Biomedical subjects

M Boaretti

Publications and source records attributed to M Boaretti.

14 recordsLinked to original sources

PubMed2007

Detection and quantification of hepatitis B virus DNA by SYBR green real-time polymerase chain reaction.

A single-round real-time polymerase chain reaction (PCR) assay based on SYBR green dye technology for the detection and quantification of hepatitis B virus (HBV) DNA in serum was evaluated and compared with a qualitative nested PCR and the Cobas Amplicor HBV Monitor assay (Roche Molecular Diagnostics, Milan, Italy). The performance of the real-time PCR assay was evaluated in a routine clinical laboratory setting with a total of 212 clinical specimens. The sensitivity of the real-time PCR corresponded to 31 IU/ml (70 copies/ml), and comparison with the qualitative nested PCR showed significant concordance for 94% of samples. The linear curve over 7 log units, spanning 10(3)-10(9) IU/ml (2.28 x 10(3) to 2.28 x 10(9) copies/ml), was observed in the quantitative determination. The interexperimental variability coefficient of the assay ranged from 0.22 to 0.39 and the intraexperimental variability coefficient from 0.24 to 0.41. By excluding values outside of the dynamic ranges of both tests, the HBV Monitor and the real-time PCR gave an agreement within +/-1 log unit for 90% of samples, while those for the remaining 10% were found to be above 1 log unit but less than 1.5 log units. When the results inside and outside the dynamic range of the HBV Monitor were examined, 90% of the results were in agreement. In conclusion, the real-time PCR based on SYBR green technology proved suitable for routine diagnostic purposes, showing good sensitivity, high specificity, high reproducibility, and good linearity over a broad dynamic range of quantification.

Benzothiazoles↗

PubMed2001

Resuscitation rate in different enterococcal species in the viable but non-culturable state.

AIMS: The viable but non-culturable (VBNC) state is a survival strategy adopted by bacteria when exposed to environmental stress. When in this state bacteria are no longer culturable on conventional growth media, but cells display metabolic activity and maintain pathogenicity factors/genes and, in some cases, resuscitation from the VBNC state has been shown. This state has been described for both human pathogens and faecal pollution indicators. In this study, we present evidence for entry of different enterococcal species into the VBNC state in an oligotrophic microcosm. METHODS AND RESULTS: The duration of the viability of the cells in the VBNC state was measured either by detecting the presence of pbp5 mRNA or by quantifying their resuscitation capability. Enterococci showed different behaviours. Enterococcus faecalis and Enterococcus hirae entered into the VBNC state within 2 weeks and remained in that state for 3 months. In the experiments described the resuscitation rate was 1:10 000 cells as soon as the cells entered the VBNC state and decreased gradually to undetectable levels over the following 3 months. Enterococcus faecium, however, remained culturable up to 4 weeks. After this time period, when the population was totally unculturable, the cells were far less resuscitable than other enterococci and only over a narrow time interval (2 weeks). CONCLUSIONS: These results suggest that Ent. faecalis and Ent. hirae enter the VBNC state but that Ent. faecium, in an oligotrophic laboratory environment, tends to die instead of entering the VBNC state. SIGNIFICANCE AND IMPACT OF THE STUDY: These experiments may mimic what happens when enterococci are released by humans and animals in natural environments.

Bacterial Proteins↗

PubMed2000

Purification of daptomycin binding proteins (DBPs) from the membrane of Enterococcus hirae.

Daptomycin binding proteins (DBPs) are membrane proteins which act as daptomycin targets. Daptomycin is a cyclic lipopeptide antibiotic which is active against Gram-positive bacteria and was shown to be the first inhibitor of lipoteichoic acid (LTA) synthesis. It was found that the antibiotic did not penetrate the bacterial cytoplasm but bound membranes with a non-covalent bond and in particular some proteins which were called DBPs. DBPs were indicated as enzymes involved in LTA synthesis whose binding and inhibition by daptomycin is responsible for the observed effect on bacterial LTA synthesis. The purification of DBPs will make it possible not only to shed light on the biosynthesis of the cell wall polymer but will also provide innovative targets for selection of new antibacterial compounds. In this study, the purification of DBPs is described. Affinity chromatography was used with daptomycin as the ligand. Final elution of DBPs from daptomycin-coupled resin was performed using either 0.1% SDS or 3 M NaCl. Polyacrylamide gel electrophoresis of the eluted protein fractions consistently showed four protein bands (ranging from 55 to 66 kDa) in denaturating conditions and two protein bands (60 and 66 kDa) in non-denaturating conditions. Isoelectrofocusing analysis of the same sample consistently revealed two bands with pIs around 5. That these purified proteins were really the desired DBPs is demonstrated by the retention of daptomycin-binding capability they displayed.

Anti-Bacterial Agents↗

PubMed1998

Peptidoglycan synthesis by Enterococcus faecalis penicillin binding protein 5.

Low-affinity penicillin binding proteins (PBPs) are a particular class of proteins involved in beta-lactam antibiotic resistance of enterococci. The activity of these PBPs is just sufficient to allow the cells to survive in the presence of high concentrations of beta-lactams that cause saturation (and inhibition) of the other PBPs. For this reason, the low-affinity PBPs are thought to be multifunctional enzymes capable of catalyzing the entire peptidoglycan synthesis. To test the validity of this claim, we analyzed the muropeptide composition by reversed-phase high-performance liquid chromatography of the peptidoglycan synthesized by PBP5 (the low-affinity PBP) of Enterococcus faecalis, in comparison with the peptidoglycan produced normally by the concerted action of the usual PBPs (namely PBPs 1, 2, and 3). Cross-linked peptidoglycan was produced. The main difference consisted in the lack of oligomers higher than trimers, thus suggesting that this oligomer cannot be used as an acceptor/donor by the transpeptidase component of PBP5. The lack of higher oligomers had little impact on total cross-linking because of the increase observed in the dimer family. This increase was distributed among the various members of the dimer family with the result that minor dimer components figured among the prevalent ones in cells in which peptidoglycan was synthesized by PBP5. This also suggests that E. faecalis PBP5 is capable of catalyzing the synthesis of a peptidoglycan that is less precise and refined than usual, and for this reason PBP5 can be considered an enzyme endowed with poor specificity for substrates, as may be expected on the basis of its survival function.

Bacterial Proteins↗

PubMed1997

Cell elongation and septation are two mutually exclusive processes in Escherichia coli.

Bacterial rod morphogenesis was studied in synchronously growing cells of Escherichia coli C600 during the reshaping process that follows the removal of mecillinam, a beta-lactam antibiotic that specifically inhibits lateral wall formation of gram-negative rods and causes transition to coccal shape. Removal of mecillinam after 30 min of action did not affect the timing of subsequent cell division, but removal after 50 min delayed resumption of cell division for approximately one generation time. In order to study the interplay between lateral wall elongation and septum formation in determining and maintaining the bacterial rod shape, we evaluated the effect of re-adding mecillinam or of adding aztreonam (a specific inhibitor of septum formation) at various stages of the reshaping process. We conclude that mecillinam was active only during the reshaping process, while aztreonam was active only later when the cells were close to dividing again. These results provide further evidence for our previous proposal according to which elongation and septation are two alternating and competing events of the cell cycle and are linked to each other to force bacterial rods to grow to a given length.

Amdinocillin↗

PubMed1996

The gene encoding for penicillin-binding protein 5 of Enterococcus faecalis is useful for development of a species-specific DNA probe.

Recently, in Escherichia coli was cloned a Sau3AI 3.4-kb fragment containing the gene encoding for penicillin-binding protein 5 (PBP5) of Enterococcus faecalis. The structural gene for the PBP of E. faecalis and the flanking regions were entirely sequenced (C. Signoretto, M. Boaretti, and P. Canepari, FEMS Microbiol. Lett. 123:99-106, 1994). When the entire cloned E. faecalis DNA insert, labeled with digoxigenin, was used as a probe to detect a homology gene in enterococci, it was observed that only DNAs of all the E. faecalis strains reacted to the probe. The same results were obtained when a HindIII fragment of 0.35 kb from the entire insert of 3.4 kb was used. In this study we tested a total of 62 clinically isolated enterococcal strains, belonging to the species E. faecalis (36 strains), E. faecium (13), E. gallinarum (6), E. bovis (2) E. avium (3), E. hirae (1), and E. casseliflavus (1). The results indicate that both the entire segment and the HindIII fragment may be useful for preparing a species-specific probe for rapid identification of E. faecalis species.

Bacterial Proteins↗

PubMed1996

Lipoteichoic acid as a target for antimicrobial action.

Daptomycin, a lipopeptide antibiotic active against gram-positive bacteria, has been found to inhibit lipoteichoic acid (LTA) synthesis as a consequence of membrane binding in the presence of Ca2+. The present study shows that among the bacterial-membrane components, daptomycin binds the protein fraction with a noncovalent bond, as suggested by the instability of the bond in the presence of an ionic detergent such as sodium dodecyl sulfate. Analysis of membrane proteins by isoelectric focusing electrophoresis reveals that 5 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 with the main inhibition observed in LTA synthesis, two-dimensional thin-layer chromatography of lipids synthesized during daptomycin treatment was performed. A 3-fold increase in diglucosyl diacylglycerol is demonstrated, while the compounds phosphatidyl-alpha-kojibiosyldiacylglycerol, glycerophosphophosphatidyl-alpha-kojibiosyldiacyl glycerol, and glycerophosphokojibiosyldiacylglycerol, which follow diglucosyl diacylglycerol in LTA synthesis, decrease progressively with time during the course of daptomycin treatment.

Anti-Bacterial Agents↗

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↗

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↗

PubMed1991

In vitro activity, beta-lactamase stability and PBP affinity of RU 51,746-2, the active metabolite of the new orally absorbed cephalosporin ester, RU 51807.

The activity of RU 51,746-2 was determined against strains of Escherichia coli producing different types of beta-lactamases or showing alterations of permeability. The production of TEM 1, OXA 2, CARB 3 and PSE 1 beta-lactamases had no influence on susceptibility to the antibiotic, whereas the synthesis of TEM 2, SHV 1 and OXA 1 beta-lactamases increased minimum inhibitory concentrations (MIC) by 2-4 times. Highly resistant to the antibiotic was a strain producing CEP 1 beta-lactamase. E. coli mutans deficient in Omp F but not in Omp C had a decreased susceptibility to RU 51,746-2. RU 51,746-2 showed a good affinity for high molecular weight penicillin binding proteins (PBPs) of E. coli. PBP 3 was found to be the target for growth inhibition, whereas the additional saturation of PBP 1 and 2 was required for obtaining the best bactericidal activity.

Carrier Proteins↗

PubMed1990

Paradoxical response of Enterococcus faecalis to the bactericidal activity of penicillin is associated with reduced activity of one autolysin.

Ten clinical isolates of Enterococcus faecalis were examined for susceptibility to the bactericidal activity of penicillin. Four of these had MBCs of penicillin equal to 2 to 4 x the MIC, and six exhibited a paradoxical response to penicillin, i.e., the bactericidal activity of the antibiotic had a concentration optimum at 2 to 4 x the MIC and decreased significantly at concentrations above this. We found that the paradoxical response to penicillin was an intrinsic and stable property of a strain, but that its phenotypic expression was not homogeneous; only a fraction of the cell population that died at low concentrations was able to survive at high penicillin concentrations. The size of this fraction increased with increasing antibiotic concentration and reached a maximum in the late-log phase of growth. All 10 strains produced a lytic enzyme that was active on Micrococcus luteus heat-killed cells, whereas only some strains lysed E. faecalis heat-killed cells. Strains producing large amounts of the latter enzyme did not show the paradoxical response to penicillin, whereas mutants of these strains that lacked this enzymatic activity paradoxically responded to the antibiotic activity. In addition, from strains that showed paradoxical response to penicillin and produced only the enzyme that was active on M. luteus, it was possible to isolate mutants that were also capable of lysing E. faecalis cells and that were killed with similar efficiency by all concentrations above the MBC. On the basis of these findings, the paradoxical response to penicillin is explained as a property of certain strains of E. faecalis; this property is genetically characterized by alterations in synthesis or activity of one autolysin but phenotypically expressed only by a few cells that are in a particular physiological condition when exposed to high concentrations of antibiotics.

Bacterial Proteins↗

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↗