Final Report Summary - CDI (Genetic and phenotipic characterization of emerging hypervirulent Clostridium difficile: role and regulation of virulence factors)
Executive Summary:
In this project, I have performed four different studies:
Study 1:
I have analysed and compared genome sequences in order to find different single genes or clusters between hypervirulent C.difficile 027 strains and epidemic 012 strains.
I found that 027 strains have some single genes such as matE and a hypothetical flagella glycosylation group of genes that are absent in 012 strains. For this reason I decided to investigate those genes and its role in virulence.
Regarding the single genes, I tried to mutate matE gene but it was not possible. It could indicate that this gene is really important for 027 strains if not vital. Further studies need to be done to confirm this hypothesis.
The hypothetical flagella glycosylation genes present in the hypervirulent 027 strains were mutated along this project and the mutants were characterized phenotypically and successfully complemented. 027 strains have a novel flagella glycosylation structure composed of 3 sugars and two additional molecules that are still uncharacterized as it has been very difficult to get them fragmentated using Mass spectrometry. An NMR study is ongoing to dilucidate the last two components of the C.difficile flagella. In this study, I have found that flagella glycosylation in C.difficile is involved in sedimentation, motility and biofilm formation. Mice experiments are still ongoing so I am not able to get any conclusions about the effect of C.difficile flagella glycosylation in vivo. Flagella glycosylation has a role in virulence in some other bacterial pathogen such as Campylobacter jejuni (Howard et al., 2009).
Study 2:
The recent emergence of new aggressive C.difficile strains also encouraged me to participate in a big epidemiological study including around 380 C.difficile strains using the Multilocus Sequence Typing Method (MLST).
C.difficile population is formed by five clades and there is evidence of microdiversity within the heterologous clade 1. This study helps to define the evolutionary origin of dominant C. difficile lineages and demonstrates that C. difficile is continuing to evolve in concert with human activity.
Study 3:
The increased prevalence of Clostridium difficile infection (CDI) has coincided with enhanced transmissibility and severity of disease. Resistance to antibiotics and disinfectants is a potential problem in managing infection control. It encouraged me to participate in another project where I assess the susceptibility of three PCR-ribotypes; 012 (epidemic strain), 017 (hypervirulent strain) and 027 (hypervirulent) to four classes of disinfectants; chlorine releasing agents (CRAs), peroxygens, quaternary ammonium compounds (QAC) and biguanide.
The oxidizing agent Peresafe is the only disinfectant consistently efficacious across all three PCR ribotypes at varying concentrations; with a consistent five log reduction in spore titre.
Study 4:
Biofilms are the most predominant state of bacteria in nature also evolved the ability to form biofilms, which provide a protection and allow quickly adaptation to changes in the environment. Biofilm formation in C.difficile is still poorly understood. I developed a method in conjunction with another colleague to study C.difficile in vitro and we found that Spo0A (spore forming protein), may play a key role in biofilm formation, as genetic inactivation of spo0A in strain R20291 exhibits decreased biofilm formation.