The Mitochondrial Isoform of FASTK Modulates Nonopsonic Phagocytosis of Bacteria by Macrophages via Regulation of Respiratory Complex I.

Phagocytosis is a pivotal process by which innate immune cells eliminate bacteria. In this study, we explore novel regulatory mechanisms of phagocytosis driven by the mitochondria. Fas-activated serine/threonine kinase (FASTK) is an RNA-binding protein with two isoforms, one localized to the mitochondria (mitoFASTK) and the other isoform to cytosol and nucleus. The mitoFASTK isoform has been reported to be necessary for the biogenesis of the mitochondrial ND6 mRNA, which encodes an essential subunit of mitochondrial respiratory complex I (CI, NADH:ubiquinone oxidoreductase). This study investigates the role and the mechanisms of action of FASTK in phagocytosis. Macrophages from FASTK─/─ mice exhibited a marked increase in nonopsonic phagocytosis of bacteria. As expected, CI activity was specifically reduced by almost 50% in those cells. To explore if decreased CI activity could underlie the phagocytic phenotype, we tested the effect of CI inhibition on phagocytosis. Indeed, treatment with CI inhibitor rotenone or short hairpin RNAs against two CI subunits (NDUFS3 and NDUFS4) resulted in a marked increase in nonopsonic phagocytosis of bacteria. Importantly, re-expression of mitoFASTK in FASTK-depleted macrophages was sufficient to rescue the phagocytic phenotype. In addition, we also report that the decrease in CI activity in FASTK─/─ macrophages is associated with an increase in phosphorylation of the energy sensor AMP-activated protein kinase (AMPK) and that its inhibition using Compound C reverted the phagocytosis phenotype. Taken together, our results clearly demonstrate for the first time, to our knowledge, that mitoFASTK plays a negative regulatory role on nonopsonic phagocytosis of bacteria in macrophages through its action on CI activity.

Molecular epidemiology of methicillin-resistant Staphylococcus aureus in a Spanish hospital over a 4-year period: clonal replacement, decreased antimicrobial resistance, and identification of community-acquired and livestock-associated clones.

This study was carried out on 189 methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates collected in a third-level hospital in Valladolid, Spain, between 2005 and 2008 in order to investigate the changes in molecular epidemiology and genetic backgrounds associated with the changes in resistance phenotypes produced over time. The MRSA isolates were classified as belonging to 10 different clones, including the identification of a novel MRSA clone, ST2422-MRSA-IV, belonging to CC121; 1 CA-MRSA strain from a USA300 clone; another from ST97-MRSA-IV, associated with clones adapted to livestock (LA-MRSA); and 2 strains belonging to a new spa type (t10258) related to the ST8-MRSA-IV clone. Sixty-two percent of the strains belonging to Spanish-prevalent MRSA sequence type ST125 harboured composite or multiple SCCmec elements including SCCmec type IV plus ccrA/B4 (ST125-SCCmec IV/VI). In the years studied, it was observed that ST125-SCCmec IV/VI replaced the multiresistant ST228-SCCmec I previously prevalent, and, as a consequence, decreased gentamicin and clindamycin resistance was further observed.

Clonal nature and diversity of resistance, toxins and adhesins genes of meticillin-resistant Staphylococcus aureus collected in a Spanish hospital.

In this study we determined the prevalence of genes coding for antimicrobial resistance, toxins, enzymes, immunoevasion and adhesins factors among 189 meticillin-resistant Staphylococcus aureus (MRSA) strains isolated from a third level hospital in Valladolid (Spain) between 2005 and 2008 in order to examine the relationship between these pathogenicity determinants, both individually and in combination, and the genetic background of main MRSA strains that are presents in Spanish hospitals. MRSA isolates were first characterised epidemiologically by a combination of molecular typing strategies like spa, SCCmec and multilocus sequence typing, and then, a cluster analysis based on pathogenicity factors genes was performed according to the hybridisation pattern of 65 virulence, 36 resistance, 15 adhesins, and 11 set/ssl genes on a Diagnostic DNA microarray (Alere StaphyType DNA microarray Jena, Germany). CC5-agr type II [ST125-SCCmecIV/VI (32.2%) or ST125-IV (19.1%), ST228-I (19.1%), ST146-IV (13.7%) and ST5- IV (0.5%)] isolates was widely distributed. CC8-agr type I [ST8-IV (11.5%), USA300 clone (0.5%), and ST239-III (1.1%)]; CC45-agr type II [ST45- IV (1.6%)], and the CC97-agr type I [ST97-IV] were also detected. We identified 42 different resistance genes profiles, 22 set/ssl genes profiles, and 91 different virulence profiles. However although the high genetic diversity of MRSA strains, mainly with respect to virulence factors genes, the results of the simultaneous assessment of resistance and virulence genes and the genetic background illustrated a correspondence relationship (p<0.001) between the different clones and same resistance and virulence genes or clusters of them. During the study period we observed changes in molecular epidemiology of MRSA isolates and as a consequence we report the changes of the resistance and virulence potential of MRSA strains produced over time in our institution.

Identification of Brucella by MALDI-TOF mass spectrometry. Fast and reliable identification from agar plates and blood cultures.

BACKGROUND: MALDI-TOF mass spectrometry (MS) is a reliable method for bacteria identification. Some databases used for this purpose lack reference profiles for Brucella species, which is still an important pathogen in wide areas around the world. We report the creation of profiles for MALDI-TOF Biotyper 2.0 database (Bruker Daltonics, Germany) and their usefulness for identifying brucellae from culture plates and blood cultures. METHODOLOGY/PRINCIPAL FINDINGS: We created MALDI Biotyper 2.0 profiles for type strains belonging to B. melitensis biotypes 1, 2 and 3; B. abortus biotypes 1, 2, 5 and 9; B. suis, B. canis, B ceti and B. pinnipedialis. Then, 131 clinical isolates grown on plate cultures were used in triplicate to check identification. Identification at genus level was always correct, although in most cases the three replicates reported different identification at species level. Simulated blood cultures were performed with type strains belonging to the main human pathogenic species (B. melitensis, B. abortus, B. suis and B. canis), and studied by MALDI-TOF MS in triplicate. Identification at genus level was always correct. CONCLUSIONS/SIGNIFICANCE: MALDI-TOF MS is reliable for Brucella identification to the genus level from culture plates and directly from blood culture bottles.

Staphylococcus aureus oxacilina resistente (MRSA): incidência de cepas adquiridas na comunidade (CA-MRSA) e importÂncia da pesquisa e descolonização em hospital / Resistent oxacilin Staphylococcus aureus (MRSA): incidence of cepas acquired in the community (CA-MRSA) and importance of research and descolonization in hospital

Staphylococcus aureus resistente à oxacilina (MRSA) é um dos maiores problemas clínicos e epidemiológicos em infecções nosocomiais. Porém, as infecções por MRSA não se limitam ao ambiente hospitalar, pois S. aureus resistente à oxacilina, comunidade adquirido, (CA-MRSA) está emergindo e pode atingir pessoas sem fatores de risco, como hospitalizações prévias. Para estimara extensão de CA-MRSA determinou-se a prevalência da colonização nasal por S. aureus em 100 amostras obtidas de uma população sem fatores de risco. As amostras foram semeadas em Agar Sangue e identificadas com provas compatíveis. Nos isolados de S. aureus aplicou-se o teste de sensibilidade aos antimicrobianos por disco-difusão (Kirby-Bauer). Encontrou-se uma freqüência de40% dessa bactéria, sendo 7,5% CA-MRSA. Quanto ao perfil de sensibilidade, S. aureus apresentou tendência à resistência aos lactâmicos e macrolídeos e sensibilidade diminuída à tetraciclina, licosaminas, aminoglicosídeos e cloranfenicol, sendo totalmente sensível aos glicopeptídeos. Os isolados de CA-MRSA apresentaram resistência apenas aos alfa-lactâmicos e macrolídeos. A prevalência da colonização por MRSA em pessoas saudáveis na comunidade potencializa a necessidade de uma maior vigilância do controle de infecção hospitalar, já que esta população que carreia CA-MRSA pode tornar-se reservatório bacteriano no ambiente hospitalar.

Resistant oxacilina Staphylococcus aureus (MRSA) is one of the biggest clinical and epidemiologic problems in nosocomial infections. However, the infections by MRSA are not limited to hospital environment; because community acquired resistant oxacilin S. aureus (CA-MRSA) is emerging and can reach people without risk factors, as previous hospitalizations. To estimate the extension of CA-MRSA prevalence nasal settling for S. aureus was determined in 100 samples of a population without risk factors. The samples were sown in Agar Blood and identified with compatible tests. In isolated of S. aureus the test of sensitivity to antimicrobials for record-diffusion was applied (Kirby-Bauer). A frequency of 40% of these bacteria, being 7.5% CA-MRSA was found. As the sensitivity profile, S. aureus presented trend to resistance to ‚-lactamics and Macrolides and sensitivity diminished to tetracycline, lycosamines, aminoglicosides and chloramphenicol, being total sensible to the glycopeptides. The isolated of CA-MRSA presented resistance only to the ‚-lactamics and macrolides. The prevalence of the settling for MRSA in healthful people in the community increases the need of a bigger control of hospital infection, once this population that carries CA-MRSA may become bacterial reservoir in hospital environment.