Tolerance percentage as a criterion for the detection of tolerant Staphylococcus aureus strains

Goessens, W. H. F.; Fontijne, P.; Raffe, M. Van; Michel, Marie-Claude

doi:10.1128/aac.25.5.575

Cited by 31 publications

(22 citation statements)

References 13 publications

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“…The differences in bacterial survival of tolerant and nontolerant strains in the thighs of mice treated with methicillin support the view that, in some cases, the results of treatment of staphylococcal infections depend not only on the MIC value of the causative agent but also on the killing rate (4).…”

Section: Methodssupporting

confidence: 55%

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

Goessens¹,

Fontijne²,

Michel³

1984

Antimicrob Agents Chemother

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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-

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Section: Methodssupporting

confidence: 55%

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

Goessens¹,

Fontijne²,

Michel³

1984

Antimicrob Agents Chemother

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“…Bradley et al (5) found that 16 of 30 strains were tolerant to oxacillin and cephalothin with 0.2 to 43% of the initial log 5-cfu/ml stationary-phase inoculum surviving after 24 h of incubation in Mueller-Hinton broth. Goessens et al (13) obtained similar results for 64 isolates, with 12.5% of the isolates showing more than 2% survivors after 24 h of incubation for a variety of antimicrobial concentrations, and termed this the tolerance percentage. Several retrospective clinical studies have suggested that the clinical response to beta-lactam therapy of antimicrobial agent-tolerant infections might be impaired (8,17,30), but tolerance did not affect the response to treatment in the rabbit model of staphylococcal endocarditis (14) or the rat model of staphylococcal pyelonephritis (16).…”

supporting

confidence: 60%

“…Clear tubes were sampled after 24 h of incubation at 35°C with a calibrated 0.01-ml loop and streaked on quadrants of a sheep blood agar plate. CFUs were counted after 18 to 24 h of incubation at 35°C, and the MBC was read as the lowest antibiotic (13,25,33,37), with cross tolerance for methicillin, oxacillin, cephalothin, penicillin G, and vancomycin but not gentamicin (25). Killing was increased by 48 h of incubation as compared with 24 h of incubation for methicillin, oxacillin, penicillin G, cephalothin, vancomycin, and gentamicin.…”

mentioning

confidence: 99%

Lack of reproducibility of macrodilution MBCs for Staphylococcus aureus

Pelletier¹

1984

Antimicrob Agents Chemother

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MBCs of methicillin, oxacillin, penicillin G, cephalothin, vancomycin, and gentamicin were determined by a standard broth macrodilution technique for 101 clinical isolates of methicillin-susceptible Staphylococcus aureus. Increased killing (more than 99.9%) was observed after 48 versus 24 h of incubation for many strains, and cross tolerance to antimicrobial bactericidal activity (less than 99.9% killing) was frequently observed among antimicrobial agents. However, these in vitro measurements of bactericidal activity against S. aureus were not reproducible.Best and colleagues (4) described a strain of Staphylococcus aureus susceptible to oxacillin by MIC criteria but resistant to the bactericidal effect of oxacillin in killing-curve experiments. Strains tolerant to the bactericidal and lytic effects of oxacillin demonstrated oxacillin-induced inhibition of staphylococcal autolytic enzymes on the surface of the organism (3). From these studies, Best et al. concluded that the usual bactericidal effect of oxacillin was due to the combined effect of antimicrobial interference with cell wall synthesis and destruction of cell wall by endogenous autolytic enzymes. Mayhall et al. (25) found oxacillin tolerance in 33 of 60 clinical S. aureus isolates by MBC testing and log 5-CFU/ml killing-curve experiments with stationary-growthphase inocula. Sabath et al. (33) defined "antimicrobial tolerance" as an MBC/MIC ratio of .32, after using standard techniques with log-growth-phase organisms to show cross tolerance among 28 of 63 blood culture isolates for the penicillins, cephalosporins, and vancomycin. Sabath and colleagues also confirmed impaired lysis and autolysin production in tolerant strains exposed to antimicrobial agents. Bradley et al. (5) found that 16 of 30 strains were tolerant to oxacillin and cephalothin with 0.2 to 43% of the initial log 5-cfu/ml stationary-phase inoculum surviving after 24 h of incubation in Mueller-Hinton broth. Goessens et al. (13) obtained similar results for 64 isolates, with 12

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“…ADPC MIC plate counts fell dramatically at the MIC and were essentially absent on plates with concentrations above the MIC as long as oxacillin action was permitted to continue. ADPC MBC plate-count patterns showed significantly less variation and inoculum phase (5,18,20,21,25,29), incubation time (1,19,22,26,34,36), and carry-over of antimicrobial agents from MIC tubes to MBC plates (12,19,31,33) have been shown to influence the measured incidence of tolerance and related phenomena for a variety of bacteria and antimicrobial agents. Less intuitively obvious has been the finding that bacteria subjected to broth dilution procedures may be inadvertently protected from the action of antimicrobial agents in broth cultures, thus causing tolerance interpretations to be spuriously high and often irregular in manifestation and reproducibility (15,26,39).…”

Section: Methodsmentioning

confidence: 99%

“…Goessens and associates (10,11,12), using a carefully controlled BDPC method, recently introduced tolerance percentage to indicate the percentage of CFU surviving antimicrobial action. They found tolerance percentage to be strain dependent for S. aureus versus oxacillin and arbitrarily defined tolerance as a tolerance percentage of 2% or greater.…”

Section: Methodsmentioning

confidence: 99%