Biomedical subjects

A Tomasz

Publications and source records attributed to A Tomasz.

At least 127 records · Page 7Linked to original sources

PubMed1994

Molecular aspects of methicillin resistance in Staphylococcus aureus.

All clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates examined so far contain the mecA gene, a 2130bp stretch of DNA of non-staphylococcal origin which, together with a larger block (up to 40-60 Kb) of 'foreign' DNA, is incorporated into the staphylococcal chromosome. mecA encodes for the 78 Kd penicillin-binding protein (PBP) 2A, which has very low affinity for beta-lactam antibiotics. The sequence of the mecA gene contains structural motifs characteristic of cell wall synthetic transpeptidases. It is generally assumed that the mecA gene product (PBP 2A) acts as a surrogate enzyme which takes over the task of cell wall synthesis from the normal complement of staphylococcal PBPs, since the latter are inhibited by relatively low (e.g. methicillin) concentrations of beta-lactam antibiotics. While direct biochemical evidence for a transpeptidase activity in PBP 2A is still missing, the essentiality of an intact mecA gene for the expression of high-level methicillin resistance has been clearly established by transposon inactivation experiments. On the other hand, it was already noted some time ago that an intact mecA and its gene product PBP 2A alone cannot be fully in control of the resistant phenotype, since all MRSA isolates, irrespective of their MIC values (from as low as 3 mg/L or as high as 1600 mg/L), were found to contain comparable amounts of PBP 2A. Such major disparities between cellular amounts of PBP 2A and the antibiotic MIC values suggested that a factor or factors of unknown nature ('factor X') other than the mecA gene product also played an essential role in the phenotypic expression of resistance. The same conclusion was reached in early genetic studies in which methicillin resistance could be reduced by insertional inactivation of a chromosomal site (omega 2003) within the so-called femA gene--(factor essential for the expression of methicillin resistance) outside the mecA determinant. More recently, several additional chromosomal sites were identified outside the mecA gene in which transposon inactivation reduced the level of beta-lactam resistance. The importance of these genes becomes clear if one realizes that it is the appropriate functioning of these determinants (in the genetic background of MRSA) rather than the quantity of PBP 2A in the cells that seems to determine the MIC value of an MRSA isolate. It is not clear at the present time how many such 'auxiliary genes' exist and exactly how these gene co-operate with the mecA gene in bringing about high-level beta-lactam resistance.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1994

Reassessment of the number of auxiliary genes essential for expression of high-level methicillin resistance in Staphylococcus aureus.

A new transposon library constructed in the background of the highly and homogeneously methicillin-resistant Staphylococcus aureus strain COL yielded 70 independent insertional mutants with reduced levels of antibiotic resistance. Restriction analysis with HindIII, EcoRV, EcoRI, and PstI and then Southern hybridization with probes for the transposon and for the femA-femB gene demonstrated that 41 of the 70 Tn551 mutants carried distinct and novel, as yet undescribed insertion sites, all of which were outside of the mecA gene and were also outside the already-characterized auxiliary genes femA, femB, femC, and femD. All previously described Tn551 mutations of this type were in genes located either on SmaI fragment A or SmaI fragment I. In contrast, inserts of the new library were located in 7 of the 16 SmaI chromosomal fragments, fragments A, B, C, D, E, F, and I. In all of the mutants, expression of methicillin resistance became heterogeneous, and the MIC for the majority of cells was reduced (1.5 to 200 micrograms ml-1) from the homogeneous methicillin MIC (1,600 micrograms ml-1) of the parental cells. Although identification of the exact number of genes inactivated through the new set of transposon inserts will require cloning and sequencing, a rough estimate of this number from mapping data suggests a minimum of at least 10 to 12 new genetic determinants, all of which are needed together with femA, femB, femC, and femD for the optimal expression of methicillin resistance.

Evaluation Studies as Topic↗

PubMed1994

Gram-positive cell walls stimulate synthesis of tumor necrosis factor alpha and interleukin-6 by human monocytes.

Purified cell walls representing a wide variety in teichoic acid and peptidoglycan structure prepared from eight different gram-positive bacterial species induced the production of tumor necrosis factor alpha (TNF-alpha) and interleukin-6 from human monocytes in the presence of 10% plasma or serum. Significant amounts of cytokines began to be produced at concentrations above 100 ng to 1 microgram of cell walls per ml, with maximal production requiring 10 to 100 micrograms of cell wall material per ml. In the absence of plasma, the cytokine-inducing capacity of cell wall preparations was lower by at least an order of magnitude. The serum-derived cofactor was inactivated by heating at 90 degrees C for 30 min, suggesting that the activity is associated with a protein. On the other hand, replacement of normal with hypogammaglobulinemic plasma, inactivation of complement (at 56 degrees C), and blockade by the monoclonal antibody MY4 of the CD14 receptors on monocytes did not inhibit the production of TNF-alpha induced by whole cell walls. Cell walls also stimulated production of TNF-alpha induced by whole cell walls. Cell walls also stimulated production of TNF-alpha in the presence of polymyxin B, and macrophages derived from the lipopolysaccharide-insensitive cell line of C3He/HeJ mice also produced this cytokine when stimulated by cell walls. Both peptidoglycan and the soluble glycan-teichoic acid component prepared by an enzymatic method from the same wall preparation exhibited a serum-dependent induction of TNF-alpha from monocytes, while stem peptides and disacharride peptides had only poor, if any, activity. Cell walls may contribute to the septic shock induced by gram-positive bacteria.

Antigens, CD↗

PubMed1994

Spread and maintenance of a dominant methicillin-resistant Staphylococcus aureus (MRSA) clone during an outbreak of MRSA disease in a Spanish hospital.

It was not until November 1989 that the 1,000-bed University-affiliated Hospital de Bellvitge "Princeps d'Espanya" in Barcelona first acquired methicillin-resistant Staphylococcus aureus (MRSA). Since that time, the outbreak of MRSA disease has continued. We have analyzed by genomic DNA fingerprinting 189 MRSA isolates collected between late 1989 and the end of 1993. The isolates include both invasive and colonizing strains as well as isolates from health-care workers and environmental sources. In addition, 52 clinical isolates of methicillin-susceptible S. aureus (MSSA) collected in the same hospital were also analyzed. Isolates were classified into clonal types on the basis of molecular typing techniques. A single MRSA clone (I::B::a) belonging to ClaI type I, pulsed-field gel electrophoretic pattern B, and Tn554 pattern a was responsible for the great majority of infections (73% of blood cultures and 79% of specimens from other clinical sources). This clone appeared at the very beginning of the outbreak, spread throughout the hospital wards, and was also carried by inpatients and health-care workers and on environmental surfaces. In contrast, no dominant lineage was apparent among MSSA isolates (33 distinct pulsed-field gel electrophoretic patterns among 52 isolates). Two MSSA isolates seem to have originated from the dominant clone by deletion of the mecA gene and some additional DNA. In several isolates, different mecA polymorphs were present in identical chromosomal backgrounds or cells with distinct chromosomal backgrounds carried the same mecA polymorph, suggesting horizontal transfer of the mecA gene.

Adult↗

PubMed1993

The peptidoglycan composition of a Staphylococcus aureus mutant selected for reduced methicillin resistance.

The peptidoglycan of a Tn551 mutant of Staphylococcus aureus (RUSA208) selected for reduced methicillin resistance was analyzed by reversed-phase high pressure liquid chromatography and mass spectrometry. RUSA208 is a member of a cluster of Tn551 mutants located on fragment A of SmaI digests but is distinct from the femA and femB class of mutants. The peptidoglycan of RUSA208 contained normal parental muropeptides but in diminished amounts only. The major muropeptides of RUSA208 were new components eluting with somewhat longer retention times from the column. Amino acid analysis of these new muropeptides showed identical compositions to the corresponding peaks in the parental strain, but mass spectrometry revealed increased molecular weights by the following mass units: 1 (in monomers), 1 or 2 (in dimers), and 2 or 3 (in trimers). These observations suggest that in RUSA208 the mutational block may be in the amidation of the stem peptide glutamate residues, resulting in the replacement of isoglutamine with free glutamic acid in one or more of the cell wall stem peptides.

Amino Acid Sequence↗

PubMed1993

Genetic control of population structure in heterogeneous strains of methicillin resistant Staphylococcus aureus.

Cultures of heterogeneous strains of methicillin resistant Staphylococcus aureus are composed of several subpopulations of cells that differ in their level of antibiotic resistance and frequencies. Experimental evidence is presented indicating that the number of these subpopulations, their resistance level and the frequency with which they are represented in a culture are strain-specific and reproducible with a surprising degree of precision from one culture to another. This implies a genetic control of population structure in these bacteria.

Electrophoresis, Gel, Pulsed-Field↗

PubMed1993

Triggering of pneumococcal autolysis by lysozyme.

Lysozyme had no effect on the rate of multiplication of growing cultures of Streptococcus pneumoniae, but it greatly reduced the lag period that precedes autolysis of these bacteria in stationary phase. Several experiments were done to understand the mechanism of this effect. Lysozyme had no hydrolytic activity on intact cell walls, and cell walls of pneumococci grown with or without lysozyme had identical composition and susceptibility to the pneumococcal autolysin. The acceleration of stationary-phase autolysis by lysozyme involved triggering of the pneumococcal autolytic enzyme since lysozyme had no detectable effect on nonautolysing (LytA-) mutants and heat-inactivated lysozyme completely lacking enzymatic activity was as effective as the nondenatured enzyme in facilitating stationary-phase autolysis. The role of lysozyme in host defense against pneumococcal infection remains elusive.

Animals↗

PubMed1993

Evidence for the introduction of a multiresistant clone of serotype 6B Streptococcus pneumoniae from Spain to Iceland in the late 1980s.

Almost all of the multiresistant pneumococci that appeared suddenly in clinical specimens in Iceland between 1989 and 1992 belonged to serogroup 6. Fifty-seven of these isolates were analyzed for serotype, penicillin-binding protein pattern, multilocus enzyme genotype, and fragmentation pattern obtained by pulsed-field electrophoretic separation of restriction enzyme digests of chromosomal DNA. All isolates were of serotype 6B and had similar or identical patterns in each molecular test. The Icelandic isolates were indistinguishable from a subgroup of multiresistant serotype 6B pneumococci that has been present with high incidence in Spain during the past two decades. The data suggest the import to Iceland of a single multiresistant clone of pneumococcus, most likely from Spain.

Bacterial Proteins↗

PubMed1993

Abnormal peptidoglycan produced in a methicillin-resistant strain of Staphylococcus aureus grown in the presence of methicillin: functional role for penicillin-binding protein 2A in cell wall synthesis.

Upon the addition of methicillin even at relatively low concentrations (5 mug/ml or 0.3% of the MIC) to the medium, methicillin-resistant staphylococci shift to the production of a new peptidoglycan with an abnormal muropeptide composition which may be the synthetic product of penicillin-binding protein 2A.

Amino Acid Sequence↗

PubMed1993

Possible role of a choline-containing teichoic acid in the maintenance of normal cell shape and physiology in Streptococcus oralis.

Streptococcus oralis ATCC 35037 took up radioactively labeled choline from growth medium. Most of the choline (80 to 90%) was incorporated into the cell wall teichoic acid, and about 10% was localized in the plasma membrane. While cells grew in choline-free medium, they did so at slow rates and produced cell walls with greatly reduced amounts of phosphate and no detectable choline. Cells grown in choline-free medium had grossly abnormal shape and size. Both biochemical and morphological abnormalities were reversible by addition of choline to the medium.

Choline↗

PubMed1993

Altered muropeptide composition in Staphylococcus aureus strains with an inactivated femA locus.

Tn551 inactivation of femA, a factor involved in methicillin resistance of Staphylococcus aureus, caused the production of peptidoglycan in which the fraction of monoglycyl- and serine-containing muropeptides was increased at the expense of pentaglycyl muropeptides. femA mutants have a specific block in the biosynthesis of pentaglycine cross bridges after the addition of the first glycine residue.

Amino Acid Sequence↗

PubMed1992

Transforming growth factor beta 2 inhibits cerebrovascular changes and brain edema formation in the tumor necrosis factor alpha-independent early phase of experimental pneumococcal meningitis.

Macrophages and granulocytes seem to play a key role in the pathogenesis of bacterial meningitis. Transforming growth factor beta (TGF-beta) leads to macrophage deactivation, as well as to inhibition of cytokine production and of endothelial granulocyte adhesion. We have investigated the influence of TGF-beta on regional cerebral blood flow (rCBF), intracranial pressure (ICP), and brain edema formation during the early phase of experimental meningitis. Rats which were inoculated intracisternally with live pneumococci or with pneumococcal cell wall hydrolyzed by the M1 muramidase (PCW-M) developed an increase of rCBF and ICP within 4 h postintracisternal challenge. A single intraperitoneal injection of TGF-beta 2 but not of TGF-beta 2 vehicle-control prevented the changes of rCBF. Furthermore, TGF-beta 2 significantly reduced the increase of ICP in rats inoculated with PCW-M. Likewise, the elevation of brain water content after intracisternal injection of pneumococci or PCW-M was blocked by pretreatment of rats with TGF-beta 2. TGF-beta 1 exhibited similar inhibitory effects in PCW-M-injected rats. The beneficial effects of TGF-beta 2 on the initial phase after pneumococcal inoculation seem to be tumor necrosis factor alpha- (TNF-alpha) independent since (a) intracisternal or intraperitoneal injection of neutralizing anti-TNF-alpha antibodies did not significantly influence rCBF, ICP, and brain water content in PCW-M-induced meningitis; and (b) TNF-alpha was only occasionally detected at low levels in cerebrospinal fluid at 4 h after PCW-M application.

Animals↗

PubMed1992

Peptidoglycan composition in heterogeneous Tn551 mutants of a methicillin-resistant Staphylococcus aureus strain.

It was shown that Tn551 inactivation of two chromosomal (so-called auxiliary) loci other than the mec gene result in a dramatic reduction of methicillin resistance and decreased cell wall turnover and autolytic capacity in a methicillin-resistant Staphylococcus aureus strain (de Jonge, B. L. M., de Lencastre, H., and Tomasz, A. (1990) J. Bacteriol. 173, 1105-1110). To understand the mechanistic basis of these phenomena we have examined the status of the autolytic enzymes and the muropeptide composition of peptidoglycan using reversed-phase high-performance liquid chromatography and mass spectral analyses. While no differences could be detected in the number of autolytic hydrolases, the mutants showed major changes in peptidoglycan composition. Nine prominent muropeptides of the parental strain each carrying a pentaglycyl substituent were missing from the cell wall of one group of mutants. The second mutant lacked four parental muropeptides which were composed of the unsubstituted disaccharide pentapeptide and its alanyl-tetraglycine derivative. The auxiliary genes are genetic determinants involved with the biosynthesis of peptidoglycan precursors, the presence of which in the cell wall may be needed for optimal cell wall turnover.

Amino Acid Sequence↗

PubMed1992

Peptidoglycan composition of a highly methicillin-resistant Staphylococcus aureus strain. The role of penicillin binding protein 2A.

All clinical isolates of methicillin-resistant Staphylococcus aureus contain an extra penicillin binding protein (PBP) 2A in addition to four PBPs present in all staphylococcal strains. This extra PBP is thought to be a transpeptidase essential for the continued cell wall synthesis and growth in the presence of beta-lactam antibiotics. As an approach of testing this hypothesis we compared the muropeptide composition of cell walls of a highly methicillin-resistant S. aureus strain containing PBP2A and its isogenic Tn551 derivative with reduced methicillin resistance, which contained no PBP2A because of the insertional inactivation of the PBP2A gene. Purified cell walls were hydrolyzed into muropeptides which were subsequently resolved by reversed-phase high-performance liquid chromatography and identified by chemical and mass spectrometric analysis. The peptidoglycan composition of the two strains were identical. Both peptidoglycans were highly cross-linked mainly through pentaglycine cross-bridges, although other, chemically distinct peptide cross-bridges were also present including mono-, tri-, and tetraglycine; alanine; and alanyl-tetraglycine. Our experiments provided no experimental data for a unique transpeptidase activity associated with PBP2A.

Amino Acid Sequence↗

PubMed1992

Geographic distribution of penicillin-resistant clones of Streptococcus pneumoniae: characterization by penicillin-binding protein profile, surface protein A typing, and multilocus enzyme analysis.

Examination of several hundred penicillin-resistant clinical isolates of Streptococcus pneumoniae has revealed extensive strain-to-strain variation in the number and molecular size of penicillin-binding proteins (PBPs). This polymorphism has been used to classify resistant isolates into groups (PBP families) that share distinct electrophoretic profiles. We describe herein properties of four such PBP families: two from Spain (and/or Ohio) and one each from Hungary and Alaska. We have discovered that representative isolates assigned to each PBP family also share capsular serotype, antibiotic resistance pattern, pneumococcal surface protein A type, and multilocus enzyme genotype. The results demonstrate independent clonal origin for strains assigned to each PBP family. Each resistant clone occurs with uniquely high incidence within specific geographic areas.

Alaska↗

PubMed1992

Multiple methicillin-resistant Staphylococcus aureus strains as a cause for a single outbreak of severe disease in hospitalized neonates.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of nosocomial infection. Outbreaks of infection caused by these pathogens are generally considered to be traceable to introduction of single strains into a hospital population. A large outbreak of bacteremic disease that recently occurred in our neonatal intensive care unit (11 episodes in 10 patients) involved 9 low birth weight infants and was associated with serious infection (4 episodes of meningitis). To determine the role of a single point source in this outbreak, isolates were characterized based on phenotypic and genotypic analyses. Phenotypic analysis included assessing hemolytic activity, phage typing, antimicrobial susceptibility testing and methicillin resistance population analysis. Genotypic analysis included assessment of plasmid profiles, dot-blot hybridization, restriction enzyme fragment pattern analysis and hybridization analysis of chromosomal DNA using a panel of staphylococcal gene probes. This analysis established that at least two distinct strains of MRSA were responsible for disease during this outbreak. This experience demonstrates the potential for MRSA to cause severe disease in the neonatal intensive care unit and indicates that the epidemiology of MRSA outbreaks is more complex than the spread of a single strain of bacteria.

Cross Infection↗

PubMed1992

A pneumococcal clinical isolate with high-level resistance to cefotaxime and ceftriaxone.

A beta-lactam-resistant serotype 23F Streptococcus pneumoniae clinical isolate from the cerebrospinal fluid of a pediatric patient from California is unusual in that the MICs of cefotaxime and ceftriaxone (2.5 micrograms/ml each) are higher than that of benzylpenicillin (0.3 micrograms/ml); the isolate also has patterns of penicillin-binding proteins and of cell wall peptides which are atypical compared with those of previously examined penicillin-resistant pneumococci.

Cefotaxime↗

PubMed1992

Role of the bacterial cell wall in middle ear inflammation caused by Streptococcus pneumoniae.

The pathogenesis of middle ear inflammation caused by Streptococcus pneumoniae was explored in the chinchilla model with different pneumococcal cell wall (CW) preparations, including isolated native CW, M1 muramidase CW (M1-CW) digest, amidase CW digest, and M1 peptidoglycan (M1-PG) digest. Inflammatory cell and lysozyme concentrations in middle ear fluid (MEF) were measured between 6 and 72 h after the middle ears were inoculated with one of the preparations or sterile saline. Middle ear histopathology was measured quantitatively at 72 h. Native CW, M1-CW digest, and amidase-CW digest caused significantly more inflammatory cell influx and lysozyme accumulation in MEF than saline did. M1-PG digest also caused more inflammatory cell influx and lysozyme accumulation in MEF than saline did but caused less inflammation than native CW or either CW digest. Epithelial metaplasia was significantly greater in ears inoculated with native CW than in ears inoculated with the CW or PG digest or with saline. Pneumococcal CW is, therefore, the principal factor that initiates middle ear inflammation in acute pneumococcal otitis media, and CW teichoication seems to be important in initiating this response.

Amidohydrolases↗