Significance of Methicillin Tolerance in Experimental Staphylococcal Endocarditis

A proportion of clinical isolates of Staphylococcus aureus exhibit resistance to bactericidal activity of certain antibiotics, despite normal susceptibility to inhibition. This phenomenon is termed "tolerance." The methodology used to determine tolerance varies greatly. To clarify the relationship between laboratory methodology and tolerance, we determined the minimal bactericidal concentrations and minimal inhibitory concentrations for 20 clinical isolates by two methods. Inocula were prepared by either 3-h growth of the organism in MuellerHinton broth or overnight (22 to 24 h) (4,9,10,13,17) have shown that the MBC, and therefore the MBC/MIC ratio, can vary for the same strains of S. aureus, depending on growth media, growth phase of the inoculum, inoculum size, and other factors.There is growing evidence that the bactericidal effects of cell wall-active antibiotic on bacteria, including S. aureus, can be suppressed in a pH-dependent manner (5,7,11,20,22,23

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confidence: 99%

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confidence: 99%

pH-dependent oxacillin tolerance of Staphylococcus aureus

Venglarcik¹,

Blair²,

Dunkle³

1983

“…The P value was 0.01 in four of the six cases and <0.05 in the other two. DISCUSSION In recent years, the relevance of tolerance of S. aureiis strains for the therapeutic response was investigated in various studies (5,6,8,9). Sabath et al (9) Guze et al (6) studied the effect of methicillin upon similar strains in a pyelonephritis model in rats.…”

Section: Methodsmentioning

confidence: 99%

“…The results reported by various authors are somewhat contradictory (2,(5)(6)(7)(8)(9). The question whether infections with tolerant strains are more difficult to treat then infections with susceptible strains can be investigated by means of an experimental infection model in animals.…”

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confidence: 99%

Responses of tolerant and nontolerant Staphylococcus aureus strains to methicillin treatment in an experimental infection in mice

Goessens¹,

Fontijne²,

Michel³

1984

Staphylococcus aureus strains can be divided into tolerant and nontolerant strains on the basis of their survival in vitro in the presence of high concentrations of methicillin (264 ,ug/ml). A strain is defined as tolerant if more than 2% of the inoculum survives under these conditions. The response of five susceptible and five tolerant S. aureus strains to treatment with methicillin was studied in an experimental thigh infection in mice. Animals were treated with one and two injections of methicillin (2.5 mg per mouse). At the end of treatment, the number of CFUs in the thigh muscles infected with the susceptible strains was found to be significantly lower than that in the thigh muscles infected with the tolerant strains.The clinical relevance of the phenomenon of tolerance has been studied repeatedly. The results reported by various authors are somewhat contradictory (2,(5)(6)(7)(8)(9) ca. 107 CFU in the left thigh muscle as stated above. Of these 18 mice, 6 were treated subcutaneously in the abdominal region with one dose of methicillin which was given 1 h after the inoculation of the bacteria. Another group of six mice received two doses of methicillin. Doses were administered at 1 and 3 h after inoculation. The remaining six mice did not receive antibiotic treatment. They were sacrificed 1 h after bacterial inoculation and served as the control group for the determination of inoculum size at the time the antibiotic treatment started. Unless stated otherwise, the doses administered were 0.15, 0.3, 0.6, 1.25, and 5 mg per mouse. Methicillin concentrations in serum. Blood specimens were obtained by orbital puncture of mice under light ether anesthesia at various intervals after the administration of methicillin. Methicillin was assayed by the aid of the agar well diffusion method, with DST agar (Oxoid Ltd., London, England) and Bacillus subtilis as an indicator organism (1).Therapeutic results. Two hours after the first and second methicillin doses (3 and 5 h after inoculation, respectively), groups of six mice (unless otherwise stated) were sacrificed by cervical dislocation. The infected thigh muscle was removed and immediately homogenized in 20 ml of ice-cold physiological saline, to which 100 RI of ,B-lactamase (15.2 U of 1-lactamase I and 2.3 U of 1-lactamase II) (Whatman Biochemicals, Ltd.) was added. The tissues were homogenized in a VirTis homogenizer for 1 min at 7,000 rpm. The homogenates were then diluted 10-fold serially in sterile saline. Each dilution (0.2 ml) was spread on nutrient agar plates, to which 0.15 U of ,B-lactamase I and 0.015 U of 1-lactamase II per ml of agar were added. After 48 h of incubation at 37°C, CFU counts were made and converted to the total number of CFUs per thigh muscle. These numbers were expressed as percentages of the mean value of the number of CFUs cultivated from muscles of untreated control mice which were sacrificed 1 h after bacterial inocu-

“…The technical difficulties associated with detecting tolerance are identical to those encountered in MBC determinations. So, tolerance is influenced by the medium (7, 12), incubation time (7,8), growth phase (6,(8)(9)(10), pH value (15), antibiotic carry-over (6,8), as well as by other technical factors (1,8,14). Another characteristic of tolerance is the heterogeneous type of resistance to the lethal effect of the antibiotic.…”

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confidence: 99%

Oscillating tolerance in synchronized cultures of Staphylococcus aureus

Holzhoffer¹,

Süßmuth²,

Haag³

1985

Cells of synchronized cultures of Staphylococcus aureus showed an oscillating MBC/MIC ratio when tested with penicillin G. Although the MICs did not differ significantly throughout the cell cycle, the MBC was at its maximum when actively dividing cells were inoculated.Antimicrobial tolerance is characterized by the fact that a susceptible strain, in terms of MIC, is killed to a 99.9% level only at concentrations equal or above 32 times the MIC (13). The technical difficulties associated with detecting tolerance are identical to those encountered in MBC determinations. So, tolerance is influenced by the medium (7, 12), incubation time (7,8), growth phase (6,(8)(9)(10), pH value (15), antibiotic carry-over (6,8), as well as by other technical factors (1,8,14). Another characteristic of tolerance is the heterogeneous type of resistance to the lethal effect of the antibiotic. Thus, population analyses of penicillin-tolerant Staphylococcus aureus showed that only a proportion of up to 7% of the initial inoculum in fact is tolerant (13). To investigate whether this tolerance percentage can be explained by a corresponding small proportion of the organisms being in a phase of the cell cycle in which they could escape killing at the time of contact with penicillin, we studied the MICs and MBCs continuously by using synchronized cultures.For synchronization of cell growth, we varied the method of Dwek et al. (2). A strain of S. aureus isolated from a patient with acute prostatitis was grown to about 108 CFU/ml in Mueller-Hinton broth and concentrated by centrifugation. A 1-ml amount of the concentrate containing 4 x 109 CFU was placed on top of a preformed Percoll gradient (Pharmacia Fine Chemicals) in Mueller-Hinton broth and centrifuged at 21,000 x g for 20 min at 37°C. The gradient was fractionated in 0.5-ml portions, the most dense of which was used to inoculate 15 ml of prewarmed Mueller-Hinton broth. The culture was continuously shaken at 37°C and one 0.2-ml sample was taken every 2 min for determination of MICs and MBCs and the CFU.MICs were determined immediately after sampling by adding 0.01 ml of the culture to each of a series of tubes containing 1 ml of twofold dilutions of penicillin G (Hoechst AG) ranging from 2 to 0.03 mg/liter in nonsupplemented Mueller-Hinton broth. After being mixed, the tubes were incubated in a shaking water bath (50 rpm) for 24 h at 37°C. The MICs were determined by visual examination. After being mixed, 0.1 ml from the clear vials were spread on Mueller-Hinton agar plates and incubated for an additional 24 h at 37°C to determine the number of surviving bacteria (antibiotic carry-over had no influence, since the CFU on Mueller-Hinton plates were comparable to those found on plates containing penicillinase). The originally measured inoculum. Determinations of CFU were performed by plating 0.05 ml of 10-fold dilutions of the sample and counting colonies after incubation for 24 h at 37°C. The experiment presented is one of five repetitions which showed only minor differences with respe...