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Biomedical subjects

K Sieradzki

Publications and source records attributed to K Sieradzki.

At least 19 recordsLinked to original sources

Evolution of nanoporosity in dealloying.

Dealloying is a common corrosion process during which an alloy is 'parted' by the selective dissolution of the most electrochemically active of its elements. This process results in the formation of a nanoporous sponge composed almost entirely of the more noble alloy constituents. Although considerable attention has been devoted to the morphological aspects of the dealloying process, its underlying physical mechanism has remained unclear. Here we propose a continuum model that is fully consistent with experiments and theoretical simulations of alloy dissolution, and demonstrate that nanoporosity in metals is due to an intrinsic dynamical pattern formation process. That is, pores form because the more noble atoms are chemically driven to aggregate into two-dimensional clusters by a phase separation process (spinodal decomposition) at the solid-electrolyte interface, and the surface area continuously increases owing to etching. Together, these processes evolve porosity with a characteristic length scale predicted by our continuum model. We expect that chemically tailored nanoporous gold made by dealloying Ag-Au should be suitable for sensor applications, particularly in a biomaterials context.

Journal Article↗

Inactivated pbp4 in highly glycopeptide-resistant laboratory mutants of Staphylococcus aureus.

Both vancomycin- and teicoplanin-resistant laboratory mutants of Staphylococcus aureus produce peptidoglycans of altered composition in which the proportion of highly cross-linked muropeptide species is drastically reduced with a parallel increase in the representation of muropeptide monomers and dimers (Sieradzki, K., and Tomasz, A. (1997) J. Bacteriol. 179, 2557-2566; and Sieradzki, K. , and Tomasz, A. (1998) Microb. Drug Resist. 4, 159-168). We now report that the distorted peptidoglycan composition is related to defects in penicillin-binding protein 4 (PBP4); no PBP4 was detectable by the fluorographic assay in membrane preparations from the mutants, and comparison of the sequence of pbp4 amplified from the mutants indicated disruption of the gene by two types of abnormalities, a 17-amino acid long duplication starting at position 305 of the pbp4 gene was detected in the vancomycin-resistant mutant, and a stop codon was found to be introduced into the pbp4 KTG motif at position 261 in the mutant selected for teicoplanin resistance. Additional common patterns of disturbances in the peptidoglycan metabolism of the mutants are indicated by the increased sensitivity of mutant cell walls to the M1 muramidase and decreased sensitivity to lysostaphin, which is a reversal of the susceptibility pattern of the parental cell walls. Furthermore, the results of high performance liquid chromatography analysis of lysostaphin digests of peptidoglycan suggest an increase in the average chain length of the glycan strands in the peptidoglycan of the glycopeptide-resistant mutants. The increased molar proportion of muropeptide monomers in the cell wall of the glycopeptide-resistant mutants should provide binding sites for the "capture" of vancomycin and teicoplanin molecules, which may be part of the mechanism of glycopeptide resistance in S. aureus.

Amino Acid Sequence↗

Electrochemical defect-mediated thin-film growth

An electrodeposition technique is described that produces atomically flat epitaxial metal overlayers of quality similar to that obtained by ultrahigh vacuum techniques at elevated temperature. In this approach, a metal of interest such as silver is co-deposited with a reversibly deposited mediator metal. The mediator is periodically deposited and stripped from the surface, and this serves to significantly increase the density of two-dimensional islands of silver atoms, promoting a layer-by-layer thin-film growth mode. In situ scanning tunneling microscopy was used to demonstrate the growth process for the heteroepitaxial system silver/gold (111) with either lead or copper as the mediator.

Journal Article↗

Inactivation of the methicillin resistance gene mecA in vancomycin-resistant Staphylococcus aureus.

Acquisition of high-level resistance to vancomycin in the laboratory mutant VM50 (vancomycin MIC increased from 1.5 to 100 microg/ml) was accompanied by the appearance of a heterogeneous phenotype and a virtual loss in methicillin resistance: in most cells of cultures of VM50 the methicillin MIC of the parental strain was reduced from 800 to 1.5 microg/ml with only a subpopulation (10(-5)) retaining methicillin resistance at near the parental level (MIC of 400 microg/ml). Interestingly, the vancomycin MIC of this subpopulation was less (25 microg/ml) than that of VM50 (100 microg/ml). A similar antagonism between methicillin and vancomycin resistance levels was observed upon introduction of an intact mecA into VM50 on a plasmid vector: methicillin resistance of the majority of cells increased from 1.5 to 100 microg/ml while the vancomycin MIC declined from 100 to 12/25 microg/ml. Membrane preparations from mutant VM50 showed no detectable penicillin-binding protein (PBP) 2A by the fluorographic assay. Sequencing of the mecA gene resident in mutant VM50 indicated the presence of a 19-bp duplication between nucleotide residues 280-298, leading to the generation of a stop codon TAA starting at nucleotide position 286.

Anti-Bacterial Agents↗

Gradual alterations in cell wall structure and metabolism in vancomycin-resistant mutants of Staphylococcus aureus.

In five vancomycin-resistant laboratory step mutants selected from the highly and homogeneously methicillin-resistant Staphylococcus aureus strain COL (MIC of methicillin, 800 microg/ml; MIC of vancomycin, 1.5 microg/ml), the gradually increasing levels of resistance to vancomycin were accompanied by parallel decreases in the levels of methicillin resistance and abnormalities in cell wall metabolism. The latter included a gradual reduction in the proportion of highly cross-linked muropeptide species in peptidoglycan, down-regulation of the production of penicillin-binding protein 2A (PBP2A) and PBP4, and hypersensitivity to beta-lactam antibiotics each with a relatively selective affinity for the various staphylococcal PBPs; the PBP2-specific inhibitor ceftizoxime was particularly effective.

Bacterial Proteins↗

Heterogeneously vancomycin-resistant Staphylococcus epidermidis strain causing recurrent peritonitis in a dialysis patient during vancomycin therapy.

Methicillin-resistant Staphylococcus epidermidis (MRSE) was recovered over a 2-month period from the dialysis fluid of a peritoneal dialysis (PD) patient who experienced recurrent episodes of peritonitis during therapeutic and prophylactic use of vancomycin. Characterization of five consecutive MRSE isolates by molecular and microbiological methods showed that they were representatives of a single strain, had reduced susceptibility to vancomycin, did not react with DNA probes specific for the enterococcal vanA or vanB gene, and showed characteristics reminiscent of the properties of a recently described vancomycin-resistant laboratory mutant of Staphylococcus aureus. Cultures of these MRSE isolates were heterogeneous: they contained-with a frequency of 10(-4) to 10(-5)-bacteria for which vancomycin MICs were high (25 to 50 microg/ml) which could easily be selected to "take over" the cultures by using vancomycin selection in the laboratory. In contrast, the five consecutive MRSE isolates recovered from the PD patient during virtually continuous vancomycin therapy showed no indication for a similar enrichment of more resistant subpopulations, suggesting the existence of an "occult" infection site in the patient (presumably at the catheter exit site) which was not accessible to the antibiotic.

Adult↗

Suppression of glycopeptide resistance in a highly teicoplanin-resistant mutant of Staphylococcus aureus by transposon inactivation of genes involved in cell wall synthesis.

The teicoplanin-resistant laboratory mutant TNM of Staphylococcus aureus strain COL (minimal inhibitory concentration for teicoplanin increased from 3 to 200 microg/ml) produced an abnormal peptidoglycan in which the proportion of cross-linked oligomeric muropeptides (pentameric and higher than pentameric species), representing approximately 60% of all muropeptide species in the parental strain, was reduced to approximately 17% in the mutant. In parallel, there was an increase in the representation of the monomeric muropeptides from 4% (in the parent) to 20% in the resistant strain. The mutant cell wall showed greatly increased porosity for the detergent extraction of cytoplasmic proteins, and this property was abolished in a Tn551 insertional derivative of TNM, which was selected for reduced (parental level) teicoplanin resistance. Transposon inactivation of the global regulatory genes Sigma-B and sar, and several genes involved in early steps of staphylococcal peptidoglycan synthesis, all caused extensive reduction of teicoplanin resistance in mutant TNM, in some cases to levels close to or below the MIC value of the parental strain.

Anti-Bacterial Agents↗

Decreased susceptibilities to teicoplanin and vancomycin among coagulase-negative methicillin-resistant clinical isolates of staphylococci.

Of 41 methicillin-resistant coagulase-negative staphylococcal clinical isolates collected during a 5-month period between late 1995 and early 1996, 28 showed tube dilution teicoplanin MICs of 4 to 8 microg/ml which increased to 16 to 32 microg/ml upon prolonged incubation. Cultures of such bacteria were heterogeneous; they contained subpopulations with frequencies of 10(-5) to 10(-4) that could grow on up to 50 microg of teicoplanin per ml. The same cultures were also heterogeneous with respect to susceptibility to vancomycin; while the MICs for the majority of cells were 2 to 4 microg/ml, subpopulations that could grow on 6 to 12 microg of vancomycin per ml were also present at frequencies of 10(-5) to 10(-7). Selective enrichment of such cultures for the resistant subpopulation occurred with relative ease under laboratory conditions. Heterogeneous phenotypes for teicoplanin (but not for vancomycin) susceptibility were also identified in several Staphylococcus epidermidis isolates collected during the preantibiotic era. The addition of half the MIC of teicoplanin inhibited autolysis and caused formation of cellular aggregates which disintegrated to individual bacteria in the stationary phase when the titer of teicoplanin in the medium fell to undetectable levels, indicating removal of the antibiotic from the culture medium by the bacteria.

Anti-Bacterial Agents↗

Suppression of beta-lactam antibiotic resistance in a methicillin-resistant Staphylococcus aureus through synergic action of early cell wall inhibitors and some other antibiotics.

We tested the effect of a number of mechanistically distinct antibacterial agents on the expression of methicillin resistance in a highly and homogeneously resistant strain of methicillin-resistant Staphylococcus aureus. The antibiotics, used at 0.25 x MIC, included inhibitors of early steps in peptidoglycan synthesis (fosfomycin, beta-chloro-D-alanine, D-cycloserine); bacitracin; teicoplanin and vancomycin; beta-lactam inhibitors chosen on the basis of their relatively selective affinities for penicillin-binding proteins 1, 2, 3 and 4 of S. aureus (imipenem, cefotaxime, cephradine and cefoxitin); compounds that inhibit various steps in protein synthesis (tetracycline, chloramphenicol, gentamicin, erythromycin and quinupristin/dalfopristin) and an inhibitor of DNA gyrase (temafloxacin). All inhibitors of early cell wall synthesis caused reduction of methicillin resistance and change from the homogeneous to the heterogeneous methicillin-resistant phenotype. Similar effects were obtained with only cephradine out of the four beta-lactams tested, and with erythromycin and quinupristin/dalfopristin as well. The other inhibitors of protein synthesis and DNA gyrase had no effect.

Anti-Bacterial Agents↗

Inhibition of cell wall turnover and autolysis by vancomycin in a highly vancomycin-resistant mutant of Staphylococcus aureus.

A highly vancomycin-resistant mutant (MIC = 100 microg/ml) of Staphylococcus aureus, mutant VM, which was isolated in the laboratory by a step-pressure procedure, continued to grow and synthesize peptidoglycan in the presence of vancomycin (50 microg/ml) in the medium, but the antibiotic completely inhibited cell wall turnover and autolysis, resulting in the accumulation of cell wall material at the cell surface and inhibition of daughter cell separation. Cultures of mutant VM removed vancomycin from the growth medium through binding the antibiotic to the cell walls, from which the antibiotic could be quantitatively recovered in biologically active form. Vancomycin blocked the in vitro hydrolysis of cell walls by autolytic enzyme extracts, lysostaphin and mutanolysin. Analysis of UDP-linked peptidoglycan precursors showed no evidence for the presence of D-lactate-terminating muropeptides. While there was no significant difference in the composition of muropeptide units of mutant and parental cell walls, the peptidoglycan of VM had a significantly lower degree of cross-linkage. These observations and the results of vancomycin-binding studies suggest alterations in the structural organization of the mutant cell walls such that access of the vancomycin molecules to the sites of wall biosynthesis is blocked.

Anti-Bacterial Agents↗

A highly vancomycin-resistant laboratory mutant of Staphylococcus aureus.

A resistant mutant with vancomycin MIC of 100 micrograms/ml was isolated relatively easily through step pressure in the laboratory from a Staphylococcus aureus strain with initial MIC of 1.5 micrograms/ml for the antibiotic. Upon addition of vancomycin (50 micrograms/ml) to the growth medium mass increase of the culture and peptidoglycan synthesis continued but cell division (daughter cell separation), cell wall turnover and autolysis were inhibited, resulting in the production of multicellular clumps of bacteria. Parallel with the increase of culture density, the concentration of vancomycin measured both by biological activity and by HPLC gradually declined in the culture medium. Cell division and wall turnover of the culture resumed with the production of cells of normal morphology at the time when the concentration of the drug in the medium decreased below 0.5-1.0 micrograms/ml. There was no detectable change in the antibiotic concentration in the culture medium during growth of a vancomycin-resistant (vanA-positive) strain of Enterococcus faecium and an intrinsically vancomycin-resistant strain of Leuconostoc. The vancomycin-resistant staphylococcal mutant gave no signal with the vanA or vanB DNA probes and contained no detectable D-lactate terminating cell wall precursors. The biochemical mechanism and clinical significance of such glycopeptide-resistant mutants remained to be established.

Anti-Bacterial Agents↗

Penicillin-resistant and multidrug-resistant Streptococcus pneumoniae in a pediatric hospital in Zagreb, Croatia.

Sixty-four penicillin-resistant Streptococcus pneumoniae isolates [benzylpenicillin minimal inhibitory concentrations (MICs) between 0.05 and 1.6 micrograms/ml] recovered at the Pediatric Hospital "Dr. Fran Mihaljevic" in Zagreb, Croatia between October 1990 and March 1993 were analyzed for serotype, antibiotic susceptibility patterns, and chromosomal relatedness using pulsed-field gel electrophoretic (PFGE) analysis of chromosomal DNA fragmented by digestion with the SmaI endonuclease. Hospital "Dr. Fran Mihaljevic" services the capital of Croatia and its vicinity. Most of the isolates were from nasopharyngeal carriage, but several isolates were from otitis media, sinusitis, and meningitis. Most isolates belonged to either serotype 23F (36/64) or 19F (12/64); the rest, including three 15C isolates, were in 11 additional distinct serotypes. The overwhelming majority (25/36) of the serotype 23F isolates had penicillin MIC values of 1-2 micrograms/ml and shared variants of a common PFGE pattern, closely related to the PFGE identified in multiresistant pneumococci of the same serotype with wide geographic spread to Spain, Portugal, France, and the United States. This group of bacteria was also resistant to tetracycline, chloramphenicol, and sulfamethoxazole/trimethoprim. In contrast to the relative genetic and phenotypic homogeneity of the more highly penicillin resistant isolates, pneumococci with penicillin MICs between 0.5 and 0.4 microgram/ml (29/64) were distributed in 13 different serotypes and as many as 20 distinct PFGE patterns.

Autolysis↗