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

## TL;DR

This study analyzed C. difficile isolates from Bulgarian patients over ten years, finding a significant rise in hypervirulent ST1 clones during the pandemic, likely due to increased antibiotic use.

## Contribution

The study identifies a 6-fold increase in hypervirulent ST1 clones during 2020–2023, linking it to antibiotic overuse in Bulgaria.

## Key findings

- 34% of isolates were ST1, the most prevalent sequence type, with a notable increase during the pandemic.
- Binary toxin-positive isolates showed 18 bp deletions in tcdC, except one ST11 isolate with a 39 bp deletion.
- Four main clonal complexes were identified, with ST1 isolates linked to increased virulence and antibiotic overuse.

## Abstract

The aim of the study was to investigate the MLST types and genes encoding Clostridioides difficile 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 cdt-negative isolates were of two clades: clade 4 (ST37), with 1 tcdA-tcdB+ isolate, and clade 1, with 64 isolates. All the cdt-positive isolates showed 18 bp deletions of tcdC, 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 cdt-negative isolates were ST3 (14%), ST2 (6%), ST42 (5%), and ST92 (5%). They were tcdA+tcdB+. 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.