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dalfopristin (Synercid®) and RPR 106 972 stability in susceptibility testing media
International Journal of Antimicrobial Agents 15 (2000) 291 – 297 www.ischemo.org
Original article
Evaluation of quinupristin/dalfopristin (Synercid®) and RPR 106 972 stability in susceptibility testing media Steven A. Marshall, Kari C. Kugler, Ronald N. Jones * Medical Microbiology Di6ision, C606 GH, Department of Pathology, Uni6ersity of Iowa College of Medicine, Iowa City, IA 52242, USA Received 18 February 2000; accepted 11 March 2000
Abstract In response to conflicting reports on the chemical stability of quinupristin/dalfopristin, a study was designed to assess the in vitro longevity and effects of media and storage conditions on this streptogramin combination. Broth microdilution trays containing parenteral (quinupristin/dalfopristin) and oral (RPR 106 972) streptogramin combinations as well as pristinomycin components (P-I and P-II) were preincubated at 35°C for 12 – 72 h before inoculation with control strains (Streptococcus pneumoniae ATCC 49 619, Haemophilus influenzae ATCC 49 247, Enterococcus faecalis ATCC 29 212, Staphylococcus aureus ATCC 29 213) and five clinical isolates with various drug resistance phenotypes. Overall, the mean quinupristin/dalfopristin activity loss was 24%/12 h, 41%/18 h, 43%/24 h, 69%/48 h and 79%/72 h with no detected loss of potency when measured by E. faecalis until 18 h. RPR 106 972 mean activity loss was 6%/12 h, 19%/18 h, 19%/24 h, 56%/48 h and 71%/72 h with no loss of potency as measured by S. aureus until 48 h. Overall, P-I components had greater stability as compared with P-II for both drug combinations. Bioassays showed similar trends in decreased activity. Bioassay differences among media types were only significant (\3 mm; greater loss of potency) for haemophilus test media for both P-II components at 72 h. The presence of an organism in the medium had no effect on stability assay results. The effect of storage temperature (4, 25°C) on quinupristin/dalfopristin and RPR 106 972 stability was also detrimental to drug potency indicating the requirement for rigid quality assurance for streptogramin diagnostic reagents when determining activity by reference or standardized susceptibility tests. © 2000 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. Keywords: Streptogramin; Quinupristin/dalfopristin; Stability
1. Introduction Streptogramin antimicrobial agents are combinations of two structurally unrelated pristinamycin components designated, P-I (streptogramin B) and P-II (streptogramin A) each derived from the Streptomyces genus. The P-I molecule is a cyclic hexadepsipeptide and the P-II molecule is a polyunsaturated macrolactone [1]. The combination of these two molecules acts synergistically and with bactericidal activity against most Grampositive organisms including methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative * Corresponding author. Tel.: +1-319-3562990; fax: + 1-3193564916. E-mail address: [email protected] (R.N. Jones).
staphylococci (CoNS), vancomycin-resistant and multiresistant Enterococcus faecium (VRE), and b-lactam-resistant streptococci [2–7]. Streptogramin combinations also have activity against some mycoplasmas, Neisseria gonorrhoeae, Haemophilus influenzae, Legionella spp. and Moraxella catarrhalis [8,9]. Quinupristin/dalfopristin (formerly RP 59 500, Synercid®) is a clinically available parenteral streptogramin combination; and RPR 106 972 is an investigational orally administered streptogramin combination [10] with activity similar to that of quinupristin/dalfopristin. There have been limited but conflicting unpublished reports on the longevity of quinupristin/dalfopristin activity once introduced into various broth or agar media. This study assesses the relative stability of these two streptogramin combinations and their respective
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S.A. Marshall et al. / International Journal of Antimicrobial Agents 15 (2000) 291–297
292
P-I and P-II components under various storage conditions and with several types of medium.
2. Materials and methods
pristin were evaluated individually as well as in combination. RPR 106 972 consists of RPR 106 950 (P-I) and RPR 112 808 (P-II), also tested individually and in combination. All compounds evaluated in this study were obtained from Rhoˆne–Poulenc Rorer (Collegeville, PA).
2.1. Antimicrobial agents 2.2. Strains Quinupristin/dalfopristin consists of RP 57 669 (quinupristin; P-I) and RP 54 476 (dalfopristin; P-II) mixed in a ratio of 30:70, respectively. Quinupristin and dalfo-
Test organisms for dilution stability and storage tests were four American Type Culture Collection (ATCC)
Table 1 Modal MICs of triplicate broth microdilution tests from pre-incubated trays containing streptogramin combinations and their pristinamycin components Organism
S. aureus ATCC 29 213
102-1151 (mecA)
E. faecalis ATCC 29 212
E. faecium E99-5 (6anA)
H. influenzae ATCC 49 247 S. pneumoniae ATCC 49 619 7-2806B S. mitis 2526A-1 2526A-2
a
Antimicrobiala
Modal MIC (mg/l) by pre-incubation time 0h
12 h
18 h
24 h
48 h
72 h
RP 59 500 P-I P-II RPR 106 972 P-I P-II RP 59 500 P-I P-II RPR 106 972 P-I P-II
0.25 2 8 1 16 16 0.5 64 4 2 \128 8
0.5 4 8 1 16 16 1 128 8 4 \128 8
0.5 4 8 1 16 16 1 128 8 4 \128 16
0.5 4 16 1 32 32 1 128 8 4 \128 16
1 8 32 2 32 64 2 128 16 8 \128 32
2 8 64 4 32 128 4 \128 32 8 \128 64
RP 59 500 P-I P-II RPR 106 972 P-I P-II
4 8 \128 2 8 \128
4 16 \128 2 8 \128
8 16 \128 2 8 \128
8 16 \128 2 8 \128
16 32 \128 4 8 \128
16 32 \128 8 8 \128
RP 59 500 P-I P-II RPR 106 972 P-I P-II
0.5 128 8 0.5 \128 8
1 128 8 0.5 \128 8
1 128 8 0.5 \128 16
1 128 8 0.5 \128 16
2 \128 16 1 \128 32
2 \128 32 2 \128 64
16 8
32 16
1 0.25 32 4
1 0.25 32 4
16 8 8 2
32 8 16 4
RP 59 500 RPR 106 972
8 2
8 2
RP 59 500 RPR 106 972 RP 59 500 RPR 106 972
0.25 50.12 8 2
0.25 50.12 8 2
RP 59 500 RPR 106 972 RP 59 500 RPR 106 972
8 4 2 1
8 4 4 1
8 4 0.5 0.25 16 2 8 4 4 1
8 4 0.5 0.25 16 2 8 4 4 1
RP 59 500 is quinupristin/dalfopristin; P-I and P-II are pristinamycin components streptogramin B and A, respectively.
S.A. Marshall et al. / International Journal of Antimicrobial Agents 15 (2000) 291–297
293
Fig. 1. Histogram of mean activity loss of quinupristin/dalfopristin and RPR 106 972 from all broth microdilution stability tests listed by pre-incubation time (35°C). Mean activity loss from all agar dilution stability tests are represented by closed (quinupristin/dalfopristin) and broken (RPR 106 972) lines.
Fig. 2. Histogram of mean activity loss of quinupristin/dalfopristin and pristinimycin components from all broth microdilution stability tests by pre-incubation time.
strains and five stock surveillance clinical isolates (University of Iowa College of Medicine, Iowa City, IA). The control strains were Enterococcus faecalis ATCC 29 212, Staphylococcus aureus ATCC 29 213, Haemophilus influenzae ATCC 49 247 and Streptococcus pneumoniae ATCC 49 619. Clinical isolates were MRSA (mec A-positive, strain c 102-1151), a VRE (6an Apositive; strain cE99-5) and three streptococcal strains (7-2806B, 2526A-1, 2526A-2) with elevated quinupristin/dalfopristin minimum inhibitory concentrations (MICs). Test organisms for agar-based bioassays included S. epidermidis HBD 523 with an elevated MIC to P-I components alone, and S. aureus HBD 511 with an elevated MIC to P-II, both obtained from Rhoˆne – Poulenc Rorer.
2.3. Stability tests Trays containing serial two-fold dilutions of quinupristin/dalfopristin and RPR 106 972 (128–0.06 mg/l) in cation-adjusted Mueller–Hinton broth (MHB), haemophilus test media, brain–heart infusion broth and MHB supplemented with 3–5% lysed horse blood broth were manufactured by PML Microbiologicals (Wilsonville, OR) and stored at −70°C until used. Additional trays containing the individual P-I and P-II streptogramin components in MHB were also utilized (PML Microbiologicals). Agar plate series containing serial two-fold dilutions of quinupristin/dalfopristin and RPR 106 972 (128–0.06 mg/ml) in Mueller–Hinton agar were freshly prepared. Trays and agar plates were
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S.A. Marshall et al. / International Journal of Antimicrobial Agents 15 (2000) 291–297
pre-incubated at 35°C in an ambient air for 72, 48, 24, 18, 12, and 0 h prior to being inoculated (in triplicate) with organisms according to reference testing method recommendations of the National Committee for Clinical Laboratory Standards (NCCLS) [11]. To assess the effect of various temperatures on streptogramin stability in media, additional broth microdilution trays were stored at 4 and 25°C for 48 h and then inoculated with selected test organisms. To evaluate the effect of media types on P-I and P-II component stability, 20 ml aliquots were taken from inoculated (S. aureus ATCC 29 213) and uninoculated pre-incubated broth microdilution tray wells containing 128, 32, and 8 mg/l of each streptogramin combination and each media type tested. Aliquots were absorbed on to blank sterile 6-mm paper disks (BD Microbiologic Systems, Cockeysville, MD). Disks were then applied to 150 mm Mueller – Hinton agar plates inoculated with bioassay organisms (HBD 511 and HBD 523) and incubated in accordance with NCCLS [12] disk diffusion method recommendations. By using the disk bioassay method, we addressed the effects of media type on the drug activity by eliminating the effect of media type on the test organism growth rate. At the same time, by using P-I or P-IIresistant test organisms, one can measure the effect of media types on the components individually. Only the inhibition zone sizes from disks containing drug concentrations of 128 mg/l are reported here.
3. Results
3.1. Broth microdilution and agar dilution stability tests Overall, both streptogramin combinations showed a
steady decline (two- to four-fold) in activity from the baseline MIC over all pre-incubation times (Table 1). An increase in MIC of all tests compared to baseline (measured as a percent decline in activity), revealed that RPR 106 972 was slightly more stable than quinupristin/dalfopristin for each time point tested (Fig. 1). Overall mean activity loss (%) at each time interval was 24%/12 h, 41%/18 h, 43%/24 h, 69%/48 h, 79%/72 h for quinupristin/dalfopristin, and 6%/12 h, 19%/18 h, 19%/24 h, 56%/48 h, 71%/72 h for RPR 106 972. Activity loss was generally first observed before 24 h for quinupristin/dalfopristin and after 24 h for RPR 106 972. Agar dilution stability tests showed declining stability trends similar to broth microdilution stability tests for both streptogramin combinations. P-I components were generally more stable than P-II components (Table 1), although analysis was limited due to off-scale dilution results. MICs generally increased two- to three-fold (P-I) or threeto four-fold (P-II) from baseline MIC over pre-incubation intervals (Fig. 2). RPR 106 972 P-I component stability was consistently higher than P-II, while quinupristin/dalfopristin P-I component stability was less than P-II prior to 24 h pre-incubation and greater than P-II after 24 h pre-incubation (Fig. 3). Notably, a two-fold increase in the baseline MIC was observed for most broth microdilution tests grown in blood product containing media compared to MHB alone (data not shown). Storage of trays at refrigerator (4°C) and room (25°C) temperatures for 48 h had a similar effect on RPR 106 972 and RP 59 500 potency, but greater stability than that observed at 35°C. Two-fold increases in MICs were generally noted after both 4 and 25°C storage (Table 2).
Fig. 3. Historgram of mean activity loss of RPR 106972 and pristinimycin components from all broth microdilution stability tests by pre-incubation time.
S.A. Marshall et al. / International Journal of Antimicrobial Agents 15 (2000) 291–297
295
Table 2 Modal MICs of streptogramin combinations and components from triplicate broth microdilution tests pre-incubated under various storage conditions for 48 h Organism
S. aureus ATCC 29 213
102-1151 (mecA)
E. faecalis ATCC 29 212
E. faecium E99-5 (6anA)
H. influenzae ATCC 49 247 S. pneumoniae ATCC 49 619
a b
Antimicrobiala
Modal MIC (mg/l) by 48-h pre-incubation condition −70°Cb
4°C
25°C
35°C
RP 59 500 P-I P-II RPR 106 972 P-I P-II RP 59 500 P-I P-II RPR 106 972 P-I P-II
0.12 1 4 0.25 8 4 0.25 64 2 1 \128 4
0.25 1 4 0.5 8 8 0.5 64 4 2 \128 8
0.25 2 8 0.5 8 16 0.5 64 4 2 \128 16
1 8 32 2 32 64 2 128 16 8 \128 32
RP 59 500 P-I P-II RPR 106 972 P-I P-II
4 8 \128 2 4 \128
4 8 \128 2 4 \128
4 8 \128 2 4 \128
16 32 \128 4 8 \128
RP 59 500 P-I P-II RPR 106 972 P-I P-II
0.5 128 4 0.25 \128 4
0.5 128 4 0.25 \128 8
0.5 128 8 0.5 \128 8
2 \128 16 1 \128 32
RP 59 500 RPR 106 972
4 2
4 2
8 4
RP 59 500 RPR 106 972
0.25 0.25
0.25 0.25
0.5 0.25
16 8 1 0.25
RP 59 500 is quinupristin/dalfopristin; P-I and P-II are pristinamycin components streptogramin B and A, respectively. Baseline MIC.
3.2. Disk bioassay stability tests
4. Conclusion
Disk bioassays also resulted in comparable patterns for component stability, thus showing a greater overall reduction in zone diameter with the P-II-susceptible test strain (Table 3). Decreases of zones were observed to be similar for both streptogramin combinations. The presence of an organism (S. aureus ATCC 29 213) in the drug/media assay had no additional effects on stability results (data not shown). The effect of medium and supplements on the streptogramin stability revealed variable results (Table 3). Zone size decreases for MHB media type was only significant (\ 3 mm) for the P-II-sensitive test strain when examining haemophilus test medium test results after 48 h pre-incubation for RPR 106 972 and after 72 h pre-incubation for quinupristin/dalfopristin.
Quinupristin/dalfopristin and RPR 106 972 streptogramin combinations were relatively unstable in unfrozen solutions or media with MICs increasing by approximately two-fold or greater per day. RPR 106 972 [10] was slightly more stable than quinupristin/ dalfopristin and the P-II components were usually more stable than P-I components. The effect of media type on streptogramin stability was minimal however, haemophilus test medium appeared most detrimental to drug stability than other media tested especially after 48 h. Also, a two-fold increase in baseline broth microdilution MIC was detected for organisms tested in blood supplemented MHB, an observation consistent with previous studies [13,14].
S.A. Marshall et al. / International Journal of Antimicrobial Agents 15 (2000) 291–297
296
Table 3 Effect of media type from pre-incubated broth microdilution trays on zone sizes of P-I-sensitive and P-II-sensitive staphylococcal bioassays Organism
Antimicrobiala and mediab
S. aureus HBD 511 (P-I-susceptible) RP 59 500 MHB LHB BHI HTM RPR 106 972 MHB LHB BHI HTM S. epidermidis HBD 523 (P-II-susceptible) RP 59 500 MHB LHB BHI HTM RPR 106 972 MHB LHB BHI HTM
Variation from Mueller–Hinton bioassay zone size (mm) by pre-incubation time 0h
12 h
18 h
24 h
48 h
72 h
16 17 18 16
16 16 16 16
15 14 15 15
15 14 14 14
13 11 11 11
11 9 9 8
19 18 20 18
17 18 17 17
17 17 17 17
16 17 17 16
14 14 15 13
13 12 13 10
23 23 24 22
21 22 21 21
21 20 20 20
21 20 20 19
18 17 16 17
16 14 13 10
22 22 23 21
20 21 20 20
18 18 19 17
18 19 19 16
16 16 16 12
14 12 11 6
a
RP 59 500 is quinupristin/dalfopristin. Mueller–Hinton broth (MHB). Mueller–Hinton broth supplemented with 5% lysed horse blood (LHB). Brain–heart infusion broth (BHI). Haemophilus test media broth (HTM). b
Unfrozen reagents containing streptogramins must be used immediately or discarded. Only frozen (− 20°C or below; −70°C preferred) storage conditions reliably maintained streptogramin potency in various susceptibility testing reagents (agar or broth), including those recommended by the NCCLS [11,12]. Special care must be exercised in testing these complex, combination antimicrobials in clinical laboratories including strict application of recommended quality control strain tests [11,12].
Acknowledgements The co-authors wish to thank the following persons for significant assistance in performing this experiment or preparing the publication: M.E. Erwin, D.J. Biedenbach, and K.L. Meyer. The research was made possible by a grant from Rhone – Poulenc Rorer.
[2]
[3] [4]
[5]
[6] [7]
References [8] [1] Porse BT, Garrett RA. Sites of interaction of streptogramin A and B antibiotics in the peptidyl transferase loop of 23 S rRNA
and the synergism of their inhibitory mechanisms. J Mol Biol 1999;286:375 – 87. Barry AL, Fuchs PC, Brown SD. Susceptibility to RPR 106 972, quinupristin/dalfopristin and erythromycin among recent clinical isolates of enterococci, staphylococci and streptococci from North American medical centers. J Antimicrob Chemother 1998;42:651 – 5. Finch RG. Antibacterial activity of quinupristin/dalfopristin: rational for clinical use. Drugs 1996;51:31 – 7. Jones RN, Ballow CH, Biedenbach DJ, Deinhart JA, Schentag JJ. Antimicrobial activity of quinupristin-dalfopristin (RP 59 500, Synercid) tested against over 28 000 recent clinical isolates from 200 medical centers in the United States and Canada. Diagn Microbiol Infect Dis 1998;31:437 – 51. Jones RN, Low DE, Pfaller MA. Epidemiologic trends in nosocomial and community-acquired infections due to antibiotic-resistant gram-positive bacteria: the role of streptogramins and other newer compounds. Diagn Microbiol Infect Dis 1999;33:101 – 12. Leclercq R, Courvalin P. Streptogramins: an answer to antibiotic resistance in gram-positive bacteria. Lancet 1998;352:591–2. Izumikawa K, Hirakata Y, Yamaguchi T, Yoshida R, Tanaka H, Takemura H, Maesaki S, Tomono K, Kaku M, Izumikawa KI, Kamihira S, Kohno S. In vitro activities of quinupristin-dalfopristin and the streptogramin RPR 106 972 against Mycoplasma pneumoniae. Antimicrob Agents Chemother 1998;42:698 – 9. Bebear C, Bouanchaud DH. A review of the in-vitro activity of quinupristin/dalfopristin against intracellular pathogens and mycoplasmas. J Antimicrob Chemother 1997;39(Suppl A):59–62.
S.A. Marshall et al. / International Journal of Antimicrobial Agents 15 (2000) 291–297 [9] Bouanchaud DH. In-vitro and in-vivo antibacterial activity of quinupristin/dalfopristin. J Antimicrob Chemother 1997;39 (Suppl A):15 – 21. [10] The Quality Control Study Group, Putnam SD, Jones RN, Johnson DM. In vitro antimicrobial activity and MIC quality control guidelines of RPR 106 972 (RPR 112 808/RPR 106 950): a novel orally administered streptogramin combination. Diagn Microbiol Infect Dis 1997;28:139–47. [11] National Committee for Clinical Laboratory Standards. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically. Approved Standard M7-A4. Wayne, PA: National Committee for Clinical Laboratory Standards, 1997.
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[12] National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Disk Susceptibility Tests. Approved Standard M2-A5. Wayne, PA: National Committee for Clinical Laboratory Standards, 1997. [13] Jones RN. Medium and supplement effects on the antimicrobial activity of quinupristin/dalfopristin tested by agar dilution and Etest methods. Diagn Microbiol Infect Dis 1996;26:99– 102. [14] King A, May J, Phillips I. Comparative activity of quinupristin/dalfopristin and RPR 106 972 and the effect of medium on in-vitro test results. J Antimicrob Chemother 1998;42:711 – 9.
Original article
Evaluation of quinupristin/dalfopristin (Synercid®) and RPR 106 972 stability in susceptibility testing media Steven A. Marshall, Kari C. Kugler, Ronald N. Jones * Medical Microbiology Di6ision, C606 GH, Department of Pathology, Uni6ersity of Iowa College of Medicine, Iowa City, IA 52242, USA Received 18 February 2000; accepted 11 March 2000
Abstract In response to conflicting reports on the chemical stability of quinupristin/dalfopristin, a study was designed to assess the in vitro longevity and effects of media and storage conditions on this streptogramin combination. Broth microdilution trays containing parenteral (quinupristin/dalfopristin) and oral (RPR 106 972) streptogramin combinations as well as pristinomycin components (P-I and P-II) were preincubated at 35°C for 12 – 72 h before inoculation with control strains (Streptococcus pneumoniae ATCC 49 619, Haemophilus influenzae ATCC 49 247, Enterococcus faecalis ATCC 29 212, Staphylococcus aureus ATCC 29 213) and five clinical isolates with various drug resistance phenotypes. Overall, the mean quinupristin/dalfopristin activity loss was 24%/12 h, 41%/18 h, 43%/24 h, 69%/48 h and 79%/72 h with no detected loss of potency when measured by E. faecalis until 18 h. RPR 106 972 mean activity loss was 6%/12 h, 19%/18 h, 19%/24 h, 56%/48 h and 71%/72 h with no loss of potency as measured by S. aureus until 48 h. Overall, P-I components had greater stability as compared with P-II for both drug combinations. Bioassays showed similar trends in decreased activity. Bioassay differences among media types were only significant (\3 mm; greater loss of potency) for haemophilus test media for both P-II components at 72 h. The presence of an organism in the medium had no effect on stability assay results. The effect of storage temperature (4, 25°C) on quinupristin/dalfopristin and RPR 106 972 stability was also detrimental to drug potency indicating the requirement for rigid quality assurance for streptogramin diagnostic reagents when determining activity by reference or standardized susceptibility tests. © 2000 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. Keywords: Streptogramin; Quinupristin/dalfopristin; Stability
1. Introduction Streptogramin antimicrobial agents are combinations of two structurally unrelated pristinamycin components designated, P-I (streptogramin B) and P-II (streptogramin A) each derived from the Streptomyces genus. The P-I molecule is a cyclic hexadepsipeptide and the P-II molecule is a polyunsaturated macrolactone [1]. The combination of these two molecules acts synergistically and with bactericidal activity against most Grampositive organisms including methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative * Corresponding author. Tel.: +1-319-3562990; fax: + 1-3193564916. E-mail address: [email protected] (R.N. Jones).
staphylococci (CoNS), vancomycin-resistant and multiresistant Enterococcus faecium (VRE), and b-lactam-resistant streptococci [2–7]. Streptogramin combinations also have activity against some mycoplasmas, Neisseria gonorrhoeae, Haemophilus influenzae, Legionella spp. and Moraxella catarrhalis [8,9]. Quinupristin/dalfopristin (formerly RP 59 500, Synercid®) is a clinically available parenteral streptogramin combination; and RPR 106 972 is an investigational orally administered streptogramin combination [10] with activity similar to that of quinupristin/dalfopristin. There have been limited but conflicting unpublished reports on the longevity of quinupristin/dalfopristin activity once introduced into various broth or agar media. This study assesses the relative stability of these two streptogramin combinations and their respective
0924-8579/00/$ - $20 © 2000 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. PII: S0924-8579(00)00180-1
S.A. Marshall et al. / International Journal of Antimicrobial Agents 15 (2000) 291–297
292
P-I and P-II components under various storage conditions and with several types of medium.
2. Materials and methods
pristin were evaluated individually as well as in combination. RPR 106 972 consists of RPR 106 950 (P-I) and RPR 112 808 (P-II), also tested individually and in combination. All compounds evaluated in this study were obtained from Rhoˆne–Poulenc Rorer (Collegeville, PA).
2.1. Antimicrobial agents 2.2. Strains Quinupristin/dalfopristin consists of RP 57 669 (quinupristin; P-I) and RP 54 476 (dalfopristin; P-II) mixed in a ratio of 30:70, respectively. Quinupristin and dalfo-
Test organisms for dilution stability and storage tests were four American Type Culture Collection (ATCC)
Table 1 Modal MICs of triplicate broth microdilution tests from pre-incubated trays containing streptogramin combinations and their pristinamycin components Organism
S. aureus ATCC 29 213
102-1151 (mecA)
E. faecalis ATCC 29 212
E. faecium E99-5 (6anA)
H. influenzae ATCC 49 247 S. pneumoniae ATCC 49 619 7-2806B S. mitis 2526A-1 2526A-2
a
Antimicrobiala
Modal MIC (mg/l) by pre-incubation time 0h
12 h
18 h
24 h
48 h
72 h
RP 59 500 P-I P-II RPR 106 972 P-I P-II RP 59 500 P-I P-II RPR 106 972 P-I P-II
0.25 2 8 1 16 16 0.5 64 4 2 \128 8
0.5 4 8 1 16 16 1 128 8 4 \128 8
0.5 4 8 1 16 16 1 128 8 4 \128 16
0.5 4 16 1 32 32 1 128 8 4 \128 16
1 8 32 2 32 64 2 128 16 8 \128 32
2 8 64 4 32 128 4 \128 32 8 \128 64
RP 59 500 P-I P-II RPR 106 972 P-I P-II
4 8 \128 2 8 \128
4 16 \128 2 8 \128
8 16 \128 2 8 \128
8 16 \128 2 8 \128
16 32 \128 4 8 \128
16 32 \128 8 8 \128
RP 59 500 P-I P-II RPR 106 972 P-I P-II
0.5 128 8 0.5 \128 8
1 128 8 0.5 \128 8
1 128 8 0.5 \128 16
1 128 8 0.5 \128 16
2 \128 16 1 \128 32
2 \128 32 2 \128 64
16 8
32 16
1 0.25 32 4
1 0.25 32 4
16 8 8 2
32 8 16 4
RP 59 500 RPR 106 972
8 2
8 2
RP 59 500 RPR 106 972 RP 59 500 RPR 106 972
0.25 50.12 8 2
0.25 50.12 8 2
RP 59 500 RPR 106 972 RP 59 500 RPR 106 972
8 4 2 1
8 4 4 1
8 4 0.5 0.25 16 2 8 4 4 1
8 4 0.5 0.25 16 2 8 4 4 1
RP 59 500 is quinupristin/dalfopristin; P-I and P-II are pristinamycin components streptogramin B and A, respectively.
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Fig. 1. Histogram of mean activity loss of quinupristin/dalfopristin and RPR 106 972 from all broth microdilution stability tests listed by pre-incubation time (35°C). Mean activity loss from all agar dilution stability tests are represented by closed (quinupristin/dalfopristin) and broken (RPR 106 972) lines.
Fig. 2. Histogram of mean activity loss of quinupristin/dalfopristin and pristinimycin components from all broth microdilution stability tests by pre-incubation time.
strains and five stock surveillance clinical isolates (University of Iowa College of Medicine, Iowa City, IA). The control strains were Enterococcus faecalis ATCC 29 212, Staphylococcus aureus ATCC 29 213, Haemophilus influenzae ATCC 49 247 and Streptococcus pneumoniae ATCC 49 619. Clinical isolates were MRSA (mec A-positive, strain c 102-1151), a VRE (6an Apositive; strain cE99-5) and three streptococcal strains (7-2806B, 2526A-1, 2526A-2) with elevated quinupristin/dalfopristin minimum inhibitory concentrations (MICs). Test organisms for agar-based bioassays included S. epidermidis HBD 523 with an elevated MIC to P-I components alone, and S. aureus HBD 511 with an elevated MIC to P-II, both obtained from Rhoˆne – Poulenc Rorer.
2.3. Stability tests Trays containing serial two-fold dilutions of quinupristin/dalfopristin and RPR 106 972 (128–0.06 mg/l) in cation-adjusted Mueller–Hinton broth (MHB), haemophilus test media, brain–heart infusion broth and MHB supplemented with 3–5% lysed horse blood broth were manufactured by PML Microbiologicals (Wilsonville, OR) and stored at −70°C until used. Additional trays containing the individual P-I and P-II streptogramin components in MHB were also utilized (PML Microbiologicals). Agar plate series containing serial two-fold dilutions of quinupristin/dalfopristin and RPR 106 972 (128–0.06 mg/ml) in Mueller–Hinton agar were freshly prepared. Trays and agar plates were
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pre-incubated at 35°C in an ambient air for 72, 48, 24, 18, 12, and 0 h prior to being inoculated (in triplicate) with organisms according to reference testing method recommendations of the National Committee for Clinical Laboratory Standards (NCCLS) [11]. To assess the effect of various temperatures on streptogramin stability in media, additional broth microdilution trays were stored at 4 and 25°C for 48 h and then inoculated with selected test organisms. To evaluate the effect of media types on P-I and P-II component stability, 20 ml aliquots were taken from inoculated (S. aureus ATCC 29 213) and uninoculated pre-incubated broth microdilution tray wells containing 128, 32, and 8 mg/l of each streptogramin combination and each media type tested. Aliquots were absorbed on to blank sterile 6-mm paper disks (BD Microbiologic Systems, Cockeysville, MD). Disks were then applied to 150 mm Mueller – Hinton agar plates inoculated with bioassay organisms (HBD 511 and HBD 523) and incubated in accordance with NCCLS [12] disk diffusion method recommendations. By using the disk bioassay method, we addressed the effects of media type on the drug activity by eliminating the effect of media type on the test organism growth rate. At the same time, by using P-I or P-IIresistant test organisms, one can measure the effect of media types on the components individually. Only the inhibition zone sizes from disks containing drug concentrations of 128 mg/l are reported here.
3. Results
3.1. Broth microdilution and agar dilution stability tests Overall, both streptogramin combinations showed a
steady decline (two- to four-fold) in activity from the baseline MIC over all pre-incubation times (Table 1). An increase in MIC of all tests compared to baseline (measured as a percent decline in activity), revealed that RPR 106 972 was slightly more stable than quinupristin/dalfopristin for each time point tested (Fig. 1). Overall mean activity loss (%) at each time interval was 24%/12 h, 41%/18 h, 43%/24 h, 69%/48 h, 79%/72 h for quinupristin/dalfopristin, and 6%/12 h, 19%/18 h, 19%/24 h, 56%/48 h, 71%/72 h for RPR 106 972. Activity loss was generally first observed before 24 h for quinupristin/dalfopristin and after 24 h for RPR 106 972. Agar dilution stability tests showed declining stability trends similar to broth microdilution stability tests for both streptogramin combinations. P-I components were generally more stable than P-II components (Table 1), although analysis was limited due to off-scale dilution results. MICs generally increased two- to three-fold (P-I) or threeto four-fold (P-II) from baseline MIC over pre-incubation intervals (Fig. 2). RPR 106 972 P-I component stability was consistently higher than P-II, while quinupristin/dalfopristin P-I component stability was less than P-II prior to 24 h pre-incubation and greater than P-II after 24 h pre-incubation (Fig. 3). Notably, a two-fold increase in the baseline MIC was observed for most broth microdilution tests grown in blood product containing media compared to MHB alone (data not shown). Storage of trays at refrigerator (4°C) and room (25°C) temperatures for 48 h had a similar effect on RPR 106 972 and RP 59 500 potency, but greater stability than that observed at 35°C. Two-fold increases in MICs were generally noted after both 4 and 25°C storage (Table 2).
Fig. 3. Historgram of mean activity loss of RPR 106972 and pristinimycin components from all broth microdilution stability tests by pre-incubation time.
S.A. Marshall et al. / International Journal of Antimicrobial Agents 15 (2000) 291–297
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Table 2 Modal MICs of streptogramin combinations and components from triplicate broth microdilution tests pre-incubated under various storage conditions for 48 h Organism
S. aureus ATCC 29 213
102-1151 (mecA)
E. faecalis ATCC 29 212
E. faecium E99-5 (6anA)
H. influenzae ATCC 49 247 S. pneumoniae ATCC 49 619
a b
Antimicrobiala
Modal MIC (mg/l) by 48-h pre-incubation condition −70°Cb
4°C
25°C
35°C
RP 59 500 P-I P-II RPR 106 972 P-I P-II RP 59 500 P-I P-II RPR 106 972 P-I P-II
0.12 1 4 0.25 8 4 0.25 64 2 1 \128 4
0.25 1 4 0.5 8 8 0.5 64 4 2 \128 8
0.25 2 8 0.5 8 16 0.5 64 4 2 \128 16
1 8 32 2 32 64 2 128 16 8 \128 32
RP 59 500 P-I P-II RPR 106 972 P-I P-II
4 8 \128 2 4 \128
4 8 \128 2 4 \128
4 8 \128 2 4 \128
16 32 \128 4 8 \128
RP 59 500 P-I P-II RPR 106 972 P-I P-II
0.5 128 4 0.25 \128 4
0.5 128 4 0.25 \128 8
0.5 128 8 0.5 \128 8
2 \128 16 1 \128 32
RP 59 500 RPR 106 972
4 2
4 2
8 4
RP 59 500 RPR 106 972
0.25 0.25
0.25 0.25
0.5 0.25
16 8 1 0.25
RP 59 500 is quinupristin/dalfopristin; P-I and P-II are pristinamycin components streptogramin B and A, respectively. Baseline MIC.
3.2. Disk bioassay stability tests
4. Conclusion
Disk bioassays also resulted in comparable patterns for component stability, thus showing a greater overall reduction in zone diameter with the P-II-susceptible test strain (Table 3). Decreases of zones were observed to be similar for both streptogramin combinations. The presence of an organism (S. aureus ATCC 29 213) in the drug/media assay had no additional effects on stability results (data not shown). The effect of medium and supplements on the streptogramin stability revealed variable results (Table 3). Zone size decreases for MHB media type was only significant (\ 3 mm) for the P-II-sensitive test strain when examining haemophilus test medium test results after 48 h pre-incubation for RPR 106 972 and after 72 h pre-incubation for quinupristin/dalfopristin.
Quinupristin/dalfopristin and RPR 106 972 streptogramin combinations were relatively unstable in unfrozen solutions or media with MICs increasing by approximately two-fold or greater per day. RPR 106 972 [10] was slightly more stable than quinupristin/ dalfopristin and the P-II components were usually more stable than P-I components. The effect of media type on streptogramin stability was minimal however, haemophilus test medium appeared most detrimental to drug stability than other media tested especially after 48 h. Also, a two-fold increase in baseline broth microdilution MIC was detected for organisms tested in blood supplemented MHB, an observation consistent with previous studies [13,14].
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296
Table 3 Effect of media type from pre-incubated broth microdilution trays on zone sizes of P-I-sensitive and P-II-sensitive staphylococcal bioassays Organism
Antimicrobiala and mediab
S. aureus HBD 511 (P-I-susceptible) RP 59 500 MHB LHB BHI HTM RPR 106 972 MHB LHB BHI HTM S. epidermidis HBD 523 (P-II-susceptible) RP 59 500 MHB LHB BHI HTM RPR 106 972 MHB LHB BHI HTM
Variation from Mueller–Hinton bioassay zone size (mm) by pre-incubation time 0h
12 h
18 h
24 h
48 h
72 h
16 17 18 16
16 16 16 16
15 14 15 15
15 14 14 14
13 11 11 11
11 9 9 8
19 18 20 18
17 18 17 17
17 17 17 17
16 17 17 16
14 14 15 13
13 12 13 10
23 23 24 22
21 22 21 21
21 20 20 20
21 20 20 19
18 17 16 17
16 14 13 10
22 22 23 21
20 21 20 20
18 18 19 17
18 19 19 16
16 16 16 12
14 12 11 6
a
RP 59 500 is quinupristin/dalfopristin. Mueller–Hinton broth (MHB). Mueller–Hinton broth supplemented with 5% lysed horse blood (LHB). Brain–heart infusion broth (BHI). Haemophilus test media broth (HTM). b
Unfrozen reagents containing streptogramins must be used immediately or discarded. Only frozen (− 20°C or below; −70°C preferred) storage conditions reliably maintained streptogramin potency in various susceptibility testing reagents (agar or broth), including those recommended by the NCCLS [11,12]. Special care must be exercised in testing these complex, combination antimicrobials in clinical laboratories including strict application of recommended quality control strain tests [11,12].
Acknowledgements The co-authors wish to thank the following persons for significant assistance in performing this experiment or preparing the publication: M.E. Erwin, D.J. Biedenbach, and K.L. Meyer. The research was made possible by a grant from Rhone – Poulenc Rorer.
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