Biomedical subjects

G Satta

Publications and source records attributed to G Satta.

At least 55 records · Page 3Linked to original sources

PubMed1993

High specificity of cphA-encoded metallo-beta-lactamase from Aeromonas hydrophila AE036 for carbapenems and its contribution to beta-lactam resistance.

The Aeromonas hydrophila AE036 chromosome contains a cphA gene encoding a metallo-beta-lactamase highly active against carbapenem antibiotics. This enzyme was induced in strain AE036 to the same extent by both benzylpenicillin and imipenem. When the cphA gene was inserted into plasmid pACYC184, used to transform Escherichia coli DH5 alpha, the MICs of imipenem, meropenem, and penem HRE664 for recombinant clone DH5 alpha(pAA20R), expressing the Aeromonas metallo-beta-lactamase, were significantly increased, but those of penicillins and cephalosporins were not. When the metallo-beta-lactamase purified from E. coli DH5 alpha(pAA20R) was assayed with several beta-lactam substrates, it hydrolyzed carbapenems but not penicillins or cephalosporins efficiently. These results demonstrate that this metallo-beta-lactamase possesses an unusual spectrum of activity compared with all the other class B enzymes identified so far, being active on penems and carbapenems only. This enzyme may thus contribute to the development of resistance to penems and carbapenems but not other beta-lactams.

Aeromonas hydrophila↗

PubMed1993

Chromosome Polymorphisms among Strains of Hansenula polymorpha (syn. Pichia angusta).

Contour-clamped homogeneous electrophoresis and an embedded-agarose method of sample preparation were combined to carry out an analysis of the chromosome sets of nine strains of Hansenula polymorpha (syn. Pichia angusta). Chromosomal DNA molecules could be separated into a series of bands ranging, approximately, from 650 up to 2,200 kb in size. Polymorphism of the electrophoretic pattern was demonstrated among the strains investigated in this study. Cross-hybridization between H. polymorpha and Saccharomyces cerevisiae ribosomal DNA was also observed.

Journal Article↗

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

Rapid access to pharmacokinetics data and correlation between antimicrobial susceptibility results and drug tissue distribution using a personal computer.

MYMIC is a computer-aided system capable of integrating antibiotic susceptibility data with the concentrations the drugs reach in various body tissues and fluids by calculating site concentration/minimal inhibitory concentration quotients. The program can be run on any low-cost personal computer operating under MS-DOS, provided it is equipped with a hard-disk drive and with a minimum of 512 kilobytes of random access memory. The use of the program does not require any knowledge of computer languages. The antibiotic susceptibility data can be entered either as minimal inhibitory concentrations or as inhibitory zone diameters; in the latter case, minimal inhibitory concentrations are automatically calculated via regression formulas. The concentrations obtained by 90 antibiotics in 51 different human tissues and fluids are recorded in a data base of over 1,000 records, obtained from roughly 700 original papers. A MYMIC sample session was simulated by mimicking infections of three different body districts (namely bone, prostate, and sputum) caused by Pseudomonas aeruginosa, Escherichia coli or Providencia stuartii.

Anti-Bacterial Agents↗

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

Mechanisms of resistance to growth inhibition and killing by beta-lactam antibiotics in enterococci.

Enterococci are characterized by an intrinsic resistance to growth inhibition by beta-lactam antibiotics. The low susceptibility of enterococci to beta-lactam antibiotics is associated with the synthesis of a particular penicillin-binding protein (PBP) that has a low affinity for beta-lactam agents. This protein appears to be capable of taking over the function of the other PBPs when they are saturated with beta-lactam molecules or inactivated by mutations. A quantitative correlation can be established between the binding of several beta-lactam molecules to the low-affinity PBP and the minimal inhibitory concentration for enterococcal strains. In contrast, the mechanism of enterococcal resistance to the bactericidal activity of beta-lactam agents that inhibit growth at relatively low concentrations appears to be associated with an alteration in the pattern of autolytic enzyme activity. In particular, lack of or poor activity of an autolytic enzyme appears to be responsible for the paradoxical bactericidal response often exhibited by clinical isolates of Enterococcus faecalis in the presence of penicillin.

Anti-Bacterial Agents↗

PubMed1992

Analysis of bacteriolytic activity patterns, a novel approach to the taxonomy of enterococci.

The bacteriolytic activities of different group D streptococcal species on various media and substrates were studied. Our results showed that all of the enterococcal species which we tested had bacteriolytic activity on at least one of the media used, while the group D nonenterococcal species had no such activity. In addition, using culture media containing different additives and different pH values, we defined seven major groups of bacteriolytic activity (lyogroups), each of which overlapped with one species (four lyogroups), two species (two lyogroups), or four species (one lyogroup). The detection of enterococcal lyogroups proved to be as reliable for species identification as the conventional methods presently in use.

Bacteriolysis↗

PubMed1992

Diffusion of meropenem and imipenem through the outer membrane of Escherichia coli K-12 and correlation with their antibacterial activities.

The outer membrane permeability to meropenem and imipenem in Escherichia coli K-12 was investigated, and its porin-deficient mutants were transformed with a constructed vector carrying the carbapenem-hydrolyzing CphA metallo-beta-lactamase gene. By using the method of Zimmermann and Rosselet, meropenem was shown to penetrate through the outer membrane of E. coli K-12 five times faster than cephaloridine but twice as slowly as imipenem. Lack of one or both porins significantly reduced the penetration of both carbapenems. No evidence of specific porin pathways of the type described in Pseudomonas aeruginosa was found. Despite its slower penetration, meropenem was two to eight times more active than imipenem against both parent and porin-defective mutants, whether harbouring CphA beta-lactamase or not. Meropenem was also more active than imipenem against E. coli DC2, a strain with a breakdown in the outer membrane permeability which made periplasmic concentrations of beta-lactams similar to the external concentrations. In this strain, meropenem caused a more than 50% reduction in cell number increase at a concentration very close to the 50% inhibitory concentration for penicillin-binding protein type 2 (PBP 2), whereas imipenem, at the same concentration, did not significantly inhibit cell growth. This result was explained by the higher affinity of meropenem for PBP 3 compared with imipenem and supports the conclusion that synergistic inhibition of both PBPs was the main mechanism in the better antibacterial activity of meropenem.

Bacteria↗

PubMed1992

Novel immunoenzymatic assay for identification of coagulase- and protein A-negative Staphylococcus aureus strains.

A purified monoclonal antibody (MAb) which specifically reacts with Staphylococcus aureus glucosaminidase was obtained. This MAb was utilized to develop an immunoenzymatic assay for the identification of S. aureus strains. The sensitivity of this assay, based on the simultaneous detection of S. aureus glucosaminidase and protein A, was evaluated by analyzing a total of 196 strains, 26 of which did not exhibit one or more of the following properties: protein A, clumping factor, and staphylocoagulase. All strains yielded positive results by the MAb-based immunoenzymatic test. The assay's ability to differentiate between S. aureus and other staphylococci was then analyzed by testing a total of 277 non-S. aureus strains that yielded negative results. Our data demonstrate that this immunoenzymatic assay can be used as a single S. aureus identification criterion, particularly useful for those strains negative for clumping factor, staphylocoagulase, or protein A.

Animals↗

PubMed1992

Modified MacConkey medium which allows simple and reliable identification of Providencia stuartii.

This work describes a modified MacConkey medium (MCP medium) enabling the simple identification of Providencia stuartii, an emerging nosocomial pathogen. A total of 813 strains, belonging to the families Enterobacteriaceae and Pseudomonadaceae, were tested on MCP medium; all P. stuartii strains were phosphatase positive, as were 97.5% of Morganella morganii strains, in contrast with all other tested organisms. A simple discriminating test, such as the ornithine or citrate test, allowed identification of strains of these species. We have also compared the reliabilities of P. stuartii identification by commercial kits (API 20E system) by using a standard MacConkey or MCP medium. Sixteen and three-tenths percent of P. stuartii strains were misidentified by using the former procedure, while with the latter all strains were correctly identified. Finally, the MCP medium was used over a 6-month period in our routine clinical laboratory. Of a total of 1,278 seeded urine samples from elderly patients, we isolated 103 P. stuartii strains which were all correctly identified by coupling MCP medium and the API 20E system. Seventeen and one-half percent of these strains were misidentified when the API 20E system was used in combination with standard MacConkey medium.

Aged↗

PubMed1991

Reversal by 5-azacytidine of the S-adenosyl-L-methionine-induced inhibition of the development of putative preneoplastic foci in rat liver carcinogenesis.

The development of gamma-glutamyltranspeptidase (GGT)-positive foci, in Wistar rats, initiated with diethylnitrosamine and subjected to selection according to 'resistant hepatocyte' protocol, was coupled, 7 weeks after initiation, with liver DNA hypomethylation and with a fall in S-adenosylmethionine/S-adenosylhomocysteine (SAM/SAH) ratio, and in 5-methylthio-adenosine (MTA) content. A 15-day treatment with SAM, started 1 week after selection, caused a dose-dependent decrease in the development of GGT-positive foci, recovery of liver SAM/SAH ratio and MTA level, and liver DNA methylation. A 12-day treatment with 20 mumol/kg per day of 5-azacytidine (AzaC), starting 1 week after selection, enhanced growth of GGT-positive foci, caused strong DNA hypomethylation, and partially counteracted the inhibition of GGT-positive foci growth, without affecting recovery of SAM/SAH ratio and MTA level, induced by SAM. These results suggest a role of DNA methylation in the antipromoting effect of SAM.

Adenosine↗

PubMed1991

The Aeromonas hydrophila cphA gene: molecular heterogeneity among class B metallo-beta-lactamases.

An Aeromonas hydrophila gene, named cphA, coding for a carbapenem-hydrolyzing metallo-beta-lactamase, was cloned in Escherichia coli by screening an Aeromonas genomic library for clones able to grow on imipenem-containing medium. From sequencing data, the cloned cphA gene appeared able to code for a polypeptide of 254 amino acids whose sequence includes a potential N-terminal leader sequence for targeting the protein to the periplasmic space. These data were in agreement with the molecular mass of the original Aeromonas enzyme and of the recombinant enzyme produced in E. coli, evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude beta-lactamase preparations followed by renaturation treatment for proteins separated in the gel and localization of protein bands showing carbapenem-hydrolyzing beta-lactamase activity by a modified iodometric technique. The deduced amino acid sequence of the CphA enzyme showed regions of partial homology with both the beta-lactamase II of Bacillus cereus and the CfiA beta-lactamase of Bacteroides fragilis. Sequence homologies were more pronounced in the regions encompassing the amino acid residues known in the enzyme of B. cereus to function as ligand-binding residues for the metal cofactor. The CphA enzyme, however, appeared to share a lower degree of similarity with the two other enzymes, which, in turn, seemed more closely related to each other. These results, therefore, suggest the existence of at least two molecular subclasses within molecular class B metallo-beta-lactamases.

Aeromonas↗

PubMed1991

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.

Acetylglucosaminidase↗

PubMed1991

New targets for the mechanism of action of antibiotics active against gram-positive cocci.

The main mechanisms of resistance to beta-lactams in gram-positive cocci include either the production of beta-lactamases or alterations in the molecular targets of these antibiotics, the penicillin binding proteins (PBPs). In spite of the appearance of new beta-lactams, more stable to the hydrolytic activity of beta-lactamases or with higher affinity for PBPs, no substantial progress in improving the activity against gram-positive has been achieved. In addition to the search for new beta-lactams it would be of interest to find molecules directed against new targets of the cell wall of gram-positive bacteria (i.e. teichoic acids and lipoteichoic acid) of which up to now no specific inhibitor is known. These two wall polymers are thought to be essential for cell survival within the host. Among new inhibitors a new antibiotic belonging to the class of acid lipopeptides called daptomycin (LY146032), and active against gram-positive seems of particular interest. Our studies demonstrate that daptomycin is a specific inhibitor of lipoteichoic acid synthesis.

Anti-Bacterial Agents↗

PubMed1991

The mechanism of staphylococci resistance to methicillin: a critical analysis of dominant opinions.

Resistance to methicillin has been reported to be constantly associated with production of a novel penicillin-binding protein (PBP), indicated as PBP2a. This PBP is always present in strains that are homogeneously resistant to the drug, but is also constantly present in the strains heterogeneously resistant, where by far the vast majority of the cells are indeed sensitive to the antibiotic. This apparent lack of correlation between the presence of PBP2a and methicillin resistance has led to the idea, shared by most scientists, that the presence of PBP2a alone cannot explain resistance of staphylococci to methicillin. This confusion has implications also for clinical microbiology. In fact in in-vitro assays, not rarely, strains that are resistant to methicillin appear sensitive to other beta-lactams. However, clinical experience has demonstrated that when infections caused by methicillin-resistant staphylococci, which, in in vitro assays, become sensitive to other beta-lactams, are treated with the latter antibiotics, the incidence of failures is higher than that observed when the same antibiotics are used to treat infections caused by staphylococci sensitive to methicillin. Also in consideration of the fact that the mechanism of resistance to methicillin is not yet understood, many authors recommend to consider methicillin-resistant staphylococci as being in-vivo resistant also to the beta-lactams to which they may become sensitive in in-vitro assays. In the opinion of the authors, the mechanism of staphylococcal resistance to methicillin is reasonably explainable at least in its fundamental aspects. The present dominant opinions concerning methicillin-resistant staphylococci will be critically analyzed on the basis both of data presented in the literature and of data obtained in the authors' laboratory.

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

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↗