Influence of technical factor variations during inoculum preparation on the agar dilution plate-count method for quantitation of Staphylococcus aureus oxacillin persisters

Abstract

The possible introduction of error into Staphylococcus aureus oxacillin intrinsic persister measurements by the agar dilution plate-count method resulting from technical factor variations associated with inoculum preparation was investigated. pH stability and exponential growth were shown to be present at the time of inoculum standardization. The use of glass or plastic tubes and the contamination of tube wails during inoculum broth culture produced no differences in test results. For S. aureus and oxaciRlin, the agar dilution plate-count method is a new and reliable approach to the quantitative study of bacterial inhibition and killing.We recently described the agar dilution plate-count (ADPC) procedure for the quantitative measurement of bacterial inhibition and killing (14). For Staphylococcus aureus and oxacillin, we found the so-called tolerance phenomenon to be artifactual and to result from the detection of high-percentage persisters. For a given test system, isolate, and time of antimicrobial action, total persister percentage appears to represent the contribution of both intrinsic and adaptive persisters. Intrinsic persister percentage appears to be strain dependent, whereas adaptive persister percentage is highly influenced by technical factor variations, the latter probably accounting for the spurious results and inaccuracy associated with broth dilution plate-count methods. Factors known to promote cell dormancy (stationary-phase inoculum, lowering of pH, drying of bacteria), phenotypic changes (cell clumping, slime or capsule production), and physical separation of bacteria from the antimicrobial agent (adherence of bacteria to container walls above the antimicrobial broth) are probably the most important inducers of adaptive persisters, and for broth dilution plate-count methods they are probably operative during the entire broth dilution phase. In the ADPC method, except for the brief inoculum preparation step, bacteria are immobilized in a single-phase agar dilution system, a situation designed to prevent the development of adaptive persisters. This study examined the possibility that technical factor variations potentially inherent in the ADPC inoculation step promote the development of adaptive persisters, thereby introducing errors into intrinsic persister measurements.S. aureus isolates were retrieved from -70°C stock storage by two 24-h passages on sheep blood agar in air at 35°C. The isolates, described in a previous study (14), had the following characteristics: R7, MIC = 0.5 ,ug/ml, 24-h persister percentage at 8 x the MIC = 0.5%; R4, MIC = 0.5 ,ug/ml, 24-h persister percentage at 8 x the MIC = 0.08%; and SPRMC (St. Paul-Ramsey Medical Center) C13, MIC = 0.5 ,ug/ml, 24-h persister percentage at 8 x the MIC = 1.2%). Material from three to five colonies on the day 2 retrieval plate was used to prepare common broth inocula as described below. * Corresponding author.The potential decrease in antibiotic effect resulting from induction of cell dormancy by the possible lowering of pH (2, 4, 10, ...

Determination of Fungicidal Activities against Yeasts and Molds: Lessons Learned from Bactericidal Testing and the Need for Standardization

“…The determination of survivors by plating an aliquot from broth-containing tubes or wells onto an agar surface may be confounded by carryover of the antimicrobial agent, resulting in continued inhibition of growth on the agar plate and an overestimation of the killing effect (53,58,61,79,88). This is more of a problem at higher concentrations of antimicrobial agent (Ͼ16ϫ MIC) and with larger transfer volumes.…”

“…These organisms may be detected as survivors during either MBC or time-kill studies. Unlike persisters, these surviving colonies demonstrate an increase in resistance, relative to the parent strain, when retested (8,24,53,88). Both selection of a preexisting resistant subpopulation and induction of a latent resistance mechanism may result in the demonstration of phenotypic resistance.…”

“…(i) Persisters. The bactericidal activity of certain antimicrobial agents, notably ␤-lactams, is directly related to the rate of bacterial growth (10,26,30,49,53,58,61,79,81,88). The lower the rate of growth, the slower the bactericidal effect.…”

“…In an in vitro test, some of the bacterial cells may be dormant or replicating slowly and thus are not killed by the otherwise bactericidal agent. These "persisters" usually constitute Ͻ0.1% of the final inoculum but will be apparent as growth on agar medium during the course of time-kill or MBC testing (8,26,49,53,79,88). Notably, when retested these persisters appear just as susceptible as the parent strain and do not constitute a resistant subpopulation (8).…”

“…Selection of a minority subpopulation of bacteria that are capable of expressing resistance to the test agent may occur during the actual performance of an in vitro susceptibility test (8,24,53,88). These organisms may be detected as survivors during either MBC or time-kill studies.…”