Restoration of Susceptibility of Intracellular Methicillin-Resistant Staphylococcus aureus to β-Lactams: Comparison of Strains, Cells, and Antibiotics

ABSTRACT Staphylococcus aureus invades eukaryotic cells. When methicillin-resistant S. aureus (MRSA) ATCC 33591 is phagocytized by human THP-1 macrophages, complete restoration of susceptibility to cloxacillin and meropenem is shown and the strain becomes indistinguishable from MSSA ATCC 25923 due to the acid pH prevailing in phagolysosomes (S. Lemaire et al., Antimicrob. Agents Chemother. 51:1627-1632, 2007). We examined whether this observation can be extended to (i) strains of current clinical and epidemiological interest (three hospital-acquired MRSA [HA-MRSA] strains, two community-acquired MRSA [CA-MRSA] strains, two HA-MRSA strains with the vancomycin-intermediate phenotype, one HA-MRSA strain with the vancomycin-resistant phenotype, and one animal [porcine] MRSA strain), (ii) activated THP-1 cells and nonprofessional phagocytes (keratinocytes, Calu-3 bronchial epithelial cells), and (iii) other β-lactams (imipenem, oxacillin, cefuroxime, cefepime). All strains showed (i) a marked reduction in MICs in broth at pH 5.5 compared with the MIC at pH 7.4 and (ii) sigmoidal dose-response curves with cloxacillin (0.01× to 100× MIC, 24 h of incubation) after phagocytosis by THP-1 macrophages that were indistinguishable from each other and from the dose-response curve for methicillin-susceptible S. aureus (MSSA) ATCC 25923 (relative potency [50% effect], 6.09× MIC [95% confidence interval {CI}, 4.50 to 8.25]; relative efficacy [change in bacterial counts over the original inoculum for an infinitely large cloxacillin concentration, or maximal effect], −0.69 log CFU [95% CI, −0.79 to −0.58]). Similar dose-response curves for cloxacillin were also observed with MSSA ATCC 25923 and MRSA ATCC 33591 after phagocytosis by activated THP-1 macrophages, keratinocytes, and Calu-3 cells. By contrast, there was a lower level of restoration of susceptibility of MRSA ATCC 33591 to cefuroxime and cefepime after phagocytosis by THP-1 macrophages, even when the data were normalized for differences in MICs. We conclude that the restoration of MRSA susceptibility to β-lactams after phagocytosis is independent of the strain and the types of cells but varies between β-lactams.

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A reference model for the combination of an arbitrary number of drugs: A generalization of the Bliss independence model

10.1101/630616 ◽

2019 ◽

Author(s):

Wim De Mulder ◽

Martin Kuiper ◽

Åsmund Flobak

Keyword(s):

Dose Response ◽

Reference Model ◽

Maximal Effect ◽

Statistical Independence ◽

Minimal Effect ◽

Response Curves ◽

Independence Model ◽

Basic Reference ◽

Dose Response Curves ◽

Bliss Independence

AbstractIt is commonplace to determine the effectiveness of the combination of drugs by comparing the observed effects to a reference model that describes the combined effect under the assumption that the drugs do not interact. Depending on what is to be understood by non-interacting behavior, several reference models have been developed in the literature. One of them is the celebrated Bliss independence model, which assimilates non-interaction with statistical independence. Intuitively, this requires the dose-response curves to have zero as minimal effect and one as maximal effect, a restriction that was indeed adopted by Bliss. However, we show how non-interaction can be interpreted in terms of statistical independence, while nevertheless allowing arbitrary values for the minimal and the maximal effect. Furthermore, our reference model allows the maximal effects of the dose-response curves to be different. In a first step, we construct a basic reference model for the case of two drugs and where the maximal effects of the two individual dose-response curves are assumed to be equal. By relying on the notion of non-interaction in terms of statistical independence, and by introducing two consistency principles, we show how a unique reference model can be derived. In a second step, a more general reference model, allowing the maximal effects to be different while still restricting to two drugs, is then easily constructed from the basic reference model. Finally, an induction step is applied to generalize the reference model to the case of an arbitrary number of drugs, allowing each dose-response curve to have a possibly different maximal effect. Although the minimal effect of the dose-response curves are restricted to be equal, which we show to be a necessary consequence of consistency rules, its value is arbitrary.Author summaryThe Bliss independence model is a very popular reference model for drug combinations, meaning that it predicts the combined effect of doses of given drugs under the assumption of non-interaction between these drugs. However, because Bliss described non-interaction as statistical independence, he thought that he had to assume that the minimal effect of all dose-response curves are zero, while the maximal effect of all dose-response curves are one. While it is acceptable that all dose-response curves have minimal effect zero, because this amounts to having a common reference state (i.e. the response when no drug at all is given), it is a severe restriction to force all dose-response curves to have maximal effect one. On the other hand, the Bliss independence model has the advantage that it relies on sound statistical theory, and the assimilation of non-interaction with statistical independence is rather intuitive. We have extended the Bliss independence model to allow the involved dose-response curves to have different maximal effects. This has been done in a rigorous way, where the statistical underlying theory that was used by Bliss remains essentially intact.

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Establishing a dose–response relationship for haloperidol decanoate

Psychiatric Bulletin ◽

10.1192/pb.29.3.104 ◽

2005 ◽

Vol 29 (3) ◽

pp. 104-107 ◽

Cited By ~ 9

Author(s):

David Taylor

Keyword(s):

Dose Response ◽

Dose Range ◽

Maximal Effect ◽

Inclusion Criteria ◽

Response Curves ◽

Haloperidol Decanoate ◽

Limited Effect ◽

Effective Doses ◽

Dose Response Curves ◽

Relapse Rates

Aims and MethodThe aim of this literature analysis was to establish the range of doses of haloperidol decanoate effective in preventing relapse in schizophrenia. Studies reporting relapse rates in patients treated for longer than 6 months were included. Relapse rate was then plotted against dose or log dose to allow drawing of dose–response curves.ResultsFifteen publications reporting 13 individual studies were identified. of these, 6 studies met inclusion criteria and were analysed. Dose–response curves indicated limited effect at 25 mg/4 weeks but near maximal effect at doses of 50 mg/4 weeks. There was no clear evidence that increasing the dose above 100 mg/4 weeks provided additional benefit in preventing relapse.Clinical ImplicationsThe recommended dose range for haloperidol decanoate (50–300 mg/ 4 weeks) does not reflect the findings of this study. Optimally effective doses appear to be around 50–100 mg/4 weeks. The use of doses above 100 mg/4 weeks is difficult to support given data available.

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The muscarinic receptor of chick embryo cells: correlation between ligand binding and calcium mobilization.

The Journal of Cell Biology ◽

10.1083/jcb.100.4.1073 ◽

1985 ◽

Vol 100 (4) ◽

pp. 1073-1081 ◽

Cited By ~ 41

Author(s):

G Oettling ◽

H Schmidt ◽

U Drews

Keyword(s):

Binding Site ◽

Dose Response ◽

Model Comparison ◽

Partial Agonist ◽

Calcium Mobilization ◽

Maximal Effect ◽

Binding Studies ◽

Response Curves ◽

Dose Response Curves ◽

Inhibition Constants

In this report we characterize muscarinic cholinergic receptor on embryonic cells. We established dose-response curves by fluorometric measurement of Ca2+ mobilization in cell suspensions of whole chick embryos stage 23/24. Ca2+ mobilization was quantitated by standardization of chlorotetracycline (CTC) fluorescence changes after stimulation with muscarinic agonists. We determined ED50 values for the agonists acetylcholine and carbachol as 3.4 X 10(-6) and 2.7 X 10(-5) M, respectively. Pilocarpine and oxotremorine were found to act as reversible competitive antagonists with inhibition constants (Kl) of 5.0 X 10(-6) and 1.4 X 10(-6) M, respectively. Bethanechol, which induced only 23% of the maximal effect obtained by acetylcholine, was a partial agonist with an ED50 of 4.8 X 10(-4) M. Its antagonistic component is expressed by an inhibition constant of 1.9 X 10(-4) M. In parallel, binding studies were performed in a competition assay with [3H]-quinuclidinylbenzilate. For the agonists acetylcholine and carbachol, binding parameters were best fitted by a "two binding-sites model." Comparison with dose-response curves indicated that Ca2+ mobilization was triggered via the high-affinity binding site. The inhibition constants of antagonists derived from the shift of dose-response curves corresponded to the fitted KD values of the binding studies when a "one binding-site model" was applied. Combination of dose-response and binding data showed close proportionality between receptor occupancy and calcium mobilization. No spare receptors were present.

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Steady-state enzyme kinetics with high-affinity substrates or inhibitors. A statistical treatment of dose–response curves

Biochemical Journal ◽

10.1042/bj1350101 ◽

1973 ◽

Vol 135 (1) ◽

pp. 101-107 ◽

Cited By ~ 52

Author(s):

Peter J. F. Henderson

Keyword(s):

Steady State ◽

Dose Response ◽

Statistical Treatment ◽

Enzyme Concentration ◽

Bacterial Respiration ◽

Maximal Effect ◽

Response Curves ◽

Kinetic Measurements ◽

Lending Library ◽

Dose Response Curves

A statistical treatment of steady-state enzyme kinetic measurements is described that allows for depletion of free substrate or free inhibitor concentrations owing to significant binding to the enzyme. Vmax., Km or Ki, enzyme concentration, the concentration of substrate or inhibitor required for a half-maximal effect and standard errors of these parameters can be calculated from dose–response measurements; the concentration of each component of the system may be estimated also. The statistically best values of the parameters are used to convert dose–response curves into convenient linear forms. The method is applied to dose–response measurements of hydroxyquinoline N-oxide inhibition of bacterial respiration and aminopterin inhibition of dihydrofolate reductase. Two FORTRAN programs for this method have been deposited as Supplementary Publication no. SUP 50019 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1973) 131, 5.

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Severe uremia depresses the ability of perifused rat pituitary cells to secrete growth hormone in response to growth hormone releasing hormone.

Journal of the American Society of Nephrology ◽

10.1681/asn.v85742 ◽

1997 ◽

Vol 8 (5) ◽

pp. 742-748

Author(s):

J Rodríguez ◽

F Santos ◽

M J García de Boto ◽

E García ◽

V Martínez ◽

...

Keyword(s):

Growth Hormone ◽

Dose Response ◽

Secretory Response ◽

Pituitary Cells ◽

Maximal Effect ◽

Response Curves ◽

Secretory Rate ◽

Releasing Hormone ◽

Gh Secretion ◽

Dose Response Curves

To examine whether growth hormone (GH) secretion is impaired by chronic renal failure (CRF) and to gain some insight into the influence of uremia itself and associated malnutrition, the GH secretory response of dispersed anterior pituitary cells perifused with GH-releasing hormone (GHRH) was investigated in 5/6 nephrectomized (UREM, N = 15) and three groups (N = 15 each) of normal renal function, sham-operated rats under three different nutritional conditions: fed "ad libitum" (SAL), pair-fed with a diet similar to the UREM group (SPF), and pair-fed with a diet similar to the UREM group in terms of protein ingestion but calorically supplemented up to intake of SAL group (SPF+). Ten days after nephrectomy, UREM rats had severe CRF, as shown by much higher (P < 0.0001) serum urea nitrogen concentrations (X +/- mean +/- SE) than sham groups (59 +/- 6 versus 8 +/- 0, 9 +/- 0, and 5 +/- 0 mmol/L, respectively), and they were growth retarded, as shown by lower gains (P < 0.0001) in weight (13.5 +/- 2.5 versus 62 +/- 2.1, 20.5 +/- 1.9, and 50.4 +/- 1.0 g) and length (2.9 +/- 0.2 versus 5.8 +/- 0.1, 3.6 +/- 0.1, and 5.6 +/- 0.1 cm). Perifusion studies showed similar basal GH secretory rate (ng/min/10(7) cells) in the four groups. A fixed sequence of progressively increasing GHRH doses resulted in a lower overall mean GH secretion in UREM rats (15.8 +/- 1.6 ng/min/10(7) cells), as compared with SAL (50.8 +/- 9.0 ng/min/10(7) cells, P < 0.01), SPF (33.0 +/- 3.3 ng/min/10(7) cells, P < 0.05), and SPF+ (49.4 +/- 5.1 ng/min/10(7) cells, P < 0.01) groups. Analysis of dose-response curves showed that the maximal secretory response was produced by the same concentration of GHRH (10 nM) in the four groups and was lower (P < 0.01) in UREM than SAL and SPF+ rats (34.9 +/- 5.0 versus 115.7 +/- 28.4 and 98.9 +/- 9.8 ng/min/10(7) cells). The concentration of GHRH that caused the half of maximal effect was identical, close to 1 nM, in the four groups of animals. This study provides direct evidence that the ability of pituitary cells to secrete GH in response to GHRH is depressed in severe CRF. The lower secretory capacity of pituitary gland is only partly dependent on caloric malnutrition associated with CRF. Data of dose-response curves suggest that decreased GH secretion may be related to a lesser number of pituitary receptors for GHRH.

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Canine gastrocnemius disuse atrophy: resistance to paralysis by dimethyl tubocurarine

Journal of Applied Physiology ◽

10.1152/jappl.1984.57.5.1502 ◽

1984 ◽

Vol 57 (5) ◽

pp. 1502-1506 ◽

Cited By ~ 13

Author(s):

G. A. Gronert ◽

R. S. Matteo ◽

S. Perkins

Keyword(s):

Dose Response ◽

Plasma Concentrations ◽

Blood Levels ◽

Maximal Effect ◽

Disuse Atrophy ◽

Response Curves ◽

Dose Response Curves ◽

Gastrocnemius Muscles ◽

The Hill

Ten dogs developed unilateral gastrocnemius disuse atrophy after unilateral hindlimb immobilization in a cast for 25 days. Dose-response curves to dimethyl tubocurarine (MTC) were determined during anesthesia with pentobarbital sodium-N2O. Bolus and continuous infusion increments of MTC every 30 min provided steady-state blood levels at each stage of paralysis. Both gastrocnemius tendons were sectioned and attached to transducers. Both sciatic nerves were stimulated every 30 min: 2 Hz for 2 s, a 15-s pause, 50 Hz for 2 s. Dose-response curves, computer calculated by nonlinear regression using a sigmoid maximal effect model of the Hill equation, were parallel for the data relating blocking of tetanus to dose of MTC. The 50% paralyzing dose (tetanus) for control vs. casted gastrocnemius muscle was 64 vs. 813 mg/kg; corresponding plasma concentrations were 0.12 vs. 2.0 micrograms/ml. Thus in vivo simultaneous tension measurements of both gastrocnemius muscles, one casted and one uncasted, demonstrated resistance to paralysis by MTC in muscle with disuse atrophy.

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Dose-response Curves in the Fibrin Plate Assay Fibrinolytic Activity of Proteases

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647705 ◽

1974 ◽

Vol 32 (02/03) ◽

pp. 356-365 ◽

Cited By ~ 12

Author(s):

F Haverkate ◽

D. W Traas

Keyword(s):

Dose Response ◽

Fibrinolytic Activity ◽

Enzyme Concentration ◽

Response Curves ◽

Agar Gel ◽

Porcine Tissue ◽

Plate Assay ◽

Fibrin Plate ◽

Different Types ◽

Dose Response Curves

SummaryIn the fibrin plate assay different types of relationships between the dose of applied proteolytic enzyme and the response have been previously reported. This study was undertaken to determine whether a generally valid relationship might exist.Trypsin, chymotrypsin, papain, the plasminogen activator urokinase and all of the microbial proteases investigated, including brinase gave a linear relationship between the logarithm of the enzyme concentration and the diameter of the circular lysed zone. A similar linearity of dose-response curves has frequently been found by investigators who used enzyme plate assays with substrates different from fibrin incorporated in an agar gel. Consequently, it seems that this linearity of dose-response curves is generally valid for the fibrin plate assay as well as for other enzyme plate bioassays.Both human plasmin and porcine tissue activator of plasminogen showed deviations from linearity of semi-logarithmic dose-response curves in the fibrin plate assay.

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BIOASSAY OF THYROID HORMONE USING HYPOTHYROID TADPOLES

Acta Endocrinologica ◽

10.1530/acta.0.0410143 ◽

1962 ◽

Vol 41 (1) ◽

pp. 143-153 ◽

Cited By ~ 2

Author(s):

U. Henriques

Keyword(s):

Thyroid Hormone ◽

Dose Response ◽

Developmental Rate ◽

Response Curves ◽

Sodium Salts ◽

Dose Response Curves

ABSTRACT A bioassay of thyroid hormone has been developed using Xenopus larvae made hypothyroid by the administration of thiourea. Only tadpoles of uniform developmental rate were used. Thiourea was given just before the metamorphotic climax in concentrations that produced neoteni in an early metamorphotic stage. During maintained thiourea neotoni, 1-thyroxine and 1-triiodothyronine were added as sodium salts to the water for three days and at the end of one week the stage of metamorphosis produced was determined. In this way identical dose-response curves were obtained for the two compounds. No qualitative differences between their effects were noted except that triiodothyronine seemed more toxic than thyroxine in equivalent doses. Triiodothyronine was found to be 7–12 times as active as thyroxine.

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