lunes, 23 de abril de 2012

MRSA epidemic linked to a quickly spreading colonization and virulence determinant : Nature Medicine : Nature Publishing Group

MRSA epidemic linked to a quickly spreading colonization and virulence determinant : Nature Medicine : Nature Publishing Group


MRSA epidemic linked to a quickly spreading colonization and virulence determinant

Journal name:
Nature Medicine
Year published:
(2012)
DOI:
doi:10.1038/nm.2692
Received
Accepted
Published online
The molecular processes underlying epidemic waves of methicillin-resistant Staphylococcus aureus (MRSA) infection are poorly understood1. Although a major role has been attributed to the acquisition of virulence determinants by horizontal gene transfer2, there are insufficient epidemiological and functional data supporting that concept. We here report the spread of clones containing a previously extremely rare3, 4 mobile genetic element–encoded gene, sasX. We demonstrate that sasX has a key role in MRSA colonization and pathogenesis, substantially enhancing nasal colonization, lung disease and abscess formation and promoting mechanisms of immune evasion. Moreover, we observed the recent spread of sasX from sequence type 239 (ST239) to invasive clones belonging to other sequence types. Our study identifies sasX as a quickly spreading crucial determinant of MRSA pathogenic success and a promising target for therapeutic interference. Our results provide proof of principle that horizontal gene transfer of key virulence determinants drives MRSA epidemic waves.

Figures at a glance

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  1. Figure 1: Spread of sasX-positive clones in China.
    Spread of sasX-positive clones in China.
    (a) A total of 807 isolates from three teaching hospitals in eastern China were analyzed for sequence type, methicillin resistance and presence of sasX. (b) Percentage of sasX-positive among MSSA and MRSA isolates. Statistical analysis is by χ2 test of all MRSA isolates (average curve, shown in black). The P value including all (MRSA and MSSA) isolates is 0.0026. (c) Percentage of sasX-positive isolates in sequence types other than ST239. Statistical analysis is by Fisher's exact test of all isolates (average curve, shown in black).
  2. Figure 2: The SasX surface protein facilitates nasal colonization.
    The SasX surface protein facilitates nasal colonization.
    (a) Detection of SasX with SasX-specific polyclonal antiserum in an indirect immunofluorescence assay. (b) Immunoblot detection of SasX in different subcellular fractions. Black arrows point to the putative SasX protein. Note that surface proteins in this assay run higher than their predicted molecular weights in the processed forms, owing to the linkage to remaining peptidoglycan parts. W, whole cell preparation; S, surface protein fraction; C, cytoplasmic fraction; E, extracellular fraction. (c) Adherence of wild-type, isogenic sasX-mutant, sasX-complemented and control strains to human nasal epithelial cells in vitro. (d) Blocking of adherence to nasal epithelial cells by recombinant GST-SasX fusion protein. Purified GST protein alone and denatured GST-SasX fusion protein were used as controls. (e) Nasal colonization model. Female imprinting control region (ICR) mice (n = 10 per group in the separate model; n = 15 per group in the competition model) were inoculated intranasally with 1 × 107 colony-forming units (CFUs) of wild-type or isogenic sasX-mutant strains, and bacteria were counted in mice that were killed at 7 d after inoculation. ST239 HS770 was used for the separate and ST239 HS663 for the competition model. Equal amounts of wild-type and deletion mutant bacteria were mixed for the competition model inoculum. Statistical analyses in ce are by unpaired t tests. All error bars show s.e.m.
  3. Figure 3: SasX promotes bacterial aggregation and mechanisms of immune evasion.
    SasX promotes bacterial aggregation and mechanisms of immune evasion.
    (a) Microscopic images of 16-h cultures of ST239 wild-type, isogenic sasX-mutant, sasX-complemented and control strains. (b) Biofilm formation. Cultures were grown for 24 h in microtiter plates. Afterward, biofilms were measured using crystal violet staining. TBS, Tris-buffered saline (buffer control). (c) Primary attachment. The same experiment was performed as in b, but with only 1 h growth. (d) Phagocytosis assay. Phagocytosis by human neutrophils of ST239 or the isogenic sasX-mutant strain was examined using microscopy. (e) Microscopic examination of phagocytosis. Slides were stained using a modified Wright-Giemsa stain. At t = 90 min, lysed neutrophils were occasionally detected in the ST239 wild-type samples. The 90-min ST239 image shows such a lysed neutrophil with adherent S. aureus bacteria. (f) Survival in human blood at t = 90 min. (g) Neutrophil killing. Bacteria from mid-logarithmic growth phase were washed and incubated with human neutrophils at a 100:1 ratio. Neutrophil lysis was determined by measuring release of lactate dehydrogenase (LDH). Statistical analyses in bd, f, g are by unpaired t tests. All error bars show s.e.m.
  4. Figure 4: SasX is a key virulence determinant during MRSA skin and lung infection.
    SasX is a key virulence determinant during MRSA skin and lung infection.
    (a) Skin infection model. Outbred, immune-competent, hairless mice were inoculated with 1 × 109 CFUs of the ST239 or ST5 wild-type and isogenic sasX-mutant strains. Developing abscess areas are shown (see representative images on the right). Differences between corresponding wild-type and mutant abscess sizes were statistically significant at every time point (unpaired t tests, P < 0.05). (b) Lung infection model. Female ICR mice were inoculated in the nose with 1 × 109 CFUs per 20 μl of the ST239 or ST5 wild-type or isogenic sasX-mutant strains. Mice were killed at day 5 after infection for subsequent analyses. Lung wet weight/body weight ratios are shown. (c) Lung infection model, concentration of TNF-α in lung tissue samples. (d) Skin infection model, H&E-stained tissue samples. (e) Lung infection model, H&E-stained tissue samples. Note increased number of infiltrating inflammatory cells in wild-type sample images in d and extensive inflammation accompanied by hemorrhagic infiltration and disruption of pulmonary architecture in wild-type sample images in e. Statistical analyses in b,c are by unpaired t tests. All error bars show s.e.m.
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