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Influence of polysorbate-80 when determining the tigecycline MIC by the reference method
Diagnostic Microbiology and Infectious Disease 58 (2007) 145 – 146 www.elsevier.com/locate/diagmicrobio
Letter to the editor Influence of polysorbate-80 when determining the tigecycline MIC by the reference method Sir, In the development of several new antimicrobial agents, the use of appropriate initial solvents and surfactant compounds has been determined to be critical for correct determination of potency (Anderegg et al., 2003; Rennie et al., 2007). Polysorbate-80 (also known as Tween-80), a surfactant, has been used in broth microdilution testing at concentrations of 0.02% to 0.002% for decades to assist dispersion of drug and bacterial cells, enhance reproducibility of end point results, and prevent antimicrobial binding to various plastics used in inoculum delivery devices or in microdilution trays. The transfer of polysorbate-80 was generally via inoculum broths or water blank solutions into microdilution panel wells. Because of limited information that has been generated on the influence of surfactants on antimicrobials other than dalbavancin or similar glycopeptides (Anderegg et al., 2003; Rennie et al., 2007), we report the impact of polysorbate-80 on the tigecycline MIC values produced by reference methods of the Clinical and Laboratory Standards Institute (CLSI) using 2 glycopeptides (teicoplanin and vancomycin) as control agents (CLSI, 2006a, 2006b). Tigecycline and the 2 comparison agents were obtained from Wyeth Pharmaceuticals, Collegeville, PA, and TREK Diagnostics, Cleveland, OH, respectively. The agents were dispersed into broth microdilution panels and frozen at 70 8C until used. A total of 12 organisms (4 Staphylococcus aureus, 3 coagulase-negative staphylococci, 1 Pseudomonas aeruginosa, and 1 Escherichia coli) were tested for 9 replicates (3 tests daily 3 days) in each participating laboratory (18 replicates per strain, or 216 total comparison MIC values). Each strain was tested without surfactant in the inoculum water blank (5 105 CFU/mL cells only) and with a 0.002% final concentration of polysorbate-80. A 2nd experiment was performed by only 1 participant and used 231 recent clinical strains from 5 major Gramnegative pathogen groups. These strains included Acinetobacter spp. (38), Burkholderia spp. (16), Enterobacteriaceae (71, 12 species), P. aeruginosa (89), and Stenotrophomonas maltophilia (17). Each organism was tested with and without polysorbate-80 (0.002%) by the reference broth 0732-8893/$ – see front matter D 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2006.11.019
microdilution method (CLSI, 2006a). All concurrently tested quality control (QC) results were within published CLSI (2006b) ranges. Table 1 illustrates the excellent intra- and interlaboratory agreement, as well as the minimal differences in tigecycline MIC results when a surfactant was added to the testing broth. A total of 98.6% of results were within the F2-fold variation of the MIC methods. Only 3 results for 1 coagulase-negative staphylococci (CoNS) strain varied by 2 log2 dilution steps occurring in triplicate results from 1 laboratory. Minimal replicates of Gram-negative bacilli (2 QC strains) were tested in this 1st experiment. Table 2 addresses results from testing 231 Gram-negative organisms (16 species or genus groups) using polysorbate-80 to determine tigecycline MIC values and comparing those results with surfactant-free cation-adjusted Mueller–Hinton broth MIC results. Again, polysorbate-80 had little effect with 99.1% of MIC results within the acceptable F1 log2 dilution range. A slight trend toward lower tigecycline MIC values with polysorbate-80 was noted with P. aeruginosa strains (Table 2), but S. maltophilia strains had slightly higher tigecycline MIC results when using the surfactant. A total of 61.0% of tigecycline MICs were identical. The results of both described investigations clearly show no significant impact of polysorbate-80 on the tigecycline MIC determinations. Also, the control results for vancomyTable 1 Comparison of tigecycline MIC results obtained with 0.002% polysorbate80 (P-80) in the cation-adjusted Mueller–Hinton broth to those MIC results produced without added surfactant (12 strains, 9 replicates per strain in 2 participant sites; 216 total tests) Organism group (no. tested)
Participant site
Tigecycline MIC variation (log2 dilutions) with P-80
S. aureus (4)
A B A B A B A B A B A, B
0 0 0 0 0 0 0 0 0 0 0
2
CoNS (3)b Enterococci (3) P. aeruginosa (1) E. coli (1) Total (12) a b
1 3 0 1 0 5 0 0 1 0 0 10a
0
+1
+2
33 36 16 23 21 27 6 8 5 7 182a
0 0 7 4 1 0 3 0 4 2 21a
0 0 3 0 0 0 0 0 0 0 3
98.6% F 1 log2 dilution step; 84.3% of results were identical. CoNS = coagulase-negative staphylococci.
146
Letter to the editor / Diagnostic Microbiology and Infectious Disease 58 (2007) 145 – 146
Cindy C. Knapp TREK Diagnostics, Cleveland OH 44131, USA
Table 2 Tigecycline MIC results tested with and without polysorbate-80 (P-80, 0.002%) for 231 Gram-negative organismsa Organism group (no. tested)
Tigecycline MIC variation (log2 dilutions) with P-80 2
Acinetobacter spp. (38) Burkholderia spp. (16) Enterobacteriaceae (71, 12 spp.) P. aeruginosa (89) S. maltophilia (17) Total (231) a b
0 0 0 0 0 0
1 1 2 13 43 2 61b
0
+1
+2
22 12 53 46 8 141b
14 2 4 0 7 27b
1 0 1 0 0 2
All tests by participant A only. 99.1% F 1 log2 dilution step; 61.0% of results were identical.
cin and teicoplanin with Gram-positive clinical and QC strains used in both investigations similarly failed to identify a significant affect of polysorbate-80 on their MIC results. All control drug results were within F1 log2 dilution step of the no-surfactant MIC, and identical MIC values were observed for 85.6% and 59.4% of vancomycin and teicoplanin tests, respectively. Tigecycline, the 1st glycylcycline used in clinical practice, has a broad spectrum of activity against Grampositive and Gram-negative pathogens (Bradford, 2004; Jones et al., 2006; Nathwani, 2005; Sader et al., 2005), and indications for treatment of serious hospital-associated infections have been established by regulatory agencies (Tygacil Package Insert, 2005). MIC breakpoints for tigecycline susceptibility have been published for indicated species (Tygacil Package Insert, 2005) and Acinetobacter spp. (Jones et al., 2007). These tigecycline MIC breakpoints can be applied with confidence only when the broth medium has been controlled for excessive oxygenation (fresh media, b 12 h from preparation) (Petersen and Bradford, 2005), but testing with or without the polysorbate-80 surfactant would be acceptable. Ronald N. Jones JMI Laboratories, North Liberty, IA 52317, USA E-mail address: [email protected]
Michael J. Dowzicky Wyeth Pharmaceuticals, Collegeville PA 19426, USA
References Anderegg TR, Biedenbach DJ, Jones RN (2003) Initial quality control evaluations for susceptibility testing of dalbavancin (BI397), an investigational glycopeptide with potent Gram-positive activity. J Clin Microbiol 41:2795 – 2796. Bradford, PA (2004) Tigecycline: a first in class glycylcycline. Clin Microbiol Newsl 26:163 – 168. CLSI (2006a) Clinical and Laboratory Standards Institute. M7-A7, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard. 7th ed. Wayne (PA)7 CLSI. CLSI (2006b) Clinical and Laboratory Standards Institute. M100-S16, Performance Standards for Antimicrobial Susceptibility Testing; Sixteenth Informational Supplement. Wayne (PA)7 CLSI. Jones RN, Stilwell MG, Sader HS, Fritsche TR Goldstein, BP (2006) Spectrum and potency of dalbavancin tested against 3,322 Grampositive cocci isolated in the United States Surveillance Program (2004). Diagn Microbiol Infect Dis 54:149 – 153. Jones RN, Ferraro MJ, Reller LB, Schreckenberger PC, Swenson JM, Sader, HS (2007) Multicenter studies of tigecycline disk diffusion susceptibility results when testing Acinetobacter spp. J Clin Microbiol 45:227–230. Nathwani, D (2005) Tigecycline: clinical evidence and formulary positioning. Int J Antimicrob Agents 25:185 – 192. Petersen PJ, Bradford, PA (2005) Effect of medium age and supplementation with the biocatalytic oxygen-reducing reagent oxyrase on in vitro activities of tigecycline against recent clinical isolates. Antimicrob Agents Chemother 49:3910 – 3918. Rennie RP, Koethe L, Jones RN, Fritsche TR, Knapp CC, Killian SB, Goldstein, BP (2007) Factors influencing broth microdilution antimicrobial susceptibility test results of dalbavancin, a new glycopeptide agent. J Clin Microbiol (in press). Sader HS, Jones RN, Stilwell MG, Dowzicky MJ, Fritsche, TR (2005) Tigecycline activity tested against 26,474 bloodstream infection isolates: a collection from 6 continents. Diagn Microbiol Infect Dis 52:181 – 186. Tygacil Package Insert (2005) Philadelphia (PA)7 Wyeth Pharmaceuticals Inc (June 2005).
Letter to the editor Influence of polysorbate-80 when determining the tigecycline MIC by the reference method Sir, In the development of several new antimicrobial agents, the use of appropriate initial solvents and surfactant compounds has been determined to be critical for correct determination of potency (Anderegg et al., 2003; Rennie et al., 2007). Polysorbate-80 (also known as Tween-80), a surfactant, has been used in broth microdilution testing at concentrations of 0.02% to 0.002% for decades to assist dispersion of drug and bacterial cells, enhance reproducibility of end point results, and prevent antimicrobial binding to various plastics used in inoculum delivery devices or in microdilution trays. The transfer of polysorbate-80 was generally via inoculum broths or water blank solutions into microdilution panel wells. Because of limited information that has been generated on the influence of surfactants on antimicrobials other than dalbavancin or similar glycopeptides (Anderegg et al., 2003; Rennie et al., 2007), we report the impact of polysorbate-80 on the tigecycline MIC values produced by reference methods of the Clinical and Laboratory Standards Institute (CLSI) using 2 glycopeptides (teicoplanin and vancomycin) as control agents (CLSI, 2006a, 2006b). Tigecycline and the 2 comparison agents were obtained from Wyeth Pharmaceuticals, Collegeville, PA, and TREK Diagnostics, Cleveland, OH, respectively. The agents were dispersed into broth microdilution panels and frozen at 70 8C until used. A total of 12 organisms (4 Staphylococcus aureus, 3 coagulase-negative staphylococci, 1 Pseudomonas aeruginosa, and 1 Escherichia coli) were tested for 9 replicates (3 tests daily 3 days) in each participating laboratory (18 replicates per strain, or 216 total comparison MIC values). Each strain was tested without surfactant in the inoculum water blank (5 105 CFU/mL cells only) and with a 0.002% final concentration of polysorbate-80. A 2nd experiment was performed by only 1 participant and used 231 recent clinical strains from 5 major Gramnegative pathogen groups. These strains included Acinetobacter spp. (38), Burkholderia spp. (16), Enterobacteriaceae (71, 12 species), P. aeruginosa (89), and Stenotrophomonas maltophilia (17). Each organism was tested with and without polysorbate-80 (0.002%) by the reference broth 0732-8893/$ – see front matter D 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2006.11.019
microdilution method (CLSI, 2006a). All concurrently tested quality control (QC) results were within published CLSI (2006b) ranges. Table 1 illustrates the excellent intra- and interlaboratory agreement, as well as the minimal differences in tigecycline MIC results when a surfactant was added to the testing broth. A total of 98.6% of results were within the F2-fold variation of the MIC methods. Only 3 results for 1 coagulase-negative staphylococci (CoNS) strain varied by 2 log2 dilution steps occurring in triplicate results from 1 laboratory. Minimal replicates of Gram-negative bacilli (2 QC strains) were tested in this 1st experiment. Table 2 addresses results from testing 231 Gram-negative organisms (16 species or genus groups) using polysorbate-80 to determine tigecycline MIC values and comparing those results with surfactant-free cation-adjusted Mueller–Hinton broth MIC results. Again, polysorbate-80 had little effect with 99.1% of MIC results within the acceptable F1 log2 dilution range. A slight trend toward lower tigecycline MIC values with polysorbate-80 was noted with P. aeruginosa strains (Table 2), but S. maltophilia strains had slightly higher tigecycline MIC results when using the surfactant. A total of 61.0% of tigecycline MICs were identical. The results of both described investigations clearly show no significant impact of polysorbate-80 on the tigecycline MIC determinations. Also, the control results for vancomyTable 1 Comparison of tigecycline MIC results obtained with 0.002% polysorbate80 (P-80) in the cation-adjusted Mueller–Hinton broth to those MIC results produced without added surfactant (12 strains, 9 replicates per strain in 2 participant sites; 216 total tests) Organism group (no. tested)
Participant site
Tigecycline MIC variation (log2 dilutions) with P-80
S. aureus (4)
A B A B A B A B A B A, B
0 0 0 0 0 0 0 0 0 0 0
2
CoNS (3)b Enterococci (3) P. aeruginosa (1) E. coli (1) Total (12) a b
1 3 0 1 0 5 0 0 1 0 0 10a
0
+1
+2
33 36 16 23 21 27 6 8 5 7 182a
0 0 7 4 1 0 3 0 4 2 21a
0 0 3 0 0 0 0 0 0 0 3
98.6% F 1 log2 dilution step; 84.3% of results were identical. CoNS = coagulase-negative staphylococci.
146
Letter to the editor / Diagnostic Microbiology and Infectious Disease 58 (2007) 145 – 146
Cindy C. Knapp TREK Diagnostics, Cleveland OH 44131, USA
Table 2 Tigecycline MIC results tested with and without polysorbate-80 (P-80, 0.002%) for 231 Gram-negative organismsa Organism group (no. tested)
Tigecycline MIC variation (log2 dilutions) with P-80 2
Acinetobacter spp. (38) Burkholderia spp. (16) Enterobacteriaceae (71, 12 spp.) P. aeruginosa (89) S. maltophilia (17) Total (231) a b
0 0 0 0 0 0
1 1 2 13 43 2 61b
0
+1
+2
22 12 53 46 8 141b
14 2 4 0 7 27b
1 0 1 0 0 2
All tests by participant A only. 99.1% F 1 log2 dilution step; 61.0% of results were identical.
cin and teicoplanin with Gram-positive clinical and QC strains used in both investigations similarly failed to identify a significant affect of polysorbate-80 on their MIC results. All control drug results were within F1 log2 dilution step of the no-surfactant MIC, and identical MIC values were observed for 85.6% and 59.4% of vancomycin and teicoplanin tests, respectively. Tigecycline, the 1st glycylcycline used in clinical practice, has a broad spectrum of activity against Grampositive and Gram-negative pathogens (Bradford, 2004; Jones et al., 2006; Nathwani, 2005; Sader et al., 2005), and indications for treatment of serious hospital-associated infections have been established by regulatory agencies (Tygacil Package Insert, 2005). MIC breakpoints for tigecycline susceptibility have been published for indicated species (Tygacil Package Insert, 2005) and Acinetobacter spp. (Jones et al., 2007). These tigecycline MIC breakpoints can be applied with confidence only when the broth medium has been controlled for excessive oxygenation (fresh media, b 12 h from preparation) (Petersen and Bradford, 2005), but testing with or without the polysorbate-80 surfactant would be acceptable. Ronald N. Jones JMI Laboratories, North Liberty, IA 52317, USA E-mail address: [email protected]
Michael J. Dowzicky Wyeth Pharmaceuticals, Collegeville PA 19426, USA
References Anderegg TR, Biedenbach DJ, Jones RN (2003) Initial quality control evaluations for susceptibility testing of dalbavancin (BI397), an investigational glycopeptide with potent Gram-positive activity. J Clin Microbiol 41:2795 – 2796. Bradford, PA (2004) Tigecycline: a first in class glycylcycline. Clin Microbiol Newsl 26:163 – 168. CLSI (2006a) Clinical and Laboratory Standards Institute. M7-A7, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard. 7th ed. Wayne (PA)7 CLSI. CLSI (2006b) Clinical and Laboratory Standards Institute. M100-S16, Performance Standards for Antimicrobial Susceptibility Testing; Sixteenth Informational Supplement. Wayne (PA)7 CLSI. Jones RN, Stilwell MG, Sader HS, Fritsche TR Goldstein, BP (2006) Spectrum and potency of dalbavancin tested against 3,322 Grampositive cocci isolated in the United States Surveillance Program (2004). Diagn Microbiol Infect Dis 54:149 – 153. Jones RN, Ferraro MJ, Reller LB, Schreckenberger PC, Swenson JM, Sader, HS (2007) Multicenter studies of tigecycline disk diffusion susceptibility results when testing Acinetobacter spp. J Clin Microbiol 45:227–230. Nathwani, D (2005) Tigecycline: clinical evidence and formulary positioning. Int J Antimicrob Agents 25:185 – 192. Petersen PJ, Bradford, PA (2005) Effect of medium age and supplementation with the biocatalytic oxygen-reducing reagent oxyrase on in vitro activities of tigecycline against recent clinical isolates. Antimicrob Agents Chemother 49:3910 – 3918. Rennie RP, Koethe L, Jones RN, Fritsche TR, Knapp CC, Killian SB, Goldstein, BP (2007) Factors influencing broth microdilution antimicrobial susceptibility test results of dalbavancin, a new glycopeptide agent. J Clin Microbiol (in press). Sader HS, Jones RN, Stilwell MG, Dowzicky MJ, Fritsche, TR (2005) Tigecycline activity tested against 26,474 bloodstream infection isolates: a collection from 6 continents. Diagn Microbiol Infect Dis 52:181 – 186. Tygacil Package Insert (2005) Philadelphia (PA)7 Wyeth Pharmaceuticals Inc (June 2005).