b
a Department of Internal Medicine I, Division of Infectious Diseases and Chemotherapy, University of Vienna, Austria; b Institute of Microbiology, School of Medicine, Comenius University, Bratislava; c Department of Computer Science, Faculty of Mathematics, Physics and Informatics, Comenius University, Bratislava, Slovak Republic
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Abstract |
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Introduction |
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In this study, we assessed the reliability of results obtained by a commercial modification of the NCCLS broth microdilution method with automated reading based on MTT tetrazolium colour growth indicator (Bel-MIDITECH, Bratislava, Slovak Republic), compared with the standard NCCLS microdilution assay results. For this purpose, a well characterized set of 527 Gram-negative and Gram-positive bacterial strains (Vienna testing set) collected and stored at the Division of Infectious Diseases and Chemotherapy (Vienna General Hospital, Austria) was used.
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Materials and methods |
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A total of 527 bacterial clinical isolates, prospectively and consecutively collected during a period of 3 years (19982000), was tested. The following Gram-positive and Gram-negative bacterial isolates were included: 40 methicillin-susceptible Staphylococcus aureus, 54 methicillin-resistant S. aureus, 33 Staphylococcus epidermidis, 74 Enterococcus faecalis, 41 Acinetobacter strains (A. baumanii, A. lwoffii, A. calcoaceticus), 26 Enterobacter aerogenes, 30 Enterobacter cloacae, 29 Citrobacter freundii, 48 Escherichia coli, 48 Klebsiella pneumoniae, 24 Klebsiella oxytoca, 30 Pseudomonas aeruginosa, 25 Proteus vulgaris and 25 Stenotrophomonas maltophilia. The bacteria were identified using API 20 E, API 20 NE, API Staph and API 20 Strep identification strips (bioMérieux, Marcy lÉtoile, France) and stored in a liquid-nitrogen freezer. The control strains used in this study were E. coli ATCC 25922 and ATCC 35218, P. aeruginosa ATCC 27853, S. aureus ATCC 29213 and E. faecalis ATCC 29212.
Antimicrobial agents
Standard antimicrobial powders obtained from various manufacturers were used. MIDITECH panels (see the assay description) were supplied dehydrated, with separate frozen indicator solution. The antimicrobial agents were tested with the following serial two-fold dilutions: ampicillin (Gram-negative 640.5 mg/L, Gram-positive 320.25 mg/L), ampicillinsulbactam (Gram-negative 64/32 0.5/0.25 mg/L, Gram-positive 32/160.25/0.125 mg/L), co-amoxiclav (Gram-negative 64/320.5/0.25 mg/L, Gram-positive 32/160.25/0.125 mg/L), cefuroxime (Gramnegative 640.5 mg/L, Gram-positive 320.25 mg/L), cefotaxime (640.5 mg/L), ceftazidime (640.5 mg/L), cefepime (320.25 mg/L), ciprofloxacin (40.125 mg/L), gentamicin (320.25 mg/L; 10008 mg/L when testing E. faecalis), imipenem (320.25 mg/L), linezolid (80.25 mg/L), meropenem (320.25 mg/L ), netilmicin (320.25 mg/L), oxacillin (40.125 mg/L; final NaCl concentration, 2% v/w), piperacillin (1281 mg/L), trimethoprimsulfamethoxazole (4/760.03/0.59 mg/L) and vancomycin (320.25 mg/L).
Inoculum preparation
Bacterial isolates were removed from storage, streaked on to Columbia agar plates supplemented with 5% sheep blood (bioMérieux) and incubated for 1824 h at 35°C in ambient air. A working bacterial suspension was prepared by suspending 35 isolated colonies in 3 mL of Mueller Hinton broth. The turbidity of this suspension was carefully adjusted photometrically to equal that of a 0.5 McFarland standard. For the test, the final inoculum was further diluted in MuellerHinton broth to achieve a final concentration of c. 5 x 105 cfu/mL.
NCCLS broth microdilution
The MICs were determined by a standardized broth microdilution method using cation-supplemented Mueller Hinton broth according to NCCLS guidelines.2 The inoculated microdilution plates were incubated for 1620 h at 35°C (24 h for oxacillin and vancomycin with staphylococci and enterococci, respectively) in ambient air. For the reading of microtitration plates, a magnifying mirror was used. Standard quality control ATCC strains with known MICs were included in each run.
MIDITECH MTT assay
The MIDITECH assay is a modification of the NCCLS broth microdilution method intended for routine testing of aerobic Gram-negative non-fastidious bacteria, staphylococci and E. faecalis.12 Instead of a traditional microtitration tray as described in the NCCLS method, this technique uses a plastic panel with multiple non-transparent nylon microchambers. The microchambers contain antibiotics and the panels are provided dehydrated. To each test microchamber was added 22 µL of bacterial suspension, prepared in MuellerHinton broth (5 x 105 cfu/mL). The panel was sealed in a plastic bag and incubated in an inverted position for the same time as recommended for the NCCLS method. At the end of the incubation time, 4 µL of MTT indicator solution was added to each microchamber and the panel was incubated for another 30 min (60 min for E. faecalis). The reactions were read visually (yellow colour = no growth, purple colour = growth), but a table scanner (610S; UMAX, Hsinchu, Taiwan) is routinely used. The panel was digitally analysed by the MIDITECH system software. The growth intensity in each chamber was compared with negative and positive controls. The collected data can be further processed by MIDITECH's interpretative reading module and a pharmacokinetic/pharmacodynamic module. In this study, only raw data produced by the reading module (MIDITECH Analyser, version 2/96) were compared with those obtained by the visual reading of the NCCLS standard broth microdilution method.
Statistical analysis
All tests were carried out in duplicate. For comparison and statistical analysis, each of these tests was considered as an individual observation; therefore, two observations were obtained for each combination of isolate and antibiotic agent.
Comparison of interpretative results (susceptible, intermediate and resistant breakpoints according to NCCLS criteria) was done by calculating minor, major, very major and essential errors and their rates according to Murray et al.13 Percentages of individual errors were calculated as follows. Minor errors: percentage of isolates interpreted by the tested method as resistant or susceptible when the isolate was categorized as intermediate by standard test, together with isolates recognized as intermediate by the tested method and recorded as resistant or susceptible by the standard test, calculated with the sum of all tested isolates as the denominator. Major errors: percentage of susceptible isolates falsely determined by the tested method as resistant, calculated with number of susceptible isolates as the denominator. Very major errors: percentage of resistant isolates falsely determined by the tested method as susceptible, calculated with the number of resistant isolates as the denominator. Essential errors: percentage of isolates with major and very major errors, calculated with the sum of susceptible and resistant isolates as the denominator. Percentage of absolute agreement was calculated as the sum of isolates with complete interpretative agreement, using the sum of all tested isolates as the denominator. Percentage of essential agreement was calculated for all isolates except those with major and very major errors, using the sum of all tested isolates as the denominator.
The degree of agreement between the MIDITECH assay and the NCCLS microdilution test was defined as the proportion of MICs determined by the MIDITECH test that fell within NCCLS results ±1 log2 dilutions (accuracy limits of the test) for each reading. MICs possibly smaller than the lowest tested concentration were all considered equal to this concentration. Similarly, MICs greater than the highest tested concentration were all considered equal to the next highest concentration. Significance of possible differences between both methods has been evaluated by the Wilcoxon signed rank directional test on the log2 results obtained by compared tests.14 The sign of the W value reflects the orientation of shifts of the MTT assay towards lower (-) or higher (+) values.
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Results |
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Gram-negative bacteria
A total of 326 Gram-negative bacteria representing 10 clinically common species were tested for susceptibility to 13 antibiotics. The results are shown in Tables 1 and 2. Table 1
shows the numbers of susceptible, intermediate and resistant isolates tested, together with the distribution of interpretative errors of the MIDITECH assay. The absolute interpretative agreement for both methods ranged from 87.12% for ampicillinsulbactam to 99.85% for meropenem. A total of 417 (4.92%) minor, three (0.05%) major and 15 (0.63%) very major errors were observed. Minor errors were highest for ampicillinsulbactam, co-amoxiclav, cefotaxime and cefuroxime. The highest number of essential errors (major and very major) was seen with trimethoprimsulfamethoxazole (1.23%) and with ampicillin sulbactam (0.86%). No essential errors were observed for ceftazidime, cefepime and gentamicin, and there were relatively few minor errors for ciprofloxacin. The best agreement was demonstrated for both carbapenems.
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Gram-positive strains
A total of 127 staphylococci and 74 E. faecalis isolates were tested in duplicate with nine antibiotics. NCCLS criteria have been applied to E. faecalis isolates only for ampicillin, gentamicin (high concentrations), ciprofloxacin, vancomycin and linezolid.2 The absolute interpretative agreement ranged from 90.80% for ciprofloxacin to 100% for vancomycin and linezolid (Table 3). A total of 81 (2.77%) minor, three (0.15%) major and eight (0.83%) very major errors were found. Two very major errors were found when testing S. aureus (one methicillin-resistant isolate) against oxacillin and two in testing for high-concentration gentamicin resistance in E. faecalis. The minor errors were highest for ciprofloxacin and for both aminoglycosides when staphylococci were tested at low antibiotic concentrations.
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Discussion |
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To assess the reliability of the MIDITECH method, we compared this technique with the standard NCCLS broth microdilution assay by using a well characterized set of Gram-negative and Gram-positive bacteria (Vienna testing set). Overall resistance rates for this set of bacteria (4.28% intermediate and 28.38% resistant) were found to be very similar to those published for the challenge susceptibility testing set of difficult-to-test bacteria from the Centers for Disease Control and Prevention.4,5 Only E. faecalis isolates could be included in this study, because of MIDITECH assay limitations for enterococci. All these isolates were susceptible to ampicillin.
Gram-negative non-fermenting bacteria (Pseudomonas species and S. maltophilia) are, in methodological studies, commonly considered as difficult to test and were evaluated separately from other Gram-negative bacilli.3,4,15 In the present study, the overall agreement between methods for these isolates (absolute agreement 70.98%, essential agreement 96.43% ± 0.64%) was very close to that for all 326 Gram-negative strains. Therefore, the data of all Gram-negative bacteria are presented as a single group.
The overall agreement of the MIDITECH colorimetric assay with the NCCLS broth microdilution test based on MICs for a total of 5751 organismantibiotic combinations tested in duplicate showed a 96.18% ± 0.67% essential agreement (±1 log2 dilution). The MIDITECH assay produced MICs differing from the NCCLS method by more than ±2 log2 dilutions in only 0.37% comparisons. The detailed statistical analysis of differences showed a significant shift in MIDITECH MICs towards lower values for nine, towards higher values for five and no significant shift for nine of 23 antibioticbacteria combinations tested. These shifts were generally within ±1 log2 dilution accuracy limit and showed no obvious relation to an individual antibiotic or resistance mechanism. Therefore, technical variables during antibiotic dilution, distribution and test procedure, rather then systemic errors, were probably responsible for the differences observed.
The absolute overall interpretative agreement observed for Gram-negative and Gram-positive bacteria was 95.42%. The essential interpretative agreement was 99.75%. The highest essential interpretative error rate for Gramnegative bacteria and trimethoprimsulfamethoxazole is obviously related to the lack of an intermediate interpretative concentration (all errors have to be referred as essential) for this antibiotic combination. An increased number of interpretative errors was documented also for ampicillinsulbactam. Despite good agreement between compared methods at ±1 log2 dilutions, we encountered 12.11% minor and 0.86% very major interpretative errors for this antibiotic combination; 11.99% of minor errors were due to lower MICs determined by the MIDITECH method. A similar situation was found for co-amoxiclav, but with fewer errors. Minor errors for other ß-lactam antibiotics in this study were almost equally distributed between higher and lower concentrations (data not shown).
To increase the sensitivity of the viability measurements, the MIDITECH test uses oxidasereductase chemistry similar to that of the Alamar method.4,5 Baker et al.,4 in comparing this test with the NCCLS technique, encountered 16.4% minor and 6.2% essential interpretative errors for ampicillinsulbactam. In their study, the Alamar method produced higher MICs of ß-lactam antibiotics if the resistant organism produced inducible or extended-spectrum ß-lactamase. The MIDITECH MICs for Gram-negative bacteria were rather underestimated, even for resistant isolates. We can only speculate that the addition of a sensitive growth indicator at the end of incubation, compared with its incorporation with the initial inoculum, reflects more precisely endpoint viability in the bacterial culture exposed overnight to an antibiotic. In the present study, we did not analyse exactly the underlying resistance mechanisms. Further studies will be necessary to elucidate the possible relationship between resistance mechanisms, induction and metabolic events in the microdilution MIC test.
The overall results for Gram-positive bacteria obtained by the MIDITECH assay were similar to those demonstrated for Gram-negative isolates. The highest minor error rate for ciprofloxacin (9.20%) was not unexpected, because almost all E. faecalis MICs were close or equal to the intermediate interpretative breakpoint for this antibiotic. Equally, MICs for many staphylococci corresponded to the netilmicin intermediate breakpoint and may explain many minor interpretative errors, despite a good essential agreement for this antibiotic. Of 74 E. faecalis isolates tested, 33 showed high-concentration gentamicin resistance by the NCCLS reference method (breakpoint 500 mg/L). Of these, the MIDITECH assay assessed one isolate incorrectly with an MIC of 250 mg/L, resulting in a 3.03% (very major) interpretative error. All five E. faecalis isolates with vancomycin MICs 32 mg/L were identified correctly by the MIDITECH test.
Oxacillin testing of staphylococci raised a special concern. The last revision of interpretative breakpoints facilitated, in particular, testing of coagulase-negtive staphylococci.16,17 Despite this, even new modifications of some commercial systems do not reach 100% agreement with the NCCLS method when testing oxacillin.3,5 In the present study, the MIDITECH assay failed to identify oxacillin resistance in one S. aureus isolate (1.25% very major error). All other S. aureus and S. epidermidis isolates were classified correctly.
The MIDITECH test was found to be user friendly, especially for large series of samples with multiple antibiotics. The principal disadvantage of this colorimetric method is the inability to test all medically relevant bacteria. However, for most clinically important aerobic pathogens, including those considered difficult to test, the MIDITECH modification of the NCCLS microdilution test provided reliable quantitative susceptibility data. The most attractive factors for the routine application of this method are its low cost and its capability for automating results. For many clinical microbiology laboratories, the need for automated reading and processing of extensive MIC data might favour the use of this method for routine work.
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Notes |
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References |
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2 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow AerobicallyFifth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA.
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.
Canton, R., Perez-Vazques, M., Oliver, A., Sanchez Del Saz, B., Gutierrez, M. O., Martinez-Ferrer, M. et al. (2000). Evaluation of the Wider System, a new computer-assisted image-processing device for bacterial identification and susceptibility testing. Journal of Clinical Microbiology 38, 133946.
4 . Baker, C. N., Banerjee, S. N. & Tenover, F. C. (1994). Evaluation of Alamar colorimetric MIC method for antimicrobial susceptibility testing of Gram-negative bacteria. Journal of Clinical Microbiology 32, 12617.[Abstract]
5 . Baker, C. N. & Ternover, F. C. (1996). Evaluation of Alamar colorimetric broth microdilution susceptibility testing method for staphylococci and enterococci. Journal of Clinical Microbiology 34, 26549.[Abstract]
6 . Ferraro, M. J. & Jorgensen, J. H. (1999). Susceptibility testing instrumentation and computerised expert systems for data analysis and interpretation. In Manual of Clinical Microbiology, 7th edn, (Murray, P. R., Baron, E. J., Pfaller, M. A., Tenover, F. C. & Yolken, R. C., Eds), pp. 15931600. American Society for Microbiology, Washington, DC.
7 . Bartlett, R. C. & Mazens, M. F. (1979). Rapid antimicrobial susceptibility testing using tetrazolium reduction. Antimicrobial Agents and Chemotherapy 15, 76974.[ISI][Medline]
8 . Johnson, T. L., Forbes, B. A., O'Connor-Scarlet, M., Machinski, A. & McClatchey, K. D. (1985). Rapid method of MIC determination utilizing tetrazolium reduction. American Journal of Clinical Pathology 83, 3748.[ISI][Medline]
9 . Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxic assays. Journal of Immunological Methods 65, 5563.[ISI][Medline]
10 . Clancy, C. J. & Nguyen, M. H. (1997). Comparison of a photometric method with standardised methods of antifungal susceptibility testing of yeasts. Journal of Clinical Microbiology 35, 287882.[Abstract]
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.
Meletiadis, J., Meis, J. F. G. M., Mouton, J. W., Donnelly, J. P. & Verweij, P. E. (2000). Comparison of NCCLS and 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) methods of in vitro susceptibility testing of filamentous fungi and development of a new simplified method. Journal of Clinical Microbiology 38, 294954.
12 . Bel-MIDITECH. (1997). MIDITECH antimiocrobial susceptibility testing system. Procedure Manual. Bel-MIDITECH, Bratislava, Slovak Republic.
13 . Murray, P. R., Niles., A. C. & Heeren, R. L. (1987). Comparison of a highly automated 5-h susceptibility testing system, the Cobas-Bact, with two reference methods: KirbyBauer disk diffusion and broth microdilution. Journal of Clinical Microbiology25, 23727.[ISI][Medline]
14 . Lehmann, E. L. (1975). Nonparametrics: Statistical Methods Based on Ranks. Holden Day, San Francisco, CA.
15 . Carroll, K. C., Cohen, S., Nelson, R., Campbell, D. M., Claridge, J. D., Garrison, M. W. et al. (1998). Comparison of various in vitro susceptibility methods for testing Stenotrophomonas maltophilia. Diagnostic Microbiology and Infectious Diseases 32, 22935.[ISI][Medline]
16 . McDonald, C. L., Maher, W. E. & Fass, R. J. (1995). Revised interpretations of oxacillin MICs for Staphylococcus epidermidis based on mecA detection. Antimicrobial Agents and Chemotherapy 39, 9824.[Abstract]
17 . National Committee for Clinical Laboratory Standards. (1999). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow AerobicallyFourth Edition: Approved Standard M7-A4. NCCLS, Wayne, PA.
Received 4 June 2001; returned 24 September 2001; revised 2 November 2001; accepted 12 November 2001