Outbreak investigation including molecular characterization of community associated methicillin-resistant Staphylococcus aureus in a primary and secondary school in Eastern Switzerland

At our tertiary children's hospital, infections with newly detected methicillin-resistant Staphylococcus aureus (MRSA) among children attending primary (age 6-12 years) and secondary school (age 13-16 years) nearly doubled in 2018 compared to previous years. This observation initiated an epidemiological outbreak investigation including phenotypic (susceptibility testing) and genotypic (whole genome sequencing) characterization of the isolates. In addition, a cross-sectional study was conducted to determine source of the outbreak, colonization frequency and to identify risk factors for transmission using a questionnaire. As a result, 49 individuals were detected with 57 corresponding isolates. Based on the case definition combined with whole genome sequencing, a core cluster was identified that shared common genetic features and a similar antimicrobial susceptibility pattern (efflux-mediated macrolide resistance, tetracycline susceptibility along with presence of Panton-Valentine leukocidin). Epidemiologic evaluation identified a distinct school as a common risk factor. However, the source of the clustered infections within that school could not be further specified. No further cases could be detected after decolonization of infected and colonized children

Myxoma virus infection promotes NK lysis of malignant gliomas in vitro and in vivo.

Myxoma virus (MYXV) is a well-established oncolytic agent against different types of tumors. MYXV is also known for its immunomodulatory properties in down-regulating major histocompatibility complex (MHC) I surface expression (via the M153R gene product, a viral E3-ubiquitin ligase) and suppressing T cell killing of infected target cells. MHC I down-regulation, however, favors NK cell activation. Brain tumors including gliomas are characterized by high MHC I expression with impaired NK activity. We thus hypothesized that MYXV infection of glioma cells will promote NK cell-mediated recognition and killing of gliomas. We infected human gliomas with MYXV and evaluated their susceptibility to NK cell-mediated cytotoxicity. MYXV enhanced NK cell-mediated killing of glioma cells (U87 cells, MYXV vs. Mock: 51.73% vs. 28.63%, P = .0001, t test; U251 cells, MYXV vs. Mock: 40.4% vs. 20.03%, P .0007, t test). Using MYXV M153R targeted knockout (designated vMyx-M153KO) to infect gliomas, we demonstrate that M153R was responsible for reduced expression of MHC I on gliomas and enhanced NK cell-mediated antiglioma activity (U87 cells, MYXV vs. vMyx-M153KO: 51.73% vs. 25.17%, P = .0002, t test; U251 cells, MYXV vs. vMyx-M153KO: 40.4% vs. 19.27, P = .0013, t test). Consequently, NK cell-mediated lysis of established human glioma tumors in CB-17 SCID mice was accelerated with improved mouse survival (log-rank P = .0072). These results demonstrate the potential for combining MYXV with NK cells to effectively kill malignant gliomas

MYXV down-regulates MHC I in U87 <i>in vivo</i>.

<p>(<b>A</b>) Immunohistochemistry was performed on paraffin sections of mice brains with established U87 tumors that were treated with indicated viruses for 48 h. Hematoxylin and Eosin (H&E) stain shows tumor, with the arrows indicating tumor location in the brain. Brown M-T7 stain represents MYXV early protein staining within the tumor. Photomicrographs were magnified 25X and 200X. (<b>B</b>) Western Blot of protein extracts from the U87 tumor-bearing hemisphere of mice brains. As control for human HLA-ABC Ab specificity, the contralateral hemisphere without tumor was used. Luciferase expression shows tumor, β-actin serves as protein loading control.</p

MYXV-enhanced NK cell-mediated clearance of U87 tumor results in prolonged survival.

<p>(<b>A</b>) Bioluminescence imaging (BLI) to monitor growth of orthotopic luciferase-expressing human U87 xenografts in CB-17 SCID mice. A total of 1.5×10⁵ IL-2-activated NK cells were administered intratumorally, 48 h (day 24 post tumor implantation) following intratumoral MYXV treatment (5×10⁶ ffu/mouse, day 22 post tumor implantation); PBS =  phosphate-buffered saline. A heat map indicating tumor size in 4 to 5 mice in each group is shown. (<b>B</b>) Luminescent intensities from U87 xenografts were quantified on day 0, 7 and 14 post-treatment (day 22, 29, and 36 post tumor implantation respectively) to monitor tumor clearance. Results show the mean fold-increase in tumor size after treatment (calculated as luminescent intensity on day 22/day 22, day 29/day 22, day 36/day 22 post tumor implantation). A statistically significant reduction in tumor size was observed in the MYXV+NK group compared with the other groups, 14 days post-treatment (36 days post tumor implantation, NK vs. MYXV+NK: 6.58±1.34 vs. 0.33±0.09 fold increase, difference = 6.25±1.34, 95% CI = 3.153 to 9.350, P1 = .0016; MYXV vs. MYXV+NK: 3.96±1.35 vs. 0.33±0.09 fold increase, difference = 3.63±1.35, 95% CI = 0.5121 to 6.750, P2 = .0277). Also, 7 days post-treatment (29 days post tumor implantation), a significant reduction was observed only in MYXV vs. MYXV+NK (3.66±0.58 vs. 0.66±0.07 fold increase, difference = 3.00±0.58, 95% CI = 1.663 to 4.344, P3 = .0009). <b>Error bars</b> represent 95% confidence intervals. (<b>C</b>) Survival curves showing that mice treated with MYXV+NK survived significantly longer than PBS (median survival for PBS-treated group vs. MYXV+NK was 51.5 days vs. 100%, hazard ratio (HR) = 30.89, 95% CI of HR = 3.333 to 286.2, log-rank P1 = .0025) or NK-treated mice (median survival for NK-treated group vs. MYXV+NK was 67 days vs. 100%, HR = 20.60, 95% CI of HR = 3.007 to 141.1, log-rank P2 = .0021) but not with MYXV-treated mice (median survival for MYXV vs. MYXV+NK-treated group was 60% vs. 100%, HR = 8.366, 95% CI of HR = 0.5188 to 134.9, log-rank P2 = .1343). Experiment was terminated on day 159 post tumor implantation.</p

MYXV down-regulates Nectin-2 via M153R.

<p>(<b>A</b>) Flow cytometric analysis to determine the role of M153R in Nectin-2 down-regulation in glioma cells. Columns represent means of triplicate of one experiment. <b>Error bars</b> represent 95% confidence intervals. (<b>B</b>) Western Blot showing down-regulation of HLA-ABC and Nectin-2 by MYXV. DV =  dead virus control; M-T7, an early gene product of MYXV, shows MYXV and vMyx-M153KO infection while β-actin serves as loading control.</p

MYXV enhances NK cell-mediated killing of glioma cells via MHC I down-regulation.

<p>M153R-mediated down-regulation of MHC I or blockade of MHC I with mAb results in enhanced NK cell-mediated killing of U87 (<b>A</b>) or U251 (<b>B</b>) target cells. For mAb blocking, mock-infected or virus-infected cells were pre-incubated with 20 µg/ml MHC class I Ab (W6/32) or isotype-matched control Ab for 30 min before co-culture with NK cells. Columns represent means of triplicate of one representative experiment. <b>Error bars</b> represent 95% confidence intervals. Supernatant was collected from 4 h co-cultures of NK cells and Mock- or MYXV- or vMyx-M153KO-infected U87 (<b>C</b>) or U251 (<b>D</b>) target cells, that were pre-incubated with either 20 µg/ml MHC class I Ab (W6/32) or isotype-matched control, and analyzed by ELISA for granzyme B release. Baseline NK cells granzyme B release was 1624 pg/ml. The result shown is granzyme B release in NK/glioma co-culture minus baseline granzyme B release by NK cells alone. Columns represent means of triplicate of one representative experiment. <b>Error bars</b> represent 95% confidence intervals.</p

M153R mediates enhanced NK cell-mediated clearance of U87 tumor with prolonged survival.

<p>(<b>A</b>) Survival study to compare oncolytic capacity of MYXV with vMyx-M153KO. Dead virus (DV) was used as control. CB-17 SCID mice with established tumors were treated with either DV (n = 4) or MYXV (5×10⁶ ffu/ml, n = 5) or vMyx-M153KO (5×10⁶ ffu/ml, n = 5) on day 19 post tumor implantation. Log-rank P1 (.0110) represents MYXV vs. DV, log-rank P2 (.0250) represents vMyx-M153KO vs. DV, log-rank P3 (.9003) represents MYXV vs. vMyx-M153KO. (<b>B</b>) A heat map indicating tumor size in 5 to 6 mice in each group is shown. CB-17 SCID mice with established tumors were treated with either NK or a combination of MYXV+NK or vMyx-M153KO+NK. NK cells (1.5×10⁵ NK cells) were administered intratumorally, 48 h (day 16 post tumor implantation) following intratumoral MYXV (5×10⁶ ffu/ml) or vMyx-M153KO (5×10⁶ ffu/ml) treatment (day 14 post tumor implantation). Images show accelerated NK clearance in MYXV+NK-treated group compared with NK or vMyx-M153KO+NK-treated group. (<b>C</b>) Survival curves showing that mice treated with MYXV+NK survived longer than NK-treated mice (HR = 31.26, 95% CI of HR = 4.097 to 238.5, log-rank P1 = .0009) and vMyx-M153KO+NK-treated mice (HR = 0.05532, 95% CI of HR = 0.006701 to 0.4567, log-rank P2 = .0072).</p

MYXV down-regulates MHC I via M153R.

<p>Dot plot showing the role of M153R in HLA-ABC down-regulation in U87 (<b>A</b>) and U251 (<b>C</b>) glioma cells and show percentage of cells infected with MYXV and vMyx-M153KO. HLA-ABC or isotype indicates number of cells stained with HLA-ABC or isotype Ab (detected on the FL2 channel; i =  percentage of cells with Ab staining); GFP indicates number of cells infected with MYXV or vMyx-M153KO (detected on the FLI channel; ii =  percentage of cells infected). Isotype control staining of mock-infected cells was used to set the gating. A representative of at least 3 independent experiments is shown. Flow cytometric analysis of U87 (<b>B</b>) and U251 (<b>D</b>) in triplicates and quantified. Columns represent means of triplicate of one representative experiment of two. <b>Error bars</b> represent 95% confidence intervals, DV =  dead virus, vMyx-M153KO =  mutated MYXV lacking M153R.</p