Oscillating tolerance in synchronized cultures of Staphylococcus aureus

Abstract

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...

Ampicillin killing curve patterns of Haemophilus influenzae type b isolates by agar dilution plate count method

“…Although the slow killing and lysis of some mutant bacterial strains, such as the Streptococcus pneumoniae mutant described by Tomasz and colleagues (23,25), have been linked to the production of autolysin inhibitors resembling lipoteichoic acids, it has not been established that similar mechanisms may be responsible for the spectrum of strain-dependent differences now established for other bacteria. A consistent observation since the early studies on penicillin action has been that the response for a given in vitro system is directly related to the rapidity of cell growth, and evidence has been presented that individual cell responses may differ greatly depending upon the stage of the cell cycle when exposed to an agent (3,12,22). On this basis, strain-dependent differences in responses might be expected if similarly prepared exponentially growing inoculum preparations contained different genetically determined proportions of responsive cells, either in various stages of their metabolic cycles or having minor differences in growth rates that are expressed as significant differences in killing dynamics.…”

Onset of penicillin-induced bacteriolysis in staphylococci is cell cycle dependent

“…Only a few methods for synchronization of staphylococci have been reported (4,5,17,20). This may be due to (i) the small sizes of staphylococci, which make it difficult to select newborn cells by density gradient centrifugation, (ii) the growth behavior of staphylococci (even in liquid cultures a considerable part of the population is present as doublets [22]), and (iii) the successive division planes being arranged perpendicular to each other (1), so that membrane elution, as described for Escherichia coli (18), is not considered a practical approach for synchronization of staphylococci.…”

Comparative kill and growth rates determined with cefdinir and cefaclor and with Streptococcus pneumoniae and beta-lactamase-producing Haemophilus influenzae

“…Since growth curves are often species and strain dependent and it is difficult to consistently determine the mid-logarithmic phase of growth unless growth curves are continuously recorded, we attempted to minimize these experimental variables by comparing kill rates with growth rates simultaneously. Moreover, several investigators have pointed out the danger of using only one 24-h pattern to assess bactericidal action and also the relative inaccuracy of using an arbitrary breakpoint (such as 0.1% survival) or artificial indices (such as the MBC or the MBC/MIC ratio) to assess bactericidal effect (4,6,19). The conditions used to assess the tolerance of clinical isolates have varied considerably, and some investigators have been able to demonstrate the phenomenon only with stationary-phase cultures (10).…”

“…There is considerable controversy about the frequency of antibiotic tolerance (17) in different species of bacteria. Technical factors, such as antibiotic carryover, adherence to test tube walls, medium composition, and pH, etc., are known to influence bactericidal activities (3,4,6,7,10,11,18,19). The organisms being tested should be in the logarithmic phase of growth, since most antibiotics act more rapidly on growing bacteria.…”