![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, June 1998, p. 1520-1523, Vol. 42, No. 6
Department of Medicine, West Campus, Beth
Israel Deaconess Medical Center, Boston, Massachusetts
02215,1
Harvard Medical School, Boston,
Massachusetts 02115,2 and
Massachusetts General Hospital, Boston, Massachusetts
021143
Received 17 November 1997/Returned for modification 26 February
1998/Accepted 1 April 1998
The in vitro activities of 13 antimicrobial agents against 30 strains of Legionella spp. were determined. Rifapentine,
rifampin, and clarithromycin were the most potent agents (MICs at which 90% of isolates are inhibited [MIC90s], The array of antimicrobial agents
useful for the treatment of serious infections caused by
Legionella spp. is limited. This is in part due to the
relative resistance of Legionella spp. to a variety of
antimicrobial agents and to the fact that these organisms are obligate
intracellular pathogens and, thus, to be effective, the drugs must be
able to penetrate into phagocytic cells (22).
Erythromycin, rifampin, and fluoroquinolones have proven in vitro and
in vivo efficacies and are used to treat clinical Legionella infections (23, 26). Mortality is still high in those with nosocomial pneumonia, especially immunocompromised and bacteremic patients (14), so there is a need for a wider range of
suitable antibiotics to treat severe Legionella infections.
This study examined the in vitro activities of several newer
antimicrobial agents, including a ketolide, two fluoroquinolones, two oxazolidinones, rifapentine, and dalfopristin-quinupristin, against
Legionella spp., an initial step in assessing their
potential usefulness as therapeutic agents.
(This work was presented in part at the 37th Interscience Conference on
Antimicrobial Agents and Chemotherapy, Toronto, Ontario, Canada, 28 September to 1 October 1997 [33]).
Thirty clinical isolates of Legionella spp. were tested,
including 21 L. pneumophila, 3 L. longbeacheae, 2 L. bozemanii, and 2 L. dumoffii isolates and one
strain each of L. micdadei and L. gormanii.
Strains were referred to our collection from various sources over
several years and were kept frozen at Antimicrobial test substances and their sources are as follows. HMR
3647, erythromycin A, clarithromycin, roxithromycin, rifampin, rifapentine, and levofloxacin were gifts from Hoechst-Marion-Roussel, Romainville, France; eperezolid (U-100592) and linezolid (U-100766) were gifts from Pharmacia & Upjohn Company Laboratories, Kalamazoo, Mich.; dalfopristin-quinupristin was provided by Rhône-Poulenc Rorer Pharmaceuticals, Collegeville, Pa.; and BAY 12-8039 was a gift of
Bayer Inc., West Haven, Conn. Clindamycin hydrochloride and doxycycline
hydrochloride were purchased from Sigma Chemical Company, St. Louis,
Mo.
Agar dilution susceptibility testing was performed on the buffered
starch-yeast extract (BSYE) agar medium described by Saito et al.
(32). Buffered charcoal-yeast extract medium (BCYE) has been
shown to impair the activities of several antimicrobial substances (i.e., macrolides, rifampin, and fluoroquinolones) in earlier studies
(3, 6, 12), so this medium was only used to subcultivate strains twice after thawing them from their To prepare inocula, several colonies were taken from BCYE plates
(Remel, Lenexa, Kans.) after 48 h of incubation and were suspended
in sterile water to a turbidity corresponding to a 0.5 McFarland
standard, which yielded a cell density of approximately 108
CFU/ml. Suspensions of bacteria were then further diluted 1:10 in
sterile water for the smaller inoculum. Final inocula of
105 and 104 CFU/spot were applied to freshly
made antibiotic-containing plates with a multiprong replicator device.
Between each antibiotic, antibiotic-free plates were stamped to avoid
carryover, and a blood agar plate was also inoculated at the end of
each run to exclude contamination by other bacteria.
Plates were incubated at 35°C in ambient air and were read after 48 and 96 h. Spots yielding the growth of single colonies and those
with a faint haze were considered to be negative.
Table 1 shows the results for the 48-h
incubation time for both inocula. For most agents, a twofold increase
in the MIC at which 90% of the isolates were inhibited
(MIC90) was observed when the plates were examined after
96 h of incubation (Table 2). Such
results may reflect either incomplete inhibition of growth at a
particular antibiotic dilution or the loss of antimicrobial potency
with prolonged incubation. Subsequent comments will be directed to
results of the 48-h readings. With the larger inoculum, all strains
grew on BSYE agar as well as on BCYE agar, whereas with the smaller
inoculum, three to six strains yielded insufficient growth on control
plates and therefore were excluded from the analysis. These findings
are consistent with results from other studies, which showed that BSYE
agar does not support the growth of some Legionella species
as well as does BCYE agar (4, 15). Table
3 compares the MICs of several
antimicrobial agents tested against Legionella spp.,
obtained in different studies using different media and methods.
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Susceptibilities of Legionella spp. to Newer
Antimicrobials In Vitro
ABSTRACT
Top
Abstract
Text
References
0.008
µg/ml). The ketolide HMR 3647 and the fluoroquinolones levofloxacin
and BAY 12-8039 (MIC90s, 0.03 to 0.06 µg/ml) were more
active than erythromycin A or roxithromycin. The MIC90s of
dalfopristin-quinupristin and linezolid were 0.5 and 8 µg/ml,
respectively. Based on class characteristics and in vitro activities,
several of these agents may have potential roles in the treatment of
Legionella infections.
TEXT
Top
Abstract
Text
References
80°C.
80°C storage
temperature and as a growth control.
TABLE 1.
Comparison of in vitro activities of 13 antimicrobial
agents against Legionella spp., determined at 48 h
of incubation
TABLE 2.
Comparison of MIC90s at 48 and 96 h of
incubation for inocula of 104 and 105 CFU/spot
TABLE 3.
Comparison of methods used and MICs of antimicrobial
agents tested against Legionella spp. in different studies
Erythromycin, probably the most widely used drug for treatment of
Legionella pneumonia (14, 27), inhibited all
strains at 0.25 and 0.5 µg/ml with the small and the large
inocula, respectively. Those data were comparable to erythromycin A
MICs obtained previously in our laboratory (7).
A new ketolide designated HMR 3004 has been shown to reach high
intracellular concentrations in phagocytes; therefore, agents of this
class may be of potential therapeutic use against intracellular pathogens like Legionella spp. (1). The ketolide
tested here, HMR 3647, inhibited 90% of all organisms at
concentrations of 0.03 µg/ml and thus showed fourfold-higher activity
than erythromycin A. These data complement a study by Bornstein et al.
(5), who found HMR 3004 to be active against
Legionella spp. with a range of MICs virtually identical to
those obtained for HMR 3647 in our study (MIC, 0.03 to 0.12 µg/ml)
when performed by the agar dilution technique on a different medium
(buffered antibiotic medium no. 1). Clarithromycin was the most potent
macrolide in our study, exhibiting an MIC90 of 0.004
µg/ml with both inocula.
Rifampin is used in combination with other drugs in severe or refractory cases of legionellosis (13). In a number of comparative studies, it was the most active drug tested (6, 10, 24). In the present study, 90% of isolates were inhibited at concentrations of 0.008 µg/ml with the larger inoculum. Rifapentine is a newly developed agent related to rifampin. The MIC90 of this drug was 0.002 µg/ml, fourfold lower than that of rifampin, with the large inoculum. All strains of L. pneumophila were inhibited at the lowest concentrations of rifampin and rifapentine tested, 0.0005 and 0.001 µg/ml, respectively. The MICs of rifampin and rifapentine for other species ranged from 0.0005 to 0.015 µg/ml and from 0.001 to 0.002 µg/ml, respectively, after 48 h of incubation.
Fluoroquinolones have been shown to be highly effective in vitro (17), and they have also been shown to inhibit the growth of legionellae in alveolar macrophage systems and in experimental treatment models of L. pneumophila pneumonia in guinea pigs (9, 16, 18). Moreover, fluoroquinolones have been used clinically for treatment of Legionella pneumonia (35). In the present study, the MIC90s for BAY 12-8039 and levofloxacin were 0.06 and 0.03 µg/ml, respectively, with the larger inoculum. The MICs for levofloxacin were two to three times higher in a study by Baltch et al. (2), but their study utilized BCYE agar, which is known to inhibit the activity of certain antimicrobial agents, especially fluoroquinolones (17). In experimental Legionnaires' disease in guinea pigs, levofloxacin appeared to be as active as ofloxacin, which was superior to ciprofloxacin and erythromycin (11, 31). Our data for BAY 12-8039 were comparable to those reported by Ruckdeschel et al. (30); in the latter study, a larger inoculum was used (108 CFU/spot).
The streptogramin combination dalfopristin-quinupristin inhibited 90% of all isolates at a concentration of 0.5 µg/ml. The overall MICs were two- to fourfold higher than those of erythromycin A, which is consistent with a report by Johnson et al. (24), in which they showed the same correlation between those two drugs with a larger inoculum (106 CFU/spot). In contrast, in a study by Dubois and Joly (10), dalfopristin-quinupristin demonstrated twofold-higher activity than erythromycin against some Legionella species. A possible role for this drug in the treatment of legionellosis is supported by reports which showed high intracellular accumulation and activity against intracellular staphylococci (8); however, the activity of dalfopristin-quinupristin against intracellular enterococci was modest (21).
The oxazolidinones linezolid (U-100766) and eperezolid (U-100592) are
recently developed antimicrobial agents which have shown therapeutic
potential based on in vitro activity against various respiratory
pathogens, including multidrug-resistant pneumococci, streptococci,
staphylococci, Haemophilus spp., and Moraxella
spp. (25, 34, 36). At the smaller inoculum, 90% of the
legionellae tested were inhibited by a 4-µg/ml concentration of
each drug, a concentration equivalent to MICs for other presumptively
susceptible organisms. However, at the larger inoculum, the
MIC90s of the agents were 8 and 
16 µg/ml, respectively.
Unless there was evidence for intracellular accumulation in phagocytes,
such in vitro data would not suggest that these specific oxazolidinones
would be likely candidates for treatment of Legionella
infections.
At the smaller and larger inocula, doxycycline inhibited 90% of strains at 2 and 8 µg/ml, respectively, after 48 h of incubation. MICs of 8 µg/ml would indicate intermediate susceptibility (7). Nevertheless, doxycycline showed activity against L. pneumophila in intracellular monocyte experiments when added at concentrations of 0.4 µg/ml (20). It was also shown to be therapeutically effective in a guinea pig model of experimental legionellosis (28) and showed clinical efficacy in the treatment of human legionellosis (14). It is known that tetracyclines accumulate in human neutrophils (19), and such discrepancies between in vitro activity and therapeutic results exemplify the potential pitfalls in predicting clinical effectiveness from in vitro data alone.
This study identified several new antimicrobial agents with in vitro activities against legionellae that were higher than that of the widely used agent erythromycin. To further explore the potential applicability of these in vitro findings to the clinical setting, intracellular susceptibility testing and animal model studies would be of interest.
| |
ACKNOWLEDGMENTS |
|---|
This study was supported by a grant from Hoechst-Marion-Roussel. Tanja Schülin was supported by a grant from Walter-Marget-Vereinigung.
| |
FOOTNOTES |
|---|
*
Corresponding author. Mailing address: Department of
Medicine
West Campus, Beth Israel Deaconess Medical Center, One
Deaconess Rd., Boston, MA 02215. Phone: (617) 632-8586. Fax: (617)
632-7442. E-mail: geliopou{at}bidmc.harvard.edu.
| |
REFERENCES |
|---|
|
Top
Abstract
Text
References
|
|---|
| 1. | Agouridas, C., A. Bonnefoy, K. Braham, P. Collette, M. Guitton, A. Hochet, P. Mauvais, and J. F. Chantot. 1995. RU 004: uptake in phagocytes, intracellular bioactivity and other immunomodulatory effects, abstr. F175, p. 143. In Abstracts of the 35th Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society for Microbiology, Washington, D.C. |
| 2. | Baltch, A. L., R. P. Smith, and W. Ritz. 1995. Inhibitory and bactericidal activities of levofloxacin, ofloxacin, erythromycin, and rifampin used singly and in combination against Legionella pneumophila. Antimicrob. Agents Chemother. 39:1661-1666[Abstract]. |
| 3. |
Barker, J., and I. D. Farrell.
1986.
The effect of charcoal on MICs for Legionella.
J. Antimicrob. Chemother.
17:127 |
| 4. |
Bornstein, N.,
C. Roudier, and J. Fleurette.
1985.
Determination of the activity on Legionella of eight macrolides and related agents by comparative testing on three media.
J. Antimicrob. Chemother.
15:17-22 |
| 5. | Bornstein, N., H. Behr, Y. Brum, and J. Fleurette. 1995. In vitro activity of RU 004 on Legionella species, abstr. F166, p. 142. In Abstracts of the 35th Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society for Microbiology, Washington, D.C. |
| 6. | Chen, S. C. A., M. L. Paul, and G. L. Gilbert. 1993. Susceptibility of Legionella species to antimicrobial agents. Pathology 25:180-183[Medline]. |
| 7. |
Collins, L. A.,
C. B. Wennersten,
M. J. Ferraro,
R. C. Moellering, Jr., and G. M. Eliopoulos.
1994.
Comparative activities of piperacillin and tazobactam against clinical isolates of Legionella spp.
Antimicrob. Agents Chemother.
38:144-146 |
| 8. | Desnottes, J. F., and N. Diallo. 1992. Cellular uptake and intracellular bactericidal activity of RP 59500 in murine macrophages. J. Antimicrob. Chemother. 30(Suppl. A):107-115. |
| 9. | Dournon, E., P. Rajagopalan, J. L. Vilde, and J. J. Pocidalo. 1986. Efficacy of pefloxacin in comparison with erythromycin in the treatment of experimental guinea pig legionellosis. J. Antimicrob. Chemother. 17(Suppl. B):41-48. |
| 10. | Dubois, J., and J. R. Joly. 1992. In vitro activity of RP 59500, a new synergistic antibacterial agent, against Legionella spp. J. Antimicrob. Chemother. 30(Suppl. A):77-81. |
| 11. |
Edelstein, P. H.,
M. A. C. Edelstein,
K. H. Lehr, and J. Ren.
1996.
In-vitro activity of levofloxacin against clinical isolates of Legionella spp., its pharmacokinetics in guinea pigs, and use in experimental Legionella pneumophila pneumonia.
J. Antimicrob. Chemother.
37:117-126 |
| 12. |
Edelstein, P. H.
1991.
Rifampin resistance of Legionella pneumophila is not increased during therapy for experimental Legionnaires disease: a study of rifampin resistance using a guinea pig model of Legionnaires disease.
Antimicrob. Agents Chemother.
35:5-9 |
| 13. | Edelstein, P. H. 1993. Legionnaires' disease. Clin. Infect. Dis. 16:741-749[Medline]. |
| 14. | Edelstein, P. H. 1995. Antimicrobial chemotherapy for Legionnaires' disease: a review. Clin. Infect. Dis. 21(Suppl. 3):S265-S276. |
| 15. |
Edelstein, P. H., and R. D. Meyer.
1980.
Susceptibility of Legionella pneumophila to twenty antimicrobial agents.
Antimicrob. Agents Chemother.
18:403-408 |
| 16. |
Edelstein, P. H.,
M. A. C. Edelstein,
J. Weidenfeld, and M. B. Dorr.
1990.
In vitro activity of sparfloxacin (CI-978; AT-4140) for clinical Legionella isolates, pharmacokinetics in guinea pigs, and use to treat guinea pigs with L. pneumophila pneumonia.
Antimicrob. Agents Chemother.
34:2122-2127 |
| 17. | Eliopoulos, G. M., E. Reiszner, M. J. Ferraro, C. B. Wennersten, and R. C. Moellering, Jr. 1988. Effect of growth medium on activity of fluoroquinolones and other antimicrobial agents against Legionella species. Rev. Infect. Dis. 10(Suppl. 1):S56. |
| 18. | Fitzgeorge, R. B., D. H. Gibson, R. Jepras, and A. Baskerville. 1985. Studies on ciprofloxacin therapy of experimental Legionnaires' disease. J. Infect. 10:194-203[Medline]. |
| 19. | Gabler, W. L. 1991. Fluxes and accumulation of tetracyclines by human blood cells. Res. Commun. Chem. Pathol. Pharmacol. 72:39-51[Medline]. |
| 20. |
Havlichek, D.,
L. Saravolatz, and D. Pohlod.
1987.
Effect of quinolones and other antimicrobial agents on cell-associated Legionella pneumophila.
Antimicrob. Agents Chemother.
31:1529-1534 |
| 21. | Herrera-Insúa, I., K. Jacques-Palaz, B. E. Murray, and R. M. Rakita. 1996. Intracellular activities of RP 59500 (quinupristin/dalfopristin) and sparfloxacin against Enterococcus faecium. Antimicrob. Agents Chemother. 40:886-890[Abstract]. |
| 22. | Horwitz, M. A., and S. C. Silverstein. 1982. Legionnaire's disease bacterium (Legionella pneumophila) multiplies intracellulary in human monocytes. J. Clin. Invest. 66:441-450. |
| 23. | Horwitz, M. A., and S. C. Silverstein. 1983. Intracellular multiplication of Legionnaires' disease bacteria (Legionella pneumophila) in human monocytes is reversely inhibited by erythromycin and rifampin. J. Clin. Invest. 71:15-28. |
| 24. | Johnson, D. M., M. E. Erwin, M. S. Barret, B. Brings Gooding, and R. N. Jones. 1992. Antimicrobial activity of ten macrolide, lincosamine and streptogramin drugs tested against Legionella species. Eur. J. Clin. Microbiol. Infect. Dis. 11:751-755[Medline]. |
| 25. | Mason, E. O., Jr., L. B. Lamberth, and S. L. Kaplan. 1996. In vitro activities of oxazolidinones U-100592 and U-100766 against penicillin-resistant and cephalosporin-resistant strains of Streptococcus pneumoniae. Antimicrob. Agents Chemother. 40:1039-1040[Abstract]. |
| 26. |
Meyer, R. D.
1991.
Role of quinolones in the treatment of legionellosis.
J. Antimicrob. Chemother.
28:623-625 |
| 27. | Nguyen, M. H., J. E. Stout, and V. L. Yu. 1991. Legionellosis. Infect. Dis. Clin. N. Am. 5:561-584[Medline]. |
| 28. | Nowicki, M., J. C. Paucod, N. Bornstein, H. Meugnier, J. Freney, P. Isoard, and J. Fleurette. 1987. Traitement par la doxycycline de la légionellose experimentale du cobaye infecté par aérosole. Pathol. Biol. 35:865-869[Medline]. |
| 29. | Pendland, S. L., S. J. Martin, C. Chen, P. C. Schreckenberger, and L. H. Danziger. 1997. Comparison of charcoal- and starch-based media for testing susceptibilities of Legionella species to macrolides, azalides, and fluoroquinolones. J. Clin. Microbiol. 35:3004-3006[Abstract]. |
| 30. | Ruckdeschel, G., and S. Lob. 1996. In vitro activity of a new 8-methoxyquinolone, BAY 12-8039, against Legionella spp. in comparison to ciprofloxacin (CIP), erythromycin (ERY) and rifampin (RIF), abstr. F7, p. 101. In Abstracts of the 36th Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society for Microbiology, Washington, D.C. |
| 31. | Saito, A., H. Koga, H. Shigeno, K. Watanabe, K. Mori, S. Kohno, Y. Shigeno, Y. Suzuyama, K. Yamaguchi, M. Hirota, et al. 1986. The antimicrobial activity of ciprofloxacin against Legionella species and the treatment of experimental Legionella pneumonia in guinea pigs. J. Antimicrob. Chemother. 10:251-260. |
| 32. |
Saito, A.,
K. Sawatari,
Y. Fukuda,
M. Nagasawa,
H. Koga,
A. Tomonaga,
H. Nakazato,
K. Fujita,
Y. Shigeno,
Y. Suzuyama,
K. Yamaguchi,
K. Izumikawa, and K. Hara.
1985.
Susceptibility of Legionella pneumophila to ofloxacin in vitro and in experimental Legionella pneumonia in guinea pigs.
Antimicrob. Agents Chemother.
28:15-20 |
| 33. | Schülin, T., C. B. Wennersten, M. J. Ferraro, R. C. Moellering, Jr., and G. M. Eliopoulos. 1997. Susceptibility of Legionella spp. to ketolide HMR 3647 and other newer antimicrobials, abstr. F-250, p. 188. In Abstracts of the 37th Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society for Microbiology, Washington, D.C. |
| 34. | Spangler, S. K., M. R. Jacobs, and P. C. Appelbaum. 1996. Activities of RPR 106972 (a new oral streptogramin), cefditoren (a new oral cephalosporin), two new oxazolidinones (U-100592 and U-100766), and other oral and parental agents against 203 penicillin-susceptible and -resistant pneumococci. Antimicrob. Agents Chemother. 40:481-484[Abstract]. |
| 35. |
Stout, J. E., and V. L. Yu.
1997.
Legionellosis.
N. Engl. J. Med.
337:682-687 |
| 36. | Zurenko, G. E., B. H. Yagi, R. D. Schaadt, J. W. Allison, S. E. Glickman, D. K. Hutchinson, M. R. Barbachyn, and S. J. Brickner. 1996. In vitro activity of U-100592 and U-100766, novel oxazolidinone antibacterial agents. Antimicrob. Agents Chemother. 40:839-845[Abstract]. |
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Clin. Vaccine Immunol. | Clin. Microbiol. Rev. |
|---|---|
| J. Clin. Microbiol. | ALL ASM JOURNALS |
