A 49-year-old previously healthy man was admitted to the hospital with a 10-day history of chills, fever, and dyspnea in August 2011. He had no history of surgery or intravenous drug use, and had no notable medical record. At the time of hospital admission, physical examination revealed a pulse rate of 62 beats per min, respiratory rate of 20 breaths per min, and blood pressure of 125/83 mmHg. A systolic murmur was best heard at the apex. He was febrile (40.7  C) and he had a minor abrasion on left foot. The rest of his physical examination was unremarkable. Pertinent laboratory investigations revealed white blood cell count of 23,100/mm3, with 94.5% neutrophils, 3.2% lymphocytes, and 2.3% mononuclear cells, platelets of 40,000/mm3 and C-reactive protein of 58.6 mg/L. The patient was hospitalized for a presumptive diagnosis of septicemia. Empirical antibiotics were started with vancomycin and levofloxacin (0.5 g q8 h) therapy.
During the next two days, echocardiography revealed large vegetation on the anterior mitral valve leaflet (3 cm x 3 cm) with moderate mitral valve regurgitation (Fig. 1A) Spiral computed tomography (CT) showed renal and splenic infarction (Fig. 1B). CT scans of the brain demonstrated multiple low-density bilateral lesions of the temporal lobes, right parietal lobe and occipital lobe, suggestive of cerebral embolism. Preliminary blood cultures grew S. aureus susceptible to ciprofloxacin, rifampicin, linezolid, vancomycin, tetracycline, sulfamethoxazole, levofloxacin and fosfomycin but resistant to penicillin, oxacillin, clindamycin, cefazolin, cefoxitin, cefuroxime, and erythromycin, as determined on the basis of CLSI disc diffusion standards. A diagnosis of acute infective endocarditis with systemic embolism caused by CA-MRSA was thus considered. Because of his impaired renal function and bacterial susceptibility profile, the patient was treated with intravenous linezolid (600 mg q12 h) and fosfomycin (8.0 g q12 h).
On day 10, his clinical status worsened with episodes of tachypnea, pink frothy sputum and oxygen saturation (SpO2) decreased rapidly to 83% with ventilator support. Furthermore, renal function deteriorated, oliguria and right lower extremity tissue necrosis appeared. Embolization of the right common iliac artery and right internal and external iliac arteries was seen on echocardiography (Fig. 1C). He subsequently developed a coma with a Glasgow Coma Scale (GCS) score of 6. His clinical condition deteriorated such that he was transferred to the ICU, and replacement of the mitral valve was accomplished with a 29-mm Carbomedics mechanical valve. Considering the presence of coma and fever (39.8  C) postoperatively, brain CT was obtained and showed multiple low-density lesions in temporal lobes, right parietal lobe, and occipital lobe, suggestive of cerebral embolism (Fig. 1D). Levofloxacin (0.75 g qd) was added to his antibiotic regimen. After 10 days of intravenous antibiotics, the patient regained consciousness (GCS score of 9) and made a good clinical recovery. On day 35, he was transferred to a secondary hospital, and linezolid therapy was continued for eight weeks. He recovered uneventfully and was well at the last follow-up in November 2013.
The isolate recovered from the vegetation was first identified by the VITEK 2 system and then identified with MicroFlex LT instrument (Bruker Daltonics). Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) based fingerprint analysis of extracted proteins yielded a pattern similar to that of confirmed S. aureus isolates, Flexcontrol 3.0 software and Biotyper 2.0 database (Bruker Daltonics) identified the isolate as S. aureus with a maximum score value of 2.166 (data not shown). In addition to MALDI-TOF-MS analysis, 16S rRNA sequencing was performed in order to identify the origin of the bacteria in the vegetation. The sequence of the PCR product was compared with sequences of closely related species in GenBank by using BLAST. Sequencing of the 16S rRNA gene of the isolates showed that there was 100% identity with the 16S rRNA gene sequence of the isolate of S. aureus (GenBank accession no. JN102565), confirming that the isolate was S. aureus.
To further investigate the genetic basis of the strain, multilocus sequence typing (MLST), a method that uses seven housekeeping genes (arcC, aroE, glpF, gmk, pta, tpi and yqiL) for genetic identification, and result was assigned by comparison with the S. aureus MLST database (http://www.mlst.net/). The staphylococcal chromosomal cassette (SCC) mec type (I-V) was also determined. The spa type was analyzed by sequencing of the PCR product of the spa gene, and the spa type was assigned using an online spa database (http://www.spaserver.ridom.de/). Detection of the accessory gene regulator (agr) allele group was according to PCR and sequencing. Likewise, the antimicrobial drug resistance genes (mecA, msrA, msrB, ermA, ermB, ermC and blaZ) were determined. The presence of gene encoding PVL (lukF/lukS) and other virulence related genes (sea, seb, sec, sed, see, seg, seh, sei, sej, sem, sen, seo, sek, sel, sep, seq, hla, hlb, hld, hlg, hlg2, eta, etb, lukE, lukM, bsaA and edin,) were investigated by PCR. The presence of adhesion genes (cna, clfA, clfB, fnbA, efb and icaA) were also determined by PCR and sequencing.
The CA-MRSA isolate was typed as sequence type (ST) 630 SCCmecV with spa-type t4549, agrI/IV and was PVL-negative. We confirmed the presence of mecA, ermC and blaZ genes by PCR and sequencing. The genome of the MRSA isolate encoded three hemolysin genes (hlb, hld and hlg2) and five adhesion genes (clfA, clfB, fnbA, efb and icaA) (Fig. 1E).