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G Orefici

Publications and source records attributed to G Orefici.

At least 55 records · Page 3Linked to original sources
PubMed1997

Non-inducible, mainly cell-associated beta-lactamase from Nocardia asteroides strain 108.

The beta-lactamase of the soil-borne strain 108 (parental strain) of Nocardia asteroides is a non-inducible enzyme mainly associated with the cells; it can be efficiently extracted by ultrasonication and SDS treatment. Crude enzyme preparations showed penicillinase and cephalosporinase activity. The kinetics of beta-lactamase production and in-vitro susceptibility to combinations of beta-lactam antibiotics plus beta-lactamase inhibitors have been studied in two stable overproducer mutants (A14 and B1) obtained by mutagenization of the parental strain with nitrosoguanidine. The cell-associated enzyme increased with bacterial growth in parental and mutant strains and was particularly abundant in stationary phase cells. The beta-lactamase inhibitors sulbactam and clavulanic acid decreased MIC values of penicillins more efficiently in the parental strain than in mutants, thus indicating some involvement of the enzyme in the resistance of N. asteroides strain 108 to beta-lactam antibiotics.

Amoxicillin↗
PubMed1997

Soluble antigens from group B streptococci induce cytokine production in human blood cultures.

Group B streptococcal antigens stimulated tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), and IL-6 production in human blood cultures in a concentration- and time-dependent fashion. The minimal concentrations of type-specific polysaccharides, lipoteichoic acid, and group-specific polysaccharide required to produce these effects were, respectively, 0.01, 1, and 10 microg/ml. Cell separation experiments indicated that monocytes were the cell type mainly responsible for cytokine production. Time course studies indicated that TNF-alpha was released before the other cytokines. TNF-alpha, however, did not appear to directly induce IL-1beta, as shown by blockade experiments with anti-TNF-alpha antibodies. IL-6 levels were moderately but significantly decreased by anti-TNF-alpha. These data indicate that several products from group B streptococci are able to directly stimulate human monocytes to release TNF-alpha, IL-1beta, and IL-6. These findings may be clinically relevant, since proinflammatory cytokines can mediate pathophysiologic changes during sepsis.

Blood↗
PubMed1997

Serological markers predicting tuberculosis in human immunodeficiency virus-infected patients.

SETTING: Human immunodeficiency virus (HIV)-positive patients retrospectively identified at the Hospital of Bari, Italy, with diagnosis of tuberculosis (TB) (n = 30) or non-tuberculous pneumonia (n = 29). Serum samples drawn at the time of diagnosis and one year before. Anti-purified protein derivative (PPD) and anti-diacyltrehalose (DAT) serum antibodies quantified by ELISA assay. OBJECTIVE: Since TB patients with HIV infection may present with elevated serum antibodies against Mycobacterium tuberculosis, we hypothesized that TB-specific antibody markers might predict TB in these subjects. DESIGN: A retrospective study was designed to assess the presence of M. tuberculosis-specific antibodies in HIV-positive patients developing TB. RESULTS: Of 30 HIV-positive TB patients, 24 (80%) had anti-PPD or anti-DAT antibodies at the time of TB diagnosis, and 20 (67%) one year before. In a sub-population of 16 of the 30 HIV-positive subjects, positivity for anti-PPD or anti-DAT antibodies one year before TB diagnosis was higher (11/16, 69%) than for the PPD skin test (4/16, 25%, P < 0.01). Antibody tests were specific for TB since positivity rates were lower both in patients with non-tuberculous pneumonia (P < 0.01) and in those with M. avium infection (P < 0.05). CONCLUSION: Antibody markers may predict TB in HIV-positive subjects, including those with negative PPD skin test.

AIDS-Related Opportunistic Infections↗
PubMed1996

Late acquisition of hyporesponsiveness to lipopolysaccharide by Mycobacterium avium-infected human macrophages in producing tumor necrosis factor-alpha but not interleukin-1 beta and -6.

To investigate whether infection with Mycobacterium avium modifies the cytokine response of human macrophages (Mphi) to lipopolysaccharide (LPS), the release of interleukin (IL)-1 beta, IL-6, and tumor necrosis factor (TNF)-alpha was determined in infected and uninfected Mphi, unstimulated or stimulated with LPS. In unstimulated Mphi, the release of IL-1 beta and IL-6 increased with the progress of infection while that of TNF-alpha progressively decreased. When Mphi were stimulated with LPS, IL-1 beta and IL-6 levels were always higher in infected than in uninfected cells, but levels of TNF-alpha significantly decreased in infected Mphi. A similar trend was obtained for TNF-alpha mRNA expression. Altogether, these results indicate that infected Mphi react to LPS stimulus with enhanced levels of IL-1 beta and IL-6 but are unable to restore the production of TNF-alpha impaired by the growth of the intracellular mycobacteria.

Gene Expression↗
PubMed1996

Protective activity of a murine monoclonal antibody against acute and chronic experimental infection with type IV group B streptococcus.

A murine IgM monoclonal antibody (MAb H11) was developed against the type polysaccharide capsular antigen of group B streptococcus (GBS), serotype IV, after intraperitoneal immunisation of BALB/c mice with heat-killed bacteria. MAb H11 reacted in immunodiffusion with the purified polysaccharide in both its sialylated and desialylated form, giving a line of identity, and opsonised type IV GBS strains in an in vitro assay. When administered at the time of intraperitoneal lethal challenge with homologous GBS, or 4 h earlier, MAb H11 protected 90% of the mice. Protection was still observed when MAb H11 was given 4 h after the challenge. This MAb was strongly effective in preventing septic arthritis induced by type IV GBS.

Acute Disease↗
PubMed1996

Type frequency and antimicrobial susceptibility of Mycobacterium avium-intracellulare complex strains isolated in Italy from AIDS and non-AIDS patients.

Typing of the glycopeptidolipid antigens performed by thin layer chromatography on 59 Mycobacterium avium-intracellulare (MAC) strains isolated in Italy from AIDS patients showed that the most frequent types were 1, 4, 3, 8, and 21 (24, 19, 14, 14 and 8% of the strains, respectively). Among non-AIDS patients, types 1, 4 and 8 were also frequently found. The antimicrobial susceptibility tested in agar and/or liquid media to a panel of drugs indicated in clofazimine and rifabutin effective agents against both AIDS and non-AIDS strains. The data obtained show that MAC type distribution in Italy appears to be different from that reported for other countries. No major differences in drug susceptibility between AIDS and non-AIDS related strains were found.

AIDS-Related Opportunistic Infections↗
PubMed1995

Structural elucidation of the novel type VII group B Streptococcus capsular polysaccharide by high resolution NMR spectroscopy.

The type VII capsular polysaccharide isolated from the newly discovered group B Streptococcus (GBS) strain contains D-glucose, D-galactose, N-acetyl-D-glucosamine and N-acetylneuraminic acid in the molar ratio 2:2:1:1. High-resolution one- and two-dimensional (1D and 2D) 1H and 13C NMR spectroscopy of the native and desialylated polysaccharides showed the type VII GBS capsular polysaccharide to contain the following branched hexasaccharide repeating unit: [formula: see text] Despite extensive structural similarity with the previously described GBS polysaccharides, the type VII polysaccharide showed no cross-reaction with the heterologous antisera.

Acetylglucosamine↗
PubMed1995

Identification of Mycobacterium tuberculosis complex, Mycobacterium avium and Mycobacterium intracellulare by selective nested polymerase chain reaction.

A nested polymerase chain reaction (PCR) procedure was devised for identification of mycobacteria. The outer reaction exploiting genus-specific sequences on the 16S rRNA gene was able to amplify specifically strains of the genus Mycobacterium. The identification of Mycobacterium tuberculosis complex, Mycobacterium avium and Mycobacterium intracellulare was accomplished by selective reamplification of the outer PCR product in three distinct inner amplifications exploiting species-specific primers mapping to a hypervariable region of mycobacterial 16S rRNA. Detection of mycobacteria, other than those for which species-specific primers were used, was accomplished by adding a supplementary genus-specific upper primer to one of the inner reactions. Specificity of amplification was confirmed for clinical isolates and reference strains of different mycobacterial species with the exception of a M. intracellulare type 7 strain which was recognized as M. avium. The amplification protocol presented thus provides a reliable and cost-effective way for identification of clinically relevant mycobacteria.

Base Sequence↗
PubMed1995

In vitro and ex vivo activities of antimicrobial agents used in combination with clarithromycin, with or without amikacin, against Mycobacterium avium.

MICs of clarithromycin, amikacin, isoniazid, rifabutin, ciprofloxacin, sparfloxacin, ethambutol, and clofazimine were determined for six isolates of Mycobacterium avium complex (MAC) from AIDS patients both by the radiometric method and by an ex vivo model of infection in human macrophages. The median MICs in macrophages were similar or slightly lower than values found in broth, except for amikacin, which had slightly higher MICs inside the cells. Combinations of clarithromycin with other antimicrobial agents showed that clarithromycin-clofazimine and clarithromycin-rifabutin were synergistic on five of six strains while clarithromycin-amikacin and clarithromycin-isoniazid were antagonistic on one and two strains, respectively. The addition of amikacin made the combinations of clarithromycin-clofazimine and clarithromycin-ethambutol synergistic against all the MAC strains. In the macrophage model, the combination of clarithromycin-clofazimine (mean survival, 21%) and clarithromycin-rifabutin (mean survival, 29%) showed a strong reduction in viable counts compared with single drugs, while clarithromycin-amikacin was less active than single drugs alone. In general, the addition of amikacin did not improve the activity of the combinations, except for clarithromycin-isoniazid-amikacin (mean survival, 19%), which was significantly more active than either clarithromycin-isoniazid or clarithromycin-amikacin. The use of the macrophage model can suggest new combinations of antimicrobial agents with anti-MAC activity which, on the basis of their in vitro effectiveness, would probably be disregarded for assay in animal models.

Amikacin↗
PubMed1995

Antigenic properties and immunoelectron microscopic localization of Mycobacterium fortuitum beta-lactamase.

Mycobacterium fortuitum is a fast-growing Mycobacterium species which produces a beta-lactamase involved in the intrinsic resistance of the microorganism to beta-lactam antibiotics. An anti-beta-lactamase serum against the purified enzyme was raised in rabbits. Antibody binding was specific for native beta-lactamase, and enzyme activity was partially inhibited by the serum; furthermore, cross-reactions with denatured class A beta-lactamases were observed. This serum was used as a probe in immunogold labeling for the localization of the cell-bound beta-lactamase in both the low-level producer ATCC 19542 (parental strain) and the overproducer mutant D316. By the combination of preembedding immunogold labeling and replica technique, it was shown that the beta-lactamase was uniformly distributed on the whole external cell surface, where it appeared to be associated with a Tween 80-removable capsule-like material. Compared with the parental strain, a much higher level of expression of surface enzyme was observed in strain D316. Surface labeling was more intense in the stationary phase of growth than in exponentially growing cells. The data obtained are interpreted in the context of the intrinsic resistance of M. fortuitum to beta-lactam antibiotics.

Antigens, Bacterial↗
PubMed1995

In vivo efficacy of azithromycin in treatment of systemic infection and septic arthritis induced by type IV group B Streptococcus strains in mice: comparative study with erythromycin and penicillin G.

We compared the activities of azithromycin, erythromycin, and penicillin G in a mouse model of systemic infection and septic arthritis induced by type IV group B streptococci (GBS). The in vitro and in vivo efficacy data for these drugs were analyzed relative to the pharmacokinetics of the drugs in sera, joints, and kidneys. Adult CD-1 mice were infected intravenously with 10(7) CFU of type IV GBS. Intraperitoneal drug administration was initiated with different dose regimens at different times after infection. A single dose of azithromycin (100 mg/kg) strongly reduced the incidence of articular lesions with respect to that with erythromycin or penicillin G. Treatment with azithromycin (three intraperitoneal administrations of 50 mg/kg at 12-h intervals) resulted in the complete prevention of arthritis. In contrast, erythromycin was poorly effective and penicillin G was effective only if inoculated 30 min after infection and at high doses (400,000 or 600,000 IU/kg). Furthermore, azithromycin was able to cure about 70% of the mice when administered 7, 8, and 9 days after GBS infection. Azithromycin was much more active than erythromycin and penicillin G with respect to bacterial killing in the joints and kidneys. In fact, cultures from these tissues were always negative no matter what treatment schedule was employed. The pharmacokinetics of azithromycin account for its superior in vivo efficacy against type IV GBS. A longer half-life and higher levels of this drug in serum and tissues with respect to those for erythromycin or penicillin G were achieved. The high affinity of azithromycin for the joints strongly supports its potential value for therapy of septic arthritis, which is a severe and frequent clinical manifestation of GBS infection.

Animals↗
PubMed1994

Electrotransformation of Streptococcus agalactiae with plasmid DNA.

A protocol for efficient electrotransformation of Streptococcus agalactiae (group B streptococcus) Lancefield's strain O90R (NTCT 9993) (an unencapsulated derivative of type Ia strain O90) was developed. The Escherichia coli-Streptococcus shuttle vector pDP28 (7.8 kb) carrying the ermB gene for resistance to erythromycin was used as donor DNA. Frozen 'electrocompetent' cells were prepared by repeated washes in 10% glycerol. A 50-microliters aliquot containing about 5 x 10(9) colony forming units of bacteria was subjected to the electric pulse. Optimal conditions for electrotransformation were determined using different media, harvesting cells at different points of the growth curve, and using different field strengths. The dose-response curve for transformation of S. agalactiae with pDP28 showed one-hit kinetics as donor DNA varied between 0.01 and 3 micrograms. The efficiency of electrotransformation for this range of amounts of donor DNA was 1.2 x 10(4) cfu micrograms-1. The transformation frequencies obtained with this electroporation protocol are high enough to allow both subcloning and shotgun cloning of streptococcal DNA in S. agalactiae.

Electroporation↗
PubMed1994

Activity of antimicrobial drugs evaluated by agar dilution and radiometric methods against strains of Nocardia asteroides isolated in Italy from immunocompromised patients.

Benzylpenicillin, amoxicillin, amoxicillin plus clavulanic acid, cephalothin, cephaloridine, cefotaxime, imipenem, erythromycin, clarithromycin, azithromycin, amikacin, ciprofloxacin and trimethoprim-sulfamethoxazole were tested in vitro by the agar dilution method against eleven strains of Nocardia asteroides isolated both from AIDS and other immunocompromised patients. Imipenem, amikacin and trimethoprim-sulfamethoxazole were shown to be the most active drugs with minimum inhibitory concentrations (MIC) values nearly always lower than concentrations achievable in blood. Ciprofloxacin, cephaloridine and cefotaxime were moderately active, while the remaining drugs were totally ineffective. When susceptibility was assessed by the radiometric method the MIC90 values were uniformly lower than those in the agar method, possibly due to lower inactivation of drugs during incubation. The two methods showed a good correlation only for imipenem, amikacin and ciprofloxacin. The results obtained by the radiometric method seem to indicate that, as for mycobacteria, this method may also give a more accurate evaluation of the antimicrobial susceptibility of Nocardiae.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Induction of IL-1 beta, IL-6, TNF-alpha, GM-CSF and G-CSF in human macrophages by smooth transparent and smooth opaque colonial variants of Mycobacterium avium.

Both smooth transparent (SmT) and smooth domed-opaque (SmD) colonial variants were obtained from a strain of Mycobacterium avium isolated from a patient with AIDS. The two variants showed similar biochemical characteristics but SmT bacteria proliferated better than SmD bacteria inside human macrophages and were much less capable than the SmD variant of inducing the release of IL-1 beta, IL-6, TNF-alpha, GM-CSF and G-CSF, after incubation for either 3 or 6 days. As cytokines are important extracellular signals for immune cells, the lack of induction observed in SmT-infected macrophages may be one of the pathogenic mechanisms of M. avium.

AIDS-Related Opportunistic Infections↗
PubMed1994

Transcription and expression analysis, using lacZ and phoA gene fusions, of Mycobacterium fortuitum beta-lactamase genes cloned from a natural isolate and a high-level beta-lactamase producer.

The gene encoding a class A beta-lactamase was cloned from a natural isolate of Mycobacterium fortuitum (blaF) and from a high-level amoxicillin-resistant mutant that produces large amounts of beta-lactamase (blaF*). The nucleotide sequences of the two genes differ at 11 positions, including two in the region upstream from the coding sequence. Gene fusions to Escherichia coli lacZ and transcription and expression analysis of the cloned genes in Mycobacterium smegmatis indicated that high-level production of the beta-lactamase in the mutant is mainly or wholly due to a single base pair difference in the promoter. These analyses also showed that transcription and translation start at the same position. A comparison of the amino acid sequence of BlaF, as predicted from the nucleotide sequence, with the determined N-terminal amino acid sequence indicated the presence of a typical signal peptide. The fusion of blaF (or blaF*) to the E. coli gene phoA resulted in the production of BlaF-PhoA hybrid proteins that had alkaline phosphatase activity. These results demonstrate that phoA can be used as a reporter gene for studying protein export in mycobacteria.

Alkaline Phosphatase↗