tam susceptibility testing of 111 Pseudomonas aeruginosa isolates collected from ... indicated that the MS-2 should not beused for testing clinical P. aeruginosa.
JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1983, p. 389-394 0095-1137/83/080389-06$02.00/0 Copyright 0 1983, American Society for Microbiology
Vol. 18, No. 2
False-Susceptible Results from the MS-2 System Used for Testing Resistant Pseudomonas aeruginosa Against Two Third-Generation Cephalosporins, Moxalactam and Cefotaxime LARRY L. STONE'* AND DONALD L. JUNGKIND2 Crozer-Chester Medical Center, Chester, Pennsylvania 19013,1 and Thomas Jefferson University Hospital,
Philadelphia, Pennsylvania 191072 Received 21 January 1983/Accepted 15 April 1983
The MS-2 system (Abbott Laboratories) was used for cefotaxime and moxalactam susceptibility testing of 111 Pseudomonas aeruginosa isolates collected from our burn treatment center. The frequency of very major errors was 52% for moxalactam and 32% for cefotaxime for all isolates requiring a minimal inhibitory concentration of -64 ,ug/ml. For isolates requiring a minimal inhibitory concentration of 64 ,ug/ml, the very major errors were 78% for moxalactam and 57% for cefotaxime. Testing of an additional 44 isolates collected from another hospital confirmed that these results were not unique to nosocomial strains from within the burn center. The ratio of errors did not change when MS-2 results were compared with disk diffusion results. Colony count testing done on cuvettes which produced false-susceptible results indicated that little numerical change occurred during the 5- to 6-h test cycle, whereas microscopy of cuvette contents revealed metabolically active ifiamentous bacilli. The high frequency of false-susceptible results indicated that the MS-2 should not be used for testing clinical P. aeruginosa isolates for resistance to the two third-generation cephalosporins currently available for the MS-2. A comparison of the disk diffusion results indicated that neither moxalactam nor cefotaxime could be reliably used for predictive, classconcept testing of P. aeruginosa.
Recently published data for cefotaxime and for moxalactam, the first two of the third-generation class of cephalosporins to be released for general use in the United States, indicated that 70 to 90%o of clinical isolates of Pseudomonas aeruginosa should be sensitive to one or both agents (6, 9, 12). Pseudomonads isolated from burn treatment centers are typically more resistant than those from other clinical sources, and P. aeruginosa has traditionally been the most difficult organism to control in burn wounds. Thus, an evaluation of the specific antipseudomonal activity of moxalactam and cefotaxime was begun on isolates from our burn treatment center immediately after the release of these antimicrobial agents. Initial testing done by the standardized agar disk diffusion method was followed with an evaluation using the MS-2 rapid susceptibility system (Abbott Laboratories). An apparent discrepancy between the results from the two methods was immediately noted. The present investigation was conducted to resolve the differences between results from the MS-2 system and those from conventional susceptibility testing methods. 389
MATERIALS AND METHODS
Organisms. P. aeruginosa isolates from patient specimens routinely submitted to our clinical microbiology laboratory by a burn treatment center (the Crozer-Chester Medical Center) were incorporated into this study. A total of 111 isolates were collected over a 5-month period. An additional 44 isolates requiring minimal inhibitory concentrations (MICs) of .8 Fag of cefotaxime or moxalactam per ml were collected from a university-affiliated (Thomas Jefferson University [TJU]) hospital not having a burn treatment center. The identity of each isolate was established according to recommended testing procedures (7). Susceptibility testing. All susceptibility testing was performed with 18-h-old subcultures grown on 5% sheep blood agar plates. Disk diffusion testing was performed in duplicate according to the procedures recommended by the National Committee for Clinical Laboratory Standards (NCCLS) (11), and duplicate results had to agree by +2 mm for acceptance. All Mueller-Hinton media and susceptibility disks were commercially supplied (BBL Microbiology Systems, Cockeysville, Md.). One of the duplicate McFarland 0.5 saline inocula prepared for disk diffusion testing was also used for inoculation of duplicate Abbott MS-2 cuvettes. All recommendations from Abbott Diagnos-
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tics for susceptibility testing with the MS-2 were rigidly followed; these included (i) use of 15 ml of freshly prepared MS-2 culture medium (Oxoid Ltd.) per cuvette, (ii) immediate inoculation with 200 ,ul of the freshly prepared inoculum, (iii) gentle agitation of the inoculated cuvettes before insertion, and (iv) testing of organisms as soon after initial isolation as possible. The two MS-2 cuvettes per isolate were each loaded with Abbott 30-,ug moxalactam elution disks in test wells 1 to 5 and with Abbott 30-,ug cefotaxime disks in wells 6 to 10. Ten results per antimicrobial agent were therefore generated for each isolate. Microdilution susceptibility testing was performed by a separate clinical laboratory (TJU). Microdilution trays were prepared with a MIC-2000 (Dynatech Industries, Alexandria, Va.). Antimicrobial agents were supplied by the respective manufacturers and were diluted in cation-supplemented Mueller-Hinton broth (15). Dilutions used were 0.5, 1, 2, 4, 8, 16, 32, and 64 ,ug/ml. Plates were frozen at -75'C until used. The inoculum was 105 organisms per ml. Quantitation and microscopy. Immediately after completion of the MS-2 test cycle, the contents of the individual test wells of selected cuvettes were aspirated for quantitative and microscopic studies. A heated 18-gauge needle was used to melt holes into the test chambers, and a smaller 25-gauge needle and tuberculin syringe were used to aspirate the specimen. This sequence averted the formation of a vacuum in the test wells and thereby prevented a draw-down of contaminating inoculum from the upper growth chamber. A prior injection of safranine dye through the filter port of the upper growth chamber provided visual assurance of the absence of any such contamination. Interpretive criteria. Food and Drug Administration (FDA) interpretive criteria were used throughout this investigation. For both moxalactam and cefotaxime, organisms requiring an MIC of
