Molecular typing of erythromycin-resistant streptococcus pyogenes strains with the M phenotype isolated in Italy.

To assess the spread of the new M phenotype, various erythromycin-resistant Streptococcus pyogenes strains from three Italian cities (Verona, Monza, Florence) were characterised. Each strain was analysed for the presence of genes ermAM and mefA, for the ability to accumulate radioactive erythromycin in the absence of sodium arsenate, for the protein T serological type, and for the DNA macrorestriction profile identified by means of pulsed-field gel electrophoresis. In a number of strains, the presence of the inducible ermAM gene was demonstrated; all these strains were negative in the efflux-pump detection assay, did not possess the mefA gene, and had similar restriction profiles. The strains with the efflux mechanism and mefA gene belonged to different serotypes. Of these, only one serotype, T4, was isolated in all three cities. The restriction profile analysis with SmaI and SfiI revealed a very close correlation between strains with the same serotype.

Comparative affinities for penicillin-binding proteins of multipolar ionic amphoteric cephalosporins in gram-negative bacteria.

Chemical modification of the highly reactive 7-aminocephalosporanic acid has yielded many compounds with improved activities and expanded clinical spectra. Introduction of an alpha-oxyimino group in C-7 position has given rise to improved activity against Gram-negative bacteria as a consequence of the high affinity of compounds carrying this substituent for the essential penicillin-binding protein (PBP) 3. The spectrum of activity of oxyimino cephalosporins has been further expanded by introduction of substituents with a quaternary nitrogen in C-3 position. These compounds have maintained their high affinity for the essential PBP 3, typical of the third generation cephalosporins and have acquired an improved ability to cross the outer membranes of Gram-negative bacteria. Among these compounds cefepime also exhibits high affinity for PBP 2, a very unusual property among cephalosporins.

Modulatory effects of hen egg-white lysozyme on immune response in mice.

The effect of her egg-white lysozyme (HEWL) on immune response was evaluated by measuring antibody-producing cells and circulating antibodies in mice inoculated with the test antigen (SRBC or BSA) and HEWL at the same time but in a separate body area. HEWL caused a premature decline in SRBC-specific plaque forming cells (PFC) and a reduction in the total amount of these cells. HEWL inhibited antibody production against BSA in the primary response, but was devoid of any effect on the secondary response elicited in the same mice by a second inoculation of the test antigen. The inhibitory effect of HEWL was dose-dependent, being maximal with 300 micrograms, required an enzymatically active protein and was not shown by other basic proteins. HEWL also abolished the enhancing effect of LPS and CFA on anti-BSA antibody production. The inhibitory activity of HEWL was further increased by hydrolyzed peptidoglycan. These results suggest that HEWL modulates the immune response in mice and performs this function through activation of non-specific suppression mechanisms.

Lysozyme inhibitors enhance immune response in mice.

The effect of hen egg-white lysozyme (HEWL) inhibitors (such as heparin, histidine methylester, chitotriose, chitobiose) on immune response was evaluated by measuring antibody-producing cells and circulating antibodies in mice inoculated with these substances and the test antigen (SRBC or BSA). It was found that these compounds have an immuno-enhancing effect which is directly proportional to their inhibitory activity on HEWL. Conversely, HEWL inhibited the immunoenhancing effect of these compounds when injected together with these and the test antigen. The results suggest that one possible mechanism by which adjuvants stimulate immune response may be the inhibition of endogenous lysozyme.

Structure-activity relationship of new 2-substituted penem antibiotics.

The antibacterial activities of three new penems with 4-hydroxyprolinamide, 1-prolinamide and N-methyl-N-2-propionamide substituents, respectively, in position 2 and of their stereoisomers were examined against Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Escherichia coli and Pseudomonas aeruginosa. All substitutes conferred a broad antibacterial spectrum on the penem moiety. Changes in stereoisomerism selectively improved the activity against E. coli, S. aureus or enterococci. The structure-activity relationships of each compound were discussed in relation to minimum inhibitory concentrations, penicillin-binding protein (PBP) affinity and outer membrane permeability coefficient in E. coli. In this microorganism, PBP 2 was the target for all compounds. Changes in stereoisomerism influenced the affinity for PBPs 1A/B and 2. All antibiotics easily permeated the outer membrane of E. coli and, within each group of compounds, the penetration rate correlated with the antibacterial activity.

Target for bacteriostatic and bactericidal activities of beta-lactam antibiotics against Escherichia coli resides in different penicillin-binding proteins.

The relationship between cell-killing kinetics and penicillin-binding protein (PBP) saturation has been evaluated in the permeability mutant Escherichia coli DC2 in which the antimicrobial activity of beta-lactams has been described as being directly related to the extent of saturation of the PBP target(s). Saturation of a single PBP by cefsulodin (PBP 1s), mecillinam (PBP 2), and aztreonam (PBP 3) resulted in a slow rate of killing (2.5-, 1.5-, and 0.8-log-unit decreases in the number of CFU per milliliter, respectively, in 6 h). Saturation of two of the three essential PBPs resulted in a marked increase in the rate of killing, which reached the maximum value when PBPs 1s and 2 were simultaneously saturated by a combination of cefsulodin and mecillinam (4.7-log-unit decrease in the number of CFU per milliliter in 6 h). Inactivation of all three essential PBPs by the combination of cefsulodin, mecillinam, and aztreonam further increased the killing kinetics (5.5-log-unit decrease in the number of CFU per milliliter), and this was not significantly changed upon additional saturation of the nonessential PBPs 5 and 6 by cefoxitin. Similar relationships between PBP saturation and killing kinetics were obtained with imipenem and meropenem at concentrations which inhibited only one PBP (PBP 2), only two PBPs (PBP 1s and 2), or all three essential PBPs. Saturation of one or more PBPs also resulted in a different rate of bacteriolysis, the highest rate being obtained by the cefsulodin-mecillinam combination and by 5 micrograms of either imipenem or meropenem per ml. All of these conditions caused saturation of PBP 2 and saturation or extensive binding of PBP 1s. However, none of these conditions caused determined the fastest possible rate of killing, which occurred only when all three essential PBPs were saturated. It was concluded that the actual killing effect of beta-lactams is reflected by killing rates that approach the fastest possible rate for the given microorganism and that the targets for the bactericidal activity are precisely those PBPs whose saturation or binding occurs under conditions.

Quinolone binding to DNA is mediated by magnesium ions.

The binding of plasmid DNA to norfloxacin, a quinolone antibacterial agent, was investigated by fluorescence, electrophoretic DNA unwinding, and affinity chromatography techniques. The amount of quinolone bound to DNA was modulated by the concentration of Mg2+. No interaction was evident in the absence of Mg2+ or in the presence of an excess of Mg2+, whereas maximum binding was observed at a Mg2+ concentration of 1-2 mM. The experimental data can be fitted to the formation of three types of Mg adducts: a binary adduct with norfloxacin and Mg2+, a binary adduct with DNA and Mg2+, and a ternary adduct with quinolone, plasmid, and Mg2+. We propose a model for the ternary complex, in which Mg acts as a bridge between the phosphate groups of the nucleic acid and the carbonyl and carboxyl moieties of norfloxacin. Additional stabilization may arise from stacking interactions between the condensed rings of the drug and DNA bases (especially guanine and adenine), which may account for the preference exhibited by quinolones for single-stranded and purine-rich regions of nucleic acids. Other possible biochemical pathways of drug action are suggested by the observation that norfloxacin binds Mg2+ under conditions that are close to physiological.

Staphylococcal endo-beta-N-acetylglucosaminidase inhibits response of human lymphocytes to mitogens and interferes with production of antibodies in mice.

The effect of a bacteriolytic enzyme, the endo-beta-N-acetylglucosaminidase excreted by Staphylococcus aureus (SaG) on the response of human lymphocytes to mitogens and on the immune response in mice has been studied. SaG inhibited incorporation of [3H]thymidine into TCA-precipitable material by human peripheral lymphocytes stimulated either by phytohemagglutinin or by concanavalin A, as well as formation of cytoplasmic immunoglobulin-containing cells by B lymphocytes treated with pokeweed mitogen. In all cases the level of inhibition first increased with the SaG concentrations reaching values of over 80% at an enzyme concentration of 100 micrograms/ml, and then decreased. Heat-inactivated SaG as well as SaG treated with both polyclonal and monoclonal specific antibodies or enzyme inhibitors such as chitotriose or hydrolyzed peptidoglycan had no effect on lymphocyte response to mitogens. In mice, SaG at a dose of 300 micrograms per mouse was found to cause a fourfold decrease in the anti-BSA antibody titer and an approximately 70-75% reduction in the immunoglobulin-containing cells in the spleens of mice injected with sheep red blood cells. SaG also completely abolished the enhancing effect of adjuvants such as muramyldipeptide, Freund's complete adjuvant, and Escherichia coli lipopolysaccharide. When SaG was injected into mice together with S. aureus peptidoglycan hydrolyzed either by SaG or by human lysozyme, the inhibitory effect on both production of anti-BSA circulating antibodies and appearance of Igc cells in the spleens of mice injected with sheep red blood cells was enhanced. As we know that (a) human tissues contain endo-beta-N-acetylglucosaminidases; (b) other human hexosaminidases (lysozymes) have previously been shown to interfere with the functions of immunocompetent cells; and (c) products of hexosaminidase hydrolysis of peptidoglycan (muropeptides) known to modulate immune response are ordinarily found in the urine of healthy persons, the possibility that hexosaminidases play a major role in the regulation of the immune response is raised and discussed.

Relevance of ionic effects on norfloxacin uptake by Escherichia coli.

The uptake of the quinolone drug norfloxacin by Escherichia coli was investigated at initial rate kinetics at different pH and monovalent/divalent metal ion concentration. The results support a simple diffusion mechanism for quinolone incorporation into cells. The uptake process decreases under acidic conditions. The presence of Na+ or K+ ions does not affect the results to an appreciable extent, whereas divalent ions cause a dramatic decrease in drug incorporation. The antibacterial activity, evaluated under identical experimental conditions, shows a direct relationship with the uptake data. As a general explanation for the above results it is suggested that the ability of the drug to penetrate into cells is a function of its net charge. The molecule in the zwitterionic form exhibits maximum permeation properties, whereas the uptake is remarkably reduced when the drug bears a net charge as a result of ionization or complex formation with bivalent ions. These results allow further insight into the mechanism of quinolone access to the intracellular compartment.

The effect of the minor groove binding agent DAPI (4,6-diamidino-2-phenyl-indole) on DNA-directed enzymes: an attempt to explain inhibition of plasmid expression in Escherichia coli [corrected].

The activity of DAPI, on a number of DNA-directed enzymes involved in DNA topology, transcription, replication and repair, is reported in this paper. DAPI was always more inhibitory than ethidium bromide, in particular against RNA polymerase and DNA ligase, which seemed to be specifically affected. While the effect on RNA polymerase is likely due to a preferential occupancy of the promoter region, that on DNA ligase could rely upon a mechanism of steric hindrance in the minor groove. These phenomena, independently from an alteration of the tertiary structure of DNA by the ligand, can account for the previously reported inhibition of plasmid expression in Escherichia coli.

Mechanisms of macrolide resistance in Ureaplasma urealyticum: a study on collection and clinical strains.

Ureaplasma urealyticum is considered as a species which is intrinsically sensitive to macrolides (MIC less than 1 microgram/ml). Nevertheless, some of the strains recently isolated in our laboratories showed moderate to high levels of resistance (MICs ranging from 2 micrograms/ml to 100 micrograms/ml). In particular, a strain (CT28) isolated from a patient with nongonococcal urethritis long treated with erythromycin revealed a MIC greater than 100 micrograms/ml for this antibiotic. In order to investigate the mechanisms of resistance, strain CT28 and ten clinical and laboratory U. urealyticum strains were compared for the sensitivity to six antibiotics including three macrolides. Moreover the amount of macrolide uptake and the specific antibiotic binding to ribosomes were studied. Strain CT28 was resistant to josamycin, erythromycin, roxithromycin, lincomycin and clindamycin but sensitive to minocycline. When compared to a sensitive strain, strain CT28 showed a six-fold reduction in intracellular macrolide influx and accumulation and a reduction in antibiotic binding to ribosomes. The mechanisms implicated in these differences may be important for macrolide resistance in U. urealyticum.

Cell fusion induced by herpes simplex is inhibited by hen egg-white lysozyme.

The formation of syncytia in cell monolayers infected with a macroplaque strain (MP) of herpes simplex virus was found to be inhibited by hen egg-white lysozyme. Inhibition was roughly proportional to the enzyme concentration. The virus titres in supernatant fluids of lysozyme-treated cultures were also reduced compared with untreated cultures. Control experiments excluded the possibility that lysozyme altered the virus viability and infectivity or impaired cell growth. Since lysozyme is a cationic protein, further experiments were performed in order to discover whether its antisyncytiogenic effect depended on its enzymatic activity or on its positive charge. Inhibition of the MP-induced polycaryocytosis was found to be caused by heat-inactivated lysozyme and three chemically-modified lysozymes with a higher positive charge (one retaining and two lacking enzymatic activity).

Lysozyme-induced inhibition of the lymphocyte response to mitogenic lectins.

Both human lysozyme (HL) and hen egg white lysozyme (HEWL) inhibited the proliferative response of peripheral blood lymphocytes to T cell mitogens such as the lectins phytohemagglutinin and concanavalin A. This inhibition was observed both when HL or HEWL was added to the lymphocyte cultures in combination with phytohemagglutinin or concanavalin A and when lymphocytes were pretreated with either lysozyme and extensively washed prior to culture with mitogens. Under both conditions, the effects were strictly dose dependent; the lysozyme concentrations yielding maximal inhibitory effect were 5 micrograms/ml for HL and 1 microgram/ml for HEWL, while both lower and higher concentrations were less effective. Specific antilysozyme rabbit sera completely prevented the inhibitory effects of both HL and HEWL on the proliferative response of lymphocytes to phytohemagglutin or concanavalin A. Chitotriose (a lysozyme inhibitor) caused a strong reduction in the inhibitory effects of the two lysozymes on the lymphocyte response to either lectin. HL and HEWL also were found to markedly inhibit the polyclonal B cell proliferation and differentiation induced by pokeweed mitogen and T cells. A less marked inhibition was also obtained when T cells, but not B cells, were pretreated with HL or HEWL. Again, as in the experiments with T cell mitogens, the effects were dose dependent and 5 micrograms/ml HL and 1 microgram/ml HEWL proved to be the most effective concentrations. The possible mechanisms by which lysozyme inhibits the lymphocyte response to mitogenic lectins are considered and discussed. The enzymatic activity seemed to perform an essential function, as shown by the loss of effect when the heat- or trypsin-inactivated lysozymes were used and by the fact that only the enzymatically active compound, among certain semisynthetic derivatives of HEWL, inhibited the lymphocyte response to the mitogens. However, the cationic properties of the lysozyme molecule appeared to be essential too, since enzymes with a similar specificity of action showed effects similar to those observed with HL or HEWL only when they carried a strong positive charge. It is suggested that lysozyme, which is naturally secreted by monocytes and macrophages, might interact with lymphocyte surface receptor sites and participate in the complex mononuclear phagocyte-lymphocyte interactions and in the modulation of lymphocyte activation.

A further insight into the mechanism of action of anthracycline antibiotics.

The uptake of Adriamycin (ADM) by eucaryotic and procaryotic cells and the interaction of the antibiotic with structurally organized DNA were investigated. The aim of this work was to understand why ADM is endowed with very low antibiotic activity in spite of being highly cytotoxic for mammalian cells, and to get a further insight into the mechanism of action of this compound. Spectrophotometric, fluorometric, chiroptical, and electron microscopic techniques were used. The drug was shown to concentrate in mammalian cells but failed to do so in bacteria and penetrated rather poorly in Candida albicans. The chromatin binding affinity of ADM appeared to be substantially reduced with respect to the affinity for free DNA. Complex stereochemistry was also influenced by the presence of nucleosomal arrangements in the polynucleotide. In addition, evidence was obtained that the antibiotic tended to accumulate at specific (linker) chromatin regions and to cause extensive compaction of organized DNA. The observed phenomena may play a relevant role in the mode of drug action in eucaryotic cells and help to explain the lack of antibacterial activity by ADM in spite of the apparent attainment of great enough intracellular levels to affect DNA template function.

Preliminary characterization of a mutant of herpes simplex virus type 1 selected for acycloguanosine resistance in vitro.

In this paper we report on the preliminary characterization of a mutant of herpes simplex virus type 1 (HSV-1) selected for acycloguanosine (acyclovir, ACV) resistance in vitro. The ACVr virus was examined for a series of parameters that include chemosensitivity assay, thymidine kinase (TK) activity, in vitro and in vivo growth, and mutation mapping. The data obtained indicate that a mutated TK gene is responsible for the ACVr phenotype. A distinctive feature of this mutant is the high level of resistance exhibited to ACV (100 microM) and the concomitant presence of a functional TK activity. Such a property makes this virus useful as a model for the study of viral resistance to nucleoside-type analogues in HSV.

DAPI-pUC8 complex: a tool to investigate biological effects of nucleic acid-drug interaction.

A complex consisting of pUC8, a 1.8 Md plasmid, and 4'-6-diamidino-2-phenylindole, a DNA-binding agent, has been performed in vitro under different conditions of ionic strength, and used to transform competent cells. A strong interference with the plasmid-coded activities, related to the P/D ratio where at the DNA-drug complex was formed, was shown to occur. Since the compound does not inhibit the uptake process neither affects plasmid activity once dissociated at high ionic strength, it is likely to be acting from inside the cell while still in the form of a DNA-adduct. This system is proposed as a useful tool to investigate the effects on target genes of drugs endowed with DNA sequence specificity.

A search for new anti-herpes virus compounds.

This study reports on the synthesis and the activity of a series of new compounds of nucleoside-type structure. They are characterized for being differently substituted in the aromatic ring of the base (deazoadenosine derivatives) or by bearing a dansyl group in the sugar moiety (dansylthymidine). One molecule belonging to this latter class of compounds (the 3'-0-dansylthymidine) is showing an anti-herpesvirus potential while being active in inhibiting the virus-encoded enzyme thymidine kinase. This finding may represent an important step for the synthesis of new enzyme inhibitors and it is discussed in terms of future developments of more active congeners.

Antitumor activity of new anthraquinone derivatives.

Seven new 9,10-anthracenedione derivatives bearing positively-charged side chains at different positions of the condensed ring system have been investigated in their interaction with DNA and their biological effects, including antitumor activity in the P 388 mouse miniscreen. The drug's affinity for DNA was found to be related to the efficacy of inhibition of cell growth and nucleic acid synthesis for a number of cell lines. Moreover, alkaline elution experiments showed a close relationship between mutagenic potential and cytotoxicity. With the exception of one, all compounds behaved like intercalating agents, as shown by the unwinding of supercoiled plasmid DNA. DNA appeared, therefore, to be the more important target for drug action. The marginal in vivo activity shown by two substances in this series suggests a number of possible structural modifications which may increase their therapeutic efficacy.

Biochemical and physical properties of the endo-beta-N-acetylglucosaminidases from Staphylococcus aureus, Staphylococcus simulans, and Staphylococcus saprophyticus.

Biochemical and physical properties of the pure bacteriolytic enzymes excreted by three different Staphylococcus species (S. aureus, S. simulans, and S. saprophyticus) were investigated. Although the three enzymes have previously been shown to share the same specificity of action (endo-beta-N-acetylglucosaminidase activity), their biochemical features clearly indicated that they were three different enzymes, confirming what had previously been suggested by the different lytic-activity patterns displayed by each species and the different procedures needed to achieve purification of each enzyme. Very different values resulted from molecular weight determination: 80,000 for the S. aureus enzyme, 45,000 for the S. simulans enzyme and 31,000 for the S. saprophyticus enzyme. Other important differences were observed in their kinetics of activity on Micrococcus luteus purified cell walls; their stability; their bacteriolytic spectrum against heat-killed cells of various microorganisms; and their response to physical and chemical factors, such as temperature, pH, ionic strength, divalent cations, chelating agents, thiol compounds, and glucose derivatives.