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P Canepari

Publications and source records attributed to P Canepari.

At least 19 recordsLinked to original sources
PubMed1996

Modified peptidoglycan chemical composition in shape-altered Escherichia coli.

Peptidoglycan synthesis and its fine chemical composition were studied in dividing cocci of Escherichia coli carrying the lov-1 mutation and in which the coccal shape was obtained either by mecillinam treatment or by transferring a pbpA mutation (penicillin-binding protein 2- phenotype), as compared to normal rods and non-dividing cocci. Synchronously dividing cocci showed peptidoglycan synthesis only in the cell cycle phase corresponding to cell septation. During the phase corresponding to lateral wall elongation, peptidoglycan synthesis was strongly reduced. This type of synthesis suggests that the dividing cocci consisted only of the two poles. Analysis of the muropeptide composition revealed a specific fourfold increase in the tetra-tetra-tetra trimer in dividing cocci as compared to non-dividing cocci or parental rods. We postulate that, in E. coli, the chemical composition of septal peptidoglycan differs from that of lateral wall peptidoglycan.

Amdinocillin↗
PubMed1996

Simpler peptidoglycan chemical composition in the highly transformant Escherichia coli DH5 alpha strain.

Artificial transformation of Escherichia coli is obtainable by treating the culture with CA2+ and other substances known to increase the permeability of the outer membrane. Nevertheless, particular strains of E. coli are more useful for transformation since the number of transformants obtained is far higher. We postulate that an additional layer of the envelope may play an important role comparable to that of the outer membrane. The chemical composition of the peptidoglycan of a highly efficient transformant E. coli strain (DH5 alpha) was analyzed in comparison with a normal and poorly transformant E. coli strain (KN126) revealing a simpler peptidoglycan chemical composition in the DH5 alpha strain. This may be responsible for the simpler architecture of the peptidoglycan which, in turn, may interfere less with the passage of the DNA across the bacterial envelope.

Bacteriolysis↗
PubMed1995

Identification of daptomycin-binding proteins in the membrane of Enterococcus hirae.

Daptomycin, a lipopeptide antibiotic active against gram-positive bacteria, was preliminarily shown to inhibit lipoteichoic acid (LTA) synthesis as a consequence of membrane binding in the presence of Ca2+ (P. Canepari, M. Boaretti, M. M. Lleó, and G. Satta, Antimicrob. Agents Chemother. 34:1220-1226, 1990). In the present study, it is shown that, along with binding bacterial-membrane components, daptomycin binds the protein fraction with a noncovalent bond, as suggested by the instability of the bond in the presence of ionic detergents such as sodium dodecyl sulfate. Analysis of membrane proteins by isoelectric focusing electrophoresis reveals that five bands with isoelectric points ranging from 5.9 to 6.2 bind radioactive daptomycin. These proteins are therefore called daptomycin-binding proteins. In an attempt to correlate these proteins to the main inhibition observed during LTA synthesis, two-dimensional thin-layer chromatography of lipids synthesized during daptomycin treatment was performed. A threefold increase in diglucosyl diacylglycerol is demonstrated, while the compounds phosphatidyl-alpha-kojibiosyldiacylglycerol, glycerophospho-phosphatidyl-alpha-kojibiosyldiacylglycerol, and glycerophospho-kojibiosyldiacylglycerol, which follow diglucosyl diacylglycerol in LTA synthesis, decrease progressively with time during the course of daptomycin treatment.

Anti-Bacterial Agents↗
PubMed1994

Cloning, sequencing and expression in Escherichia coli of the low-affinity penicillin binding protein of Enterococcus faecalis.

Low-affinity penicillin binding proteins are particular membrane proteins, in several Gram-positive bacteria, which are involved in beta-lactam antibiotic resistance. The structural gene for the low-affinity penicillin binding protein 5 (PBP5) of Enterococcus faecalis was cloned and sequenced. From the sequence of the 3378 bp, a 2040 bp coding region was identified. From biochemical analysis it emerges that E. faecalis PBP5 is a type II membrane protein with an uncleaved N-terminal and is composed of 679 amino acids with a molecular weight of 74055. This protein showed 48 and 33% of identity with Enterococcus hirae PBP5 and Staphylococcus aureus PBP2a, both low-affinity PBPs involved in beta-lactam resistance. Anti-PBP5 antibodies cross-reacted with a membrane protein present in other species of enterococci, but the entire gene fragment cloned hybridized only with DNAs of E. faecalis strains, thus suggesting that genes coding for low-affinity PBPs of enterococci are not strictly homologous. In this experiment digoxigenin-labelled E. faecalis DNA was used.

Amino Acid Sequence↗
PubMed1994

Lack of correlation between salivary Streptococcus mutans and lactobacilli counts and caries in IDDM children.

In a previous clinical study regarding the incidence of caries and the periodontal health, a group of young patients with various levels of glyco-metabolic control was studied and the results showed that the decayed-missing-filled teeth (DMFT) index was higher in insulin dependent diabetes mellitus (IDDM) type 1 patients with a poor glyco-metabolic balance than in a control group or in IDDM patients with sufficient glyco-metabolic balance. In light of these results, the purpose of this study was to find an explanation for these clinical observations by searching at a microbiological level. The results indicate that salivary counts of Streptococcus mutans and lactobacilli were higher in patients with active caries whether or not they be diabetic, than in people with no active caries, but the count of S. mutans was not directly correlated to the DMFT index. No significant alterations were found in salivary flow, pH, buffer capacity and glucose concentration in all the groups in this study. We conclude that the salivary count of S. mutans is not sufficient alone to account for the higher susceptibility to active caries of young IDDM patients with poor glyco-metabolic control.

Adolescent↗
PubMed1994

[Microbiological contamination in the dental office and its possible decrease].

A microbiological analysis of the environment after dental work is presented in this paper. Detection of oral streptococci in the air is used as an index of the presence of salivary aerosol in consequence of the use of dental tools at high spin. This salivary aerosol may be considered a very important cause for the transmission of infectious diseases in the dental surgery. The real efficacy of a tool for the production of a dry aerosol of phenols or clorexidine with the purpose of environmental disinfection, is evaluated. Among possible parameters has been considered both the spray ability of the tool and the bactericidal activity of the aerosol at variable length from the source. Data here presented demonstrate the real utility of such an instrument for the disinfection of the dental surgery to be applied daily at the end of the work, not only in reducing environment microbial counts but also in totally eliminating salivary microorganisms.

Aerosols↗
PubMed1994

Rapid detection of PCR products of hepatitis C virus (HCV) with a non-radioactive oligoprobe.

Dot-blot for rapid detection of PCR amplification products of hepatitis C virus (HCV) using a digoxigenin (DIG)-labelled oligoprobe was developed and its sensitivity compared with Southern blot hybridization. The specificity and sensitivity of the DIG-labelled probe were identical to those of the 32P-labelled when the DIG-labelled nucleic acids were detected by enzyme-catalyzed chemiluminescent reaction. The lack of radioactivity makes this procedure suitable for routine use in diagnostic laboratories.

Base Sequence↗
PubMed1993

The activity of daptomycin on Enterococcus faecium protoplasts: indirect evidence supporting a novel mode of action on lipoteichoic acid synthesis.

The effect of daptomycin, an acidic lipopeptide antibiotic active against Gram-positives, was studied in Enterococcus faecium protoplasts. This antibiotic killed 99% of the protoplasts within 60 minutes of treatment, while vancomycin was ineffective, thus excluding peptidoglycan synthesis as the only target of the action of daptomycin. As previously seen with whole cells, in protoplasts lipoteichoic acid synthesis was the earliest and most strongly inhibited among types of macro-molecular synthesis. Radioactive daptomycin tightly bound only to the cytoplasmic membrane, in which the enzymes involved in lipoteichoic acid synthesis are located. These conclusions strongly support our previous proposal that daptomycin, though active against peptidoglycan synthesis, primarily inhibits lipoteichoic acid synthesis.

Anti-Bacterial Agents↗
PubMed1993

The reshaping process of Klebsiella pneumoniae cells after removal of mecillinam, an antibiotic that causes transition from rod to coccal shape.

The process of bacterial morphogenesis that leads to rod shape formation was studied in synchronous cells during the reshaping process after removal of mecillinam, a beta-lactam antibiotic which, by specifically inhibiting lateral wall formation of rods, cause rod-to-sphere transition in Gram-negative rods. The addition of mecillinam for 50 min of the cell cycle made the cells to skip a division, while the addition of the antibiotic for 30 min (or less), allowed the cells to divide regularly. In order to study the interplay between lateral wall elongation and septum formation in reacquisition of rod shape, we evaluated the effect of re-adding mecillinam or adding piperacillin, a specific inhibitor of septum formation, at various stages of the reshaping process. It was found that mecillinam was active only when added within the first 30 min of the reshaping process, while piperacillin was active only after 30 min when the cells were close to starting to divide again. These findings provide further support for our previous proposal that, in bacterial rods, elongation and septation are two alternating and competing events of the cell cycle, and are linked to each other in such a way as to force bacterial rods to grow to a given length.

Amdinocillin↗
PubMed1993

Peptidoglycan synthesis and its fine chemical composition in dividing and not dividing Klebsiella pneumoniae cocci.

Peptidoglycan synthesis and its fine chemical composition were studied in dividing and in non-dividing Klebsiella pneumoniae cocci and compared with rods. The beta-lactam mecillinam, a specific inhibitor of lateral wall elongation which causes rod-to-sphere transition in rods, showed 50% inhibition of the peptidoglycan in normal rods of the parent Mir A12 only if added at an early stage of the cell cycle and no effect if added later or during septation. In the rods of the mutant Mir M7, mecillinam was shown to inhibit 50% of peptidoglycan synthesis until rods become cocci, and thereafter to be absolutely devoid of effects. On the contrary, piperacillin, a specific inhibitor of septum formation, was active on all strains regardless of their cell shape, only if added at 20 and removed at 40 min of the cell cycle. As regards the analysis of peptidoglycan fine chemical composition, bacteria dividing as cocci showed alterations in the muropeptide composition consisting in a 50-fold increase in the tetramer family. This alteration was not seen in the cocci that did not divide as such. These results confirm our previous claim that septum formation and lateral wall elongation are mutually exclusive in normal rods and that septum formation requires the synthesis of a peptidoglycan of different chemical composition.

Amdinocillin↗
PubMed1992

In vitro activity of cefpirome (HR 810) against enterococci and staphylococci.

The inhibitory activity of cefpirome (HR 810), a new cephalosporin derivative for parenteral use, was tested by agar dilution methods against Enterococcus faecalis (100 strains), Staphylococcus aureus (40 strains) and coagulase-negative staphylococcal species (60 strains) in comparison with other beta-lactam antibiotics. For E. faecalis, the cefpirome minimum inhibitory concentration (MIC) range was 2-128 micrograms/ml, with an MIC50 of 8 micrograms/ml, and an MIC90 of 64 micrograms/ml. The optimal bactericidal activity against strains with MICs of < or = 8 micrograms/ml occurred at 2-4 times the MIC, and the reduction in the initial inoculum was 99.9-99.7% after 24 h incubation at these concentrations. Mec gene-negative staphylococci (both S. aureus and coagulase-negative species) had cefpirome MICs of 0.25-2 micrograms/ml (MIC50 0.5 microgram/ml, MIC90 1 microgram/ml). Mec gene-positive strains had MICs of 0.5-128 micrograms/ml (MIC50 2 micrograms/ml, MIC90 32 micrograms/ml). Strains with borderline resistance to oxacillin which did not harbor the mec gene and which were susceptible to cefpirome maintained their susceptibility even when high-density inocula were used and after several passages in media containing the antibiotic. These studies present some potential advantages of cefpirome over other cephalosporins in the inhibitory activity against Gram-positive cocci.

Anti-Bacterial Agents↗
PubMed1990

Mechanisms of resistance of enterococci to beta-lactam antibiotics.

Two mechanisms are responsible for resistance of enterococci to beta-lactam antibiotics: alterations of penicillin-binding proteins and production of a beta-lactamase. The latter has been found in a few clinical isolates of Enterococcus faecalis, whereas the former appears to account for resistance in most strains. A correlation has been established between the amount of a particular penicillin-binding protein which has a low affinity for penicillin and the level of resistance. The higher activity of some penicillins, as compared to cephalosporins, has been related to the relatively higher affinity for these penicillins of the penicillin-binding protein involved in the mechanism of resistance. Alterations in the autolytic enzyme pattern have been associated with the paradoxical response to bactericidal activity of penicillin often exhibited by Enterococcus faecalis clinical isolates.

Anti-Bacterial Agents↗
PubMed1990

Lipoteichoic acid as a new target for activity of antibiotics: mode of action of daptomycin (LY146032).

Daptomycin at the MIC allowed the cell mass increase of enterococcal strains and Bacillus subtilis to continue for 2 to 3 h at rates comparable to those of the controls. During this time the cell shape of the former changed to a rod configuration and that of the latter changed to long rods. In these bacteria, in which cell mass continued to increase, the MIC of daptomycin inhibited peptidoglycan synthesis by no more than 20% after 20 min of incubation and by roughly 50% after 2 h of incubation. Other macromolecules, such as DNA, RNA, and proteins, were only slightly affected. In contrast, incorporation of [14C]acetate into lipids was reduced by about 50% in the various strains after 20 min of treatment with daptomycin at the MIC. When the effect of the major lipid-containing polymers on synthesis was evaluated in detail, it was found that under conditions in which peptidoglycan and the other macromolecules mentioned above were inhibited only slightly (20%) and total lipid synthesis was inhibited by 50%, synthesis of teichoic and lipoteichoic acid was inhibited by 50 and 93%, respectively. Daptomycin was not found to enter the cytoplasm of either bacterial or mammalian cells. It bound, in the presence of calcium ions only, to whole bacterial cells, cell walls (both those that contained and those that did not contain membranes), and isolated membranes of bacterial and mammalian cells. Washing with EDTA removed daptomycin from all cells mentioned above and cell fractions except the bacterial membrane. It is concluded that lipoteichoic acid is most likely the primary target of daptomycin.

Acetates↗
PubMed1990

Bacterial cell shape regulation: testing of additional predictions unique to the two-competing-sites model for peptidoglycan assembly and isolation of conditional rod-shaped mutants from some wild-type cocci.

The two-competing-sites model for peptidoglycan assembly for bacterial cell shape regulation suggests that in rods, bacterial cell shape depends on the balance between two reactions (sites), one responsible for lateral wall elongation and the other responsible for septum formation. The two reactions compete with each other so that no lateral wall can be formed during septum formation and vice versa. When the site for lateral wall elongation overcomes that for septum formation, long rods or filaments are formed and cell division may be blocked. When the reaction leading to septum formation is hyperactive compared with the other, coccobacilli or cocci are formed. Other bacteria carry only one site for peptidoglycan assembly and can grow only as cocci. The two-competing-sites model predicts that two different types of cocci exist (among both morphology mutants and wild-type strains); one carries only the site for septum formation, whereas the other also carries the site for lateral wall elongation, the former site predominating over the latter. As a consequence of the inhibition (by antibiotics or by mutations) of septum formation in wild-type cocci of various species and in coccoid morphology mutants, some cocci are expected to undergo transition to rod shape and others are not. We have evaluated these predictions and show that they are in agreement. In fact, we found that among wild-type cocci belonging to 13 species, those of 6 species formed rods, whereas the remaining organisms maintained their coccal shape when septa were inhibited by antibiotics. Some coccoid morphology mutants of rod-shaped bacteria underwent coccus-to-rod transition after septum inhibition by antibiotics, whereas others maintained their coccal shape. When a mutation that causes septum inhibition was expressed in a morphology mutant of Klebsiella pneumoniae grown as a coccus, transition to rod shape was observed. A total of 914 mutants unable to form colonies at 42 degrees C were isolated from the coccoid species mentioned above. Between 75 and 95% of the mutants isolated from the species that formed rods when septum formation was inhibited by antibiotics but none of those isolated from the others underwent coccus-to-rod transition upon incubation at the nonpermissive temperature.

Anti-Bacterial Agents↗
PubMed1989

In vitro activity of lomefloxacin (SC-47111) against enterobacteriaceae, enterococci and staphylococci.

The activity of lomefloxacin, a new difluorinated quinolone, was tested against 190 Enterobacteriaceae strains (belonging to 23 different species), 70 enterococci and 70 staphylococci. As regards Enterobacteriaceae, the activity of lomefloxacin was the same as that of norfloxacin in 9 out of the 23 species tested, and only slightly lower in further 8 species. Minimum inhibitory concentrations (MIC) values for 90% of strains were 0.5 microgram/ml in 2 species, 0.25 microgram/ml in 6, 0.125 microgram/ml in 4, and lower than 0.125 microgram/ml in 8. Slightly higher values were obtained for Serratia marcescens (2 micrograms/ml), whilst, as already reported for the other new quinolones, the susceptibility of the Providencia genus was very poor, with MIC values up to 128 micrograms/ml for the vast majority of strains. Lomefloxacin proved bactericidal at the MIC in all the Enterobacteriaceae strains tested but 20. In the latter strains, however, bactericidal activity could be appreciated at values slightly exceeding MIC. As regards enterococci, the MIC for 90% of strains was 32 micrograms/ml. Minimum bactericidal concentration (MBC) was the same as the MIC for 78% of the strains tested and was only twofold higher in all the others. The new drug was also active against staphylococci having an MIC50 and MIC90 of 0.5 and 2 micrograms/ml, respectively. It was bactericidal at the MIC for 62% of the strains and at twofold the MIC for all the others.

4-Quinolones↗
PubMed1988

Evaluation of bactericidal activity of cefotaxime and other beta-lactams by a novel method.

In previous studies on Streptococcus faecium we proposed that the minimum beta-lactam concentration killing 99.9% of a bacterial population within 3 hours be defined as the minimum directly bactericidal concentration (MDBC) of that drug. In the present study we first evaluated the kinetics of cellular killing by various beta-lactams as related to penicillin-binding-protein (PBP) binding in Escherichia coli DC2, a hyperpermeable mutant. We concluded that in E. coli the MDBC for beta-lactams coincides with the minimum concentration capable of saturating PBPs 1b, 2 and 3. Of the antibacterial drugs we studied, cefsulodin, mecillinam and aztreonam had a much greater affinity for one essential PBP (PBP 1b, 2 and 3, respectively) than for all others, whereas cefotaxime had close affinities for all the above PBPs. MDBC values of greater than 500, 500, greater than 50, 10 and 1.5 mg/L were obtained for cefsulodin, mecillinam, aztreonam, ampicillin and cefotaxime, respectively. On the basis of the pharmacokinetic properties of these drugs, our results indicate that mecillinam, ampicillin and cefsulodin may be bactericidal in urine but not at other body sites; aztreonam is probably bactericidal in urine and blood, but not elsewhere; and cefotaxime is bactericidal in all the biological fluids we studied.

Amdinocillin↗
PubMed1987

Bacteriostatic and bactericidal activities of beta-lactams against Streptococcus (Enterococcus) faecium are associated with saturation of different penicillin-binding proteins.

The MICs and MBCs of benzylpenicillin, ampicillin, cefotaxime, and methicillin were evaluated against a Streptococcus (Enterococcus) faecium wild-type strain and against three mutants hyperproducing PBP 5 in cells incubated at both optimal and suboptimal temperatures. In the wild-type strain grown at optimal temperature, the MBCs of all beta-lactams were significantly greater than the MICs (bacteriostatic effect). As opposed to this, in the same cells grown at suboptimal temperature and in the mutants hyperproducing PBP 5 at all temperatures, the MICs of all antibiotics coincided with the MBCs (bactericidal effect). Under all conditions in which the MIC and MBC were the same, with all antibiotics, growth inhibition occurred only at the minimal concentration saturating all penicillin-binding proteins (PBPs) (or at higher concentrations). On the contrary, under conditions in which the MIC was lower than the MBC, only some of the PBPs were saturated (or bound) at both the MIC and the MBC, PBP 5 in no case being either saturated or bound. Under all conditions in which saturation of all PBPs was needed for growth inhibition, cells died at all antibiotic MBCs with kinetics which were much faster than those with which they died at the MBCs under conditions in which not all PBPs were saturated (or bound). In addition, under the former conditions, antibiotic concentrations above the MBCs did not significantly accelerate cell death kinetics, while under the latter conditions there was an acceleration in kinetics with increasing antibiotic concentrations up to full saturation of PBPs. It is suggested that the killing that occurs when all PBPs are saturated is a direct consequence of inactivation of PBP functions, while killing occurring when only some of them are saturated or bound is also (or mainly) an indirect consequence of inability of cells to grow and that, in S. faecium, the targets for growth inhibition and cell killing reside in different PBPs: for the latter effect, inactivation of one (or more) of the high-molecular-weight PBPs is sufficient, whereas in the former case inactivation of PBP 5 is necessary (after saturation of all other PBPs).

Anti-Bacterial Agents↗