Liberec Regional Hospital is a tertiary health care facility providing specialized care in the Liberec Region, in the north of the Czech Republic, which is inhabited by approximately 500,000 people. The hospital has about 1,200 beds, with 200 beds for follow-up care. As a nonuniversity hospital, it does not have state-funded research activities but has been listed as a research organization since 2019. The average number of patient-bed days in Liberec Regional Hospital in the years 2017–2018 was 224,039.

Samples for our study were collected over 13 months—from June 2017 till June 2018. In this period, 556 stool samples from hospitalized patients (n = 493) were sent to the microbiological laboratory of Liberec Regional Hospital. In this group, 46.9% (n = 261) of samples were from males and 53.1% (n = 295) from females. The 70–79 age group was the most represented, accounting for 32.7% (n = 182) of the sample. Internal medicine was the most represented specialty with 35.3% (n = 196). A more detailed overview is presented in Table 1. A duplicate sample, which was defined as a sample collected within 10 days of the first collection, was not included in the study.

QuikChek Complete (TechLab, USA) was used for CDI screening and basic toxinotyping C. difficile. After the CDI screening, GDH-positive samples were selected for stool cultivation. The stool samples were exposed to 96% ethanol (1 mL or a small pea-sized portion of stool in 1:1 mixture with alcohol) for 30 min. Two drops (approximately 50–75 µL) of deposit were inoculated onto Brazier’s Clostridium difficile Selective agar (Oxoid, Czechia) and spread to obtain single colonies. The inoculated plates were immediately transferred to an anaerobic environment and incubated at 37 °C for 48 h. The identification of C. difficile colonies was performed according to the characteristic color and shape of the colonies (irregular with grey to white color) and by Gram-stained microscopy and subsequent toxinotyping by PCR.

DNA was isolated from the C. difficile suspension (1–3 colonies in 500 µLµ nuclease free water) using a PathogenFree DNA Isolation Kit (GeneProof, Czechia) and used for PCR reaction focused on the genes encoding the toxins (cdtA, cdtB, tcdA, and tcdB). Mastermix was 12.5 µL Hotstart Mastermix (Qiagen, Netherlands), 5 µL primer mix (see Table 2 for more details), 5 µL nuclease free water, and 2.5 µL sample DNA. The cycling program was activation for 15 min at 94 °C, followed by 35 cycles of denaturation for 45 s at 94 °C, annealing for 45 s at 50 °C, and elongation for 1 min at 72 °C, with the final elongation step for 30 min at 72 °C. The obtained amplicons were detected by capillary electrophoresis in a MultiNa (Shimadzu, Japan) instrument.

This was based on the amplification of an intergene locus between the 16S and 23S rDNA genes. Each ribotype has a specific number of intergene locus with a different length. Amplification thus produced multiple fragments with different lengths. This fragment profile is specific for each ribotype. For ribotyping, the PCR mastermix was 25 µL Hotstar Mastermix (Qiagen, Netherlands), 0.3 µL of each primer (see Table 2 for more details), 22.4 µL nuclease free water, and 2 µL sample DNA. The cycling program was activation for 15 min at 95 °C, followed by 35 cycles of denaturation for 1 min at 94 °C, annealing for 1 min at 60 °C, and elongation for 1 min at 72 °C, with the final elongation step for 30 min at 72 °C. PCR fragments were then analyzed in an ABI310 automatic genetic analyzer (Thermo Fisher Scientific, Waltham, MA, USA) in a 50 cm capillary loaded with POP4 polymer (Thermo Fisher Scientific, Waltham, MA, USA). LIZ600 was used as the size standard (Thermo Fisher Scientific, Waltham, MA, USA). The length of each fragment was determined using the GeneMapper software (Thermo Fisher Scientific, Waltham, MA, USA). The resulting profile was then uploaded to the WEBRIBO database (https://webribo.ages.at/).