Molecular Epidemiology of Toxigenic Clostridioides difficile Isolated from Bulgarian Patients and the Prevalence of Hypervirulent ST1 Clone

The aim of the study was to investigate the MLST types and genes encoding <i>Clostridioides difficile</i> toxins from fecal clinical samples of patients from two large Bulgarian cities. Overall, 100 toxigenic isolates were obtained during a 10-year period from five hospitals in Sofia and Varna, Bulgaria. Toxin gene patterns of the isolates were determined with conventional polymerase chain reaction, and multilocus sequence typing (MLST) types were determined according to Griffith's scheme. The evolutionary relatedness of ST types was analyzed through PHYLOViZ. Binary toxin-positive isolates accounted for 35% (35/100), and the most prevalent sequence type (ST) type was ST1 (clade2) with 34%. One binary toxin-positive isolate belonged to ST11 (clade5). The <i>cdt-</i>negative isolates were of two clades: clade 4 (ST37), with 1 <i>tcdA</i>-<i>tcdB</i>+ isolate, and clade 1, with 64 isolates. All the <i>cdt-</i>positive isolates showed 18 bp deletions of <i>tcd</i>C, except a ST11 isolate exhibiting a 39 bp deletion. Importantly, there was more than a 6-fold increase in ST1 isolates in the 2020-2023 period versus 2014-2019. The main <i>cdt-</i>negative isolates were ST3 (14%), ST2 (6%), ST42 (5%), and ST92 (5%). They were <i>tcdA</i>+<i>tcdB</i>+. Four main clonal complexes (with one allele difference) were found. The first one encompassed 19 isolates and included ST3 and ST42; the second included ST8, ST16, ST52, ST92 and ST36 (11 isolates); the third consisted of 13 isolates (ST2, ST49, ST13 and ST110 clones); and the fourth included ST10 and ST44 (5 isolates). The significance of our work is the detection of a high frequency of hypervirulent isolates during the COVID-19 pandemic period, which we attribute to the national consumption of systemic antibiotics, which increased during the second period, unlike the trend in other European countries. The results highlight the need for enhanced infection control measures and strict compliance with antibiotic stewardship programs.