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A Tomasz

Publications and source records attributed to A Tomasz.

At least 163 records · Page 9Linked to original sources
PubMed1989

New mechanism for methicillin resistance in Staphylococcus aureus: clinical isolates that lack the PBP 2a gene and contain normal penicillin-binding proteins with modified penicillin-binding capacity.

Seventeen clinical isolates of Staphylococcus aureus (from the United States and Europe) selected for low (borderline)-level methicillin resistance (MIC of methicillin, 2 to 4 micrograms/ml; MIC of oxacillin, 0.5 to 8 micrograms/ml) were examined for their mechanisms of resistance. Five strains were typical of heterogeneous S. aureus: they gave positive reactions with a DNA probe specific for mec and contained a small fraction (10(-6] of highly resistant cells (MIC, greater than 100 micrograms/ml). The rest of the 12 strains were homogeneous with respect to their methicillin resistance: the MIC of methicillin for all cells was 2 to 4 micrograms/ml, and no cells for which MICs were 50 micrograms/ml or higher were detectable (less than 10(-9]. None of these strains reacted with the mec-specific DNA probe. One representative strain of each group was characterized in more detail. Strain CDC-1, prototype of heterogeneous methicillin-resistant S. aureus, contained penicillin-binding protein (PBP) 2a; its DNA could transform a methicillin-susceptible and novobiocin-resistant recipient to methicillin resistance with ca. 35% linkage to Novr. Introduction of the "factor X" determinant (K. Murakami and A. Tomasz, J. Bacteriol. 171:874-879, 1989) converted strain CDC-1 to high, homogeneous resistance. Strain CDC-6, prototype of the second group of isolates, showed completely homogeneous MICs of methicillin, oxacillin, and cefotaxime. The strain contained modified "normal" PBPs: PBPs 1 and 2 showed low drug reactivity (and/or cellular amounts), and PBP 4 was present in elevated amounts. No PBP 2a could be detected. DNA isolated from strain CDC-6 could transform the methicillin-susceptible and novobiocin-resistant strain to methicillin resistance in a multistep fashion, but this resistance showed no genetic linkage to the Nov marker. We suggest that staphylococci with borderline resistance may contain at least three different classes of mechanism: heterogeneous, methicillin-resistant S. aureus, PBPs of modified drug reactivities, and the previously reported hyperproduction of beta-lactamase (L.K. McDougal and C. Thornsberry, J. Clin Microbiol. 23:832-839, 1986).

Bacterial Proteins↗
PubMed1989

Mechanism of pneumococcal cell wall degradation in vitro and in vivo.

We compared the products of autolytic amidase-catalyzed wall degradation in vivo (in penicillin-induced lysis) and in vitro. Pneumococci labeled in their cell wall stem peptides by radioactive lysine were treated with penicillin, and the nature of wall degradation products released to the medium during lysis of the bacteria was determined. At early times of lysis (20% loss of wall label), virtually all the radioactive peptides released (greater than 94%) were of high molecular size and were still attached to glycan and teichoic acid. At times of more extensive bacterial lysis (56%), progressively larger and larger fractions of the released peptides became free, i.e., detached from glycan and teichoic acid. Analysis of the nondegraded residual wall material by high-resolution high-pressure liquid chromatography revealed that this in vivo-triggered autolysis did not involve selective hydrolysis of some of the chemically distinct stem peptides. Parallel in vitro experiments yielded completely different results. Purified pneumococcal cell walls labeled with radioactive lysine were treated in vitro with low concentrations of pure amidase, and the nature of wall degradation products released during limited hydrolysis and after more extensive degradation was determined. In sharp contrast to the in vivo experiments, the main products of in vitro hydrolysis were free peptides. After a short treatment with amidase (resulting in a 20% loss of label), the material released was enriched for the monomeric stem peptides. At all times of hydrolysis (including the time of extensive degradation), only a relatively small fraction of the released wall peptides was covalently attached to glycan and teichoic acid components (17% as compared with 40% in the intact cell wall). We propose that the in vivo-triggered amidase activity first attacks the amide bonds in some strategically located (or unprotected) stem peptides that hold large segments of cell wall material together. The observations indicate that the in vivo activity of the pneumococcal autolysin is under topographic constraints.

Amidohydrolases↗
PubMed1989

Involvement of multiple genetic determinants in high-level methicillin resistance in Staphylococcus aureus.

A methicillin-susceptible, novobiocin-resistant strain of Staphylococcus aureus (RN2677; methicillin MIC, 0.8 micrograms/ml) was transformed with DNA prepared from highly and homogeneously methicillin-resistant S. aureus strains (methicillin MIC, greater than or equal to 400 micrograms/ml) or from heterogeneous strains in which the majority of cells had a low level of resistance (methicillin MIC, 6.3 micrograms/ml). All methicillin-resistant transformants showed low and heterogeneous resistance (methicillin MIC, 3.1 micrograms/ml) irrespective of the resistance level of DNA donors. All transformants examined produced normal amounts of the low-affinity penicillin-binding protein (PBP) 2a, and methicillin resistance and the capacity to produce PBP 2a showed the same degree of genetic linkage to the novobiocin resistance marker with both homogeneous and heterogeneous DNA donors. Next, we isolated a methicillin-susceptible mutant from a highly and homogeneously resistant strain which had a Tn551 insertion near or within the PBP 2a gene and thus did not produce PBP 2a. With this mutant used as the recipient, genetic transformation of the methicillin resistance gene was repeated with DNA isolated either from highly and homogeneously resistant strains or from heterogeneous (low-resistance) strains. All transformants obtained expressed high and homogeneous resistance and produced PBP 2a irrespective of the resistance level of the DNA donors. Our findings suggest that (i) the methicillin resistance locus is identical to the structural gene for PBP 2a, (ii) although the ability to produce PBP 2a is essential for resistance, the MICs for the majority of cells are not related to the cellular concentration of PBP 2a, and (iii) high MICs and homogeneous expression of resistance require the products of other distinct genetic elements as well.

DNA Transposable Elements↗
PubMed1989

DNA probe for identification of Streptococcus pneumoniae.

A total of 287 clinical isolates of Streptococcus pneumoniae (pneumococcus) were tested for their ability to undergo autolysis when treated with sodium deoxycholate. The test was positive for all but one isolate, strain DOC-1. This autolysis required the activity of an enzyme which is unique and characteristic of S. pneumoniae: a choline-dependent N-acetylmuramoyl-L-alanine amidase, the gene product of the lytA gene. We used lytA as a DNA probe to test the distribution of the autolysin gene among clinical isolates of S. pneumoniae. In dot blot hybridization experiments our probe reacted with the DNA of 60 of 60 strains tested, including the autolysis-deficient clinical isolate DOC-1. No hybridization occurred when strains of Streptococcus sanguis, Streptococcus mutans, Streptococcus pyogenes, Streptococcus (Enterococcus) faecalis, Streptococcus (Enterococcus) faecium, Streptococcus agalactiae, and Streptococcus bovis were tested. The lytA gene appears to be an ideal candidate for use as a DNA probe for the identification of S. pneumoniae.

Amidohydrolases↗
PubMed1989

Variation in penicillin-binding protein patterns of penicillin-resistant clinical isolates of pneumococci.

A large number of pneumococcal isolates (over 80 strains) from a variety of geographic locales and representing a spectrum of resistance levels from a penicillin MIC of 0.003 microgram/ml up to an MIC of 16 micrograms/ml were analyzed for their penicillin-binding protein (PBP) patterns. With a few exceptions, the great majority of strains with penicillin MICs up to about 0.05 microgram/ml contained the same set of five PBPs with molecular sizes typical of those of susceptible pneumococci. In strains with penicillin MICs of about 0.1 microgram/ml and up, virtually all isolates showed two common features: (i) all isolates showed loss of PBP 1A (98 kilodaltons) with or without a parallel appearance of a "new" PBP that ranged in molecular size between 96 and 97 kilodaltons; and (ii) in strains with penicillin MICs of 0.5 microgram/ml or more, PBP 2B could not be detected on the fluorograms even with very high concentrations of radioactive penicillin. Beyond these two common features, resistant strains with similar penicillin MICs showed a surprising variety of PBP profiles (i.e., in the number and molecular sizes of PBPs), each characteristic of a given isolate. We suggest that in pneumococci remodeling of critical PBPs in more than one way may result in comparable levels of penicillin resistance.

Bacterial Proteins↗
PubMed1988

A sandwich adhesion on Streptococcus pneumoniae attaching to human oropharyngeal epithelial cells in vitro.

Streptococcus pneumoniae attach to human pharyngeal epithelial cells through the specific interaction of bacterial surface adhesins with glycoconjugate receptors. The present study defines the adhesin as a molecule bridging between an anchoring site in the bacterial cell wall and the epithelial cell receptor. The nature of the adhesin was defined in three ways: First, the attachment of whole bacteria was reduced by trypsin, periodate and heat. Second, heat treatment of whole bacteria was shown to release material, which was able to reconstitute the adherence. The heat extract bound to epithelial cells, as shown by fluorescence labelling, and agglutinated latex beads covalently coupled with receptor oligosaccharide. Active material could be extracted by heat from both high and low adhering strains, but could reconstitute only attaching strains. Third, the bacterial component binding the adhesin was localized to protoplasts and cell wall fractions obtained by mechanical or deoxycholate induced lysis of pneumococci. Isolated pneumococcal surface components, which did not inhibit attachment, included peptidoglycan, C polysaccharide, Forssmann antigen, capsular polysaccharide and a phenol extract produced in analogy to streptococcal lipoteichoic acid. The procedure used to extract the adhesin was previously used to prepare the competence factor. The competence deficient mutant RA7- attached poorly compared to the competent R6 parent. The possible relatedness of attachment to competence is discussed.

Adhesins, Bacterial↗
PubMed1988

The contribution of pneumococcal cell wall to the pathogenesis of experimental otitis media.

We studied the contribution of pneumococcal cell wall to the pathogenesis of otitis media in chinchillas after middle ear inoculation of killed, encapsulated type 7F Streptococcus pneumoniae; killed, unencapsulated R6 S. pneumoniae; and isolated R6 pneumococcal cell wall. Ears inoculated with encapsulated and unencapsulated pneumococci had significantly higher concentrations of polymorphonuclear and mononuclear leukocytes and lysozyme in middle ear fluid and developed more epithelial metaplasia and granulation tissue than did saline-inoculated ears. The mean concentration of lysozyme in middle ear fluid was higher in ears inoculated with killed, unencapsulated than encapsulated pneumococci. The middle ear mucoperiosteum of ears inoculated with pneumococcal cell wall showed significantly more polymorphonuclear leukocytes, epithelial metaplasia, subepithelial congestion, and granulation tissue than did control ears. Because nonviable, unencapsulated pneumococci and pneumococcal cell wall caused middle ear inflammation in the chinchilla model of otitis media, it is possible that cell envelope and cell wall components released during bacterial lysis may contribute to chronic otitis media with effusion in humans.

Acute Disease↗
PubMed1988

Penicillin resistance and defective lysis in clinical isolates of pneumococci: evidence for two kinds of antibiotic pressure operating in the clinical environment.

Seventy percent of clinical isolates of penicillin-resistant pneumococci also exhibit defective lysis when treated with penicillin exceeding the minimal inhibitory concentration (MIC). To provide a possible explanation for the frequent association of these two traits, we exposed penicillin-susceptible pneumococci to two kinds of antibiotic pressures in the laboratory. Treatment of cultures with cycles of high concentrations of penicillin (20 X MIC) followed by growth of the survivors in drug-free medium selected for lysis-defective mutants that died only slowly during antibiotic treatment but had unchanged MICs. Exposure to sustained, low levels of penicillin produced resistant mutants, with elevated MICs, that lysed normally with penicillin. We suggest that the cyclic antibiotic exposure generally used in the clinical setting may select primarily for enhanced survival. From these survivors a second type of antibiotic exposure--sustained antibiotic concentrations just above the MIC (concentrations that may be restricted to the tail-end trough of a dosing interval)--selects for penicillin-resistant mutants.

Bacteriolysis↗
PubMed1988

Insertional inactivation of the major autolysin gene of Streptococcus pneumoniae.

The lytA gene encoding the major pneumococcal autolysin (N-acetylmuramoyl-L-alanine amidase) was inactivated by inserting the 2-kilobase MspI fragment of pE194 containing the staphylococcal ermC gene. Stable autolysis-deficient (Lyt-) mutants and their isogenic Lyt+ parents were used in experiments designed to test possible physiological functions of the amidase. No autolysis could be induced in the mutants grown at 37 degrees C by deoxycholate, by incubation in stationary phase, or by treatment with penicillin. On the other hand, the Lyt- mutants exhibited normal growth rates and yields and normal adaptive responses during shifts from one growth temperature or nutritional condition to another. There was no evidence for impeded cell separation (chain formation). Colonies of Lyt- insertional mutants produced normal hemolytic zones on blood agar; they showed normal (high) levels of competence for genetic transformation. Lyt- mutants were also able to produce type 3 and 6 capsular polysaccharides, and such strains showed the same degree of virulence in mice as did the isogenic Lyt+ parent. The physiological function(s) of the amidase remains a puzzle.

Amidohydrolases↗
PubMed1988

Autolysis-resistant peptidoglycan of anomalous composition in amino-acid-starved Escherichia coli.

Nongrowing Escherichia coli deprived of an essential amino acid continued to produce peptidoglycan at a rate approximately 30% of that of growing cells. The composition of this peptidoglycan was very different from that of growing cells and resembled that of peptidoglycan left undegraded during partial autolysis of the bacteria. Synthesis of this peptidoglycan of anomalous composition began at once upon the removal of the amino acid from the medium. Fifteen minutes of amino acid deprivation was sufficient to virtually completely prevent penicillin-induced autolytic wall degradation in vivo. During this time, although the specific activities of soluble and membrane-bound hydrolytic transglycosylases and endopeptidases remained high, the peptidoglycan produced showed decreased sensitivity to degradation in vitro. After more extensive (2-h) starvation, triggering of autolysis by chaotropic agents was also blocked. Autolysis in growing cells may be selective for peptidoglycan representing the cylindrical portion of the sacculus. It is suggested that at least part of the mechanism of the well-known lysis resistance of nongrowing E. coli is related to the deposition of structurally anomalous and relatively autolysin-resistant peptidoglycan at some strategically located sites on the bacterial surface.

Amino Acids↗
PubMed1988

Altered peptidoglycan structure in a pneumococcal transformant resistant to penicillin.

A series of isogenic pneumococcal transformants differing in their levels of penicillin resistance and containing altered penicillin-binding proteins were compared for their cell wall structures by using a recently developed technique that can resolve the peptidoglycan stem peptides of Pneumococcus strains to over 40 components (J. F. Garcia-Bustos, B. T. Chait, and A. Tomasz, J. Biol. Chem. 32:15400-15405). The stem peptides from the highly resistant transformants differed strikingly from those of the susceptible recipient strain, and the peptide patterns were almost identical to that of the DNA donor. Four peptides representing the major components in the walls of susceptible cells were replaced by six new peptides that were only minor components of susceptible cell walls. A remarkable common feature of these new species was their high alanine content. Amino acid analysis, sequencing, and mass spectrometry allowed the assignment of the extra alanine residues to dialanine or alanylserine cross bridges in the six new stem peptides. The common feature of the four peptide species that were present as major components in the susceptible walls, but became minor species in the resistant cells, was the absence of a cross bridge in at least one of the stem peptide components. We suggest that the extensive remodelling of cell wall synthetic enzymes that accompanies acquisition of penicillin resistance eventually also alters the reactivity of these proteins towards their natural substrates in cell wall synthesis. As a result, highly penicillin-resistant pneumococci will shift from the use of wall precursors with linear stem peptides to a preferential use of precursors containing the more-hydrophobic peptides carrying dialanyl or alanylserine cross bridges.

Alanine↗
PubMed1987

Structure of the peptide network of pneumococcal peptidoglycan.

The peptide network of Streptococcus pneumoniae cell walls was solubilized using the pneumococcal autolytic amidase (N-acetylmuramoyl-L-alanine amidase, EC 3.5.1.28). The peptide material was fractionated into size classes by gel filtration followed by reverse-phase high-performance liquid chromatography which resolved the peptide population into over 40 fractions. About 40% of the lysines present participate in cross-links between stem peptides. The main components (3 monomers, 5 dimers, and 2 trimers), accounting for 77% of all the wall peptides, were purified. Their structures were determined using a combination of amino acid and end-group analysis, mass spectrometry, and gas-phase sequencing. Two different types of cross-links between stem peptides were found. In the most abundant type there is an alanylserine cross-bridge between the alanine in position 4 of the donor stem peptide and the lysine at position 3 of the acceptor peptide, as in type A3 peptidoglycan. In the second type of cross-link there is no intervening cross-bridge, as in the type A1 peptidoglycan of Gram-negative bacteria. The data indicate that pneumococcal peptidoglycan has a structural complexity comparable to that recently shown in some Gram-negative species.

Cell Wall↗
PubMed1987

Nonsteroidal anti-inflammatory agents in the therapy for experimental pneumococcal meningitis.

An increased inflammatory mass in the subarachnoid space during bacterial meningitis may correlate with a poor outcome of disease. Using a rabbit model of pneumococcal meningitis, we sought to reduce this inflammatory process. The ability of the pneumococcal cell wall to cause death and to generate leukocytosis and abnormal chemistry in cerebrospinal fluid was prevented when animals were treated with inhibitors of cyclooxygenase pathway of arachidonate metabolism. Bacterial lysis by ampicillin led to release of cell wall that caused a significant, transient increase in meningeal inflammation. This inflammatory burst was also prevented by administering cyclooxygenase inhibitors concurrently with the antibiotic.

Ampicillin↗
PubMed1987

A mutant of Streptococcus pneumoniae that exhibits thermosensitive penicillin tolerance and the paradoxical effect.

Mutants of Streptococcus pneumoniae that contain active autolysin and yet cannot be induced to lyse during treatment with penicillin (Lyt+Tol+ mutants) have been described. We have now shown that these mutants are temperature dependent (32 degrees C); at 37 degrees C these bacteria underwent penicillin-induced lysis. In addition, mutants at the lysis-permissive temperature showed the so-called 'paradoxical response' to penicillin. Temperature shift experiments indicated that the change from tolerant to lytic response or vice versa is a fast process. No differences were detected in autolysin specific activity or in the kinetics of inhibition of protein, peptidoglycan and teichoic acid syntheses in cells treated with penicillin at 32 and 37 degrees C. The results of genetic crosses indicated that the thermosensitivity of penicillin-induced autolysis in the Lyt+Tol+ mutants is not a property of the autolytic enzyme itself. The observations suggest that the thermosensitive process in the mutants represents either a step(s) in autolysin regulation or involves some difference in the structure of the cell walls produced at 32 degrees C versus 37 degrees C.

Bacteriolysis↗
PubMed1987

Teichoic acid-containing muropeptides from Streptococcus pneumoniae as substrates for the pneumococcal autolysin.

Pneumococcal cell walls in which the normal phosphorylcholine component of the wall teichoic acids is replaced with phosphorylethanolamine cannot absorb the homologous autolytic enzyme and are completely resistant to autolytic degradation (S. Giudicelli and A. Tomasz, J. Bacteriol. 158:1188-1190, 1984). We have now isolated and characterized soluble teichoic acid-containing muropeptides from such cell walls and tested them as substrates for the pneumococcal autolytic enzyme. Both choline- and ethanolamine-containing muropeptides were hydrolyzed to the same extent by the enzyme. Furthermore, free choline concentrations that totally inhibited the digestion of pneumococcal cell walls in vivo and in vitro were without effect when the soluble substrates were used.

Amidohydrolases↗
PubMed1986

Construction of a penicillin-tolerant laboratory mutant of Staphylococcus aureus.

Numerous conflicting reports describing clinical penicillin tolerant Staphylococcus aureus isolates have raised questions concerning the reproducibility and genetic homogeneity of such isolates. The purpose of our study was to construct a tolerant mutant which would then serve as a reference strain and could also be used for testing the potential significance of antibiotic tolerance during chemotherapy of staphylococcal infections. A penicillin sensitive laboratory mutant strain was mutagenized and exposed to cycles of penicillin selection. A culture was then obtained which lost about 1 log of viability during exposure to up to 1000 times the MIC of penicillin. The same conditions caused a 3-4 log decline in the viability of the parent cell. The tolerance property was stable for at least 150 generations of nonselective growth and was reproducible in randomly picked colonies. The superior survival during treatment was also demonstrable in cultures grown in rabbit serum or cerebrospinal fluid. Penicillin tolerant cultures could also be obtained by selection without mutagenesis, suggesting that such a mutant may indeed be selected for in the clinical environment.

Drug Tolerance↗
PubMed1986

Conversion of a homogeneously methicillin-resistant strain of Staphylococcus aureus to heterogeneous resistance by Tn551-mediated insertional inactivation.

Plasmid pRN3208, thermosensitive for replication, and carrying the erythromycin transposon Tn551, was used for insertional inactivation of methicillin resistance in a highly and homogeneously resistant strain of Staphylococcus aureus. Two kinds of insertionally inactivated cells were obtained. Cultures of the major class contained highly methicillin resistant cells with a frequency of about 10(-3) to 10(-4), produced DNA with methicillin resistance transforming activity, and also produced penicillin binding protein 2a, the 78 kd low affinity penicillin binding protein characteristic of methicillin resistant Staphylococcus aureus, in apparently normal quantities. The single member of class B had no detectable methicillin resistant cells (less than 10(-8)) with an MIC greater than 1 micrograms/ml, contained no DNA with methicillin resistant transforming activity and no penicillin binding protein 2a. The data suggest that in the class A cells insertional inactivation did not affect the structural gene(s) of methicillin resistance but a regulatory locus or loci needed for the homogeneous expression of resistance.

Bacterial Proteins↗