Genomic and transcriptomic differences in community acquired methicillin resistant Staphylococcus aureus USA300 and USA400 strains

BMC Genomics Genomic and transcriptomic differences in community acquired methicillin resistant Staphylococcus aureus USA300 and USA400 strains Marcus B Jones 0 Christopher P Montgomery Susan Boyle-Vavra Kenneth Shatzkes Rosslyn Maybank Bryan C Frank Scott N Peterson Robert S Daum 0 J. Craig Venter Institute , 4120 Capricorn Lane, La Jolla, CA 92037 , USA Background: Staphylococcus aureus is a human pathogen responsible for substantial morbidity and mortality through its ability to cause a number of human infections including bacteremia, pneumonia and soft tissue infections. Of great concern is the emergence and dissemination of methicillin-resistant Staphylococcus aureus strains (MRSA) that are resistant to nearly all -lactams. The emergence of the USA300 MRSA genetic background among community associated S. aureus infections (CA-MRSA) in the USA was followed by the disappearance of USA400 CA-MRSA isolates. Results: To gain a greater understanding of the potential fitness advantages and virulence capacity of S. aureus USA300 clones, we performed whole genome sequencing of 15 USA300 and 4 USA400 clinical isolates. A comparison of representative genomes of the USA300 and USA400 pulsotypes indicates a number of differences in mobile genome elements. We examined the in vitro gene expression profiles by microarray hybridization and the in vivo transcriptomes during lung infection in mice of a USA300 and a USA400 MRSA strain by performing complete genome qRT-PCR analysis. The unique presence and increased expression of 6 exotoxins in USA300 (12- to 600-fold) compared to USA400 may contribute to the increased virulence of USA300 clones. Importantly, we also observed the up-regulation of prophage genes in USA300 (compared with USA400) during mouse lung infection (including genes encoded by both prophages Sa2usa and Sa3usa), suggesting that these prophages may play an important role in vivo by contributing to the elevated virulence characteristic of the USA300 clone. Conclusions: We observed differences in the genetic content of USA300 and USA400 strains, as well as significant differences of in vitro and in vivo gene expression of mobile elements in a lung pneumonia model. This is the first study to document the global transcription differences between USA300 and USA400 strains during both in vitro and in vivo growth. - Background Methicillin-resistant Staphylococcus aureus (MRSA) isolates have emerged as a leading cause of infectious diseases [1-6]. Once confined primarily to hospitals and patients with defined risk factors, infections caused by communityassociated MRSA (CA-MRSA) isolates have become epidemic in the United States and now frequently occur among previously healthy individuals without these risk factors [7,8]. Although the majority of CA-MRSA infections are mild, some CA-MRSA infectious syndromes such as complicated skin and soft tissue infections, necrotizing pneumonia, bacteremia, and sepsis can be life-threatening [7-10]. After the first report of CA-MRSA deaths in children in it became recognized that the strain type was USA400 [11-14]. During the early emergence of CAMRSA, isolates of both MRSA and MSSA with the USA400 pulsed field pattern were identified as causes of severe sepsis and death characterized by rapidly progressive clinical deterioration, with necrotizing pneumonia and multiple-organ-system involvement and bilateral adrenal hemorrhage characteristic of the WaterhouseFriderichsen syndrome [14]. However, among community associated MRSA infections, USA400 has been nearly completely replaced by another CA-MRSA background, called USA300 [15-23]. The reasons for the replacement of USA400 by USA300 are not known. Many have speculated that the dominance of USA300 is evidence of altered fitness. In support of this notion, USA300 strains are hypervirulent, compared with USA400 strains, in several animal models of skin infection and pneumonia [24]. Although the relationship between fitness and virulence is not clear, understanding the mechanisms of the extraordinary virulence of USA300 strains will shed light onto the reasons for its emergence. The virulent nature of S. aureus is mediated by a wide array of cell surface proteins, secreted toxins and mobile genetic elements [15, 25-31]. As such there are at least two possible explanations for the virulence of USA300. One is that USA300 strains have acquired novel genomic content that enhances its fitness and/or virulence. For example, among sequenced S. aureus isolates, the arginine catabolic mobile element (ACME) is present in most USA300 strains circulating in the United States, but not USA400 isolates [22]. ACME is a large mobile genetic element also found in some Staphylococcus epidermidis isolates encoding at least 33 open reading frames [22,32]. Deletion of ACME from USA300 enhanced survival in a rabbit model of bacteremia [32], but ACME was not necessary for virulence in rodent models of pneumonia or skin infection [33]. However, ACME encodes an arginine deiminase gene, called arcA that allows for enhanced survival in acidic environments [34]. Interestingly, this process drives the synthesis of host polyamines that are toxic to S. aureus. It is likely not a coincidence that ACME therefore also encodes a spermine acetyl transferase, speG, which counteracts the toxic effects of polyamines. In this regard, ACME appears to at least partially account for the observed pathogenesis of USA300 in skin infection [35,36]. Nevertheless, ACME-deficient USA300 clinical isolates have been described supporting the data from animal models that ACME is not a necessary component of pathogencity or survival in humans. An alternative explanation is that USA300 has evolved to alter the expression/activity of S. aureus regulatory genes that are part of the core genome. In support of this idea, USA300 isolates have increased expression of the global regulatory systems agr and saeRS, and attendant increased expression of downstream toxins such as the Panton-Valentine leukocidin (PVL, encoded by lukSFPV), the alpha-hemolysin (Hla, encoded by hla), and phenol soluble modulins (PSMs), compared to USA400 [24,37,38]. Although there is a strong epidemiologic association of PVL carriage with severe disease caused by CA-MRSA, its role in pathogenesis is controversial. Since PVL is present in both USA300 and USA400 isolates, it is not likely that its presence accounts for the differences in virulence between the genomic backgrounds. In contrast, agr, saeRS, hla and the PSMs which are core genes present in almost all S. aureus strains, clearly advance the pathogenesis of USA300 in animal models [37,39,40]. Although it is known that USA300 has altered regulation of selected global regulators and virulence genes compared with USA400, it is not known whether there are global differences in the transcription profiles between USA300 and USA400. By comparing the in vitro transcriptional profiles of USA300 and USA400 using DNA (...truncated)