Abstract
The in vitro activity of daptomycin against 337 gram-positive European clinical isolates with known resistance genes was determined. The MIC ranges for Staphylococcus aureus, enterococci, pneunococci, and streptococci were 0.03 to 1, 0.25 to 8, 0.12 to 1, and 0.06 to 8 μg/ml, respectively. For only one streptococcus isolate and seven enterococcus isolates was the MIC 8 μg/ml.
The emergence of multiple-antibiotic-resistant gram-positive isolates continues to provide challenges. Daptomycin is a novel lipopeptide that exhibits rapid in vitro bactericidal activity against gram-positive pathogens (3, 8, 12, 13, 16, 19, 22), probably by disrupting bacterial membrane potential (1, 2, 4). The objective of this study was to determine the in vitro activities of daptomycin and other antibiotics against 337 antibiotic-resistant gram-positive clinical isolates with genetically characterized resistance determinants.
The isolates were collected between 1997 and 2000 from 23 European centers as described previously (6). No center or country provided a disproportionate number of isolates.
PCR analysis and, if necessary, DNA sequencing were used to identify the resistance determinants. In staphylococci, the mecA gene and mutations associated with quinolone resistance were determined as described previously (7, 18). The tetracycline resistance genes in Staphylococcus aureus and Streptococcus pneumoniae were detected as described by Warsa et al. (21). Fluoroquinolone resistance genes and macrolide, lincosamide, and streptogramin resistance genes in S. pneumoniae and other streptococci were detected as described earlier (9-11, 15, 18, 20). In enterococci, primers used to identify the ant(4′)-Ia gene have been described previously (17). Primers used to identify aac(6′)-Ie+aph(2") were 5′-GAACATGAATTACACGAGGG and 5′-CCATTTTCGATAAATTCCTG; the primers used for the detection of the aph(3′)-IIIa gene were 5′-AAATGACGGACAGCCGGTAT and 5′-CGATGGAGTGAAAGAGCCTG. Vancomycin resistance encoded by vanA in enterococci was determined as described previously (5).
MICs were determined by National Committee for Clinical Laboratory Standards methodology (14). Antimicrobial agents and frozen microtiter plates containing antibiotic solutions and physiologic concentrations of Ca2+ (50 μg/ml) were supplied by TREK Diagnostic Systems, Inc. (Westlake, Ohio).
For 38 mecA-positive, methicillin-resistant S. aureus isolates, the MIC range for daptomycin was 0.03 to 0.5 μg/ml (Table 1), with MICs at which 50 and 90% of the isolates tested are inhibited (MIC50 and MIC90, respectively) of 0.25 and 0.5 μg/ml, respectively. Four types of amino acid changes were detected in the quinolone resistance-determining region of grlA/gyrA of 49 S. aureus isolates. The MIC range for daptomycin against these resistant isolates was 0.06 to 0.5 μg/ml (Table 1).
TABLE 1.
MIC range and defined resistance determinants of various gram-positive pathogens
| Species | Resistance determinant(s) | n | Antibiotic | MIC range (μg/ml) |
|---|---|---|---|---|
| Staphylococcus aureus | mecA | 38 | Daptomycin | 0.03-0.5 |
| Oxacillin | >8->8 | |||
| Ser80Phe/Glu88Lysa | 14 | Daptomycin | 0.12-0.5 | |
| Ciprofloxacin | 8->8 | |||
| Ser80Phe/Ser84Leu | 13 | Daptomycin | 0.12-0.5 | |
| Ciprofloxacin | 4->8 | |||
| Ser80Tyr/Glu88Lys | 9 | Daptomycin | 0.12-0.5 | |
| Ciprofloxacin | 8->8 | |||
| Ser80Tyr/Ser84Leu | 13 | Daptomycin | 0.06-0.5 | |
| Ciprofloxacin | 8->8 | |||
| tetK | 18 | Daptomycin | 0.06-1 | |
| tetM | 18 | Daptomycin | 0.06-0.25 | |
| tetK+tetM | 7 | Daptomycin | 0.12-0.5 | |
| Enterococcus faecalis | vanA | 7 | Daptomycin | 0.5-4 |
| Teicoplanin | 32->64 | |||
| Vancomycin | 256->256 | |||
| aph(3′)IIIa | 4 | Daptomycin | 0.5-1 | |
| Gentamicin | >500->500 | |||
| aac(6′)-aph2" | 11 | Daptomycin | 0.5-2 | |
| Gentamicin | >500->500 | |||
| aac(6′)-aph2" + aph(3′)IIIa | 17 | Daptomycin | 0.5-8 | |
| Gentamicin | >500->500 | |||
| Enterococcus faecium | vanA | 27 | Daptomycin | 0.25-8 |
| Teicoplanin | 8->64 | |||
| Vancomycin | 64->256 | |||
| aph(3′)IIIa | 2 | Daptomycin | 4-4 | |
| Gentamicin | >500->500 | |||
| aac(6′)-aph2" | 5 | Daptomycin | 4-8 | |
| Gentamicin | >500->500 | |||
| aac(6′)-aph2" + aph(3′)IIIa | 9 | Daptomycin | 0.5-8 | |
| Gentamicin | >500->500 | |||
| Streptococcus pneumoniae | nc/nc/nc/ncb | 2 | Daptomycin | 0.12-0.12 |
| Levofloxacin | 1-1 | |||
| nc/nc/nc/Ile460Val | 5 | Daptomycin | 0.12-1 | |
| Levofloxacin | 1 | |||
| Ser81Phe/Ser79Phe/nc/nc | 12 | Daptomycin | 0.12-0.25 | |
| Levofloxacin | 4->8 | |||
| Ser81Tyr/Ser79Phe/nc/Ile460Val | 1 | Daptomycin | 0.12 | |
| Levofloxacin | 4 | |||
| Ser81Phe/Asp83Asn/nc/nc | 1 | Daptomycin | 0.25 | |
| Levofloxacin | 8 | |||
| Ser81Phe/Asp83Asn/nc/Ile460Val | 1 | Daptomycin | 0.06 | |
| Levofloxacin | 8 | |||
| tetM | 12 | Daptomycin | 0.12-1 | |
| ermB | 9 | Daptomycin | 0.12-0.12 | |
| Clindamycin | >1->1 | |||
| Erythromycin | >2->2/PICK> | |||
| mefE | 7 | Daptomycin | 0.12-0.25 | |
| Erythromycin | >2->2 | |||
| tetM+ermB | 7 | Daptomycin | 0.12-0.25 | |
| Erythromycin | >2->2 | |||
| tetM+mefE | 6 | Daptomycin | 0.06-0.25 | |
| Erythromycin | >2->2 | |||
| Streptococcus mitis | nc/nc/nc/ncc | 3 | Daptomycin | 0.5-1 |
| Levofloxacin | 1-1 | |||
| Erythromycin | 0.06->2 | |||
| nc/nc/Ser494Thr/nc | 5 | Daptomycin | 1-8 | |
| Levofloxacin | 1-1 | |||
| Erythromycin | 0.06->2 | |||
| Ser81Phe/Ser79Ile/Ser494Thr/nc | 1 | Daptomycin | 0.5 | |
| Levofloxacin | 4 | |||
| Erythromycin | >2 | |||
| Ser81Phe/Ser79Phe/nc/nc | 1 | Daptomycin | 2 | |
| Levofloxacin | 0.5 | |||
| Erythromycin | >2 | |||
| Ser81Tyr/Ser79Ile/nc/nc | 1 | Daptomycin | 0.25 | |
| Levofloxacin | 8 | |||
| Erythromycin | 2 | |||
| Ser81Tyr/Ser79Ile/Ser494Thr/nc | 1 | Daptomycin | 1 | |
| Levofloxacin | 2 | |||
| Erythromycin | 2 | |||
| Streptococcus sanguis | nc/nc/nc/nc | 1 | Daptomycin | 2 |
| Levofloxacin | 1 | |||
| Erythromycin | 0.03 | |||
| nc/nc/Ser494/Thrnc | 2 | Daptomycin | 1-1 | |
| Levofloxacin | 1-1 | |||
| Erythromycin | 0.03->2 | |||
| Ser81Phe/Ser79Ile/nc/nc | 1 | Daptomycin | 0.5 | |
| Levofloxacin | 1 | |||
| Erythromycin | 0.03 | |||
| Streptococcus agalactiae | ermA | 1 | Daptomycin | 0.25 |
| Clindamycin | ≤0.25 | |||
| Erythromycin | >2 | |||
| ermA+ermTR | 2 | Daptomycin | 0.25-0.5 | |
| Clindamycin | ≤0.25->1 | |||
| Erythromycin | 2->2 | |||
| ermA+ermC+ermTR | 1 | Daptomycin | 0.25 | |
| Clindamycin | >1 | |||
| Erythromycin | >2 | |||
| ermB | 3 | Daptomycin | 0.06-0.25 | |
| Clindamycin | ≤0.25->1 | |||
| Erythromycin | 1->2 | |||
| Streptococcus bovis | ermB | 11 | Daptomycin | 0.03-0.12 |
| Clindamycin | 1->1 | |||
| Erythromycin | >2->2 | |||
| ermB+ermC | 1 | Daptomycin | 0.06 | |
| Clindamycin | >1 | |||
| Erythromycin | >2 | |||
| ermB+ermTR | 1 | Daptomycin | 0.03 | |
| Clindamycin | >1 | |||
| Erythromycin | >2 | |||
| Streptococcus equisimilis | ermB+ermTR | 1 | Daptomycin | 0.06 |
| Clindamycin | >1 | |||
| Erythromycin | >2 | |||
| ermC+ermTR | 1 | Daptomycin | 0.03 | |
| Clindamycin | ≤0.25 | |||
| Erythromycin | >2 | |||
| Streptococcus intermedius | ermB+ermTR | 1 | Daptomycin | 1 |
| Clindamycin | >1 | |||
| Erythromycin | >2 | |||
| Streptococcus mitis | ermA+mefA | 1 | Daptomycin | 0.5 |
| Clindamycin | ≤0.25 | |||
| Erythromycin | 2 | |||
| ermB | 5 | Daptomycin | 0.5-2 | |
| Clindamycin | ≤0.25->1 | |||
| Erythromycin | >2->2 | |||
| mefA | 5 | Daptomycin | 0.25-1 | |
| Clindamycin | ≤0.25-≤0.25 | |||
| Erythromycin | >2->2 | |||
| Streptococcus oralis | mefA | 4 | Daptomycin | 0.5-2 |
| Clindamycin | ≤0.25-≤0.25 | |||
| Erythromycin | 2->2 | |||
| Streptococcus pyogenes | ermB | 2 | Daptomycin | 0.03-0.06 |
| Clindamycin | ≤0.25->1 | |||
| Erythromycin | 2->2 | |||
| ermTR | 2 | Daptomycin | 0.06-0.06 | |
| Clindamycin | ≤0.25-≤0.25 | |||
| Erythromycin | >2->2 | |||
| mefA | 1 | Daptomycin | 0.03 | |
| Clindamycin | ≤0.25 | |||
| Erythromycin | >2 | |||
| Streptococcus salivarius | ermB | 2 | Daptomycin | 0.12-0.12 |
| Clindamycin | 1->1 | |||
| Erythromycin | 2->2 | |||
| mefA | 1 | Daptomycin | 0.12 | |
| Clindamycin | ≤0.25 | |||
| Erythromycin | >2 |
Mutations in grlA/gyrA of S. aureus, respectively.
Mutations in gyrA/gyrB/parC/parE of S. pneumoniae, respectively. nc, no change in amino acids
Mutations in gyrA/gyrB/parC/parE of other streptococci, respectively.
A tetracycline-resistant phenotype in S. aureus was caused by the presence of either tetK (n = 18), tetM (n = 18), or both genes (n = 7). The MIC ranges of daptomycin for these isolates were 0.06 to 1, 0.06 to 0.25, and 0.12 to 0.5 μg/ml, respectively (Table 1).
Seven Enterococcus faecalis and 27 Enterococcus faecium vanA-positive isolates were tested for sensitivity to a number of antibiotics (Table 1). The MICs for daptomycin ranged from 0.5 to 4 μg/ml for E. faecalis and from 0.25 to 8 μg/ml for E. faecium; the MIC50s for these species were 0.5 and 4 μg/ml, respectively; and the MIC90 was 4 μg/ml for both species.
A total of 32 E. faecalis and 16 E. faecium isolates exhibited high-level gentamicin resistance. The daptomycin MICs ranged from 0.5 to 8 μg/ml for both species (Table 1); the MIC50s for E. faecalis and E. faecium were 2 and 4 μg/ml, respectively; and the MIC90s were 2 and 8 μg/ml, respectively.
In this study, daptomycin MICs for E. faecium appeared slightly higher than those in other published data. A recent study reported MIC50s and MIC90s of 2 μg/ml for 25 E. faecium isolates (both vancomycin resistant and susceptible) at physiologic calcium concentrations (50 μg/ml) (22). One explanation for these results may be the limited number of isolates tested in our study.
The MICs of daptomycin and other antibiotics were determined for pneumococci with known resistance mechanisms for fluoroquinolones, tetracycline, clindamycin, and erythromycin. Two fluoroquinolone-susceptible isolates lacking amino acid changes in GyrA, ParC, GyrB, or ParE were tested, and for both, the daptomycin MIC was 0.12 μg/ml (Table 1).
Twenty isolates, categorized into four different groups according to amino acid changes associated with quinolone resistance, were tested for activity against a number of antibiotics (Table 1). The daptomycin MICs for the 20 isolates ranged from 0.12 to 1 μg/ml, and the MIC50s and MIC90s were 0.12 and 0.25 μg/ml, respectively.
The S. pneumoniae isolates were divided into five groups based on the presence of tetracycline and erythromycin/clindamycin resistance determinants (Table 1). The lowest daptomycin MIC obtained in four of the five groups of isolates was 0.12 μg/ml; the tetM mefE group had the lowest MIC, 0.06 μg/ml. The highest daptomycin MIC obtained in the five groups was 1.0 μg/ml for isolates with tetM, compared with 0.12 μg/ml for ermB-containing isolates or 0.25 μg/ml for isolates in the remaining three groups.
Daptomycin and a number of comparator agents were tested for activity in 12 Streptococcus mitis and four Streptococcus sanguis isolates containing an assortment of different amino acid changes associated with quinolone resistance (Table 1). The daptomycin MICs ranged from 0.25 to 8 μg/ml. The daptomycin MIC of 8 μg/ml occurred with a single S. mitis isolate and was somewhat higher than what was observed in other streptococcal species.
The activities of daptomycin and three additional antibiotics were tested in eight streptococcal species (n = 46) with different clindamycin and/or erythromycin resistance-encoding genes (Table 1). The daptomycin MICs ranged from 0.03 to 2 μg/ml. No major differences were observed between streptococcal isolates with characterized resistance determinants, with the exception of a single fluoroquinolone-resistant S. mitis isolate.
The results were generally comparable with other data on European and North American antibiotic-resistant clinical isolates that were phenotypically but not genetically characterized (3, 8, 12, 16, 21).
The Food and Drug Administration has defined susceptibility interpretive criteria for the approved indications. Streptococcus pyogenes, Streptococcus agalactiae, and Streptococcus dysgalactiae subsp. equisimilis isolates as well as all S. aureus isolates are susceptible to a MIC of ≤1 μg/ml, whereas vancomycin-susceptible E. faecalis are susceptible to MICs of ≤4 μg/ml. Based on these breakpoints, all isolates belonging to these species tested were susceptible to daptomycin. Furthermore, daptomycin also shows excellent activity against the other resistant gram-positive isolates.
In conclusion, daptomycin exhibits broad in vitro activity against a wide range of antibiotic-resistant, gram-positive pathogens containing different resistance determinants.
REFERENCES
- 1.Alborn, W. E., Jr., N. E. Allen, and D. A. Preston. 1991. Daptomycin disrupts membrane potential in growing Staphylococcus aureus. Antimicrob. Agents Chemother. 35:2282-2287. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Allen, N. E., W. E. Alborn, Jr., and J. N. Hobbs, Jr. 1991. Inhibition of membrane potential-dependent amino acid transport by daptomycin. Antimicrob. Agents Chemother. 35:2639-2642. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Barry, A. L., P. C. Fuchs, and S. D. Brown. 2001. In vitro activities of daptomycin against 2,789 clinical isolates from 11 North American medical centers. Antimicrob. Agents Chemother. 45:1919-1922. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Canepari, P., M. Boaretti, M. del Mar Lleó, and G. Satta. 1990. Lipoteichoic acid as a new target for activity of antibiotics: mode of action of daptomycin (LY146032). Antimicrob. Agents Chemother. 34:1220-1226. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Dutka-Malen, S., S. Evers, and P. Courvalin. 1995. Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J. Clin. Microbiol. 33:24-27. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Fluit, A. C., M. E. Jones. F.-J. Schmitz, J. Acar, R. Gupta, J. Verhoef, and the SENTRY Participants Group. 2000. Antimicrobial susceptibility and frequency of occurrence of clinical blood isolates in Europe from the SENTRY Antimicrobial Surveillance Program, 1997 and 1998. Clin. Infect. Dis. 30:454-460. [DOI] [PubMed] [Google Scholar]
- 7.Fluit, A. C., C. L. C. Wielders, J. Verhoef, and F.-J. Schmitz. 2001. Epidemiology and susceptibility of 3,051 Staphylococcus aureus isolates from 25 university hospitals participating in the European SENTRY study. J. Clin. Microbiol. 39:3727-3732. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Fuchs, P. C., A. L. Barry, and S. D. Brown. 2002. In vitro bactericidal activity of daptomycin against staphylococci. J. Antimicrob. Chemother. 49:467-470. [DOI] [PubMed] [Google Scholar]
- 9.Janoir, C., V. Zeller, M.-D. Kitzis, N. J. Moreau, and L. Gutmann. 1996. High-level fluoroquinolone resistance in Streptococcus pneumoniae requires mutations in parC and gyrA. Antimicrob. Agents Chemother. 40:2760-2764. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Jones, M. E., D. F. Sahm, N. Martin, S. Scheuring, P. Heisig, C. Thornsberry, K. Köhrer, and F.-J. Schmitz. 2000. Prevalence of gyrA, gyrB, parC, and parE mutations in clinical isolates of Streptococcus pneumoniae with decreased susceptibilities to different fluoroquinolones and originating from worldwide surveillance studies during the 1997-1998 respiratory season. Antimicrob. Agents Chemother. 44:462-466. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Kataja, J., H. Seppälä, M. Skurnik, H. Sarkkinen, and P. Huovinen. 1998. Different erythromycin resistance mechanisms in group C and group G streptococci. Antimicrob. Agents Chemother. 42:1493-1494. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.King, A., and I. Phillips. 2001. The in vitro activity of daptomycin against 514 gram-positive aerobic clinical isolates. J. Antimicrob. Chemother. 48:219-223. [DOI] [PubMed] [Google Scholar]
- 13.Lamp, K. C., M. J. Rybak, E. M. Bailey, and G. W. Kaatz. 1992. In vitro pharmacodynamic effects of concentration, pH, and growth phase on serum bactericidal activities of daptomycin and vancomycin. Antimicrob. Agents Chemother. 36:2709-2714. (Erratum, 37:930, 1993.) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.National Committee for Clinical Laboratory Standards. 2000. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 5th ed. Approved standard M7-A5. National Committee for Clinical Laboratory Standards, Wayne, Pa.
- 15.Pan, X.-S., J. Ambler, S. Mehtar, and L. M. Fisher. 1996. Involvement of topoisomerase IV and DNA gyrase as ciprofloxacin targets in Streptococcus pneumoniae. Antimicrob. Agents Chemother. 40:2321-2326. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Rybak, M. J., E. Hershberger, T. Moldovan, and R. G. Grucz. 2000. In vitro activities of daptomycin, vancomycin, linezolid, and quinupristin-dalfopristin against staphylococci and enterococci, including vancomycin-intermediate and -resistant strains. Antimicrob. Agents Chemother. 44:1062-1066. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Schmitz, F. J., A. C. Fluit, M. Gondolf, R. Beyrau, E. Lindenlauf, J. Verhoef, H. P. Heinz, and M. E. Jones. 1999. The prevalence of aminoglycoside resistance and corresponding resistance genes in clinical isolates of staphylococci from 19 European hospitals. J. Antimicrob. Chemother. 43:253-259. [PubMed] [Google Scholar]
- 18.Schmitz, F.-J., M. E. Jones, B. Hofmann, B. Hansen, S. Scheuring, M. Lückefahr, A. Fluit, J. Verhoef, U. Hadding, H.-P. Heinz, and K. Köhrer. 1998. Characterization of grlA, grlB, gyrA, and gyrB mutations in 116 unrelated isolates of Staphylococcus aureus and effects of mutations on ciprofloxacin MIC. Antimicrob. Agents Chemother. 42:1249-1252. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Snydman, D. R., N. V. Jacobus, L. A. McDermott, J. R. Lonks, and J. M. Boyce. 2000. Comparative in vitro activities of daptomycin and vancomycin against resistant gram-positive pathogens. Antimicrob. Agents Chemother. 44:3447-3450. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Sutcliffe, J., T. Grebe, A. Tait-Kamradt, and L. Wondrack. 1996. Detection of erythromycin-resistant determinants by PCR. Antimicrob. Agents Chemother. 40:2562-2566. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Warsa, U. C., M. Nonoyama, T. Ida, R. Okamoto, T. Okubo, C. Shimauchi, A. Kuga, and M. Inoue. 1996. Detection of tet(K) and tet(M) in Staphylococcus aureus of Asian countries by the polymerase chain reaction. J. Antibiot. (Tokyo) 49:1127-1132. [DOI] [PubMed] [Google Scholar]
- 22.Wise, R., J. M. Andrews, and J. P. Ashby. 2001. Activity of daptomycin against gram-positive pathogens: a comparison with other agents and the determination of a tentative breakpoint. J. Antimicrob. Chemother. 48:563-567. [DOI] [PubMed] [Google Scholar]