Search PubMed⌕ Search

Biomedical subjects

H F Chambers

Publications and source records attributed to H F Chambers.

At least 37 records · Page 2Linked to original sources
PubMed1996

Gyrase mutations in laboratory-selected, fluoroquinolone-resistant mutants of Mycobacterium tuberculosis H37Ra.

To characterize mechanisms of resistance to fluoroquinolones by Mycobacterium tuberculosis, mutants of strain H37Ra were selected in vitro with ofloxacin. Their quinolone resistance-determining regions for gyrA and gyrB were amplified and sequenced to identify mutations in gyrase A or B. Three types of mutants were obtained: (i) one mutant (TKp1) had no mutations in gyrA or gyrB; (ii) mutants that had single missense mutations in gyrA, and (iii) mutants that had two missense mutations resulting in either two altered gyrase A residues or an altered residue in both gyrases A and B. The TKp1 mutant had slightly reduced levels of uptake of [14C]norfloxacin, which was associated with two- to fourfold increases in the MICs of ofloxacin, ciprofloxacin, and sparfloxacin. Gyrase mutations caused a much greater increase in the MICs of fluoroquinolones. For mutants with single gyrA mutations, the increases in the MICs were 4- to 16-fold, and for mutants with double gyrase mutations, the MICs were increased 32-fold or more compared with those for the parent. A gyrA mutation in TKp1 secondary mutants was associated with 32- to 128-fold increases in the MICs of ofloxacin and ciprofloxacin compared with the MICs for H37Ra and an eight-fold increase in the MIC of sparfloxacin. Sparfloxacin was the most active fluoroquinolone tested. No sparfloxacin-resistant single-step mutants were selected at concentrations of > 2.5 micrograms/ml, and high-level resistance (i.e., MIC, > and = 5 micrograms/ml) was associated with two gyrase mutations. Mutations in gyrB and possibly altered levels of intracellular accumulation of drug are two additional mechanisms that may be used by M. tuberculosis in the development of fluoroquinolone resistance. Because sparfloxacin is more active in vitro and selection of resistance appears to be less likely to occur, it may have important advantage over ofloxacin or ciprofloxacin for the treatment of tuberculosis.

Anti-Infective Agents↗
PubMed1996

Evaluation of Vitek GPS-SA card for testing of oxacillin against borderline-susceptible staphylococci that lack mec.

Fifty-one Staphylococcus aureus strains lacking mec for which oxacillin MICs were 1 to 8 micrograms/ml were tested against oxacillin and the combination of oxacillin and clavulanic acid with the Vitek GPS-SA card, the reference broth microdilution method, and the oxacillin agar screen plate. Of the 51 strains, 44 (86%) did not grow on the oxacillin agar screen plate, broth microdilution MICs were 1 to 2 micrograms/ml, and GPS-SA card MICs were < or = 2 micrograms/ml, with the exception of 3 strains that failed to grow in the card on repeated attempts. Another seven strains did grow on the oxacillin agar screen plate. For four of the latter group of strains, oxacillin broth microdilution MICs were > 4 micrograms/ml and GPS-SA card MICs were > or = 4 micrograms/ml; for the other 3 strains, corresponding MICs were 4 and < or = 2 micrograms/ml, respectively. The GPS-SA card classified 86% of strains as oxacillin susceptible.

Bacterial Proteins↗
PubMed1995

Ampicillin, sulbactam, and rifampin combination treatment of experimental methicillin-resistant Staphylococcus aureus endocarditis in rabbits.

Ampicillin or amoxicillin at 625-800 mg/kg/day, in combination with a beta-lactamase inhibitor, each is as effective as vancomycin in animal models of methicillin-resistant Staphylococcus aureus endocarditis. Studies were done to determine whether the addition of rifampin would permit lowering the dose of ampicillin into the range recommended for use in humans without loss of efficacy. The efficacy of ampicillin/sulbactam (300/150 or 150/75 mg/kg/day intramuscularly, in three divided doses) in combination with rifampin (5 mg/kg intramuscularly, three times daily) was compared with that of vancomycin (25 mg/kg intravenously, twice daily, or 30 mg/kg intramuscularly, three times daily) in the rabbit model of methicillin-resistant S. aureus aortic valve endocarditis. Neither ampicillin/sulbactam nor rifampin alone was effective. The ampicillin/sulbactam/rifampin regimen was as effective as vancomycin. This regimen may be an alternative to vancomycin in treatment of methicillin-resistant S. aureus infections.

Ampicillin↗
PubMed1995

Point mutations in Staphylococcus aureus PBP 2 gene affect penicillin-binding kinetics and are associated with resistance.

In Staphylococcus aureus, penicillin-binding protein 2 (PBP 2) has been implicated in non-PBP 2a-mediated methicillin resistance. The PBP 2 gene (pbpB) was cloned from an expression library of a methicillin-susceptible strain of S. aureus (209P), and its entire sequence was compared with that of the pbpB gene from strains BB255, BB255R, and CDC6. Point mutations that resulted in amino acid substitutions near the conserved penicillin-binding motifs were detected in BB255R and CDC6, two low-level methicillin-resistant strains. Penicillin binding to PBP 2 in both BB255R and CDC6 is altered, and kinetic analysis indicated that altered binding of PBP 2 by penicillin was due to both lower binding affinity and more rapid release of bound drug. These structural and biochemical changes may contribute to the strains' resistance to beta-lactam antibiotics.

Amino Acid Sequence↗
PubMed1995

Can penicillins and other beta-lactam antibiotics be used to treat tuberculosis?

An increase in the number of tuberculosis cases caused by multiple-drug-resistant strains of Mycobacterium tuberculosis has stimulated search for new antituberculous agents. Beta-lactam antibiotics, traditionally regarded as ineffective against tuberculosis, merit consideration. Four major penicillin-binding proteins (PBPs) with approximate molecular sizes of 94, 82, 52, and 37 kDa were detected by fluorography of [3H]penicillin-radiolabeled membrane proteins prepared from M. tuberculosis H37Ra. The presence of membrane-associated beta-lactamase precluded the use of membranes for assaying the binding affinities of beta-lactam antibiotics. Therefore, ampicillin affinity chromatography was used to purify these four PBPs from crude membranes in order to assay the binding affinities of beta-lactam antibiotics. Ampicillin, amoxicillin, and imipenem, beta-lactam antibiotics previously reported to be active in vitro against M. tuberculosis, bound to M. tuberculosis PBPs at therapeutically achievable concentrations. Binding of the 94-, 82-, and 52-kDa PBPs, but not the 37-kDa PBP, was associated with antibacterial activity, suggesting that these PBPs are the critical targets. Studies of mycobacterial cell wall permeability, which was assayed with a panel of reference cephalosporins and penicillins with different charge positivities, indicated that the rate of penetration of beta-lactam antibiotics to the target PBPs could not account for resistance. Resistance could be reversed with the beta-lactamase inhibitors clavulanate or sulbactam or could be circumvented by the use of a beta-lactamase-stable drug, imipenem, indicating that mycobacterial beta-lactamase, probably in conjunction with slow penetration, is a major determinant of M. tuberculosis resistance to beta-lactam antibiotics. These findings confirm in vitro data that M. tuberculosis is susceptible to some beta-lactam antibiotics. Further evaluation of these drugs for the treatment of tuberculosis in animal models and in clinical trials is warranted.

Animals↗
PubMed1995

In vitro and in vivo antistaphylococcal activities of L-695,256, a carbapenem with high affinity for the penicillin-binding protein PBP 2a.

L-695,256 is a synthetic carbapenem beta-lactam antibiotic that binds with a high degree of affinity to penicillin-binding protein (PBP) PBP 2a, the protein that mediates staphylococcal resistance to methicillin. The concentration of L-695,256 that inhibited binding of radiolabeled [3H]penicillin to PBP 2a by 50% was 1.2 micrograms/ml, whereas they were 14 and 68 micrograms/ml for penicillin and imipenem, respectively. Cell wall synthesis, determined by incorporation of [14C]N-acetylglucosamine into whole cells, was inhibited by 50% at concentrations of 1.3, 26, and 132 micrograms/ml for L-695,256, penicillin, and imipenem, respectively, for the methicillin-resistant strain COL. Growth of cells of each of two homogeneously resistant strains, COL and 76, was completely inhibited by 4 micrograms of L-695,256 per ml, whereas growth was inhibited by 100 micrograms or more of penicillin or imipenem per ml. The efficacies of L-695,256 (10 mg/kg given three times daily [t.i.d.]), imipenem (37.5 mg/kg t.i.d.), penicillin (300,000 units/kg t.i.d.), and vancomycin (25 mg/kg given twice daily) were compared in the rabbit model of aortic valve endocarditis established with these homogeneous strains. After 4 days of treatment, mean bacterial densities in aortic valve vegetations were reduced by 4.0 to 5.8 log10 CFU/g for L-695,256, 1.0 to 1.8 log10 CFU/g for imipenem, -1.1 to 3.9 log10 CFU/g for penicillin, and 1.1 to 3.0 log10 CFU/g for vancomycin in comparison to the densities of controls. Compounds such as L-695,256 that are bound by PBP 2a with a high degree of affinity are likely to be extremely effective in the treatment of infections caused by methicillin-resistant staphylococci.

Animals↗
PubMed1995

Quantitative culture of Mycobacterium tuberculosis from clinical sputum specimens and dilution endpoint of its detection by the Amplicor PCR assay.

The minimum number of Mycobacterium tuberculosis CFU detectable in clinical sputum specimens by the Amplicor PCR test was estimated by performing the test on duplicate samples of quantitatively cultured serial dilutions of sputum. Positive PCR test results were obtained for all samples that contained 42 CFU of M. tuberculosis. The detection limits of the PCR assay for decontaminated (N-acetyl-L-cysteine [NALC]-NaOH) and nondecontaminated (NALC only) specimens were equivalent, even though the number of CFU cultured from decontaminated samples was only 11 to 20% of the number cultured from nondecontaminated samples. Thus, the 42 CFU that could be detected in nondecontaminated specimens by the Amplicor PCR test correspond to the approximately 8 CFU (0.20 x 42) that could be recovered in culture after decontamination with NALC-NaOH.

Bacteriological Techniques↗
PubMed1994

Kinetics of penicillin binding to penicillin-binding proteins of Staphylococcus aureus.

Reduced affinity of penicillin-binding proteins (PBPs) for binding penicillin has been proposed as a mechanism of beta-lactam antibiotic resistance in staphylococci. Penicillin binding by PBPs of three penicillin-susceptible and two penicillin-resistant strains of Staphylococcus aureus was studied in kinetic assays to determine rate constants, drug concentrations at which PBPs were bound and the relationship between concentrations that bound PBPs and concentrations that inhibited bacterial growth. PBPs 1 and 2 of the resistant strains exhibited slower acylation and more rapid deacylation than susceptible strains. In contrast PBP 4, a naturally low-affinity PBP, was modified such that it exhibited a lower rate of deacylation. The concentrations of penicillin at which modified PBPs were bound correlated with concentrations that inhibited growth of the resistant strains. Acquisition of penicillin resistance in these strains of S. aureus results, at least in part, from structural modifications affecting binding of multiple PBPs and appears to include recruitment of a non-essential PBP, PBP 4.

Bacterial Proteins↗
PubMed1994

Altered production of penicillin-binding protein 2a can affect phenotypic expression of methicillin resistance in Staphylococcus aureus.

Altered production of penicillin-binding protein 2a (PBP 2a) may affect the phenotypic expression of resistance in methicillin-resistant Staphylococcus aureus (MRSA). COL, an MRSA strain that constitutively produces PBP 2a, was transformed with a recombinant plasmid containing the two beta-lactamase regulatory genes, blaI and blaR1, with either the beta-lactamase gene, blaZ, or a truncated blaZ. Both of the transformed MRSA strains now produced an inducible PBP 2a, and the MICs of nafcillin, methicillin, and imipenem for these strains were similar to those for the parental strain. A mutation in blaR1 that resulted in the complete repression of PBP 2a production altered the phenotypic expression of methicillin resistance in that strain, as evidenced by efficiency-of-plating experiments. Rather than being homogeneously resistant like COL, the blaR1 mutant strain now appeared to have a small resistant subpopulation. Gene products that regulate PBP 2a production may contribute to the organism's expression of methicillin resistance, but additional chromosomally located factors are required.

Carrier Proteins↗
PubMed1993

Effect of thrombocytopenia on the early course of streptococcal endocarditis.

Although platelets are a major factor in the pathogenesis of endocarditis, it is unclear if these cells promote or limit disease progression. To address this issue, the effects of thrombocytopenia on the early course of endovascular infection were examined. Aortic valve endocarditis was produced in rabbits by using Streptococcus sanguis M99. Thrombocytopenia was induced by intravenous administration of antiplatelet serum. Compared with controls (infected rabbits given nonimmune serum), thrombocytopenic rabbits had higher densities of streptococci within vegetations (mean log10 cfu/g, 9.78 vs. 8.11, P < .002) and a higher total number of bacteria per valve (mean log10 total cfu/valve, 8.96 vs. 7.43, P < .004). When tested for its interactions with platelets in vitro, strain M99 bound, activated, and aggregated rabbit platelets extensively and was rapidly killed by platelet microbicidal protein. These results indicate that platelets can limit disease progression in endocarditis. The host defense properties of platelets may in part be mediated by platelet microbicidal protein.

Animals↗
PubMed1993

Efficacy of cefoperazone in combination with sulbactam in experimental Staphylococcus aureus endocarditis in rabbits.

The activity of cefoperazone with and without sulbactam was studied in vitro and in vivo against strains of methicillin-resistant and methicillin-susceptible Staphylococcus aureus. Cefoperazone with or without sulbactam was inactive in vitro against the methicillin-resistant strain and was bound by penicillin-binding protein 2a with an IC50 of 190 mg/L (the concentration that reduced radio-labelling with 3H-penicillin by 50%). Cefoperazone was hydrolysed by beta-lactamase in vitro but sulbactam improved cefoperazone activity in a rabbit model of aortic valve endocarditis caused by a beta-lactamase producing methicillin-susceptible strain.

Animals↗
PubMed1993

blaI and blaR1 regulate beta-lactamase and PBP 2a production in methicillin-resistant Staphylococcus aureus.

For Staphylococcus aureus, it is hypothesized that two genes located upstream of the beta-lactamase gene, blaZ, are required for the inducible expression of beta-lactamase. blaR1 is predicted to encode a signal-transducing membrane protein, and blaI is predicted to encode a repressor protein. These same two genes may also regulate the production of penicillin-binding protein 2a (PBP 2a), a protein essential for expression of methicillin resistance. To confirm that these two genes encode products that can control both beta-lactamase and PBP 2a production, blaI, blaR1, and blaZ with a 150-nucleotide deletion at the 3' end were subcloned from a 30-kb staphylococcal beta-lactamase plasmid and three beta-lactamase-negative strains of methicillin-resistant S. aureus were transformed with the recombinant plasmid containing that insert. The production of PBP 2a and a nonfunctional beta-lactamase was detected by fluorography and by immunoblots with polyclonal antisera directed against each of the proteins. Whereas the parent strains did not produce beta-lactamase and constitutively produced PBP 2a, PBP 2a and a truncated beta-lactamase were now inducible in the transformants. Therefore, two plasmid-derived genes regulate the production of both PBP 2a and beta-lactamase.

Bacterial Proteins↗
PubMed1993

Detection of Mycobacterium tuberculosis in sputum samples by polymerase chain reaction using a simplified procedure.

A repetitive sequence of Mycobacterium tuberculosis DNA was amplified by polymerase chain reaction (PCR), from sputum samples, for the diagnosis of pulmonary tuberculosis. The method of heating the sample in a boiling water bath to break down the bacterial cell wall and to release the DNA was compared with that of enzymatic lysis of bacteria and then phenol-chloroform extraction of DNA. Heating the sample was the better method with a sensitivity of approximately 10 microorganisms. A total of 78 sputum specimens prepared by heating were examined by PCR, and the results were compared with the results of acid-fast stained smears, cultures, and clinical data. M. tuberculosis was detected by PCR in all smear- and culture-positive and smear-negative, culture-positive cases. Additionally, PCR was capable of detecting four of nine cases which were smear and culture negative but clinically suspected of tuberculosis. DNA amplification by PCR is a sensitive and specific method for the diagnosis of tuberculosis, and with this simplified DNA isolation procedure it can be used in routine clinical practice.

Bacteriological Techniques↗
PubMed1993

Detection of methicillin-resistant staphylococci.

Susceptibility tests commonly used in the clinical laboratory for detection of methicillin-resistant strains of staphylococci are discussed. The biochemical and genetic basis of methicillin resistance, including heterogeneous expression of resistance, is summarized. The conditions that can affect results of disk diffusion, broth dilution, and agar screen tests and sensitivity and specificity of these tests are reviewed. DNA detection methodologies, which, with their greater accuracy, may replace susceptibility testing, also are discussed.

Methicillin Resistance↗
PubMed1993

Short-course combination and oral therapies of Staphylococcus aureus endocarditis.

The experimental basis for use of 2-week semisynthetic penicillin plus aminoglycoside combination therapy for Staphylococcus aureus endocarditis is reviewed. The use of oral therapy is examined. Short-course combination of oral regimens can be effective and may be suitable for selected patients with S. aureus endocarditis.

Administration, Oral↗
PubMed1992

Studies of RP 59500 in vitro and in a rabbit model of aortic valve endocarditis caused by methicillin-resistant Staphylococcus aureus.

The activity of RP 59500 against methicillin-resistant Staphylococcus aureus was studied in vitro and in a rabbit model of aortic valve endocarditis. Three strains, 67-O, 529, and Du, ranging from relatively resistant to susceptible to RP 59500 in vitro (mean agar dilution MICs of 0.65, 0.21, and 0.12 mg/L respectively) were used to establish endocarditis, which was treated either with RP 59500, 20 mg/kg im four times a day for four days or with vancomycin, 25 mg/kg iv twice a day for four days. RP 59500 was ineffective for the most resistant strain, 67-O. RP 59500 was effective for the intermediately susceptible strain 529, but vancomycin was more effective. RP 59500 was slightly more effective than vancomycin against the most susceptible strain, Du, but the difference was not statistically significant. These results suggest that strains inhibited by RP 59500 at a concentration of greater than or equal to 0.5 mg/L should probably be considered resistant in this model of infection. RP 59500 was effective in vivo against the two susceptible strains, but overall, vancomycin was the more active drug.

Animals↗
PubMed1992

Characterization of penicillin-binding protein 2 of Staphylococcus aureus: deacylation reaction and identification of two penicillin-binding peptides.

Penicillin-binding protein (PBP) 2 is the major PBP of five that have been identified in susceptible strains of Staphylococcus aureus. Beta-lactam antibiotic binding to PBP 2 is important for the antibacterial effect. Antibiotic binding to PBP 2 in strain 209P was examined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis in competition assays using [3H]penicillin as the radiolabel. Clavulanic acid, which is specifically bound by PBP 2, and cefaclor, which is specific for PBP 3, were studied. Cefaclor, which alone appeared not to bind PBP 2, in combination inhibited PBP 2 binding of clavulanic acid. By varying the temperature during radiolabeling with [3H]penicillin in cefaclor competition assays and in direct radiolabeling assays with [3H]cefaclor, it was shown that cefaclor was bound by PBP 2 with high affinity (50% inhibitory concentration, less than or equal to 0.1 microgram/ml) and that the apparent low-affinity binding (50% inhibitory concentration, greater than 10 micrograms/ml) in competition assays performed at 37 degrees C was due to rapid deacylation. Two penicillin-binding peptides of PBP 2 also were identified in fluorographs of PBPs separated by nonequilibrium pH gradient gel and two-dimensional electrophoresis. Rapid deacylation for some antibiotics and the presence of two penicillin-binding peptides are two properties of PBP 2 that should be considered when correlating results of binding assays with effects of beta-lactam antibiotics on S. aureus.

Anti-Bacterial Agents↗