Establishment of a genotyping scheme for Coxiella burnetii.

Coxiella burnetii is the causative agent of Q fever. The bacterium is highly infectious and is classified as a category B biological weapon. The tools of molecular biology are of utmost importance in a rapid and unambiguous identification of C. burnetii in naturally occurring Q fever outbreaks, or in cases of a deliberate release of the infectious agent. In this work, development of a multiple locus variable number tandem repeats (VNTR) analysis (MLVA) for the characterization of C. burnetii is described. Sixteen C. burnetii isolates and five passage history/laboratory variants were characterized. The VNTR markers revealed many polymorphisms resulting in nine unique MLVA types that cluster into five different clusters. This proves that the MLVA system is highly discriminatory. The selected VNTR markers were stable. The MLVA method developed in this report is a promising tool for the characterization of C. burnetii isolates and their epidemiological study.

Multilocus sequence typing scheme for Enterococcus faecium.

A multilocus sequence typing (MLST) scheme has been developed for Enterococcus faecium. Internal fragments from seven housekeeping genes of 123 epidemiologically unlinked isolates from humans and livestock and 16 human-derived isolates from several outbreaks in the United States, the United Kingdom, Australia, and The Netherlands were analyzed. A total of 62 sequence types were detected in vancomycin-sensitive E. faecium (VSEF) and vancomycin-resistant E. faecium (VREF) isolates. VSEF isolates were genetically more diverse than VREF isolates. Both VSEF and VREF isolates clustered in host-specific lineages that were similar to the host-specific clustering obtained by amplified fragment length polymorphism analysis. Outbreak isolates from hospitalized humans clustered in a subgroup that was defined by the presence of a unique allele from the housekeeping gene purK and the surface protein gene esp. The MLST results suggest that epidemic lineages of E. faecium emerged recently worldwide, while genetic variation in both VREF and VSEF was created by longer-term recombination. The results show that MLST of E. faecium provides an excellent tool for isolate characterization and long-term epidemiologic analysis.