# P-1003. Evolutionary Differences within the Epidemic Clostridioides difficile Strain Identified as PCR-Ribotype 027

## TL;DR

This study tracks the evolution of a Clostridioides difficile strain over 40 years, showing increasing antibiotic resistance and genetic mutations.

## Contribution

The study provides new insights into the genetic and antimicrobial resistance evolution of the C. difficile RT027 strain over decades.

## Key findings

- MICs for several antibiotics increased significantly in RT027 isolates over 40 years.
- Fluoroquinolone resistance mutations emerged in the 2000s but were absent in earlier isolates.
- Recent isolates showed rpoB mutations linked to resistance to fidaxomicin.

## Abstract

In the early 2000s, the epidemic strain identified as PCR Ribotype 027 (RT027) and multilocus sequence type (MLST) 1 was the dominant strain group accounting for >70% of CDI in some institutions. Notably, the strain group was fluroquinolone resistant (FQR) which likely contributed to the spread of the group strain. However, there is a paucity of data comparing historic clinical isolates prior to the spread of the epidemic strain.Table 1.Minimum Inhibitory Concentration for 10 antibiotics for 91 isolates identified at PCR-RT 027MIC: Minimum Inhibitory Concentration; RT: RibotypeFigure 1.Bayesian time scale phylogenetic analysis with minimum inhibitory concentration changes

Minimum Inhibitory Concentration for 10 antibiotics for 91 isolates identified at PCR-RT 027

MIC: Minimum Inhibitory Concentration; RT: Ribotype

Bayesian time scale phylogenetic analysis with minimum inhibitory concentration changes

We determined the antimicrobial minimum inhibitory concentration (MIC) for azithromycin (AZM), ceftriaxone (CRO), clindamycin (CLI), moxifloxacin (MXF), and vancomycin (VAN) in 91 clinical isolates previously identified as RT027 collected from 1984 – 2023 from unique patients. Whole genome sequencing was conducted on all 91 isolates to assess for mutations associated with antimicrobial resistance and complete a Bayesian time scale phylogenetic analysis.Figure 1.Bayesian time scale phylogenetic analysis with key antimicrobial gene mutations

Bayesian time scale phylogenetic analysis with key antimicrobial gene mutations

There was a significant uptrend in the MIC for AZM, CRO, MXF, and VAN over the 40-year period. Notably, the MIC for AZM increased from 3.18 µg/mL to 933.60 µg/mL, the MIC for CRO increased from 17.96 µg/mL to 55.71 µg/mL, and the MXF MIC increased from 0.50 µg/mL peaking from 2001-2009 at 45.25 µg/mL (p< 0.01, for all comparisons). [Table 1] The gyrA/B mutations associated with FQ resistance were not present in the historical cohort. From 2001 – 2020, the gyrA/B mutation were commonly found with a valine-to-leucine mutation at position 497 (Figure 1/2). Notably, the FQS isolates had only a modest association with FQR isolates from 2001 - 2020, but they do appear to be associated with FQR isolates from 2021 – 2023. These more contemporary FQR isolates more commonly had an rpoB mutation which could confer resistance to fidaxomicin

Despite the prevalence of RT027 decreasing over the past 25 years, it still harbors significant mutations which confer antimicrobial resistance. While accrual of mutations can lead to a significant fitness cost to bacteria, continued surveillance is required to ensure that new outbreaks associated with RT027 do not occur.

Andrew M. Skinner, MD, Recursion Pharmaceuticals: Advisor/Consultant