Whole genome sequence (WGS)-based strain typing finds increasing use in the epidemiologic analysis of bacterial pathogens in both public health as well as more localized infection control settings. This minireview describes methodologic approaches that have been explored for WGS-based epidemiologic analysis and considers the challenges and pitfalls of data interpretation. Personal collection of relevant publications. When applying WGS to study the molecular epidemiology of bacterial pathogens, genomic variability between strains is translated into measures of distance by determining single nucleotide polymorphisms in core genome alignments or by indexing allelic variation in hundreds to thousands of core genes, assigning types to unique allelic profiles. Interpreting isolate relatedness from these distances is highly organism specific, and attempts to establish species-specific cutoffs are unlikely to be generally applicable. In cases where single nucleotide polymorphism or core gene typing do not provide the resolution necessary for accurate assessment of the epidemiology of bacterial pathogens, inclusion of accessory gene or plasmid sequences may provide the additional required discrimination. As with all epidemiologic analysis, realizing the full potential of the revolutionary advances in WGS-based approaches requires understanding and dealing with issues related to the fundamental steps of data generation and interpretation.

A highly stable strain of Staphylococcus aureus with a pulsed-field gel electrophoresis type of USA300 and multilocus sequence type 8 has been isolated from patients residing in diverse geographic regions of the United States. This strain, designated USA300-0114, is a major cause of skin and soft tissue infections among persons in community settings, including day care centers and correctional facilities, and among sports teams, Native Americans, men who have sex with men, and military recruits. The organism is typically resistant to penicillin, oxacillin, and erythromycin (the latter mediated by msrA ) and carries SCC mec type IVa. This strain is variably resistant to tetracycline [mediated by tet (K)]; several recent isolates have decreased susceptibility to fluoroquinolones. S. aureus USA300-0114 harbors the genes encoding the Panton-Valentine leucocidin toxin. DNA sequence analysis of the direct repeat units within the mec determinant of 30 USA300-0114 isolates revealed differences in only a single isolate. Plasmid analysis identified a common 30-kb plasmid that hybridized with blaZ and msrA probes and a 3.1-kb cryptic plasmid. A 4.3-kb plasmid encoding tet (K) and a 2.6-kb plasmid encoding ermC were observed in a few isolates. DNA microarray analysis was used to determine the genetic loci for a series of virulence factors and genes associated with antimicrobial resistance. Comparative genomics between USA300-0114 and three other S. aureus lineages (USA100, USA400, and USA500) defined a set of USA300-0114-specific genes, which may facilitate the strain's pathogenesis within diverse environments.

Methicillin-resistant Staphylococcus aureus (MRSA) PFGE strain type USA300 (multilocus sequence type 8, clonal complex 8, staphylococcal cassette chromosome mec type IV) was first reported in the USA as a cause of skin and soft issue infection among college football players in Pennsylvania and among prisoners in Missouri in 2000. Over the next 5 years, USA300 became the predominant community-associated MRSA strain in the USA. It was the most common PFGE type recovered from skin and soft tissue infections in persons presenting to 11 emergency departments across the USA, and caused outbreaks in Native American populations, children in daycare centres, military recruits, prison inmates and among men who have sex with men. Although predominantly a cause of skin and soft issue infection, USA300 isolates also have been recovered from cases of invasive disease including bacteraemia, endocarditis, severe necrotizing pneumonia and osteomyelitis. Isolates of USA300 usually carry the genes encoding the Panton–Valentine leucocidin and the arginine catabolic mobile element, but rarely carry staphylococcal enterotoxin genes. USA300 isolates are becoming more resistant to antimicrobial agents, including erythromycin, levofloxacin, mupirocin and tetracycline, and have spread to Europe, South America and Australia. The emergence of the MRSA USA300 strain type represents a unique biological success story.

Antibiotic resistance among nosocomial pathogens is a cause of major concern.^1-3 Three aspects of this problem have been particularly challenging: the frequent emergence of resistance to the newest antibiotics; the presence of antibiotic resistance genes on bacterial plasmids, which may be transferred among different bacterial species; and the spread of resistant bacteria among patients not only in the hospital but also in the community.

Multidrug-resistant nosocomial pathogens present a major burden for hospitals. Rapid cluster identification and pathogen profiling, i.e., of antibiotic resistance and virulence genes, are crucial for effective infection control. Methicillin-resistant Staphylococcus aureus (MRSA), in particular, is now one of the leading causes of nosocomial infections. In this study, whole-genome sequencing (WGS) was applied retrospectively to an unusual spike in MRSA cases in two intensive care units (ICUs) over the course of 4 weeks. While the epidemiological investigation concluded that there were two separate clusters, each associated with one ICU, S. aureus protein A gene ( spa ) typing data suggested that they belonged to single clonal cluster (all cases shared spa type t001). Standardized gene sets were used to extract an allele-based profile for typing and an antibiotic resistance and toxin gene profile. The WGS results produced high-resolution allelic profiles, which were used to discriminate the MRSA clusters, corroborating the epidemiological investigation and identifying previously unsuspected transmission events. The antibiotic resistance profile was in agreement with the original clinical laboratory susceptibility profile, and the toxin profile provided additional, previously unknown information. WGS coupled with allelic profiling provided a high-resolution method that can be implemented as regular screening for effective infection control.

Pulsed-field gel electrophoresis (PFGE) has become the gold standard of molecular methods in epidemiological investigations. In spite of its high resolving power, use of the method has been hampered by inadequate laboratory-to-laboratory reproducibility. In the project described here we have addressed this problem by organizing a multilaboratory effort in which the same bacterial strains (subtype variants of the Iberian and Brazilian methicillin-resistant Staphylococcus aureus —MRSA—clones) were analyzed by twenty investigators in thirteen different laboratories according to an indentical protocol, which is reproduced here in detail. PFGE patterns obtained were analyzed at a central laboratory in order to identify specific technical problems that produced substandard macrorestriction patterns. The results including the specific technical problems and their most likely causes are described in this communication. Also listed are seven major epidemic clones of MRSA which have been characterized by molecular fingerprinting techniques and the prototypes of which have been deposited at the American Type Culture Collection, from where they will be available for interested investigators for the purpose of typing MRSA isolates. It is hoped that this communication will contribute to the improvement of the reproducibility and technical/aesthetic quality of PFGE analysis.