Use of the Phoenix automated system for identification of Streptococcus and Enterococcus spp.

The Phoenix system (Becton Dickinson Diagnostic Systems, Sparks, MD) was evaluated for identification (ID) to the species level of streptococci and enterococci. Two hundred clinical isolates were investigated: beta-hemolytic streptococci (n = 50), Streptococcus pneumoniae organisms (n = 46), viridans group streptococci (n = 31), Enterococcus faecium (n = 36), Enterococcus faecalis (n = 25), and other catalase-negative cocci (n = 12). The API system (bioMérieux, Marcy l'Etoile, France) was used as a comparator. Molecular methods (sequencing of 16S rRNA and zwf and gki genes and ddl gene amplification) were used to investigate discordant results. Upon resolution of discrepancies, correct species ID was achieved by the Phoenix system for 121/129 (93.8%) streptococci and 63/70 (90.0%) enterococci. Excellent results were obtained for S. pneumoniae (45/45) and beta-hemolytic streptococci (49/50). With regard to viridans streptococci, the accuracy of the Phoenix system was 83.9%. Among the latter organisms, the best performance was obtained with isolates of the Streptococcus sanguinis group and Streptococcus anginosus group; problems were instead encountered with the Streptococcus mitis group. Four E. faecium and three E. faecalis isolates were misidentified as Enterococcus casseliflavus/Enterococcus gallinarum or Enterococcus durans. Thus, these isolates were identified only at the genus level. Compared with commercially available systems, the Phoenix system appears a reliable diagnostic tool for identifying clinically relevant streptococci and enterococci. The SMIC/ID-2 panel proved particularly effective for beta-hemolytic streptococci and pneumococci.

Failure of levofloxacin treatment in community-acquired pneumococcal pneumonia.

BACKGROUND: Streptococcus pneumoniae is the leading cause of community-acquired pneumonia (CAP). High global incidence of macrolide and penicillin resistance has been reported, whereas fluoroquinolone resistance is uncommon. Current guidelines for suspected CAP in patients with co-morbidity factors and recent antibiotic therapy recommend initial empiric therapy using one fluoroquinolone or one macrolide associated to other drugs (amoxicillin, amoxicillin/clavulanate, broad-spectrum cephalosporins). Resistance to fluoroquinolones is determined by efflux mechanisms and/or mutations in the parC and parE genes coding for topoisomerase IV and/or gyrA and gyrB genes coding for DNA gyrase. No clinical cases due to fluoroquinolone-resistant S. pneumoniae strains have been yet reported from Italy. CASE PRESENTATION: A 72-year-old patient with long history of chronic obstructive pulmonary disease and multiple fluoroquinolone treatments for recurrent lower respiratory tract infections developed fever, increased sputum production, and dyspnea. He was treated with oral levofloxacin (500 mg bid). Three days later, because of acute respiratory insufficiency, the patient was hospitalized. Levofloxacin treatment was supplemented with piperacillin/tazobactam. Microbiological tests detected a S. pneumoniae strain intermediate to penicillin (MIC, 1 mg/L) and resistant to macrolides (MIC >256 mg/L) and fluoroquinolones (MIC >32 mg/L). Point mutations were detected in gyrA (Ser81-Phe), parE (Ile460-Val), and parC gene (Ser79-Phe; Lys137-Asn). Complete clinical response followed treatment with piperacillin/tazobactam. CONCLUSION: This is the first Italian case of community-acquired pneumonia due to a fluoroquinolone-resistant S. pneumoniae isolate where treatment failure of levofloxacin was documented. Molecular analysis showed a group of mutations that have not yet been reported from Italy and has been detected only twice in Europe. Treatment with piperacillin/tazobactam appears an effective means to inhibit fluoroquinolone-resistant strains of S. pneumoniae causing community-acquired pneumonia in seriously ill patients.

Poliovirus type 1 infection of murine PRNP-knockout neuronal cells.

Transfection of the prion protein gene (Prnp) into prion-deficient mouse cells was shown to reduce the replication of coxsackievirus B3, an enterovirus. Because mice can be susceptible to poliovirus infection by parenteral routes, the authors tested the susceptibility to poliovirus-1 (PV-1) of a panel of murine neuronal cell lines differing in their ability to express Prnp. The investigated cell lines (prionless HpL3.4 cells, HpL3.4 cells transfected with a Prnp vector, HpL3.4 cells transfected with a void vector, wild-type Hw3.5 Prnp(+/+) cells) expressed the murine homologue (Tage4) of human poliovirus receptor (CD155/hPVR). PV-1 infection of Prnp(-/-) HpL3.4 cells resulted in the production of high viral titers, though viral antigens could be detected in only 0.5% to 2% of cells. Wild-type Prnp(+/+) cells and prionless cells transfected with the Prnp gene were not permissive to PV-1. Results of viral titration and immunofluorescence were confirmed by conventional polymerase chain reaction (PCR) and quantitative real-time PCR. Exposure to PV-1 had no influence on the gene expression profile of Prnp(+/+) cells. In contrast, PV-1 infection was associated with upregulation of several genes in permissive Prnp(-/-) cell cultures: type I interferon (IFN) genes, IFN-related developmental regulator 1 (IFNRD1), tumor necrosis factor superfamily member 13b (TNFSF13b), interleukin (IL) - 7, granulocyte/macrophage colony-stimulating factors (CSFs), hepatocyte growth factor (HGF), vascular endothelial growth factor-A, transforming growth factors beta1 and beta3 (TGFb1, TGFb3), as well as a variety of bone morphogenetic proteins endowed with neuroprotective activity. Distinction of permissive from nonpermissive neuronal cells on the basis of Prnp expression suggests that prion-deficient mice could represent an extraordinarily sensitive animal model for poliovirus infection.

Culture of skeletal myoblasts from human donors aged over 40 years: dynamics of cell growth and expression of differentiation markers.

BACKGROUND: Local myogenesis, neoangiogenesis and homing of progenitor cells from the bone marrow appear to contribute to repair of the infarcted myocardium. Implantation into heart tissues of autologous skeletal myoblasts has been associated with improved contractile function in animal models and in humans with acute myocardial ischemia. Since heart infarction is most prevalent in individuals of over 40 years of age, we tested whether culture methods available in our laboratory were adequate to obtain sufficient numbers of differentiated skeletal myoblasts from muscle biopsy specimens obtained from patients aged 41 to 91. METHODS AND RESULTS: No matter of donor age, differentiated skeletal muscle cells could be produced in vitro in amounts adequate for cellular therapy (>/=300 millions). Using desmin as a cytoplasmic marker, about 50% cultured cells were differentiated along myogenic lineages and expressed proteins proper of skeletal muscle (myosin type I and II, actin, actinin, spectrin and dystrophin). Cytogenetic alterations were not detected in cultured muscle cells that had undergone at least 10 population doublings. Molecular methods employed for the screening of persistent viral infections evidenced that HCV failed to replicate in muscle cells cultured from one patient with chronic HCV infection. CONCLUSION: The proposed culture methods appear to hold promise for aged patients not only in the field of cardiovascular medicine, but also in the urologic and orthopedic fields.

Proliferative activity of extracellular HIV-1 Tat protein in human epithelial cells: expression profile of pathogenetically relevant genes.

BACKGROUND: Tat is being tested as a component of HIV vaccines. Tat activity has been mainly investigated on cells of lymphoid/hematopoietic lineages. HIV-1, however, is known to infect many different cells of both solid organs and mucosal surfaces. The activity of two-exon (aa 1-101) and synthetic (aa 1-86) Tat was studied on mammary and amniotic epithelial cells cultured under low serum conditions. RESULTS: small concentrations of Tat (100 ng/ml) stimulated cell proliferation. Tat antibodies neutralized the mitogenic Tat activity. Changes of gene expression in Tat-treated cells were evaluated by RT-PCR and gene-array methods. Within 4 hours of treatment, exposure to Tat is followed by up-regulation of some cell cycle-associated genes (transcription factors, cyclin/cdk complexes, genes of apoptotic pathways) and of genes relevant to HIV pathogenesis [chemokine receptors (CXCR4, CCR3), chemotactic cytokines (SDF-1, RANTES, SCYC1, SCYE1), IL6 family cytokines, inflammatory cytokines, factors of the TGF-beta family (TGFb, BMP-1, BMP-2)]. Up-regulation of anti-inflammatory cytokines (IL-10, IL-19, IL-20), a hallmark of other persistent viral infections, was a remarkable feature of Tat-treated epithelial cell lines. CONCLUSION: extracellular Tat is mitogenic for mammary and amniotic epithelial cells and stimulates the expression of genes of pathogenetic interest in HIV infection. These effects may favor virus replication and may facilitate the mother-to-child transmission of virus.