**5. New Therapeutic Strategies**

With this close link between the microbiome and the onset of *Clostridium difficile* infection, several lines of research are currently underway aimed at developing new therapeutic strategies (Table 4).

Despite all this new evidence, there is currently still not full unanimity on defining the microbial species associated with asymptomatic colonization versus those related to the onset of CDI, which remains one of the current challenges to be addressed [47,63].

One of the proposed strategies is to be able to specifically identify which types of microbiota alterations are most at risk for the onset of *CDI* [38].

This field is particularly promising due to the fact that we are increasingly witnessing significant technological development, with reduced sequencing costs.

In this case, it is the evolution of science in a broad sense, understood as the development of new technologies that are increasingly within the reach of researchers that could also prove decisive in this specific area of study.

Other authors, on the other hand, suggest how diet may influence the composition of the microbiota and consequently its interaction with various pathological conditions, including CDI, thus looking for a correlation between diet and the development of this disease [64].

This aspect also appears to be much studied for other diseases such as metabolic syndrome and chronic inflammatory bowel disease, less so for CDI.

From the pathophysiological point of view, the connection between diet and the microbiome is very clear.

However, the occurrence of *CDI*, as we have seen, also calls into question several other factors, and that is why it makes this field of study very difficult, albeit very promising.

Meanwhile, new therapeutic frontiers make use of probiotics and prebiotics.

Probiotics are "Live microorganisms that, when administered in adequate amounts, confer a health benefit upon the host" [65], while prebiotics are "A selectively fermented ingredient that allows specific changes, both in the composition and/or activity in the gastrointestinal microflora that confers benefits upon host well-bring and health" [66].

Meanwhile, "synbiotic" means the administration of a prebiotic together with a specific probiotic to enhance the engraftment and development of that specific microbe [67].

A study was recently conducted in which treatment with live purified Firmicutes bacterial spores was successfully proposed for patients with recurrent CDI [68].

An increase in the concentration of secondary bile acids compared to primary bile acids is one of the factors inhibiting the germination of CD spores.

For this very reason, spore-forming *Firmicutes* bacteria were grafted with the specific purpose of increasing the concentration of secondary bile acids to thus inhibit spore germination and subsequent CD growth [68].

Here, we have an important finding of how an accurate understanding of this complex individual–host interaction can provide us with important new therapeutic weapons.

Some of the latest research involves in vitro experiments and makes use of synbiotics, bacterial secreted compounds that inhibit the activity of *C. difficile* toxins [67].

These research studies, though currently relegated to the early stages, appear to be very promising.

Live recombinant biotherapeutic products (LBPs) are defined as microorganisms that have been genetically modified by the targeted addition, deletion, or modification of genetic material [69].

Several clinical trials on CDI are currently underway: one is biotherapeutic, whose potential use in CDI relapses is being studied [70], while another one involves the use of Gram-positive selective-spectrum antimicrobials [71] for *CDI* patients.

Again, efforts are being made to counteract this disease by looking at the factors that regulate host immunity.

Other researchers have focused on studying the interaction between the onset of CDI and the presence of valerate, a short-chain fatty acid produced by amino acid fermentation in the microbiota.

Valerate appears to be one of the factors that inhibit the growth of *C. difficile*; in fact, its levels increase after fecal microbiota transplantation [72].

Some of the most surprising research involves the use of bacteriophages.

Bacteriophages are viruses that can go to specific targets, going on to infect and replicate within the designated host [69].

Their potential and undisputed strength would be to go against *C. difficile* without the use of antibiotics [3,69].

There has also been speculation that this particular therapeutic weapon could be used against *C. difficile*, but without obtaining important evidence at present.

A very promising paper was recently published, in which ADS024, a newly characterized strain of *Bacillus velezensis*, was identified that appears to have strong activity against *C. difficile* with negligible impact on the rest of the bacterial flora, and also having protease activity directed against TcdA and TcdB toxins which we have seen to be responsible for the clinical manifestations of this disease [73].

We fully share the enthusiasm shown by Khanna [74] in a recent article in which he summarized the four ongoing trials of capsule therapies (CP101, RBX7455, SER-109, VE303) plus the one based on the use of enema (RBX2660), again related to remodeling the microbiota to cope with *CDI*.

In conclusion, we also point out an excellent paper by Rusha et al. [75] who expounded an excellent summary of probiotic strains used in human clinical trials to treat CDAD.

**Table 4.** New strategies for the prevention of *Clostridium difficile* infection through its interaction with the gut microbiota.


#### **Table 4.** *Cont.*


#### **6. Conclusions**

In this article, we carefully explored aspects concerning the complex interaction between *C. difficile* and gut microbiota.

The microbiome is a complex system whose fundamental importance we understand more and more, both for the health and disease of the individual.

It will still take a long time to fully understand it, for multiple reasons, including its extreme variability in different individuals and its involvement in so many different functions.

*C. difficile* infection is one of the great public health challenges.

It is one of the most frequent infections, where the role of its interaction with the microbiota is crucial.

From these assumptions, several therapeutic strategies to deal with CDI have arisen and are being studied.

The most intuitive one is to go after the microorganisms themselves that cause this disease, as is currently done with the use of antibiotics, or by experimenting with new and different innovative methods, such as bacteriophages.

The other strategy, on the other hand, is to work on the substrate that promotes the pathogenesis of *CDI*.

In this regard, we mentioned fecal microbiota transplantation and all the other numerous therapeutic strategies that are still being researched or tested.

We also saw how it is not enough to eliminate the bacteria that cause CDI since in the absence of a protective substrate from the microbiome, the risk of recurrence is extremely high.

It is precisely in order to prevent these recurrences that the use of fecal transplantation, one of the latest therapeutic frontiers involving the gut microbiota, is being employed.

As we discussed extensively, research on these issues is more alive than ever, and we hope it will continue to be so that we can have new therapeutic weapons against this infection.

**Author Contributions:** Conceptualization, A.P. and M.C. (Marcello Candelli); methodology, A.P. and F.R.; software, F.M. and C.Z.; validation, A.G., F.F. and V.O.; formal analysis, A.P. and G.S.; investigation, F.R., F.M. and G.P.; resources, G.P.; data curation, F.M., C.Z. and G.S.; writing original draft preparation, A.P., F.R. and F.M.; writing—review and editing, M.C. (Marcello Covino), F.F. and A.G.; visualization, M.C. (Marcello Covino) and V.O.; supervision, A.G., F.F. and M.C. (Marcello Candelli). All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki. Ethical review and approval were waived for this study because it is a review.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.
