TIPIZZAZIONE MOLECOLARE DI CLOSTRIDIUM DIFFICILE Vincenza Romano, PhD vincenza.romano@uniparthenope.it

Denève et al., 2009

del locus di patogenicità Tossine A e B TcdA e TcdB Mappa parziale del locus di patogenicità Rupnik et. al. 1997. Rispettivamente citotossina ed enterotossina. Codificate a livello di uno stesso Locus genico: PaLoc Mutazioni nei geni per le tossine danno origine ai diversi tossinotipi.

TcdA e TcdB Large Clostridial toxins Major virulence factors Intracellular Glucosyltransferases Glucosylating enzymatic domain Autocatalityc Processing Translocating Binding

Tossina Binaria CDT cdtA componente catalitica e cdtB componente di legame. E’ stato dimostrato che questa tossina è presente solo nei campioni con modificazioni nei geni per le tossine A e B, fatta eccezione per il tossinotipo VIII. (Stubbs et al. 2000).

CDT Introduction

Identificazione e profilo tossigenico Tecniche molecolari Identificazione e profilo tossigenico Toxinotyping Ribotyping

PCR per tossina A (con e senza delezione) PCR per GluD e tossina B PCR per tossina A (con e senza delezione) MD PhD E. J . Kuijper Lieden University Medical Center, Olanda Toxinotyping http://www.mf.uni-mb.si/tox/ MD PhD Maja Rupnik University of Maribor, Slovenia

Identificazione e profilo tossigenico tcdA tcdB and gluD Duplex PCR gene tossina A (con e senza delezione). Deletion tcdA (314 bp) tcdA (369 bp) Duplex PCR per gene GluD e gene tossina B tcdB (200 bp) gluD (158 bp)

PCR per la Tossina binaria (Stubbs et al) cdtA cdtB Binary toxin genes 327 bp PCR separate: 451 bp

TOXINOTYPING This technique allows to the identification of 32 toxinotypes designated with 0 (if identical to the reference strain VPI 10463), and with I to XXXI if presenting variations in the toxin genes. Toxinotyping is a restriction fragment length polymorphism (RFLP) based method detecting the polymorphism of the PaLoc through the amplification and digestion of six of its regions(from A1 to A3 and from B1 to B3), using restriction enzymes. Regions A3 and B1 are the most variable and through their amplification and restriction is possible to establish if a strain belongs to a known or a new toxinotype.

RIBOTYPING Analisi dei profili mediante Gel Compare (Bionumerics) ITS

Correlazione tra profilo tossigenico e suddivisione ottenuta tramite PCR ribotipo

METODI Più di 220 ribotipi

Ribotipi più comuni 126 050 044 005 066 010 013 001 A+B+CDT- 002 003 I 002 003 I 012 014/020 015 017 A-B+CDT- VIII 018 023 A+B+CDT+ IV 027 III 029 046 053 056 XII 070 XIII 075 078 V 081 087 095   106 131 Ribotipi più comuni 126 A+B+CDT+ V 050 A+B+CDT- 044 005 066 010 Non tossigenico NT 013

Most isolated ribotypes in CDI-cases In 2008, the European Center for Disease prevention and Control (ECDC) commissioned an incidence survey in 34 European countries in order to establish the incidence and mortality of CDI in those countries and the ribotypes involved in the outbreaks. Sixty-four different C. difficile PCR ribotypes were found (Fig. 12), among them ribotypes 014/020 (15%), 001 (10%), and 078 (8%) were most prevalent, whereas the prevalence of ribotype 027 was just 5%. This hypervirulence was correlated with its ability to produce higher concentration of toxins in vitro (Warny et al., 2005), to the higher rates of sporulation and to the production of the binary toxin, although the role in pathogenicity of this toxin is still under investigation. Geographical distribution of C. difficile PCR ribotypes in European countries in November 2008 (From Bauer et al., 2011)

Grazie per l’attenzione