In vitro studies of pharmacodynamic properties of vancomycin against Staphylococcus aureus and Staphylococcus epidermidis

The bactericidal activities of vancomycin against two reference strains and two clinical isolates of Staphylococcus aureus and Staphylococcus epidermidis were studied with five different concentrations ranging from 2x to 64x the MIC. The decrease in the numbers of CFU at 24 h was at least 3 log10 CFU/ml for all strains. No concentration-dependent killing was observed. The postantibiotic effect (PAE) was determined by obtaining viable counts for two of the reference strains, and the viable counts varied markedly: 1.2 h for S. aureus and 6.0 h for S. epidermidis. The determinations of the PAE, the postantibiotic sub-MIC effect (PA SME), and the sub-MIC effect (SME) for all strains were done with BioScreen C, a computerized incubator for bacteria. The PA SMEs were longer than the SMEs for all strains tested. A newly developed in vitro kinetic model was used to expose the bacteria to continuously decreasing concentrations of vancomycin. A filter prevented the loss of bacteria during the experiments. One reference strain each of S. aureus and S. epidermidis and two clinical isolates of S. aureus were exposed to an initial concentration of 10x the MIC of vancomycin with two different half-lives (t1/2s): 1 or 5 h. The post-MIC effect (PME) was calculated as the difference in time for the bacteria to grow 1 log10 CFU/ml from the numbers of CFU obtained at the time when the MIC was reached and the corresponding time for an unexposed control culture. The difference in PME between the strains was not as pronounced as that for the PAE. Furthermore, the PME was shorter when a t1/2 of 5 h (approximate terminal t1/2 in humans) was used. The PMEs at t1/2s of 1 and 5 h were 6.5 and 3.6 h, respectively, for S. aureus. The corresponding figures for S. epidermidis were 10.3 and less than 6 h. The shorter PMEs achieved with a t1/2 of 5 h and the lack of concentration-dependent killing indicate that the time above the MIC is the parameter most important for the efficacy of vancomycin.     

Structural and dynamical characterization of a biologically active unfolded fibronectin-binding protein from Staphylococcus aureus

A 130-residue fragment (D1-D4) taken from a fibronectin-binding protein of Staphylococcus aureus, which contains four fibronectin-binding repeats and is unfolded but biologically active at neutral pH, has been studied extensively by NMR spectroscopy. Using heteronuclear multidimensional techniques, the conformational properties of D1-D4 have been defined at both a global and a local level. Diffusion studies give an average effective radius of 26.2 +/- 0.1 A, approximately 75% larger than that expected for a globular protein of this size. Analysis of chemical shift, 3JHNalpha coupling constant, and NOE data show that the experimental parameters agree well overall with values measured in short model peptides and with predictions from a statistical model for a random coil. Sequences where specific features give deviations from these predictions for a random coil have however been identified. These arise from clustering of hydrophobic side chains and electrostatic interactions between charged groups. 15N relaxation studies demonstrate that local fluctuations of the chain are the dominant motional process that gives rise to relaxation of the 15N nuclei, with a persistence length of approximately 7-10 residues for the segmental motion. The consequences of the structural and dynamical properties of this unfolded protein for its biological role of binding to fibronectin have been considered. It is found that the regions of the sequence involved in binding have a high propensity for populating extended conformations, a feature that would allow a number of both charged and hydrophobic groups to be presented to fibronectin for highly specific binding.     

Increased overall antibiotic susceptibility in Staphylococcus aureus femAB null mutants

The staphylococcal pentaglycine side chain of the peptidoglycan is reduced to one glycine in femAB null mutants. This is associated with increased susceptibility to methicillin and to a whole range of unrelated antibiotics as well. Genetic evidence suggests that femAB null mutants are only viable because of a compensatory mutation in an unlinked site.     

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.     

Examination of methicillin-resistant and methicillin-susceptible Staphylococcus aureus mutants with low-level fluoroquinolone resistance

For Staphylococcus aureus, stepwise mutations result in high-level quinolone resistance. Methicillin-resistant and -susceptible quinolone-resistant, first-step mutants generated in vitro were obtained and found to be no different than those recovered from murine abscesses. Approximately 10% of all first-step mutants were resistant to ethidium bromide, and selected strains had mutations that mapped to flqB. NorA-mediated resistance among first-step mutants may be more prevalent than previously reported.     

Some biochemical properties of Acholeplasma laidlawii mutants defective in glucose carrier

Some membrane properties of glucose transport system of Acholeplasma laidlawii mutants uncapable of uptaking glucose as well as those of its non-metabolizing analogue, 3-0-methylglucose are studied. In contrast to the wild strain the mutant PTL-S cells fail to uptake carbohydrates with the saturation. There is no competition between carbohydrates during transport in this mutant no sugar exit is observed, and the sensitivity of transport to thiol reagents and pronase B is considerably affected. All other investigated membrane functions were intact. The data given indicate the presence of a specific defect in glucose transport system.     

Attenuated mutants of Staphylococcus aureus with two temperature-sensitive lesions: isolation and characterization

The feasibility of constructing attenuated mutants of Staphylococcus aureus with two temperature-sensitive (ts) lesions for ultimate development of a live-attenuated strain was investigated. Temperature-sensitive S. aureus strain G/1/2, which grows well at 31 degrees C but does not replicate at 37 degrees C, was subjected to chemical mutagenesis. After two enrichment cycles, fifteen mutants able to grow at 25 degrees C but unable to grow at 31 degrees C, were identified. Growth curves with temperature shifts from 25 to 31 degrees C, and from 31 to 37 degrees C confirmed that these were mutants with two lesions (dts), each with a different cut-off temperature. The reversion frequency of mutant G/1/2 at 37 degrees C was 2 x 10(-6) whereas those of several dts mutants were much lower (dts7: 7 x 10(-9) and dts12: 1 x 10(-9)). There was no increase in ts mutation reversion rate in response to prolonged incubation at 37 degrees C. The data support the further development of these mutants for use as a stable attenuated vaccine.     

Cytokine expression in a rat model of Staphylococcus aureus endophthalmitis

PURPOSE: To examine the ability of viable Staphylococcus aureus to induce the production of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, cytokine-induced neutrophil chemoattractant (CINC), and interferon (IFN)-gamma after intravitreal injection. METHODS: Experimental rat eyes were injected with a 25-microl volume of approximately 80 colony-forming units of viable S. aureus; control eyes received sterile saline. Eyes were graded daily for signs of clinical inflammation and were removed 6, 24, 48, and 72 hours after injection. One group was prepared for histologic analysis, and vitreous was removed from the other group for cytokine analysis, using standard enzyme-linked immunosorbent assay procedures. RESULTS: TNF-alpha, IL-1beta, CINC, and IFN-gamma were detected in experimental vitreous samples at increased levels that peaked at 24 hours. TNF-alpha, IL-1beta, and CINC declined at 48 hours, but IFN-gamma remained elevated. At 72 hours, levels returned to baseline. Statistically significant elevations of TNF-alpha, IL-1beta, and CINC were detected in experimental samples at 24, but not at 6 and 48 hours compared with levels in saline control samples (P < 0.03). A statistically significant increase in IFN-gamma was detected at 24 and 48 hours compared with control levels (P < 0.03). In experimental animals, clinical inflammation and inflammatory cells peaked at 24 hours, persisted at 48 hours, and began to decline thereafter. Neutrophils were the predominant inflammatory cell detected at 24 (72.3% of cells) and 48 (60.1%) hours. By 72 hours, the total number of inflammatory cells had decreased by 75.0%, and the cellular infiltrate had changed so that neutrophils equaled monocytes-macrophages. CONCLUSIONS: S. aureus induced the expression of TNF-alpha, IL-1beta, CINC, and IFN-gamma. The time course of these cytokine levels could account for the clinical inflammatory responses and the entry and decline of vitreous cells in this model of bacterial endophthalmitis.     

Multiple classes of yeast mutants are defective in vacuole partitioning yet target vacuole proteins correctly

In Saccharomyces cerevisiae the vacuoles are partitioned from mother cells to daughter cells in a cell-cycle-coordinated process. The molecular basis of this event remains obscure. To date, few yeast mutants had been identified that are defective in vacuole partitioning (vac), and most such mutants are also defective in vacuole protein sorting (vps) from the Golgi to the vacuole. Both the vps mutants and previously identified non-vps vac mutants display an altered vacuolar morphology. Here, we report a new method to monitor vacuole inheritance and the isolation of six new non-vps vac mutants. They define five complementation groups (VAC8-VAC12). Unlike mutants identified previously, three of the complementation groups exhibit normal vacuolar morphology. Zygote studies revealed that these vac mutants are also defective in intervacuole communication. Although at least four pathways of protein delivery to the vacuole are known, only the Vps pathway seems to significantly overlap with vacuole partitioning. Mutants defective in both vacuole partitioning and endocytosis or vacuole partitioning and autophagy were not observed. However, one of the new vac mutants was additionally defective in direct protein transport from the cytoplasm to the vacuole.     

Some biochemical properties of the components of Staphylococcus aureus binding to human platelets

The purpose of this study was to ascertain whether pulmonary function in children who were lifetime residents of the highly polluted district of Teplice in northern Bohemia was lower than that for children who were lifetime residents of the cleaner district of Prachatice in southern Bohemia. Forced expiratory spirometry was measured twice (February/March and April) in approximately 235 eighth-grade students in each district. On both testing occasions, height-adjusted forced expiratory volume in 1 s and forced expiratory flow between 25% and 75% forced vital capacity were significantly lower (p < .001) in children from Teplice than in those from Prachatice. These differences were not associated with parental smoking habits, presence of pets, heating/cooking fuels, private home/apartment residency, or rural/urban residency. In Teplice, no differences were observed between lung functions measured at the end of the high pollution season (February/March) and those measured after the children breathed much cleaner air for a 4-wk period (April). This result was suggestive of a condition of chronically depressed lung function. No differences across times were observed in Prachatice, indicating that our measurements were reliable.     

Genetic analysis of type 5 capsular polysaccharide expression by Staphylococcus aureus

Capsules are produced by over 90% of Staphylococcus aureus strains, and approximately 25% of clinical isolates express type 5 capsular polysaccharide (CP5). We mutagenized the type 5 strain Reynolds with Tn918 to target genes involved in CP5 expression. From a capsule-deficient mutant, we cloned into a cosmid vector an approximately 26-kb EcoRI fragment containing the transposon insertion. In the absence of tetracycline selection, Tn918 was spontaneously excised, thereby resulting in a plasmid containing 9.4 kb of S. aureus DNA flanking the Tn918 insertion site. The 9.4-kb DNA fragment was used to screen a cosmid library prepared from the wild-type strain. Positive colonies were identified by colony hybridization, and a restriction map of one clone (pJCL19 with an approximately 34-kb insert) carrying the putative capsule gene region was constructed. Fragments of pJCL19 were used to probe genomic DNA digests from S. aureus strains of different capsular serotypes. Fragments on the ends of the cloned DNA hybridized to fragments of similar sizes in most of the strains examined. Blots hybridized to two fragments flanking the central region of the cloned DNA showed restriction fragment length polymorphism. A centrally located DNA fragment hybridized only to DNA from capsular types 2, 4, and 5. DNA from pJCL19 was subcloned to a shuttle vector for complementation studies. A 6.2-kb EcoRI-ClaI fragment complemented CP5 expression in a capsule-negative mutant derived by mutagenesis with ethyl methanesulfonate. These experiments provide the necessary groundwork for identifying genes involved in CP5 expression by S. aureus.     

Structural characterization of an abnormally cross-linked muropeptide dimer that is accumulated in the peptidoglycan of methicillin- and cefotaxime-resistant mutants of Staphylococcus aureus

Laboratory mutants of Staphylococcus aureus strain ATCC 8325 (27S) selected for increased minimal inhibitory concentration (MIC) values to methicillin and cefotaxime showed increased rates of cell wall turnover and detergent-induced autolysis in virtual parallel with the increasing MIC for the antibiotic. Also in parallel with the increasing MICs for the particular antibiotic used in the selection was the gradual accumulation of an unusual muropeptide in the peptidoglycan of the mutants, muropeptide 12, which is a minor component of the cell wall of the parental strain. Analysis of muropeptide 12, its peptide derivative, and its lysostaphin degradation products by high pressure liquid chromatography, Edman degradation, and mass spectrometry suggests that muropeptide 12 is a dimer in which the two monomeric components are interlinked by two pentaglycyl cross-bridges, thus generating a 14-member macrocyclic ring structure. This unusual cross-linked structure may be the product of the abnormal activity of penicillin-binding protein 2 which has grossly reduced antibiotic binding capacity in the mutant staphylococci.     

Molecular characterization of the gene operon of heat shock proteins HSP60 and HSP10 in methicillin-resistant Staphylococcus aureus

Methicillin-resistance of S.aureus (MRSA) was diminished or depressed at 44 degrees C. In order to investigate whether bacterial heat shock response is correlated with methicillin resistance, we examined the inducibility of the heat shock proteins (HSPs) in MRSA, and cloned and sequenced of their genes. A temperature shift from 37 degrees C to 46 degrees C enhanced the production of at least 8 kinds of cytoplasmic proteins as measured with two-dimensional PAGE. The induced protein profile was almost the same as methicillin sensitive S.aureus, and stress conditions due to ethanol, cadmium or low pH. Two of these proteins were HSP60 and HSP10. Their N-terminal amino acid sequences were 79%, and 83%, homologous with thermobacterium PS3, respectively. A positively hybridized 4.2 kbp DNA fragment encoding both proteins was isolated from the chromosomal DNA of MRSA. The resulting sequence revealed two reading frames and showed high homology to those of hsp60 (groEL) and hsp10 (groES) of bacteria (E.coli) and several other species. The genes of HSP60 and HSP10 in S.aureus comprised an operon as in E.coli. The relationship between those HSPs and PBP2' is currently under investigation.     

Generation of conditional mutants in higher eukaryotes by switching between the expression of two genes

A regulatory system for the in-depth study of gene functions in higher eukaryotic cells has been developed. It is based on the tetracycline-controlled transactivators and reverse tTA, which were remodeled to discriminate efficiently between two different promoters. The system permits one to control reversibly the activity of two genes, or two alleles of a gene, in a mutually exclusive way, and also allows one to abrogate the activities of both. This dual regulatory circuit, which can be operated by a single effector substance such as doxycycline, overcomes limitations of conventional genetic approaches. The conditional mutants that can now be generated will be useful for the study of gene function in vitro and in vivo. In addition, the system may be of value for a variety of practical applications, including gene therapy.     

Thermodynamic studies on the equilibrium properties of a series of recombinant betaW37 hemoglobin mutants

In human hemoglobin (Hb) the beta37 tryptophan residue (betaW37), located at the hinge region of the alpha1beta2 interface, forms many contacts with alpha subunit residues of the opposite dimer, in both the T and R quaternary structures. We have carried out equilibrium O2 binding studies on a series of recombinant Hbs that have mutations at this residue site: betaW37Y, betaW37A, betaW37G, and betaW37E. Binding isotherms measured at high concentrations of these mutants were found to be shifted toward increased affinity and decreased cooperativity from that of the normal HbA0 tetramer. Analysis of these binding isotherms indicated that amino acid substitutions at the beta37 position could both destabilize the tetrameric form of the mutants relative to their constituent dimers and also alter cooperativity of the intact tetrameric species. These alterations from wild-type function are dependent on the particular side chain substituted, with the magnitude of change increasing as Trp is substituted by Tyr, Ala, Gly, and Glu. The dimer to tetramer assembly free energy of deoxy-betaW37E, the most perturbed mutant in the series, was measured using analytical gel chromatography to be 9 kcal/tetramer less favorable than that of deoxy HbA0. Stabilizing the betaW37E tetramer by addition of IHP, or by cross-linking at the alphaK99 positions, does not restore normal O2 binding behavior. Thermodynamic parameters of all the mutants were found to correlate with their CO binding rates and with their high-resolution X-ray crystal structures (see accompanying papers: Kwiatkowski et al. (1998) Biochemistry 37, 4325-4335; Peterson & Friedman (1998) Biochemistry 37, 4346-4357; Kavanaugh et al. (1998) Biochemistry 37, 4358-4373].