No abstract

We evaluated the use of mannitol salt agar with oxacillin for use as a primary screening medium for the simultaneous detection and identification of methicillin-resistant Staphylococcus aureus in clinical surveillance specimens. Oxacillin agar dilution susceptibility tests with mannitol salt agar and Mueller-Hinton agar were performed in parallel with disk-agar diffusion testing on 95 oxacillin-susceptible and 105 oxacillin-resistant S. aureus stock isolates. MICs were found to be comparable, showing distinct separation of susceptible and resistant isolates into two groups with MICs of-2 and .32 ,ig/ml, respectively. In accord with these findings, 4 ,ug of oxacillin per ml was selected for use in the screening medium. For performance evaluation, mannitol salt agar with 4 ,ig of oxacillin per ml was compared with mannitol salt agar without oxacillin by performing parallel screening tests on 153 clinical surveillance specimens. For detection of methicillin-resistant S. aureus, mannitol salt agar with 4 ,ug of oxacillin per ml was as sensitive as mannitol salt agar without oxacillin and required significantly fewer confirmatory tests. For primary identification of methicillin-resistant S. aureus, mannitol salt agar with 4 ,ug of oxacillin per ml was 6.4% false-positive and 1.1% false-negative, with a 93.6% positive predictive value. These findings indicate that mannitol salt agar with 4 ,ug of oxacillin per ml can be used as a reliable and cost-effective screening medium for the simultaneous detection and identification of methicillin-resistant S. aureus in clinical surveillance specimens.

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, ...

The bactericidal dynamics of oxacillin against four Staphylococcus aureus isolates with known 24-h "persister" percentages were studied by using the agar dilution plate count method. Isolates were selected to provide a representative spectrum whose individual 24-h trough intrinsic persister percentages ranged from greater than 1 to less than 0.01%. Resultant agar dilution plate count method killing curve patterns were found to be reproducible and served to characterize each isolate. The paradoxical effect was observed for each isolate, with paradoxical peaks tending to develop and diminish sequentially during the course of oxacillin action. The observed strain-dependent dynamics of oxacillin killing underscore the artifactual nature of the so-called tolerance phenomenon and negate the usefulness of MBCs and MBC/MIC ratios for the characterization of S. aureus isolates.

The AutoMicrobic system (AMS) (Vitek Systems, Inc., Hazelwood, Mo.) was compared with the API-20E system for the identification of gram-negative bacilli by using 380 stock clinical isolates and 377 immediately encountered fresh clinical isolates. For the stock isolates, with Enterobacteriaceae-Plus Biochemical Cards and automated interpretation, 364 (95.8%) were in agreement to the species level. For the fresh clinical isolates, agreement at the genus and species levels was 89.7 and 85.9%, respectively, when Enterobacteriaceae-Plus Cards were interpreted by the AMS. Manual interpretation of Enterobacteriaceae-Plus Biochemical Cards improved species level agreement to 91.0%. Subsequent retesting of all discrepant isolates with the Gram-Negative Identification Card resulted in significant improvement of results, and for the stock and fresh clinical isolates, species level agreement was 98.7 and 97.3%, respectively. AMS susceptibility testing was evaluated by comparing ampicillin and cephalothin MICs determined in parallel by AMS and a reference broth microdilution test for stock isolates, and by comparison of AMS and standardized disk agar diffusion test results for fresh clinical isolates. For the stock isolates, AMS mean integer MICs approximated microdilution mean integer MICs with AMS, providing excellent MIC replicability. For ampicillin and cephalothin, 50 and 46.8%, respectively, of AMS integer MICs were within + 1 ,ug/ml of the reference values, and 89.3 and 63.1% of AMS integer MICs were within ±2 ,ug/ml of the reference values. For the fresh clinical isolates, AMS and reference results were in disagreement for 4.5% of the antimicrobial agents tested, with 2.3% as a combination of "major" and "very major" errors.