Clostridium difficile, dubbed the CD superbug by the tabloid press, is the most frequent cause of nosocomial diarrhea worldwide. C. difficile associated disease (CDAD) ranges from mild to chronic diarrhea and sometimes life-threatening disease of the gut termed pseudomembranous colitis. CDAD occurs when the bacteria that normally reside in the gut and protect it from harmful infection are killed by antibiotic treatment. This leaves the gut available for colonization by C. difficile once the antibiotic treatment finishes. The population at risk is substantial, including not only patients on antimicrobial therapy, but also the immunocompromised and the elderly. Since broad-spectrum antibiotics exacerbate the condition, treatment is difficult, especially for these susceptible groups, and the relapse rate is high (≤55%). Beyond the morbidity and mortality caused, CDAD is a substantial economic problem that can lead to patient isolation, ward closures and in extreme cases, hospital closure. More disturbingly, the reported incidence of CDAD has increased significantly in the last decade and a new hypervirulent strain is causing outbreaks of increased severity in North America and Europe Citation[1]. The origin of this strain, termed the NAP1/027 hypervirulent strain, is uncertain, although it has been proposed that increased use of fluoroquinolones may provide a selective advantage for this epidemic strain Citation[1].
In the UK, CDAD is a notifiable disease and for patients over 65 years of age alone, the reported incidence in 2006 was 51,690 cases. This represents a 17.2% increase on the previous year‘s figures and a 30-fold increase in the number of reported cases 10 years ago. The numbers of reported C. difficile cases are now three-times those for the notorious methicillin-resistant Staphylococcus aureus (MRSA) pathogen and the mortality rates for C. difficile last year far exceeded those for MRSA infection. Despite the importance of C. difficile, the pathogen does not have the profile of other pathogens and has yet to seep into the public consciousness. Given the continued use of broad-spectrum antibiotics, the aging population, increasing rates of hospitalization and the emergence of the hypervirulent strain, the incidence of CDAD is likely to continue to rise. Clearly this is alarming – but what is being done to stem the flood of C. difficile infection? The answer is, very little. Attempts to improve hospital hygiene are valiant, but as with MRSA this is not the long-term solution. A programme of research dedicated to understanding what makes the C. difficile microorganism tick at the molecular level is required. Through information derived from such studies the rational design of long-term interventions can be implemented to reduce the burden of disease (this is also true for other hospital superbugs). It is scandalous how very little government funds go into basic research on hospital superbugs – the public would be shocked Citation[2]. The Healthcare Commission in the UK, under the direction of the Secretary of State for Health, published a report in July 2006 on a large outbreak of C. difficile infection in the Stoke Mandeville Hospital, Buckinghamshire, UK, which occurred in 2004/2005. The report was highly critical of control measures used and highlighted the unacceptably high number of deaths in the outbreak. This may be true, but blaming hygiene measures and hospital staff is a convenient excuse and does not address the underlying problem of the emergence of the hypervirulent strains.
So what is known about C. difficile? It produces two related toxins, designated A and B. However, production of these toxins cannot completely explain C. difficile pathogenesis. In recent years, increasing numbers of strains have been reported that have truncated versions of toxin A and/or B, or that lack the toxins altogether. Thus, remarkably little is understood about how the organism causes disease and it is still unknown why C. difficile is almost unique in its ability to colonize the host after disturbance of the bowel microflora.
Despite the emergence of the hypervirulent strains and our lack of basic knowledge of CDAD, there is new hope on the horizon with three recent developments on the molecular characterization of the organism. These include the completion of the C. difficile genome sequence, the availability of C. difficile microarrays and the ability to construct defined mutants to expedite the identification of potential virulence determinants.
As a result of the medical and economic importance of CDAD, and the difficulties in studying the genetics of C. difficile, in 1998 a group of UK scientists proposed sequencing a representative C. difficile strain. The strain chosen, 630, is multidrug resistant and was isolated from a patient with severe pseudomembranous colitis that had spread to several other patients on the same ward. Thus, strain 630 has the genetic attributes of a fully virulent, highly transmissible, drug-resistant strain. The recently finished complete genome sequence of strain 630 revealed that a large proportion (11%) consists of mobile genetic elements Citation[3]. These mobile elements are responsible for the acquisition by C. difficile of an extensive array of genes involved in virulence, host interaction and the production of surface structures. The genome sequence of C. difficile 630 also revealed a bacterium with a number of adaptations to a gastrointestinal lifestyle, including an adaptable metabolic capacity, and a versatile genome content, with a wide range of mobile elements including conjugative transposons, phage, insertion sequence (IS) elements and IStrons. Strain 630 also encodes multiple antibiotic-resistance determinants largely mediated through conjugative transposons. The ease with which C. difficile can acquire antibiotic resistance determinants serves as a timely reminder on restricting the use of antibiotics given orally and developing alternative therapies against the increasing problem of CDAD. Disturbingly, the genome also provided some evidence that resistance to vancomycin, the main antibiotic of choice against C. difficile, may be imminent.
The genome sequence is a huge milestone in C. difficile research as it provides all the pieces in the jigsaw puzzle, and now major efforts are being directed to determine the constitution of this important pathogen. However, the 630 sequence represents a single C. difficile strain, which may not be representative of the species. There are at least two further C. difficile genome sequencing efforts in progress focusing on the hypervirulent strains, including the one that was responsible for the Stoke Mandeville outbreak. However, a wider comparison of strains from diverse sources and geographical locations is required for an in-depth analysis of the origins, transmission and evolution of the species. Carefully designed DNA microarrays can provide such information.
During the finishing stages of the 630 genome project, a C. difficile DNA microarray was designed and constructed at the Bacterial Pathogen Microarray facility at St George‘s, London, UK. This was used to apply comparative phylogenomics (whole genome comparisons using DNA microarrays combined with Bayesian phylogenies) to model the phylogeny of C. difficile. The sample included 75 diverse isolates comprising hypervirulent, toxin variable and animal strains. The analysis identified four distinct statistically supported clusters comprising all hypervirulent strains in one clade, a toxin A-B+ clade, and two clades with human and animal isolates Citation[4]. Genetic differences among clades revealed several genetic islands relating to virulence and niche adaptation, including antibiotic resistance, motility, adhesion and enteric metabolism. Surprisingly, only 19.7% of genes were shared by all strains, confirming that this enteric species undergoes extreme genetic exchange. The study has provided insight into the possible origins of C. difficile and its evolution that may have implications in disease control strategies. For example, the finding of strains from livestock (particularly pigs) within the same clade as human isolates, suggests a possible zoonotic link for the transmission of C. difficile. This finding supports other molecular typing studies. Perhaps in some instances C. difficile should be considered a foodborne pathogen and if this is proven to be the case, it is possible pork should be excluded from hospital menus.
One of the main reasons why C. difficile research has been slow is that in contrast to other enteric organisms such as Escherichia coli, Salmonella and Shigella spp., well-developed mutagenesis systems for use in C. difficile are lacking. Recently, after decades of intensive research, substantial headway has been made in the Minton and Mullany laboratories in developing techniques to construct defined C. difficile mutants. This advance will enable researchers to dissect out the determinants important in the pathogenesis of C. difficile.
Constructing defined mutants coupled with information from genome sequences and microarray studies bodes well for the characterization of this particularly intractable microbe. Clearly, there has never been a better time to study this important, yet frequently neglected pathogen. So, funding permitting, we are all tooled up and ready to go. Perhaps soon Clostridium difficile will no longer be ‘C‘est difficile‘.
Bibliography
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- Bacteria not welcome at the MRC? [Editorial]. Nat. Rev. Microbiol.4(1) , 2 (2006).
- Sebaihia M , WrenBW, MullanyP et al.: The multidrug-resistant human pathogen Clostridium difficile has a highly mobile, mosaic genome.Nat. Genet.38(7) , 779–786 (2006).
- Stabler R , GerdingDDN, SongerG et al.: Comparative phylogenomics of Clostridium difficile reveals clade specificity and microevolution of hypervirulent strains. J. Bacteriology (2006) (In press).