**4.** *Clostridium difficile* **Infection**

## *4.1. Disruption of the Microbiome and Clostridium difficile Infection Risk Factors*

Clearly, the most well-known risk factor for developing *CDI* is antibiotic use, both short- and long-term because of its impact on microbiota diversity. In fact, a healthy microbial community gut is capable of interfering with *C. difficile* spores, and it does not necessarily result in disease. However, every change in the microbial environment could bring spore germination, CD growth, and toxin production [48].

Another known risk factor is increasing age. In elderly people, the structure of the microbiome undergoes changes, is less diverse, and shows a decrease in protective species such as *Bifidobacteria* and some *Firmicutes*, as well as an increase in *Bacteroidetes* and *Proteobacteria* [49].

That change also influences the immune system of an elderly person which becomes weaker and more fragile. As a matter of fact, the rate of *C. difficile* infection is higher for people aged 65. Advancing age is also associated with hospitalization, use of antibiotics, and development of different diseases [50–54].

Another risk factor is proton pump inhibitors (PPIs), which increase the gastric pH and modulate microbiota, influencing above all *Lactobacillus* [51].

Gastrointestinal pathologies such as inflammatory bowel disease (IBD) may impact *C. difficile* susceptibility. As some studies show, we observed in subjects suffering from IBD a decreased diversity of *Firmicutes* and *Bacteroidetes* and the presence of pathogenic bacteria such as the *Proteobacteria* phylum [52].

Moreover, its inflammatory products in IBD (antimicrobial peptides lipocalin-2 and calprotectin) potentially impact the growth of surrounding microbes [53].

Differently, as a protective factor, we can safely say that a good composition of microbial communities' gut and a strong immune response could be useful against CDI.

Serum IgG antibodies against toxins A and B have been associated with protection in some studies [54].

So immunization, both active and passive, could be a good strategy to study for CDI treatment [55].

Therefore, a full understanding of all these risk factors is of paramount importance for the development of new preventive strategies to avoid the occurrence of this dangerous infection.

#### *4.2. Recurrent Clostridium difficile Infection and the Incomplete Recovery of the Microbiota*

The most common complication of *CDI* is incomplete recovery and recurrent infection.

The rates are about 20.30% after an initial infection and up to 60% after three infections [56]. Some studies notice that each episode of CDI brings an increasing chance of recurrence, also due to an increase in antibiotic use and disease severity [57].

However, it is also hypothesized that antibiotic treatment interferes with the ability of the gut microbiota to recover fully and re-establish colonization resistance in some individuals. Alternatively, recurrence could reflect the failure of the host to mount a protective immune response against *C. difficile* [58].

This is how an imbalance, an acquired rupture of this delicate balance between the host and the individual, can degenerate into an almost irreparable situation.

We can safely assert that being able to prevent one of the most frequent complications, namely the recurrence of this widespread infection, is one of the great challenges of public health.
