A strain of C. difficile that is highly resistant to clindamycin was responsible for large outbreaks of diarrhea in four hospitals in different states. The use of clindamycin is a specific risk factor for diarrhea due to this strain. Resistance to clindamycin further increases the risk of C. difficile-associated diarrhea, an established complication of antimicrobial use.

SummaryClostridium difficile is an emerging nosocomial pathogen of increasing importance and virulence but our ability to study the molecular mechanisms underlying the pathogenesis of C. difficile-associated disease has been limited because of a lack of tools for its genetic manipulation. We have now developed a reproducible method for the targeted insertional inactivation of chromosomal C. difficile genes. The approach relies on the observation that an Escherichia coli-Clostridium perfringens shuttle vector is unstable in C. difficile and can be used as a form of conditional lethal vector to deliver gene constructs to the chromosome. We have used this methodology to insertionally inactivate two putative response regulator genes, rgaR and rgbR, which encode proteins with similarity to the toxin gene regulator, VirR, from C. perfringens. Transcriptomic analysis demonstrated that the C. difficile RgaR protein positively regulated four genes, including a putative agrBD operon. The RgaR protein was also purified and shown to bind specifically to sites that contained two consensus VirR boxes located just upstream of the putative promoters of these genes. The development of this methodology will significantly enhance our ability to use molecular approaches to develop a greater understanding of the ability of C. difficile to cause disease.

Clostridium difficile is a nosocomial pathogen that causes a range of chronic intestinal diseases, usually as a result of antimicrobial therapy. Macrolidelincosamide-streptogramin B (MLS) resistance in C. difficile is encoded by the Erm B resistance determinant, which is thought to be located on a conjugative transposon, Tn5398. The 9630 bp Tn5398 element has been cloned and completely sequenced and its insertion site determined. Analysis of the resultant data reveals that Tn5398 is not a classical conjugative transposon but appears to be a mobilizable non-conjugative element. It does not carry any transposase or site-specific recombinase genes, nor any genes likely to be involved in conjugation. Furthermore, using PCR analysis it has been shown that isolates of C. difficile obtained from different geographical locations exhibit heterogeneity in the genetic arrangement of both Tn5398 and their Erm B determinants. These results indicate that genetic exchange and recombination between these determinants occurs in the clinical and natural environment.

Mobilisable transposons are transposable genetic elements that also encode mobilisation functions but are not in themselves conjugative. They rely on coresident conjugative elements to facilitate their transfer to recipient cells. Clostridial mobilisable transposons include Tn4451 and Tn4452 from Clostridium perfringens, and Tn4453a and Tn4453b from Clostridium difficile, all of which are closely related, and Tn5398 from C. difficile. The Tn4451 group of elements encodes resistance to chloramphenicol and is unusual in that transposition is dependent upon a large resolvase protein rather than a more conventional transposase or integrase. This group of elements also encodes the mobilisation protein TnpZ that, by acting at the RS(A) or oriT site located on the transposon, and in the presence of a coresident conjugative element, promotes the movement of the nonreplicating circular intermediate and of plasmids on which the transposon resides. The erythromycin resistance element Tn5398 is unique in that it encodes no readily identifiable transposition or mobilisation proteins. However, the element is still capable of intraspecific transfer between C. difficile isolates, by an unknown mechanism. The detailed analysis of these mobilisable clostridial elements provides evidence that the evolution and dissemination of antibiotic resistance genes is a complex process that may involve the interaction of genetic elements with very different properties.

The ErmB macrolide-lincosamide-streptogramin B (MLS) resistance determinant from Clostridium difficile 630 contains two copies of an erm(B) gene, separated by a 1.34-kb direct repeat also found in an Erm(B) determinant from Clostridium perfringens. In addition, both erm(B) genes are flanked by variants of the direct repeat sequence. This genetic arrangement is novel for an ErmB MLS resistance determinant.