Journal of Antimicrobial Chemotherapy
Efficacy of several antibiotic combinations against Brucella melitensis
Rev 1 experimental infection in BALB/c mice
M. J. Grillo ´1*, M. J. De Miguel1, P. M. Mun ˜oz1, C. M. Marı ´n1, J. Ariza2and J. M. Blasco1
1Unidad de Sanidad Animal, Centro de Investigacio ´n y Tecnologı ´a Agroalimentaria (CITA) de Arago ´n,
50.080 Zaragoza, Spain;2Departamento de Enfermedades Infecciosas, Hospital
Universitario de Bellvitge, Universidad de Barcelona, Spain
Received 28 March 2006; returned 3 May 2006; returned 12 June 2006; accepted 23 June 2006
Objectives: The objective of the present study was to compare the efficacy of gentamicin given alone or
combined with doxycycline with that of standard combination therapies in BALB/c mice experimentally
infected with the Brucella melitensis vaccine strain Rev 1.
Methods: A standard broth microdilution method was applied to determine the susceptibility of strain
Rev 1 to the clinically most relevant aminoglycosides. Eight groups of BALB/c mice were inoculated
intraperitoneally (ip) with 1 · 106cfu/mouse of strain Rev 1. While one group remained untreated, the
other seven groups were treated 10 days later once a day for 14 days with (i) doxycycline given orally at 2
mg/day; (ii) streptomycin given ip at 0.4 mg/day; (iii) gentamicin given ip at 0.4 mg/day; (iv) rifampicin
given orally at 0.5 mg/day; (v) doxycycline plus streptomycin; (vi) doxycycline plus gentamicin; and (vii)
doxycycline plus rifampicin. The number of cfu per spleen and clearance of Rev 1 were assessed 34 days
Results: With the exception of streptomycin, strain Rev 1 was susceptible to all aminoglycosides tested.
As expected, the combination doxycycline/streptomycin was ineffective against Rev 1 infection. In
contrast, the combinations doxycycline/gentamicin and doxycycline/rifampicin were effective in the
clearance of Rev 1 infection, but only the former improved significantly the therapeutic efficacy as
compared with that of the antibiotics given alone.
Conclusions: Gentamicin may be used along with doxycycline when the classical combination is
considered the first choice in the treatment of patients with brucellosis due to B. melitensis vaccine
strain Rev 1.
Keywords: Human brucellosis, B. melitensis Rev 1 vaccine strain, antimicrobial therapy
Brucellosis is an important public health problem, whose
occurrence in humans is related directly with the prevalence of
the infection in animals, and particularly in domestic ruminants.
In endemic situations, vaccination is the only suitable way for
controlling brucellosis in ruminants.1Brucella melitensis is the
main causative agent of brucellosis in both humans and small
ruminants. The most effective and widely used vaccine against
brucellosis in sheep and goats is the attenuated B. melitensis Rev 1
live vaccine strain.2However, this vaccine strain can cause
abortion and excretion in milk when sheep and goats are vaccina-
ted during pregnancy in mass-vaccination programmes, increasing
the risk of human infections due to Rev 1.1Moreover, accidental
Rev 1 inoculations are not rare in veterinarians and shepherds
during the vaccination campaigns.3–5
The treatment of choice of human brucellosis caused by
B. melitensis field strains is a classical combination of long-acting
tetracyclines and streptomycin.6However, other aminoglycosides
such as gentamicin and netilmicin have shown better in vitro
activity than streptomycin and clinical trials have demonstrated
a similarly good efficacy.7In the past years, some authors have
recommended the use of gentamicin in these combination
schedules based on its more favourable profile.8
doxycycline/aminoglycoside combinations have been considered
the most effective treatment for human brucellosis, whereas the
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Journal of Antimicrobial Chemotherapy Advance Access published July 18, 2006
oral association of doxycycline and rifampicin may be a good
alternative because of its more comfortable administration for
non-complicated forms of the disease.6
ineffective in human infections caused by Rev 1,4probably due
to the resistance of this vaccine strain to streptomycin as a logical
consequence of its isolation procedure.2,9Thus, in the case of
being diagnosed, these infections due to Rev 1 are empirically
treated with the combination doxycycline/rifampicin. Surpris-
ingly, no clinical information exists to the possible use of other
aminoglycosides in these patients, probably because of the
assumption among clinicians of the existence of cross-resistances
between streptomycin and other aminoglycosides. Based on the
current knowledge of the mechanisms of resistance of Rev 1 strain
to streptomycin, we speculated that other aminoglycosides may be
active against this infection and therefore that the combination
doxycycline–gentamicin may also be suitable for the treatment of
patients infected with Rev 1.
In a first trial, and to exclude the possibility of cross-resistance,
we have determined the susceptibility of B. melitensis Rev 1
vaccine strain against a selection of aminoglycosides in vitro.
As the clinically most relevant aminoglycoside, the efficacy of
gentamicin given alone or combined with doxycycline was
determined in comparison with standard therapies in a second trial
using BALB/c mice experimentally infected with the B. melitensis
Rev 1 strain.
Material and methods
Bacterial strain and suspensions
The B. melitensis Rev 1 reference strain was kept freeze-dried in the
Brucella Culture Collection of the CITA (Zaragoza, Spain). Before its
use, B. melitensis Rev 1 cells were rehydrated in sterile Buffered
Saline Solution (BSS; 0.85% NaCl, 0.1% KH2PO4, 0.2% K2HPO4;
pH 6.85) and cultured onto Blood Agar Base (BAB No. 2; Difco,
Becton Dickinson, USA) plates at 37?C for 5 days. The resulting
culture was assessed for purity and absence of dissociation by the
White and Wilson method10and selected Rev 1 colonies subcultured
in BAB for 24–48 h at 37?C. Cultures were harvested in BSS and
adjusted by spectrophotometry to 109cfu/mL (OD600= 0.170) either
in Mueller–Hinton broth (M–H; Difco, Becton Dickinson, USA)—for
MIC determination—or in BSS—for inoculation in mice. For MIC
assays, the bacterial suspension was properly diluted in the M–H
broth to obtain a suspension containing 5 · 104cfu/mL. For mice
inoculations, two serial 10-fold dilutions of the adjusted suspension
were performed in BSS to obtain a final bacterial suspension
containing 1 · 107cfu/mL.
A standard broth microdilution method was applied to determine
the susceptibility of the B. melitensis Rev 1 vaccine strain to
aminoglycosides other than streptomycin: kanamycin, amikacin,
tobramycin, gentamicin, netilmicin and spectinomycin.11Tetracy-
clines (tetracycline and doxycycline) and rifampicin (all obtained
from Sigma-Aldrich, Spain) were used as controls. For this, 2-fold
dilutions ranging from 256 to 0.25 mg/L of each antibiotic were
prepared in M–H broth. A total of 0.1 mL of each antibiotic dilution
plus 0.1 mL of the corresponding B. melitensis Rev 1 suspension
containing 5 · 104cfu/mL were dispensed in each of 96 wells on
sterile polystyrene microplates (Maxisorp Nunc?, Denmark). After
homogenization, plates were incubated at 37?C for 5–7 days and the
bacterial growth assessed by direct observation of absence of
Seven-week-old female BALB/c mice were obtained from Charles
River Laboratories (Barcelona, Spain). Animals were kept in cages,
in groups of 10 mice per cage, for 2 weeks before the start of the
experiments, with water and food ad libitum and accommodated
under biosafety conditions in the restricted-access facility at the
CITA (Zaragoza, Spain; registration number ES 50297012005).
The experimental procedures on mice and the facilities used to hold
the experimental animals are in accordance with the current European
(Directive 86/609/EEC, in DOCE number 358), National (Real
Decreto 233/1988, in BOE number 67) and Regional (Ley 11/2003,
in BOA number 35) laws. A total of 56 mice were inoculated
intraperitoneally (ip) with 1 · 106cfu/mouse of B. melitensis Rev 1
strain in 0.1 mL of the adequate bacterial suspension (see above). Ten
days later, mice were allotted randomly in eight groups each
composed of seven mice. One group of mice was kept untreated as
control and the remaining groups were treated once a day for 14 days,
with the following antibiotics: (i) doxycycline (Vibravenosa?
100 mg, Pfizer S.A., Madrid, Spain) given orally at 2 mg/day;
(ii) streptomycin (Sulfato de estreptomicina Reig Jofre ´?1 g,
Laboratorios Reig Jofre ´, S.A., Barcelona, Spain) given ip at
0.4 mg/day; (iii) gentamicin (Gevramycin?20 mg, Schering Plough,
Madrid, Spain) given ip at 0.4 mg/day; (iv) rifampicin (Rifaldin?
20 mg/mL, Aventis Pharma, Madrid, Spain) given orally at 0.5 mg/
day; (v) doxycycline/streptomycin; (vi) doxycycline/gentamicin;
(vii) doxycycline/rifampicin. Combined treatments were applied
with the same doses and routes as the corresponding antibiotics
given alone. The selection of the antibiotic doses was made according
to previous experimental brucellosis studies in mice.12–14To avoid
inhibitory effects due to tissue persistence of the antibiotics, the
numbers of cfu/spleen of the B. melitensis Rev 1 strain were deter-
mined 10 days after the last treatment (i.e. 34 days after inoculation),
as described elsewhere.15Briefly, the spleens were aseptically
removed, individually weighed, blended and diluted in 9 volumes
(1:10; w:v) of BSS. Serial 10-fold dilutions of each spleen were
performed in the same diluent, and 0.1 mL of the homogenate and of
each dilution were smeared in triplicate onto BAB culture plates. The
number of Rev 1 cfu was determined after incubation of plates
at 37?C in air for 5–7 days. The mean and SD (n = 7) of the log
cfu/spleen for each group of mice as well as the ratio of mice cured
(no colonies isolated in the first dilution of the spleen; limit of
detection is <4 cfu/spleen) were calculated. A value of 1 cfu was
considered when no cfu were detected in 0.3 mL of the spleen
homogenate seeded. The statistical comparisons between mean
values (n = 7) of log cfu/spleen were made using the one-way
ANOVA and the Fisher’s Protected Least Significant Differences
(PLSD) tests. The percentages of mice cured according to the
different treatments were statistically compared using the c2test with
the Fisher–Yates correction when required.
As expected, the Rev 1 strain was not inhibited at concentrations
of streptomycin as high as 2 mg/L, but it was at concentrations
of 4 mg/L. Moreover, the Rev 1 strain was susceptible to the
lowest concentration (0.25 mg/L) of the remaining aminoglyco-
sides tested, thus showing no evidence for a common pattern
of resistance of Rev 1 to the different aminoglycosides.
Grillo ´ et al.
Page 2 of 5
The tetracyclines used as controls (tetracycline and doxycycline)
inhibited the growth of Rev 1 at the lowest concentrations (0.25
mg/L) tested. The MIC for rifampicin, used also as a control, was
The results of the antibiotic treatments applied in BALB/c
mice experimentally infected with B. melitensis Rev 1 are
summarized in Table 1. When given alone, streptomycin was
the only antibiotic unable to induce a significant reduction in the
level of splenic infection with respect to that obtained in
the untreated control mice. Moreover, all mice treated with
only streptomycin were found to be infected at the end of
the experiment, and this was also the case in untreated controls. In
contrast, doxycycline, gentamicin or rifampicin given alone
induced a significant reduction in the number of cfu per spleen
of the Rev 1 vaccine strain (P < 0.0001) with respect to untreated
controls, and no statistical differences were found in the log cfu of
Rev 1 per spleen between these three treatment groups. However,
none of these antibiotics given alone was able to clear the Rev 1
infection in all treated mice, although treatment with rifampicin
resulted in significantly (P < 0.03) more cured mice than single
treatments with either doxycycline or gentamicin. The combined
doxycycline–streptomycin therapy induced a significant (P <
0.0001) reduction in the log cfu of Rev 1 per spleen with respect
to both untreated controls and mice treated with streptomycin
treatment resulted in no significant differences in the log cfu of
Rev 1 per spleen with respect to mice treated with doxycycline
alone, indicating that the positive therapeutic effect was due
exclusively to doxycycline. Moreover, only four of the seven mice
treated with this combination were fully cleared of Rev 1
infection. In contrast, being equally effective in reducing the log
cfu of Rev 1/spleen, treatment with doxycycline combined with
either rifampicin or gentamicin resulted in a complete clearance
of Rev 1 infection in all treated mice. The doxycycline–rifampicin
combination improved the results obtained with doxycycline
alone but resulted in similar efficacy to rifampicin given alone.
In contrast, the combined treatment with doxycycline and
gentamicin resulted in significantly better efficacy than that of
either antibiotic given alone.
Antibiotic therapy for human brucellosis has been the objective of
many studies but little attention has been directed to the infections
induced by the attenuated B. melitensis Rev 1 vaccine strain.
Doxycycline is one of the most widely used antibiotics for
treating human brucellosis, but relapse rates are very high when it
is used as monotherapy. For this reason, a combination with
aminoglycosides is recommended in order to increase the efficacy
of treatments and avoid relapses. While streptomycin has been the
aminoglycoside most frequently used, gentamicin offers a better
Owing to the resistance of Rev 1 strain to streptomycin, the
treatment of this infection can be problematic.4Since a cross-
resistance with other aminoglycosides has been assumed among
clinicians, and in the case of being adequately diagnosed, Rev 1
infected patients are empirically treated with an oral combination
of doxycycline and rifampicin, which is a good alternative, but
less effective in complicated or focal forms of disease.6
Clinical failures in brucellosis treatment are not related to
the development of antimicrobial resistance, but are a conse-
quence of the ability of Brucella to survive within the cells.16This
particular intracellular location protects brucellae against anti-
microbial agents and probably explains why this bacterium has
not required the evolutionary development of antimicrobial
resistance as has occurred in most microorganisms. However,
it has been reported that NorMI multidrug efflux protein in
B. melitensis confers resistance to gentamicin and other
antimicrobial agents and the substrate specificity of NorMI is
highly similar to that of YdhE protein of Escherichia coli, which
confers resistance to aminoglycosides such as kanamycin and
It has been reported that B. melitensis Rev 1 strain carries a
mutation in the rpsL gene encoding the small subunit of the
ribosomal protein S12, leading to an amino acid Pro-to-Leu
change at codon 91.19In fact, amino acid changes leading to
chromosomally acquired streptomycin resistance in other bacteria
have been reported at similar locations, conferring low-levels
of resistance.20,21In Salmonella Typhimurium, streptomycin-
resistant mutants having this rpsL mutation acquire compensatory
mutations, mainly in the rpsD, rpsE and rplS genes encoding the
ribosomal proteins S4, S5 and L19, respectively,22but the
existence of compensatory mutations in B. melitensis Rev 1 strain
remains unknown. In ribosomal S12 mutants of E. coli there was
no cross-resistance between streptomycin and the other amino-
glycosides.23In the present study we confirm similar findings for
Table 1. Efficacy of the several antibiotic combinations tested
against B. melitensis Rev 1 strain in experimentally infected
(mean – SD)1
No. of cured/
Doxycycline + streptomycin
Doxycycline + rifampicin
Doxycycline + gentamicin
3.90 – 0.46
1.40 – 0.37a
3.45 – 0.34
1.55 – 1.22a
0.96 – 0.77a
1.28 – 0.85a,c
0.62 – 0.05a,b,d
0.68 – 0.05a,b,c,d
*Mice were inoculated intraperitoneally with 1 · 106B. melitensis
Rev 1/mouse. Ten days after, all groups (n = 7) but one kept as untreated
control were treated once a day for 14 days with doxycycline (2 mg/day,
orally), streptomycin (0.4 mg/day, ip), gentamicin (0.4 mg/day, ip),
rifampicin (0.5 mg/day, orally), doxycycline-streptomycin,
doxycycline-gentamicin or doxycycline-rifampicin (given at the same
doses and routes as when used alone). All mice were killed 10 days
after the last treatment (i.e. 34 days after infection) to determine
the cfu/spleen of the Rev 1 strain.
1Statistical differences between mean levels of Rev 1 splenic infections by the
Fisher’s PLSD test:aP < 0.0001 versus untreated control;bP < 0.015 versus
doxycycline alone;cP < 0.015 versus the corresponding antibiotic but
doxycycline given alone;dP < 0.04 versus doxycycline-streptomycin
treatment.2One mouse was considered cured when no Rev 1 colonies were
isolated from the first spleen decimal dilution cultured (<4 cfu/spleen).
Statistical differences between the percentage of cured mice by the c2test:
eP < 0.002 versus doxycycline;fP < 0.02 versus gentamicin given alone;
gP < 0.05 versus doxycycline-streptomycin treatment;hP < 0.05 versus
untreated controls and streptomycin given alone.
Antibiotic therapy of B. melitensis Rev 1 infection in mice
Page 3 of 5
B. melitensis Rev 1 strain since all the aminoglycosides tested
except streptomycin were effective in inhibiting the growth of
this vaccine strain in vitro. The susceptibility against the other
aminoglycosides and doxycycline (MIC ? 0.25 mg/L) and
rifampicin (MIC 1 mg/L) used as controls was in agreement
with the results obtained from B. melitensis field strains.24
However, the in vitro efficacy of some aminoglycosides does not
necessarily correlate with their in vivo efficacy, because of the
limited penetration of these antibiotics into eukaryotic cells, the
niche of brucellae. As the comparatively high MIC of strepto-
mycin for the vaccine strain suggests, treatment of mice with
streptomycin given alone even at relatively high doses (0.4 mg/
day) was fully ineffective against Rev 1 infection (Table 1).
Accordingly, the moderate therapeutic effect obtained after
treatment with the combination doxycycline–streptomycin should
be exclusively attributable to the efficacy of doxycycline. This
result was in agreement with that reported in Rev 1 infected
To the best of our knowledge, no reports concerning the
antibiotic therapy of Rev 1 infection in mice are available. The
mouse model used was based on preliminary experiments
conducted with virulent Brucella strains,12but adapted to the
particular kinetics of the infection induced by the attenuated
Rev 1 strain. Based on preliminary reports in mice,14,25and
the effective use of gentamicin in clinical practice,7we selected
gentamicin as the aminoglycoside of choice to be tested in
mice. In this experiment we used a high dose of gentamicin of
0.4 mg/day, increased with respect to that used in previous
experiments, showing that gentamicin doses of around 0.1–
0.2 mg/day and pharmacokinetic parameters similar to those
obtained with usual doses in humans are subtherapeutic for
treating experimental brucellosis in mice.14,25However, this gen-
tamicin dose was equivalent to that used for streptomycin but in
contrast to streptomycin when given alone reduced significantly
the Rev 1 cfu with respect to untreated controls (Table 1).
Treatments with doxycycline or rifampicin resulted also in a
significant reduction of the levels of Rev 1 infection, but none of
the antibiotics given alone was able to clear the infection in all
animals treated (Table 1). The complete clearance of the bacteria
after antibiotic treatment is a good predictor for avoiding relapses
and, accordingly, should be considered as determinant for
selecting the most adequate treatment.
The mean splenic counts were below the limit of detection
of the method in both experimental groups and, thus, varying
exclusively according the spleen weights. The extrapolation
of these therapeutic results to human disease should be
made with caution, and the goal of the present study was not to
compare the efficacy of doxycycline–gentamicin combination
with doxycycline–rifampicin combination in the infection due to
Rev 1 vaccine strain, but to prove the absence of cross-resistance
between streptomycin and other aminoglycosides in vitro and
in the mouse model. Our results strongly suggest that gentamicin
may be used along with doxycycline when the classical
combination of a tetracycline with an aminoglycoside is consid-
ered the first choice in the treatment of patients with brucellosis
due to B. melitensis Rev 1 vaccine strain.
This research was supported in part by the Ministerio de
Educacio ´n y Ciencia of Spain (Comisio ´n Interministerial de
Ciencia y Tecnologı ´a; Project AGL2004-07088-C03-03), the
Instituto de Salud Carlos III (Red Tema ´tica de Investigacio ´n en
Brucellosis, G03/204), Gobierno de Arago ´n (Grupo Consolidado
para investigacio ´n en Brucelosis) and the European Commission
(Research Contract QLK2-CT-2002-00918). Fellowship support
to P. M. M. from the Ministerio de Educacio ´n y Ciencia (Spain) is
also gratefully acknowledged.
None to declare.
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