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Antimicrobial Agents and Chemotherapy, May 1999, p. 1298-1300, Vol. 43, No. 5
Department of Medicine, Section of Infectious
Diseases, Hacettepe University School of Medicine, 06100 Ankara,
Turkey
Received 17 September 1998/Returned for modification 7 January
1999/Accepted 20 February 1999
Brucellae survive acidic pHs in phagolysosomes. Azithromycin,
streptomycin, and quinolones were active against Brucella
melitensis at pH 7.0 but not at pH 5.0; rifampin and doxycycline
retained activity at pH 5.0. Regardless of pH, azithromycin-rifampin
and ofloxacin-rifampin showed less synergy than established
streptomycin-doxycycline and rifampin-doxycycline combinations.
Brucellosis occurs worldwide
but is most frequent in the Mediterranean basin and South
America (13). Because the bacteria are intracellular,
successful treatment requires antibiotics with good cellular
penetration: combinations of doxycycline with either rifampin or an
aminoglycoside usually are effective, but administration for 6 weeks is
required and relapse is frequent (9). Consequently, new
treatments are sought. Fluoroquinolones and newer macrolides have good
anti-Brucella activity in vitro (1, 9, 12) and reach high intracellular concentrations, but their in vitro activity may predict efficacy poorly, since brucellae survive in compartments that are inaccessible or hostile to antimicrobial activity. These include the phagolysosomes of macrophages, where the pH may be as low
as 5.0 (8). Acidity impairs the activities of quinolones and macrolides.
Moreover, as new antimicrobials may be used in combination, their
interactions with established anti-Brucella agents need assessment. We therefore evaluated the in vitro activities of doxycycline, rifampin, streptomycin, quinolones, erythromycin, and
azithromycin alone and in combination against Brucella
melitensis at pH 5.0 and pH 7.0.
Bacteria.
The 43 B. melitensis isolates were
collected between 1991 and 1994 from blood or bone marrow cultures of
individual inpatients with acute brucellosis at Hacettepe University
Hospital. They were identified to the species level by conventional
methods, on the basis of not requiring CO2 and not
producing H2S. A class II biological safety cabinet was used.
MICs at different pH values.
In vitro activities of
doxycycline (Sigma, St. Louis, Mo.), streptomycin (Sigma), rifampin
(Sigma), ofloxacin (Hoechst Marion Roussel,
Istanbul, Turkey), ciprofloxacin (Bayer, Istanbul, Turkey), erythromycin (Sigma), and azithromycin (Pfizer, Istanbul, Turkey) were
determined by microdilution. Mueller-Hinton broth (Oxoid, Basingstoke,
Hants, United Kingdom), supplemented with 1% PoliVitex (BioMèrieux, Marcy l'Etoile, France) and adjusted to pH 7.0 or pH 5.0, was used. The inoculum was 105 to
106 CFU per well, and the trays were incubated at
35°C. MICs were evaluated after 48 h. Escherichia
coli ATCC 25922 and Staphylococcus aureus ATCC 29213 served as controls.
Combination studies.
Twenty of the 43 isolates were chosen
randomly for the combination studies. The activities of
azithromycin-rifampin, ofloxacin-rifampin, doxycycline-rifampin,
and doxycycline-streptomycin were tested by checkerboard titration
(5) at pH 7.0 or pH 5.0. The media, inocula, and
conditions were the same as those for MIC tests. Fractional
inhibitory concentrations (FICs) were calculated as (MIC of
antibiotic in combination)/(MIC of antibiotic alone) and summed to give
Activities of individual antibiotics.
All the antibiotics
except erythromycin had good activities against most isolates at pH
7.0, with MICs at which 90% of the isolates were inhibited
(MIC90s) below standard National Committee for
Clinical Laboratory Standards breakpoints (10). The
activity of rifampin was increased two- to eightfold at pH 5.0, but
the MIC50s of ofloxacin, ciprofloxacin, erythromycin,
streptomycin, and azithromycin increased to well above their
breakpoints. Doxycycline MICs were increased at pH 5.0, but the
MIC90 remained below the breakpoint of 4 µg/ml (Table
1).
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
In Vitro Activities of Antibiotics Alone and in Combination
against Brucella melitensis at Neutral and Acidic
pHs
ABSTRACT
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FIC indices, which were classified as follows: 
0.75, synergistic;
0.75 to 1, additive; 1 to 2, indifferent; 
2, antagonistic
(11).
TABLE 1.
In vitro activities of antibiotics against B. melitensis isolates (n = 43) in relation to pH
Activities of antimicrobial combinations. The activities of combinations at pH 7.0 and pH 5.0 are summarized in Fig. 1A and B, respectively. The established rifampin-doxycycline and streptomycin-doxycycline combinations were synergistic against almost all (17 or 18 of 20) the isolates at pH 7.0. Synergism was observed with rifampin-doxycycline for 17 of 20 isolates at pH 5.0, whereas the streptomycin-doxycycline combination was synergistic for 7, additive for 12, and indifferent for 1. The rifampin-azithromycin combination was indifferent for activity against most (16 to 17 of 20) isolates at both pH levels, whereas the rifampin-ofloxacin combination was antagonistic for activity against 17 isolates at pH 7.0 but only for activity against 7 isolates at pH 5.0, with synergy apparent for four organisms.
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7.0, such as the serum. Streptomycin likewise loses activity against
brucellae at pH 5.0 (Table 1) but still is useful in combination
therapy for brucellosis (4). We therefore tested azithromycin and ofloxacin combined with rifampin, which has good intracellular penetration and is active at acidic pHs.
Rifampin-doxycycline and streptomycin-doxycycline were tested
for comparison. Acidic pHs did not compromise the synergy between
rifampin and doxycycline, and although the synergy between streptomycin
and doxycycline was reduced at pH 5.0, the combination remained
synergistic or additive. No antagonism was seen with these
established combinations. By contrast, virtually no synergy was seen
with the new combinations.
Despite in vitro antagonism, we have obtained successful
cures of human brucellosis with ofloxacin-rifampin, at rates
comparable to those with doxycycline-rifampin (2). Ofloxacin
and rifampin may achieve their highest anti-Brucella
activities at different sites in vivo, perhaps evading the antagonism
seen in vitro. Moreover, intracellular killing of brucellae by
lysosome-tropic antibiotics such as macrolides may be augmented by host
factors, and National Committee for Clinical Laboratory Standards
breakpoints may not be appropriate to intracellular pathogens. The
azithromycin-plus-rifampin combination has not been evaluated in vivo
but may deserve study in animals, considering the poor correlation
between in vivo and in vitro data for other combinations. Nevertheless,
based on the present results, there is little reason to discard
established regimens.
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ACKNOWLEDGMENTS |
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D.M.L. is grateful to the British Council for supporting a Link Program between Hacettepe University and The London Hospital Medical College, where he worked at the time of this study. This program financed the visits during which this study was partly undertaken.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Medicine, Section of Infectious Diseases, Hacettepe University School of Medicine, Ankara 06100, Turkey. Phone: 90-312-311 1271. Fax: 90-312-310 4179. E-mail: ma08-k{at}tr-net.net.tr.
Present address: Department of Pediatrics, Clinical Microbiology
Laboratory, Hacettepe University School of Medicine, Ankara, Turkey.
Present address: Antibiotic Resistance Monitoring and Reference
Laboratory, Central Public Health Laboratory, Colindale, London NW9
5HT, United Kingdom.
§ Present address: Department of Microbiology and Clinical Microbiology, Hacettepe University School of Medicine, Ankara, Turkey.
Present address: Pfizer Ilaçlari A.
.,
Ortaköy 80840, Istanbul, Turkey.
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Antimicrobial Agents and Chemotherapy, May 1999, p. 1298-1300, Vol. 43, No. 5
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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