Title: Emerging Clostridioides difficile strains belonging to PCR ribotype955 in Serbia are distinct from metronidazole resistant RT955 outbreak isolates from the UK

Introduction. End 2023, the UK Health Security Agency sent an alert about a new hypervirulent Clostridioides difficile PCR ribotype, ribotype 955 (RT955), causing slowly progressing infection clusters in hospitals in the Midlands. We have previously identified RT955 strains isolated in southern Serbia dating back to 2018. Here, we report on the clinical and microbiological findings of RT955 in southern Serbia during the COVID-19 period.Methods. Between March 2018 and February 2022, CDI surveillance was performed in southern Serbia with participation of Community Health Center Nis, various hospitals, military hospitals and one clinical center in the city of Nis. These centers provide medical services for approximately 750,000 inhabitants. Using the ECDC recommended protocol, clinical and epidemiological data were collected and stool samples that were PCR-positive were cultured for the presence of C. difficile. Isolates were sent to Leiden for further characterization by PCR ribotyping, core genome multilocus sequence typing (cgMLST) and whole genome sequencing with SNP typing.Results. C. difficile RT955 was identified in 27 (7%) of 383 surveyed patients with CDI. Of 27 patients, 16 (59%) was older than 60 years and 19 (70%) were male. CDI was always associated with previous antibiotic therapy and had a hospital onset in 23 (85%) patients. Of 27 patients, 16 (59%) had COVID-19 as concomitant disease. CDI was categorised as severe in 3 (11%) patients. At 90 days follow up, no CDI related mortality was found. Of 15 patients treated with metronidazole, 9 patients switched to vancomycin. All sequenced strains belonged to multilocus sequence type (ST) 1 and were highly similar, with 0-1 alleles differences in a cgMLST analysis. The strains differed clearly from the UK RT955 outbreak strain by whole genome sequencing and phenotypic resistance to lincosamides and rifampicin. Both the UK and Serbian RT955 strains contained gyrA_p.T82I associated with resistance to fluoroquinolone antimicrobials and carried the PnimBG promoter mutation, suggestive for haem-dependent metronidazole resistance.Conclusion. Since 2018, C. difficile RT955 is present in Serbia, without presentation of large outbreaks. The Serbian RT955 strains differed clearly from a representative UK cluster strain, but shared its haem dependent metronidazole resistance.