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Journal of Clinical Microbiology, March 2004, p. 1386-1387, Vol. 42, No. 3
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.3.1386-1387.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

LETTER TO THE EDITOR

Quality Control Guidelines for MIC Susceptibility Testing of Omiganan Pentahydrochloride (MBI 226), a Novel Antimicrobial Peptide

Top Letter References

 
Antimicrobial cationic peptides are thought to be an important self-defense mechanism against infectious agents for plants, animals, and bacterial species (2). While cationic peptides vary in their mechanisms of action, most are reactive with bacterial cytoplasmic membranes (2). Omiganan pentahydrochloride (formerly MBI 226), a novel topical mammal-derived antimicrobial peptide (sequence: ILRWPWWPWRRK-amide),acts by disrupting the cytoplasmic membranes of both gram-positive and -negative bacteria, resulting in depolarization and cell death (D. Dugourd, C. Pasetka, D. Erfle, E. Rubinchik, K. Lee, and H. D. Friedland, Abstr. 102nd Gen. Meet. Am. Soc. Microbiol., abstr. A-47, 2002). Omiganan also demonstrated a dose-dependent inhibition of DNA, RNA, and protein synthesis at the macromolecular level, providing another mechanism of action (D. Dugourd, C. Pasetka, D. Erfle, E. Rubinchik, M. Guarna, and P. McNichol, Abstr. 102nd Gen. Meet. Am. Soc. Microbiol., abstr. A-46, 2002).

Omiganan is currently in phase III clinical trials as a prophylactic topical agent to minimize catheter-related bloodstream infections (D. J. Hoban, E. Witwicki, G. G. Zhanel, L. Palatnick, H. D. Friedland, and J. McBride, Abstr. 42nd Intersci. Conf. Antimicrob. Agents Chemother., abstr. E-1647, 2002). The spectrum of activity for omiganan encompasses gram-positive bacteria and yeast. To determine the accurate assessment of the in vitro testing profiles of clinical isolates, quality control (QC) guidelines for omiganan are required (3).

A seven-laboratory consortium participated in a MIC QC study for omiganan by following the National Committee for Clinical Laboratory Standards (NCCLS)-recommended guidelines (3), test methods (4, 5), and interpretive criteria (6) and using common American Type Culture Collection (ATCC) QC strains. The reference frozen-form broth microdilution panels were prepared by TREK Diagnostics (Cleveland, Ohio) and contained three lots of cation-adjusted Mueller-Hinton broth (CA-MHB) (BBL, Sparks, Md.; Difco, Detroit, Mich.; Oxoid, Hampshire, England) supplemented or not with 5% lysed horse blood, three lots of cation-unadjusted Mueller-Hinton broth (MHB) (Oxoid [two lots] and Difco) supplemented or not with 5% lysed horse blood, and three lots of RPMI 1640 broth (Sigma [two lots], St. Louis, Mo.; Irvine Scientific, Santa Ana, Calif.). All panels were stored at -70°C until used. The omiganan standard powder was obtained from Micrologix Biotech, Inc. (Vancouver, Canada). The internal QC agents used in the study were levofloxacin (Ortho-McNeil, Rahway, N.J.) and vancomycin (Sigma Chemical) for the bacterial QC strains and flucytosine and amphotericin B from Sigma Chemical for the yeast QC strains. Each laboratory tested the following seven ATCC QC strains: Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, S. pneumoniae ATCC 49619, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Candida krusei ATCC 6258, and Candida parapsilosis ATCC 22019 over a 10-day period. In summary, each organism was tested once daily over 10 days in three medium lots by seven laboratories generating 210 (10 x 3 x 7) MIC results per QC strain.

Concurrent testing using vancomycin as the internal control for S. aureus ATCC 29213, E. faecalis ATCC 29212, and S. pneumoniae ATCC 49619, using levofloxacin as the internal control for E. coli ATCC 25922 and P. aeruginosa ATCC 27853, and using flucytosine and amphotericin B as the internal controls for C. krusei ATCC 6258 and C. parapsilosis ATCC 22019 showed that 99.8% of all participant MIC results (1,399 values) were within published NCCLS guidelines (5). Inoculum colony counts were performed from the broth microdilution panels by subculturing in a quantitative manner onto drug-free plates. The inoculum counts for the bacterial QC testing ranged from 1.0 x 10⁵ to 5.3 x 10⁵ CFU/ml (average inoculum, 3.1 x 10⁵ CFU/ml), and those for the yeast QC tests ranged from 5.5 x 10² to 4.5 x 10³ CFU/ml (average inoculum, 2.1 x 10³ CFU/ml).

Proposed QC ranges were optimized to encompass 95% of all results as recommended by the NCCLS M23-A2 guideline (3). MIC results for each tested antimicrobial agent were tabulated and compared by intra- and interlaboratory analysis and by medium lots. The bacterial QC strains were tested in CA-MHB and MHB, and these values were compared.

The results for E. faecalis ATCC 29212 did not show any shift due to the medium divalent cation differences (CA-MHB versus MHB). The modal value for both medium types was 64 µg/ml, with 75.5% of the total omiganan MIC results in CA-MHB and 90.0% of the total results in MHB achieving this value. The proposed omiganan MIC QC ranges for both CA-MHB and MHB were 32 to 128 µg/ml and encompassed 100% of all participant results. For S. aureus ATCC 29213, 57.1% of all results using CA-MHB and 58.1% of all results using MHB were at the modal value of 16 µg/ml. However, the proposed omiganan MIC QC ranges for CA-MHB (8 to 64 µg/ml) versus MHB (4 to 32 µg/ml) varied by 1 log₂ dilution step (4 log₂ dilution ranges).

S. pneumoniae ATCC 49619 and E. coli ATCC 25922 had similar medium-specific shifts, with MICs being 1 log₂ dilution lower for the MHB than for CA-MHB. S. pneumoniae ATCC 49619 had a modal value of 64 µg/ml (75.7% of total results) in the CA-MHB versus a value of 32 µg/ml (92.4% of total results) in the MHB. Thus, the proposed 3 log₂ dilution omiganan MIC QC range saw a twofold shift for CA-MHB (32 to 128 µg/ml) versus MHB (16 to 64 µg/ml). Both ranges included all of the reported results. E. coli ATCC 25922 had a modal value of 32 µg/ml (75.7% of total results) in the CA-MHB compared to a 16 µg/ml (80.5% of total results) in the MHB. The proposed omiganan MIC QC ranges also were 1 log₂ dilution higher for CA-MHB when E. coli ATCC 25922 was used (Table 1).

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TABLE 1. Proposed MIC QC ranges for omiganan listed by medium typea

P. aeruginosa ATCC 27853 QC trials exhibited a 2-log₂-dilution difference in the modal omiganan values and proposed MIC QC ranges for CA-MHB and MHB. The modal value in CA-MHB was 128 µg/ml (86.7% of total results) compared to MICs in MHB of 32 µg/ml (60.0% of total results). The proposed omiganan MIC QC range for CA-MHB was 64 to 256 µg/ml, and that for MHB was 8 to 64 µg/ml. The cations in the media did not affect all QC strains in the same manner, so it is important to note the differences that medium selection makes for some QC strains, most notably P. aeruginosa ATCC 27853.

Table 1 also shows the distribution of omiganan MICs for the two yeast QC strains. A total of 49.5% of the results for C. parapsilosis ATCC 22019 were at the modal value of 64 µg/ml. The proposed omiganan MIC QC range of 32 to 128 µg/ml included 99.0% of all reported results. The modal value for C. krusei ATCC 6258 was 32 µg/ml (53.8% of results). The proposed omiganan MIC QC range of 16 to 64 µg/ml for C. krusei ATCC 6258 includes all reported results.

This study established QC results from a NCCLS M23-A2 (3) study design for omiganan tested by broth microdilution methods (4-6). Three log₂ dilution ranges (mode ± 1 log₂ dilution) were established for nine QC organism-medium ranges. Only on three occasions was it necessary to assign a 4-log₂-dilution range, where nearly equal numbers of omiganan MICs occurred at two adjacent dilution steps (S. aureus ATCC 29213 in CA-MHB and MHB and P. aeruginosa ATCC 27853 in MHB). As omiganan advances through phase III clinical trials and beyond, the MIC QC ranges established during this study will permit accurate laboratory susceptibility testing as an aid in assessing the value of the compound against contemporary cutaneous bacterial isolates or for detection of emerging resistances as part of local or regional epidemiology programs. The latter applications would be similar to those currently used in in vitro tests of mupirocin, another topical agent, to confirm resistances in clinically refractory strains or to select topical agents for formulary addition (1).

 
Members of the Quality Control Working Group are as follows: G. Hall (Cleveland Clinic Foundation, Cleveland, Ohio), H. S. Sader (The Jones Group/JMI Laboratories, North Liberty, Iowa), C. Knapp (TREK Diagnostics, Cleveland, Ohio), R. Rennie (University of Alberta, Edmonton, Alberta, Canada), A. Wanger (University of Texas, Houston), A. Limaye (University of Washington, Seattle), and D. Hardy (Strong Memorial Hospital, Rochester, N.Y.).

 
Members of The Quality Control Working Group are listed in the acknowledgments.

Top Letter References

 

1
1. Finlay, J. E., L. A. Miller, and J. A. Poupard. 1997. Interpretive criteria for testing susceptibility of staphylococci to mupirocin. Antimicrob. Agents Chemother. 41:1137-1139.[Abstract]
2
2. Friedrich, C. L., D. Moyles, T. J. Beveridge, and R. E. W. Hancock. 2000. Antibacterial action of structurally diverse cationic peptides on gram-positive bacteria. Antimicrob. Agents Chemother. 44:2086-2092.[Abstract/Free Full Text]
3
3. National Committee for Clinical Laboratory Standards. 2001. Development of in vitro susceptibility testing criteria and quality control parameters. Approved standard M23-A2, 2nd ed. National Committee for Clinical Laboratory Standards, Wayne, Pa.
4
4. National Committee for Clinical Laboratory Standards. 2002. Reference method for broth microdilution antifungal susceptibility testing of yeasts. Approved standard, M27-A2, 2nd ed. National Committee for Clinical Laboratory Standards, Wayne, Pa.
5
5. National Committee for Clinical Laboratory Standards. 2003. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard M7-A6, 6th ed. National Committee for Clinical Laboratory Standards, Wayne, Pa.
6
6. National Committee for Clinical Laboratory Standards. 2003. Performance standards for antimicrobial susceptibility testing. Informational supplement M100-S13, 12th ed. National Committee for Clinical Laboratory Standards, Wayne, Pa.

						Tamara R. Anderegg* Thomas R. Fritsche Ronald N. Jones The Jones Group/JMI Laboratories North Liberty, IA 52317 The Quality Control Working Group,
						* Phone: (319) 665-3370Fax: (319) 665-3371E-mail: tamara-anderegg{at}jmilabs.com

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Journal of Clinical Microbiology, March 2004, p. 1386-1387, Vol. 42, No. 3
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.3.1386-1387.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Fritsche, T. R., Rhomberg, P. R., Sader, H. S., Jones, R. N. (2008). Antimicrobial activity of omiganan pentahydrochloride tested against contemporary bacterial pathogens commonly responsible for catheter-associated infections. J Antimicrob Chemother 61: 1092-1098 [Abstract] [Full Text]  
• Fritsche, T. R., Rhomberg, P. R., Sader, H. S., Jones, R. N. (2008). Antimicrobial Activity of Omiganan Pentahydrochloride against Contemporary Fungal Pathogens Responsible for Catheter-Associated Infections. Antimicrob. Agents Chemother. 52: 1187-1189 [Abstract] [Full Text]  
• Levy, O. (2004). Antimicrobial proteins and peptides: anti-infective molecules of mammalian leukocytes. J. Leukoc. Biol. 76: 909-925 [Abstract] [Full Text]  

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