiotic treatment. Depending on the degree of C. difficile risk induction, the antibiotics were divided into three groups: high (fluoroquinolones, 2nd and 3rd generation cephalosporins, clindamycin, ampicillin, broad-spectrum penicillins with inhibitors, except for ticarcillin with clavulanate, and piperacillin with tazobactam), moderate (macrolides, trimethoprim/sulfamethoxazole, other penicillins, and sulfonamides) and low risk (aminoglycosides, bacitracin, carbapenems, chloramphenicol, daptomycin, metronidazole, rifampicin, teicoplanin, tigecycline, tetracycline, and vancomycin) (Kukla et al., 2020). Current standard treatment for CDI involves treatment with antibiotics such as metronidazole, vancomycin, or fidaxomicin (Mills et al., 2018; Gnocchi et al., 2020). Vancomycin is the first-line antibiotic therapy for both first episode of infection and fulminant infections in adults (Esposito et al., 2015; Wang et al., 2020). Unfortunately, vancomycin is a strong disruptor of gut microbiota, while the rate of CDI recurrence after treatment cessation occurs in 20–30% of patients (Lessa et al., 2015; Winston and Theriot, 2016). On the other hand, metronidazole is used especially in the first episodes of mild acute CDI and less for severe disease because the concentrations in the colon become readily undetectable due to the fact that it is absorbed very quickly (Gnocchi et al., 2020). The rapid absorption from the gut is also reflected in a negligible effect on normal microbiota (Lessa et al., 2015; Chilton et al., 2018). Fidaxomicin can be used in C. difficile non-severe and also severe infections treatment due to the fact that it is poorly absorbed at the intestinal level, ensuring the persistence of killing concentration in the gut and has a narrow antimicrobial spectrum (e.g., Gram-positive and Gram-negative anaerobes and facultative aerobes) unaffecting the equilibrium of the normal intestinal microbiota (Tannock et al., 2010; Louie et al., 2011, 2012; Gnocchi et al., 2020). Moreover, in another study, vancomycin and metronidazole treatment, but not fidaxomicin were associated with the potentially pathogenic fungal operational taxonomic units’ emergence as well as with bacterial functions enriched for xenobiotic metabolism that could contribute to dysbiosis that could favor the occurrence, persistence and recurrence of CDI (Lamendella et al., 2018).

Adjunctive therapies are frequently used due to the important role of gut microbiota disturbances in C. difficile pathogenesis (Table 1 and Figure 1). Thus, specific manipulation of the microbiota to ameliorate dysbiotic changes and restore intestinal microbiota homeostasis could represent an essential part of the therapy (Table 2).

One way to modulate microbiota is using “biotics” with beneficial impact on resident microbiota that confer health benefits for the host, such as probiotics (e.g., Saccharomyces boulardii, Lactobacillus, Bifidobacterium, and probiotic mixtures), prebiotics, symbiotics or postbiotics (Surawicz et al., 2000; Na and Kelly, 2011; Maziade et al., 2015; Collins and Auchtung, 2017). According to International Scientific Association for Probiotics and Prebiotics (ISAPP)¹, prebiotics are substrates different from fibers, that are selectively metabolized by host microorganisms. Synbiotics are complementary/synergistic mixtures comprising live microorganisms (probiotics) and prebiotics. In case of synergistics synbiotics the two components taken individually do not have to meet criteria for prebiotic or probiotic. Postbiotics are cellular fractions or structures prepared from inactivated microbes. The postbiotic preparations exclude filtrates or live cultures individual components, while inactivated probiotics are not considered automatically postbiotics, unless a health benefit is demonstrated.

Most data regarding the protective role of commensal bacteria against C. difficile infection were obtained by studying the effects of different probiotics. For example, Bifidobacterium breve (YH68), widely used in the field of food fermentation and biomedicine, has shown antibacterial activity against C. difficile, by inhibiting the growth, spore production, toxigenesis and virulence gene expression (Valdes-Varela et al., 2016; Yang and Yang, 2019), potentiating the effect of anti-C. difficile antibiotics in vitro (Yang and Yang, 2018) or preventing the occurrence of clinical manifestations in vivo (Yun et al., 2017). Probiotic use has been shown to decrease CDI incidence in high-risk populations by as much as 50%, especially when they are combined with prebiotics (Shen et al., 2017).

It was demonstrated that the administration of non-living bacteria or microbial components (e.g., proteins, lipids, or nucleic acids) has an immunostimulatory effect proving that the beneficial impact on the host health is due to the physical interaction of specific microbial components, but in order to be effective for a long period, these need continuous administration.

Many bacterial strains such as Bacillus clausii and Lactobacillus reuteri have been shown to secrete soluble compounds that directly inhibit C. difficile (Khalaf et al., 2012; Deng and Swanson, 2015; Antonara and Leber, 2016). Organisms that produce secondary bile acids, such as Clostridium scindens, enhance C. difficile colonization resistance (Winston and Theriot, 2016).

Fecal microbiota transplantation (FMT) is considered the most effective microbiota-targeted intervention for the treatment of antibiotic-refractory CDI (Arbel et al., 2017), but however, the long-term effects, including the risk of other diseases, are not known (Schaffler and Breitruck, 2018).

Another therapeutic approach is the administration of non-toxigenic C. difficile strains or a mixture of spore-forming commensals, which act by providing nutritional niche competition. Despite their efficiency in decreasing CDI recurrence, there is the risk of switching to the toxigenic phenotype (Brouwer et al., 2013; Khanna et al., 2016).

The available studies suggest that C. difficile colonization and infection are influenced by the presence, absence or abundance of certain bacteria in the human gut, which could generate favorable conditions for germination, proliferation and production of clostridial toxins which, on their turn, will alter the integrity of the intestinal mucosa. Therefore, the clinical manifestations and severity of CDI are linked to gut dysbiosis, that could have multiple causes, among which the administration of high-risk antibiotics. The presence of protective microbial species, together with the particularities of the immune system and lack of receptors for clostridial toxins could explain the fact that in children, despite the high carriage rate, the symptomatic and severe cases are rare. However, if there is a gut microbiota composition predisposing to C. difficile asymptomatic carriage or clinical infection still needs clarification. Also, the mechanisms involved in C. difficile crosstalk with the commensal microbiota and/or particular soluble compounds remain only partially explained. Adjunctive microbiota-targeting therapies based on probiotics, prebiotics, postbiotics, synbiotics, non-toxigenic bacteria or fecal microbiota transplantation proved to be very useful for the therapeutic management of CDI.

All authors contributed equally to this manuscript with the conception and design of the study, literature review and analysis, drafting and critical revision and editing, and final approval of the final version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.