Pre-epidemic evolution of the MRSA USA300 clade and a molecular key for classification.

Introduction: USA300 has remained the dominant community and healthcare associated methicillin-resistant Staphylococcus aureus (MRSA) clone in the United States and in northern South America for at least the past 20 years. In this time, it has experienced epidemic spread in both of these locations. However, its pre-epidemic evolutionary history and origins are incompletely understood. Large sequencing databases, such as NCBI, PATRIC, and Staphopia, contain clues to the early evolution of USA300 in the form of sequenced genomes of USA300 isolates that are representative of lineages that diverged prior to the establishment of the South American epidemic (SAE) clade and North American epidemic (NAE) clade. In addition, historical isolates collected prior to the emergence of epidemics can help reconstruct early events in the history of this lineage. Methods: Here, we take advantage of the accrued, publicly available data, as well as two newly sequenced pre-epidemic historical isolates from 1996, and a very early diverging ACME-negative NAE genome, to understand the pre-epidemic evolution of USA300. We use database mining techniques to emphasize genomes similar to pre-epidemic isolates, with the goal of reconstructing the early molecular evolution of the USA300 lineage. Results: Phylogenetic analysis with these genomes confirms that the NAE and SAE USA300 lineages diverged from a most recent common ancestor around 1970 with high confidence, and it also pinpoints the independent acquisition events of the of the ACME and COMER loci with greater precision than in previous studies. We provide evidence for a North American origin of the USA300 lineage and identify multiple introductions of USA300 into South and North America. Notably, we describe a third major USA300 clade (the pre-epidemic branching clade; PEB1) consisting of both MSSA and MRSA isolates circulating around the world that diverged from the USA300 lineage prior to the establishment of the South and North American epidemics. We present a detailed analysis of specific sequence characteristics of each of the major clades, and present diagnostic positions that can be used to classify new genomes.

Molecular Characterization of Haemaphysalis Species and a Molecular Genetic Key for the Identification of Haemaphysalis of North America.

Haemaphysalis longicornis (Acari: Ixodidae), the Asian longhorned tick, is native to East Asia, but has become established in Australia and New Zealand, and more recently in the United States. In North America, there are other native Haemaphysalis species that share similar morphological characteristics and can be difficult to identify if the specimen is damaged. The goal of this study was to develop a cost-effective and rapid molecular diagnostic assay to differentiate between exotic and native Haemaphysalis species to aid in ongoing surveillance of H. longicornis within the United States and help prevent misidentification. We demonstrated that restriction fragment length polymorphisms (RFLPs) targeting the 16S ribosomal RNA and the cytochrome c oxidase subunit I (COI) can be used to differentiate H. longicornis from the other Haemaphysalis species found in North America. Furthermore, we show that this RFLP assay can be applied to Haemaphysalis species endemic to other regions of the world for the rapid identification of damaged specimens. The work presented in this study can serve as the foundation for region specific PCR-RFLP keys for Haemaphysalis and other tick species and can be further applied to other morphometrically challenging taxa.