Clostridioides difficile infection (CDI) is one of the leading causes of healthcare-associated infections, presenting high morbidity and mortality rates [1,2]. Antibiotic use is the major risk factor for developing CDI, in addition to ageing, presence of comorbidities and immunosuppression [2,3]. The etiologic agent, C. difficile, is an obligate anaerobic, gram positive bacillus that inhabits the intestinal microbiota of about 4–15 % healthy adults [3,4]. This species is well known for its capacity to form spores that are extremely resilient against antimicrobial substances and hostile environments for extended periods, favoring its transmission within healthcare facilities [4]. Once ingested, the highly resistant spores gain entry into the intestinal mucosa, where germination is triggered by environmental factors such as bile salts and amino acids. Colonization is further hampered by the normal microbiota and the immune system defense mechanisms [4,5]. The primary virulence factors of C. difficile include toxin A (TcdA), toxin B (TcdB), and a binary toxin called C. difficile transferase (CDT) [6]. In a scenario of gut dysbiosis induced by broad-spectrum antibiotics, the uncontrolled colonization and proliferation of C. difficile will result in toxin production, biofilm formation, and sporulation, with damage of the intestinal mucosa and disease manifestation [5].

The oral cavity is the main gateway of C. difficile entry to the lower gastrointestinal tract. The complex oral ecosystem provides a wide variety of colonizing niches, harboring an incredibly diverse microbiota that frequently resides within multispecies biofilms [7,8]. Dysbiosis of the dental biofilm is the primary cause of the main oral diseases in humans, caries and periodontal diseases [7]. In particular, periodontitis is a destructive form of inflammatory disease, characterized by loss of periodontal attachment and alveolar bone that support the teeth [9]. The periodontal lesion foster a rich multispecies biofilm, comprising mainly gram-negative anaerobic species [8]. Evidence has shown that the periodontitis-associated biofilm may also harbor numerous pathogens of medical relevance, including multidrug resistant enterococci, staphylococci, Helicobacter pylori and species of the families Pseudomonadales and Enterobacterales [10]. In this context, the anaerobic dysbiotic subgingival biofilm could also provide a niche for colonization by C. difficile. This pathogen was reclassified into the Peptostreptococcaceae family [11], which comprises taxa that are highly abundant in the subgingival microbiota of patients with periodontal diseases [12]. Conceivably, persistence of C. difficile vegetative cells and/or spores within the subgingival biofilm could limit the efficacy of CDI treatment with antimicrobials, becoming the periodontal lesion a potential source of oral-fecal reinfection through saliva. In previous studies, we have found C. difficile in the biofilm of advanced forms of periodontitis by using whole genomic DNA probes and the checkerboard method [13,14]. However, cross-reaction with close-related species may occur when using this methodology. In the current study, we used a specific multiplex PCR for C. difficile genes to screen a large number of oral and fecal samples from systemically healthy individuals with different periodontal conditions. We aimed to search for relevant correlations between the presence of these specific genes and periodontal disease severity.