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Toxinotyping of Toxinotyping of C. difficileC. difficile strains strains
1 2 3 4 5 6 7
B1 PCR fragment1 2 3 4 9 5 5 6 7 7 8
A3 PCR fragment
PCR method for screening changes in PaLoc
markers for toxinotyping
B1
B2
B3
PL3
A1
A2
A3
PL4
tcdR tcdB tcdAtcdE tcdC
2 kb
2.5 kb 3 kb 2 kb 3 kb 3 kb
2.1 kb 2 kb 1.5 kb 2 kb 1.6 kb
HN Hc Hc H H
RH
REc P X
h h SE E
PL2
PL1
M. Rupnik Institute of Public Health Maribor Medical Faculty, University of Maribor © www.clostridia.net
Types of A-B+ C. difficile strains
toxinotype
characteristics
molecular basis for TcdA non-production
number of strains
VIII 1.8 deletion in tcdA stop codon at aa
position 47 >100
X 6 kb deletion in tcdA rearrangement in
PaLoc 1
XVI similar to
toxinotype V (A+B+) not known 1
XVII similar to
toxinotype X not known 1
0-like identical to VPI 10463
not known 1
V-like identical to
toxinotype V not known 1
C. difficile typing – mostly used methods
Ribotyping REA PFGE
(Europe) (USA) (North America)
PCR of 16S-23S rDNA intergenic spacer region160 ribotypes
Stubbs, JCM 1999
Bidet, JCM 2000
HindIII restriction of whole DNA
>100 REA groups(Rea Types)
Gerding D., Chicago, USA
SmaI restriction of whole DNA
no large international collection
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Large clostridial toxins (LCT)Large clostridial toxins (LCT)
C. difficile C. difficile (TcdA, TcdB) (TcdA, TcdB)
C. sordellii C. sordellii (TcsH, TcsL) (TcsH, TcsL)
C. novyi C. novyi (Tcn(Tcn))
• size (250-300 kDa) size (250-300 kDa)
• cytotoxicitycytotoxicity
• glycosyltransferasesglycosyltransferases
• autocatalytic proteolysisautocatalytic proteolysis
G T P aza
G T P aza
R h o /R as
R h o /R as
T h r
T h r
+
+
g lc
g lc
U D P
U D P
to k s in
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C. difficile C. difficile pathogenicity locus pathogenicity locus (PaLoc)(PaLoc)
tcd Btcd D tcd E tcd Ctcd A
H c H H c H c H H c P P H c H
cdu1
cdu1
cdu2cdu2 ' cdd1
cdd1
cdd2cdd3
cdd4
PP
11 5 b p
PaLoc1 9 k b
C . difficile tox-
C . difficile V PI 10463
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