Bactericidal effect of antibiotics on Bartonella and Brucella spp.: clinical implications

Jean-Marc Rolain, Max Maurin and Didier Raoult*

a Unité des Rickettsies CNRS UPRES-A 6020, Faculté de Médecine, Université de la Méditerranée, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The species Bartonella and Brucella are phylogenetically closely related bacteria, both of which can produce chronic infections in humans that are difficult to cure with antibiotics. MICs of antibiotics for both species correlate poorly with the in vivo efficacy of the antibiotics. In this study we have determined MBCs of several antibiotics for this group of pathogens. Only the aminoglycosides were bactericidal, and this correlates well with the usefulness of these antibiotics for the therapy of human brucellosis and chronic Bartonella spp. infections such as endocarditis. Our data indicate that current clinical experience in treating brucellosis may help to define better the optimum antibiotic therapy for Bartonella-related diseases.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bartonella and Brucella spp. belong to the {alpha}2 subgroup of Proteobacteria (Figure 1). The genus Bartonella includes 12 validated species, five of which have been associated with human infections: Bartonella henselae, Bartonella quintana, Bartonella elizabethae, Bartonella clarridgeiae and Bartonella bacilliformis. The spectrum of diseases caused by Bartonella spp. includes cat scratch disease, bacillary angiomatosis, peliosis hepatis, endocarditis, trench fever, bacteraemia in homeless people and Carrion's disease which manifests as Oroya fever or verruga peruana.1 Infections caused by Brucella spp. are common zoonoses in many parts of the world and may present with a broad spectrum of clinical manifestations.2

These pathogens are Gram-negative bacteria that can be grown in vitro in axenic blood-enriched media. MICs have been determined using methods adapted to their fastidious growth and have shown that many antibiotics are bacteriostatic in vitro against Brucella and Bartonella spp.3,4 However, MICs are poorly correlated with the in vivo efficacy of antibiotics in patients suffering from either brucellosis3 or Bartonella-related infections.4 We hypothesized that bactericidal activity of antibiotics against this group of pathogens may be more critical in predicting their efficacy in humans, especially in chronic or relapsing infections. Thus, we have determined MBCs of several antibiotics for these bacteria and tentatively correlate our results with current clinical experience.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains are listed in the TableGo. All bacteria were grown on Columbia agar (bioMérieux, Lyon, France), in a 5% CO2-enriched atmosphere at 37°C, except for B. bacilliformis which was grown at 30°C without CO2.


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Table. MICs and MBCs (mg/L) for Brucella, Bartonella henselae, Bartonella quintana and Bartonella bacilliformis
 
Drugs

The antibiotics tested were amoxycillin (Beecham–Sevigne, Paris, France), gentamicin (Dakota Pharm, Creteil, France), streptomycin (Diamant, Puteaux, France), ciprofloxacin (Bayer Pharma, Sebs, France), erythromycin (Abbott, Rungis, France), rifampicin (Cassenne, Puteaux, France) and doxycycline (Pfizer, Neuilly, France).

MIC determination

A modified version of the antibiotic agar dilution method of the National Committee for Clinical Laboratory Standards (NCCLS) was used for determination of MICs, as described previously.4,5 The optimum time for visualization of bacterial growth was 3 days for Brucella spp., 5 days for B. quintana and B. henselae, and 6 days for B. bacilliformis. The MIC was defined as the lowest concentration of the antibiotic tested giving complete inhibition of bacterial growth as compared with a drug-free control.

MBC determination

The bactericidal activity of antibiotics was determined in a broth assay with Schaedler medium (bioMérieux) supplemented with either 5% or 10% sheep blood, respectively, for Brucella and Bartonella spp. A series of glass tubes were filled with 0.9 mL of a bacterial inoculum of 106 cfu/mL and 0.1 mL of two-fold serial dilutions of each antibiotic tested. One tube receiving 0.1 mL of saline served as a growth control. Tubes were incubated for 24 h, according to NCCLS guidelines, at 30°C for B. bacilliformis or 37°C for other species. After incubation for 24 h, 10-fold serial dilutions of the different bacterial suspensions were then plated on to blood agar (bioMérieux) and reincubated for 3–6 days before enumeration of colonies. The MBC was defined as the lowest concentration of the antibiotic inducing a 99.9% decrease in bacterial inocula following the 24 h incubation period, as compared with the primary inoculum dose.

Controls

Escherichia coli C.I.P. 53126 and Staphylococcus aureus C.I.P. 103811 (Institut Pasteur, Marnes-la-Coquette, France) were used as controls. The medium used was either Mueller–Hinton or Columbia agar (bioMérieux) enriched with 10% horse blood. MICs were evaluated after 1, 3, 5 and 6 days of incubation. For determination of MBCs, bacterial suspensions (106 cfu/mL) were incubated for 24 h in liquid Schaedler medium at 37°C, with or without horse blood, and in the presence or absence of CO2. After 1 day of exposure to antibiotics, 10-fold serial dilutions of each bacterial suspension were plated on to agar (Mueller–Hinton or Columbia) and colonies were counted after various incubation times.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
MICs and MBCs for the E. coli and S. aureus control strains, as determined in Mueller–Hinton agar, were similar to those determined by the Pasteur Institute and conditions of culture did not influence the MIC and MBC results more or less than one dilution. Brucella and Bartonella spp. strains showed similar antibiotic susceptibilities (TableGo). All strains were highly susceptible to the ß-lactams amoxycillin and ceftriaxone, aminoglycosides, doxycycline, rifampicin, erythromycin and ciprofloxacin (TableGo). In contrast, in our experimental conditions, only the aminoglycosides, and to a lesser extent doxycycline were bactericidal (TableGo).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We found in this study that both the Brucella and Bartonella spp. were highly susceptible to most antibiotics tested, which is consistent with previous reports.3,4,6 However, MICs do not correlate well with clinical experience.1,3 Failures of monotherapy using a ß-lactam, a macrolide, a tetracycline, rifampicin or a fluoroquinolone for either disease have been reported.1,3,4 Relapses are also frequent following withdrawal of treatment. Such discrepancies between in vitro and clinical data may be explained by the lack of bactericidal activity of most antibiotics against these pathogens. In our study only aminoglycosides displayed a bactericidal activity against both Brucella and Bartonella spp. strains tested at concentrations achievable in human serum. MBC results for B. henselae were consistent with a previous report from our team.7 Rubinstein et al.6 also reported that only streptomycin exhibited a bactericidal activity against Brucella spp. within the first 24 h of antibiotic exposure, whereas a bactericidal activity was shown with minocycline, rifampicin and ciprofloxacin after 48 h of antibiotic exposure. Fluoroquinolones have been shown to be bactericidal against Brucella spp. in vitro,6 but these results are controversial.8 Bartonella and Brucella spp. share a common ability to multiply within eukaryotic cells, which allows them to be protected against antibiotics. The in vitro activity of aminoglycosides against intracellular Brucella sp. has been demonstrated previously.3 Moreover, we have shown that only aminoglycosides were bactericidal against B. henselae within a cell system.7

Current knowledge indicates that brucellosis and Bartonella-related infections share comparable microbiological, pathophysiological and clinical findings. Brucellosis and Bartonella spp. infections are characterized mainly by two clinical forms: an acute and a chronic stage. There is a broad spectrum of chronic infections due to Bartonella or Brucella spp.: endocarditis, cutaneous tumour (verruga peruana due to B. bacilliformis and bacillary angiomatosis due to B. henselae and B. quintana) and osteoarticular damage. Combinations of doxycycline plus streptomycin or rifampicin are recommended for human brucellosis,3 and only streptomycin is considered consistently effective for the treatment of verruga peruana.9 Aminoglycosides, especially gentamicin, are also used to treat patients with Bartonella-related endocarditis. In our experience (unpublished results) as well as in that reported in the literature,1 all patients treated with a combination of tetracycline for 6 weeks and aminoglycoside for 2 weeks recovered fully without any relapse, although valve replacement was required for most of the patients, because of extensive valvular damage.10

In conclusion, because human infections with Brucella and Bartonella spp. share many similarities discussed previously, we propose that our current knowledge in treating human brucellosis may help to define the optimum treatment for infections due to Bartonella spp. In particular, the use of an aminoglycoside seems critical in patients with endocarditis.



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Figure. Phylogenetic relationships in the {alpha}2 subgroup of Proteobacteria and bactericidal activity of antibiotics.

 

    Acknowledgments
 
The authors thank Richard and Emma Birtles for reviewing the manuscript.


    Notes
 
* Corresponding author. Tel: +33-4-91-32-43-75; Fax: +33-4-91-38-77-72; E-mail: Didier.Raoult{at}medecine.univ-mrs.fr Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Spach, D. H. & Koehler, J. E. (1998). Bartonella-associated infections. Infectious Diseases Clinics of North America 12, 137–55.

2 . Corbel, M. J. (1997). Brucellosis: an overview. Emerging Infectious Diseases 3, 213–21.[ISI][Medline]

3 . Hall, W. H. (1990). Modern chemotherapy for brucellosis in humans. Review of Infectious Diseases 12, 1060–99.[ISI][Medline]

4 . Maurin, M., Gasquet, S., Ducco, C. & Raoult, D. (1995). MICs of 28 antibiotic compounds for 14 Bartonella (formerly Rochalimaea) isolates. Antimicrobial Agents and Chemotherapy 39, 2387–91.[Abstract]

5 . Sobraques, M., Maurin, M., Birtles, R. J. & Raoult, D. (1999). In vitro susceptibilities of four Bartonella bacilliformis strains to 30 antibiotic compounds. Antimicrobial Agents and Chemotherapy 43, 2090–2.

6 . Rubinstein, E., Lang, R., Shasha, B., Hagar, B., Diamanstein, L., Joseph, G. et al. (1991). In vitro susceptibility of Brucella melitensis to antibiotics. Antimicrobial Agents and Chemotherapy 35, 1925–7.[ISI][Medline]

7 . Musso, D., Drancourt, M. & Raoult, D. (1995). Lack of bactericidal effect of antibiotics except aminoglycosides on Bartonella (Rochalimaea) henselae. Journal of Antimicrobial Chemotherapy 36, 101–8.[Abstract]

8 . Garcia-Rodriguez, J. A., Garcia Sanchez, J. E. & Trujillano, I. (1991). Lack of effective bactericidal activity of new quinolones against Brucella spp. Antimicrobial Agents and Chemotherapy 35, 756–9.[ISI][Medline]

9 . Vargas, C. M. (1998). In Bartonellosis or Carrion Disease. New Aspects of an Old Disease, 1st edn, pp. 117–22. A.F.A. Editores Importadores S.A., Lima, Peru.

10 . Drancourt, M., Mainardi, J. L., Brouqui, P., Vandenesch, F., Carta, A., Lehnert, F. et al. (1995). Bartonella (Rochalimaea) quintana endocarditis in three homeless men. New England Journal of Medicine 332, 419–23.[Abstract/Free Full Text]

Received 15 February 2000; returned 11 May 2000; revised 20 June 2000; accepted 5 July 2000