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.
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 (slpAST), 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, slpAST, 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, slpAST, 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.
Killgore
George
G
Centers for Disease Control and Prevention, Atlanta, GA 30333, USA.
Thompson
Angela
A
Johnson
Stuart
S
Brazier
Jon
J
Kuijper
Ed
E
Pepin
Jacques
J
Frost
Eric H
EH
Savelkoul
Paul
P
Nicholson
Brad
B
van den Berg
Renate J
RJ
Kato
Haru
H
Sambol
Susan P
SP
Zukowski
Walter
W
Woods
Christopher
C
Limbago
Brandi
B
Gerding
Dale N
DN
McDonald
L Clifford
LC
D054458:Amplified Fragment Length Polymorphism Analysis / Q000379:methods; D001426:Bacterial Proteins / Q000235:genetics; D001427:Bacterial Toxins / Q000235:genetics; D015373:Bacterial Typing Techniques / Q000379:methods*; D002170:Canada; D016360:Clostridioides difficile / Q000145:classification*; D004269:DNA, Bacterial / Q000235:genetics*; D004196:Disease Outbreaks; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods; D004761:Enterocolitis, Pseudomembranous / Q000453:epidemiology / Q000382:microbiology; D005838:Genotype; D006801:Humans; D018598:Minisatellite Repeats; D017720:Molecular Epidemiology / Q000379:methods*; D009426:Netherlands; D000091402:Prohibitins; D015203:Reproducibility of Results; D015183:Restriction Mapping / Q000379:methods; D021521:Ribotyping / Q000379:methods; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods; D006113:United Kingdom; D014481:United States
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004269:DNA, Bacterial; C000717331:PHB2 protein, human; D000091402:Prohibitins; C070683:surface layer protein A, Bacteria