Comparison of Seven Techniques for Typing International Epidemic Strains of Clostridium difficile : Restriction Endonuclease Analysis, Pulsed-Field Gel Electrophoresis, PCR-Ribotyping, Multilocus Sequence Typing, Multilocus Variable-Number Tandem-Repeat Analysis, Amplified Fragment Length Polymorphism, and Surface Layer Protein A Gene Sequence Typing

Author:

Killgore George1,Thompson Angela1,Johnson Stuart2,Brazier Jon3,Kuijper Ed4,Pepin Jacques5,Frost Eric H.5,Savelkoul Paul6,Nicholson Brad7,van den Berg Renate J.4,Kato Haru8,Sambol Susan P.2,Zukowski Walter2,Woods Christopher7,Limbago Brandi1,Gerding Dale N.2,McDonald L. Clifford1

Affiliation:

1. Centers for Disease Control and Prevention, Atlanta, Georgia

2. Hines VA Hospital, Hines, Illinois

3. Anarobe Reference Laboratory, National Public Health Service for Wales, Microbiology Cardiff University Hospital of Wales, Cardiff, United Kingdom

4. Department of Medical Microbiology, Center for Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands

5. Department of Microbiology and Infectious Diseases, University of Sherbrooke, Sherbrooke, Quebec, Canada

6. Department of Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, The Netherlands

7. Division of Infectious Diseases, Department of Medicine, Duke University School of Medicine, Durham, North Carolina

8. Department of Bacterial Pathogenesis and Infection Control, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama, Tokyo, Japan

Abstract

ABSTRACT Using 42 isolates contributed by laboratories in Canada, The Netherlands, the United Kingdom, and the United States, we compared the results of analyses done with seven Clostridium difficile typing techniques: multilocus variable-number tandem-repeat analysis (MLVA), amplified fragment length polymorphism (AFLP), surface layer protein A gene sequence typing ( slpA ST), PCR-ribotyping, restriction endonuclease analysis (REA), multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). We assessed the discriminating ability and typeability of each technique as well as the agreement among techniques in grouping isolates by allele profile A (AP-A) through AP-F, which are defined by toxinotype, the presence of the binary toxin gene, and deletion in the tcdC gene. We found that all isolates were typeable by all techniques and that discrimination index scores for the techniques tested ranged from 0.964 to 0.631 in the following order: MLVA, REA, PFGE, slpA ST, PCR-ribotyping, MLST, and AFLP. All the techniques were able to distinguish the current epidemic strain of C. difficile (BI/027/NAP1) from other strains. All of the techniques showed multiple types for AP-A (toxinotype 0, binary toxin negative, and no tcdC gene deletion). REA, slpA ST, MLST, and PCR-ribotyping all included AP-B (toxinotype III, binary toxin positive, and an 18-bp deletion in tcdC ) in a single group that excluded other APs. PFGE, AFLP, and MLVA grouped two, one, and two different non-AP-B isolates, respectively, with their AP-B isolates. All techniques appear to be capable of detecting outbreak strains, but only REA and MLVA showed sufficient discrimination to distinguish strains from different outbreaks.

Publisher

American Society for Microbiology

Subject

Microbiology (medical)

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