Characterization of Clostridium difficile isolates using capillary gel electrophoresis-based PCR ribotyping

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Clostridium difficile infections (CDI) require accurate strain typing for epidemiological surveillance.
• Conventional PCR ribotyping methods can present challenges in inter-laboratory reproducibility.
• Standardized and efficient typing methods are crucial for managing CDI outbreaks.

Purpose of the Study:

• To develop and evaluate a novel Clostridium difficile PCR ribotyping method utilizing capillary gel electrophoresis (CGE).
• To compare the performance of CGE-based PCR ribotyping with conventional methods.
• To assess the utility of CGE for subtyping and accurate identification of C. difficile isolates.

Main Methods:

• Development of a CGE-based PCR ribotyping protocol.
• Analysis of 146 C. difficile isolates, including reference strains and clinical isolates from Austria.
• Comparison of CGE results with conventional PCR ribotyping and multiple-locus variable-number tandem-repeat (MLVA) analysis.
• Utilisation of a web-based software for pattern allocation and identification.

Main Results:

• The CGE method generated up to 11 fragments per isolate, resulting in 47 distinct ribotype patterns.
• Reference strain PCR ribotypes were accurately reflected in the CGE chromatograms.
• CGE successfully subtyped 24 isolates of PCR ribotype 014 into seven distinct subgroups, aiding in precise identification.
• The web-based software facilitated correct identification of isolates by allocating subgroup patterns to established ribotypes.

Conclusions:

• Capillary gel electrophoresis-based PCR ribotyping offers a robust and reproducible method for Clostridium difficile typing.
• This technique addresses limitations in inter-laboratory comparisons and significantly reduces hands-on time.
• CGE-based PCR ribotyping is a valuable tool for accurate epidemiological surveillance and outbreak investigation of C. difficile.