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Safi, M., B. Albalaa & A. Al-Mariri, 2013. Prophylactic efficacy of some antibiotic com-
binations against Brucella melitensis 16M in BALB/c mice. Bulg. J. Vet. Med., 16, No 3,
198−207.
Brucellosis is an endemic zoonosis in Syria, affecting both humans and animals. Data regarding
suitable antibiotic combinations in post-exposure prophylaxis against Brucella melitensis infections
are rare. Prophylactic effects of some antibiotic combinations were assessed in BALB/c mice, to limit
or control infection by B. melitensis 16M. Antibiotics were administrated prior to (for 7 days), after or
at the same time as (for 5 days) the bacterial administration. When a concentration of 104 CFU of
bacteria was injected, doxycycline-rifampicin combination reduced the bacterial counts in the spleens
of infected mice in all mice groups either 48 h our 30 days after the cessation of antibiotic treatment;
whereas, all other combinations had almost good efficacy only 30 days after the cessation of
antibiotic treatment. On another hand, only doxycycline-rifampicin and rifampicin-levofloxacin com-
binations had good efficacy 48 hours after the cessation of antibiotic treatment, when a concentration
of 107 CFU of bacteria was injected. In conclusion, these results suggest that doxycycline-rifampicin
combination, and may be doxycycline-ciprofloxacin and rifampicin-levofloxacin combinations, had
good prophylactic efficacy against B. melitensis infections and may provide protection against these
infections.
Key words: antibiotic combinations, Brucella melitensis, prophylaxis
INTRODUCTION
Brucellosis remains the commonest an- nymous, 1986) has released recommen-
thropozoonosis worldwide (Pappas et al., dations for use of doxycycline, combined
2006). B. melitensis is the major global with either rifampicin or streptomycin for
cause of human disease, followed by B. treating human brucellosis. Different
abortus and B. suis. It is transmitted to regimens have been universally applied in
humans through direct contact with in- clinical practice (Ariza et al., 2007). Al-
fected animals, consumption of dairy pro- though Brucella isolates are generally
ducts, or inhalation of aerosols. considered susceptible to the antibiotics
Brucellosis is still hyperendemic in the recommended by the WHO, sporadic ca-
Mediterranean basin, Middle East, South- ses of a kind of antibiotic resistance have
west Asia and parts of Latin America been reported (Baykam et al., 2004;
(Black, 2004). In 1986, the WHO (Ano- Lopez-Merino et al., 2004). Despite all
M. Safi, B. Albalaa & A. Al-Mariri
these regimens, a small percentage of one traditional drug with one quinolone
relapses are still seen, ranging from 5% to against B. melitensis infection.
15% in uncomplicated cases. Risk factors This study aimed to assess the pro-
for relapse have been assessed (Ariza et phylaxis with doxycycline-rifampicin, do-
al., 1995; Solera et al., 1998), but it xycycline-ciprofloxacin, doxycycline-levo-
remains unclear what is the best regimen floxacin, rifampicin-ciprofloxacin, and ri-
to be used in their presence. fampicin-levofloxacin combinations against
The high incidence of relapses and B. melitensis infection in BALB/C mice.
therapeutic failures, in addition to the side
effects of drug combination strategies, has
MATERIALS AND METHODS
led to the investigation of new treatment
schemes of the disease. Fluoroquinolones,
may serve as alternative drug choices Bacteria
(Kilic et al., 2008). Despite that clinical B. melitensis strain 16M, obtained from
experience with fluoroquinolones, such as the Laboratory of Microbiology and
ciprofloxacin, for the treatment of Immunology URBM (University of Na-
brucellosis has been disappointing, this mur, Belgium), was used as the challenge
therapeutical group could be potentially strain in this study. Brucella were grown
useful for prophylaxis of Brucella infec- for 48 h in 2YT agar (peptone, 16 g/L;
tion. The efficacy of ciprofloxacin and yeast extract, 10 g/L; NaCl, 5 g/L; agar,
levofloxacin against Brucella spp. has 13 g/L [GibcoBRL]) at 37 °C.
been determined in vitro in a number of Bacteria were harvested into 20 mL of
studies that include reported MIC90 values sterile phosphate-buffered saline (PBS)
(minimum inhibitory concentration for and the bacterial suspension was stan-
90% of the organisms) of 0.19 µg/mL dardised to 1010 colony-forming units
(Turkmani et al., 2006; Turan et al., (CFU)/mL prior to dilution to appropriate
2007), 0.25 µg/mL for ciprofloxacin (Bo- concentrations of inoculates. The concen-
dur et al., 2003); and 0.5 µg/mL (Trujil- trations were determined retrospectively
lano-Martin et al., 1999) for levofloxacin. by enumeration of ten-fold dilutions of the
Data are lacking regarding suitable inoculates on 2YT plates.
post exposure antibiotic prophylaxis, All experiments with live Brucella we-
which would ideally be a single-agent, re performed in biosafety level 2 facilities.
short-course, and oral regimen (Atkins et
al., 2010). However, ciprofloxacin admi- Antibiotics
nistered with doxycycline or rifampicin Doxycycline (Sigma, St. Louis, USA), ri-
appears to show some efficacy against fampicin (Sigma), ciprofloxacin (Bayer,
brucellosis in humans (Agalar et al., Istanbul, Turkey), and levofloxacin (Sig-
1999). Reports concerning the efficacy of ma) were dissolved as per manufacturer
ciprofloxacin (Shasha et al., 1992; Atkins recommendations to a working concent-
et al. 2009a) and ofloxacin (Shasha et al., ration of 8 mg/mL. Antibiotics were pre-
1992) in the protection against brucellosis pared freshly each day and sterilised
in murine model were disappointing. To through a 0.2 µm filter.
our knowledge, in literature, no reports
were found concerning the prophylactic
role of antibiotic combination between
Table 1. Number of mice groups depending on the injection protocol and the time of sacrifice
Number of mice groups challenged with 104 cfu
B. melitensis/mouse
48 h prior to at the time 24 h after control group
challenge of challenge challenge
Animals culled 48 h after the final 5 5 5 1
antibiotic administration
Animals culled 30 days after the 5 5 5 1
final antibiotic administration
Number of mice groups challenged with 107 cfu
B. melitensis/mouse
48 h prior to at the time 24 h after control group
challenge of challenge challenge
Animals culled 48 h after the final 5 5 5 1
antibiotic administration
Animals culled 30 days after the 5 5 5 1
final antibiotic administration
RESULTS
Log10 CFU/Spleen
Brucella infection 30 days after the
cessation of treatment in all groups, when
a concentration of 104 CFU of B. meli-
tensis 16M was injected. However, the
*
doxycycline-rifampicin combination was
relatively more effective in prior to
exposure and 24 h after exposure groups CON D+R D+C D+L R+C R+L
(Fig. 2B, C, P<0.0001) than other combi-
nations. Significant protection was B
observed 48 hours after the cessation of 24 h after challenge
antibiotic treatment in mice treated with Log10 CFU/Spleen
doxycycline-rifampicin combination in all
groups, i.e. either prior to exposure, at the
same time as exposure or 24 h after
exposure (Fig. 1, P<0.0001). However, do-
xycycline-ciprofloxacin combination pro- * *
tection was observed 48 hours after the
CON D+R D+C D+L R+C R+L
cessation of antibiotic treatment in at the
same time as exposure and prior to
exposure groups (Fig. 1A, C, P<0.01 and C
Log10 CFU/Spleen
A A
Same day as challenge Same day as challenge
Log10 CFU/Spleen
Log10 CFU/Spleen
*
*
Log10 CFU/Spleen
Log10 CFU/Spleen
* *
CON D+R D+C D+L R+C R+L CON D+R D+C D+L R+C R+L
C C
48 h prior to challenge 48 h prior to challenge
Log10 CFU/Spleen
Log10 CFU/Spleen
*
*
DISCUSSION
A
Same day as challenge Antibiotic therapy for human brucellosis
Log10 CFU/Spleen
24 h after challenge
aminoglycoside most frequently used,
gentamicin offers a better efficacy–toxici-
ty profile. Clinicians and laboratory re-
searchers have performed several micro-
* * biological and clinical studies of the
possible use of quinolones in the
treatment of human brucellosis. The intra-
CON D+R D+C D+L R+C R+L cellular penetration and excellent in vitro
activity of the fluoroquinolones make
C them attractive in treating intracellular
48 h prior to challenge infections such as brucellosis (Qadri et al.,
Log10 CFU/Spleen
the need for a regimen that would xacin for 14 days or 21 days do not
eliminate disease relapse further necessita- eliminate a B. melitensis infection. On the
ted the use of quinolones. other hand, data reported by Atkins et al.
In literature, only some data regarding (2009a) indicated the relatively poor
suitable antibiotic combinations post- efficacy of ciprofloxacin for treating bru-
exposure prophylaxis in murine models cellosis compared with doxycycline, but
are available. The first experimental highlight the ability of ciprofloxacin po-
results showed that antibiotic combina- tentially to provide a low level of protec-
tions therapy with streptomycin plus tion. In another two studies, Atkins et al.
aureomycin, terramycin, or sulfadiazine suggested that, comparing with doxycyc-
were definitely superior to any monothe- line, neither trovafloxacin nor grepafloxa-
rapy by one of these drugs. Such com- cin (Atkins et al., 2010), neither moxiflo-
bined therapy completely eradicated xacin nor gatifloxacin (Atkins et al., 2009b)
Brucella from the spleens of all but 1 of would likely be valuable for post exposure
100 mice treated with any of these prophylaxis of Brucella infection.
combinations (Shaffer et al., 1953). The Our data indicate that when the
results of Lang et al. (1993) demonstrated infection was performed with a high
that the combinations doxycycline-strepto- concentration of B. melitensis 16M (107
mycin and rifampicin-streptomycin are CFU), all used combinations, with the
synergistic against B. melitensis, while the exception of doxycycline-rifampicin and
combination streptomycin-ciprofloxacin is rifampicin-levofloxacin combinations in
indifferent and ineffective in the mana- the groups that killed 48 h after the
gement of acute murine brucellosis. The cessation of antibiotic treatment, had no
results also appear to support the clinical prophylactic efficacy against B. melitensis
superiority of combination drug therapy infection. On the contrary, doxycycline-
over monotherapy. On another hand, ciprofloxacin and rifampicin-levofloxacin
Grillo et al. (2006) found that the combi- combinations had almost the same good
nations doxycycline-gentamicin and efficacy as doxycycline-rifampicin combi-
doxycycline-rifampicin were effective in nation when a low concentration of B.
the clearance of Rev 1 infection, but only melitensis 16M (104 CFU) was used. In
doxycycline-gentamicin combination im- addition, the doxycycline-levofloxacin
proved significantly the therapeutic effica- combination showed a moderate prophy-
cy as compared with that of the antibiotics lactic effect. Finally, the rifampicin-cipro-
given alone. As a prophylactic agent floxacin combination showed relatively
against bioterrorism organisms, ciproflo- good activity only in the groups that killed
xacin is recommended for post-exposure 30 days after the cessation of antibiotic
prophylaxis against Yersinia pestis (Rus- treatment.
sell et al., 1996), tularaemia (Russell et Nevertheless, Al Sibai et al. (1992), in
al., 1998), and systemic anthrax (Steward a prospective study, reported high
et al., 2004). Therefore, ciprofloxacin, and probabilities of brucellosis relapse after
fluoroquinolones in general, might also be monotherapy with ciprofloxacin (26.7%).
potentially useful for prophylaxis of Also, in 480 patients with various forms
Brucella infection (Atkins et al., 2009a). of brucellosis, Aygen et al. (2002)
Data reported by Shasha et al. (1992) revealed that the probabilities of relapse
indicate that mice treated with ciproflo- for the various treatment regimens were
ciprofloxacin and doxycycline against ex- the spine. Neurosurgery, 33, 838–844.
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Journal of Infection, 36, 85–92. Correspondence:
Steward, J., M. S. Lever, A. J. Simpson, A. M.
Mazen Safi M.D., PhD
Sefton & T. J. Brooks, 2004. Post-
Department of Molecular Biology
exposure prophylaxis of systemic anthrax
and Biotechnology,
in mice and treatment with fluoroquino-
Atomic Energy Commission,
lones. Journal of Antimicrobial Chemo-
P.O. Box 6091, Damascus, Syria
therapy, 54, 95–99.
Tel: +963.11.213580; Fax: +963.11.6112289
Tekkok, I. H., M. Berker, O. E. Ozcan, T. e-mail: ascientific@aec.org.sy
Ozgen & E. Akalin, 1993. Brucellosis of