Background: The continued rise of Clostridium difficile infections worldwide has been accompanied by the rapid emergence of a highly virulent clone designated PCR-ribotype 027. To understand more about the evolution of this virulent clone, we made a three-way genomic and phenotypic comparison of an 'historic' non-epidemic 027 C. difficile (CD196), a recent epidemic and hypervirulent 027 (R20291) and a previously sequenced PCR-ribotype 012 strain (630). Results: Although the genomes are highly conserved, the 027 genomes have 234 additional genes compared to 630, which may contribute to the distinct phenotypic differences we observe between these strains relating to motility, antibiotic resistance and toxicity. The epidemic 027 strain has five unique genetic regions, absent from both the non-epidemic 027 and strain 630, which include a novel phage island, a two component regulatory system and transcriptional regulators. Conclusions: A comparison of a series of 027 isolates showed that some of these genes appeared to have been gained by 027 strains over the past two decades. This study provides genetic markers for the identification of 027 strains and offers a unique opportunity to explain the recent emergence of a hypervirulent bacterium.

Clostridium difficile has rapidly emerged as the leading cause of antibiotic-associated diarrheal disease, with the transcontinental spread of various PCR ribotypes, including 001, 017, 027 and 078. However, the genetic basis for the emergence of C. difficile as a human pathogen is unclear. Whole genome sequencing was used to analyze genetic variation and virulence of a diverse collection of thirty C. difficile isolates, to determine both macro and microevolution of the species. Horizontal gene transfer and large-scale recombination of core genes has shaped the C. difficile genome over both short and long time scales. Phylogenetic analysis demonstrates C. difficile is a genetically diverse species, which has evolved within the last 1.1–85 million years. By contrast, the disease-causing isolates have arisen from multiple lineages, suggesting that virulence evolved independently in the highly epidemic lineages.

Clostridium difficile persists in hospitals by exploiting an infection cycle that is dependent on humans shedding highly resistant and infectious spores. Here we show that human virulent C. difficile can asymptomatically colonize the intestines of immunocompetent mice, establishing a carrier state that persists for many months. C. difficile carrier mice consistently shed low levels of spores but, surprisingly, do not transmit infection to cohabiting mice. However, antibiotic treatment of carriers triggers a highly contagious supershedder state, characterized by a dramatic reduction in the intestinal microbiota species diversity, C. difficile overgrowth, and excretion of high levels of spores. Stopping antibiotic treatment normally leads to recovery of the intestinal microbiota species diversity and suppresses C. difficile levels, although some mice persist in the supershedding state for extended periods. Spore-mediated transmission to immunocompetent mice treated with antibiotics results in self-limiting mucosal inflammation of the large intestine. In contrast, transmission to mice whose innate immune responses are compromised (Myd88 ^−/− ) leads to a severe intestinal disease that is often fatal. Thus, mice can be used to investigate distinct stages of the C. difficile infection cycle and can serve as a valuable surrogate for studying the spore-mediated transmission and interactions between C. difficile and the host and its microbiota, and the results obtained should guide infection control measures.

Staphylococcus aureusis the most common cause of hospital-acquired infection. In healthy hosts outside of the health care setting,S. aureusis a frequent colonizer of the human nose but rarely causes severe invasive infection such as bacteremia, endocarditis, or osteomyelitis. To identify genes associated with community-acquired invasive isolates, regions of genomic variability, and theS. aureuspopulation structure, we compared 61 community-acquired invasive isolates ofS. aureusand 100 nasal carriage isolates from healthy donors using a microarray spotted with PCR products representing every gene from the sevenS. aureussequencing projects. The core genes common to all strains were identified, and 10 dominant lineages ofS. aureuswere clearly discriminated. Each lineage carried a unique combination of hundreds of “core variable” (CV) genes scattered throughout the chromosome, suggesting a common ancestor but early evolutionary divergence. Many CV genes are regulators of virulence genes or known or predicted to be expressed on the bacterial surface and to interact with the host during nasal colonization and infection. Within each lineage, isolates showed substantial variation in the carriage of mobile genetic elements and their associated virulence and resistance genes, indicating frequent horizontal transfer. However, we were unable to identify any association between lineage or gene and invasive isolates. We suggest that theS. aureusgene combinations necessary for invasive disease may also be necessary for nasal colonization and that community-acquired invasive disease is strongly dependent on host factors.

The majority of individuals infected with TB develop a latent infection, in which organisms survive within the body while evading the host immune system. Such persistent bacilli are capable of surviving several months of combinatorial antibiotic treatment. Evidence suggests that stationary phase bacteria adapt to increase their tolerance to environmental stresses. We have developed a unique in vitro model of dormancy based on the characterization of a single, large volume fermenter culture of M. tuberculosis, as it adapts to stationary phase. Cells are maintained in controlled and defined aerobic conditions (50% dissolved oxygen tension), using probes that measure dissolved oxygen tension, temperature, and pH. Microarray analysis has been used in conjunction with viability and nutrient depletion assays to dissect differential gene expression. Following exponential phase growth the gradual depletion of glucose/glycerol resulted in a small population of survivors that were characterized for periods in excess of 100 days. Bacilli adapting to nutrient depletion displayed characteristics associated with persistence in vivo, including entry into a non-replicative state and the up-regulation of genes involved in beta-oxidation of fatty acids and virulence. A reduced population of non-replicating bacilli went on to adapt sufficiently to re-initiate cellular division.