System with Predried Panels

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1991, p /91/ $02.00/0 Copyright 1991, American Society for Microbiology Vol. 29, No. 2 Pseudoresistance of Pseudomonas aeruginosa Resulting from Degradation of Imipenem in an Automated Susceptibility Testing System with Predried Panels ROGER L. WHITE,'* MICHAEL B. KAYS,' LAWRENCE V. FRIEDRICH,' EVE W. BROWN,2 AND JAMES R. KOONCE' College of Pharmacy' and Department of Pathology and Laboratory Medicine, Division of Clinical Microbiology,2 Medical University of South Carolina, 171 Ashley Avenue, Charleston, South Carolina Received 19 July 1990/Accepted 5 November 1990 During the use of a single lot of custom breakpoint panels (Sensititre; Radiometer America Inc., Westlake, Ohio), imipenem susceptibility declined from 70 to 44% for clinical isolates of Pseudomonas aeruginosa. With a new lot, susceptibility increased to 73%. Subsequent evaluations with P. aeruginosa ATCC revealed a similar susceptibility pattern and an increase in the MIC of imipenem when determined in panels with increasing ages. Imipenem concentrations were determined by high-performance liquid chromatography by using 11 different lots of MIC and breakpoint panels (139 to 893 days of age). The amount of imipenem remaining declined from 94.4 to 13.8% (r = ) over the age range of the panels. These data suggest that imipenem in Sensititre MIC and breakpoint panels degrades over time and that the decrease in imipenem may be largely responsible for the decline in P. aeruginosa susceptibility. The in vitro activity of imipenem encompasses a broad range of bacteria, including Pseudomonas aeruginosa (2-5, 15). Imipenem has been shown to be effective in the treatment of serious bacterial infections (12, 16, 19, 20); however, cure rates for infections caused by P. aeruginosa have been lower than those for infections caused by other organisms, primarily because of the development of resistance during therapy (6, 9, 10, 19, 20). During review of our quarterly antibiograms, we observed a precipitous decline in the susceptibility of clinical isolates of P. aeruginosa to imipenem. Over 12 months, susceptibility declined from 70 to 44% with the use of a single lot of gram-negative breakpoint panels. With a new lot, susceptibility increased to 73%. Because of the apparent relationship between imipenem susceptibility and age of the panels, our objectives were to evaluate further the effect of the age of our panels on the susceptibility of P. aeruginosa to imipenem and to determine the actual concentrations of imipenem in microdilution panels of various ages. (This work was presented at the 90th Annual Meeting of the American Society for Microbiology, Anaheim, Calif. [18].) Susceptibility of clinical isolates of P. aeruginosa and P. aeruginosa ATCC to imipenem was determined according to the guidelines of the manufacturer by using custom gram-negative breakpoint panels (Sensititre; Radiometer America Inc., Westlake, Ohio). Susceptibility data were reviewed retrospectively from February 1988 to March 1989 and concurrently from April 1989 to December The breakpoints of the National Committee for Clinical Laboratory Standards were used to categorize the data as susceptible, moderately susceptible, or resistant (7). All microdilution panels used during this period and those used in subsequent analyses were stored at room (air-conditioned) temperature. Imipenem MICs for P. aeruginosa ATCC were * Corresponding author. 398 determined by using gram-negative MIC panels obtained from the manufacturer. The age of each panel was determined by subtracting the date of manufacture from the study date. Imipenem concentrations were analyzed by high-performance liquid chromatography (HPLC) by using a Waters model 510 pump, a Waters WISP 710B automatic sample injector, a Waters Lambda-Max model 481 LC spectrophotometer (wavelength, 299 nm), and a Shimadzu C-R3A integrator with a Zorbax tetramethylsilane reversed-phase column (Dupont, Wilmington, Del.). The mobile phase consisted of 2.5% acetonitrile in water (adjusted to ph 7.2), and the flow rate was 2 ml/min. The injection volume was 20 RI, and the mean retention time was 3.2 min. Because of its known instability, a preliminary degradation study was performed to ascertain the stability of imipenem during HPLC analysis. Imipenem (16 Fg/ml) was prepared in potassium phosphate buffer (10 mm, ph 7.2), and samples were injected continuously over 12 h. The degradation rate was determined by plotting the natural logarithm of the peak area versus time. All subsequent standard and unknown peak areas were corrected for degradation during analysis by the following equation: peak area/[e-(kdt), where Kd is the degradation rate constant ( h-'), and t is the time (in hours) from preparation of the standard or from reconstitution of the panels to analysis of the sample (<11 h for all samples). Panels with the silica desiccant of the desired blue color were reconstituted by pipetting 100 RI of buffer into the wells containing imipenem (expected range, 4 to 64,ug/ml). The panels were allowed to stand at ambient temperature for 10 min, and the contents were thoroughly mixed by repeatedly aspirating and expelling the buffer in the well. All samples were injected in duplicate, and the means were used in the analysis. Immediately before use, two standard curves were prepared in buffer by using standard laboratory imipenem powder (911,ug/mg; lot 8036R; Merck Sharp & Dohme, West Point, Pa.). Standard concentrations ranged from 1 to 32,ug/ml and from 0.25 to 4,ug/ml (detector set at 0.05 and 0.01 Downloaded from on July 19, 2018 by guest

2 VOL. 29, 1991 NOTES Q bl't :i U A ra.d cn * QUARTER (CALENDAR YEAR) FIG. 1. Susceptibility trends for clinical isolates of P. aeruginosa (a) and P. aeruginosa ATCC (L) to imipenem over time. The asterisk indicates a new lot of the Sensititre breakpoint panels. absorbance units, full scale, respectively). Both curves were constructed by using peak areas and were linear over their respective concentration ranges (r = for both curves). The intraday coefficient of variation was --4% at all concentrations. The percentage of imipenem remaining in each well was determined by the following equation: (assayed concentration/expected concentration) x 100. The susceptibilities of clinical isolates of P. aeruginosa and P. aeruginosa ATCC to imipenem are displayed in Fig. 1. The number of clinical isolates in each quarter from April 1988 to December 1989 was 92, 91, 76, 99, 64, 63, and 71, respectively. Over 12 months, susceptibility declined from 70 to 44% when a single lot of custom gram-negative breakpoint panels was used. The percentage of moderately susceptible and resistant results steadily increased over time. With the new lot, susceptibility increased to 73%, followed by a similar decline with time. The number of quality control tests performed during each quarter was 57, 90, 39, 18, 13, 41, 45, and 46, respectively. Similar to the clinical isolates, susceptibility of P. aeruginosa ATCC declined from 67 to 8%, while resistance increased from 2 to 30%. With the new lot of panels, susceptibility increased to 56% and declined to 7% in the fourth quarter of During this time, the amount of resistant results increased from 2 to 33%. Thirty-three MICs were determined by using panels from nine different lots ranging from 113 to 722 days of age. The MIC of imipenem for the quality control organism increased from 4 to 32,ug/ml over this age range. Panels assayed for imipenem consisted of nine lots of gram-negative MIC panels, one lot of a gram-positive MIC panel, and one lot of a custom gram-negative breakpoint panel. Panel ages ranged from 139 to 893 days, and with the exception of two lots, the panels were within their stated expiration dates. The percentage of imipenem remaining in each well versus the age of the panel is displayed in Fig. 2. The amount of imipenem remaining declined from 94.4 to 13.8% over the age of the panels and appeared to decline in a linear fashion (r = ). The stability of imipenem in various liquid and solid media has been described previously (1, 8, 14, 17). When incorporated into Mueller-Hinton broth and stored at -70 C, imipenem retained its antimicrobial activity for up to 1 year (8). When stored at temperatures of : -250C, the MICs of imipenem increased over time, suggesting degradation of the drug (1, 8). When stored at 4 C in Mueller-Hinton agar, the MIC of imipenem increased eightfold in only 14 days (17). Using predried microdilution panels (Sensititre), Staneck (13) compared imipenem MICs with those obtained by a reference agar dilution method. The results were within ±1 twofold dilution for 94% of 226 clinical isolates. Also, the MIC of imipenem was determined for P. aeruginosa ATCC on each day of testing, and the values were within ± 1 twofold dilution of the median MIC (2,ug/ml) for 97% of the repetitions. Although these data suggest that imiperem remained stable over the study period, MICs may be an insensitive test for evaluating drug stability because sigrificant degradation may occur before an increase in MIC would be observed. In our susceptibility data for P. aeruginosa ATCC 27853, tli AGE OF PANELS (DAYS) FIG. 2. Percentage of imipenem remaining in Sensititre panels, as determined by an HPLC assay (y = x; r = ). Downloaded from on July 19, 2018 by guest

3 400 NOTES we noted a gradual shift from susceptible to moderately susceptible to resistant using both lots of Sensititre breakpoint panels. These changes suggested imipenem degradation sufficient to cause at least a two- to fourfold decline in susceptibility. A subsequent HPLC assay revealed declining imipenem concentrations with increasing panel age. Although this phenomenon was suggested by quality control testing, the recommended susceptibility of this organism to imipenem during this period was listed as "variable" in the Sensititre Technical Product Information booklet (11). Therefore, moderately susceptible and resistant results, although heavily scrutinized, were considered within the quality control guidelines and were not immediately investigated. In conclusion, imipenem in Sensititre breakpoint and MIC panels degrades over time, even though it was handled and stored as recommended by the manufacturer. Substantial decreases in imipenem concentrations may occur in these panels prior to their respective expiration dates. This decline was most likely responsible for the dramatic decrease in the susceptibilities of our clinical isolates of P. aeruginosa. This work was supported in part by a grant from Merck Sharp & Dohme. 1. Baron, E. J., and J. A. Hindler Bioactivity of imipenem as a function of medium, time, and temperature. Antimicrob. Agents Chemother. 25: Barry, A. L., R. N. Jones, C. Thornsberry, L. W. Ayers, and R. Kundargi Imipenem (N-formimidoyl thienamycin): in vitro antimicrobial activity and P-lactamase stability. Diagn. Microbiol. Infect. Dis. 3: Cohn, D. L., L. G. Reimer, and L. B. Reller Comparative in-vitro activity of MK0787 (N-formimidoyl thienamycin) against 540 blood culture isolates. J. Antimicrob. Chemother. 9: Edwards, J. R., P. J. Turner, C. Wannop, E. S. Withnell, A. J. Grindey, and K. Nairn In vitro antibacterial activity of SM-7338, a carbapenem antibiotic with stability to dehydropeptidase I. Antimicrob. Agents Chemother. 33: Kropp, H., J. G. Sundelof, J. S. Kahan, F. M. Kahan, and J. Birnbaum MK0787 (N-formimidoyl thienamycin): evaluation of in vitro and in vivo activities. Antimicrob. Agents Chemother. 17: Lynch, M. J., G. L. Drusano, and H. L. T. Mobley Emergence of resistance to imipenem in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 31: National Committee for Clinical Laboratory Standards J. CLIN. MICROBIOL. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. M7-T2. National Committee for Clinical Laboratory Standards, Villanova, Pa. 8. Nickolai, D. J., C. J. Lammel, B. A. Byford, J. H. Morris, E. B. Kaplan, W. K. Hadley, and G. F. Brooks Effects of storage temperature and ph on the stability of eleven P-lactam antibiotics in MIC trays. J. Clin. Microbiol. 21: Ogle, J. W., L. B. Reller, and M. L. Vasil Development of resistance in Pseudomonas aeruginosa to imipenem, norfloxacin, and ciprofloxacin during therapy: proof provided by typing with a DNA probe. J. Infect. Dis. 157: Quinn, J. P., E. J. Dudek, C. A. DiVincenzo, D. A. Lucks, and S. A. Lerner Emergence of resistance to imipenem during therapy for Pseudomonas aeruginosa infections. J. Infect. Dis. 154: Radiometer America Inc. Sensititre technical product information booklet (S/SPI). Radiometer America Inc., Westlake, Ohio. 12. Rapp, R. P., B. Young, K. Bertch, P. Tibbs, and T. S. Foster Clinical outcome of nosocomial pneumonia following imipenem/cilastatin therapy. Drug Intell. Clin. Pharm. 21: Staneck, J. L Imipenem susceptibility testing with a commercially prepared dry-format microdilution tray. J. Clin. Microbiol. 23: Swanson, D. J., C. DeAngelis, I. L. Smith, and J. J. Schentag Degradation kinetics of imipenem in normal saline and in human serum. Antimicrob. Agents Chemother. 29: Tally, F. P., N. V. Jacobus, and S. L. Gorbach In vitro activity of N-formimidoyl thienamycin (MK0787). Antimicrob. Agents Chemother. 18: Trumbore, D., R. Pontzer, M. E. Levison, D. Kaye, M. Cynamon, C. Liu, D. R. Hinthorn, J. S. Tan, and T. M. File Multicenter study of the clinical efficacy of imipenem/cilastatin for treatment of serious infections. Rev. Infect. Dis. 7(Suppl. 3):S476-S Turgeon, P. L., and C. Desrochers Stability of imipenem in Mueller-Hinton agar stored at 4 C. Antimicrob. Agents Chemother. 28: White, R. L., E. W. Brown, L. V. Friedrich, and M. B. Kays Abstr. Annu. Meet. Am. Soc. Microbiol. 1990, C-269, p Winston, D. J., M. A. McGrattan, and R. W. Busuttil Imipenem therapy of Pseudomonas aeruginosa and other serious bacterial infections. Antimicrob. Agents Chemother. 26: Zajac, B. A., M. A. Fisher, G. A. Gibson, and R. R. MacGregor Safety and efficacy of high-dose treatment with imipenemcilastatin in seriously ill patients. Antimicrob. Agents Chemother. 27: Downloaded from on July 19, 2018 by guest

4 LETTER TO THE EDITOR External Factors Affecting Imipenem Performance in Dried Microdilution MIC Plates I am writing in reference to the articles "Pseudoresistance of Pseudomonas aeruginosa Resulting from Degradation of Imipenem in an Automated Susceptibility Testing System with Predried Panels" (6) and "False Resistance to Imipenem with a Microdilution Susceptibility Testing System" (4) Ȯn the basis of our extensive research with imipenem, we at Sensititre believe that while slow deterioration is inevitable with imipenem, the widely discrepant results reported by O'Rourke et al. and White et al. may be attributed more to external effects such as those described throughout this letter than to moderate losses of potency during storage. Initially we could not duplicate the shift in the imipenem susceptibility results obtained by O'Rourke et al. and White et al. As a result of our inability to fully explain the variability between the acceptable results obtained with reserve batches of plates in our hands and the results obtained by White and O'Rourke et al., we worked in conjunction with the manufacturers of imipenem, Merck, Sharp and Dohme, to resolve the problem. In the interim, we changed our manufacturing process to ensure greater stability of imipenem in our plates. The authors of both these articles (4, 6) were kept informed of the progress of our work and of the interim steps taken to protect the integrity of our product. We also reduced the shelf life of imipenem to 12 months as a safeguard to our customers. In the course of our intensive investigation, additional factors that give a much more complex picture than was described in either publication emerged. It was discovered that the following variables also affect the stability of imipenem. (i) Moisture. The MIC for the quality control (QC) organism Pseudomonas aeruginosa ATCC was measured by using 166 batches of plates from retention stocks held-by the manufacturer. These covered plates stored for up to 3 years. Imipenem MICs for only 1.6% (6 of 374) of the strains were outside the National Committee for Clinical Laboratory Standards QC range of 1 to 4 jj.g/ml, indicating that the panels performed correctly when the self-indicating desiccant was blue (showing that there had not been ingress of moisture), whereas the MIC results with the small number of plates which had pink silica gel sachets upon opening were aberrant for 53.2% of isolates tested. (ii) Broth. It has been reported (3) that different lots of Mueller-Hinton broth obtained from different commercial vendors can result in unacceptably high imipenem MICs with the QC strain P. aeruginosa ATCC This was not observed with other QC strains, including Escherichia coli ATCC and Staphylococcus aureus ATCC Broth TABLE 1. Imipenem MICs obtained by using broths from 2 vendors No. of strains with the following MIC (pg/ml): A B TABLE 2. Strain Imipenem MICs obtained by using different strains of P. aeruginosa No. of strains with the following MIC (pug/ml): A B Work undertaken by Sensititre substantiates this observation. The data shown in Table 1 were obtained by using plates dosed with the same inoculum ofp. aeruginosa ATCC which had been assayed in broths from two vendors. The expected MIC is 1 to 4,ug/ml. Recently reported findings by White et al. (5) confirm this observation. White et al. observed a shift in the modal MIC of imipenem for P. aeruginosa ATCC from 2 to 8,ug/ml in different lots of broth. Time-kill studies showed that this was due to differences in the rate of degradation of imipenem in broth, in the time to regrow, and in the concentration of organism after 24 h of incubation. They also presented data showing that acceptable QC results can be obtained with P. aeruginosa ATCC on Sensititre panels stored for up to 18 months when dosed with appropriate broth. A recent report (2) has shown that the zinc content in lots of Mueller-Hinton agar varies from vendor to vendor. Higher zinc content was associated with higher MICs of imipenem for P. aeruginosa but not for other gram-negative bacilli. Further studies are required to see whether this observation explains the effect observed by White et al. (5). (iii) Organism. The condition of the P. aeruginosa ATCC QC strain can affect the MIC of imipenem by 1 to 2 doubling dilutions. The results shown in Table 2 were obtained from 100 determinations of a strain freshly obtained from the American Type Culture Collection (strain B) compared with a strain that had had multiple passaging (strain A). In general, the best results are obtained with strains that have had minimal subculturing. 1. Baron, E. J., and J. A. Hindler Bioactivity of imipenem as a function of medium, time, and temperature. Antimicrob. Agents Chemother. 25: Cooper, G., A. Louie, R. Chu, R. P. Smith, W. Ritz, and M. Franke The role of zinc (Zn) on the activity of imipenemcilastatin (IM) against Pseudomonas aeruginosa, abstr. C-145, p Abstr. 91st Gen. Meet. Am. Soc. Microbiol American Society for Microbiology, Washington, D.C. 3. Nadler, H., D. Pitkin, and W. Sheikh The effect of culture medium source on antimicrobial activity of carbapenems. Abstr. 29th Intersci. Conf. Antimicrob. Agents Chemother., abstr O'Rourke, E. J., K. G. Lambert, K. C. Parsonnet, A. B. Macone, and D. A. Goldman False resistance to imipenem with a microdilution susceptibility testing system. J. Clin. Microbiol. 29: White, R. L., M. B. Kays, and L. V. Friedrich Effect of media and drug degradation during in vitro testing on the activity of imipenem, abstr. A-20, p. 4. Abstr. 91st Gen. Meet. Am. Soc. Microbiol American Society for Microbiology, Washington, D.C.

5 536 LETTER TO THE EDITOR 6. White, R. L., M. B. Kays, L. V. Friedrich, E. W. Brown, and J. R. Koonce Pseudoresistance of Pseudomonas aeruginosa resulting from degradation of imipenem in an automated susceptibility testing system with predried panels. J. Clin. Microbiol. 29: Roger Grist Sensititre Ltd. The Manor Manor Royal Crawley West Sussex RHIO 2PY United Kingdom Author's Reply We would like to acknowledge that the findings cited in our previous report may only apply under the conditions that existed in our institution during the time of the study. We suggest that our original report be read in its entirety. A number of details mentioned by Dr. Grist were addressed in the manuscript (3). As emphasized by Dr. Grist, the complexity of this issue made it quite difficult to isolate the various factors involved. However, we and others (1, 3) have demonstrated degradation of imipenem indirectly through the use of MIC and breakpoint testing and directly through assay of panels by both microbiologic and high-performance liquid chromatographic methods. Although the external effects suggested by Dr. Grist may have contributed to the discrepancies, degradation of approximately 50% over 1 year represents a substantial loss of potency. The "acceptable" results found by Sensititre with reserve batches may be due to the controlled conditions of their study and especially to the lack of exposure to shipping conditions. We investigated the variables which may affect the results of microbiologic testing mentioned by Dr. Grist (with the exception of the zinc studies). As stated in our report, we only used panels with blue dessicants, suggesting that moisture was not a factor in our findings. As suggested by Sensititre, we obtained a fresh QC strain of P. aeruginosa ATCC However, we were able to duplicate our previous results obtained with the strain that had been subcultured. As indicated by Dr. Grist, we reported the effects of broth on the in vitro activity of imipenem (2). It is important to note that we found that some lots of broth produced MIC results within acceptable QC limits despite the fact that panels in which only 40% of the stated potency of imipenem remained were used. However, the discrepancies due to imipenem degradation were obtained with broth supplied by Sensititre that was within its stated expiration date at the time of the study. In summary, we tested for many of the factors that may contribute to variability during in vitro susceptibility testing and demonstrated that drug degradation substantially contributed to a decline in P. aeruginosa susceptibility. Our initial and interim findings were communicated to Sensititre. Although it was initially suggested that imipenem degradation was an insignificant factor, the change in the manufacturing process and the reduction in the shelf life suggest otherwise. We applaud the recent efforts by Sensititre, especially those of Dr. Grist, in addressing this problem. 1. O'Rourke, E. J., K. G. Lambert, K. C. Parsonnet, A. B. Macone, and D. A. Goldmann False resistance to imipenem with a microdilution susceptibility testing system. J. Clin. Microbiol. 29: White, R. L., M. B. Kays, and L. V. Friedrich Effect of media and drug degradation during in vitro testing on the activity of imipenem, abstr. A-20, p. 4. Abstr. 91st Gen. Meet. Am. Soc. Microbiol American Society for Microbiology, Washington, D.C. 3. White, R. L., M. B. Kays, L. V. Friedrich, E. W. Brown, and J. R. Koonce Pseudoresistance of Pseudomonas aeruginosa resulting from degradation of imipenem in an automated susceptibility testing system with predried panels. J. Clin. Microbiol. 29: Roger L. White Michael B. Kays Lawrence V. Friedrich James R. Koonce College of Pharmacy Medical University of South Carolina Charleston, S.C J. CLIN. MICROBIOL. Eve W. Brown Department of Pathology and Laboratory Medicine Division of Clinical Microbiology Medical University of South Carolina Charleston, S.C