toxinotyping of c. difficile strains 1234567 b1 pcr fragment 12349556778 a3 pcr fragment pcr method...

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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