Comparison of Virulence in Community-Associated Methicillin-Resistant Staphylococcus aureus Pulsotypes USA300 and USA400 in a Rat Model of Pneumonia

MAJOR ARTICLE Comparison of Virulence in Community-Associated Methicillin-Resistant Staphylococcus aureus Pulsotypes USA300 and USA400 in a Rat Model of Pneumonia Christopher P. Montgomery,1 Susan Boyle-Vavra,2 Patricia V. Adem,3,a Jean C. Lee,4 Aliya N. Husain,3 Julia Clasen,4 and Robert S. Daum2 From the 1Department of Pediatrics, Section of Critical Care Medicine, the 2Department of Pediatrics, Section of Infectious Diseases, and the 3 Department of Pathology, University of Chicago, Illinois, and the 4Channing Laboratory, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts Disease caused by community-associated methicillinresistant Staphylococcus aureus (CA-MRSA) can be se- Received 26 December 2007; accepted 5 March 2008; electronically published 3 July 2008. Potential conflicts of interest: R.S.D. reports serving on paid advisory boards for Clorox, Sanofi Pasteur, GlaxoSmithKline, Pfizer, and the MRSA National Faculty Meeting (sponsored by Astellas and Theravance); receiving lecture fees from Nabi Biopharmaceuticals and Pfizer; and receiving grant support from Clorox, Pfizer, Sage Products, and Sanofi Pasteur. Presented in part: Gordon Conference on the Biology of Acute Respiratory Tract Infections, Ventura, California, 19 –24 March 2008; Pediatric Academic Societies’ Annual Meeting, Honolulu, Hawaii, 2– 6 May 2008 (6430.2). Financial support: National Institute of Child Health and Human Development (Research Training in Pediatrics grant 5K12HD043387-04 to C.P.M.); National Institute of Allergy and Infectious Diseases (R01AI40481 to R.S.D. and S.B.-V., 1R01AI067584 to R.S.D. and S.B.-V., and R01AI29040 to J.C.L.); Centers for Disease Control and Prevention (R01CCR 523379 to R.S.D. and S.B.-V., R01CI000373 to R.S.D. and S.B.-V., and U01-CI00384 to R.S.D.); Grant HealthCare Foundation (to R.S.D. and S.B.-V.); Heinrich-Hertz Foundation (to J.C.). a Present affiliation: Centers for Disease Control and Prevention, Atlanta, Georgia. Reprints or correspondence: Christopher P. Montgomery, University of Chicago, Dept. of Pediatrics, Section of Critical Care, 5841 S. Maryland Ave., MC 1145, Chicago, IL 60637 (). The Journal of Infectious Diseases 2008; 198:561–70 © 2008 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2008/19804-0015$15.00 DOI: 10.1086/590157 Background. The predominant genetic background of community-associated methicillin-resistant Staphylococcus aureus has transitioned from USA400 to USA300 in most US communities. The explanation for this shift is unclear. We hypothesized that USA300 must be more pathogenic—specifically, that USA300 would have increased virulence when compared with USA400 in an animal model. Methods. Rats were inoculated intratracheally with 1 of 6 S. aureus isolates from the USA300 and USA400 backgrounds. We assessed mortality, in vivo bacterial growth, and histopathology. We assessed the in vitro expression of capsule and of selected genes believed to be important in virulence in S. aureus, including agr, saeRS, sarA, ␣-toxin (hla), and Panton-Valentine leukocidin (pvl). Results. USA300 isolates were more lethal, produced more severe pneumonia, and had higher in vivo bacterial density in the lung than did USA400 isolates. In vitro expression of agr, saeRS, sarA, hla, and pvl were greater in USA300 isolates. USA300 isolates were unencapsulated, whereas 2 of 3 USA400 isolates produced capsule. Conclusions. USA300 isolates were more virulent than USA400 isolates in a model of necrotizing pneumonia. The explanation for this is unclear, but it likely results from increased expression of S. aureus regulatory systems (e.g., agr, saeRS, and sarA) and the resultant upregulation of key virulence factors including ␣-toxin and PVL. vere, especially when it manifests as necrotizing pneumonia or severe sepsis [1–3]. When epidemic CA-MRSA disease was first described in several Midwestern medical centers, including Chicago [4], a genetic background now called USA400 [5] (as determined by pulsed-field gel electrophoresis [PFGE]) or multilocus sequence type (MLST) 1, was the major circulating clone [6]. A shift has occurred, however, and the current CA-MRSA background predominant in most US locales is USA300, or MLST 8 [7–9]. It is unclear why USA300 has become the predominant CA-MRSA background responsible for symptomatic infection in most of the United States. Inspection of the published genome sequences of representative USA300 and USA400 strains [10 –12] has revealed that methicillin resistance in both lineages is encoded by the mecA gene within the mobile genetic element staphylococcal cassette chromosome mec (SCCmec) type IV [13]. Also, these sequenced genomes contain the genes lukS-PV and lukF-PV, which encode the Panton-Valentine leukocidin (PVL) carried by nearly identical integrated prophages [10 –12]. Based in part on this strong epidemiologic association, it has been Comparison of Virulence in USA300 and USA400 ● JID 2008:198 (15 August) ● 561 Table 1. Source and characteristics of bacterial strains used in this study. Strain Clinical syndrome Site of isolation MLST Pulsotype SCCmec type Reference MW2 649 2672 LAC 923 2145 Severe sepsis Necrotizing pneumonia and severe sepsis Necrotizing pneumonia and severe sepsis Unknown Soft tissue infection Pneumonia Blood Respiratory tract Respiratory tract Unknown Abscess Respiratory tract 1 1 1 8 8 8 USA400 USA400 USA400 USA300 USA300 USA300 IV IV Negative IV IV IV [15] [2] [1] [15] Present study Present study NOTE. MLST, multilocus sequence type; SCCmec, staphylococcal cassette chromosome mec. 562 ● JID 2008:198 (15 August) ● Montgomery et al. tures identical to the fatal, necrotizing pneumonia that occurs in patients. METHODS. Isolate selection. Clinical isolates representing the MLST 1 lineage (presumed to represent USA400) and the MLST 8 lineage (presumed to represent USA300) were selected for evaluation (table 1) from the isolate library at the University of Chicago, which contains more than 1000 clinical S. aureus isolates categorized by clinical characteristics, antibiotic susceptibility data, and information from molecular typing, including evaluation for SCCmec type and MLST. The isolates selected were recovered from patients with a variety of CAMRSA disease syndromes, including necrotizing pneumonia, severe sepsis, and abscesses. All isolates contained the lukS-PV and lukF-PV genes, and all USA300 isolates contained the arcA gene from ACME (see below). Strains MW2 (USA400) and LAC (USA300-0114) were kindly provided by Michael Otto (National Institutes of Health) [15]. PFGE. Whole cell DNA-containing agarose plugs were prepared and digested with SmaI, as described elsewhere [5]. Restriction fragments were resolved on a CHEF DR-III PFGE system (Bio-Rad). A SmaI digest of S. aureus strain 8325-4 (ATCC 35556) served as the molecular size standard. Strain LAC served as a standard for U (...truncated)