Bartonella Infection: Treatment and Drug Resistance

Silpak Biswas; Jean-Marc Rolain

Disclosures

Future Microbiol. 2010;5(11):1719-1731. 

In This Article

Antibiotic Treatment of Bartonellosis

The treatment of Bartonella infections with antibiotics depends on the clinical presentation of the disease and the immune status of the patient, thus the current recommendations for treatment must be adapted to each clinical situation.[3]

Antibiotic Susceptibility Testing Techniques

Agar Dilution Method The agar dilution method is used for in vitro antibiotic susceptibility testing for Bartonella isolates, as described previously by Maurin et al..[90] Bartonella strains were grown on Columbia agar supplemented with 5% sheep blood. The cultures were additionally supplemented with twofold serial dilutions of the antibiotic of interest. The cells were harvested after 5 days of incubation and were suspended in phosphate buffer (pH 7.4). Tenfold dilution of bacterial suspensions at a concentration equivalent to that of the McFarland 0.5 standard were used for the antibiotic assays; the concentration corresponds to approximately 106 colony-forming unit/ml, as determined using the colony-forming unit technique. A total of 10µl of each bacterial suspension was plated onto blood-supplemented agar. The plates were incubated at 37°C in a 5% CO2 atmosphere. Bacterial growth was evaluated after 5 days of incubation by comparison with growth on antibiotic-free agar controls. The minimum inhibitory concentration (MIC) value is defined as the first antibiotic concentration allowing inhibition of growth after 5 days of incubation.

Etest Assay Etest assay has been recently used to evaluate susceptibility to antibiotics in Bartonella. The gradient of antibiotic covers a stable, continuous and exponential gradient of antibiotic concentration directly underneath the strip. After incubation, when bacterial growth becomes visible, a symmetrical inhibition ellipse centered along the strip is seen. The MIC (in µg/ml) is read directly from the scale where the ellipse edge intersects the strip. Bartonella isolates are grown on Columbia 5% sheep blood agar plates, and the antibiotic susceptibility testing of all Bartonella isolates is carried out using the available Etest strips for different antibiotics, as recommended by the manufacturer. The MIC is measured after an incubation of 5–12 days[91–93] (Figure 1). A previous study has shown that the MIC results obtained using Etest were reliable and correlated well with the results using agar dilution method.[91]

Figure 1.

Antibiotic susceptibility testing using the Etest assay for Bartonella henselae with rifampin Estrip showing minimum inhibitory concentration value. (A) E-strip. (B) Zone of growth inhibition showing minimum inhibitory concentration value.

Antibiotic Susceptibility Results

Based on in vitro testing, Bartonella species are susceptible to many antibiotics, including penicillin and other cephalosporin-based compounds, (e.g., aminoglycosides, chloramphenicol, tetracyclines, macrolide compounds, rifampin, fluoroquinolones and co-trimoxazole). However, the in vitro sensitivity results do not consistently correlate with the in vivo patient data; for instance, penicillin has no in vivo efficacy despite the very low MICs observed in vitro.[3,90,94] Agar-based susceptibility studies also demonstrated that many antibiotics are only bacteriostatic against Bartonella species in vitro.[95–97] Previous studies demonstrated that, in vitro, aminoglycosides are the only class of antibiotics that are bactericidal against Bartonella species grown either in liquid medium or in endothelial cells.[98,99]

Mechanisms of Resistance to Antibiotics in Bartonella

The chief mechanisms by which antimicrobial agents act are interference with nucleic acid synthesis, binding to the ribosome, and the inhibition of cell wall synthesis and folate metabolism.[100] Bacteria can develop resistance to antibiotics by two genetic processes. First, mutation and selection (vertical gene transfer) and second, exchange of genes between strains and species (horizontal gene transfer). For intracellular bacteria including Bartonella species, antibiotic resistance is mainly due to spontaneous mutations or intrinsic mutations in the target genes (i.e., vertical gene transfer), which are reviewed later in this article. However, we have recently demonstrated for the first time the possibility of lateral gene transfer of a conjugative plasmid between Bartonella rattaustraliani and other bacteria including B. henselae or rhizobiales.[101] This may suggest that antibiotic resistance genes could be transferred laterally and that this should be further investivated in the future.

Natural Antibiotic Resistance Heterogeneity of susceptibility of 20 new Bartonella isolates to fluoroquinolones isolated from Australian mammals has recently been investigated in one of our studies.[102] In this study we found that ciprofloxacin was more effective in vitro than ofloxacin. This heterogeneity was linked to a natural mutation, Ser 83→Ala, in the quinolone resistance determining region (QRDR) of gyrA. Interestingly, an in silico genome analysis study[103] revealed a natural mutation at position 83 of the QRDR region (Ser 83→Ala) of gyrA present in three Bartonella species (B. bacilliformis, B. quintana and B. henselae). Many studies have demonstrated that species naturally bearing a serine residue at position 83 of gyrA are usually susceptible to fluoroquinolones, while the presence of an alanine at this critical position usually corresponds to natural resistance to these antibiotics.[104–106]

Similarly, an A2059G transition in the 23S rRNA-encoding gene responsible for erythromycin resistance was detected in one of the 15 lymph nodes from patients with CSD.[107] This node was excised from a 10-year-old female who was not treated with antibiotics prior to excision, suggesting that naturally occurring erythromycin-resistant strains may infect humans.

In vitro Antibiotic Resistance Recently, specific antibiotic resistance mutations have been characterized in B. henselae, B. quintana and B. bacilliformis, selected by in vitro serial passages (Table 1).[107–109]

In Bartonella species, different mechanisms of erythromycin resistance (Figure 2A) have been reported in in vitro studies. Previously, we have demonstrated that the fully erythromycin-resistant strain of B. quintana obtained after the 16th passage in vitro harbored a repeat insertion of 27 bases in the ribosomal protein L4, resulting in an insertion of nine repeated amino acids between amino acids R71 and A72 in the highly conserved region of the protein.[110] Recently, we have reported several mutations in the 23S rRNA encoding gene and the L4 ribosomal protein in B. henselae strain Marseille and in other B. henselae erythromycin-resistant mutants in vitro. Most of the mutations in the 23S rRNA-encoding gene (e.g., A2058G, A2058C and C2611T) have been previously shown to confer erythromycin resistance in other bacteria. We found amino acid mutations at two different positions (G71R and H75Y) in the L4 ribosomal protein in erythromycin-resistant mutants of B. henselae.[107] An A2058G mutation in an erythromycin-resistant strain of B. bacilliformis has also been reported by our team.[109] A more recent study demonstrated that azithromycin was effective only until the second passage for B. henselae isolates obtained from cats.[111] Compared with the parental strain, each azithromycin-resistant B. henselae mutant had a homogeneous single nucleotide substitution at position 2058 (A2058G, Escherichia coli numbering) in the 23S rRNA encoding gene..[111,112]

Figure 2.

Molecular mechanisms of antibiotic resistance in Bartonella spp. (A) Mechanism of macrolide resistance due to change in 50S ribosomal subunit and (B) mechanism of aminoglycoside resistance due to change in 30S ribosomal subunit. (C) Mechanism of rifampin resistance due to change in rpoB gene in RNA polymerase and (D) mechanism of fluoroquinolone resistance due to change in gyrA gene in DNA gyrase.

We have also selected a gentamicin-resistant strain of B. henselaein vitro.[113] The 16S rRNA-encoding gene, the candidate gene for gentamicin resistance (Figure 2A), was characterized by sequence analysis. The gentamicin-resistant mutant of B. henselae carried an A1408G mutation in the 16S rRNA-encoding gene, as depicted by the double A/G peak. Additionally, this mutation is the most frequently found mutation in gentamicin-resistant clinical isolates in other bacterial species.[114–117] Although we obtained a gentamicin-resistant mutant in vitro, this mutant was obtained after nine passages (18 weeks), suggesting that the selection of such gentamicin-resistant strains is not likely to occur in vivo.[113]

Fluoroquinolones have been widely used for the treatment of Bartonella infections in humans and in veterinary medicine. However, fluoroquinolones alone should not be used for the treatment of bartonellosis since there is an intrinsic low level of resistance due to the gyrA mutation. Moreover, high level of resistance to fluoroquinolones is easily obtained in vitro. Alteration of the target enzymes appears to be the most dominant factor in the development of resistance to quinolones (Figure 2B).[103,118] The small region from codons 67–106 of gyrA in E. coli was designated the QRDR. Variations in the QRDR region were found in species with natural resistance to fluoroquinolones.[119–123] In 2003, Minnick et al. isolated and characterized B. bacilliformis mutants that were resistant to ciprofloxacin.[124] In 2007, we obtained a ciprofloxacin-resistant strain of B. bacilliformis in vitro; the strain contained a transition from C to T at position 549 (E. coli numbering) of the gyrA gene, encoding the predicted amino acid change Asp 87→Asn in gyrA.[109] This same mutation (Asp 87→Asn) has also been recently found in ciprofloxacin-resistant strains of B. henselae and B. quintana (Table 1).[108]

Another recent study demonstrated that B. henselae isolates obtained from cats became resistant to pradofloxacin and enrofloxacin (both are fluoroquinolones that are mainly used in veterinary medicine) after different numbers of passages.[112] Compared with the parental B. henselae strains, the pradofloxacin- and enrofloxacin-resistant mutants had an amino acid change from serine to valine at position 83 (E. coli numbering) in gyrA.[112] The Ser 83→Val mutation found in pradofloxacin- and enrofloxacin-resistant mutants in this study had been reported previously by Tavío et al.[125] in a fluoroquinolone-resistant E. coli isolate.

Finally, amino acid substitutions in the RNA polymerase and point mutations in the rpoB gene have been demonstrated after in vitro selection of rifampin-resistant (Figure 2B) strains of B. bacilliformis[109] and B. quintana[126] by our group. These strains showed a mutation at serine 531 (Ser→Phe) in the rifampin-resistance determining region of the rpoB gene. Amino acid 531 is one of the most frequently mutated sites conferring rifampin resistance in other bacterial species (Table 1).[127–129]

Treatment of bartonellosis in Animals

No antibiotics have been shown to be fully effective against Bartonella infections in cats and dogs. In previous studies by Kordick et al., doxycycline and enrofloxacin appeared to be effective against Bartonella infection in cats. In the study, 22.7 mg of enrofloxacin was given orally every 12 h and 25 mg of doxycycline was given orally every 12 h; the treatment duration was for 14–28 days. Bacteremia in naturally infected cats with chronic infection was successfully cleared from nine out of 14 cats treated with enrofloxacin and from only two out of eight cats treated with doxycycline.[130] Interestingly, azithromycin, a macrolide compound with good intracellular penetration, has seemingly become the drug of choice to treat cats and dogs with B. henselae infections. However, relapses after antibiotic withdrawal have also been reported for this treatment.[4,131,132] Doxycycline and enrofloxacin may be used for the treatment of cats and fluoroquinolones with doxycycline or azithromycin may be used for the treatment of dogs.[3,130,201] However, because many different treatment regimens have been tested, it is difficult to draw any conclusion about the efficacy of fluoroquinolone alone or in combination. Finally, treatment of cats against fleas is also critical to avoid human transmission.

Treatment of Bartonellosis in Humans

The treatment recommendations for infections caused by Bartonella species are described in Table 2. Doxycycline and erythromycin are the most frequently recommended antibiotics used for the treatment of Bartonella infection in humans, however, clinical improvement has been reported following the use of penicillin, gentamicin, ceftriaxone, ciprofloxacin and azithromycin.[3,133]

Cat scratch disease does not typically respond well to antibiotic therapy.[3] Numerous reports have evaluated the effectiveness of many antimicrobial agents for the treatment of typical, uncomplicated CSD.[71] Most investigators have observed no benefit with antibiotic treatment, whereas anecdotal reports have indicated that ciprofloxacin, rifampin and co-trimoxazole may be effective.[133] In a prospective, randomized, double-blinded, placebo-controlled study that addressed antibiotic treatment of CSD in humans performed by Bass et al.,[134] azithromycin administered orally for 5 days was considered to be effective in decreasing lymph node size over the first 4 weeks of therapy. However, enlargement of different lymph nodes or an increase in the size of the original lymph node occurred in some study subjects despite azithromycin therapy.

In typical CSD, an antibiotic treatment is not recommended even if azithromycin could be useful for patients with large and bulky lymphadenopathy.[134] For atypical presentation of CSD antibiotic treatment is needed and a combination of doxycycline and rifampin has been proposed for neuroretinitis and encephalopathy.[3]

During World War I, soldiers with trench fever cleared the infection in the absence of antibiotic treatment. However, successful treatment of some trench fever patients with tetracycline or chloramphenicol was reported after World War II, although these data remain anecdotal.[135] A randomized clinical trial by Foucault et al. reported that homeless people with episodes of B. quintana bacteremia should be treated with a combination of gentamicin and doxycycline.[136] Results showed eradication of bacteremia in seven out of nine treated patients compared with two out of 11 untreated controls. Patients with acute B. quintana bacteremia could be treated with gentamicin in combination with doxycycline for 28 days.[3]

Treatment of Bartonella endocarditis is critical since the death rate and valvular surgery are higher in these patients.[77,81] Raoult et al. have reported that the recovery rate of patients was higher when aminoglycosides were used in combination with β-lactam or with other antibiotics. Thus, the recommendation for patients with Bartonella endocarditis is doxycycline for 6 weeks plus gentamicin for 14 days.[77,81]

Bacillary angiomatosis and PH should be treated with erythromycin for 3–4 months as the first-line antibiotic treatment.[3] Although erythromycin has an antibiotic activity against Bartonella, it has been demonstrated that erythromycin also had an anti-angiogenic effect on endothelial cells that may contribute to its good in vivo activity. Doxycycline may be used as an alternative regimen. The duration of treatment with erythromycin is critical (3 months for bacillary angiomatosis and 4 months for PH) to limit relapses.

Penicillin G, chloramphenicol, tetracycline, streptomycin and erythromycin have been used for the treatment of Oroya fever, which is caused by B. bacilliformis.[3] Fluoroquinolones have been successfully used for the treatment of Oroya fever but we do not recommend their use alone, since there is an intrinsic low level of resistance to fluoroquinolones in the Bartonella genus due to an intrinsic mutation in DNA gyrase. As an alternative treatment, chloramphenicol alone or in combination with a β-lactam or ciprofloxacin could be used.

Since 1975, rifampin has become the drug of choice for the treatment of verruga peruana.[137] However, failure of rifampin treatment has also been reported,[54] which could be due to resistant strains that are easily obtained in vitro.[109] In one of our recent studies, we recommended doxycycline combined with gentamicin as the preferred regimen for the treatment of the chronic phase of Carrion's disease.[109]

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